Reviewer #3 (Public Review):

D-type retroviruses, which include M-PMV assemble in the cytosol, however, do not efficiently start their maturation before membrane binding. There is very little known about the structural changes leading to maturation of D-type retroviruses and this manuscript presents compelling structural changes of the M-PMV matrix domain in mutations abrogating the myristol exposure or mutation which reasonably argue that myristol group is exposed (The relationship between these mutants and myristol exposure is argued based on structure of the matrix and liposome binding, however is not directly shown in structure). Assuming that the authors are correct about their mutations affect on myristol exposure, they have measured very interesting M-PMV matrix domain conformational changes which exposes the MAPP site to the protease.

Oligomerization of the matrix is probed by formation of disulfide bridges in a matrix mutant on liposomes with engineered cystine where authors suspect monomers of the matrix would be touching each other. The oligomerization data is very weak, does not directly support trimer formation and since 2D diffusion on liposomes would increase matrix-matrix interactions, can be non-specific, a point supported by presence of a stronger dimer band than trimer and tetramer. The main issue with the manuscript is that the authors do not show any evidence that the proposed mechanism actually works in the context of full M-PMV assembled particles.

Reviewer #3 (Public Review):

The study by Li et al investigates the role of type I interferon in suppressing ADAM17-mediated release of EGFR, the pathway previously implicated by this group in photosensitive skin reactions. Understanding the relevance of lupus murine models to the human disease is very important and the studies address this important gap in the knowledge. The most significant findings are: 1) the same high IFN and low Langerhans cell (LC) signatures seen in a lupus patient's skin, exist in the non-lesional skin of lupus mouse models; 2) IFN-Is and IFN-I signaling suppress ADAM17 activity in LCs in vitro and in vivo; 3) Blocking IFN-I signaling ameliorates photosensitive reactions, in an EGFR-dependent manner. These three conclusions are largely supported by the presented evidence but could be distilled as well as strengthened by additional data.

One of the strengths of the study is that the authors defined the relevance of lupus skin mouse models to human disease in the context of the Interferon-LC axis. The extensive computational approaches represent useful tools to compare cellular and molecular signatures across samples as well as species. This is highly relevant to the studies of lupus, a highly complex disease, for which the relevance of murine models has remained undefined. Major strengths related to the Aims of the study are that the authors established a role of interferon in suppressing Adam17 activity in the skin and showed that blocking interferon can reduce sunlight-induced skin inflammation in the lupus murine models. Interestingly, the authors observed that blocking IFN signaling in the absence of a high IFN-signature worsened sunlight-induced skin injury. The specificity of Adam17 in LCs for TNFR1 shedding provides an elegant approach to probing Adam17 activity in these cells.

While the three conclusions stated above are largely supported by the presented evidence, the data supporting a direct role of ADAM17 in IFN-triggered photosensitive reactions could be strengthened. Some of the concerns are outlined below:<br /> (1) Computational analyses in Figures 1 and 2 emphasize the co-occurrence of a high IFN-I signature and a low LC and/or DC signatures. It is not clear if the downregulation of the DC gene set indicates diminished presence of LCs in the non-lesional skin of the lupus mouse models or "reflects decreased LC function" as the authors suggest.

(2) Given the hypothesis that IFN-I may be the cause of a decreased DC signature in the mouse skin, it would be relevant to ask if this signature is also decreased in the IMQ model, which is a known model of IFN-induction as confirmed by the authors. Likewise, asking how anti-IFNAR treatment affects the DC signature / LC numbers would be important, in the absence and presence of UV. The authors indicate in Fig. 5I that IMQ reduces LC numbers.

(3) Decreased inflammation in LCad17 mice in the IMQ+UV model is unexpected. Previous studies by this group showed increased UV-induced inflammation in the absence of LC-ADAM17 (Shipman et al 2018). Therefore, it is not surprising that anti-IFNAR did not have an impact in these mice as ADAM17 deficiency appears to have normalized the response. These results are not addressed in the context of the previously published findings.

(4) Including the data that demonstrate the specificity of LCs for Adam17 expression in the epidermis and shedding of TNFR1 as a readout of LC-ADAM17-specific activity in the main figures would be important.

(5) UV light is an important inducer of IFN. Authors have previously shown that UV also induces Adam17 expression. Therefore, the question remains whether a high baseline IFN signature in lupus skin suppresses UV-induced Adam17 expression?

(6) A direct mechanistic link between high IFN-I and loss of Adam17 activity driving photosensitive reactions could be strengthened. Would blocking Adam17 with a blocking antibody suppress photosensitive reactions in lupus mouse models? Would treating LCAd17 mice with IFN fail to enhance or diminish UV-induced inflammation?

Reviewer #3 (Public Review):

This study used novel ingestible pills to measure pH and other gastric signals, and related these measures to self-report ratings of emotions induced by video clips. The main finding was that when participants viewed videos of disgust, there was an association between gastric pH and feelings of disgust and fear, and (in the opposite direction) happiness. These findings may be the first to relate objective measures of gastric physiology to emotional experience. The methods open up many new questions that can be addressed by future studies and are thus likely to have an impact on the field.

