Reviewer #1 (Public Review):

Summary:

This is an important study that tests the effects of using neurofeedback, in the form of reward delivery when large sharp wave-ripples (SWRs) are detected, on neurophysiological and behavioral measures. The authors report that the rate of SWRs ripples increased prior to reward delivery, but this increased rate of SWRs had no significant effect on memory performance. They also found that compensatory decreases in SWR rate occurred in the period after reward delivery such that the overall SWR rate remained stable.

Strengths:

The study has many strengths. The paradigm of closed loop detection of SWRs and reward delivery is powerful and provides an innovative way to causally test the effects of increasing SWR rates. Other studies could adopt this method to test other hypotheses or to assess the effects of increasing SWR rates prior to reward delivery in rodent models of brain disorders. The methods and results are clearly explained. The results are presented in a transparent way.

Weaknesses:

In the linear mixed effects model analysis used in Figure 2, and statistics reported in the figure legend, an interaction effect showing that neurofeedback differentially affected the SWR rate and count pre- and post-award seems to be missing in the reported statistics.

In the Discussion, the authors write, "Further, because subjects learn to modulate SWR rate, rather than simply generating a single suprathreshold event on command, it is likely that they learn to engage a SWR-permissive state during the targeted interval in which brain-wide neural activity and neuromodulatory tone also enter a SWR-permissive realm". This seems to imply that the neurofeedback is directly modulating neural activity. However, it is unclear from the paper exactly how the neurofeedback is modulating the SWR rate. Considering that SWRs occur during immobility, is it possible that the animals are learning to remain more immobile and modulating the SWR rate in that way?

Reviewer #1 (Public Review):

Summary:<br /> The paper presents a nice study investigating differences in biological motion perception in participants with ADHD in comparison with controls. Motivated by the idea that there is a relationship between biological motion perception and social capabilities, the authors investigated local and global (holistic) biological motion perception, the group, and several additional behavioral variables that are affected in ADHS (IQ, social responsiveness, and attention/impulsivity). As well as local global biological motion perception is reduced in ADHD participants. In addition, the study demonstrates a significant correlation between local biological motion perception skills and the social responsiveness score in the ADHD group, but not the controls. A path analysis in the ADHD data suggests that general performance in biological motion perception is influenced mainly by global biological motion perception performance and attentional and perceptual reasoning skills.

Strengths:<br /> It is true that there exists not much work on biological motion perception and ADHD. Therefore, the presented study contributes an interesting new result to the biological motion literature and adds potentially also new behavioral markers for this clinical condition. The design of the study is straightforward and technically sound, and the drawn conclusions are supported by the presented results.

Weaknesses:<br /> Some of the claims about the relationship between genetic factors and ADHD and the components of biological motion processing have to remain speculative at this point because genetic influences were not explicitly tested in this paper.

Reviewer #1 (Public Review):

This work describes a new and powerful approach to a central question in ecology: what are the relative contributions of resource utilisation vs interactions between individuals in the shaping of an ecosystem? This approach relies on a very original quantitative experimental set-up whose power lies in its simplicity, allowing an exceptional level of control over ecological parameters and of measurement accuracy.

In this experimental system, the shared resource corresponds to 10^12 copies of a fixed single-stranded target DNA molecule to which 10^15 random single-stranded DNA molecules (the individuals populating the ecosystem) can bind. The binding process is cycled, with a 1000x-PCR amplification step between successive binding steps. The composition of the population is monitored via high-throughput DNA sequencing. Sequence data analysis describes the change in population diversity over cycles. The results are interpreted using estimated binding interactions of individuals with the target resource, as well as estimated binding interactions between individuals and also self-interactions (that can all be directly predicted as they correspond to DNA-DNA interactions). A simple model provides a framework to account for ecosystem dynamics over cycles. Finally, the trajectory of some individuals with high frequency in late cycles is traced back to the earliest cycles at which they are detected by sequencing. Their propensities to bind the resource, to form hairpins, or to form homodimers suggest how different interaction modes shape the composition of the population over cycles.

The authors report a shift from selection for binding to the resource to interactions between individuals and self-interactions over the course of cycles as the main drivers of their ecosystem. The outcome of the experiment is far from trivial as the individual-resource binding energy initially determines the relative enrichment of individuals, and then seems to saturate. The richness of the population dynamics observed with this simple system is thus comparable to that found in some natural ecosystems. The findings obtained with this new approach will likely guide the exploration of natural ecosystems in which parameters and observables are much less accessible.

My review focuses mainly on the experimental aspects of this work given my own expertise. The introduction exposes very convincingly the scientific context of this work, justifying the need for such an approach to address questions pertaining to ecology. The manuscript describes very clearly and rigorously the experimental set-up. The main strengths of this work are (i) the outstanding originality of the experimental approach and (ii) its simplicity. With this setup, central questions in ecology can be addressed in a quantitative manner, including the possibility of running trajectories in parallel to generalize the findings, as reported here. Technical aspects have been carefully implemented, from the design of random individuals bearing flanking regions for PCR amplification, binding selection and (low error) amplification protocols, and sequencing read-out whose depth is sufficient to capture the relevant dynamics. One missing aspect in the data analysis is the quantification of the effect of PCR amplification steps in shaping the ecosystem (to be modeled if significant). In addition, as it stands the current work does not fully harness the power of the approach. For instance, with this setup, one can tune the relative contributions of binding selection vs amplification for instance (to disentangle forces that shape the ecosystem). One can also run cycles with new DNA individuals, designed with arbitrarily chosen resource binding vs self-binding, that are predicted to dominate depending on chosen ecological parameters.

Reviewer #1 (Public Review):

In this study, the authors obtained multiple, novel and compelling datasets to better understand the relationship between histone H1 and RNA-directed DNA methylation in plants. Most of the authors' claims concerning H1 and RNA polymerase V (Pol V) are backed by convincing and independent lines of evidence. However, the authors also make some overly broad conclusions, for which additional experiments/data analyses should be explored to improve confidence in their findings. Furthermore, Pol V produces noncoding transcripts that act as scaffold RNAs, which AGO4-bound siRNAs recognize in plant chromatin to mediate RNA-directed DNA methylation. Detection of Pol V transcript products at sites of Pol V redistribution in h1 mutants would significantly enhance the impact of this manuscript. Below I have listed several strengths and weaknesses of the manuscript.

Strengths<br /> 1. The authors report high-quality NRPE1 ChIP-seq data, allowing them to directly test how and where Pol V occupancy depends on histone H1 function in Arabidopsis.<br /> 2. nrpe1 mutants generated via CRISPR/Cas9 in the h1 mutant background (nrpe1 h1.1-1 h1.2-1 triple mutants), allow the authors to study the role of Pol V in ectopic DNA methylation in H1-deficient plants.<br /> 3. Pol V recruitment via ZincFinger-DMS3 expression (a modified version of Pol V's DMS3 recruitment factor) sends Pol V to new genomic loci and thus provides the authors with an innovative dataset for understanding H1 function at these sites.

Weaknesses<br /> 1. The manuscript does not include detection or quantification of Pol V transcripts generated at ectopic sites in the h1 mutant background.<br /> 2. Statistical tests are missing throughout and are needed to support several of the authors' claims.<br /> 3. The SUVH1-3xFLAG ChIP-seq analyses in Fig. 6 require additional controls and are not fully explained in the results. The broad conclusions drawn (based on those experiments) are thus not convincing.

Previous studies have charted the relationship between H1 function and RNA-directed DNA methylation (RdDM) via analyses of Pol IV-dependent 24 nt siRNAs and factors that recruit Pol IV (Choi et al., 2021 and Papareddy et al., 2020). Harris and colleagues have extended this work and shown that histone H1 function also antagonizes Pol V occupancy in the context of constitutive heterochromatin. The authors thus provide important evidence to show that H1 limits the encroachment of both polymerases Pol IV and Pol V into plant heterochromatin.

Reviewer #1 (Public Review):

People can perform a wide variety of different tasks, and a long-standing question in cognitive neuroscience is how the properties of different tasks are represented in the brain. The authors develop an interesting task that mixes two different sources of difficulty, and find that the brain appears to represent this mixture on a continuum, in the prefrontal areas involved in resolving task difficulty. While these results are interesting and in several ways compelling, they overlap with previous findings and rely on novel statistical analyses that may require further validation.

Strengths<br /> 1. The authors present an interesting and novel task for combining the contributions of stimulus-stimulus and stimulus-response conflict. While this mixture has been measured in the multi-source interference task (MSIT), this task provides a more graded mixture between these two sources of difficulty.

2. The authors do a good job triangulating regions that encoding conflict similarity, looking for the conjunction across several different measures of conflict encoding. These conflict measures use several best-practice approaches towards estimating representational similarity.

3. The authors quantify several salient alternative hypothesis and systematically distinguish their core results from these alternatives.

4. The question that the authors tackle is important to cognitive control, and they make a solid contribution.

Concerns<br /> 1. The evidence from this previous work for mixtures between different conflict sources makes the framing of 'infinite possible types of conflict' feel like a strawman. The authors cite classic work (e.g., Kornblum et al., 1990) that develops a typology for conflict which is far from infinite. I think few people would argue that every possible source and level of difficulty will have to be learned separately. This work provides confirmatory evidence that task difficulty is represented parametrically (e.g., consistent with the n-back, MOT, and random dot motion literature).

2. The degree of Stroop vs Simon conflict is perfectly negatively correlated across conditions. This limits their interpretation of an integrated cognitive space, as they cannot separately measure Stroop and Simon effects. The author's control analyses have limited ability to overcome this task limitation. While these results are consistent with parametric encoding, they cannot adjudicate between combined vs separated representations.

Reviewer #1 (Public Review):

Summary:<br /> The authors developed computational models that capture the electrical and Ca2+ signaling behavior in mesenteric arterial cells from male and female mice. A baseline model was first formulated with eleven transmembrane currents and three calcium compartments. Sex-specific differences in the L-type calcium channel and two voltage-gated potassium channels were then tuned based on experimental measurements. To incorporate the stochastic ion channel openings seen in smooth muscle cells under physiological conditions, noise was added to the membrane potential and the sarcoplasmic Ca2+ concentration equations. Finally, the models were assembled into 1D vessel representations and used to investigate the tissue-level electrical response to an L-type calcium channel blocker.

Strengths:<br /> A major strength of the paper is that the modeling studies were performed on three different scales: individual ionic currents, whole-cell, and 1D tissue. This comprehensive computational framework can help provide mechanistic insight into arterial myocyte function that might be difficult to achieve through traditional experimental methods.

The authors aimed to develop sex-specific computational models of mesenteric arterial myocytes and demonstrate their use in drug-testing applications. Throughout the paper, model behavior was both validated by experimental recordings and supported by previously published data. The main findings from the models suggested that sex-specific differences in membrane potential and Ca2+ handling are attributable to variability in the gating of a small number of voltage-gated potassium channels and L-type calcium channels. This variability contributes to a higher Ca2+ channel blocker sensitivity in female arterial vessels. Overall, the study successfully met the aims of the paper.

Weaknesses:<br /> A main weakness of the paper, as addressed by the authors, is the simplicity of the 1D vessel model; it does not take into account various signaling pathways or interactions with other cell types which could impact smooth muscle electrophysiology. Another potential shortcoming is the use of mouse data for optimizing the model, as there could be discrepancies in signaling behavior that limit the translatability to human myocyte predictions.

Reviewer #1 (Public Review):

The authors previously showed in cell culture that Su(H), the transcription factor mediating Notch pathway activity, was phosphorylated on S269 and they found that a phospho-deficient Su(H) allele behaves as a moderate gain of Notch activity in flies, notably during blood cell development. Since a downregulation of Notch signaling was proposed to be important for the production of a specialized blood cell types (lamellocytes) in response to wasp parasitism, the authors hypothesized that Su(H) phosphorylation might be involved in this cellular immune response.

Consistent with their hypothesis, the authors show that Su(H)S269A knock-in flies display a reduced response to wasp parasitism and that Su(H) is phosphorylated upon infestation. Using in vitro kinase assays and a genetic screen, they identify the PKCa family member Pkc53E as the putative kinase involved in Su(H) phosphorylation and they show that Pkc53E can bind Su(H). They further show that Pkc53E deficit or its knock-down in larval blood cells results in similar blood cell phenotypes as Su(H)S269A, including a reduced response to wasp parasitism, and their epistatic analyses indicate that Pkc53E acts upstream of Su(H).

Strengths<br /> The manuscript is well presented and the experiments are sound, with a good combination of genetic and biochemical approaches and several clear phenotypes which back the main conclusions. Notably Su(H)S269A mutation or Pkc53E deficiency strongly reduces lamellocyte production and the epistatic data are convincing.

Weaknesses<br /> The phenotypic analysis of larval blood cells remains rather superficial. Looking at melanized cells is a crude surrogate to quantify crystal cell numbers as it is biased toward sessile cells (with specific location) and does not bring information concerning the percentage of blood cells differentiated along this lineage.

In Su(H)S269A knock-in or Pkc53E zygotic mutants, the increase in crystal cells in uninfected conditions and the decreased capacity to induce lamellocytes following infection could have many origins which are not investigated. For instance, premature blood cell differentiation could promote crystal cell differentiation and reduce the pool of lamellocytes progenitors. These mutations could also affect the development and function of the posterior signaling center in the lymph gland, which plays a key role in lamellocyte induction. Similarly, the mild decrease on resistance to wasp infestation (Fig. 2A) could reflect a constitutive reduction in blood cell numbers in Su(H)S269A larvae rather than a defective down-regulation of Notch activity.<br /> Whereas the authors also present targeted-knock down/inhibition of Pkc53E suggesting that this enzyme is required in blood cells to control crystal cell fate (Fig. 6), it is somehow misleading to use lz-GAL4 as a driver in the lymph gland and hml-GAL4 in circulating hemocytes as these two drivers do not target the same blood cell populations/steps in the crystal cell development process.

In addition, the authors do not present evidence that Pkc55E function (and Su(H) phosphorylation) is required specifically in blood cells to promote lamellocyte production in response to infestation.

Finally, the conclusion that Pkc53E is (directly) responsible for Su(H) phosophorylation needs to be strengthened. Most importantly, the authors do not demonstrate that Pkc53E is required for Su(H) phosphorylation in vivo (i.e. that Su(H) is not phosphorylated in the absence of Pkc53E following infestation). In addition, the in vitro kinase assays with bacterially purified Pkc53E (in the presence of PMA or using an activated variant of Pkc53E) only reveal a weak activity on a Su(H) peptide encompassing S269 (Fig. 4). Moreover, while the authors show a coIP between an overexpressed Pkc53E and endogenous Su(H) (Fig. 7) (in the absence of infestation), it has recently been reported that Pkc53E is a cytoplasmic protein in the eye (Shieh et al. 2023), calling for a direct assessment of Pkc53E expression and localization in larval blood cells under normal conditions and upon infestation. Furthermore, the effect of the PKCa agonist PMA on Su(H)-induced reporter gene expression in cell culture and crystal cell number in vivo is somehow consistent with the authors hypothesis, but some controls are missing (notably western blots to show that PMA/Staurosporine treatment does not affect Su(H)-VP16 level) and it is unclear why STAU treatment alone promotes Su(H)-VP16 activity (in their previous reports, the authors found no difference between Su(H)S269A-VP16 and Su(H)-VP16) or why PMA treatment still has a strong impact on crystal cell number in Su(H)S269A larvae.

Joint Public Review:

The study has many limitations which need to be addressed and there is a lack of functional explanation of carriage. These limitations are: a) the lack of inclusion of non-Black patients; and b) the lack of appropriate explanation if results are false-positive since APOL1 provides risk for chronic renal disease (CRD) and patients with CRD are predisposed to sepsis. Sepsis occurred in 565 Black subjects, of whom 105 (29% ) had APOL1 high-risk genotype and 460 had-low risk genotype. Importantly, the risk for sepsis associated with APOL1 HR variants was no longer significant after adjusting for subjects pre-existing severe renal disease or after excluding these subjects. Thus, the susceptibility pathway seems to be: APOL1 variants > CKD > sepsis diathesis.

Reviewer #1 (Public Review):

In this study, Fang H et al. describe a potential pathway, ITGB4-TNFAIP2-IQGAP1-Rac1, that may involve in the drug resistance in triple negative breast cancer (TNBC). Mechanistically, it was demonstrated that TNFAIP2 bind with IQGAP1 and ITGB4 activating Rac1 and the following drug resistance. The present study focused on breast cancer cell lines with supporting data from mouse model and patient breast cancer tissues. The study is interesting. The experiments were well controlled and carefully carried out. The conclusion is supported by strong evidence provided in the manuscript. The authors may want to discuss the link between ITGB4 and Rac 1, between IQGAP1 and Rac1, and between TNFAIP2 and Rac1 as compared with the current results obtained. This is important considering some recent publications in this area (Cancer Sci 2021, J Biol Chem 2008, Cancer Res 2023). In addition, some key points need to be addressed in order to support their conclusion in full.

Reviewer #1 (Public Review):

Proposed significance: Targeted therapy in general has miraculous results.<br /> Good and detailed study of molecular characteristics and microenvironment of tumor of PCCs .However molecular classification system based on limited number of cases is not acceptable.<br /> Early diagnosis is of utmost importance in patient care and the next important is classification of tumor for treatment purposes.<br /> Further research is needed to develop Molecular signature of tumor types . This will help in targeted therapy and precision medicine.

Strength: Molecular characterisation of tumor

Weakness: The sample size is very small from a statistical point of view to derive a conclusion. Only Observations can be recorded<br /> Transcriptome profiles of 11 tumor tissues were studied but they belong to the same 5 patients.

Validation of tumor tissue: comparison is made with adjacent normal tissue (n=5 )<br /> Chromogranin IHC marker is used for identifying tumor cells. However, chromogranin marker positivity is also seen in normal adrenal medulla /chromaffin cells.<br /> Any better evidence of Validation of tumor tissue?

Tumor microenvironment:<br /> CD8+T cells: it is mentioned in the article that there is lack of CD8+ Tcells in both types of PCC, (Page 5, line 16)

However Figures 7 D, E and F show presence of CD8+T cells. Needs clarification or quantification.

Tumor heterogeneity : Page 7 Line 5<br /> PASS system is used by authors for predicting malignant potential and tumor heterogeneity.<br /> Molecular methods need to be used for evaluating tumor heterogeneity rather than histomorphology.

Ground of comparision is not valid. PASS system is based on histomorphology and present study/attempt at classification is based on molecular studies. So they cannot be compared .

Page 5 ,Line 18: HLA downregulation is observation and its regulation by RET is a possibility. Its involvement in tumor progression needs solid proof. So targeting kinase pathway for therapy is only a possibility.

Reviewer #1 (Public Review):

This manuscript proposes a complex unclear model involving Ca2+ signaling in inflammasome activation. The experimental approaches used to study the calcium dynamics are problematic and the results shown are of inadequate quality. The major claims of this manuscript are not adequately substantiated.

Major concerns:

1. The analysis of lysosomal Ca2+release is being carried out after many hours of treatment. Such evidence is not meaningful to claim that PA activates Ca2+ efflux from lysosome and even if this phenomenon was robust, it is not doubtful that such kinetics are meaningful for the regulation of inflammasome activation. Furthermore, the evidence for lysosomal Ca2+ release is indirect and relies on a convoluted process that doesn't make any conceptual sense to me. In addition to these major shortcomings, the indirect evidence of perilysosomal Ca2+ elevation is also of very poor quality and from the standpoint of my expertise in calcium signaling, the data are incredulous. The use of GCaMP3-ML1, *transiently transfected* into BMDMs is highly problematic. The efficiency of transfection in BMDMs is always extremely low and overexpression of the sensor in a few rare cells can lead to erroneous observations. The overexpression also results in gross mislocalization of such membrane-bound sensors. The accumulation of GCaMP3-ML1 in the ER of these cells would prevent any credible measurements of perilysosomal Ca2+ signals. A meaningful investigation of this process in primary macrophages requires the generation of a mouse line wherein the sensor is expressed at low levels in myeloid cells, and shown to be localized almost exclusively in the lysosomal membrane. The mechanistic framework built around these major conceptual and technical flaws is not especially meaningful and since these are foundational results, I cannot take the main claims of this study seriously.

2. The cytosolic Ca2+ imaging shown in Figure 1C doesn't make any sense. It looks like a snapshot of basal Ca2+ many hours after PA treatment - calcium elevations are highly dynamic. Snapshot measurements are not helpful and analyses of Calcium dynamics requires a recording over a certain timespan. Unfortunately, this technical approach has been used throughout the manuscript. Also, BAPTA-AM abrogates IL-1b secretion because IL-1b transcription is Ca2+ dependent - the result shown in figure 1D does not shed light on anything to do with inflammasome activation and it is misleading to suggest that.

3. Trpm2-/- macrophages are known to be hyporesponsive to inflammatory stimuli - the reduced secretion of IL-1b by these macrophages is not novel. From a mechanistic perspective, this study does not add much to that observation and the proposed role of TRPM2 as a lysosomal Ca2+ release channel is not substantiated by good quality Ca2+ imaging data (see point 3 above). Furthermore, the study assumes that TRPM2 is a lysosomal ion channel. One paper reported TRPM2 in the lysosomes but this is a controversial claim, with no replication or further development in the last 14 years. This core assumption can be highly misleading to readers unfamiliar with TRPM2 biology and it is necessary to present credible evidence that TRPM2 is functional in the lysosomal membrane of macrophages. Ideally, this line of investigation should rest on robust demonstration of TRPM2 currents in patch-clamp electrophysiology of lysosomes. If this is not technically feasible for the authors, they should at least investigate TRPM2 localization on lysosomal membranes of macrophages.

4. Apigenin and Quercetin are highly non-specific and their effects cannot be attributed to CD38 inhibition alone. Such conclusions need strong loss of function studies using genetic knockouts of CD38 - or at least siRNA knockdown. Importantly, if indeed TRPM2 is being activated downstream of CD38, this should be easily evident in whole cell patch clamp electrophysiology. TRPM2 currents can be resolved using this technique and authors have Trpm2-/- cells for proper controls. Authors attempted these experiments but the results are of very poor quality. If the TRPM2 current is being activated through ADPR generated by CD38 (in response to PA stimulation), then it is very odd that authors need to include 200 uM cADPR to see TRPM2 current (Fig. 3A). Oddly, even these data cast great doubt on the technical quality of the electrophysiology experiments. Even with such high concentrations of cADPr, the TRPM2 current is tiny and Trpm2-/- controls are missing. The current-voltage relationship is not shown, and I feel that the results are merely reporting leak currents seen in measurements with substandard seals. Also 20 uM ACA is not a selective inhibitor of TRPM2 - relying on ACA as the conclusive diagnostic is problematic.

5. TRPM2 is expressed in many different cell lines. The broad metabolic differences observed by the authors in the Trpm2-/- mice cannot be attributed to macrophage-mediated inflammation. Such a conclusion requires the study of mice wherein Trpm2 is deleted selectively in macrophages or at least in the cells of the myeloid lineage.

6. The ER-Lysosome Ca2+ refilling experiments rely on transient transfection of organelle-targeted sensors into BMDMs. See point #1 to understand why I find this approach to be highly problematic. Furthermore, the data procured are also not convincing and lack critical controls (localization of sensors has not been demonstrated and their response to acute mobilization of Ca2+ has not been shown to inspire any confidence in these results).

7. Authors claim that SCOE is coupled to K+ efflux. But there is no credible evidence that SOCE is activated in PA stimulated macrophages. The data shown in Fig 4 supp 1 do not investigate SOCE in a reliable manner - the conclusion is again based on snapshot measurements and crude non-selective inhibitors. The correct way to evaluate SOCE is to record cytosolic Ca2+ elevations over a period of time in absence and presence of extracellular Ca2+. However, even such recordings can be unreliable since the phenomenon is being investigated hours after PA stimulation. So, the only definitive way to demonstrate that Orai channels are indeed active during this process is through patch clamp electrophysiology of PA stimulated cells.

Reviewer #1 (Public Review):

Summary:<br /> The authors of this manuscript characterize new anion conducting that is more red-shifted in its spectrum than prior variants called MsACR1. An additional mutant variant of MsACR1 that is renamed raACR has a 20 nm red-shifted spectral response with faster kinetics. Due to the spectral shift of these variants, the authors proposed that it is possible to inhibit the expression of MsACR1 and raACR with lights at 635 nm in vivo and in vitro. The authors were able to demonstrate some inhibition in vitro and in vivo with 635 nm light. Overall the new variants with unique properties should be able to suppress neuronal activities with red-shifted light stimulation.

Strengths:<br /> The authors were able to identify a new class of anion conducting channelrhodopsin and have variants that respond strongly to lights with wavelength >550 nm. The authors were able to demonstrate this variant, MsACR1, can alter behavior in vivo with 635 nm light. The second major strength of the study is the development of a red-shifted mutant of MsACR1 that has faster kinetics and 20 nm red-shifted from a single mutation.

Weaknesses:<br /> The red-shifted raACR appears to work much less efficiently than MsACR1 even with 635 nm light illumination both in vivo (Figure 4) and in vitro (Figure 3E) despite the 20 nm red-shift. This is inconsistent with the benefits and effects of red-shifting the spectrum in raACR. This usually would suggest raACR either has a lower conductance than MsACR1 or that the membrane/overall expression of raACR is much weaker than MsACR1. Neither of these is measured in the current manuscript.

There are limited comparisons to existing variants of ACRs under the same conditions in the manuscript overall. There should be more parallel comparison with gtACR1, ZipACR, and RubyACR in identical conditions in cultured cell lines, cultured neurons, and in vivo. This should be in terms of overall performance, efficiency, and expression in identical conditions. Without this information, it is unclear whether the effects at 635 nm are due to the expression level which can compensate for the spectral shift.

There should be more raw traces from the recordings of the different variants in response to short pulse stimulation and long pulse stimulation to different wavelengths. It is difficult to judge what the response would be like when these types of information are missing.

Despite being able to activate the channelrhodopsin with 635 nm light, the main utility of the variant should be transcranial stimulation which was not demonstrated here.

Figure 3B is not clearly annotated and is difficult to match the explanation in the figure legend to the figure. The action potential spikings of neurons expressing raACR in this panel are inhibited as strongly as MsACR1.

For many characterizations, the number of 'n's are quite low (3-7).

Reviewer #1 (Public Review):

In this paper, the authors attempt to overcome the "fundamental limitations" of Lempel-Ziv complexity by developing and testing a complexity estimator based on state-space modelling (CSER) that they argue allows higher temporal resolution and spectral decomposition of entropy directly. They test the performance of this approach using MEG, EEG, and ECoG data from monkeys and humans. Although in principle, these developments might be useful for those already using LZ complexity in their analyses, these developments ignore much of the non-LZ entropy community which has already developed related solutions to the same issues. It is thus not clear currently whether this approach is necessary or unique per se:

• As the authors intimate, LZ is a relatively crude but efficient estimator; it leverages a simple binarization of time points above and below the time series mean to look at patterns (in turn disregarding the magnitude of the signal itself). The unique benefit of LZ in and of itself is not at all clear to this reviewer. It is nearly guaranteed that LZ will be extremely highly correlated with various other common measures of "discrete" entropy (especially permutation entropy, which ranks all time-series points prior to computing motifs/patterns rather than anchor anything by the mean (as does LZ), but nevertheless ignores the value range of the signal). The general appeal of the authors' intended developments to further improve LZ specifically would dramatically boost should they be able to make a case that LZ is somehow special, to begin with.

• Beyond this, we can now turn to the authors' rationale for the LZ developments proposed. Despite the authors' statement in the abstract that LZ complexity is "the most widely used approach complexity of neural dynamics," to my knowledge, sample entropy (and its multiscale variant, MSE) is much more commonly used in cognitive neuroscience. Such measures of entropy already enjoy several benefits over LZ. First, the continuous magnitude of the signal is relevant in sample entropy (i.e., it is not discrete in the same way as LZ because the values of each data point matter prior to the estimation of patterns). This is important for people in that community because electrophysiologists/neuroimagers often assume the values of the signal to matter (e.g., for ERPs, the magnitude of power, etc.). Ignoring the magnitude of signal values altogether, as in LZ, is a somewhat dramatic choice, especially if the authors then end up arguing that the spectral decomposition of entropy itself is valuable (after signal value ranges have been ignored!). In any case, as far as I know, LZ has never been shown the be more sensitive than e.g., sample entropy/MSE in relation to any outcome variable, but perhaps the authors can provide evidence for this and argue what LZ should practically do that is unique. Second, the use of MSE more easily allows (although not without its challenges) to directly compare spectral power and single/multiscale entropy straight away, which has been done in quite some depth already without the need for a state-space model of any kind (e.g., Kosciessa et al., 2020, PLOS CB). Instead of using a standard spectral power approach and comparing to entropy, the authors propose the spectrally decompose CSER entropy time series directly. Why? What should this do over standard multi-scale entropy approaches (like MSE, which estimate "fast" and "slow" complexity dynamics), which do not require a Fourier? And if they already believe that the spectrum cannot capture entropy (hence rationalizing the use of LZ-type measures in general), why do they want to invoke spectral estimation assumptions into the estimation of entropy when they could just compare the standard spectrum to entropy to begin with, without any complex modelling in between? I just don't see the need for a lot of what is proposed here; the authors provide solutions to problems that (at least for several in this community) may not exist at all.

• Figure 2: the authors show results descriptively comparing LZ and CSER, but without comparing the two measures directly. The patterns overall look extremely similar; why not correlate the values from the two measures in each dataset to make a case for what CSER is adding here? By eye test, it appears they will be extremely highly correlated, which leaves the reader wondering what CSER (with all of its model complexities and assumptions) has added.