My main concern is with the reliability of the results. The study associates many measures (pH, temperature, pressure, EGG) in stomach, small bowel, and large bowel with multiple emotion ratings. This amounts to many statistical tests. Only one of these measures (pH in the stomach) shows a significant effect. Furthermore, the key findings, as displayed in Figure 4 do not look particularly convincing. Perhaps this is a display issue, but the relations between stomach pH and Vas ratings of disgust, fear, and happiness were not apparent from the scatter plot and may be influenced by outliers (e.g., happiness).

Reviewer #3 (Public Review):

In this manuscript, the authors seek to shed light on the role of awake hippocampal replay during memory tasks that are claimed to be short-term memory. For this, they make use of a real-time detection and disruption system of awake hippocampal ripples, which are used as a proxy for awake neuronal replay. The manuscript describes extensively the tasks as well as the disruption system and controls used during the experiments. The authors present numerous and solid analyses of the behavioral data acquired during the tasks. Nonetheless, the current version of the manuscript is lacking a more complete discussion in which the results are contrasted to previous similar findings, as well as mentioning the role of the awake ripple in the stabilization of hippocampal maps. Some extra analyses are also suggested below. The manuscript would also be enriched if the authors suggested alternative mechanisms for memory rehearsal. Finally, some claims of "we are first" seem inappropriate when compared to the previous literature.

Major comments:

How does one define short-term memory (STM) in rodents? The examples and papers cited in the first paragraphs refer mostly to human working memory tasks, from which it is known that a non-rehearsed STM lasts typically 20-30 seconds. Could the authors mention how this concept is translated to rodents? Could you clarify until what point memory is considered STM and what is the criteria to consider it has turned into long-term memory or when is it simply working memory or habit/skill? Further, why should these tasks be classified as testing STM while Jadhav et al. tasks are working memory or as they now mention in this article rule learning? In humans, the retention of memory after a certain time is achieved by retrieving a long-term memory. How do we know if the considerable training the rats received has not allowed the use of a long-term memory strategy which allows the rats to perform well even in the absence of rehearsal (replay)? These are conceptual explanations that would help understand the key concept of STM in greater detail.

Further, claims of "first" should be adjusted, since I do not see a large difference between the w (m) maze of Jadhav and these tasks. The main difference between the two projects would rather be that Jadhav tests when animals are still newer to the task while here overtrained animals are used. In Jadhav, it's unlikely that just rule learning is affected since the inbound component is not affected by disruption, which also tests rule learning. Therefore, it is still likely that the effect seen in Jadhav et al is a deficit in working memory/short-term memory. And here it is more likely, that no effect was seen since with overtrained animals other strategies (cortical, striatal, etc) were used. The authors should compare in more detail how overtrained animals were in these different projects as well as in the articles they cite for replay analysis.

The main conclusion of the authors is that hippocampal replay is not the rehearsal mechanism expected in STM given that its disruption doesn't lead to behavioral changes. Could the authors hypothesize in their discussion what other neural mechanisms different from hippocampal replay may be involved in this rehearsal? The discussion also lacks closure with respect to how the findings fit in the study of STM in human memory. This would make the article more interesting to a larger audience and highlight its translational aspect.

The results describe deeply the behavioral performance of the rats and the validation of the ripple detection/disruption system. However, one important aspect missing is how the hippocampal activity and its encoding of space may be affected by the awake ripple disruption. The authors don't cite the work by Roux et al., Nature Neuroscience. 2017 where optogenetic stimulation of hippocampal neurons provided evidence that neuronal activity associated with awake hippocampal ripples during goal-directed behavior is required for both stabilizing and refining hippocampal place fields, while memory performance was not affected during ripple-locked stimulations compared to a ripple-delayed stimulation control (See supplementary Figure 7 of the mentioned article). I would like the authors to comment on their own findings and contrast them with those of Roux et al.

Line 64: Could the authors clarify what they mean by "indirect" causal evidence when discussing the contribution of papers by Jadhav, Igata, and Fernandez? Is it the fact that rodents' learning speed changed instead of showing a complete absence of learning? Or is it the fact that the disruption/prolongation is done on the hippocampal ripple and not strictly in the replay sequence? I would also highlight this latter difference, given that the above-mentioned authors describe their methodological approaches in terms of ripples and not in terms of replay content. For example, the use of "replay" instead of "ripple" in Line 61 results in methodological inaccurate terms such as replay disruption and replay prolongation.

Despite its apparent lack of statistical significance, the reported mean ripple detection rate during the trial and non-trial periods tend to be always higher in the disruption condition of all tasks by observing the median of the boxplots in Figure 1J, Figure 2H, and Figure 3J. It is worth investigating this further using the same linear regression method as Girardeau et al. Journal of Neuroscience, 2014 which may reduce the variability and allow comparing slopes of a cumulative number of ripples over time. This may reveal a compensatory homeostatic-like increase in the rate of ripples during the disrupted sessions, which may suggest a need for the ripple/replay occurrence in spite of it not having an effect on the rats' performance during the task.

In line 425, the authors report a median relative delay of 52.9 of their disruption system. Such a value would indicate that only around 47% of the ripple is being blocked. Is there any data from the authors or others that could reassure the reader that the 52.9% of the ripple that "leaks" is not enough for the replay phenomenon to occur? Considering the findings of Fernandez-Ruiz et al. 2019 on large-duration ripples, could the authors report the relative delay for both short and long ripples (>100 ms) separately? Line 494: The authors define long ripples as (>120 ms) but this doesn't coincide with the 100ms threshold from Fernandez Ruiz et al. 2019.

The online ripple detector used filtered the traces in the 135-255 Hz range. This is a narrower frequency range compared to online detectors used by Jadhav et al. 2012 (100-400 Hz) and Fernandez-Ruiz et al. 2019 (80-300 Hz). What motivated the use of this narrow range? Would the omittance of ripples below 135 Hz have implications in the results? Could the authors add to the supplement a figure similar to Figure 4B (FDR vs TPR) using a wider frequency range similar to the authors above in the offline detection of ripples?

It is unclear what criterion was used to train the rats in the NMTS task. Line 216 specifies a learning criterion of 80% fully correct trials in one session for three days in a row, while the methods in line 852 mention an average performance below 50% for at least three days in a row.

In the methods section, it is not mentioned if there was a specific region in the cortex where the tetrode was placed (Line 908). The detections in this tetrode were used to mark events as "false positives". The authors should be careful in line 933 when they make the statement "ripples are not present in the cortex". There have been recent publications that challenge this affirmation. See Khodagholy, Science. 2017, Nitzan, Nature Comm. 2020.

Reviewer #3 (Public Review):

This is a potentially quite interesting paper that defines E2 and E3 genes in Drosophila that can impact the accumulation of the Q72-GFP protein in the fly eye. The authors then focus on the eff gene, showing which human homolog can rescue fly knockdown. They extend to skeletal muscle, from the htt protein, to show that eff by TMT mass spec decreases with age normally in the fly muscle and that there is a significant overlap of proteins that are disrupted with eff knockdown in young animals in muscle vs aged animals normally in muscle.

Overall these data suggest eff decrease with age may contribute to the increase in ubiquitinated proteins in muscle with age, and that upregulation of eff activity might be of interest to extending lifespan. Because eff function can be performed by a human homologue, the findings may also apply to human situations of aging.

These data are overall interesting and are of relevance for those interested in neurodegenerative disease and aging, although a number of points from the figures seem confusing and need more explanation or clarity.

Reviewer #3 (Public Review):

Summary: The paper aims at providing a neurocomputational account on how social perception translates in prosocial behaviors. Participants first completed a novel social perception task during fMRI scanning, in which were asked to judge the merit or need of people depicted in different situations. Second , a separate altruistic choice task was used to examine how the perception of merit and need influences the weights people place on themselves, others and fairness when deciding to provide help. Finally, a link between perception and action was drawn in those participants who completed both tasks.

Strengths: The paper is overall very well written and presented, leaving the reader at ease when describing complex methods and results. The approach used by the author is very compelling, as it combines computational modeling of behavior and neuroimaging data analyses. Despite not being able to comment on the computational model, I find the approach used (to disentangle sensitivity and biases, for merit and need) very well described and derived from previous theoretical work. Results are also clearly described and interpreted.

Weaknesses: in the social perception task, merit and need are evaluated by means of very different cues that rely on different cognitive processes (more abstract thinking for merit than need). Despite this limitation of the task, the authors were able to argue convincingly in the revised version about the solidity of their findings. Sample size is quite small for study 2, nevertheless the results provide convincing evidence.

Reviewer #3 (Public Review):

Summary:

Amacrine cells are a heterogeneous collection of retinal interneurons. Most are inhibitory, and like inhibitory neurons in other neural circuits, strongly shape retinal function. With a few exceptions, the role of amacrine cells in retinal signaling is poorly understood. This paper introduces an approach to study a set of wide-field amacrine cells that extend processes over large regions of the retina.

Strengths:

A substantial strength of the paper is the combination of genetic manipulations, electrophysiology, optogenetics and electron microscopy used to study these cells. As a result of that broad set of techniques, the results cover many properties of how the cells work and provide a nice overview. The paper is also (with a few exceptions below) clearly presented and the experiments look to be carefully executed with clean results.

Weaknesses:

My largest concern with the paper is that overall the results provided an initial view of an interesting set of issues about the function of these cells, but the interesting initial results are not pursued in more depth.

Spatial spread of signals in neurites<br /> An immediate question about axonless WACs is the extent of spread of signals along their processes, and hence whether they act as a collection of independent or semi-independent elements. This bears directly on interpretation of the responses to oriented stimuli for example. Did you do any experiments that might provide additional information about this issue? For example, if you stimulate one of the WACs peripherally do you see a strong modulation of the somatic voltage? Or in the imaging experiments, if you mask a region of the processes so that it is not receiving a stimulus, do you see responses "leak" into that occluded region from surrounding stimulated regions?