• On the logic of and evidence for the use of CSER: The use of a state space model to allow estimation of "prediction errors" appears to be akin to a latent autocorrelation model with a lag/step size of 1 time-point, and trained only on prestim baseline data. When a successive time point is "deviant" from that autocorrelative function, the authors argue that this provides a measure of instantaneous entropy. This seems simple at first glance, but it is very difficult for this reviewer to wrap their head around. This approach anchors stim-related entropy estimation to prestim entropy for every subject, disallowing the direct comparison of values across subjects during the stimulus phase itself. This does not directly provide a measure of instantaneous task-related entropy, but a mixture of pre and post stim sources based on a state-space model. Does it need to be this complicated? Why does a simple window-based function not suffice to generate temporal dynamics of entropy without coupling the task-based signal to the prestim period? There are many such approaches already existing in the field.

• Figure 3: The authors show that gamma-band CSER is the most sensitive. Isn't it true that this is the exact inverse of the dominance of typical spectral effects under such conditions (that across the literature in psychedelics, sleep, and anaesthesia, there are dominant shifts in low-frequency spectral power)? Although low-frequency power is expected to be a dominant determinant of entropy in the entire signal (see Kosciessa et al., 2020, PLOS CB), something else appears to be happening here. At face value, because gamma is the spectral band with the lowest power in every imaging modality we know of, there is inherently less repeatability/autocorrelation in that same signal, which necessarily should produce more "prediction error/instantaneous entropy" in any condition. When the authors then take the "mean difference" of gamma-based entropy values from each of the two conditions in each sample, any condition-based shift in entropy should inherently be easier to detect. In any case, why not simply show these CSER spectral results next to a standard spectrum over the same conditions and then directly compare the unique utility of e.g., gamma power to CSER gamma? And if you compute something like the percent change between conditions for each spectral band, do you maintain gamma dominance?

Reviewer #2 (Public Review):

DeKraker et al. propose a new method for hippocampal registration using a novel surface-based approach that preserves the topology of the curvature of the hippocampus and boundaries of hippocampal subfields. The surface-based registration method proved to be more precise and resulted in better alignment compared to traditional volumetric-based registration. Moreover, the authors demonstrated that this method can be performed across image modalities by testing the method with seven different histological samples. This work has the potential to be a powerful new registration technique that can enable precise hippocampal registration and alignment across subjects, datasets, and image modalities.

Reviewer #1 (Public Review):

In this manuscript, "Diminishing neuronal acidification by channelrhodopsins with low proton conduction" by Hayward and colleagues, the authors report on the properties of novel optogenetic tools, PsCatCh2.0 and ChR2-3M, that minimize photo-induced acidification. The authors point out that acidification is an undesirable side-effect of many optogenetic approaches that could be minimized using the new tools. ChRs are known to acidify cells, while Arch are known to alkalize cells. This becomes particularly important when optical stimulation is prolonged and pH changes can become significant. pH is known to affect neuronal excitability, vesicular release, and more. To develop novel optogenetic tools with minimal proton conductances, the authors combined channelrhodopsin stimulation with a red-shifted pH sensor to measure pH during optogenetic stimulation. The authors report that optogenetic activation of CheRiff caused slow cellular acidification. 150 seconds of illumination caused a 3-fold increase in protons or approximately a 0.6 unit pH change that returned to baseline very slowly. They also found that pH changes occurred more rapidly, and recovered more rapidly, in dendrites. The authors go on to robustly characterize PsCatCh2.0 and ChR2-3M in terms of their proton conductances, photocurrent, kinetics, and more. They convincingly show that these constructs induced reduced acidification while maintaining robust photocurrents. In sum, this manuscript shows important findings that convincingly characterizes 2 optogenetic tools that have reduced pH artifacts that may be of broad interest to the field of neuroscience research and optogenetic therapies.

Reviewer #1 (Public Review):

Summary:

The work studies functional connectivity gradients using advanced resting-state analyses in fetuses and sheds light on pre-existing functional topographies and their continued development during the third trimester of gestation.

Strengths:

The work is novel, and applies state of the art connectomic mapping techniques to study fetal brain organization. The work capitalizes on the existence of large, open access datasets, and shows interesting and impactful findings on the presence of functional topographies from 25GW onwards.

Weaknesses:

To better understand underlying factors in cortical functional organization, the authors could add additional exploratory analyses to assess the role of cortical microstructure/myelin and thalamic connectivity.

Reviewer #1 (Public Review):

Summary: Direction selectivity (DS) in the visual system is first observed in the radiating dendrites of starburst amacrine cells (SACs). Studies over the last two decades have aimed to understand the mechanisms that underlie these unique properties. Most recently, a 'space-time' model has garnered special attention. This model is based on two fundamental features of the circuit. First, distinct anatomical types of bipolar cells (BCs) are connected to proximal/distal regions of each of the SAC dendritic sectors (Kim et al., 2014). Second, that input across the length of the starburst is kinetically diverse, a hypothesis that has been only recently demonstrated experimentally using iGluSnFR imaging (Srivastava et al., 2022). However, the stark kinetic distinctions, i.e., the sustained/transient nature of BC input to SACs dendrites appear to be present mainly in responses to stationary stimuli. When BC receptive field properties are probed using white noise stimuli, the kinetic differences between BCs are relatively subtle or nonexistent (Gaynes et al., 2022; Strauss et al., 2022, Srivastava et al., 2022). Thus, if and how BCs contribute to direction selectivity driven by moving spots that are commonly used to probe the circuit remains to be clarified. To address this issue, Gaynes et al., combine evolutionary computational modeling (Ankri et al., 2020) with two-photon iGluSnFR imaging to address to what degree BCs contribute to the generation of direction selectivity in the starburst dendrites in response to stimuli that are commonly used experimentally.

Strengths:

Combining theoretical models and iGluSnFR imaging is a powerful approach as it first provides a basic intuition on what is required for the generation of robust DS, and then tests the extent to which the experimentally measured BC output meets these requirements.

The conclusion of this study builds on the previous literature and comprehensively considers the diverse BC receptive field properties that may contribute to DS (e.g. size, lag, rise time, decay time).

By 'evolving' bipolar inputs to produce robust DS in a model network, these authors provide a sound framework for understanding which kinetic properties could potentially be important for driving downstream DS. They suggest that response delay/decay kinetics, rather than the center/surround dynamics are likely to be most relevant (albeit the latter could generate asymmetric responses to radiating/looming stimuli).

Weaknesses: Finally, these authors report that the experimentally measured BC responses are far from optimal for generating DS. Thus, the BC-based DS mechanism does not appear to explain the robust DS observed experimentally (even with mutual inhibition blocked). Nevertheless, I feel the comprehensive description of BC kinetics and the solid assessment of the extent to which they may shape DS in SAC dendrites, is a significant advancement in the field.

Reviewer #1 (Public Review):

Guglielmo et al. characterized addiction-like behaviors in more than 500 outbred heterogeneous stock (HS) rats using extended access to cocaine self-administration (6 h/daily) and analyzed individual differences in escalation of intake, progressive-ratio (PR) responding, continued use despite adverse consequence (contingent foot shocks), and irritability-like behavior during withdrawal. By principal component analysis, they found that escalation of intake, progressive ratio responding, and continued use despite adverse consequences loaded onto the same factor, whereas irritability-like behaviors loaded onto a separate factor. Characterization of rats in four categories of resilient, mild, moderate, and severe addiction-like phenotypes showed that females had higher addiction-like behaviors, particularly due to a lower number of resilient individuals, than males. The authors suggest that escalation of intake, continued use despite adverse consequences, and progressive ratio responding are highly correlated measures of the same psychological construct and that a significant proportion of males, but not females may be resilient to addiction-like behaviors. The amount of work in this study is impressive, and the results are interesting. However, there are several issues that need to be addressed to improve their manuscript. In particular, the language should be toned down and the statistical analysis approach could be improved.

Strengths: Large dataset. Males and females included.

Weaknesses: Language and statistical analysis can be improved.

Reviewer #1 (Public Review):

The present work establishes 14-3-3 proteins as binding partners of Spastin and suggests that this binding is positively regulated by phosphorylation of Spastin. The authors show evidence that 14-3-3 - Spastin binding prevents Spastin ubiquitination and final proteasomal degradation, thus increasing the availability of Spastin. The authors measured microtubule severing activity in cell lines and axon regeneration and outgrowth as a prompt to Spastin activity. By using drugs and peptides that separately inhibit 14-3-3 binding or Spastin activity, they show that both proteins are necessary for axon regeneration in cell culture and in vivo models in rats.

The following is an account of the major strengths and weaknesses of the methods and results.

Major strengths<br /> -The authors performed pulldown assays on spinal cord lysates using GST-spastin, then analyzed pulldowns via mass spectrometry and found 3 peptides common to various forms of 14-3-3 proteins. In co-expression experiments in cell lines, recombinant Spastin co-precipitated with all 6 forms of 14-3-3 tested.<br /> -By protein truncation experiments they found that the Microtubule Binding Domain of Spastin contained the binding capability to 14-3-3. This domain contained a putative phosphorylation site, and substitutions that cannot be phosphorylated cannot bind to Spastin.<br /> -Spastin overexpression increased neurite growth and branching, and so did the phospho null spastin. On the other hand, the phospho mimetic prevents all kinds of neurite development.<br /> -Overexpression of GFP-Spastin shows a turn-over of about 12 hours when protein synthesis is inhibited by cycloheximide. When 14-3-3 is co-overexpressed, GFP-Spastin does not show a decrease by 12 hours. When S233A is expressed, a turn-over of 9 hours is observed, indicating that the ability to be phosphorylated increases the stability of the protein.<br /> -In support of that notion, the phospho-mimetic S233D makes it more stable, lasting as much as the over-expression of 14-3-3.<br /> -Authors show that Spastin can be ubiquitinated, and that in the presence of ubiquitin, Spastin-MT severing activity is inhibited.<br /> -By combining FCA with Spastazoline, the authors claim that FCA increased regeneration is due to increased Spastin Activity in various models of neurite outgrowth and regeneration in cell culture and in vivo, the authors show impressive results on the positive effect of FCA in regeneration, and that this is abolished when Spastin is inhibited.

Major weaknesses<br /> -However convincing the pull-downs of the expressed proteins, the evidence would be stronger if a co-immunoprecipitation of the endogenous proteins were included.<br /> -To better establish the impact of Spastin phosphorylation in the interaction, there is no indication that the phosphomimetic (S233D) can better bind Spastin, and this result is contradicting to the conclusion of the authors that Spastin-14-3-3 interaction is necessary for (or increases) Spastin function<br /> -To fully support the authors' suggestion that 14-3-3 and Spastin work in the same pathway to promote regeneration, I believe that some key observations are missing.<br /> 1-There is no evidence showing that 14-3-3 overexpression increases the total levels of Spastin, not only its turnover.<br /> 2- There is no indication that increasing the ubiquitination of Spastin decreases its levels. To suggest that proteasomal activity is affecting the levels of a protein, one would expect that proteasomal inhibition (with bortezomib or epoxomycin), would increase its levels.<br /> 3- Authors show that S233D increases MT severing activity, and explain that it is related to increased binding to 14-3-3. An alternative explanation is that phosphorylation at S233 by itself could increase MT severing activity. The authors could test if purified Spastin S233D alone could have more potent enzymatic activity.<br /> -Finally, I consider that there are simpler explanations for the combined effect of FC-A and spastazoline. FC-A mechanism of action can be very broad, since it will increase the binding of all 14-3-3 proteins with presumably all their substrates, hence the pathways affected can rise to the hundreds. The fact that spastazoline abolishes FC-A effect, may not be because of their direct interaction, but because Spastin is a necessary component of the execution of the regeneration machinery further downstream, in line with the fact that spastizoline alone prevented outgrowth and regeneration, and in agreement with previous work showing that normal Spastin activity is necessary for regeneration.

In summary, the evidence of the interaction of 14-3-3 and Spastin is solid, but it is weak with respect to showing evidence for the binding of endogenous proteins in neurons. Another strength of the manuscript is the important recovery of function after spinal cord injury after stimulation of 14-3-3 interactions. Although it is experimentally difficult to demonstrate that the effect of FC-A is due to the prevention of Spastin ubiquitination, the effect itself is very robust and remarkable in vivo.

Reviewer #1 (Public Review):

Summary: In this study, Franke et al. explore and characterize the color response properties across the primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The authors use awake-behaving 2P imaging to define the spectral response properties of visual interneurons in Layer 2/3. They find that opponent responses are more prominent at photopic light levels, and diversity in color opponent responses exists across the visual science, with green ON/ UV OFF responses being stronger represented in the upper visual field. This is argued to be relevant for detecting certain features that are more salient when the chromatic space is used, possibly due to noise reductions.

Strengths: The work is well crafted and written and provides a thorough characterization that reveals an uncharacterized diversity of visual properties in V1. I find this characterization important because it reveals how strongly chromatic information can modulate the response properties in V1. In the upper visual field, 25% of the cells differentially relay chromatic information, and one may wonder how this information will be integrated and subsequently used to aid vision beyond the detection of color per see. I personally like the last paragraph of the discussion that highlights this fact.

Weaknesses:

One major point highlighted in this paper is the fact that Green ON/UV OFF responses are not generated in the retina. But glancing through the literature, I saw this is not necessarily true. Fig 1. of Joesch & Meister, a paper cited, shows this can be the case. Thus, I would not emphasize that this wasn't present in the retina. This is a minor point, but even if the retina could not generate these signals, I would be surprised if the diversity of responses would only arise through feed-forward excitation, given the intricacies of cortical connectivity. Thus, I would argue that the argument holds for most of the responses seen in V1; they need to be further processed by cortical circuitries. This takes me to my second point, defining center and surround. The center spot is 37.5 deg of visual angle, more than 1 mm of the retinal surface. That means that all retinal cells, at least half and most likely all of their surrounds will also be activated. Although 37.5 deg is roughly the receptive field size previously determined for V1 neurons, the one-to-one comparison with retinal recording, particularly with their center/surround properties, is difficult. This should be discussed. I assume that the authors tried a similar approach with sparse or dense checker white noise stimuli. If so, it would be interesting if there were better ways of defining the properties of V1 neurons on their complex/simple receptive field properties to define how much of their responses are due to an activation of the true "center" or a coactivation of the surround. Interestingly, at least some of the cells (Fig. 1d, cells 2 and 5) don't have a surround. Could it be that in these cases, the "center" and "surround" are being excited together? How different would the overall statistics change if one used a full-filed flicker stimulus instead of a center/surround stimulus? How stable are the results if the center/surround flicker stimulus is shifted? These results won't change the fact that chromatic coding is present in the VC and that there are clear differences depending on their position, but it might change the interpretation. Thus, I would encourage you to test these differences and discuss them.

Reviewer #1 (Public Review):

In this work, Dasguta et al. have dissected the role of Sema7a in fine tuning of a sensory microcircuit in the posterior lateral line organ of zebrafish. They attempt to also outline the different roles of a secreted verses membrane-bound form of Sema7a in this process. Using genetic perturbations and axonal network analysis, the authors show that loss of both Sema7a isoforms causes abnormal axon terminal structure with more bare terminals and fewer loops in contact with presynaptic sensory hair cells. Further, they show that loss of Sema7a causes decreased number and size of both the pre- and post-synapse. Finally, they show that overexpression of the secreted form of Sema7a specifically can elicit axon terminal outgrowth to an ectopic Sema7a expressing cell. Together, the analysis of Sema7a loss of function and overexpression on axon arbor structure is fairly thorough and revealed a novel role for Sema7a in axon terminal structure. However, the connection between different isoforms of Sema7a and the axon arborization needs to be substantiated. Furthermore, an autocrine role for Sema7a on the presynaptic cell is not ruled out as a contributing factor to the synaptic and axon structure phenotypes. Finally, critical controls are absent from the overexpression paradigm. These issues weaken the claims made by the authors including the statement that they have identified differential roles for the GPI-anchored verses secreted forms of Sema7a on synapse formation and as a chemoattractant for axon arborization respectively. The manuscript itself would benefit from the inclusion of details in the text to help the reader interpret the figures, tools, data, and analysis.

Reviewer #1 (Public Review):

Summary:<br /> The authors report an fMRI investigation of the neural mechanisms by which selective attention allows capacity-limited perceptual systems to preferentially represent task-relevant visual stimuli. Specifically, they examine competitive interactions between two simultaneously-presented items from different categories, to reveal how task-directed attention to one of them modulates the activity of brain regions that respond to both. The specific hypothesis is that attention will bias responses to be more like those elicited by the relevant object presented on its own, and further that this modulation will be stronger for more dissimilar stimulus pairs. This pattern was confirmed in univariate analyses that measured the mass response of a priori regions of interest, as well as multivariate analyses that considered the patterns of evoked activity within the same regions. The authors follow these neuroimaging results with a simulation study that favours a "tuning" mechanism of attention (enhanced responses to highly effective stimuli, and suppression for ineffective stimuli) to explain this pattern.

Strengths:<br /> The manuscript clearly articulates a core issue in the cognitive neuroscience of attention, namely the need to understand how limited perceptual systems cope with complex environments in the service of the observer's goals. The use of a priori regions of interest, and the inclusion of both univariate and multivariate analyses as well as a simple model, are further strengths. The authors carefully derive clear indices of attentional effects (for both univariate and multivariate analyses) which makes explication of their findings easy to follow.

Weaknesses:<br /> There are some relatively minor weaknesses in presentation, where the motivation behind some of the procedural decisions could be clearer. There are some apparently paradoxical findings reported -- namely, cases in which the univariate response to pairs of stimuli is greater than to the preferred stimulus alone -- that are not addressed. It is possible that some of the main findings may be attributable to range effects: notwithstanding the paradox just noted, it seems that a floor effect should minimise the range of possible attentional modulation of the responses to two highly similar stimuli. One possible limitation of the modelled results is that they do not reveal any attentional modulation at all under the assumptions of the gain model, for any pair of conditions, implying that as implemented the model may not be correctly capturing the assumptions of that hypothesis.

Reviewer #1 (Public Review):

Jafarinia et al. have made an interesting contribution to unravelling the molecular mechanisms underlying pathological phenotypes of repeat expansion of the C9orf72 gene. The repeat expression leads to the expression of polyPR proteins. Using coarse-grained molecular dynamics simulations, the authors identify putative binding partners involved in nucleocytoplasmic transport (NCT), and that conjecture that polyPR affects essential processes by binding to NCT-related proteins. The results are well-reported, but only putative, and need experimental support to be more conclusive. Also, a comparison with results from all-atom MD simulations in explicit water could help verify the results. But even without these, the work is very useful as a first step to unravel the role of polyPR and related peptides.

Reviewer #1 (Public Review):

The goal of the current study was to evaluate the effect of neuronal activity on blood-brain barrier permeability in the healthy brain, and to determine whether changes in BBB dynamics play a role in cortical plasticity. The authors used a variety of well-validated approaches to first demonstrate that limb stimulation increases BBB permeability. Using in vivo-electrophysiology and pharmacological approaches, the authors demonstrate that albumin is sufficient to induce cortical potentiation and that BBB transporters are necessary for stimulus-induced potentiation. The authors include a transcriptional analysis and differential expression of genes associated with plasticity, TGF-beta signaling, and extracellular matrix were observed following stimulation. Overall, the results obtained in rodents are compelling and support the authors' conclusions that neuronal activity modulates the BBB in the healthy brain and that mechanisms downstream of BBB permeability changes play a role in stimulus-evoked plasticity. These findings were further supported with fMRI and BBB permeability measurements performed in healthy human subjects performing a simple sensorimotor task. While there are many strengths in this study, there is literature to suggest that there are sex differences in BBB dysfunction in pathophysiological conditions. The authors only used males in this study and do not discuss whether they would also expect to sex differences in stimulation-evoked BBB changes in the healthy brain. Another minor limitation is the authors did not address the potential impact of anesthesia which can impact neurovascular coupling in rodent studies. The authors could have also better integrated the RNAseq findings into mechanistic experiments, including testing whether the upregulation of OAT3 plays a role in cortical plasticity observed following stimulation. Overall, this study provides novel insights into how neurovascular coupling, BBB permeability, and plasticity interact in the healthy brain.

Reviewer #1 (Public Review):

Summary:

Exposure to cranial irradiation (IR) leads to cognitive deficits in the survivors of brain cancer. IR upregulates miR-206-3p, which in turn reduces the PAK3-LIMK1 axis leading to the loss of F and G-actin ratio and, thereby, mature dendritic spine loss. Silencing miR-206-3p reverses these degenerative consequences.

Strengths:<br /> The authors show compelling data indicating a clear correlation between PAK3 knockdown and the loss of mature dendritic spine density. In contrast, overexpression of PAK3 in the irradiated neurons restored mature spine types and recovered the F/G ratio. These in vitro results support the authors' hypotheses that PAK3 and LIMK1-mediated downstream signaling impact neuronal structure and reorganization in vitro. These data were supported by similar experiments using differentiated human neurons. Importantly, silencing miR-206-30 using antagonist miR also reverses IR-induced downregulation of the PAK3-LIMK1 axis, preventing spine loss and cognitive deficits.

Weaknesses:

All the miR-206-3p data are presented from in vitro cortical neurons or human stem cell-derived neuron cultures. This data (IR-induced elevation of miR-206-3p) should also be confirmed in vivo using an irradiated mouse brain to correlate the cognitive dysfunction timepoint.

Antago-miR-206-3p reversed Ir-induced upregulation of miR-206 (in vitro), and prevent reductions in PAK3 and downstream markers. Importantly, it reversed cognitive deficits induced by IR. This data should be supported by in vivo staining for important dendritic markers, including cofillin, p-cofilin, PSD-95, F- and G-actin within the hippocampal and PFC regions.

Other neuronal and non-neuronal targets of miR-206-3p should be discussed and looked into as a downstream impact of IR-induced functional and physiological impairments in the brain.

Reviewer #1 (Public Review):

The authors investigate pleiotropy in the genetic loci previously associated to a range of neuropsychiatric disorders: Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Parkinson's disease, and schizophrenia. The local statistical fine-mapping and variant colocalisation approaches they use have the potential to uncover not only shared loci but also shared causal variants between these disorders. There is existing literature describing the pleiotropy between ALS and these other disorders but here the authors apply state of the art, local genetic correlation approaches to further refine any relationships.

Complex disease and GWAS is not my area of expertise but the authors managed to present their methods and results in a clear, easy to follow manner. Their results statistically support several correlations between the disorders and, for ALS and AD, a shared variant in the vicinity of the lead SNP from the original ALS GWAS. Such findings could have important implications for our understanding of the mechanisms of such disorders and eventually the possibility of managing and treating them.

The authors have built a useful pipeline that plugs together all the gold-standard, existing software to perform this analysis and made it openly available which is commendable. However, there is little discussion of what software is available to perform global and local correlation analysis and, if there are multiple tools available, why they consider the ones they selected to be the gold-standard.

There is some mention of previous findings of genetic pleiotropy between ALS and these other disorders in the introduction, and discussion of their improved ALS-AD evidence relative to previous work. However, detailed comparisons of their other correlations to what was described before for the same pairs of disorders (if any) is missing. Adding this would strengthen the impact of this paper.

Finally, being new to this approach I found the abstract a little confusing. Initially, the shared causal variant between ALS and AD is mentioned but immediately in the following sentence they describe how their study "suggested that disease- implicated variants in these loci often differ between traits". After reading the whole paper I understood that the ALS-AD shared variant was the exception but it may be best to restructure this part of the abstract. Additionally, in the abstract the authors state that different variants "suggests the role of distinct mechanisms across diseases despite shared loci". Is it not possible that different variants in the same regulatory region or protein-coding parts of a gene could be having the same effect and mechanism? Or does the methodology to establish that different variants are involved automatically mean that the variants are too distant for this to be possible?

Reviewer #1 (Public Review):

Summary: Chang et al. provide glutamate co-expression profiles in the central noradrenergic system and test the requirement of Vglut2-based glutamatergic release in respiratory and metabolic activity under physiologically relevant gas challenges. Their experiments show that conditional deletion of Vglut2 in NA neurons does not impact steady-state breathing or metabolic activity in room air, hypercapnia, or hypoxia. Their observations challenge the importance of glutamatergic signaling from Vglut2 expressing NA neurons in normal respiratory homeostasis in conscious adult mice.

Strengths: The comprehensive Vglut1, Vglut2, and Vglut3 co-expression profiles in the central noradrenergic system and the combined measurements of breathing and oxygen consumption are two major strengths of this study. Observations from these experiments provide previously undescribed insights into (1) expression patterns for subtypes of the vesicular glutamate transporter protein in the noradrenergic system and (2) the dispensable nature of Vglut2-dependent glutamate signaling from noradrenergic neurons to breathing responses to physiologically relevant gas challenges in adult conscious mice.

Weaknesses: Although the cellular expression profiles for the vesicular glutamate transporters are provided, the study fails to document that glutamatergic-based signaling originating from noradrenergic neurons is evident at the cellular level under normal, hypoxic, and/or hypercapnic conditions. This limits the reader's understanding of why conditional Vglut2 knockdown is dispensable for breathing under the conditions tested.

Reviewer #1 (Public Review):

Qin et al., demonstrate, convincingly, that plasticity of ocular dominance of binocular neurons in the visual thalamus persists in adulthood. The adult plasticity is similar to that described in critical period juveniles in that it is absent in transgenic mice with the deletion of the GABA a1 receptor in thalamus, which also blocks ocular dominance plasticity in primary visual cortex. However, the adult plasticity is not dependent on feedback from primary visual cortex, an important difference from juveniles. These findings are an important contribution to a growing body of work identifying plasticity in the adult visual system, and identifies the visual thalamus as a potential target for therapies to reverse adult amblyopia.

Reviewer #1 (Public Review):

This manuscript by Xu and colleagues addresses the important question of how multi-modal associations are encoded in the rodent brain. They use behavioral protocols to link stimuli to whisker movement and discover that the barrel cortex can be a hub for associations. Based on anatomical correlations, they suggest that structural plasticity between different areas can be linked to training. Moreover, they provide electrophysiological correlates that link to behavior and structure. Knock-down of nlg3 abolishes plasticity and learning.

This study provides an important contribution as to how multi-modal associations can be formed across cortical regions.

Reviewer #1 (Public Review):

Meta-cognition, and difficulty judgments specifically, is an important part of daily decision-making. When facing two competing tasks, individuals often need to make quick judgments on which task they should approach (whether their goal is to complete an easy or a difficult task).

In the study, subjects face two perceptual tasks on the same screen. Each task is a cloud of dots with a dominating color (yellow or blue), with a varying degree of domination - so each cloud (as a representation of a task where the subject has to judge which color is dominant) can be seen an easy or a difficult task. Observing both, the subject has to decide which one is easier.

It is well-known that choices and response times in each separate task can be described by a drift-diffusion model, where the decision maker accumulates evidence toward one of the decisions ("blue" or "yellow") over time, making a choice when the accumulated evidence reaches a predetermined bound. However, we do not know what happens when an individual has to make two such judgments at the same time, without actually making a choice, but simply deciding which task would have stronger evidence toward one of the options (so would be easier to solve).

It is clear that the degree of color dominance ("color strength" in the study's terms) of both clouds should affect the decision on which task is easier, as well as the total decision time. Experiment 1 clearly shows that color strength has a simple cumulative effect on choice: cloud 1 is more likely to be chosen if it is easier and cloud 2 is harder. Response times, however, show a more complex interactive pattern: when cloud 2 is hard, easier cloud 1 produces faster decisions. When cloud 2 is easy, easier cloud 1 produces slower decisions.

The study explores several models that explain this effect. The best-fitting model (the Difference model is the paper's terminology) assumes that the decision-maker accumulates evidence in both clouds simultaneously and makes a difficulty judgment as soon as the difference between the values of these decision variables reaches a certain threshold. Another potential model that provides a slightly worse fit to the data is a two-step model. First, the decision maker evaluates the dominant color of each cloud, then judges the difficulty based on this information.

Importantly, the study explores an optimal model based on the Markov decision processes approach. This model shows a very similar qualitative pattern in RT predictions but is too complex to fit to the real data. Possibly, the fact that simple approaches such as the Difference model fit the data best could suggest the existence of some cognitive constraints that play a role in difficulty judgments and could be explored in future research.

The Difference model produces a well-defined qualitative prediction: if the dominant color of both clouds is known to the decision maker, the overall RT effect (hard-hard trials are slower than easy-easy trials) should disappear. Essentially, that turns the model into the second stage of the two-stage model, where the decision maker learns the dominant colors first. The data from Experiment 2 impressively confirms that prediction and provides a good demonstration of how the model can explain the data out-of-sample with a predicted change in context.

Overall, the study provides a very coherent and clean set of predictions and analyses that advance our understanding of meta-cognition. The field would benefit from further exploration of differences between the models presented and new competing predictions (for instance, exploring how the sequential presentation of stimuli or attentional behavior can impact such judgments). Finally, the study provides a solid foundation for future neuroimaging investigations.

Reviewer #1 (Public Review):

The authors focused on genetic variability in relation to insulin resistance. They used genetically different lines of mice and exposed them to the same diet. They found that genetic predisposition impacts the overall outcome of metabolic disturbances. This work provides a fundamental novel view on the role of genetics and insulin resistance.