Orientation tuning and connectivity<br /> The most developed functional results in the paper relate to the sensitivity of the WAC processes to oriented stimuli. Interpretation of these results depends on a few factors. First is the spread of signals in the WAC processes - as noted above. Second is connectivity. The paper shows that the B/K WAC activity increases inhibitory input to Off-delta and On-alpha ganglion cells. These cells, as noted in the paper, are not orientation tuned. But the orientation tuned ganglion cells stratify in a similar location within the IPL, and hence are situated in an appropriate place to receive input from the B/K WACs. Did you focus exclusively on connections to the Off-delta and On-alpha cells (along with the Off-alpha) or did you look at any other ganglion cell types? This should at least get discussed in more detail.

In several places it is unclear whether the paper intends to be a methods paper or a basic research paper. One example is the last sentence of the abstract. If it is intended to be a basic research paper (which is my overall impression) then I suggest shifting the emphasis in some of those key locations towards results and away from methods.

Reviewer #3 (Public Review):

Summary:<br /> In the current work, Howell et al studied the connectivity between the cortex and thalamus using DTI tractorgraphy per parcel to all voxels in the thalamus. Following they performed various dimensional reduction techniques to uncover how differences in connectivity to the thalamus vary across cortical parcels. Following they explore the spatial correlation of these variations with cortical myelin and functional organization, thalamic nuclei, gene expression derived core-matrix cell differentiation, and extend the model towards macaques. Overall, the authors find a differentiation between sensory and association areas in terms of the association with the thalamus, which reflects differences in cortical microstructure and function, and links to core-matrix differences and can be replicated in macaques.

Strengths:<br /> A clear strength of the current work is the combination of different models and approaches to study the link between the cortex and the thalamus. This approach nicely bridges different approaches to describe the role of the thalamus in cortical organisation using a diffusion-based approach. Especially the extension of the model to the macaque is quite nice.

Weaknesses:<br /> Potential weaknesses of the study are that it seems to largely integrate aspects of the thalamus that have been already described before. The differentiation between sensory and association systems across thalamic subregions is something that has been described before (see: Oldham and Ball, 2023; Zheng et al., 2023; Yang et al., 2020 Mueller, 2020; Behrens, 2003).

Appraisal:<br /> However, the aim of the study: 'to investigate the spatial extent of anatomical connectivity patterns within the thalamus in both humans and non-human primates and determine if such patterns differ between sensorimotor and association cortical areas' has been met.

Discussion:<br /> Overall, I think the study is a nice addition to the growing literature studying the anatomical connectivity between the thalamus and cortex and its functional implications.

Reviewer #3 (Public Review):

Summary:<br /> The authors proposed a normative principle for how the brain's internal estimate of an observed sensory variable should be updated during each individual observation. In particular, they propose that the update size should be inversely proportional to the variance of the variable. They then proposed a microcircuit model of how such an update can be implemented, in particularly incorporating two types of interneurons and their subtractive and divisive inhibition onto pyramidal neurons. One type should represent the estimated mean while another represents the estimated variance. The authors used simulations to show that the model works as expected.

Strengths:<br /> The paper addresses two important issues: how uncertainty is represented and used in the brain, and the role of inhibitory neurons in neural computation. The proposed circuit and learning rules are simple enough to be plausible. They also work well for the designated purposes. The paper is also well-written and easy to follow.

Weaknesses:<br /> I have concerns with two aspects of this work.

(1) The optimality analysis leading to Eq (1) appears simplistic. The learning setting the authors describe (estimating the mean of a stationary Gaussian variable from a stream of observations) is a very basic problem in online learning/streaming algorithm literature. In this setting, the real "optimal" estimate is simply the arithmetic average of all samples seen so far. This can be implemented in an online manner with \hat{\mu}_{t} = \hat{\mu}_{t-1} +(s_t-\hat{\mu}_{t-1})/t. This is optimal in the sense that the estimator is always the maximum likelihood estimator given the samples seen up to time t. On the other hand, doing gradient descent only converges towards the MLE estimator after a large number of updates. Another critique is that while Eq (1) assumes an estimator of the mean (\hat{mu}), it assumes that the variance is already known. However, in the actual model, the variance also needs to be estimated, and a more sophisticated analysis thus needs to take into account the uncertainty of the variance estimate and so on. Finally, the idea that the update should be inverse to the variance is connected to the well-established idea in neuroscience that more evidence should be integrated over when uncertainty is high. For example, in models of two-alternative forced choices it is known to be optimal to have a longer reaction time when the evidence is noisier.

(2) While the incorporation of different inhibitory cell types into the model is appreciated, it appears to me that the computation performed by the circuit is not novel. Essentially the model implements a running average of the mean and a running average of the variance, and gates updates to the mean with the inverse variance estimate. I am not sure about how much new insight the proposed model adds to our understanding of cortical microcircuits.