Reviewer #1 (Public Review):

Summary:<br /> The authors measured the oxygen stable isotope ratios in six orangutan teeth using a state-of-the-art micro-sampling technique (SHRIMP SI) to gather substantial multi-year isotopic data for six modern and five fossil orangutan individuals from Borneo and Sumatra. This fine-scale sampling technique allowed them to address the fundamental question of whether breastfeeding affects the oxygen isotope ratios in teeth forming in the first one to two years of life, during which orangutans are assumed to largely depend on breastmilk. The authors provide compelling evidence that the consumption of milk does not appear to affect the overall isotopic profile in early-forming teeth. They conclude that this allows us to use these teeth as terrestrial/arboreal isotopic proxies in paleoenvironmental research, which would provide an invaluable addition to otherwise largely marine climate records in these regions.

Strengths:<br /> The overall large sample size of orangutan dental isotope records as well as the rigorous dating of the fossil specimens provide a strong dataset for addressing the outlined questions. The direct comparison of modern and fossil orangutan specimens provides a valuable evaluation of the use of these modern and past environmental proxies, with some discussion of the implications for the environmental conditions during the expansion of early modern humans into this region of the world.

Weakness:<br /> Although the overall conclusions of this paper are well supported and discussed, one important aspect could have more detailed consideration: the ecology and behavior of orangutans. As one example, orangutans are almost exclusively (~96%) arboreal creatures foraging for plant foods in the forest canopy, and as such they mostly meet their water requirements from the plants they eat, only very rarely drinking surface water (Ashbury et al. 2015). As a result, all orangutan water and foods are strongly affected by the so-called canopy effect, which could have found stronger consideration in this study. The canopy effect in primate plant foods has been demonstrated to easily exceed 5‰ within the same forest canopy and even within the same tree, mainly depending on stratigraphy/height (Lowry et al. 2021). This variation may explain the noise in the isotopic data within a given orangutan tooth, which lies well within this 5% range, and could easily obscure any possible breastfeeding effect in dental isotope ratios. If the canopy effect may indeed introduce so much noise in the oxygen isotope data, this should be also considered in the use of the data as a climate proxy. The question arises if a terrestrial long-lived mammal species may be a more suitable proxy than an arboreal one.

Reviewer #1 (Public Review):

Summary:<br /> Radial spokes (RS) are made of >20 proteins and are believed to be a transducer to coordinate axonemal dyneins to enable the beating motion of motile cilia. While the atomic structure of RS from green algae Chlamydomonas and H. Sapience has been solved by single particle cryo-EM recently, this work by Bicka et al. provided the atomic structure of RS from ciliate Tetrahymena. They identified component proteins of Tetrahymena RS, which correspond to those in the atomic structure of Chlamydomonas and human RS. These proteins were likely already guessed as RS components, based on sequence similarity, but in this work experimentally identified for the first time. Furthermore, they discovered novel isoforms of RS proteins and characterized them structurally and functionally. RSP3 has three isoforms (A, B, and C). They are distributed specifically in the three radial spokes within the repeating unit as proved by mutant analysis, cryo-EM, and proteomics. By high-speed video microscopy, they proved the essential roles of RSP3B for ciliary beating. These isoforms have never been reported in past works and this demonstrates the novelty of this work.

Strength:<br /> Their discovery of RSP3 isoforms is unexpected and, although it is still not clear why Tetrahymena needs to have these isoforms, will evoke future research. The authors characterized the multi-facet aspects of these proteins precisely, structurally by cryo-EM, functionally by waveform and velocity analysis, and in terms of protein networking by co-IP and bioID studies.

Weakness:<br /> While the first half of this manuscript about RSP3 isoforms is very well organized and described (this reviewer still has some advice to make this story convincing and attractive), the later part has room for improvement. Some results are presented in the current manuscript without mentioning figures or tables, for example in "250: The components of the Tetrahymena radial spoke stalks" no figure/table is cited. There is also inconsistency - in 327 RSP9 is mentioned as a MORN protein, but in Fig.6 Sup.3 Table.1, it is mentioned as "unknown".

Reviewer #1 (Public Review):

The authors aim to theoretically explain the wide range of time scales observed in cortical circuits in the brain -- a fundamental problem in theoretical neuroscience. They propose that the variety of time scales arises in recurrent neural networks with heterogeneous units that represent neuronal assemblies of different sizes that transition through sequences of high- and low-activity metastable states. When transitions are driven by intrinsically generated noise, the heterogeneity leads to a wide range of escape times (and hence time scales) across units. As a mathematically tractable model, they consider a recurrent network of heterogeneous bistable rate units in the chaotic regime. The model is an extension of the previous model by Stern et al (Phys. Rev. E, 2014) to the case of heterogeneous self-coupling parameters. Biologically, this heterogeneous parameter is interpreted as different assembly sizes. The chaoticity acts as intrinsically generated noise-driving transitions between bistable states with escape times that are indeed widely distributed because of the heterogeneity. The distribution is successfully fitted to experimental data. Using previous dynamic mean-field theory, the self-consistent auto-correlation function of the driving noise in the mean-field model is computed (I guess numerically). This leaves the theoretical problem of calculating escape times in the presence of colored noise, which is solved using the unified colored-noise approximation (UCNA). They find that the log of the correlation time of a given unit increases quadratically with the self-coupling strength of that unit, which nicely explains the distribution of time scales over several orders of magnitude. As a biologically plausible implementation of the theory, they consider a spiking neural network with clustered connectivity and heterogeneous cluster sizes (extension of the previous model by Mazzucato et al. J Neurosci 2015). Simulations of this model also exhibit a quadratic increase in the log dwell time with cluster size. Finally, the authors demonstrate that heterogeneous assemblies might be useful to differentially transmit different frequency components of a broadband stimulus through different assemblies because the assembly size modulates the gain.

I found the paper conceptually interesting and original, especially the analytical part on estimating the mean escape times in the rate network using the idea of probe units and the UCNA. It is a nice demonstration of how chaotic activity serves as noise-driving metastable activity. Calculating the typical time scales of such metastable activity is a hard theoretical problem, for which the authors made considerable advancement. The conclusions of this paper are mostly well supported by simulations and mathematical analysis, but some aspects need to be clarified and extended, especially concerning the biological plausibility of the rate network model and its relation to the spiking neural network model as well as the analytical calculation of the mean dwell time.

1) The theory is based on a somewhat unbiological network of bistable rate units. It seems to only loosely apply to the implementation with a spiking neural network with clustered architecture, which is used as a biological justification of the rate model. In the spiking model, a wide distribution of time scales also emerges as a consequence of noise-induced escapes in combination with heterogeneity. Apart from this analogy, however, the mechanisms for metastability seem to be quite different: firstly, the functional units in the spiking neural network are presumably not bistable themselves but multistability only emerges as a network effect, i.e. from the interaction with other assemblies and inhibitory neurons. (This difference yields anti-correlations between assemblies in the spiking model, in marked contrast to the independence of bistable rate units (if N is large).) Secondly, transitions between metastable states are presumably not driven by chaotic dynamics but by finite-size fluctuations (e.g. Litwin-Kumar & Doiron 2012). The latter is also strongly dependent on assembly size. More precisely, the mechanism of how assembly size shapes escape times T seems to be different: in the rate model the self-coupling ("assembly size") predominantly affects the effective potential, whereas in the spiking network, the assembly size predominantly affects the noise.

Furthermore, the prediction of the rate model is a quadratic increase of log(T), however, the data shown in Fig.5b do not seem to strongly support this prediction. More details and evidence that the data "was best fit with a quadratic polynomial" would be necessary to test the theoretical prediction. Therefore, the correspondence between the rate model and the spiking model should probably be regarded in a looser sense than presented in the paper.

2) The time scale of a bistable probe unit driven by network-generated "noise" is taken to be the mean dwell time T (mean escape time) in a metastable state. It seems that the expressions Eq.4 and Eq.21 for this time are incorrect. The mean dwell time is given by the mean first-passage time (MFPT) from one potential minumum to the opposite one including the full passage across the barrier. At least, the final point for the MFPT should be significantly beyond the barrier to complete the escape. However, the authors only compute the MFPT to a location -x_c slightly before the barrier is reached, at which point the probe unit has not managed to escape yet (e.g. it could go back to -x_2 after reaching -x_c instead of further going to +x_2). It is not clear whether the UCNA can be applied to such escape problems because it is valid only in regions, where the potential is convex, and thus the UCNA may break down near the potential barrier. Indeed, the effective potential is not defined near the barrier (see forbidden zone in Fig.4b), and hence it is not clear how to calculate the mean escape time. Nonetheless, the incomplete MFPT computed by the authors seems to qualitatively predict the dependence on the self-coupling parameter s, at least in the example of Fig.4c. However, if the incomplete MFPT is taken as a basis, then the incomplete MFPT should also be used for the white-noise case for a fair comparison. It seems that the corresponding white-noise case is given by Eq.4 with tau_1=0, which still has the same dependence on the self-coupling s_2, contrary to what is claimed in the paper (it is unclear how the curve for the white-noise case in Fig.4 was obtained). Note that the UCNA has been designed such that it is valid for both small and large tau_1 (thus, it is also unclear why the assumption of large tau_1 is needed).

3) The given argument that the time-scale separation arises as network effect is not very clear. Apart from the issue of a fair comparison of colored and white noise raised in point 1 above, an external colored noise with matched statistics that drives a single bistable unit would yield the same MFPT and thus would be an alternative explanation independent of the network dynamics.

4) The UCNA has assumptions and regimes of validity that are not stated in the paper. In particular, it assumes an Ornstein-Uhlenbeck noise, which has an exponential auto-correlation function, and local stability (region where potential is convex). Because the self-consistent auto-correlation function is generally not exponential and the probe unit also visits regions where the potential is concave, the validity of the UCNA is not clear. On the other hand, the assumption of large correlation time might be dropped as the UCNA's main feature is that it works for both large and small correlation times.

Reviewer #1 (Public Review):

In this work, the authors were aiming to probe why enhancers tend to have multiple binding sites for the same transcription factor (TF). As a test bed, they use the snail distal enhancer, which drives a band of expression in the early Drosophila embryo and is composed of multiple, generally weak binding sites for several activating TFs. Using the MS2-MCP reporter system, the authors characterize the live mRNA dynamics driven by the wild-type and mutant enhancers, in which individual or pairs of binding sites have been deleted. They complement these experimental measurements with two computational models - a simple thermodynamic model to explore the cooperativity of TF binding to enhancers and a Hidden Markov Model to analyze the kinetic parameters of their dynamic measurements. The key finding from the experiments is that ablating any of several TF binding sites individually or in pairs dramatically reduces the expression levels, though not the spatial extent, of the snail distal enhancer. This effect holds true in a ~600 bp minimal enhancer and a ~1800 bp extended enhancer. The bulk of this effect is due to a marked decrease in transcriptional amplitude. A simple thermodynamic model confirms the intuition that synergy between the TF binding sites can explain the experimental results and further analysis shows that the modest decline in transcriptional burst duration in mutant enhancers is likely due to more frequent dissociation of the enhancer-promoter complex.

The paper's strengths include the use of well-established measurement and analysis techniques to uncover the surprisingly dramatic effect of single TF binding site mutations, even in the extended enhancer which contains ~20 TF binding sites. This work starts to chip away at the question of why multiple TF binding sites are so frequently observed in enhancers and complement studies of other similar enhancers. It is likely to be of interest to the enhancer biology community. It also sets the stage to explore whether this observation will generalize to other enhancers with different properties, e.g. those with stronger TF binding sites or whose activity is more strongly shaped by repressive TFs.

Reviewer #1 (Public Review):

In this manuscript, the authors investigated plausible circuit mechanisms for their recently reported effect of NMDAR antagonists on the synchrony of prefrontal neurons in a cognitive task. On the basis of previously proposed computational network models of spiking excitatory and inhibitory neurons and their mean-field and linear stability analysis descriptions, they show that a specific network configuration set close to the onset of instability of the asynchronous state can replicate qualitatively key empirical observations. For such a network, a small increase in external drive causes a large increase in neuronal synchrony, and this is not happening if NMDAR-dependent transmission is reduced. This shows parallelism with the empirical data thus representing its first neural network explanation.

The paper provides valuable insights into possible mechanisms related to cortical dysfunction under NMDAR hypofunction, a topic of importance for several neuropsychiatric disorders. However, the fact that the manuscript remains at a rather abstract level and does not attempt a closer match to the experimental data is a limitation of the study.

1) The manuscript is strongly based on state diagrams and parametric descriptions of neural dynamics in a computational model that has been extensively studied before (Brunel, Wang 2003). Many of the parametric dependencies of this model shown here were already reported before, although not specifically altering concurrently external inputs and NMDAR-dependent transmission as done now. The main novelty of the study is the application of this framework to a specific empirical dataset of great scientific relevance. However, the manuscript emphasizes the model exploration in relation to a limited set of effects in the data (changes in synchrony immediately before motor response) and not so much the comparison to the neural recordings more generally (for instance, firing rates, other time periods in the task, etc.)

2) As discussed in the introduction, empirical data available suggests that 0-lag synchrony in prefrontal networks is affected by manipulations that reduce NMDAR function (Zick et al. 2018) and by manipulations that enhance NMDAR function (Zick et al. 2021). The computational model presented in this manuscript does not show this U-shaped behavior and the discussion does not mention this. It should be discussed whether the model can accommodate this or not.

Reviewer #1 (Public Review):

Esmaily and colleagues report two experimental studies in which participants make simple perceptual decisions, either in isolation or in the context of a joint decision-making procedure. In this "social" condition, participants are paired with a partner (in fact, a computer), they learn the decision and confidence of the partner after making their own decision, and the joint decision is made on the basis of the most confident decision between the participant and the partner. The authors found that participants' confidence, response times, pupil dilation, and CPP (i.e. the increase of centro-parietal EEG over time during the decision process) are all affected by the overall confidence of the partner, which was manipulated across blocks in the experiments. They describe a computational model in which decisions result from a competition between two accumulators, and in which the confidence of the partner would be an input to the activity of both accumulators. This model qualitatively produced the variation in confidence and RTs across blocks.

The major strength of this work is that it puts together many ingredients (behavioral data, pupil and EEG signals, computational analysis) to build a picture of how the confidence of a partner, in the context of joint decision-making, would influence our own decision process and confidence evaluations. Many of these effects are well described already in the literature, but putting them all together remains a challenge. However, the construction is fragile in many places: the causal links between the different variables are not firmly established, and it is not clear how pupil and EEG signals mediate the effect of the partner's confidence on the participant's behavior.

Finally, one limitation of this setting is that the situation being studied is very specific, with a joint decision that is not the result of an agreement between partners, but the automatic selection of the most confident decisions. Thus, whether the phenomena of confidence matching also occurs outside of this very specific setting is unclear.

Joint Public Review

This manuscript utilizes Drosophila melanogaster as a model system to functionally characterize the role of genes previously associated with obstructive pulmonary disease (COPD) in epithelial barrier function. Using genetic and imaging approaches, the authors characterised a previously unrecognised role of intestinal Acetylcholine receptor (AchR) signalling, in the regulation of epithelial barrier function. The working model proposes that Acetylcholine (Ach) produced by enteroendocrine cells (EEs) and enteric neurons signals to AchR in enterocytes (ECs). This signalling activates the secretion of the Peritrophic membrane (PM) through the regulation of the exocytic protein Syt4. In this way, Ach/AchR signalling works to protect epithelial barrier function and organismal tolerance to ingested damaging agents, such as those causing oxidative stress.

Overall, the data presented support the main model of the paper: EC AchR activation is necessary to maintain epithelial barrier function. The evidence, however, on the mechanisms downstream of AchR, namely, the involvement of this signalling pathway in the regulation of Syt4 is weak.

The work in this manuscript represents an important proof of concept for the use of the Drosophila midgut as a model to functionally interrogate genes from human genetic association studies in pathologies affecting epithelial homeostasis.

Reviewer #1 (Public Review):

Mano et. al. use a combination of behavioral, genetic silencing, and functional imaging experiments to explore the temporal properties of the optomotor response in Drosophila. They find a previously unreported inversion of the behavior under high contrast and luminance conditions and identify potential pathways mediating the effect.

Strengths:<br /> Quantifications of optomotor behavior have been performed for many decades. Despite a large number of previous studies, the authors still find something fundamentally novel: under high contrast conditions and extended stimulation periods, the behavior becomes dynamic over time. The turning response shows an initial transient positive following response. The amplitude of the behavior then decreases and even inverts such that animals show an anti-directional rotation response. The authors systematically explore the stimulation feature space, including large ranges of spatial and temporal frequencies and conditions with high and low contrast. They also test two wild-type fly species and even compare experiments across two different labs and setups. From these data, it seems clear that the behavior is robust and largely depends on the brightness of the stimulation, rearing conditions, and genetic background. The authors discuss that these effects have not clearly been reported elsewhere beforehand, and convincingly argue why this may be the case.

In general, the presented behavioral quantifications illustrate the importance of further experimental studies of the temporal dynamics of behavior in response to dynamically varying stimulus features, across different stimulus types, genetic backgrounds, and model animal systems. It also illustrates the importance of relating the conditions that animals experience in the laboratory to the ones they would experience in the wild. As the authors mention, the brightness during a sunny day can reach values as high as 4000 cd/m2, while experimental stimulation in the lab has so far often been orders of magnitude below that.

The study then systematically explores potential neural elements involved in the behavior. Through a set of silencing experiments, they find that T4 and T5 neurons, as expected, are required for motion behaviors. On the other hand, silencing HS cells largely abolishes the 'classical' syn-directional response but leaves anti-directional turning intact. On the other hand, silencing CH cells abolishes the anti-directional response but leaves the syn-directional behavior intact. Through functional imaging in T4, T5, HS, and CH neurons, the authors could show that none of these neurons shows a response inversion depending on contrast level. Together, these experiments nicely illustrate that the dynamics do not seem to be computed within the early parts of visual processing, but they must happen on the level of the lobula plate or further downstream.

Weaknesses:<br /> While the authors have already explored various parameters of the experiment, it would have been nice to see additional experiments regarding the initial adaptation phase. The experiments in Figure 2e, where the authors show front-to-back or back-to-front gratings before the rotation phase, are a good start. What would the behavioral dynamics look like if they had exposed animals to long periods of static high or low contrast gratings, whole field brightness, or darkness? Such experiments would surely help to better understand the stimulus features on which the adaptation elements operate. It would be interesting to explore to what degree such static stimuli impact the subsequent behavioral dynamics.

Given the dynamics of the behavior, it would probably also be worth looking at the turning dynamics after the stimulus has stopped. If direction-selective adaptation mechanisms are regulating the turning response, one may find long-lasting biases even in the absence of stimulation. If the authors have more data after the stimulus end, it would be good to further expand the time range by a few seconds to show if this is the case or not (for example, in Figure 1b).

Another important experiment could be to initially perform experiments in a closed-loop configuration, and then quickly switch to open-loop. The closed-loop configuration should allow the motion computing circuitry to adapt to the chosen environmental conditions. Explorations of the changes in turning response dynamics after such treatments should then enable further dissections of the mechanisms of adaptation. Closed-loop experiments under different contrast conditions have already been performed (for example, Leonhardt et al. 2016), which also showed complex response dynamics after stimulus on- and offset. It would be great to discuss the current open-loop experiments, and maybe some new closed-loop results, in relation to the previous work.

The authors mention the different rearing conditions, and there is one experiment in Figure S2 which mentions running experiments at 25 deg C. But it is not clear from the Methods at which temperature all other experiments have been performed. It is also not clear at which temperature the shibire block experiments were performed. As such experiments require elevated temperatures, I assume that all behavioral experiments have been performed at such levels? How high were those?

What does the fly see before and after the stimulus (i.e. the gray boxes in all figures)? Are these periods of homogenous gray levels or are these non-moving gratings with the luminance and contrast of the subsequent stimulus? It would be important to add this information to the methods and to the figure illustrations or legends.

It would be nice to discuss the potential location where the motion adaptation may be implemented in the brain. A small model scheme as an additional figure could further help to discuss how such computations may be mechanistically implemented, helping readers to think about future experimental dissections of the behavior.

For setting up similar experiments in other labs, the authors need to better describe how they measured the luminance of the arena. Do they simply report the brightness delivered by the Lightcrafter system, or did they measure this with a lux-meter? If so, at which distance was the measurement performed and with which device? Given that the behavior is sensitive to the specific properties of the stimulus, it will be important to report these numbers carefully to enable other groups to reproduce effects.

Reviewer #1 (Public Review):

In this work, Cheikh et al. develop a novel method to probe tissue mechanics in vivo, with particular application to the early Drosophila embryo. The method is based on filling a pulled micropipette with a mixture of fluorescent dye and PDMS, which is cured and allowed to harden. Etching away the tip of the glass micropipette leaves exposed the PDMS core, which, like the bristles held in a brush handle, is easily deformed. Calibration of the stiffness of the PDMS tip allows for direct measurement of forces through the tip displacement. Apart from the particular application here, this method should prove to be widely useful in biological physics.

The authors then inserted this force probe into Drosophila embryos at the stage when cellularization has occurred, and demonstrate the ability to deform the tissue (visualized by fluorescently labelled cell walls). Crucially, the time course of the deformation can be controlled by the rate at which the pipette is translated, allowing for the study of potential viscous or viscoelastic effects.

The authors find from their experiments and extensive computational analysis of mechanical models of the embryo that there must be a significant difference between the mechanical properties of the apical and basal sides of the tissue.

This is a very well executed paper that provides compelling evidence for the utility of the experimental method and the particular issues in Drosophila mechanics. A strength of the paper is the clear and simple focus on a particular deformation and its experimental and theoretical analysis. The computational section is a bit less clearly connected to the observations, in the sense that some kind of very simplified model incorporating the apicobasal differences is lacking.

Reviewer #1 (Public Review):

In this study, the authors investigate the interactions between Plasmodium falciparum RH5, an essential ligand mediating erythrocyte invasion by the malaria parasite, and its cognate receptor basigin. Based on published observations that basigin forms complexes with the plasma membrane Ca2+-ATPase PMCA1/4 or monocarboxylate transporter MCT1, the authors asked whether RH5 can interact with basigin complexed with PMCA or MCT1, whether this modulates the function of PMCA and whether these interactions may explain the mechanism of action of neutralising antibodies targeting RH5. The objectives and rationale of the study are very clear.

Using size exclusion chromatography, 2D blue native PAGE, antibody shift, and depletion assays, the authors demonstrate that native basigin in human erythrocytes is essentially found in heteromeric complexes with either PMCA4 or MCT1. They measured the binding of PfRH5 to purified basigin-PMCA and basigin-MCT1 complexes by surface plasmon resonance and found that RH5 interacts with complexed basigin with higher affinity than with isolated basigin. RH5 did not alter the ATPase activity of PMCA, either in purified PMCA-basigin complexes or in CHO cells expressing human basigin and PMCA4, leading the authors to rule out RH5-mediated alteration of PMCA-mediated calcium export as a mechanism underlying the changes in calcium flux at the interface between the erythrocyte and the invading parasite. Finally, the authors used structural modelling to show that growth-inhibitory antibodies sterically block the binding of RH5 to basigin-PMCA and basigin-MCT1 complexes, providing a molecular explanation for why most potent anti-RH5 neutralising antibodies do not prevent RH5 binding to isolated basigin.

The paper is well-written and the claims are well-supported by the data. The study provides new insight into an essential interaction during blood-stage malaria and reveals the mode of action of growth-inhibitory antibodies, with potential implications for the design of RH5-based malaria vaccines. The study does not address whether PMCA and MCT1 are required during erythrocyte invasion by P. falciparum merozoites, and does not provide direct evidence to completely rule out a role of RH5-PMCA interaction in calcium flux modulation in the context of erythrocyte invasion by the parasite.

Reviewer #1 (Public Review):

Summary:<br /> The study assesses the impact of testing contacts of cases in school classes when identified, rather than at the end of quarantine, on various outcomes such as secondary infections, tracing delay, and identification of the possible source of infection. The authors find that the intervention likely reduced tracing delay and increased the number of possible infection sources. However, due to unmeasured confounding, it remains unclear if secondary transmission actually decreased. The analysis requires clarification and further explanation in parts.

Major strengths and weaknesses:<br /> The study benefits from the assessment of various outcomes in contact tracing in addition to changes in transmission, such as tracing delay, and the identification of putative infectors; however the assumption that other cases found in households are infectors of the index case rather than putative infectees, may introduce significant bias, but this is not mentioned in the Discussion despite being significant. It is difficult to understand the intervention in Figure 1 due to unclear labelling and incomplete descriptions in the caption. The authors mention that the same school class could be included multiple times for multiple outbreaks - was there a time cutoff for inclusion? I had a lot of trouble interpreting or reproducing the values given in Table 1. Firstly, the methods used to produce the RRs given are not described in the methods section of the paper. What are the outcomes - "classes" and "indexes" are poroly defined. Is this output from univariate or multivariate regression model, and what is the link function? I was also unable to reproduce the RRs listed in the table despite attempting several methods. The closest numbers I achieved were by crudely dividing the risks (e.g. for the RR for known infection source I took the ratio of indexes for which a school contact was suspected pre and post-intervention (644/1175)/(146/429) = 1.61), but if this is the case then the unknown class is by definition not the reference category. This is the same for the other RRs stated in the table. The methods used should be clarified and results updated if erroneous. The mediation analysis components and their relevance to the study could be better explained in the methods and results.

Achievement of aims and support for conclusions:<br /> The authors partially achieved their aims by demonstrating a likely decrease in tracing delay and an increase in possible infection sources. However, the study's inability to determine if secondary transmission decreased due to unmeasured confounding limits the conclusiveness of the findings. The authors should reiterate the main numerical results in the first few paragraphs of the discussion.

Impact on the field and utility of methods and data:<br /> This study has the potential to impact the field by highlighting the benefits of testing contacts earlier in school classes. The findings on reduced tracing delay and increased identification of infection sources can inform future strategies and interventions. However, clarity on the analysis methods, as well as the results, are necessary to ensure the utility and reliability of the findings.

Reviewer #1 (Public Review):

Briggs et al use a combination of mathematical modelling and experimental validation to tease apart the contributions of metabolic and electronic coupling to the pancreatic beta cell functional network. A number of recent studies have shown the existence of functional beta cell subpopulations, some of which are difficult to fully reconcile with established electrophysiological theory. More generally, the contribution of beta cell heterogeneity (metabolism, differentiation, proliferation, activity) to islet function cannot be explained by existing combined metabolic/electrical oscillator models. The present studies are thus timely in modelling the islet electrical (structural) and functional networks. Importantly, the authors show that metabolic coupling primarily drives the islet functional network, giving rise to beta cell subpopulations. The studies, however, do not diminish the critical role of electrical coupling in dictating glucose responsiveness, network extent as well as longer-range synchronization. As such, the studies show that islet structural and functional networks both act to drive islet activity, and that conclusions on the islet structural network should not be made using measures of the functional network (and vice versa).

Strengths:

- State-of-the-art multi-parameter modelling encompassing electrical and metabolic components.

- Experimental validation using advanced FRAP imaging techniques, as well as Ca2+ data from relevant gap junction KO animals.

- Well-balanced arguments that frame metabolic and electrical coupling as essential contributors to islet function.

- Likely to change how the field models functional connectivity and beta cell heterogeneity.

Weaknesses:

- Limitations of FRAP and electrophysiological gap junction measures not considered.

- Limitations of Cx36 (gap junction) KO animals not considered.

- Accuracy of citations should be improved in a few cases.

Reviewer #1 (Public Review):

Notwithstanding that the molecular underpinnings of the mechanistic target of rapamycin complex 1 (mTORC1) signaling are relatively well understood, quantitative data pertinent to mTORC1-dependent integration of a variety of stimuli is lacking. To address this question, Sparta et al., developed a series of fluorescent reporters that in combination with live cell microscopy allowed them to determine responses of mTORC1 to several stimuli including glucose, amino acids, and insulin at the single cell resolution. Considering the central role of mTORC1 in homeostasis and its dysregulation across a variety of pathological states, it was thought that this study should be of broad interest to a wide spectrum of biomedical disciplines ranging from biochemistry, molecular and cellular biology to neurobiology and cancer research.

Strengths: This study employs powerful approach based on use of live cell imaging of multiple fluorescent reports that are indicative of alterations in mTORC1 activity. In contrast to traditional approaches based on querying phosphorylation status of mTORC1 substrates by Western blotting this approach allows time-resolved measurement of mTORC1 activity at the single cell resolution. Using this approach, the authors provide solid evidence to corroborate a model of graded activation of mTORC1 by amino acids, insulin, and combination thereof.

Weaknesses: The major weaknesses were thought to be related to the interpretation of the current model of mTORC1 regulation as AND gate and reliance on a single cell line. Some minor technical issues were also observed pertinent to the lack of controls demonstrating the effectiveness of manipulations of nutrients and/or insulin as well as the effects of such manipulation on the expression of reporters used to monitor mTORC1 activity.

There are multiple degrees/levels of communication that any one person will run into within every day of their lives. First, and most importantly, is intrapersonal communication. Intrapersonal communication is a broad concept of communicative phenoms that occur within the individual mind. For many, it can resemble traditonal forms of external communication where a "voice" is acting as a guide to understanding and internalizing the world around us. Intrapersonal communication is hard to conceptualize, and can vary due to biological factors effecting each individuals brains to create unique mental environments. Interpersonal communication, which is the next most intimate form of communication, is when two people exchange messages or share a dialogue. This only occurs with one other person, as the dynamic within a conversation completely changes on a psychological level when more people are there. Then there are multiple interactions of group communication, going from small, to public settings, and larger. All of these, albeit intrapersonal, can be done through verbal, nonverbal, and mediated communication.

Communication Competence refers to the values that differentiate successful, and non successful speakers. it involves a balance of both appropriate and effective communication, where omitting one or the other characteristics could damage peoples perceptions of your communication skills. Gauging these aspects to tailor to certain people and situations is the most effective strategy; communication that is successful in one context may go awfully in another dependent on culture, personal morals of your listener, etc. The way you successfully communicate with a child would not bode well if used during a college conference.