Reviewer #3 (Public Review):

Summary:

Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine the activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during the approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength of their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of learning and memory. The conclusions of this manuscript are mostly well supported by the data, but some additional analysis and/or experiments may be required to fully support the author's conclusions.

Weaknesses:

(1) During teleportation between familiar to novel environments the authors report a decrease in the freezing ratio when combining the mice in the two experimental groups (Figure 3aiii). A major conclusion from the manuscript is the difference in VTA and LC activity following environment change, given VTA and LC activity were recorded in separate groups of mice, did the authors observe a similar significant reduction in freezing ratio when analyzing the behavior in LC and VTA groups separately?

(2) The authors satisfactorily apply control analyses to account for the unequal axon numbers recorded in the LC and VTA groups (e.g. Figure 1). However, given the heterogeneity of responses observed in Figures 3c, 4b and the relatively low number of VTA axons recorded (compared to LC), there are some possible limitations to the author's conclusions. A conclusion that LC-CA1 axons, as a general principle, heighten their activity during novel environment presentation, would require this activity profile to be observed in some of the axons recorded in most all LC-CA1 mice. Additionally, if the general conclusion is that VTA-CA1 axons ramp activity during the approach to reward, it would be expected that this activity profile was recorded in the axons of most all VTA-CA1 mice. Can the authors include an analysis to demonstrate that each LC-CA1 mouse contained axons that were activated during novel environments and that each VTA-CA1 mouse contained axons that ramped during the approach to reward?

(3) A primary claim is that LC axons projecting to CA1 become activated during novel VR environment presentation. However, the experimental design did not control for the presentation of a familiar environment. As I understand, the presentation order of environments was always familiar, then novel. For this reason, it is unknown whether LC axons are responding to novel environments or environmental change. Did the authors re-present the familiar environment after the novel environment while recording LC-CA1 activity?

Reviewer #3 (Public Review):

Summary:<br /> In the manuscript titled "Large pan-cancer cell screen coupled to (phospho-)proteomics underscores high-dose vitamin C (VitC) as a potent anti-cancer agent," the authors use a combination of proteomics and cell viability assays to understand the effect of Vitamin C on different solid tumor models in 51 different cancer cell lines. They found that many cancer cell lines are sensitive to high-dose Vitamin C, with IC50 values in the micromolar to millimolar range. Given that Vitamin C, when administered intravenously, can reach 20mM, this suggests that Vitamin C could provide some benefits to patients. The authors also generate and analyze bulk proteomic data for all 51 cell lines. They perform statistical analysis of these data to identify proteins that are up or downregulated in sensitive vs resistant cell lines in the same tumor and commonly across tumors. They then focus on PDAC cell lines and measure bulk and phosphoproteomics of PDAC cell lines 2, 4 and 24 hours after Vitamin C treatment.

Strengths: The strengths of the study are the rather large datasets accumulated on bulk proteomics of 51 different cancer cell lines. The IC50 values of these cell lines in response to Vitamin C is also useful.

Weaknesses:<br /> Though identifying targets to sensitize cancer cells to Vitamin C treatment is interesting, I felt the manuscript delved too much into listing off genes they found, with speculation on why the particular protein would be enriched in sensitive or resistant cell lines without testing any key claims experimentally.

Major Issues

(1) The overall premise of the study is that proteins that are enriched in Vitamin C-sensitive cell lines point to mechanisms of sensitivity and those enriched in Vitamin C resistant lines underlie mechanisms of resistance. Yet this is never directly tested. To show that the authors would need to knockdown/knockout a gene enriched in resistant lines and show this sensitizes cells to Vitamin C treatment or overexpress a protein associated with resistance and show that this leads to resistance in an otherwise sensitive cell line.

(2) One of the key strengths of this study is the large datasets generated, namely the proteomics data for 51 different cell lines. Yet the data is not included as a supplement or uploaded to a public repository.

Reviewer #3 (Public Review):

Summary:<br /> In this study, Brenes and colleagues carried out proteomic analysis of several human induced pluripotent (hiPSC) and human embryonic stem cell (hESC) lines. The authors found quantitative differences in the expression of several groups of cytoplasmic and mitochondrial proteins. Overall, hiPSC expressed higher levels of proteins such as glutamine transporters, mitochondrial metabolism proteins, and proteins related to lipid synthesis. Based on the protein expression differences, the authors propose that hiPSC lines differ from hESC in their growth and metabolism.

Strengths:<br /> The number of generated hiPSC and hESC lines continues to grow, but potential differences between hiPSC and hESC lines remain to be quantified and explained. This study is a promising step forward in understanding of the differences between different hiPSC and hESC lines.

Weaknesses:<br /> It is unclear whether changes in protein levels relate to any phenotypic features of cell lines used. For example, the authors highlight that increased protein expression in hiPSC lines is consistent with the requirement to sustain high growth rates, but there is no data to demonstrate whether hiPSC lines used indeed have higher growth rates.

The authors claim that the cell cycle of the lines is unchanged. However, no details of the method for assessing the cell cycle were included so it is difficult to appreciate if this assessment was appropriately carried out and controlled for.

Details and characterisation of iPSC and ESC lines used in this study were overall lacking. The lines used are merely listed in methods, but no references are included for published lines, how lines were obtained, what passage they were used at, their karyotype status, etc. For details of basic characterisation, the authors should refer to the ISSC Standards for the use of human stem cells in research. In particular, the authors should consider whether any of the changes they see may be attributed to copy number variants in different lines.

The expression data for markers of undifferentiated state in Figure 1a would ideally be shown by immunocytochemistry or flow cytometry as it is impossible to tell whether cultures are heterogeneous for marker expression.

TEM analysis should ideally be quantified.

All figure legends should explicitly state what graphs are representing (e.g. average/mean; how many replicates (biological or technical), which lines)? Some data is included in Methods (e.g. glutamine uptake), but not for all of the data (e.g. TEM).

Validation experiments were performed typically on one or two cell lines, but the lines used were not consistent (e.g. wibj_2 versus H1 for respirometry and wibj_2, oaqd_3 versus SA121 and SA181 for glutamine uptake). Can the authors explain how the lines were chosen?

The authors should acknowledge the need for further functional validation of the results related to immunosuppressive proteins.

Differences in H1 histone abundance were highlighted. Can the authors speculate as to the meaning of these differences?

Reviewer #3 (Public Review):

This reviewer appreciates the responses to previous notes. The authors attempted to address concerns mostly in writing, avoiding performing some of the experiments suggested in my previous review. Although some of the points were clarified, and the revised manuscript presents valuable insights into the implications of YBR238C and RMD9 on cellular function and yeast aging, my major concern still needs to be addressed. The gene expression signature significantly changes under different metabolic conditions. The media condition under which samples are collected for RNAseq analyses should match the media condition under which the lifespans of those KO strains are tested. This is the major confounding effect, and the conclusions are not informative based on the analysis done in this study.

To avoid experiments, the authors responded that yeast culture results in low optical density and does not reach the stationary phase under rapamycin treatment conditions; however, the simple solution is to grow the yeast cells until they reach the stationary phase and then rapamycin treatment can be done for certain hours - collect the cells for transcriptomics analysis then it can be compared to the CLS gene set.

Another example is chromosome copy number alteration, which can be easily analyzed using transcriptome data, and it is an important aspect to understand whether observed expression changes are also affected by this alteration in YBR238C KO cells. However, the authors ignore this important point as well.

After all, this is an interesting study "limited by subfield" and will be of general interest in the yeast aging field, again considering the lack of homology of the genes of interest in higher eukaryotes.

Reviewer #3 (Public Review):

Summary:<br /> Kobayashi et al identify MER21C as a common promoter of GPR1-AS/Liz in Euarchontoglires, which establishes a somatic DMR that controls ZFDB2 imprinting. In mice, MER21C appears to have diverged significantly from its primate counterparts and is no longer annotated as such.

Strengths:<br /> The authors used high-quality cross-species RNA-seq data to characterise GPR1-AS-like transcripts, which included generating new data in five different species. The association between MER21C/B elements and the promoter of GPR1-AS in most species is clear and convincing. The expression pattern of MER21C/B elements overall further supports their role in enabling correct temporal expression of GPR1-AS during embryonic development.

Weaknesses:<br /> A deeper comparison of syntenic regions to the GPR1-AS promoter could be performed to provide a clearer picture of how the MER21C/B element evolved. The use of alternative TE annotation software may also be helpful. These analyses would be particularly useful to drive home the conclusion that the mouse (Liz) promoter is derived from the same insertion.

Reviewer #3 (Public Review):

Summary:<br /> The authors use several quantitative approaches to characterize the feeding ecologies of bohaiornithid enantiornithines, including allometric data, mechanical advantage and finite element analyses of the jaw, and morphometric analyses of the claws. The authors combine their results with data for other enantiornithines collected from the literature to shed new insight on the ecological evolution of Enantiornithes as a clade.

Although the authors have taken steps to improve their paper, I generally find improvements unsatisfying, especially regarding my comments.

My remaining concerns:

Teeth: My concern here is not whether having teeth limits available niche space compared to having a keratinous beak. Rather, my concern regards how exploitation of the same niche space might be differently reflected in parameter space between birds with teeth and birds with beaks. Can we reliably expect two species that both eat seeds to occupy the same parameter space if, for example, distribution of stress/strain is across a series of teeth vs. across a more uniform beak? In this manuscript, the authors are clearly making this assumption, but that assumption is not made explicit, let alone justified. The authors should discuss this.