Although general applications of communication vary; individuals who have a degree of communication competence generally share similar personal values and skills that help achieve greater communication. Skillfullness, or the ability to take note of and work around/correctly use situational cues to approach a conversation the best they can, is one of these characteristics. The ability to work and adapt under stressful and pressured situations is another important skill to a competent communicator. Having a general idea of possible sources of error, and being able to switch on the fly to make light of those failed situations can be just as important as having a line of communication go exactly as planned. Involvement and understanding the audience tend to go hand in hand, where involvement ties into persuasiveness and excitability of the audience, and understanding is more of an emotional tie-in that creates a deeper, empathetic bond between speaker and audience to tap into their feelings to attempt to seem as genuine and "human" as possible. The last two characteristics, cognitive complexity and self-monitoring, both are skills that typically take practice and a lot of inward thinking to achieve. Cognitive complexity means saying the same thing in different ways. It can either mean changing verbiage to different synonyms, or completely altering a sentence while still maintaining the original meaning. Providing different way to comprehend the same message opens a wider range of people to interact and find your message valuable. Self Monitoring is the hardest of these characteristics to get, as it means turning inwards to focus on your behaviors and manipulate said behavior for your advantage. This can mean having pre-meditated points of conversation to follow, being very aware of the flow of the conversation, having a "meta" sense of your self and paying attention to things like body language, etc.

Reviewer #1 (Public Review):

This paper presents extensive numerical simulations using a model that incorporates up to second-order epistasis to study the joint effects of microscopic epistasis and clonal interference on the evolutionary dynamics of a microbial population. Previous works that explicitly modeled microscopic epistasis typically assumed strong selection & weak mutation (SSWM), a condition that is generally not met in real-life evolutionary processes. Alternatively, another class of models coarse-grained the effects of microscopic epistasis into a generic distribution of fitness effects. The framework introduced in this paper represents an important advance with respect to these previous approaches, allowing for the explicit modeling of microscopic epistasis in non-SSWM scenarios. The modeling framework presented promises to be a valuable tool to study microbial evolution in silico.

Reviewer #1 (Public Review):

This article is interested in how butterfly, or more precisely, butterfly wing scale precursor cells, each make precisely patterned ultrastructures made of chitin.

To do this, the authors sought to use the butterfly Parides eurimedes, a papilionid swallowtail, that carries interesting, unusual structures made of 1) vertical ridges, that lack a typical layered stacking arrangement; and 2) deep honeycomb-like pores (rather than. These two features make the organism chosen a good point of comparison with previous studies, including classic papers that relied on electronic microscopy (SEM/TEM), and more recent confocal microscopy studies.

The article shows good microscopy data, including detailed, dense developmental series of staining in the Parides eurimedes model. The mix of cell membrane staining, chitin precursor, and F-actin staining is well utilized and appropriately documented with the held of 3D-SIM, a microscopy technique considered to provide super-resolution (here needed to visualize sub-cellular processes).

The key message from this article is that F-actin filaments are later repurposed, in papilionid butterflies, to finish the patterning of the inter-ridge space, elaborating new structures (this was not observed so far in other studies and organisms). The model proposed in Figure 6 summarized these findings well, with F-actin reshaping itself into a tulip that likely pulls down a chitin disk to form honeycombs. These interpretations of the microscopy data are interesting and novel.

There are two other points of interest, that deserve future investigation:

1) The authors performed immunolocalizations of Arp2 and pharmacological inhibitions of Arp2/3, and found some possible effect on honeycomb lattice development. The inter-ridge region of the butterfly Papilio polytes, which lacks these structures, did not seem to be affected by drug treatments. Effects were time-dependent, which makes sense. These data provide circumstantial evidence that Arp2/3 is involved in the late role of F-actin formation or re-organisation.

2) The authors perform a comparative study in additional papilionids (Fig. 6 in particular). I find these data to be quite limited without a dense sampling, but they are nonetheless interesting and support a second-phase role of F-actin re-organisation.

The article is dense, well produced and succinctly written. I believe this is an interesting and insightful study on a complex process of cell biology, that inspires us to look at basic phenomena in a broader set of organisms.

Reviewer #1 (Public Review):

Funabiki et al, performed a co-evolutionary analysis of Lsh/HELLS and CDCA7, two factors with links to DNA methylation pathways in mammals, amphibia and fish. The authors suggest that conserved roles for the two factors in DNA methylation maintenance pathways can be traced back to the last eukaryotic common ancestor. Overall, the findings are important and the results could be useful for researchers studying DNA methylation pathways in many different organisms.

Comments on current version:

In the revised version of this manuscript the authors addressed all previously raised issues. I would like to thank them for that. The data is now clearly presented and interpreted and more experimental detail has been added. Thus, the manuscript is much improved and provides an interesting basis for experimental follow-up and further functional investigations.

Reviewer #1 (Public Review):

In chicken embryos, the counter-rotating migration of epiblast cells on both sides of the forming primitive streak (PS), a process referred to as polonaise movements, has attracted longstanding interest as a paradigm of morphogenetic cell movements. However, the association between these cell movements and PS development is still controversial. This study investigated PS development and polonaise movements separately at their initial stage, showing that both could be uncoupled (at least at the initial phase), being activated via Vg1 signaling.

Strengths of this study

Polonaise movements, i.e., the circular cell migration of epiblast cells on both sides of the forming PS in avian embryos, have been the subject of research through live imaging and promoted the development of new tools to analyze quantitatively such movements. However, conclusions from previous studies remain controversial, at least partly due to the nature of perturbations to PS development and polonaise movements.

This study performed the challenging technique of electroporation to successfully mark and manipulate Wnt/PCP pathways in unincubated chicken embryo cells at the initiation phase of these two processes. In addition, the authors separately altered PS development and polonaise movements: PS development was perturbed by inhibiting either the Wnt/PCP pathway or DNA synthesis using aphidicolin, while polonaise movements were modified by the development of a second PS after engrafting Vg1-expressing COS cells located at the opposite end of the blastoderm. The study concluded that Vg1 elicits both PS development and polonaise movements, which occur in a parallel and are not inter-dependent.

To support these conclusions, particle image velocimetry (PIV) of cell trajectories captured by live imaging was performed. These tools delineated visually appealing cell movements and gave rise to vorticity profiles, adding more value to this study.

Weaknesses of this study

Engrafted Vg1-expressing COS cells located at the anterior end of the blastoderm elicited both the development of a second PS and marked bilateral polonaise movements while perturbing these movements along the original PS. How do polonaise movements along the second PS dominate over those along the normal PS? The authors suggested a model in which Vg1 acts in a graded or dose-dependent manner since engrafted COS cells over-expressed Vg1. This model can be tested by reducing the mass of engrafted COS cells. Although the authors propose performing this analysis in further investigations, it would be preferable to incorporate into this study for better consistency.

The authors claim that chicken embryo development is representative of "amniotes," but it does not hold for all groups. Avian and mammal species are exceptional among amniotes in the sense they develop a PS (e.g., Coolen et al. 2008). Moreover, in certain mammalian embryos like mouse embryos, cells laterally to the PS do not move much (Williams et al. 2012). The authors should avoid the generalization that chicken embryos unequivocally represent amniotes as opposed to the observed in non-amniote embryos. The observations in chicken embryos as they stand are significant enough.

References:<br /> Coolen M, et al. (2008). Molecular characterization of the gastrula in the turtle Emys orbicularis: an evolutionary perspective on gastrulation. PLoS One. 3(7):e2676. doi: 10.1371/journal.pone.0002676

Williams M, et al. (2012). Mouse primitive streak forms in situ by initiation of epithelial to mesenchymal transition without migration of a cell population. Dev Dyn. 241(2):270-283. doi: 10.1002/dvdy.23711

Reviewer #1 (Public Review):

Summary:<br /> The authors explored correlations between taste features of botanical drugs used in ancient times and therapeutic uses, finding some potentially interesting associations between intensity and complexity of flavors and therapeutic potential, plus some more specific associations described in the discussion sections. I believe the results could be of potential benefit to the drug discovery community, especially for those scientists working in the field of natural products.

Strengths:<br /> Owing to its eclectic and somehow heterodox nature, I believe the article might be of interest to a general audience. In fact, I have enjoyed reading it and my curiosity was raised by the extensive discussion.

The idea of revisiting a classical vademecum with new scientific perspectives is quite stimulating.

The authors have undertaken a significant amount of work, collecting 700 botanical drugs and exploring their taste and association with known uses via eleven trained panelists.

Weaknesses:<br /> I have some methodological concerns. Was subjective bias within the panel of participants explored or minimized in any manner? Were the panelists exposed to the drugs blindly and on several occasions to assess the robustness of their perceptions? Judging from the total number of taste assessments recorded and from Supplementary Material, it seems that not every panelist tasted every drug. Why? It may be a good idea to explore the similarity in the assessments of the same botanical drug by different volunteers. If a given descriptor was reported by a single volunteer, was it used anyway for the statistical analysis or filtered out?

The idea of "versatility" is repeatedly used in the manuscript, but the authors do not clearly define what they call "versatile".

The introduction should be expanded. There are plenty of studies and articles out there exploring the evolution of bitter taste receptors, and associating it with a hypothetical evolutionary advantage since bitter plants are more likely to be poisonous. Since plant secondary metabolites are one of the most important sources of therapeutic drugs and one of their main functions is to protect plants from environmental dangers (e.g., animals), this evolutionary interplay should be at least briefly discussed in the introductory section. Since the authors visit some classical authors, Parecelsus' famous quote "All things are poison and nothing is without poison. Solely the dose determines that a thing is not a poison" may be relevant here. Also note that some authors have explored the relationship between taste receptors and pharmacological targets (e.g., Bioorg Med Chem Lett. 2012 Jun 15;22(12):4072-4).

Reviewer #1 (Public Review):

Summary:<br /> The overall analysis and discovery of the common motif are important and exciting. Very few human/primate ribozymes have been published and this manuscript presents a relatively detailed analysis of two of them. The minimized domains appear to be some of the smallest known self-cleaving ribozymes.

Strengths:<br /> The manuscript is rooted in deep mutational analysis of the OR4K15 and LINE1 and subsequently in modeling of a huge active site based on the closely-related core of the TS ribozyme. The experiments support the HTS findings and provide convincing evidence that the ribozymes are structurally related to the core of the TS ribozyme, which has not been found in primates prior to this work.

Weaknesses:<br /> 1. Given that these two ribozymes have not been described outside of a single figure in a Science Supplement, it is important to show their locations in the human genome, present their sequence and structure conservation among various species, particularly primates, and test and discuss the activity of variants found in non-human organisms. Furthermore, OR4K15 exists in three copies on three separate chromosomes in the human genome, with slight variations in the ribozyme sequence. All three of these variants should be tested experimentally and their activity should be presented. A similar analysis should be presented for the naturally-occurring variants of the LINE1 ribozyme. These data are a rich source for comparison with the deep mutagenesis presented here. Inserting a figure (1) that would show the genomic locations, directions, and conservation of these ribozymes and discussing them in light of this new presentation would greatly improve the manuscript. As for the biological roles of known self-cleaving ribozymes in humans, there is a bioRxiv manuscript on the role of the CPEB3 ribozyme in mammalian memory formation (doi.org/10.1101/2023.06.07.543953), and an analysis of the CPEB3 functional conservation throughout mammals (Bendixsen et al. MBE 2021). Furthermore, the authors missed two papers that presented the discovery of human hammerhead ribozymes that reside in introns (by de la PeÃ{plus minus}a and Breaker), which should also be cited. On the other hand, the Clec ribozyme was only found in rodents and not primates and is thus not a human ribozyme and should be noted as such.

2. The authors present the story as a discovery of a new RNA catalytic motif . This is unfounded. As the authors point out, the catalytic domain is very similar to the Twister Sister (or "TS") ribozyme. In fact, there is no appreciable difference between these and TS ribozymes, except for the missing peripheral domains. For example, the env33 sequence in the Weinberg et al. 2015 NCB paper shows the same sequences in the catalytic core as the LINE1 ribozyme, making the LINE1 ribozyme a TS-like ribozyme in every way, except for the missing peripheral domains. Thus these are not new ribozymes and should not have a new name. A more appropriate name should be TS-like or TS-min ribozymes. Renaming the ribozymes to lanterns is misleading.

3. In light of 2) the story should be refocused on the fact the authors discovered that the OR4K15 and LINE1 are both TS-like ribozymes. That is very exciting and is the real contribution of this work to the field.

4. Given the slow self-scission of the OR4K15 and LINE1 ribozymes, the discussion of the minimal domains should be focused on the role of peripheral domains in full-length TS ribozymes. Peripheral domains have been shown to greatly speed up hammerhead, HDV, and hairpin ribozymes. This is an opportunity to show that the TS ribozymes can do the same and the authors should discuss the contribution of peripheral domains to the ribozyme structure and activity. There is extensive literature on the contribution of a tertiary contact on the speed of self-scission in hammerhead ribozymes, in hairpin ribozyme it's centered on the 4-way junction vs 2-way junction structure, and in HDVs the contribution is through the stability of the J1/2 region, where the stability of the peripheral domain can be directly translated to the catalytic enhancement of the ribozymes.

5. The argument that these are the smallest self-cleaving ribozymes is debatable. LÃ1/4nse et al (NAR 2017) found some very small hammerhead ribozymes that are smaller than those presented here, but the authors suggest only working as dimers. The human ribozymes described here should be analyzed for dimerization as well (e.g., by native gel analysis) particularly because the authors suggest that there are no peripheral domains that stabilize the fold. Furthermore, Riccitelli et al. (Biochemistry) minimized the HDV-like ribozymes and found some in metagenomic sequences that are about the same size as the ones presented here. Both of these papers should be cited and discussed.

6. The authors present homology modeling of the OR4K15 and LINE1 ribozymes based on the crystal structures of the TS ribozymes. This is another point that supports the fact that these are not new ribozyme motifs. Furthermore, the homology model should be carefully discussed as a model and not a structure. In many places in the text and the supplement, the models are presented as real structures. The wording should be changed to carefully state that these are models based on sequence similarity to TS ribozymes. Fig 3 would benefit from showing the corresponding structures of the TS ribozymes.

Reviewer #1 (Public Review):

The authors have performed extensive work generating reporter mice and performing single-cell analysis combined with in situ hybridization to arrive at 14 clusters of enterochromaffin (EC) cells. Then, they focus on Piezo channel expression in distal EC cells and find that these channels might play a role in regulating colonic motility. Overall, this is an informative study that comprehensively classifies EC cells in different regions of the small and large intestine. From a functional point of view, however, the authors seem to ignore the fact that the expression of Piezo-2-IRES-Cre is broad, which would raise concerns regarding their physiological conclusions.

The authors may wish to consider the following specific points:

It is surprising that the number of ileal EC cells is less than that of the distal colon, and it would be interesting to know whether the authors can comment about ileal EC cells. It is unclear why ileal ECs were not included in the study, even though they are mentioned in the diagram (Fig. 2c).

Based on their analysis, there are 10 EC cell clusters in SI while there are only 4 clusters in the colon. The authors should comment on whether this is reflective of lesser diversity among colonic ECs or due to the smaller number of colonic ECs collected.

The authors previously described that distal colonic EC cells exhibit various morphologies (Kuramoto et al., 2021). Do Ascl1(+) EC cells particularly co-localize with EC cells with long basal processes? Also, to validate the RNA seq data, the authors might show co-localization between Piezo2/Ascl1/Tph1 in distal EC cells. It would be interesting to see whether Ascl1-CreER (which is available in Jax) specifically labels distal colonic EC cells as this could provide a good genetic tool to specifically manipulate distal colonic EC cells.

The authors used Piezo2-IRES-Cre mice, whose expression is rather broad. They might examine the distribution of Chrm3-mCitrine in the intestine (IF/IHC would be straightforward). And if the expression is in other cell types (which is most likely the case), they should justify that the observed phenotype derives from Piezo2-expressing EC cells. Alternatively, they could use Piezo2-Cre;ePetFlp (or Vil-Flp);Chrm3 to specifically express DREADD receptors in distal colonic EC cells. Also, what does 5HT release look like in jejunal EC cells in Piezo-CHRM3 mice?

For the same reasons as above, DTR experiments may also be non-specific. For example, based on the IF staining (Fig. 6b,d), there seems to be a loss of Tph1+ cells in the proximal colon of Piezo2-DTR mice, so the effects of the Piezo2-DTR likely extend beyond the distal colon.

It is unclear why the localized loss of Piezo2 in Piezo2-DTR mice alters small intestinal transit (Fig. 6g,h). The authors should discuss the functional differences observed between Piezo2-DTR (intraluminal app) and Vil1-Piezo2 KO mice i.e., small intestinal transit, 5HT release, etc. Are these differences due to the residual Piezo2 expression in Piezo2 KO mice? In this context, the authors may want to discuss their findings in the context of recent papers, such as those from the Patapoutian and Ginty groups.

Reviewer #1 (Public Review):

Summary:<br /> This study aims to further resolve the history of speciation and introgression in Heliconius butterflies. The authors break the data into various partitions and test evolutionary hypotheses using the Bayesian software BPP, which is based on the multispecies coalescent model with introgression. By synthesizing these various analyses, the study pieces together an updated history of Heliconius, including a multitude of introgression events and the sharing of chromosomal inversions.

Strengths:<br /> Full-likelihood methods for estimating introgression can be very computationally expensive, making them challenging to apply to datasets containing many species. This study provides a great example of how to apply these approaches by breaking the data down into a series of smaller inference problems and then piecing the results together. On the empirical side, it further resolves the history of a genus with a famously complex history of speciation and introgression, continuing its role as a great model system for studying the evolutionary consequences of introgression. This is highlighted by a nice Discussion section on the implications of the paper's findings for the evolution of pollen feeding.

Weaknesses:<br /> The analyses in this study make use of a single method, BPP. The analyses are quite thorough so this is okay in my view from a methodological standpoint, but given this singularity, more attention should be paid to the weaknesses of this particular approach. Additionally, little attention is paid to comparable methods such as PhyloNet and their strengths and weaknesses in the Introduction or Discussion. BPP reduces computational burden by fixing certain aspects of the parameter space, such as the species tree topology or set of proposed introgression events. While this approach is statistically powerful, it requires users to make informed choices about which models to test, and these choices can have downstream consequences for subsequent analyses. It also might not be as applicable to systems outside of Heliconius where less previous information is available about the history of speciation and introgression. In general, it is likely that most modelling decisions made in the study are justified, but more attention should be paid to how these decisions are made and what the consequences of them could be, including alternative models.

• Co-estimating histories of speciation and introgression remains computationally challenging. To circumvent this in the study, the authors first estimate the history of speciation assuming no gene flow in BPP. While this approach should be robust to incomplete lineage sorting and gene tree estimation, it is still vulnerable to gene flow. This could result in a circular problem where gene flow causes the wrong species tree to be estimated, causing the true species tree to be estimated as a gene flow event. This is a flaw that this approach shares with summary-statistic approaches like the D-statistic, which also require an a-priori species tree. Enrichment of particular topologies on the Z chromosome helps resolve the true history in this particular case, but not all datasets will have sex chromosomes or chromosome-level assemblies to test against.

• The a-priori specification of network models necessarily means that potentially better-fitting models to the data don't get explored. Models containing introgression events are proposed here based on parsimony to explain patterns in gene tree frequencies. This is a reasonable and common assumption, but parsimony is not always the best explanation for a dataset, as we often see with phylogenetic inference. In general, there are no rigorous approaches to estimating the best-fitting number of introgression events in a dataset. Likewise, the study estimates both pulse and continuous introgression models for certain partitions, though there is no rigorous way to assess which of these describes the data better.

• Some aspects of the analyses involving inversions warrant additional consideration. Fewer loci were able to be identified in inverted regions, and such regions also often have reduced rates of recombination. I wonder if this might make inferences of the history of inverted regions vulnerable to the effects of incomplete lineage sorting, even when fitting the MSC model, due to a small # of truly genealogically independent loci. Additionally, there are several models where introgression events are proposed to explain the loss of segregating inversions in certain species. It is not clear why these scenarios should be proposed over those in which the inversion is lost simply due to drift or selection.

Reviewer #1 (Public Review):

Summary:

This is a very well written and performed study describing a TOPBP1 separation of function mutation, resulting in defective MSCI maintenance but normal sex body formation. The phenotype differs from that of a previous TOPBP1 null allele, in which both MSCI and sex body formation were defective. Additional defects in CHK phosphorylation and SETX localization are also described.

Strengths:

The study is very rigorous, with a remarkably large number of MSCI marks assayed, phosphoproteomics (leading to the interesting SETX discovery) and 10X RNAseq, allowing the MSCI phenotype to be further deconvolved. The approaches in most cases are robust.

Weaknesses:

There aren't many; please find list below:

1. The authors are committed to the idea that maintenance of MSCI is the major defect here. However, based on the data, an alternative would be that some cells achieve sex body formation and MSCI normally, while others do not. It would only take a small percentage of cells exhibiting MSCI failure to kill all the cells in the same germinal epithelium, so this could still explain the complete pachytene block. This isn't a major point...this phenotype is clearly different to the TOPBP1 KO, but a broader discussion of possibilities in the discussion would help. I raise this in the context of both the cytology and 10X analysis:

a) The assessment that sex body formation is normal is based on cytology in Supp 8 and 9, but a more rigorous approach would be to assess condensation of the XY pair in stage-matched spread cells (maybe they have that data already) by measuring distances between the X and Y centromere, or looking at stage IV of the seminiferous cycle, where all cells should have oval sex bodies but sex body mutants have persistent elongated XY pairs (see work of Namekawa and Turner). The authors do actually mention that gH2AX spreading is defective in many cells....and if this is true, condensation to form a sex body would almost certainly not have taken place in those cells.

b) Regarding the 10X data, the finding that expression of some XY genes is elevated and others are not is also consistent with a "partial" phenotype (some cells have normal XY bodies and MSCI, others fail in both). In Fig 6E, X expression looks to be elevated in B5 vs wt at all stages...if this were a maintenance issue, shouldn't it be equal to that in wt and then elevate later?

2. How is the quantitation showing impaired localization of select markers (e.g. SETX) normalized? How do we know that the antibody staining simply didn't work as well on the mutant slides?

3. Is testis TOPBP1 protein expression reduced in the B5 mutant?

4. 10X analysis: how were the genes on the y-axis in Supp 24 arranged? Is this by location on the X chromosome?

5. The final analyses in Fig 7: X-genes are subdivided based on their behavior (up, down, unchanged). What isn't clear to me is whether the authors have considered the fact that there are global changes in gene expression during meiosis (very low in lep , zyg and early pach, then ramps up hugely from mid pach). In other words, is this normalized to autosomal gene expression?

6. Again regarding the 10X analysis, my prediction would be that not ALL X and Y gene would increase in pach if MSCI were ablated...we should remember that XY genes have been subject to MSCI for some 160 million years of evolution, and this will mean that many enhancers that originally drove their expression prior to the evolution of MSCI will now be lost. This has been our experience: many XY genes aren't elevated at pach even in mutants in which MSCI is totally defective. I'd urge the authors to consider this possibility when they use XY gene expression patterns to diagnose the severity or timing of the MSCI phenotype. This could be a discussion point.

Reviewer #1 (Public Review):

The study by Kahraman et al describes the application of a reaction-based probe "diacetylated Zinpyr1" (DA-ZP1) that was developed for the enrichment of human islet beta cells (Lee et al. 2020 to purify human cadaveric alpha cells. The probe binds zinc with high enough affinity to allow the authors to separate beta cells from alpha cells based on the fluorescence intensity; beta cells had high intensity and alpha cells had medium intensity. FACs sorting of cells with intermediate fluorescent intensity were enriched for glucagon expression indicating they were alpha cells. They went on to reaggregate the purified alpha cells into pseudo-islets to test for viability, proliferation, ability to secrete glucagon and transcriptome analysis. These studies demonstrated that the pseudo-alpha cell islets were able to be maintained in culture for up to 10 days without losing their function and with only minor changes in gene expression.

The strengths of the manuscript include:<br /> 1. The description and characterization of a novel tool with which to purify human islet alpha cells<br /> 2. The ability to use the same DA-ZP1 probe to purify both human alpha and beta cells<br /> 3. The functional analysis to show that purified alpha cells retain their identity and maintain function even after in vitro culturing.<br /> 4. Providing a comparison of the transcriptome between whole islets, unsorted islets and sorted alpha cell pseudo-islets. The data is strengthened by the use of four donor islets and several timepoints for the transcriptomic analysis.<br /> 5. The quality of the data and data presentation

Weaknesses include:<br /> 1. Lack of a comparison with other published methods to purify human alpha cells<br /> 2. Unbiased transcriptome analysis of the sorted "high" vs. "medium" fluorescent populations to assess the degree of cross contamination between the 2 populations<br /> 3. Use of only one donor islet for functional analyses

Overall, this study represents a solid characterization of a new tool for purifying cadaveric human alpha cells that will be useful to researchers in the islet biology and diabetes fields.

Reviewer #1 (Public Review):

The authors primary objective in this study was to identify differences between patients with preeclampsia and normal patients with respect to the placental syncytiotrophoblast extracellular vesicle proteome.

One of the strengths of this study is that it is one of only a few studies that investigated the difference in proteome between patients with preeclampsia and those with normal pregnancies using placental extracellular vesicles obtained by an ex-vivo dual lobe placenta perfusion technique.

The main weaknesses of this study are:

1. The small sample size in that there were only 12 cases.<br /> 2. The study patients and control population of normal pregnancies were not matched for gestational age at delivery.

The authors were able to achieve their study aims and the results support their conclusions.

These findings could be used in future studies of the disease mechanisms and as biomarkers for prediction of preeclampsia. As such, they may be very useful for the identification of women at risk for preeclampsia well before the onset of disease.

Reviewer #1 (Public Review):

Summary:

How plants perceive their environment and signal during growth and development is of fundamental importance for plant biology. Over the last few decades, nano domain organisation of proteins localised within the plasma-membrane has emerged as a way of organising proteins involved in signal pathways. Here, the authors addressed how a non-surface localised signal (viral infection) was resisted by PM localised signalling proteins and the effect of nano domain organisation during this process. This is valuable work as it describes how an intracellular process affects signalling at the PM where most previous work has focused on the other way round, PM signalling effecting downstream responses in the plant. They identify CPK3 as a specific calcium dependent protein kinase which is important for inhibiting viral spread. The authors then go on to show that CPK3 diffusion in the membrane is reduced after viral infection and study the interaction between CPK3 and the remorins, which are a group of scaffold proteins important in nano domain organisation. The authors conclude that there is an interdependence between CPK3 and remorins to control their dynamics during viral infection in plants.

Strengths:

The dissection of which CPK was involved in the viral propagation was masterful and very conclusive. Identifying CPK3 through knockout time course monitoring of viral movement was very convincing. The inclusion of overexpression, constitutively active and point mutation non functioning lines further added to that.

Weaknesses:

My main concerns with the work are twofold.<br /> 1) Firstly, the imaging described and shown is not sufficient to support the claims made. The PM localisation and its non-PM localised form look similar and with no PM stain or marker construct used to support this. The sptPALM data conclusions are nice and fit the narrative. However, no raw data or movie is shown, only representative tracks. Therefore the data quality cannot be verified and in addition, the reporting of number of single particle events visualised per experiment is absent, only number of cells imaged is reported. Therefore it is impossible for the reader to appreciate the number of single molecule behaviours obtained and hence the quality of the data.

2) Secondly, remorins are involved in a lot of nano domain controlled processes at the PM. The authors have not conclusively demonstrated that during viral infection the remorin effects seen are solely due to its interaction with CPK3. The sptPALM imaging of REM1.2 in a cpk3 knockout line goes part way to solve this but more evidence would strengthen it in my opinion. How do we not know that during viral infection the entire PM protein dynamics and organisation are altered? Or that CPK3 and REM are at very distant ends of a signalling cascade. Negative control experiments are required here utilising other PM localised proteins which have no role during viral infection. In addition, if the interaction is specific, the transiently expressed CPK3-CA construct (shown to from nano domains) should be expressed with REM1.2-mEOS to show the alterations in single particle behaviour occur due to specific activations of CPK3 and REM1.2 in the absence of PIAMV viral infection and it is not an artefact of whole PM changes in dynamics during viral infection.

In addition, displaying more information throughout the manuscript (such as raw particle tracking movies and numbers of tracks measured) on the already generated data would strengthen the manuscript further.

Overall, I think this work has the potential to be a very strong manuscript but additional reporting of methods and data are required and additional lines of evidence supporting interaction claims would significantly strengthen the work and make it exceptional.

Reviewer #1 (Public Review):

Summary of the major findings -

1. The authors used saturation mutagenesis and directed evolution to mutate the highly conserved fusion loop (98 DRGWGNGCGLFGK 110) of the Envelope (E) glycoprotein of Dengue virus (DENV). They created 2 libraries with parallel mutations at amino acids 101, 103, 105-107, and 101-105 respectively. The in vitro transcribed RNA from the two plasmid libraries was electroporated separately into Vero and C6/36 cells and passaged thrice in each of these cells. They successfully recovered a variant N103S/G106L from Library 1 in C6/36 cells, which represented 95% of the sequence population and contained another mutation in E outside the fusion loop (T171A). Library 2 was unsuccessful in either cell type.