Cranial kinesis: As with teeth, my concern here regards our ability to compare data between birds with and without a flexible beak to mitigate forces when foraging. I appreciate that the functional complexity of the kinetic neognath skull precludes our ability to account for it in analyses such as these, but when comparisons are made using these analyses *specifically among neognaths*, we can reliably assume that we are comparing like to like - that is, we can assume that both have kinetic skulls, and so kinesis is reflected similarly in the data for each bird. Similarly, even if a comparison between two neognaths focuses exclusively on the mandible - in which cranial kinesis is not directly reflected - we can assume that those mandibles serve as comparisons between functionally similar systems. However, we cannot necessarily make those same assumptions when comparing the kinetic skull of a neognath to the akinetic skull of an enantiornithine. Indeed, even when focusing just on the mandible, can we reliably assume that data collected from an akinetic enantiornithine reflect the same comparative context as data collected from kinetic neognaths? I appreciate that the authors added a call for better functional understanding of bird cranial kinesis - a call I enthusiastically endorse - but the authors should still discuss how that current lack of understanding impacts interpretations of the comparisons they draw.

Finally, I still find the discussion to be overly long and lacking clear focus and organization. I again urge the authors to minimally consider adding subheadings to better allow the reader to follow the flow of ideas, and I second Reviewer #1's suggestion to add a "Conclusions" section.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Coberski et al describes a combined experimental and computational study aimed to shed light on the catalytic mechanism in a methyltransferase that transfers a methyl group from S-adenosylmethionine (SAM) to a substrate adenosine to form N6-methyladenosine (m6A).

Strengths:<br /> The authors determine crystal structures in complex with so-called bi-substrate analogs that can bridge across the SAM and adenosine binding sites and mimic a transition state or intermediate of the methyl-transfer reaction. The crystal structures suggest dynamical motions of the substrate(s) that are examined further using classical MD simulations. The authors then use QM/MM calculations to study the methyl-transfer process. Together with biochemical assays of ligand/substrate binding and enzyme turnover, the authors use this information to suggest what the key steps are in the catalytic cycle. The manuscript is in most places easy to read.

Weaknesses:<br /> After revising the manuscript, there are few weaknesses beyond those listed in the paper.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors use inhibitors and mimetics of juvenile hormone (JH) to demonstrate that JH has a key role in late embryonic development in Thermobia, specifically in gut and eye development but also resorption of the extraembryonic fluid and hatching. They then exogenously apply JH early in development (when it is not normally present) to examine the biological effects of JH at these stages. This causes a plethora of defects including developmental arrest, deposition of chitin, limb development, and enhanced muscle differentiation. The authors interpret these early effects on development as JH being important for the shift from morphogenetic growth to differentiation - a role that they speculate may have facilitated the evolution of metamorphosis (hemi- and holo-metaboly). This paper will be of interest to insect evo-devo researchers, particularly those with interests in the evolution of metamorphosis.

Strengths:

The experiments are generally conducted very well with appropriate controls and the authors have included a very detailed analysis of the phenotypes.<br /> The manuscript significantly advances our understanding of Thermobia development and the role of JH in Thermobia development.<br /> The authors interpret this data to present some hypotheses regarding the role of JH in the evolution of metamorphosis, some aspects of which can be addressed by future studies.

Weaknesses:

The results are based on using inhibitors and mimetics of JH and there was no attempt to discern immediate effects of JH from downstream effects. The authors show, for instance, that the transcription of myoglianin is responsive to JH levels, it would have been interesting to see if any of the phenotypic effects are due to myoglianin upregulation/suppression (using RNAi for example). These kinds of experiments will be necessary to fully work out if and how the JH regulatory network has been co-opted into metamorphosis.

The results generally support the authors' conclusions. However, the discussion contains a lot of speculation and some far-reaching conclusions are made about the role of JH and how it became co-opted into controlling metamorphosis. There are some interesting hypotheses presented and the author's speculations are consistent with the data presented. However, it is difficult to make evolutionary inferences from a single data point as although Thermobia is a basally branching insect, the lineage giving rise to Thermobia diverged from the lineages giving rise to the holo- and hemimetabolous insects approx.. 400 mya and it is possible that the effects of JH seen in Thermobia reflect lineage-specific effects rather than the 'ancestral state'. The authors ignore the possibility that there has been substantial rewiring of the networks that are JH responsive across these 400 my. I would encourage the authors to temper some of the discussion of these hypotheses and include some of the limitations of their inferences regarding the role of JH in the evolution of metamorphosis in their discussion.

Reviewer #3 (Public Review):

Summary:<br /> After the previous identification that the Streptococcus agalactiae MprF enzyme can synthesize also lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), besides the already known lysyl-phosphatidylglycerol (Lys-PG), the authors aim for the current manuscript was to investigate the molecular determinants of MprF lipid substrate specificity in a variety of bacterial species.

Strengths:<br /> - In general, the manuscript is well constructed and easy to follow, especially taking into account the multidisciplinary aspect of it (computational machine learning combined with lipid biology).<br /> -The added value of the Restricted Boltzmann machines (RBM) approach, in comparison to standard computational pairwise sequence statistics, becomes evident. This is exemplified by a successful, although not perfect, classification and categorization of MprF activity.<br /> - The MS analysis (monoisotopic mass, plus fragmentation pattern), convincingly shows the identification of a novel lipid species Lys-Glc2-DAG.