2. The fusion loop mutant virus called D2-FL (N103S/G106L) was created through reverse genetics. Another variant called D2-FLM was also created, which in addition to the fusion loop mutations, also contains a previously published, evolved, and optimized prM-furin cleavage sequence that results in a mature version of the virus (with lower prM content). Both D2-FL and D2-FLM viruses grew comparably to wild type virus in mosquito (C6/36) cells but their infectious titers were 2-2.5 log lower than wildtype virus when grown in mammalian (Vero) cells. These viruses were not compromised in thermostability, and the mechanism for attenuation in Vero cells remains unknown.

4. Next, the authors probed the neutralization of these viruses using a panel of monoclonal antibodies (mAbs) against fusion loop and domain I, II and III of E protein, and against prM protein. As intended, neutralization by fusion loop mAbs was reduced or impaired for both D2-FL and D2-FLM, compared to wild type DENV2. D2-FLM virus was equivalent to wild type with respect to neutralization by domain I, II, and III antibodies tested (except domain II-C10 mAb) suggesting an intact global antigenic landscape of the mutant virion. As expected, D2-FLM was also resistant to neutralization by prM mAbs (D2-FL was not tested in this batch of experiments).

5. Finally, the authors evaluated neutralization in the context of polyclonal serum from convalescent humans (n=6) and experimentally infected non-human primates (n=9) at different time points (27 total samples). Homotypic sera (DENV2) neutralized D2-FL, D2-FLM, and wild type DENV similarly, suggesting that the contribution of fusion loop and prM epitopes is insignificant in a serotype-specific neutralization response. However, heterotypic sera (DENV4) neutralized D2-FL and D2-FLM less potently than wild type DENV2, especially at later time points, demonstrating the contribution of fusion loop- and prM-specific antibodies to heterotypic neutralization.

Impact of the study-

1. The engineered D2-FL and D2-FLM viruses are valuable reagents to probe antibodies targeting the fusion loop and prM in the overall polyclonal response to DENV.

2. Though more work is needed, these viruses can facilitate the design of a new generation of DENV vaccine that does not elicit fusion loop- and prM-specific antibodies, which are often poorly neutralizing and lead to antibody-dependent enhancement effect (ADE).

3. This work can be extended to other members of the flavivirus family.

4. A broader impact of their work is a reminder that conserved amino acids may not always be critical for function and therefore should not be immediately dismissed in substitution/mutagenesis/protein design efforts.

Appraisal of the results -

The data largely support the conclusions, but some improvements and extensions can benefit the work.

1. In Figure 3A, the authors concluded that the engineered dengue virus fusion loop mutant viruses are insensitive to monoclonal antibodies (mAbs) targeting the fusion loop. However, the reduction in neutralization sensitivity varied depending on the mAb tested. The contribution of the optimized prM cleavage site (D2-FLM) to sensitivity to fusion loop mAbs also varied.

a) Are the epitopes known for these mAbs? It would be useful to discuss how the epitope of 1M7 differs from the other mAbs. What are the critical residues?<br /> d) Maybe the D2-FL mutant can be further evolved with selection pressure with fusion loop mAbs 1M7 +/-1N5 and/or other fusion loop mAbs.

2. It would have been useful to include D2-M for comparison (with evolved furin cleavage sequence but no fusion loop mutations).

3. Data for polyclonal serum can be better discussed. Table 1 is not discussed much in the text.

Suggestions for further experiments-

1. It would be interesting to see the phenotype of single mutants N103S and G106L, relative to double mutant N103S/G106L (D2-FL).<br /> 2. The fusion capability of these viruses can be gauged using liposome fusion assay under different pH conditions and different lipids.<br /> 3. Correlative antibody binding vs neutralization data would be useful.

Reviewer #1 (Public Review):

This study investigated an important question in human reproduction: why most fully aneuploid embryos is incompatible with normal fetal development. Specifically, the authors investigated the cellular responses to aneuploidy through analysis of gene expression in a set of donated human blastocysts. The samples included uniform aneuploid embryos of meiotic origin and mosaic aneuploid embryos from the SAC inhibitor reversine treatment. The authors relied mainly on low-input RNA sequencing and immunofluorescence staining. Pathway analysis with RNA-seq data of trophectoderm cells suggested activation of p53 and possibly apoptosis, and this cellular signature appeared to be stronger in TE cells with a higher degree of aneuploidy. Immunostaining also found some evidence of apoptosis, increased expression of HSP70 and autophagy in some aneuploid cells. With combinational OCT4 and GATA4 as lineage markers, it appeared that aneuploidy could alter the second lineage segregation and primitive endoderm formation in particular.

Although this study is largely descriptive, it generated valuable RNA-seq data from a set of aneuploid TE cells with known karyotypes. Immunostaining results in general were consistent with findings in mouse embryos and human gastruloids.

While there is a scarcity of human embryo materials for research, the lack of single cell level data limits further extension of the presented data on the consequences of mosaic embryos. A major concern is that the gene list used for pathway analysis is not FDR controlled. It is also unclear how the many plots generated with the "supervised approach" were actually performed. The authors also appear to have ignored the possibility that high-dosage group could have a higher mitotic defects. Assuming a fully aneuploid embryo, why do only some cells display p53 and autophagy marker? The conclusion about proteotoxic stress was largely based on staining of HSP70. It appears from Figure 3 d,h that the same cells exhibited increased HSP70 and CASP8 staining. Since HSP70 is known to have anti-apoptotic effect, could the increased expression of Hsp70 be an anti-apoptotic response?

Reviewer #1 (Public Review):

Summary:<br /> Chen et al. describe the bacterial and fungal composition of cervical samples from women with/without Cesarean-section scar diverticulum (CSD) using whole metagenomic sequencing. Also, they report the metabolomic profile associated with CSD and built correlation networks at the taxonomical and taxonomic-metabolic levels to establish potential bacteria-fungi interactions. These interactions could be used, long-term, as therapeutic options to treat or prevent CSD.

Strengths:<br /> - The authors have used advanced techniques in shotgun sequencing which is a powerful tool able to characterize the microbiome at the species (or lower) level and metabolomics.<br /> - These are novel results showing the interaction of bacteria and fungi and present a wider view of the role of the microbiome in female infertility.

Weaknesses:<br /> - This is a pilot study with only 24 cases and 24 controls. Because the human microbiota entails individual variability, this work should be confirmed with a higher sample size to achieve enough statistical power.<br /> - The authors do not report here the use of blank controls. The use of this type of control is important to "subtract" the potential background from plasticware, buffer or reagents from the real signal. Lack of controls may lead to microbiome artefacts in the results. This can be seen in the results presented where the authors report some bacterial contaminants (Agrobacterium tumefaciensis, Aequorivita lutea, Chitinophagaceae, Marinobacter vinifirmus, etc) as part of the most common bacteria found in cervical samples.<br /> - Samples used for this study were collected from the cervix. Why not collect samples from the uterine cavity and isthmocele fluid (for cases)? In their previous paper using samples from the same research protocol ((IRB no. 2019ZSLYEC-005S) they used endometrial tissue from the patients, so access to the uterine cavity was guaranteed.<br /> - Through the use of shotgun genomics, results from all the genomes of the organisms present in the sample are obtained. However, the authors have only used the metagenomic data to infer the taxonomical annotation of fungi and bacteria.

Reviewer #1 (Public Review):

Understanding the ecology including the dietary ecology of enantiornithines is challenging by all means. This work explores the possible trophic diversity of the "opposite-bird" enantiornithines by referring to the body mass, jaw mechanical advantage, finite element analysis of the jaw bones, and morphometrics of the claws and skull of both fossil and extant avian species. By incorporation of the dietary information of longipterygids and pengornithinds, the authors predicted a wide variety of foods for enantiornithine ancestors. This indicates the evolutionary successes of enantiornitine during Cretaceous is very likely to have been driven by the wide range of recipes. I believe this work represented the most comprehensive analysis of enantiornithines' diet and trophic diversity by far and the first systematic dietary analysis of bohaiornithids, though the analysis themselves are largely based on the indirect evidence including jaw bone morphologies and claw and skull morphometrics. Anyway, I believe the authors did most the paleontologists could do, and I do not know whether the conclusions could be further supported by incorporating some geochemical data, as most of the specimens the authors analyzed were recovered from a small geographic area. The results also indicate that the developmental trajectories of enantiornithines, at least for jaw bones, might also have been diverse to some extent in response to the diverse ecological niches they adapted. My only concern regarding the analysis is to what extent the conclusions are convincing by comparing specimens representing various ontogenetic stages.

Reviewer #1 (Public Review):

In this work, Veseli et al. present a computational framework to infer the functional diversity of microbiomes in relation to microbial diversity directly from metagenomic data. The framework reconstructs metabolic modules from metagenomes and calculates the per-population copy number of each module, resulting in the proportion of microbes in the sample carrying certain genes. They applied this framework to a dataset of gut microbiomes from 109 inflammatory bowel disease (IBD) patients, 78 patients with other gastrointestinal conditions, and 229 healthy controls. They found that the microbiomes of IBD patients were enriched in a high fraction of metabolic pathways, including biosynthesis pathways such as those for amino acids, vitamins, nucleotides, and lipids. Hence, they had higher metabolic independence compared with healthy controls. To an extent, the authors also found a pathway enrichment suggesting higher metabolic independence in patients with gastrointestinal conditions other than IBD indicating this could be a signal for a general loss in host health. Finally, a machine learning classifier using high metabolic independence in microbiomes could predict IBD with good accuracy. Overall, this is an interesting and well-written article and presents a novel workflow that enables a comprehensive characterization of microbiome cohorts.

Reviewer #1 (Public Review):

This study by Sokač et al. entitled "GENIUS: GEnome traNsformatIon and spatial representation of mUltiomicS data" presents an integrative multi-omics approach which maps several genomic data sources onto an image structure on which established deep-learning methods are trained with the purpose of classifying samples by their metastatic disease progression signatures. Using published samples from the Cancer Genome Atlas the authors characterize the classification performance of their method which only seems to yield results when mapped onto one out of four tested image-layouts.

A few remaining issues are unclear to me:

1) While the authors have now extended the documentation of the analysis script they refer to as GENIUS, I assume that the following files are not part of the script anymore, since they still contain hard-coded file paths or hard-coded gene counts:

• https://github.com/mxs3203/GENIUS/blob/master/GenomeImage/make_images_by_chr.py
• https://github.com/mxs3203/GENIUS/blob/master/GenomeImage/randomize_normal_imgs.py
• https://github.com/mxs3203/GENIUS/blob/master/GenomeImage/utils.py

If these files are indeed not part of the script anymore, then I would recommend removing them from the GitHub repo to avoid confusion. If, however, they are still part of the script, the authors failed to remove all hard-coded file paths and the software will fail when users attempt to use their own datasets.

2) The authors leave most of the data formatting to the user when attempting to use datasets other than their own presented for this study:

Script arguments:

• a. clinical_data: Path to CSV file that must contain ID and label column we will use for prediction
• b. ascat_data: Path to output matrix of ASCAT tool. Check the example input for required columns
• c. all_genes_included: Path to the CSV file that contains the order of the genes which will be used to create Genome Image
• d. mutation_data: Path CSV file representing mutation data. This file should contain Polyphen2 score and HugoSymbol
• e. gene_exp_data: Path to the csv file representing gene expression data where columns=sample_ids and there should be a column named "gene" representing the HugoSymbol of the gene
• f. gene_methyl_data: Path to the csv file representing gene methylation data wherecolumns=sample_ids and there should be a column named "gene1" representing the HugoSymbol of the gene

While this suggests that users will have a difficult time adjusting this analysis script to their own data, this issue is exacerbated by the fact that their analysis script has almost no internal checks whether data format standards were met. Thus, the user will be left with cryptic error messages and will likely give up soon after. I therefore strongly recommend adding internal data format checks and helpful error or warning messages to their script to guide users in the input data adoption process.

Reviewer #1 (Public Review):

Using a pharmacological and knock-down approach, the authors could demonstrate that ROCK activity is required for the normal development of the larval skeleton. The presence of ROCK in the pluteus stage depends on the activity of VEGF that is responsible for the formation of the tubular syncytial sheath of the calcifying primary mesenchyme cells in which the skeleton forms. The importance of ROCK in skeleton formation was confirmed in cell culture experiments, demonstrating that ROCK inhibition leads to decreased elongation and abnormal branching of spicules. µCT analyses underline this finding demonstrating that the inhibition of ROCK mainly affects the elongation of spicules while growth in girth is little affected. F-actin labeling experiments could demonstrate that ROCK inhibition interferes with the organization of the actomyosin network in the early phase of skeleton formation, while f-actin organization in the tips of the elongating spicule is unaffected by the pharmacological inhibition of ROCK. Finally, ROCK inhibition strongly affects the expression of major regulatory and calcification-related genes in the calcifying cells. Based on these findings the authors propose a model for the regulatory interaction between the skeletogenic GRN, ROCK, and the f-actin system relevant for skeletogenesis.

I reviewed this paper previously for submission to another Journal. I emphasize again, that this is an interesting and important work that aims to uncover the interaction between the Rho-associated Kinase, ROCK, the actomyosin network, and its relevance for the formation of the calcitic skeleton of the sea urchin larva. I carefully went through the revised manuscript. In their new version, the authors rearranged the figures to provide a more direct comparison between the in vivo and cell culture experiments which mitigates the criticism of collateral effects by the inhibitors on the whole organism. The authors also performed an additional experiment localizing the F-Actin signal in spicules of PMC cell cultures under ROCK inhibition. This experiment strengthens the concept that ROCK activity is important for tip dominance rather than CaCO3 deposition rates. The results section was substantially reorganized and only very minor changes were made to the introduction and discussion.

I think that this work has great potential to provide seminal insights into an understudied aspect of the biomineralization process - the role and regulation of the cytoskeleton in calcifying cells. As I mentioned in my previous review there are some gaps in this work that need to be answered to provide a conclusive dataset on the role of ROCK and the actomyosin system in the mineralization process. The manuscript in its current form provides evidence for the interaction of ROCK with the actomyosin system in the sea urchin larva and that perturbation of this system affects skeletogenesis. However, it is missing an explanation regarding the mechanism by which ROCK affects skeleton formation. No difference in f-actin localization was found at the spicule tips in control and ROCK-inhibited larvae. A slight hint was found in the difference in vesicle size and f-actin organization within calcifying cells, but it remains unresolved if ROCK activity impacts the trafficking of calcification vesicles. The authors provide an interesting discussion on the involvement of f-actin and ROCK on vesicular trafficking, and exocytosis based on existing knowledge from animal and plant models. But for the sea urchin larva, this important link between ROCK, f-actin, and the biomineralization process remains unanswered. In their previous work by Winter et al. 2021, the authors demonstrated excellent technologies to monitor vesicular dynamics in the calcifying cells. This tool would be ideal to investigate the role of ROCK and the actomyosin network on the trafficking dynamics of Ca2+-rich vesicles. These experiments (among others suggested in the following review) may help to uncover the critical mechanism to resolve the missing gap in this manuscript.

Major comments<br /> One MASO led to reduced skeleton formation while the other one additionally induced ectopic branching. How was the optimum concentration for the MASOs determined? Did the authors perform a dose-response curve? What is the reason for this difference? Which of the two MASOs can be validated by reduced ROCK protein abundance? Since the ROCK antibody works, I would like to see a control experiment on Rock protein abundance in control and ROCK MO injected larvae which is the gold-standard for validating the knock-down.

L212 "Together, these measurements show that ROCK is not required for the uptake of calcium into cells." But what about trafficking and exocytosis? As mentioned earlier, I think this is a really important point that needs to be confirmed to understand the function of ROCK in controlling calcification. In their previous study (reference 45) the authors demonstrated that they have superior techniques in measuring vesicle dynamics in vivo. Here an acute treatment with the ROCK inhibitor would be sufficient to test if calcein-positive vesicle motion, including the observed reduction in velocity close to the tissue skeleton interface, is affected by the inhibitor.

Is there a colocalization of ROCK and f-actin in the tips of the spicules? This would support the mechano-sensing-hypothesis by ROCK.

L 283. "F-actin is enriched at the tips of the spicules independently of ROCK activity" The results of this paragraph clearly demonstrate that ROCK inhibition has no effect on the localization of f-actin at the tips of the growing spicules. In addition, the new cell culture experiments underline this observation. Still, the central question that remains is, what is the interaction between ROCK, f-actin, and the mineralization process, that leads to the observed deformations? What does the f-actin signal look like in a branched phenotype or in larvae that failed to develop a skeleton (inhibition from Y20)?

Immunohistochemical analyses on f-actin localization and abundance should be additionally performed with ROCK knock-down phenotypes to confirm the pharmacological inhibition.

L 365 "...supporting its role in mineral deposition..." "...Overall, our studies indicate that ROCK activity....is essential for the formation of the spicule cavity......which could be essential for mineral deposition..." I think the authors need to do a better job in clearly separating between the potential processes impacted by ROCK perturbation. Is it stabilization and mechano-sensing in the spicule tip or the intracellular trafficking and deposition of the ACC? If the dataset does not allow for a definite conclusion, I suggest clearly separating the different possibilities combined with thorough discussion-based findings from other mineralizing systems where the interaction between ROCK and F-actin has been described.

Reviewer #1 (Public Review):

Summary:<br /> This study applied pattern similarity analyses to intracranial EEG recordings to determine how neural drift is related to memory performance in a free recall task. The authors compared neural similarity within and across lists, in order to contrast signals related to contextual drift vs. the onset of event boundaries. They find that within-list neural differentiation in the lateral temporal cortex correlates with the probability of word recall; in contrast, across-list pattern similarity in the medial parietal lobe correlates with recall for items near event boundaries (early-list serial positions). This primacy effect persists for the first three items of a list. Medial parietal similarity is also enhanced across lists for end-of-list items, however, this effect then predicts forgetting. The authors do not find that within- or across-list pattern similarity in the hippocampus is related to recall probability.

Strengths:<br /> The authors use a large dataset of human intracranial electrophysiological recordings, which gives them high statistical power to compare neural activity and memory across three important memory encoding regions. In so doing, the authors also address a timely and important question about the neural mechanisms that underlie the formation of memories for events.

The use of both within and across event pattern similarity analyses, combined with linear mixed effects modeling, is a marriage of techniques that is novel and translatable in principle to other types of data.

Weaknesses:<br /> In several instances the paper does not address apparent inconsistencies between the prior literature and the findings. For example, the first main finding is that recalled items have more differentiated lateral temporal cortex representations within lists than not recalled items. This seems to be the opposite of the prediction from temporal context models that are used to motivate the paper-context models would predict that greater contextual similarity within a list should lead to greater memory through enhanced temporal clustering in recall. This is what El-Kalliny et al (2019) found, using a highly similar design (free recall, intracranial recordings from the lateral temporal lobe). The authors never address this contradiction in any depth in order to reconcile it with the previous literature and with the motivating theoretical model.

The way that the authors conduct the analysis of medial parietal neural similarity at boundaries leads to results that cannot be conclusively interpreted. The authors report enhanced similarity across lists for the first item in each list, which they interpret as reflecting a qualitatively distinct boundary signal. However, this finding can readily be explained by contextual drift if one assumes that whatever happens at the start of each list is similar or identical across lists (for example, a get ready prompt or reminder of instructions). The authors do not include analyses to rule this out, which undermines one of the main findings.

Although several previous studies have linked hippocampal fMRI and electrophysiological activity at event boundaries with memory performance, the authors do not find similar relationships between hippocampal activity, event boundaries, and memory. There are potential explanations for why this might be the case, including the distinction between item vs. associative memory, which has been a prominent feature of previous work examining this question. However, the authors do not address these potential explanations (or others) to explain their findings' divergence from prior work -this makes it difficult to interpret and to draw conclusions from the data about the hippocampus' mechanistic role in forming event memories.

There is a similar absence of interpretation with respect to the previous literature for the data showing enhanced boundary-related similarity in the medial parietal cortex. The authors' interpretation seems to be that they have identified a boundary-specific signal that reflects a large and abrupt change in context, however, another plausible interpretation is that enhanced similarity in the medial parietal cortex is related to a representation of a schema for the task structure that has been acquired across repeated instances.

The authors do not directly compare their model to other models that could explain how variability in neural activity predicts memory. One example is the neural fatigue hypothesis, which the authors mention, however there are no analyses or data to suggest that their data is better fit by a boundary/contextual drift mechanism as opposed to neural fatigue.

Reviewer #1 (Public Review):

Summary

This paper summarises responses from a survey completed by around 5,000 academics on their manuscript submission behaviours. The authors find several interesting stylised facts, including (but not limited to):

- Women are less likely to submit their papers to highly influential journals (*e.g.*, Nature, Science and PNAS).<br /> - Women are more likely to cite the demands of co-authors as a reason why they didn't submit to highly influential journals.<br /> - Women are also more likely to say that they were advised not to submit to highly influential journals.

Recommendation

This paper highlights an important point, namely that the submissions' behaviours of men and women scientists may not be the same (either due to preferences that vary by gender, selection effects that arise earlier in scientists' careers or social factors that affect men and women differently and also influence submission patterns). As a result, simply observing gender differences in acceptance rates---or a lack thereof---should not be automatically interpreted as as evidence of for or against discrimination (broadly defined) in the peer review process. I do, however, make a few suggestions below that the authors may (or may not) wish to address.

Major comments

## What do you mean by bias?

In the second paragraph of the introduction, it is claimed that "if no biases were present in the case of peer review, then 'we should expect the rate with which members of less powerful social groups enjoy successful peer review outcomes to be proportionate to their representation in submission rates." There are a couple of issues with this statement.<br /> - First, the authors are implicitly making a normative assumption that manuscript submission and acceptance rates *should* be equalised across groups. This may very well be the case, but there can also be important reasons why not -- e.g., if men are more likely to submit their less ground-breaking work, then one might reasonably expect that they experience higher rejection rates compared to women, conditional on submission.<br /> - Second, I assume by "bias", the authors are taking a broad definition, i.e., they are not only including factors that specifically relate to gender but also factors that are themselves independent of gender but nevertheless disproportionately are associated with one gender or another (e.g., perhaps women are more likely to write on certain topics and those topics are rated more poorly by (more prevalent) male referees; alternatively, referees may be more likely to accept articles by authors they've met before, most referees are men and men are more likely to have met a given author if he's male instead of female). If that is the case, I would define more clearly what you mean by bias. (And if that isn't the case, then I would encourage the authors to consider a broader definition of "bias"!)

## Identifying policy interventions is not a major contribution of this paper

In my opinion, the survey evidence reported here isn't really strong enough to support definitive policy interventions to address the issue and, indeed, providing policy advice is not a major -- or even minor -- contribution of your paper, so I would not mention policy interventions in the abstract. (Basically, I would hope that someone interested in policy interventions would consult another paper that much more thoughtfully and comprehensively discusses the costs and benefits of various interventions!)

Minor comments

- What is the rationale for conditioning on academic rank and does this have explanatory power on its own---i.e., does it at least superficially potentially explain part of the gender gap in intention to submit?

Reviewer #1 (Public Review):

Summary:<br /> This paper addresses the mechanisms positioning microtubule asters in Drosophila explants. Taking advantage of a genetic mutant, blocking the cell cycle in early embryos, the authors generate embryos with centrosomes detached from nuclei and then study the positioning mechanisms of such asters in explants. They conclude that asters interact via pushing forces. While this is an artificial system, understanding the mechanics of asters positioning, in particular, whether forces are pushing or pulling is an important one.

Strengths:<br /> The major strength of this paper is the series of laser cutting experiments supporting that asters position via pushing forces acting both on the boundary (see below for a relevant comment) and between asters. The combination of imaging, data analysis and mathematical modeling is also powerful.

Weaknesses:<br /> This paper has weaknesses, mainly in the presentation but also in the quality of the data which do not always support the conclusions satisfactorily (this might in part be a presentation issue).

In Figure 2, it is difficult for me to understand what is being tracked. I believe that the authors track the yolk granules (visible as large green blobs) and not lipid droplets. There is some confusion between the text, legends and methods so I could not tell. If the authors are tracking yolk granules as a proxy for hydrodynamics flows it seems appropriate to cite previous papers that have used and verified these methods. More notably, this figure is somewhat disconnected with the rest of the paper. I find the analysis interesting in principle but would urge the authors to propose some interpretation of the experiments in the context of their big-picture message. At this point, I cannot understand what the Figure adds.

In Figure 3, it is not surprising that the aster-aster interactions are different from interactions with the boundary which is likely more rigid. It is also hard to understand why the force and thus velocity should scale as microtubule length. This Figure should be better conceptualized. I think that it becomes clear at the end of the paper that the authors are trying to derive an effective potential to use in a mathematical model in Figure 5 to test their hypotheses. I think that should be told from the start, so a reader understands why these experiments are being shown.

The experiments in Figure 4 are very nice in supporting a pushing model. However, it would help if the authors could speculate what the single aster is pushing against in this experiment. The experiments reported in Figure 1 seemed to suggest that the aster mainly pushed against the boundary. In the experiments in Figure 4 do the individual asters touch the boundary on both sides? I think that readers need more information on what the extract looks like for those experiments.

Figure 4F could use some statistics. I doubt that the acceleration in the pink curves would be significant. I believe that the deceleration is and that is probably the most crucial result. Since the authors present only 3 asters pairs it is important to be sure that these conclusions are solid.

Reviewer #1 (Public Review):

The authors use a previously established reporter comprising a slow- and a fast-folding fluorescent protein fused to a randomly-generated library of penta-peptides at its amino-terminus and a signal sequence for import into the endoplasmic reticulum (ER). They then determine the stability of these constructs in a high throughput FACS-sorting procedure and identify a set of peptides that route the construct to proteasomal degradation. Increasing the copy number of one of these peptides further decreases the stability of the construct. This polypeptide resembles a "degron" for ER proteins, because it also targets other ER proteins with different topological and folding properties for degradation. It only works when placed at the amino-terminus of a protein and utilizes components of the Hrd1 ubiquitin ligase complex, a well-established quality control ubiquitin ligase in the ER membrane. Importantly, the degron also targets ER-proteins in mammalian cells.

The authors convincingly show that fusion of their newly identified degron to the amino terminus of ER-resident proteins with different topology suffices to target them for proteasomal degradation. The data for this are well-founded and contain appropriate controls. While technically sound, the study does only give superficial information on general properties of the degron and its recognition by cellular factors. Further simple experiments would have addressed a number of important points. The authors only provide data about the composition of the identified amino acid sections from the high-throughput approach and the statistical preference for certain amino acids at individual positions. They do not study degron composition experimentally by substituting individual amino acids with other residues and analyzing protein stability. Increasing the numbers of the initially identified degron pentamer increases substrate turnover, but the basis for this remains unclear. Each copy may be actively involved in better recognition, elongation of the degron may facilitate accessibility by recognition factors or multiplying the short amino acid stretch may generate new signatures at the amino-terminus that are more readily recognized by a quality control machinery. Consequently, this study does not allow conclusions to be drawn about general properties of degron composition and/or structure. The degron also functions with cytoplasmic proteins, suggesting that similar characteristics of a polypeptide attract the attention of quality control systems also in other cellular compartments. However, the authors did not pursue this finding further, e.g. by identifying factors for degron recognition in the cytoplasm. It would have been particularly interesting to test whether the degron would initiate degradation when placed at cytoplasmically-exposed amino termini of membrane-bound ER proteins. Information on degron properties is required to better understand principles of substrate recognition by protein quality control pathways and to design constructs for targeting endogenous proteins via proteolysis targeting chimeras (PROTACs).

Reviewer #1 (Public Review):

This study by Park et al. describes an interesting approach to disentangle gene-environment pathways to cognitive development and psychotic-like experiences (PLEs) in children. They have used data from the ABCD (Adolescent Brain Cognitive Development) study and have included phenotypes polygenic scores (PGS) of educational attainment (EA) and cognition, environmental exposure data, cognitive performance data and self-reported PLEs. The study has several strengths, including its large sample size, interesting approach and comprehensive statistical model,

One remaining concern is the authors' conflation of PLEs and schizophrenia. They stated, for example, that it is necessary to adjust for schizophrenia PGS. Even though studies have found a statistical relationship between schizophrenia PGS and PLEs, this relationship is not very strong (although statistically significant) and other studies have found no relationship. Similarly, having PLEs increases the risk of developing psychosis, but that does not necessarily mean that this risk is substantial or specific. I think this needs more nuance in the manuscript and the term 'schizophrenia' should be used sparsely and very carefully as the paper has focused on PLEs.

Reviewer #1 (Public Review):

Summary:<br /> This Research Advance is an extension of this group's prior paper published in 2022 on the conserved roles of the Hippo pathway effector Yorkie in C. owczarzaki (PMID: 35659869). This species is an amoeba that holds an important phylogenetic position as a close relative of multicellular animals. The prior study used genome editing to delete the C. owczarzaki Yki (termed coYki) and found that Yki is not required for proliferation but instead regulates cell contractility and cell aggregation. In the current study, the authors wanted to address whether Hippo pathway kinases - coHippo (coHpo) and coWarts (coWts) - regulate coYki and whether they are dispensable for proliferation but instead regulate cell contractility and cell aggregation. They used genome editing to delete coHpo and coWts singly in C. owczarzaki. Both mutant strains had increased nuclear location of transfected coYki (tagged with Scarlet), suggesting that Hippo kinase pathway regulation of Yki is conserved in this organism. Neither kinase is required for proliferation. Either kinase mutant strain had a significantly increased percentage of cells that were elongated, which was relatively rare in a control population. The incident of elongation could be enhanced in both kinase-mutant and in control cells when myosin inhibitors were added to the media. coHpo and coWts-mutant aggregates were more tightly packed than control cell aggregates, which they hypothesize is due to the increased contractility seen in kinase-mutant cells. They could reduce the density of packing in kinase-mutant aggregates when they treated the cells with myosin or F-actin inhibitors. To test whether the effects observed in kinase-mutant strains were due to increased Yki activation, they generated a coYki with four S to A substitutions (termed coYki4SA), which should produce a dominant-active Yki impervious to phosphorylation and hence inactivation by Hippo kinases. Control C. owczarzaki cells transfected with coYki4SA had increased cell density in aggregates and elongation in adherent cells. These results support their conclusions that coHpo and coWts regulate cell contractility and cell packing through coYki.

Strengths:<br /> The major strengths of the paper include high quality data, robust analyses of the data, and a well-written manuscript. The combination of genome editing in C. owczarzaki, transfection of C. owczarzaki, and time-lapse movies of adherent cells generally support the conclusions (1) that control of cell density is an ancient function of the Hippo pathway; (2) that Hippo pathway control of cytoskeletal properties and contractile behavior underlie its regulation of cell density, and (3) that Hippo kinase control of Yki localization is likely an ancient function of the pathway.

Weaknesses:<br /> There are only minor weaknesses. (1) Fig. 3C needs the "still" for the movie of control C. owczarzaki (in Movie S1). (2) The elongated cell shape is seen infrequently in control cells, and I wonder whether these events are transient inactivation of coHpo or coWts in these cells. Perhaps the authors could comment on this in the discussion. (3) Does C. owczarzaki normally aggregate or this is a lab-specific phenotype? For example, the slime mold Dictyostelium discoideum forms aggregates during its life cycle. Could some additional information about C. owczarzaki be added to the introduction?

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to provide information about the likely function of uncharacterised genes in fission yeast. The authors highlight the bias in the literature to well-studied genes/proteins and the fact that the functions of many proteins that are conserved from yeast to humans remain unknown. Initial functional characterisation could provide the impetus for researchers to dedicate time and resources to detailed investigations of protein function. The authors subject the fission yeast deletion set to a battery of perturbations (drug treatments etc) and measured the resultant colony size. In total, 131 conditions were analysed for nearly 3,500 mutants, representing a rich dataset. Clustering analysis was then used to identify common phenotype patterns and thereby infer protein functions using a "guilt by association approach. To assign potential GO terms to uncharacterised proteins, the authors developed a new computational approach (NET-FF) which combined two previous approaches, which they validated against curated annotations on the S. pombe database Pombase. Finally, the authors chose a group of genes which their analysis predicted to be involved in cellular ageing for experimental validation, cross-validating a priority unstudied novel gene (SPAC23C4.09c) to be involved in this process. Overall, the functional analysis performed in this manuscript is rigorous, thorough and incorporates some novel approaches leading to new insights and predicted protein functions. It will be an important resource for the fission yeast community.

Reviewer #1 (Public Review):

Summary:

In this paper, Jeong et al. investigate the prevalence and cause of TADs that are preserved in eukaryotic cells after cohesin depletion. The authors perform an extensive analysis of previously published Hi-C data, and find that roughly 15% of TADs are preserved in both mouse liver cells and in HCT-116 cells. They confirm previous findings that epigenetic mismatches across the boundaries of TADs can cause TAD preservation. However, the authors also find that not all preserved TADs can be explained this way. Jeong et al. provide an argument based on polymer simulations that "physical boundaries" in 3D structures provide an additional mechanism that can lead to TAD preservation. However, in its current form, we do not find the argumentation and evidence that leads to this claim to be fully compelling.

Strengths:

We appreciate the extensive statistical analysis performed by the authors on the extent to which TAD's are preserved; this does seem like a novel and valuable contribution to the field.

Weaknesses:

1. As the authors briefly note, the fact that compartmentalization due to epigenetic mismatches can cause TAD-like structures upon cohesin depletion has already been discussed in the literature; see for example Extended Data Figure 8 in (Schwarzer et al., 2017) or the simulation study (Nuebler et al., 2018). We are hence left with the impression that the novelty of this finding is somewhat overstated in this manuscript.<br /> 2. It is not quite clear what the authors conceptually mean by "physical boundaries" and how this could offer additional insight into preserved TADs. First, the authors use the CCM model to show that TAD boundaries correlate with peaks in the single cell boundary probability distribution of the model. This finding is consistent with previous reports that TAD-like structures are present in single cells, and that specific TAD boundaries only arise as a population average. The finding based on the CCM simulations hence seems to be that preserved TADs also arise as a population average in cohesin-depleted cells, but we do not follow what the term "physical boundaries" refers to in this context. The authors then use the Hi-C data to infer a maximum-entropy-based HIPPS model. They find that preserved TADs often have boundaries that correspond to peaks in the single cell boundary probabilities of the inferred model. The authors seem to imply that these peaks in the boundary probability correspond to "physical boundaries" that cause the preservation of TADs. This argument seems circular; the model is based on inferring interaction strengths between monomers, such that the model recreates the input Hi-C map. This means that the ensemble average of the model should have a TAD boundary where one is present in the input Hi-C data. A TAD boundary in the Hi-C data would then seem to imply a peak in the model's single-cell boundary probability. (The authors do display two examples where this is not the case in Fig.3h, but looking at these cases by eye, they do not seem to correspond to strong TAD boundaries.) "Physical boundaries" in the model are hence a consequence of the preserved TADs, rather than the other way around, as the authors seem to suggest. At the very least the boundary probability in the HIPPS model is not an independent statistic from the Hi-C map (on which their model is constrained), so we have concerns about using the physical boundaries idea to understand where some of the preserved TADs come from.

References:<br /> Nuebler, J., Fudenberg, G., Imakaev, M., Abdennur, N., & Mirny, L. A. (2018). Chromatin organization by an interplay of loop extrusion and compartmental segregation. Proceedings of the National Academy of Sciences of the United States of America, 115(29), E6697-E6706. https://doi.org/10.1073/PNAS.1717730115/SUPPL_FILE/PNAS.1717730115.SAPP.PDF

Schwarzer, W., Abdennur, N., Goloborodko, A., Pekowska, A., Fudenberg, G., Loe-Mie, Y., Fonseca, N. A., Huber, W., Haering, C. H., Mirny, L., & Spitz, F. (2017). Two independent modes of chromatin organization revealed by cohesin removal. Nature 2017 551:7678, 551(7678), 51-56. https://doi.org/10.1038/nature24281

Joint Public Review:

This paper by Castello-Serrano et al. addresses the role of lipid rafts in trafficking in the secretory pathway. By performing carefully controlled experiments with synthetic membrane proteins derived from the transmembrane region of LAT, the authors describe, model and quantify the importance of transmembrane domains in the kinetics of trafficking of a protein through the cell. Their data suggest affinity for ordered domains influences the kinetics of exit from the Golgi. Additional microscopy data suggest that lipid-driven partitioning might segregate Golgi membranes into domains. However, the relationship between the partitioning of the synthetic membrane proteins into ordered domains visualised ex vivo in GPMVs, and the domains in the TGN, remain at best correlative. Additional experiments that relate to the existence and nature of domains at the TGN are necessary to provide a direct connection between the phase partitioning capability of the transmembrane regions of membrane proteins and the sorting potential of this phenomenon.

The authors have used the RUSH system to study the traffic of model secretory proteins containing single-pass transmembrane domains that confer defined affinities for liquid ordered (lo) phases in Giant Plasma Membrane derived Vesicles (GPMVs), out of the ER and Golgi. A native protein termed LAT partitioned into these lo-domains, unlike a synthetic model protein termed LAT-allL, which had a substituted transmembrane domain. The authors experiments provide support for the idea that ER exit relies on motifs in the cytosolic tails, but that accelerated Golgi exit is correlated with lo domain partitioning.

Additional experiments provided evidence for segregation of Golgi membranes into coexisting lipid-driven domains that potentially concentrate different proteins. Their inference is that lipid rafts play an important role in Golgi exit. While this is an attractive idea, the experiments described in this manuscript do not provide a convincing argument one way or the other. It does however revive the discussion about the relationship between the potential for phase partitioning and its influence on membrane traffic.

Our detailed comments are listed below:

ER exit:<br /> The experiments conducted to identify an ER exit motif in the C-terminal domain of LAT are straightforward and convincing. This is also consistent with available literature. The authors should comment on whether the conservation of the putative COPII association motif (detailed in Fig. 2A) is significantly higher than that of other parts of the C-terminal domain. One cause of concern is that addition of a short cytoplasmic domain from LAT is sufficient to drive ER exit, and in its absence the synthetic constructs are all very slow. However, the argument presented that specific lo phase partitioning behaviour of the TMDs do not have a significant effect on exit from the ER is a little confusing. This is related to the choice of the allL-TMD as the 'non-lo domain' partitioning comparator. Previous data has shown that longer TMDs (23+) promote ER export (eg. Munro 91, Munro 95, Sharpe 2005). The mechanism for this is not, to my knowledge, known. One could postulate that it has something to do with the very subject of this manuscript- lipid phase partitioning. If this is the case, then a TMD length of 22 might be a poor choice of comparison. A TMD 17 Ls' long would be a more appropriate 'non-raft' cargo. It would be interesting to see a couple of experiments with a cargo like this.

Golgi exit:<br /> For the LAT constructs, the kinetics of Golgi exit as shown in Fig. 3B are surprisingly slow. About half of the protein remains in the Golgi at 1 h after biotin addition. Most secretory cargo proteins would have almost completely exited the Golgi by that time, as illustrated by VSVG in Fig. S3. There is a concern that LAT may have some tendency to linger in the Golgi, presumably due to a factor independent of the transmembrane domain, and therefore cannot be viewed as a good model protein. For kinetic modeling in particular, the existence of such an additional factor would be far from ideal. A valuable control would be to examine the Golgi exit kinetics of at least one additional secretory cargo.

Comments about the trafficking model<br /> 1. In Figure 1E, the export of LAT-TMD from the ER is fitted to a single-exponential fit that the authors say is "well described". This is unclear and there is perhaps something more complex going on. It appears that there is an initial lag phase and then similar kinetics after that - perhaps the authors can comment on this?

2. The model for Golgi sorting is also complicated and controversial, and while the authors' intention to not over-interpreting their data in this regard must be respected, this data is in support of the two-phase Golgi export model (Patterson et al PMID:18555781). Furthermore contrary to the statement in lines 200-202, the kinetics of VSVG exit from the Golgi (Fig. S3) are roughly linear and so are NOT consistent with the previous report by Hirschberg et al. Moreover, the kinetics of LAT export from the Golgi (Fig. 3B) appear quite different, more closely approximating exponential decay of the signal. These points should be described accurately and discussed.

Relationship between membrane traffic and domain partitioning:<br /> 1. Phase segregation in the GPMV is dictated by thermodynamics given its composition and the measurement temperature (at low temperatures 4degC). However at physiological temperatures (32-37degC) at which membrane trafficking is taking place these GPMVs are not phase separated. Hence it is difficult to argue that a sorting mechanism based solely on the partitioning of the synthetic LAT-TMD constructs into lo domains detected at low temperatures in GPMVs provide a basis (or its lack) for the differential kinetics of traffic of out of the Golgi (or ER). The mechanism in a living cell to form any lipid based sorting platforms naturally requires further elaboration, and by definition cannot resemble the lo domains generated in GPMVs at low temperatures.

2. The lipid compositions of each of these membranes - PM, ER and Golgi are drastically different. Each is likely to phase separate at different phase transition temperatures (if at all). The transition temperature is probably even lower for Golgi and the ER membranes compared to the PM. Hence, if the reported compositions of these compartments are to be taken at face value, the propensity to form phase separated domains at a physiological temperature will be very low. Are ordered domains even formed at the Golgi at physiological temperatures?

3. The hypothesis of 'lipid rafts' is a very specific idea, related to functional segregation, and the underlying basis for domain formation has been also hotly debated. In this article the authors conflate thermodynamic phase separation mechanisms with the potential formation of functional sorting domains, further adding to the confusion in the literature. To conclude that this segregation is indeed based on lipid environments of varying degrees of lipid order, it would probably be best to look at the heterogeneity of the various membranes directly using probes designed to measure lipid packing, and then look for colocalization of domains of different cargo with these domains.

4. In the super-resolution experiments (by SIM- where the enhancement of resolution is around two fold or less compared to optical), the authors are able to discern a segregation of the two types of Golgi-resident cargo that have different preferences for the lo-domains in GPMVs. It should be noted that TMD-allL and the LATallL end up in the late endosome after exit of the Golgi. Previous work from the Bonafacino laboratory (PMID: 28978644) has shown that proteins (such as M6PR) destined to go to the late endosome bud from a different part of the Golgi in vesicular carriers, while those that are destined for the cell surface first (including TfR) bud with tubular vesicular carriers. Thus at the resolution depicted in Fig 5, the segregation seen by the authors could be due to an alternative explanation, that these molecules are present in different areas of the Golgi for reasons different from phase partitioning. The relatively high colocalization of TfR with the GPI probe in Fig 5E is consistent with this explanation. TfR and GPI prefer different domains in the GPMV assays yet they show a high degree of colocalization and also traffic to the cell surface.

Reviewer #1 (Public Review):

Summary:<br /> This important study from Godneeva et al. establishes a Drosophila model system for understanding how the activity of Tif1 proteins is modified by SUMO. The authors nicely show that Bonus, like homologous mammalian Tif1 proteins, is a repressor, and that it interacts with other co-repressors Mi-2/NuRD and setdb1 in Drosophila ovaries and S2 cells. They also show that Bonus is SUMOylated by Su(var)2-10 on at least one lysine at its N-terminus to promote its interaction with setdb1. By combining nice biochemistry with an elegant reporter gene approach, they show that SUMOylation is important for Bonus interaction with setdb1, and that this SUMO-dependent interaction triggers high levels of H3K9me3 deposition and gene silencing. While there are still major questions of how SUMO molecularly promotes this process, this study is a valuable first step that opens the door for interesting future experimentation.

Major Point:<br /> The RNAseq and ChIPseq data is not available. This is critical for the review of the paper and would help the readers and reviewers interpret the Bonus mutant phenotype and its mechanism of repressing genes.

1) The author's conclusion that Bonus SUMOylation is "essential for its chromatin localization" is not supported by the data. Figure 5F shows less 3KR mutant in the chromatin fraction but there is still significant signal.<br /> 2) The author's conclusion that Bonus is SUMOylated at a single site close to its N-terminus is not necessarily true. In several SUMO and Bonus blots throughout the paper (5B, 6C, S4A), there are >2 differentially migrating species that could represent more than one SUMO added to Bonus. While the single K20R mutation eliminates all of these species in Fig 5C, it is possible that K20R SUMOylation is required for additional SUMOylation events on other residues. One way to determine if Bonus is SUMOylated on multiple sites is to add recombinant SUMO protease to the extract and see if multiple higher molecular weight bands collapse into a single migrating species (implying multiple SUMOs) or multiple migrating species (implying something else is altering gel migration).<br /> 3) The authors state that most upregulated genes in BonusGLKD are not highly enriched in H3K9me3. The heatmap in figure 3D is not an ideal presentation of this argument. The authors should show an example of what the signal on a highly enriched gene looks like for comparison. The authors also argue that because most upregulated genes in BonusGLKD are not highly enriched in H3K9me3, they must be indirectly repressed. Another possibility is that bonus-mediated H3K9me3 is only important (and present) during early nurse cell differentiation and is later lost and dispensable during the rapid endocycles. After bonus establishes repression though H3K9me3, it might be maintained through bonus-Mi2/Nurd, something else, or nothing at all. The authors could discuss this possibility or perform H3K9me3 ChIP during cyst formation and early nurse cell differentiation rather than in whole ovaries, which are enriched for later stages.<br /> 4) The BonusGLKD RNAseq analysis is underwhelming. The conclusion that "Bonus represses tissue-specific genes" has limited value. Every gene that is not expressed in ovaries is "tissue-specific." What subset of tissue-specific genes does Bonus repress? What common features do these genes have and how do they compare to other sets of tissue-specific genes, such as those reportedly repressed by setdb1, Polycomb proteins, small ovary, l(3)mbt, and stonewall (among others in female germ cells). Comparing these available data sets could help the authors understand the mechanism of Bonus repression and how BonusGLKD leads to sterility. The authors could also further analyze the differences between nos-Gal4 and MT-Gal4 to better understand why nos- but not MT-driven knockdown is sterile.

Main Study Limitations:<br /> 1) It is unclear which genes are directly vs indirectly regulated by bonus, which makes it difficult to understand Bonus's repressive mechanism. Several lines of experiments could help resolve this issue. 1) Bonus ChIPseq, which the authors mentioned was difficult. 2) RNAseq of BonusGLKD rescued with KR3 mutation. This would help separate SUMO/setdb1-dependent regulation from Mi-2 dependent regulation. Similarly, comparing differentially expressed genes in Su(var)2-10GLKD, setdb1GLKD, 3KR rescue, and MI-2 GLKD could identify overlapping targets and help refine how bonus represses subsets of genes through these different corepressors.

2) The paper falls short in discussing how SUMO might promote repression. This is important when considering the conservation (of lack thereof) of SUMOylation sites in Tif1 proteins in distantly related animals. One piece of data that was not discussed is the apparent localization of SUMOylated bonus in the cytoplasmic fraction of the blot in Figure 5F. Su(var)2-10 is mostly a nuclear protein, so is bonus SUMOylated in the nucleus and then exported to the cytoplasm? Also, setdb1 is a nuclear protein, so it is unlikely that the SUMOylated bonus directly interacts with setdb1 on target genes. Together with Fig 5E (unSUMOylatable Bonus aggregates in the nucleus), one could make a model where SUMO solubilizes bonus (perhaps by disassembling aggregates) and indirectly allows it to associate with setdb1 and chromatin. It is also important to note that in Figure 5I, the K3R mutation appears to lessen but not eliminate Bonus interaction with setdb1. This data again disfavors a model where SUMO establishes an interaction interface between setdb1 and Bonus. To determine which form of Bonus interacts with setdb1, the authors could perform a setdb1 pulldown and monitor the SUMOylation state of coIPed Bonus through mobility shift. If mostly unSUMOylated bonus interacts with setdb1, and SUMO indirectly promotes Bonus interaction with setdb1 (perhaps by disassembling Bonus aggregates), then the precise locations of Bonus SUMOylation sites could more easily shift during evolution, disfavoring the author's convergent evolution hypothesis.

Reviewer #1 (Public Review):

In the study by Venkat et al. the authors expand the current knowledge of allosteric diversity in the human kinome by c-terminal splicing variants using as a paradigm DCLK1. In this work, the authors provide evolutionary and some mechanistic evidence about how c-terminal isoform specific variants generated by alternative splicing can regulate catalytic activity by means of coupling specific phosphorylation sites to dynamical and conformational changes controlling active site and substrate pocket occupancy, as well as interfering with protein-protein interacting interfaces that altogether provides evidence of c-terminal isoform specific regulation of the catalytic activity in protein kinases.

The paper is overall well written, the rationale and the fundamental questions are clear and well explained, the evolutionary and MD analyses are very detailed and well explained. Overall I think this is a study that brings some new aspects and concepts that are important for the protein kinase field, in particular the allosteric regulation of the catalytic core by c-terminal segments, and how evolutionary cues generate more sophisticated mechanisms of allosteric control in protein kinases.

Current submission: I have read and gone through the revised manuscript and the rebuttal letter and I confirm that the authors did an excellent job answering all the comments satisfactorily.

Communication Is Symbolic/Arbitrary * symbols, which are marks/objects that represent something else by association, are something that are taught and can be highly subjective depending on multiple aspects. words are visual symbols, and thus can have major forms of differential meanings. Meaning can change from contextual changes, cultural changes, physical body language, etc. Symbols are arbitrary; there is no reason why dog means the large quadrapedal canine creature. You can change the symbol and the true nature would not be affected.

Communication Is Shared Meaning * Symbols many be "meaningless" in a conceptual and disconnected sense, but they carry heavy meaning when individuals have an agreed sense and deliberate identity to it. Culture is a major form of how important group consensus ties to meaning both linguistically and socially. Beyond culture a major part of communication deals with experiences from an abstract intimate sense of self. Even members of the same culture will have different perceptions of an experience, which can make sharing difficult. human interpretation lends to answers objectively "correct" in one persons mind, but false in another. These are connotative definitions vs denotative definitions.

Communication Involves Intentionality

• we are in a constant state of communication, whether intended or not, but verbal communication almost always comes with a premediate sense of conviction and intention. Direct intention towards a shared topic makes communication more effective. Body language, is inversely related in that many times it can be an unconscious decision without intention.

Dimensions of Communication

• there are dimensions, or levels of interpretation during communication. Relational dimension describes how different relationships allow for specific tones, like friendship, cordial business, etc, and using specific tones for wrong situations could be seen as inappropriate. without having a clear understanding of the relationship you have with a person could make the reception of a message clouded or confusing. Content dimension refers to explicit information and the wordage used to convey a certain message.

Communication Is a Process

• communication is ongoing and dynamic, and even if a relationship with an individual start at one level of communication can mean it cant grow to be a "deeper" level where you can communicate more freely. Think of inside jokes.

Communication Is Culturally Determined

• Culture is learned and alters our perceptions of the world and what is considered "normal". Religion is a major aspect of culture that defines morality, values, etc. These perceptions are hard to pinpoint until interacting with someone who doesn't share those values.

Communication Occurs in a Context

• external forces are a major influence in how we interact/commun. yelling out "fire!" at a movie theatre vs yelling that as a lyric at a concert carry two very context dependent results.

Reviewer #1 (Public Review):

This carefully done research paper presents a fundamental model of techniques that are useful for the elucidation of kinase substrates. The paper utilizes state-of-the-art approaches to define a kinetic phosphoproteome and how to integrate that data with complementary approaches using a chemical probe (in this case KTPyS, a triphosphate) to find these substrates. Using these approaches TgCDPK1 was demonstrated to affect microneme secretion via a direct interaction with a HOOK complex (defined as a HOOK protein TGG1_289100, an FTS TGGT1_264050 and 2 other proteins TGGT1_316650 and 306920).

This work is carefully controlled and the analysis pathways are logical and provide paradigms for how to approach the question of identifying substrates of kinases using proteomic approaches employing genetic and chemical strategies.

The authors succeeded in the identification of candidate substrates for TgCDPK1. Validation of the results was provided by previous studies in the literature that characterized some of these substrates as well as the experiments in this manuscript on the characterization of the HOOK complex that is phosphorylated by CDPK1.

The HOOK complex (defined as a HOOK protein TGG1_289100, an FTS TGGT1_264050, and 2 other proteins TGGT1_316650 and 306920) was clearly demonstrated to be involved in invasion via its role in microneme trafficking.

Reviewer #1 (Public Review):

In this nice study, the authors set out to investigate the role of the canonical circadian gene Clock in the rhythmic biology of the basal metazoan Nematostella vectensis, a sea anemone, which might illuminate the evolution of the Clock gene functionality. To achieve their aims the team generated a Clock knockout mutant line (Clock-/- ) by CRISPR/Cas9 gene deletion and subsequent crossing. They then compared wild-type (WT) with Clock-/- animals for locomotor activity and transcriptomic changes over time in constant darkness (DD) and under light/dark cycles to establish these phenotypes under circadian control and those driven by light cycles. In addition, they used Hybridization Chain Reaction-In situ Hybridization (HCR-ISH) to demonstrate the spatial expression of Clock and a putative circadian clocl-controlled gene Myh7 in whole-mounted juvenile anemones.

The authors demonstrate that under LD both WT and Clock-/- animals were behaviourally rhythmic but under DD the mutants lost this rhythmicity, indicating that Clock is necessary for endogenous rhythms in activity. With altered LD regimes (LD6:6) they show also that Clock is light-dependent. RNAseq comparisons of rhythmic gene expression in WT and Clock-/- animals suggest that clock KO has a profound effect on the rhythmic genome, with very little overlap in rhythmic transcripts between the two phenotypes; of the rhythmic genes in both LD and DD in WT animals (220- termed clock-controlled genes, CCGS) 85% were not rhythmic in Clock-/- animals in either light condition. In silico gene ontology (GO) analysis of CCGS reflected process associated with circadian control. Correspondingly, those genes rhythmic in KO animals under DD (here termed neoCCGs) were not rhythmic in WT, lacked upstream E-box motifs associated with circadian regulation, and did not display any GO enrichment terms. 'Core' circadian genes (as identified in previous literature) in WT and Clock-/- animals were only rhythmic under entrainment (LD) conditions whilst Clock-/- displayed altered expression profiles under LD compared to WT. Comparing CCGs with previous studies of cycling genes in Nematostellar, the authors selected a gene from 16 rhythmic transcripts. One of these, Myh7 was detectable by both RNAseq and HCR-ISH and considered a marker of the circadian clock by the authors.

The authors claim that the study reveals insights into the evolutionary origin of circadian timing; Clock is conserved across distant groups of organisms, having a function as a positive regulator of the transcriptional translational feedback loop at the heart of daily timing, but is not a central element of the core feedback loop circadian system in this basal species. Their behavioural and transcriptomic data largely support the claims that Clock is necessary for endogenous daily activity but that the putative molecular circadian system is not self-sustained under constant darkness (this was known already for WT animals)- rather it is responsive to light cycles with altered dynamics in Clock-/- specimens in some core genes under LD. In the main, I think the authors achieved their aims and the manuscript is a solid piece of important work. The Clock-/- animal is a useful resource for examining time-keeping in a basal metazoan.

The work described builds on other transcriptomic-based works on cnidaria, including Nematostellar, and does probe into the molecular underpinnings with a loss-of-function in a gene known to be core in other circadian systems. The field of chronobiology will benefit from the evolutionary aspect of this work and the fact that it highlights the necessity to study a range of non-model species to get a fuller picture of timing systems to better appreciate the development and diversity of clocks.

Strengths:<br /> The generation of a line of Clock mutant Nematostellar is a very useful tool for the chronobiological community and coupled with a growing suite of tools in this species will be an asset. The experiments seem mostly well conceived and executed (NB see 'weaknesses'). The problem tackled is an interesting one and should be an important contribution to the field.

Weaknesses:<br /> I think the claims about shedding light on the evolutionary origin of circadian time maintenance are a little bold. I agree that the data do point to an alternative role for Clock in this animal in light responsiveness, but this doesn't illuminate the evolution of time-keeping more broadly in my view. In addition, these are transcriptomic data and so should be caveated- they only demonstrate the expression of genes and not physiology beyond that. The time-course analysis is weakened by its low resolution, particularly for the RAIN algorithm when 4-hour intervals constrain the analysis. I accept that only 24h rhythms were selected in the analysis from this but, it might be that detail was lost - I think a preferred option would be 2 or 3-hour resolution or 2 full 24h cycles of analysis.

The authors discount the possibility of the observed 12h rhythmicity in Clock-/- animals by exposing them to LD6:6 cycles before free-running them in DD. I suggest that LD cycles are not a particularly robust way to entrain tidal animals as far as we know. Recent papers show inundation/mechanical agitation are more reliable cues (Kwiatkowski ER, et al. Curr Biol. 2023, 2;33(10):1867-1882.e5. doi: 10.1016/j.cub.2023.03.015; Zhang L., et al Curr Biol. 2013, 23;19, 1863-1873 doi.org/10.1016/j.cub.2013.08.038.) and might be more effective in revealing endogenous 12h rhythms in the absence of 24h cues.

Reviewer #1 (Public Review):

In their manuscript, Laporte et al. analyze the process of formation of the quiescent-cell nuclear microtubule (Q-nMT) bundle, a set of parallel MTs that emanate from the nuclear side of the spindle pole bodies (SPBs) upon the entry of Saccharomyces cerevisiae cells in quiescence. Based on their results, the authors propose that Q-nMT bundle formation is a multistep process that comprises three distinct sequential phases. The authors further evaluate the role of different factors during the growth of the Q-nMT bundle upon quiescence entry, as well as during the disassembly of this structure once cells resume their proliferation.

The Q-nMT is an interesting cellular structure whose physiological function is still widely unknown. Hence, providing new insights into the dynamics of Q-nMT bundle formation and identifying new factors involved in this process is an interesting topic of relevance in the field. The authors made a substantial effort in order to evaluate Q-nMT bundle formation and provide a considerable amount of data, mainly obtained from microscopy analyses. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented.

Despite the interest in the study, there are important issues that could affect the validity of the conclusions drawn in the manuscript. In this way, regarding the analysis of the dynamics of Q-nMT bundle formation, the experimental set up described in some of the experiments raises certain concerns, which mostly derive from the nocodazole treatments and the use of this microtubule-depolymerizing agent as the only approach to evaluate the stability of the Q-nMT bundle. On the other hand, regarding the factors involved in Q-nMT formation, the differences in microtubule length with the wild-type strain, despite being statistically significant, are really subtle for many of the mutants analyzed (e.g., bir1, slk19, etc.). Additionally, there are proteins that are proposed to participate in the process of Q-nMT formation and whose expression during quiescence needs to be demonstrated. Finally, although the cell viability defects observed for some of the mutants in these factors could be certainly associated with the lack of expression or mutation of the specific gene under evaluation, in none of the cases can they be directly attributed to a defect in aberrant Q-nMT bundle formation.

Based on the aforementioned reasons, and despite the considerable effort by the authors, it is my impression that many of the conclusions of the manuscript are not sufficiently justified by the data provided. Additional evidence, including the incorporation of key experimental controls that are currently missing, would be required in order to more strongly support the conclusions of the manuscript.

Reviewer #1 (Public Review):

In their study, Aman et al. utilized single cell transcriptome analysis to investigate wild-type and mutant zebrafish skin tissues during the post-embryonic growth period. They identified new epidermal cell types, such as ameloblasts, and shed light on the effects of TH on skin morphogenesis. Additionally, they revealed the important role of the hypodermis in supporting pigment cells and adult stripe formation. Overall, I find their figures to be of high quality, their analyses to be appropriate and compelling, and their major claims to be well-supported by additional experiments. Therefore, this study will be an important contribution to the field of vertebrate skin research.

Reviewer #1 (Public Review):

Polymorphisms in genes in the human leukocyte antigen (HLA) class II region comprise the most important inherited risk factors for many autoimmune diseases including type 1 diabetes (T1D) and celiac disease (CD). The paper focuses on the novel triad ((SNPs): rs3135394, rs9268645, and rs3129877) finding quite interesting results. The paper suggests further studies at the molecular and structural level to increase our fundamental knowledge of the etiology of autoimmune deceases.

Reviewer #1 (Public Review):

Summary:

Parkinson and colleagues address an interesting and important question, i.e., whether the bumblebee Bombus terrestris can perceive field-realistic concentrations of different pesticides in a sugar solution mimicking nectar. The study directly follows up on a previous study conducted by the same team (Kessler et al. 2015, Nature), which was partly questioned by another more recent study (Arce et al. 2018, Proc. R. Soc. B). The authors apply a combination of electrophysiological measurements and behavioral feeding tests to answer this question. Their results strongly suggest that B. terrestris workers are not able to perceive field-realistic doses of pesticides in a sugar solution. They additionally show that B. terrestris can physiologically differentiate between solutions varying in sugar composition.

Strengths:

Sophisticated methodology, a combination of approaches, clear and precise language

Weaknesses:

Topic and study implications could be discussed more broadly, the statistical approach is not fully clear to me.

Reviewer #1 (Public Review):

This work investigates the function of the PTB domain containing adaptor protein Numb in skeletal muscle structure and function. In particular, the effects of reduced Numb expression in aging muscle is proposed as a mechanism for reduced contractile function associated with sarcopenia. Using ex-vivo analysis of conditional Numb and Numblike knockout muscle the authors demonstrate that loss of Numb but not the related Numblike expression perturbs muscle muscle force generation. In order to explore the molecular mechanisms involved, Numb interacting proteins were identified in C2C12 cell cultured myotubes by immunoprecipitation and LC-MS/MS. The authors identify Septin 7 as a Numb binding protein and demonstrate that loss of Numb/Numblike in myofibers causes changes in Septin 7 subcellular localization. Several questions remain. The authors could provide further clarity on the expression of Numb and Numb-like proteins and the specificity of antibodies used in this study since some Numb antibodies recognize both Numb and Numblike. The authors focus on septin 7 amongst the list of potential Numb interactions identified by AP-MS. Of note, septin 2, 9 and 10 were also identified in the AP-MS experiment. Whether these septins form a complex or are also disrupted by Numb/Numblike loss remains an interesting area for further investigation. Additional investigation of the specificity and mapping of the Numb-Septin 7 (or another Septin) interaction would be of interest and provide an approach for future studies to demonstrate the biological relevance and specificity of the Numb-Septin 7 interaction in skeletal muscle.

Reviewer #1 (Public Review):

Summary:

Pineda et al investigate the association of the hypothesis that Dux4, an embryonic transcription factor, expression in tumor cells is associated with immune evasion and resistance to immunotherapy. They analyze existing cohorts of bulk RNAseq sequenced tumors across cancer types to identify Dux4 expression and association with survival. They find that Dux4 expression is detected in a higher proportion of metastatic tumors compared to primary tumors, is associated with decreased immune infiltrate and a variety of immune metrics and previously nominated immune signatures, and do an in depth evaluation of a cohort of metastatic urothelial cell carcinoma, finding that Dux4 expression is associated with a more immunodeficient tumor microenvironment (desert or excluded microenvironment) and worse survival in this aPDL1 treated cohort. They then find that Dux4 expression is a major independent predictor of survival in this cohort using different types of survival analyses (KM, Cox PH, and random survival forests). With prior existing biological data supporting the hypothesis (in prior work, the senior author has demonstrated Dux4 expression causally suppresses MHC-I expression in interferon-gamma treated cell lines), the current work links Dux4 expression with less immune activity in clinical tumor samples and with survival in ICI treated urothelial carcinomas, and demonstrates that Dux4 expression provides independent information towards survival including other molecular and clinical characteristics (TMB, ECOG PS as the other strongest markers), and provides interesting resolution on landmark analyses with TMB and Dux4 expression providing greater informativeness at later survival landmarks (e.g. 1 year and later), while ECOG PS has strong informativeness already at earlier time points. This work provides impetus towards more mechanistic and functional dissection of the mechanism of Dux4-associated changes with the tumor microenvironment (e.g. in vivo mouse studies) as well as potential interventional studies (e.g. Dux4 as a target in combination therapies). What the work does not provide is additional resolution on the mechanism of how Dux4 may be associated with a more immunodeficient microenvironment.

The conclusions are generally well supported, but there are issues that would benefit from clarification and extension:

- The finding that Dux4 expression is detected in a higher proportion of metastatic tumors and at higher levels compared to primaries (Fig 1BC) is striking. However, at least for one tumor type (melanoma), the "primary" samples are sourced as n=400+ tumors from TCGA, but the TCGA melanoma cohort is comprised of mostly metastatic (n=81 primary and 367 metastatic tumors in the PanCan Atlas), so it is unclear whether this is correctly interpreted. The analysis of tumors with matched FFPE and flash frozen samples with hybrid probe capture and polyA sequencing, respectively is a nice validation to show that the difference in Dux4 expression is not due to differences in preservation of starting material/sequencing in the metastatic samples vs primary samples (S1BC). However, the cited work from which this data arises (D. Robinson et al 2015) is a study of a cohort of prostate cancers with polyA bulk RNAseq sequencing and at least in that work does not seem to have matched FFPE sequencing, making the provenance of this data unclear at a minimum.

- The findings that Dux4 expression in the metastatic urothelial carcinoma setting is associated with a more immunodeficient microenvironment (Figure 2) is clear and unambiguous using multiple lines of data and analyses (bulk RNAseq, DUX4-positive vs DUX4-negative tumors, different immune cell and cytokine signatures; IHC showing an association with immune deserts and immune excluded phenotypes). However, this is an association and does not demonstrate causality.

- The survival analyses (Fig 3,4,5) show fairly convincingly that Dux4 provide independent predictive information beyond clinical variables and TMB towards survival in the aPDL1 treated metastatic urothelial carcinoma cohort, however, there are different choices of Dux4 expression categorization where the rationale is not clearly justified (e.g. Dux4 expression < 0.5 TPM and > 1 TPM in Fig 3, < 0.25 TPM and > 1 TPM in Fig 4 and 5) by either the underlying distribution (e.g. a bimodal distribution) or some type of percentile split.

- The authors demonstrate that adding Dux4 to clinical markers and TMB results in an improved predictive model for survival, but there are a few questions regarding this model as a clinical biomarker<br /> o Is Dux4 expression better than other correlated immune signatures/markers (e.g. interferon gamma, T effector signature, overall immune infiltrate) in providing additional information?<br /> o Since Dux4 expression is categorized to < 0.25 TPM and > 1 TPM, not all patients are included in the model (i.e. between 0.25 TPM and 1 TPM). How many patients this excludes is unclear, and is important to know if this is to be a clinically relevant biomarker.

- The use of random survival forests to quantify the (predictive) marginal effect of Dux4+ vs Dux4- expression on survival in a non-parametric model as well as shed light on association with survival at different landmark times using Shapley values is quite interesting and well conducted.

Reviewer #1 (Public Review):

In this study, the researchers aimed to investigate the cellular landscape and cell-cell interactions in cavernous tissues under diabetic conditions, specifically focusing on erectile dysfunction (ED). They employed single-cell RNA sequencing to analyze gene expression patterns in various cell types within the cavernous tissues of diabetic individuals. The researchers identified decreased expression of genes associated with collagen or extracellular matrix organization and angiogenesis in several cell types, including fibroblasts, chondrocytes, myofibroblasts, valve-related lymphatic endothelial cells, and pericytes. They also discovered a newly identified marker, LBH, that distinguishes pericytes from smooth muscle cells in mouse and human cavernous tissues. Furthermore, the study revealed that pericytes play a role in angiogenesis, adhesion, and migration by communicating with other cell types within the corpus cavernosum. However, these interactions were found to be significantly reduced under diabetic conditions. The study also investigated the role of LBH and its interactions with other proteins (CRYAB and VIM) in maintaining pericyte function and highlighted their potential involvement in regulating neurovascular regeneration. Overall, the manuscript is well-written and the study provides novel insights into the pathogenesis of ED in patients with diabetes and identifies potential therapeutic targets for further investigation.

Joint Public Review:

In this manuscript, the authors proposed an approach to systematically characterise how heterogeneity in a protein signalling network affects its emergent dynamics, with particular emphasis on drug-response signalling dynamics in cancer treatments. They named this approach Meta Dynamic Network (MDN) modelling, as it aims to consider the potential dynamic responses globally, varying both initial conditions (i.e., expression levels) and biophysical parameters (i.e., protein interaction parameters). By characterising the "meta" response of the network, the authors propose that the method can provide insights not only into the possible dynamic behaviours of the system of interest but also into the likelihood and frequency of observing these dynamic behaviours in the natural system.

The authors studied the Early Cell Cycle (ECC) network as a proof of concept, specifically focusing on PI3K, EGFR, and CDK4/6, with particular interest in identifying the mechanisms that cancer could potentially exploit to display drug resistance. The biochemical reaction model consists of 50 equations (state variables) with 94 kinetic parameters, described using SBML and computed in Matlab. Based on the simulations, the authors concluded the following main points: a large number of network states can facilitate resistance, the individual biophysical parameters alone are insufficient to predict resistance, and adaptive resistance is an emergent property of the network. Finally, the authors attempt to validate the model's prediction that differential core sub-networks can drive drug resistance by comparing their observations with the knock-out information available in the literature. The authors identified subnetworks potentially responsible for drug resistance through the inhibition of individual pathways. Importantly, some concerns regarding the methodology are discussed below, putting in doubt the validity of the main claims of this work.

While the authors proposed a potentially useful computational approach to better understand the effect of heterogeneity in a system's dynamic response to a drug treatment (i.e., a perturbation), there are important weaknesses in the manuscript in its current form:

(1) It is unclear how the random parameter sets (i.e., model instances) and initial conditions are generated, and how this choice biases or limits the general conclusions for the case studied. Particularly, it is not evident how the kinetic rates are related to any biological data, nor if the parameter distributions used in this study have any biological relevance.<br /> (2) Related to this problem, it is not clear whether the considered 100,000 random parameter samples sufficiently explore parameter space due to the combinatorial explosion that arises from having 94 free parameters, nor 100,000 random initial conditions for a system with 50 species (variables).<br /> (3) Moreover, the authors filter out all the cases with stiff behaviour. This filtering step appears to select model parameters based on computational convenience, rather than biological plausibility.<br /> (4) Also, it is not clear how exactly the drug effect is incorporated into the model (e.g., molecular inhibition?), nor how it is evaluated in the dynamic simulations (e.g., at the beginning of the simulation?). Moreover, in a complex network, the results may differ depending on whether the inhibition is applied from the start or after the network has reached a stable state.<br /> (5) On the same line, the conclusions need to be discussed in the context of stability, particularly when evaluating the role of initial conditions. As stable steady states are determined by the model parameters, once again, the details of how the perturbation effect is evaluated on the simulation dynamics are critical to interpret the results.<br /> (6) The presented validation of the model results (Fig. 7) is only qualitative, and the interpretation is not carefully discussed in the manuscript, particularly considering the comparison between fold-change responses without specifying the baseline states.

Reviewer #1 (Public Review):

Wang and all present an interesting body of work focused on the effects of high altitude and hypoxia on erythropoiesis, resulting in erythrocytosis. This work is specifically focused on the spleen, targeting splenic macrophages as central cells in this effect. This is logical since these cells are involved in erythrophagocytosis and iron recycling. The results suggest that hypoxia induces splenomegaly with decreased number of splenic macrophages. There is also evidence that ferroptosis is induced in these macrophages, leading to cell destruction. However, additional data demonstrates that RBC clearance is increased, aka shortening the RBC lifespan, calling into question whether splenic function is impaired in hypoxia or whether the spleen enlargement is compensatory, leading to increased erythropoiesis; similarly, increased iron in the spleen provides potential evidence of enhanced erythrophagocytosis with iron release. Many of the reviewers' prior comments are not addressed or only superficially addressed and the additional experimental results and text to the background and discussion sections in the revised manuscript does not increase enthusiasm or clarity. Taken together, there are many issues with the presented results, with somewhat superficial data, with overstated conclusions, decreasing confidence that the hypotheses and observed results are directly causally related to hypoxia in the way that the authors propose.

Reviewer #1 (Public Review):

It is well established that tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a leading cause of mortality and morbidity worldwide. However, the only vaccine licensed against tuberculosis is Bacille Calmette Guerin (BCG), has been around for nearly a century, and has limited efficacy in adults. Herein, the authors sought to investigate the effectiveness of a nanoparticle-based formulation of a subunit vaccine composed of Mtb lipid and protein antigens. The authors found that they were able to load the lipid, mycolic acid, into their nanoparticles without disrupting the architecture and that the loaded particles activated T cells both in vitro and in vivo. Moreover, when they vaccinated with particles loaded with both lipid and protein antigens, they found that the lipid antigen persisted, and mycolic acid-specific T cells were able to be activated 6 weeks post-vaccination, in contrast to peptide-specific T cells. The authors investigated further and found that persistence required the nanoparticle encapsulation, rather than free lipid, and that it was independent of route (intratracheal, intravenous, or subcutaneous) of administration. To address the mechanisms underlying antigen persistence, the authors loaded the nanoparticles with a dye and demonstrated that the nanoparticle encapsulated lipid antigen was primarily stored in lung alveolar macrophages and that CD1b+ dendritic cells presented the antigen to mycolic acid specific T cells. Finally, the authors conducted mixed bone marrow chimera studies to examine the phenotype of the mycolic acid specific T cells and found that the memory T cell population phenotypically resembled T follicular helper, regulatory T cells, and exhausted T cells. Interestingly, while a large percentage of these lipid antigen specific T cells in the lymph nodes, lung and spleen were CXCR5+PD1+, the cells were still proliferating (Ki67+). Overall, this is a comprehensive study that has the potential to significantly enhance the field.

Reviewer #1 (Public Review):

Murphy, Fancy and Skene performed a reanalysis of snRNA-seq data from Alzheimer Disease (AD) patients and healthy controls published previously by Mathys et al. (2019), arriving at the conclusion that many of the transcriptional differences described in the original publication were false positives. This was achieved by revising the strategy for both quality control and differential expression analysis. I believe the authors' intention was to show the results of their reanalysis not as a criticism of the original paper (which can hardly be faulted for their strategy which was state-of-the-art at the time and indeed they took extra measures attempting to ensure the reliability of their results), but primarily to raise awareness and provide recommendations for rigorous analysis of sc/snRNA-seq data for future studies.

STRENGTHS:

The authors demonstrate that the choice of data analysis strategy can have a vast impact on the results of a study, which in itself may not be obvious to many researchers.

The authors apply a pseudobulk-based differential expression analysis strategy (essentially, adding up counts from all cells per individual and comparing those counts with standard RNA-seq differential expression tests), which is (a) in line with latest community recommendations, (b) different from the "default options" in most popular scRNA-seq analysis suites, and (c) explains the vastly different number of DEGs identified by the authors and the original publication. The recommendation of this approach together with a detailed assessment of the DEGs found by both methodologies could be a useful finding for the research community. Unfortunately, it is currently not fully substantiated and is confounded with concurrent changes in QC measures (see weaknesses).

The authors show a correlation between the number of DEGs and the number of cells assessed, which indicates a methodological shortcoming of the original paper's approach (actually, the authors of the original paper already acknowledged that the lesser number of DEGs for rare cell types was a technical artefact). To be educational for the reader it would be important to provide more information about the DEGs that were "found" and those that were "lost". Given vast inter-individual heterogeneity in humans, it is likely that the study was underpowered to find weaker differences using the pseudobulks (Fig. 1B shows that only genes with more than 4-fold change were found "significant").

All code and data used in this study are publicly available to the readers.

WEAKNESSES:

The authors interpret the fact that they found fewer DEGs with their method than the original paper as a good thing by making the assumption that all genes that were not found were false positives. However, they do not prove this, and it is likely that at least some genes were not found due to a lack of statistical power and not because they were actually "incorrect". The original paper also performed independent validations of some genes that were not found here.

I am concerned that the only DEGs found by the authors are in the rare cell types, foremost the rare microglia (see Fig. 1f). It is unclear to me how many cells the pseudo-bulk counts were based on for these cells types, but it seems that (a) there were few and (b) there were quite few reads per cells. If both are the case, the pseudobulk counts for these cell populations might be rather noisy and the DEG results are liable to outliers with extreme fold changes.

The authors claim they improved the quality control of the dataset. While I do not think they did anything wrong per se, the authors offer no objective metric to assess this putative improvement. This is another major weakness of the paper as it confounds the results of the improved (?) differential analysis strategy and dilutes the results. I detail this weakness in the two following points:

Removing low-quality cells: The authors apply a new QC procedure resulting in the removal of some 20k more cells than in the original publication. They state "we believe the authors' quality control (QC) approach did not capture all of these low quality cells" (l. 26). While all the QC metrics used are very sensible, it is unclear whether they are indeed "better". For instance, removal with a mitochondrial count of <5% seems harsh and might account for a large proportion of additional cells filtered out in comparison to the original analysis. There is no blanket "correct cutoff" for this percentage. For instance, the "classic" Seurat tutorial https://satijalab.org/seurat/articles/pbmc3k_tutorial.html uses the 5% threshold chosen by the authors, an MAD-based selection of cutoff arrived at 8% here https://www.sc-best-practices.org/preprocessing_visualization/quality_control.html, another "best practices" guide choses by default 10% https://bioconductor.org/books/3.17/OSCA.basic/quality-control.html#quality-control-discarded, etc. Generally, the % of mitochondrial reads varies a lot between datasets. As far as I can tell, the original paper did not use a fixed threshold but instead used a clustering approach to identify cells with an "abnormally high" mitochondrial read fraction. That also seems reasonable. Overall, I cannot assess whether the new QC is really more appropriate than the original analysis and the authors do not provide any evidence in favor of their strategy.

Batch correction: "Dataset integration has become a standard step in single-cell RNA-Seq protocols" (l. 29). While it is true that many authors now choose to perform an integration step as part of their analysis workflow, this is by no means uncontroversial as there is a risk of "over-integration" and loss of true biological differences. Also, there are many different methods for dataset integration out there, which will all have different results. More importantly, the authors go on "we found different cell type proportions to the authors (Fig. 1a) which could be due to accounting for batch effects" but offer no support for the claim that the batch effects are indeed related to the observed differences. An alternative explanation would be a selective loss/gain of certain cell types during quality control. The original paper stated concerns about losing certain cell types (microglia, which do not seem to be differentially abundant in the original paper / new analysis).

Relevant literature is incompletely cited. Instead of referring to reviews of best practices and benchmarks comparing methods for batch correction and or differential analysis, the authors only refer to their own previous work.

Due to a lack of comparison with other methods and due to the fact that the author's methodology was only applied to a single dataset, the paper presents merely a case study, which could be useful but falls short of providing a general recommendation for a best practice workflow.

APPRAISAL:

The manuscript could help to increase awareness of data analysis choices in the community, but only if the superiority of the methodology was clearly demonstrated. The recommended pseudobulk differential expression approach along with the indication of drastic differences that this might have on the results is the main output of the current manuscript, but it is difficult to assess unequivocally how this influenced the results because the differential analysis comes after QC and cell type annotation, which have also been changed in comparison to the original publication. In my opinion, the purpose of the paper might be better served by focusing on the DE strategy without changing QC and instead detailing where/how DEGs were gained/lost and supporting whether these were false positives.

Reviewer #1 (Public Review):

Summary: Cullinan et al. explore the hypothesis that the cytoplasmic N- and C-termini of ASIC1a, not resolved in x-ray or cryo-EM structures, form a dynamic complex that breaks apart at low pH, exposing a C-terminal binding site for RIPK1, a regulator of necrotic cell death. They expressed channels tagged at their N- and C-termini with the fluorescent, non-canonical amino acid ANAP in CHO cells using amber stop-codon suppression. Interaction between the termini was assessed by FRET between ANAP and colored transition metal ions bound either to a cysteine reactive chelator attached to the channel (TETAC) or metal-chelating lipids (C18-NTA). A key advantage to using metal ions is that they are very poor FRET acceptors, i.e. they must be very close to the donor for FRET to occur. This is ideal for measuring small distances/changes in distance on the scales expected from the initial hypothesis. In order to apply chelated metal ions, CHO cells were mechanically unroofed, providing access to the inner leaflet of the plasma membrane. At high pH, the N- and C- termini are close enough for FRET to be measured, but apparently too far apart to be explained by a direct binding interaction. At low pH, there was an apparent increase in FRET between the termini. FRET between ANAP on the N-and C-termini and metal ions bound to the plasma membrane suggests that both termini move away from the plasma membrane at low pH. The authors propose an alternative hypothesis whereby close association with the plasma membrane precludes RIPK1 binding to the C-terminus of ASIC1a.

Strengths: The findings presented here are certainly valuable for the ion channel/signaling field and the technical approach only increases the significance of the work. The choice of techniques is appropriate for this study and the results are clear and high quality. Sufficient evidence is presented against the starting hypothesis.

Weaknesses: I have a few questions about certain controls and assumptions that I would like to see discussed more explicitly in the manuscript.

--My biggest concern is with the C-terminal citrine tag. Might this prevent the hypothesized interaction between the N- and C-termini? What about the serine to cysteine mutations? The authors might consider a control experiment in channels lacking the C-terminal FP tag.

--Figure 2 supplement 1 shows apparent read-through of the N-terminal stop codons. Given that most of the paper uses N-terminal ANAP tags, this figure should be moved out of the supplement. Do N-terminally truncated subunits form functional channels? Do the authors expect N-terminally truncated subunits to co-assemble in trimers with full-length subunits? The authors should include a more explicit discussion regarding the effect of truncated channels on their FRET signal in the case of such co-assembly.

--As the epitope used for the western blots in Figure 2 and supplements is part of the C-terminal tag, these blots do not provide an estimate of the fraction of C-terminally truncated channels (those that failed to incorporate ANAP at the stop codon). What effect would C-terminally truncated channels have on the FRET signal if incorporated into trimers with full-length subunits?

--Some general discussion of these results in the context of trimeric channels would be helpful. Is the putative interaction of the termini within or between subunits? Are the distances between subunits large enough to preclude FRET between donors on one subunit and acceptor ions bound on multiple subunits?

--The authors conclude that the relatively small amount of FRET between the cytoplasmic termini suggests that the interaction previously modeled in Rosetta is unlikely. Is it possible that the proposed structure is correct, but labile? For example, could it be that the FRET signal is the time average of a state in which the termini directly interact (as in the Rosetta model) and one in which they do not?

Reviewer #1 (Public Review):

In this manuscript, Nagel et al. sought to comprehensively characterize the composition of urinary compounds, some of which are putative chemosignals. They used urines from adult males and females in three different strains, including one wild-derived strain. By performing mass spectrometry of two classes of compounds: volatile organic compounds and proteins, they found that urines from inbred strains are qualitatively similar to those of a wild strain. This finding is significant because there is a high degree of genetic diversity in wild mice, with chemosensory receptor genes harboring many polymorphisms.

In the second part of this work, the authors used calcium imaging to monitor the pattern of vomeronasal neuron responses to these urines. By performing pairwise comparisons, the authors found a large degree of strain-specific response and a relatively minor response to sex-specific urinary stimuli. This is a finding generally in agreement with previous calcium imaging work by Ron Yu and colleagues in 2008. The authors extend the previous work by using urines from wild mice. They further report that the concentration diversity of urinary compounds in different urine batches is largely uncorrelated with the activity profiles of these urines. In addition, the authors found that the patterns of vomeronasal neuron response to urinary cues are not identical when measured using different recipient strains. This fascinating finding, however, requires an additional control to exclude the possibility that this is not due to sampling error.

There are several weaknesses in this manuscript, including the lack of analysis of the compositions of sulfated steroids and other steroids, which have been proposed to be the major constituents of vomeronasal ligands in urines and the indirect (correlational) nature of their mass spectrometry data and activity data.

Overall, the major contribution of this work is the identification of specific molecules in mouse urines. This work is likely to be of significant interest to researchers in chemosensory signaling in mammals and provides a systematic avenue to exhaustively identify vomeronasal ligands in the future.

Reviewer #1 (Public Review):

In this study, the authors build upon previous research that utilized non-invasive EEG and MEG by analyzing intracranial human ECoG data with high spatial resolution. They employed a receptive field mapping task to infer the retinotopic organization of the human visual system. The results present compelling evidence that the spatial distribution of human alpha oscillations is highly specific and functionally relevant, as it provides information about the position of a stimulus within the visual field.

Using state-of-the-art modeling approaches, the authors not only strengthen the existing evidence for the spatial specificity of the human dominant rhythm but also provide new quantification of its functional utility, specifically in terms of the size of the receptive field relative to the one estimated based on broad band activity.

The present manuscript currently omits the complementary view that the retinotopic map of the visual system might be related to eye movement control. Previous research in non-human primates using microelectrode stimulation has clearly shown that neuronal circuits in the visual system possess motor properties (e.g. Schiller and Styker 1972, Schiller and Tehovnik 2001). More recent work utilizing Utah arrays, receptive field mapping, and electrical stimulation further supports this perspective, demonstrating that the retinotopic map functions as a motor map. In other words, neurons within a specific area responding to a particular stimulus location also trigger eye movements towards that location when electrically stimulated (e.g. Chen et al. 2020).

Similarly, recent studies in humans have established a link between the retinotopic variation of human alpha oscillations and eye movements (e.g., Quax et al. 2019, Popov et al. 2021, Celli et al. 2022, Liu et al. 2023, Popov et al. 2023). Therefore, it would be valuable to discuss and acknowledge this complementary perspective on the functional relevance of the presented evidence in the discussion section.

Reviewer #1 (Public Review):

This study investigates the impact of recurrent connections on grid fields generated in networks trained by adjusting the strength of feedforward spatial inputs. The main result is that if the recurrent connections in the network are given a 1D continuous attractor architecture, then aligned grid firing patterns emerge in the network following training. Detailed analyses of the low dimensional dynamics of the resulting networks are then presented. The simulations and analyses appear carefully carried out.

The feedforward model investigated by the authors (previously introduced by Kropff & Treves, 2008) is an interesting and important alternative to models that generate grid firing patterns through 2-dimensional continuous attractor network (CAN) dynamics. However, while both classes of model generate grid fields, in making comparisons the manuscript is insufficiently clear about their differences. In particular, in the CAN models grid firing is a direct result of their 2-D architecture, either a torus structure with a single activity bump (e.g. Guanella et al. 2007, Pastoll et al. 2013), or sheet with multiple local activity bumps (Fuhs & Touretzky, Burak & Fiete, 2009). In these models, spatial input can anchor the grid representations but is not necessary for grid firing. By contrast, in the feedforward models neurons transform existing spatial inputs into a grid representation. Thus, the two classes of model implement different computations; CANs path integrate, while the feedforward models transform spatial representations. A demonstration that a 1D CAN generates coordinated 2D grid fields would be surprising and important, but it's less clear why coordination between grids generated by the feedforward mechanism would be surprising. As written, it's unclear which of these claims the study is trying to make. If the former, then the conclusion doesn't appear well supported by the data as presented, if the latter then the results are perhaps not so unexpected, and the imposed attractor dynamics may still not be relevant.

Whichever claim is being made, it could be helpful to more carefully evaluate the model dynamics given predictions expected for the different classes of model. Key questions that are not answered by the manuscript include:

- At what point is the 1D attractor architecture playing a role in the models presented here? Is it important specifically for training or is it also contributing to computation in the fully trained network?

- Is an attractor architecture required at all for emergence of population alignment and gridness? Key controls missing from Figure 2 include training on networks with other architectures. For example, one might consider various architectures with randomly structured connectivity (e.g. drawing weights from exponential or Gaussian distributions).

- In the trained models do the recurrent connections substantially influence activity in the test conditions? Or after training are the 1D dynamics drowned out by feedforward inputs?

- What is the low dimensional structure of the input to the network? Can the apparent discrepancy between dimensionality of architecture and representation be resolved by considering structure of the inputs, e.g. if the input is a 2 dimensional representation of location then is it surprising that the output is too?

- What happens to representations in the trained networks presented when place cells remap? Is the 1D manifold maintained as expected for CAN models, or does it reorganise?

Reviewer #1 (Public Review):

Summary:<br /> Mitochondria is the power plant of the cells including neurons. Thomas et al. characterized the distribution of mitochondria in dendrites and spines of L2/3 neurons from the ferret visual cortex, for which visually driven calcium responses of individual dendritic spines were examined. The authors analyzed the relationship between the position of mitochondria and the morphology or orientation selectivity of nearby dendrite spines. They found no correlation between mitochondrion location and spine morphological parameters associated with the strength of synapses, but correlation with the spine-somatic difference of orientation preference and local heterogeneity in preferred orientation of nearby spines. Moreover, they reported that the spines that have a mitochondrion in the head or neck are larger in size and have stronger orientation selectivity. Therefore, they proposed that "mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs."

Strengths:<br /> This paper attempted to address a fundamental question: whether the distribution of the mitochondria along the dendrites of visual cortical neurons is associated with the functions of the spines, postsynaptic sites of excitatory synapses. Two state of the art techniques (2 photon Ca imaging of somata and spines and EM reconstructions of cortical pyramidal neurons) had been used, which provides a great opportunity to examine and correlate the function of spine ultrastructure and spatial distribution of dendritic mitochondria.

Weaknesses:<br /> Overall, the findings are interesting. However, the study lacks the data providing insights into either the mechanisms or the functional meaning of the pattern of mitochondrion distribution along the dendrites, which restricts the significance of the study. It also suffers from small correlation coefficients and small sample sizes (60-121 spines in 4 neurons) as well as missing some important analysis.

Reviewer #1 (Public Review):

Summary<br /> In this manuscript, Hagihara et al. characterized the relationship between the changes in lactate and pH and the behavioral phenotypes in different animal models of neuropsychiatric disorders at a large-scale level. The authors have previously reported that increased lactate levels and decreased pH are commonly observed in the brains of five genetic mouse models of schizophrenia (SZ), bipolar disorder (BD), and autism spectrum disorder (ASD). In this study, they expanded the detection range to 109 strains or conditions of animal models, covering neuropsychiatric disorders and neurodegenerative disorders. Through statistical analysis of the first 65 strains/conditions of animal models which were set as exploratory cohort, the authors found that most strains showed decreased pH and increased lactate levels in the brains. There was a significant negative correlation between pH and lactate levels both at the strain/condition level and the individual animal level. Besides, only working memory was negatively correlated with brain lactate levels. These results were successfully duplicated by studying the confirmative cohort, including 44 strains/conditions of animal models. In all strains/conditions, the lactate levels were not correlated with age, sex, or storage duration of brain samples.

Strengths<br /> 1. The manuscript is well-written and structured. In particular, the discussion is really nice, covering many potential mechanisms for the altered lactate levels in these disease models.<br /> 2. Tremendous efforts were made to recruit a huge number of various animal models, giving the conclusions sufficient power.

Weaknesses<br /> 1. The biggest concern of this study is the limited novelty. The point of "altered pH and/or lactate levels in the brains from human and rodent animals of neuropsychiatric disorders" has been reported by the same lab and other groups in many previous papers.<br /> 2. This study is mostly descriptive, lacking functional investigations. Although a larger cohort of animal models were studied which makes the conclusion more solid, limited conceptual advance is contributed to the relevant field, as we are still not clear about what the altered levels of pH and lactate mean for the pathogenesis of neuropsychiatric disorders.<br /> 3. The experiment procedure is also a concern. The brains from animal models were acutely collected without cardiac perfusion in this study, which suggests that resident blood may contaminate the brain samples. The lactate is enriched in the blood, making it a potential confounded factor to affect the lactate levels as well as pH in the brain samples.<br /> 4. The lactate and pH levels may also be affected by other confounded factors, such as circadian period, and locomotor activity before the mice were sacrificed. This should also be discussed in the paper.<br /> 5. Another concern is the animal models. Although previous studies have demonstrated that dysfunctions of these genes could cause related phenotypes for certain disorders, many of them are not acknowledged by the field as reliable disease models. Besides, gene deficiency could also cause many known or unknown unrelated phenotypes, which may contribute to the altered levels of lactate and pH, too. In this circumstance, the conclusion "pH and lactate levels are transdiagnostic endophenotype of neuropsychiatric disorders" is somewhat overstated.<br /> 6. The negative correlationship between pH and lactate is rather convincing. However, how much the contribution of lactate to pH is not tested. In addition, regarding pH and lactate, which factor contributes most to the pathogenesis of neuropsychiatric disorders is also unclear. These questions may need to be addressed in the future study.<br /> 7. The authorship is open to question. Most authors listed in this paper may only provide mice strains or brain samples. Maybe it is better just to acknowledge them in the acknowledgments section.<br /> 8. The last concern is about the significance of this study. Although the majority of strains showed increased lactate, some still showed decreased lactate levels in the brains. These results suggested that lactate or pH is an endophenotype for neuropsychiatric disorders, but it is hard to serve as a good diagnostic index as the change is not unidirectional in different disorders. In other words, the relationship between lactate level and neuropsychiatric disorders is not exclusive.

Reviewer #1 (Public Review):

This study by Hormigo et al. examines the relationship between activity in the zona incerta (ZI) and behavior. The authors aim to assess the hypothesis that the ZI might mediate a general behavioral function, namely the distribution of information about ongoing movement to other brain areas that regulate behavior. Given the heterogeneity of prior literature on the ZI, this topic is important and interesting. The study employs a strong diversity of technical approaches, spanning electrophysiological recordings, calcium imaging, optogenetics, virally-mediated cell-type ablation, and several behavioral assays. The output is a large dataset where each experiment is useful and interesting, and together, the results could be interpreted as consistent with the prospect of the ZI mediating a general function. However, there are notable weaknesses in the current version of this paper. First, it is unclear whether the experiments and analyses were set up to be able to rule out more specific candidate functions of the ZI. Second, many important details of the experiments and their results are hard to decipher given the current descriptions and presentations of the data.

The paper could be significantly strengthened by including more details from each experiment, stronger justifications for the limited behaviors and experimental analyses performed, and, finally, a broader analysis of how the recorded activity in the ZI relates to behavioral parameters.

(1) Anatomical specification: The ZI contains many distinct subdivisions--each with its own topographically organized inputs/outputs and putative functions. The current manuscript doesn't reference these known divisions or their behavioral distinctions, and one cannot tell exactly which portion(s) of the ZI was included in the current study.

Moreover, the elongated structure of the ZI makes it very difficult to specifically or completely infect virally. The data could be better interpreted if the paper included basic information on the locations of recordings, the extent of the AAV spread in the ZI in each viral experiment, and what fraction of infected neurons were inside versus outside ZI.

(2) Electrophysiological recording on the treadmill: The authors are commended for this technically very difficult experiment. The authors do not specify, however, how they knew when they were recording in ZI rather than surrounding structures, particularly given that recording site lesions were only performed during the last recording session. A map of the locations of the different classes of units would be valuable data to relate to the literature.

(3) The rationale of the analysis of activity with respect to "movement peak": It is unclear why the authors did not assess how ZI activity correlates with a broad set of movement parameters, but rather grouped heterogeneous behavioral epochs to analyze firing with respect to "movement peaks".

(4) The display of mean categorical data in various figures is interesting, however, the reader cannot gather a very detailed view of ZI firing responses or potential heterogeneity with so little information about their distributions.

(5) Somatosensory firing responses in ZI: It is unclear why the authors chose the specific stimuli used in the study. How often did they evoke reflexive motor responses? What was the latency of sensory-evoked responses in ZI activity and the latency of the reflexive movement?

(6) It would be valuable to see example traces in Figure 3 to get a better sense of the time course and contexts under which Ca signals in ZI tracks movement. What is the typical latency? What is the typical range of magnitudes of responses? Does the Ca signal track both fast and slow movements? How are the authors sure that there are no movement artifacts contributing to the calcium imaging? It seems there is more information in the dataset that could be valuable.

(7) Figure 4: The rationale for quantifying the F/Fo responses over a 6-second window, rather than with respect to discrete movement parameters, is not well explained. What types of movement are binned in this approach and might this broad binning hinder the ability to detect more specific relationships between activity and movement?

(8) Separation of sensory and motor responses in Figure 5: The current data do not adequately differentiate whether the responses are sensory or motor given the high correlation of the sensory inputs driving motor responses. Because isoflurane can diminish auditory responses early in the auditory pathway, this reviewer is not convinced the isoflurane experiments are interpretable.

(9) Given the broad duration of the mean avoidance response (Fig. 6 C, bottom), it would be useful to know to what extent this plot reflects a prolonged behavior or is the result of averaging different animals/trials with different latencies. Given that the shapes of the F/Fo responses in ZI appear similar across avoids and escapes (Fig. 6D), despite their apparent different speeds and movement durations (Fig 6C), it would be valuable to know how the timing of the F/Fo relates to movement on a trial-by-trial basis.

(10) Lesion quantification: One cannot tell what rostral-caudal extent of ZI was lesioned and quantified in this experiment. It would be easier to interpret if also plotted for each animal, so the reader can tell how reliable the method is. The mean ablation would be better shown as a normalized fraction of cells. Although the authors claim the lesions have little impact on behavior, it appears the incompleteness of the lesions could warrant a more conservative interpretation.

(11) Optogenetics: the location of infected neurons is poorly described, including the rostral-caudal extent and the fraction of neurons inside and outside of ZI. Moreover, it is unclear how strongly the optogenetic manipulations in this study are expected to affect neuronal activity in ZI.

Reviewer #1 (Public Review):

In this paper, the interocular/binocular combination of temporal luminance modulations is studied. Binocular combination is of broad interest because it provides a remarkable case study of how the brain combines information from different sources. In addition, the mechanisms of binocular combination are of interest to vision scientists because they provide insight into when/where/how information from two eyes is combined.

This study focuses on how luminance flicker is combined across two eyes, extending previous work that focused mainly on spatial modulations. The results appear to show that temporal modulations are combined in different ways, with additional differences between subcortical and cortical pathways.

The manuscript has been revised to address prior reviewers' comments. It now provides more justification for the empirical choices made by the authors, and a better illustration of the methods. That said, the paper would still benefit from an expanded rationale for significance beyond this specific area. There were no substantive changes made to the abstract or introduction, and only little to the discussion.

Reviewer #1 (Public Review):

In this manuscript Rubin and Aso provide important new tools for the study of learning and memory in Drosophila. In flies, olfactory learning and memory occurs at the Mushroom Body (MB) and is communicated to the rest of the brain through Mushroom Body Output Neurons (MBONs). Previously, typical MBONs were thoroughly studied. Here, atypical MBONs that have dendritic input both within the MB lobes and in adjacent brain regions are studied. The authors describe new cell-type-specific GAL4 drivers for the majority of atypical MBONs (and other MBONs) and using an optogenetic activation screen they examined their ability to drive behaviors and learning.

The experiments in this manuscript were carefully performed and the results are clear. The tools provided in this manuscript are of great importance to the field.

Reviewer #1 (Public Review):

The goal of the authors is to use whole-exome sequencing to identify genomic factors contributing to asthenoteratozoospermia and male infertility. Using whole-exome sequencing, they discovered homozygous ZMYND12 variants in four unrelated patients. They examined the localization of key sperm tail components in sperm from the patients. To validate the findings, they knocked down the ortholog in Trypanosoma brucei. They further dissected the complex using co-immunoprecipitation and comparative proteomics with samples from Trypanosoma and Ttc29 KO mice. They concluded that ZMYND12 is a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.

The major strengths are that the authors used the cutting-edge technique, whole-exome sequencing, to identify genes associated with male infertility, and used a new model organism, Trypanosoma brucei to validate the findings, together with other high-throughput tools, including comparative proteomics to dissect the protein complex essential for normal sperm formation/function. The major weakness is that limited samples could be collected from the patients for further characterization by other approaches, including western blotting and TEM.

In general, the authors achieved their goal, and the conclusion is supported by their results. The findings not only provide another genetic marker for the diagnosis of asthenoteratozoospermia but also enrich the knowledge of cilia/flagella.

Reviewer #1 (Public Review):

It has been shown previously that maternal aging in mice is associated with an increase in accumulation of damaged mitochondria and activation of parkin-mediated autophagy (see DOI: 10.1080/15548627.2021.1946739). It has also been shown that C-natriuretic peptide (CNP) regulates oocyte meiotic arrest and that its use during in vitro oocyte maturation can improve parameters associated with decreased oocyte quality. Here the authors tested whether use of CNP treatment in vivo could improve oocyte quality and fertility of aged mice, for which they provided convincing evidence. They also attempted to determine how CNP improves oocyte developmental competence. They showed a correlation between CNP use in vivo and the appearance (and some functional qualities) of cytoplasmic organelles more closely approximating those of oocytes from young mice. However, this correlation could not be interpreted to imply causation. Additional experiments performed using CNP during in vitro maturation were not properly controlled and so are not possible to interpret.

A strength of the manuscript is that the authors use an in vivo treatment to improve oocyte quality rather than just using CNP during oocyte maturation in vitro as has been done previously. This strategy provides more potential for improving oocyte quality - over the course of oocyte growth and maturation - rather than just the final few hours of maturation alone. This strategy also has the potential to be translated into a more generally useful clinical therapeutic method that using CNP during in vitro maturation. However, it is difficult to glean information regarding how CNP might have its effects in vivo. A range of models are used in the manuscript with a mix of in vivo studies with in vitro experiments, which results in some disconnect between systemic CNP and its reported intrafollicular action as well as in the short-term versus longer-term actions of CNP on oocyte quality. Specifically, CNP was shown to be reduced in the plasma of aged mice, but this was not shown in the granulosa cells, which are the reported source of CNP that acts on oocytes. Whether the ovarian source of CNP is reduced in aged females was not demonstrated, and CNP is not known to act on oocytes through an endocrine effect. In vivo treatments with CNP by i.p. injection were performed, but the dose (120 ug/kg) and time (14 days) of treatment were not validated by any prior experiments to give them physiological relevance.

Weaknesses:

1. The Results section is not always clear regarding what CNP treatment was done - in vivo injections or in vitro maturation. For example, what is the difference, if any, between Figures 2C-D and Figures S2A-B?

This remains unclear in the revised manuscript.

2. Immature oocytes from aged females (~1 year) were treated with a two-step culture system with a pre-IVM step with CNP. Controls included oocytes from young (6-8 weeks) females or oocytes from aged females treated by conventional IVM. The description of these methods suggests that control oocytes did not receive an equivalent pre-IVM culture, hence the relevance of comparisons of CNP-treated versus control oocyte is questionable. This concern has not been addressed in the revised manuscript. It was observed that aged oocytes pre-cultured in CNP improved polar body extrusion rates and meiotic spindle morphology compared to oocytes in conventional IVM, as has been well established. The description of statistical methods does not make clear whether the PBE rate in CNP-treated old oocytes remained significantly lower than young controls.

This concern has not been addressed in the revised manuscript.

3. The main effect of the CNP 2-week treatment appears to be increasing the number of follicles that grow into secondary and antral stages, but there is no attempt made to discover the mechanism by which this occurs and therefore to understand why there might be an increase in the number of ovulated eggs, quality of the eggs, and litter size. It is also not clear how an intraperitoneal injection can guarantee its effectiveness because the half-life of CNP is very short, only a few minutes.

This concern has not been addressed in the revised manuscript.

4. Meiotic spindle morphology, as well as a number of putative markers of cytoplasmic maturation are also suggested to be improved after pre-culture with CNP. In each case a subjective interpretation of "normal" morphology of these markers is derived from observations of the young controls and the proportions of oocytes with normal or abnormal appearance is evaluated. However, parameters that define abnormal patterns of these markers appear to be subjective judgements, and whether these morphological patterns can be mechanistically attributed to the differences in developmental potential cannot be concluded.

This concern has not been addressed in the revised manuscript.

5. In addition to the localization patterns of mitochondria, the mitochondrial membrane potential, oocyte ATP content and ROS levels were assessed through more objective quantitative methods. These are well known to be defective in oocytes of aged females and CNP treatment improved these measures. Mitochondrial dysfunction is the most obvious link between oocyte apoptosis, autophagy, cytoplasmic organelle miss-localization and aberrant spindle morphology. Among the most intriguing results is the finding that CNP mediated a cAMP-dependent protein kinase (PKA) dependent reduction in mitochondrial autophagy mediators PINK and Parkin and reduced the recruitment of Parkin to mitochondria in oocytes. However, it may not be possible to directly link this observation to the improvements in IVM oocyte quality, since PINK/Parkin assessments were performed in oocytes from cultured follicles treated with CNP for 6 days.

This weakness has not been addressed in the revised manuscript.

6. The gold standard assay for oocyte quality is embryo transfer and live birth. The authors assessed the impact of maturing oocytes in vitro in the presence of CNP on oocyte quality by less robust assays (e.g., preimplantation embryo development in vitro), so the impact on oocyte quality is less certain.

This weakness has not been addressed in the revised manuscript.

7. The numbers of embryos should have been corrected for the number of eggs fertilized as a starting point so that the percentage that developed to each stage could be expressed as a percentage of successfully fertilized eggs rather than overall percentages. As currently shown in the Figures and described in the Legend, there is no information regarding what the percentage on the y-axis means. For example, does Figure 4B show the number of 2C embryos divided by the number of eggs inseminated? Or is it divided by the number of successfully fertilized eggs, and if so, how was that assessed?

There is no additional information provided in the revised manuscript to address these concerns.

8. When fewer eggs are fertilized, the numbers of embryos per group are lower and so the impact of culturing multiple embryos together is lost. As a result, it is possible that culture conditions rather than oocyte quality drove the differences in the numbers of embryos that achieved each stage of development.

This concern has not been addressed in the revised manuscript. Similar numbers of oocytes were cultured together, but not similar numbers of fertilized oocytes, or embryos.

9. Not all claims in the Discussion are supported by the evidence provided. For example, "In addition, the findings demonstrated that CNP improved cytoplasmic maturation events by maintaining normal CG, ER and Golgi apparatus distribution and function in aged oocytes" but it was never demonstrated that the altered distribution had any functional impact.

This concern has not been addressed in the revised manuscript.

10. Incompleteness and errors in the Methods section reduce confidence in many of the results reported.

This concern has not been addressed in the revised manuscript.

11. The methods used for Statistical Analysis are never explained in either the Methods or the Figure legends. It is unclear whether appropriate analyses were done, and it is frequently unclear what was the sample size and how many times a particular experiment was repeated. These weaknesses detract from confidence in the data.

This concern has not been addressed adequately in the revised manuscript.

Reviewer #1 (Public Review):

This manuscript describes a series of experiments documenting trophic egg production in a species of harvester ant, Pogonomyrmex rugosus. In brief, queens are the primary trophic egg producers, there is seasonality and periodicity to trophic egg production, trophic eggs differ in many basic dimensions and contents relative to reproductive eggs, and diets supplemented with trophic eggs had an effect on the queen/worker ratio produced (increasing worker production).

The manuscript is very well prepared and the methods are sufficient. The outcomes are interesting and help fill gaps in knowledge, both on ants as well as insects, more generally. More context could enrich the study and flow could be improved.

Reviewer #1 (Public Review):

The authors report a study, where they have sequenced whole genomes of four individuals of an extinct species of butterfly from western North America (Glaucopsyche xerces), along with seven genomes of a closely related species (Glaucopsyche lygdamus), mainly from museum specimens, several to many decades old. They then compare these fragmented genomes to a high-quality, chromosome-level assembly of a genome of a European species in the same genus (Glaucopsyche alexis). They find that the extinct species shows clear signs of declining population sizes since the last glacial period and an increase in inbreeding, perhaps exacerbating the low viability of the populations and contributing to the extinction of the species.

The study really highlights how museum specimens can be used to understand the genetic variability of populations and species in the past, up to a century or more ago. This is an incredibly valuable tool, and can potentially help us to quickly identify whether current populations of rare and declining species are in danger due to inbreeding, or whether at least their genetic integrity is in good condition and other factors need to be prioritised in their conservation. In the case of extinct species, sequencing museum specimens is really our only window into the dynamics of genomic variability prior to extinction, and such information can help us understand how genetic variation is related to extinction.

I think the authors have achieved their goal admirably, they have used a careful approach to mapping their genomic reads to a related species with a high-quality genome assembly. They might miss out on some interesting genetic information in the unmapped reads, but by and large, they have captured the essential information on genetic variability within their mapped reads. Their conclusions on the lower genetic variability in the extinct species are sound, and they convincingly show that Glaucopyche xerces is a separate species to Glaucopsyche lygdamus (this has been debated in the past).

Reviewer #1 (Public Review):

First, I agree with the authors of this manuscript that conformational changes in the XFEL structures with 2.8 A resolution are not reliable enough for demonstrating the subtle changes in the electron transfer events in this bacterial photosynthesis system. Actually, the data statistics in the paper by Dods et al. showed that the high-resolution range of some of the XFEL datasets may include pretty high noise (low CC1/2 and high Rsplit) so the comparison of the subtle conformational changes of the structures is problematic.

The manuscript by Gai Nishikawa investigated time-dependent changes in the energetics of the electron transfer pathway based on the structures by Dods et al. by calculating redox potential of the active and inactive branches in the structures and found no clear link between the time-dependent structural changes and the electron transfer events in the XFEL structures published by Dods, R.et al. (2021). This study provided validation for the interpretation of the structures of those electron-transferring proteins.

The paper was well prepared.

Comments on latest version:

The revisions the authors have made have improved the manuscript.

Reviewer #1 (Public Review):

Summary: This paper performs fine-mapping of the silkworm mutants bd and its fertile allelic version, bdf, narrowing down the causal intervals to a small interval of a handful of genes. In this region, the gene orthologous to mamo is impaired by a large indel, and its function is later confirmed using expression profiling, RNAi, and CRISPR KO. All these experiments are convincingly showing that mamo is necessary for the suppression of melanic pigmentation in the silkworm larval integument.

The authors also use in silico and in vitro assays to probe the potential effector genes that mamo may regulate.

Strengths: The genotype-to-phenotype workflow, combining forward (mapping) and reverse genetics (RNAi and CRISPR loss-of-function assays) linking mamo to pigmentation are extremely convincing.

Weaknesses:

1) The last section of the results, entitled "Downstream target gene analysis" is primarily based on in silico genome-wide binding motif predictions.<br /> While the authors identify a potential binding site using EMSA, it is unclear how much this general approach over-predicted potential targets. While I think this work is interesting, its potential caveats are not mentioned. In fact the Discussion section seems to trust the high number of target genes as a reliable result. Specifically, the authors correctly say: "even if there are some transcription factor-binding sites in a gene, the gene is not necessarily regulated by these factors in a specific tissue and period", but then propose a biological explanation that not all binding sites are relevant to expression control. This makes a radical short-cut that predicted binding sites are actual in vivo binding sites. This may not be true, as I'd expect that only a subset of binding motifs predicted by Positional Weight Matrices (PWM) are real in vivo binding sites with a ChIP-seq or Cut-and-Run signal. This is particularly problematic for PWM that feature only 5-nt signature motifs, as inferred here for mamo-S and mamo-L, simply because we can expect many predicted sites by chance.

2) The last part of the current discussion ("Notably, the industrial melanism event, in a short period of several decades ... a more advanced self-regulation program") is flawed with important logical shortcuts that assign "agency" to the evolutionary process. For instance, this section conveys the idea that phenotypically relevant mutations may not be random. I believe some of this is due to translation issues in English, as I understand that the authors want to express the idea that some parts of the genome are paths of least resistance for evolutionary change (e.g. the regulatory regions of developmental regulators are likely to articulate morphological change). But the language and tone is made worst by the mention that in another system, a mechanism involving photoreception drives adaptive plasticity, making it sound like the authors want to make a Lamarckian argument here (inheritance of acquired characteristics), or a point about orthogenesis (e.g. the idea that the environment may guide non-random mutations).<br /> Because this last part of the current discussion suffers from confused statements on modes and tempo of regulatory evolution and is rather out of topic, I would suggest removing it.

In any case, it is important to highlight here that while this manuscript is an excellent genotype-to-phenotype study, it has very few comparative insights on the evolutionary process. The finding that mamo is a pattern or pigment regulatory factor is interesting and will deserve many more studies to decipher the full evolutionary study behind this Gene Regulatory Network.

Minor Comment :

The gene models presented in Figure 1 are obsolete, as there are more recent annotations of the Bm-mamo gene that feature more complete intron-exon structures, including for the neighboring genes in the bd/bdf intervals. It remains true that the mamo locus encodes two protein isoforms.<br /> An example of the Bm-mamo locus annotation, can be found at : https://www.ncbi.nlm.nih.gov/gene/101738295<br /> RNAseq expression tracks (including from larval epidermis) can be displayed in the embedded genome browser from the link above using the "Configure Tracks" tool.

Based on these more recent annotations, I would say that most of the work on the two isoforms remains valid, but FigS2, and particularly Fig.S2C, need to be revised.

Reviewer #1 (Public Review):

This manuscript presents an important study that contributes to our understanding of the reliability of ancient environmental DNA (aeDNA) extracted from sediment cores. The authors address the potential biases and challenges associated with using aeDNA to infer past ecosystems, specifically focusing on the case of mammoths and woolly rhinoceroses in the Yamal peninsula, West Siberia.

The introduction provides an overview of the significance of sedimentary deposits as archives of past ecosystem changes and illustrates the remarkable insights gained from previous studies using aeDNA, highlighting its potential for reconstructing paleoecology, phylogeography, and understanding extirpation and extinction events of keystone taxa. The authors then report the detection of DNA and near complete mitochondrial genomes of multiple mammoth and woolly rhinoceros individuals in the sampled sediment cores (which are dated to the last few centuries). The authors then employed additional methods to confirm the presence of ancient DNA from mammoths in these sediment cores. Conventional PCR and Sanger sequencing of a mammoth COI fragment confirmed the amplification of mammoth DNA. Mammal metabarcoding and droplet digital PCR (ddPCR) further supported the detection of mammoth DNA in both cores.

The hybridisation enrichment experiment results showed high read counts assigned to Mammuthus, ranging from 2,852 to 72,919 reads per library in core LK-001. Negative controls did not produce any reads assigned to mammals, indicating the absence of contamination. The study also revealed the presence of woolly rhinoceros sequences in the sediment cores, with 12 out of 23 libraries producing more than 100 reads assigned to woolly rhinoceros. The total number of woolly rhinoceros reads was 2,737, and the cumulative mitogenome coverage reached 44%.

The authors carefully addressed the incongruity between the temporal occurrence of these extinct species and the presence of their DNA in recent sediments. They proposed several mechanisms that could explain the recovery of Pleistocene megafaunal DNA in the sediment cores. The minor amount of ancient DNA post mortem damage observed in the mammoth sequences indicates exceptional preservation, consistent with an origin from (recent) permafrost. The dynamics of permafrost thawing and redeposition in the study area provide a plausible explanation for the presence of ancient DNA in the sediments. The authors discuss potential mechanisms for the redistribution of Late Pleistocene material in the sediments, including thermo-denudation processes, methane emissions from degrading permafrost, and the formation of taliks and methane seepage. These processes can disturb the stratigraphy of lake sediments and potentially mix ancient material within the modern sediments. I believe the conclusions are supported by the data and the manuscript is well-written and clear to follow for the reader.

Reviewer #1 (Public Review):

This manuscript by Neininger-Castro and colleagues presents a novel automatic image analysis method for assessing sarcomeres, the basic units of myofibrils and validates this tool in a couple of experimental approaches that interfere with sarcomere assembly in iPSC-cardiomyocytes (iPSC-CM).

Automatic quantification of sarcomeres is definitely something that is useful to the field. I am surprised that there is no reference in the manuscript to SarcTrack, published by Toepfer and colleagues in 2019 (PMID 30700234), which has exactly the same purpose. The advantage of the image analysis software presented in the current manuscript appears to me to be that it can cover both mature sarcomeres and nascent sarcomeres in premyofibrils effectively.

Reviewer #1 (Public Review):

The authors use electrophysiological and behavioral measurements to examine how animals could reliably determine odor intensity/concentration across repeated experiences. Because stimulus repetition leads to short-term adaptation evidenced by reduced overall firing rates in the antennal lobe and firing rates are otherwise concentration-dependent, there could be an ambiguity in sensory coding between reduced concentration or more recent experience. This would have a negative impact on the animal's ability to generate adaptive behavioral responses that depend on odor intensities. The authors conclude that changes in concentration alter the constituent neurons contributing to the neural population response, whereas adaptation maintains the 'activated ensemble' but with scaled firing rates. This provides a neural coding account of the ability to distinguish odor concentrations even after extended experience. Additional analyses attempt to distinguish hypothesized circuit mechanisms for adaptation but are inconclusive. A larger point that runs through the manuscript is that overall spiking activity has an inconsistent relationship with behavior and that the structure of population activity may be the more appropriate feature to consider.

To my knowledge, the dissociation of effects of odor concentration and adaptation on olfactory system population codes was not previously demonstrated. This is a significant contribution that improves on any simple model based on overall spiking activity. The primary result is most strikingly supported by visualization of a principal components analysis in Figure 4. However, there are some weaknesses in the data and analyses that limit confidence in the overall conclusions.

1) Behavioral work interpreted to demonstrate discrimination of different odor concentrations yields inconsistent results. Only two of the four odorants follow the pattern that is emphasized in the text (Figure 1F). Though it's a priori unlikely that animals are incapable of distinguishing odor concentrations at any stage in adaptation, the evidence presented is not sufficient to reach this conclusion.<br /> 2) While conclusions center on concepts related to the combination of activated neurons or the "active ensemble", this specific level of description is not directly demonstrated in any part of the results. We see individual neural responses and dimensional reduction analyses, but we are unable to assess to what extent the activated ensemble is maintained across experience.<br /> 3) There is little information about the variance or statistical strength of results described at the population level. While the PCA presents a compelling picture, the central point that concentration changes and adaptation alter population responses across separable dimensions is not demonstrated quantitatively. The correlation analysis that might partially address this question is presented to be visually interpreted with no additional testing.<br /> 4) Results are often presented separately for each odor stimulus or for separate datasets including two odor stimuli. An effort should be made to characterize patterns of results across all odor stimuli and their statistical reliability. This concern arises throughout all data presentations.<br /> 5) The relevance of the inconclusive analysis of inferred adaptation mechanisms in Figure 2d-f and the single experiment including a complex mixture in Figure 7 to the motivating questions for this study are unclear.<br /> 6) Throughout the description of the results, typical standards for statistical reporting (sample size, error bars, etc.) are not followed. This prevents readers from assessing effect sizes and undermines the ability to assign a confidence to any particular conclusion.

DOI: 10.1371/journal.pbio.3002223

Resource: RRID:ZFIN_ZDB-ALT-160830-1

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-160830-1

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