Weaknesses:<br /> -In many of the analyzed strains, the presence of the lipid species Lys-PG, Lys-Glc-DAG, and Lys-Glc2-DAG is correlated to the presence of the MprF enzyme(s), but one should keep in mind that a multitude of other membrane proteins are present that in theory could be involved in the synthesis as well. Therefore, there is no direct evidence that the MprF enzymes are linked to the synthesis of these lipid species. Although, it is unlikely that other enzymes are involved, this weakens the connection between the observed lipids and the type of MprF.<br /> -Related to this, in a few cases MprF activity is tested, but the manuscript does not contain any information on protein expression levels. Heterologous expression of membrane proteins is in general challenging and due to various reasons, proteins end up not being expressed at all. As an example, the absence of activity for the E. faecalis MprF1 and E. faecium MprF2 could very well be explained by the entire absence of the protein.

Overall, the authors largely achieved their goals, as the applied RBM approach led to specific sequence determinants in MprF enzymes that could categorize the specificity of these enzymes. The experimental data could largely confirm this categorization, although a stronger connection between synthesized lipids and enzyme activity would have further strengthened the observations.

The work now focuses only on MprF enzymes, but could in theory be expanded to other categories of lipid-synthesizing enzymes. In other words, the RBM approach could have an impact on the lipid synthesis field, if it would be a tool that is easily applicable. Moreover, the lipids synthesized by MprF (Lys-PG, but also other cationic lipids) play an important role in bacterial resistance against certain antibiotics.

Reviewer #3 (Public Review):

The revised study provides better evidence to suggest that loss of Aprt activity in Drosophila provides a model for the loss of HGPRT activity in humans, which is causative for LND. Analysis of Drosophila Aprt mutations and RNAi-mediated knockdown reveals similar phenotypes to LND, particularly neurological defects, reduced nighttime sleep, and potentially seizures. LND is currently resistant to treatments and screening of a limited number of compounds in Drosophila has not identified a compound that can reduce all of the associated phenotypes. It is appropriate, therefore, that claims to have developed a clinically exploitable model for human LND have been toned down. Future drug screening may well prove profitable, but currently the evidence that Drosophila Aprt will be a suitable model for LND remains speculative.

The second approach adopted is to express a 'humanised mutated' form of HGPRT in Drosophila, which holds more promise for the development of a pharmacological screen. In particular, the locomotor defect is recapitulated but the seizure-like activity, whilst reported as being recapitulated, is debatable. A recovery time of 2.3 seconds is very much less than timings for typical seizure mutants. Nevertheless, the SING behaviour could be sufficient to screen against. However, this is not explored. With respect the short seizure duration, the authors cite similar findings for porin loss of function, but the cited study similarly did not employ anti-seizure drug exposure to validate that this phenotype is seizure related.

In summary, this is a largely descriptive study reporting the behavioural effects of an Aprt loss-of-function mutation. RNAi KD and rescue expression studies suggest that a mix of neuronal (particularly dopaminergic and possibly adenosinergic signalling pathways) and glia are involved in the behavioural phenotypes affecting locomotion, sleep and seizure. There remains insufficient evidence to have full confidence that the Arpt fly model will prove valuable for understanding / treating LND.

Reviewer #3 (Public Review):

Summary:<br /> This paper aims to investigate how the human brain represents different forms of value and uncertainty that participate in active inference within a free-energy framework, in a two-stage decision task involving contextual information sampling, and choices between safe and risky rewards, which promotes a shift from exploration to exploitation. They examine neural correlates by recording EEG and comparing activity in the first vs second half of trials and between trials in which subjects did and did not sample contextual information, and perform a regression with free-energy-related regressors against data "mapped to source space." Their results show effects in various regions, which they take to indicate that the brain does perform this task through the theorised active inference scheme.

Strengths:<br /> This is an interesting two-stage paradigm that incorporates several interesting processes of learning, exploration/exploitation, and information sampling. Although scalp/brain regions showing sensitivity to the active-inference-related quantities do not necessarily suggest what role they play, it can be illuminating and useful to search for such effects as candidates for further investigation. The aims are ambitious, and methodologically it is impressive to include extensive free-energy theory, behavioural modelling, and EEG source-level analysis in one paper.

Weaknesses:<br /> Though I could surmise the above general aims, I could not follow the important details of what quantities were being distinguished and sought in the EEG and why. Some of this is down to theoretical complexity - the dizzying array of constructs and terms with complex interrelationships, which may simply be part and parcel of free-energy-based theories of active inference - but much of it is down to missing or ambiguous details.

In general, an insufficient effort has been made to make the paper accessible to readers not steeped in the free energy principle and active inference. There are critical inconsistencies in key terminology; for example, the introduction states that aim 1 is to distinguish the EEG correlates of three different types of uncertainty: ambiguity, risk, and unexpected uncertainty. But the abstract instead highlights distinctions in EEG correlates between "uncertainty... and... risk" and between "expected free energy .. and ... uncertainty." There are also inconsistencies in mathematical labelling (e.g. in one place 'p(s|o)' and 'q(s)' swap their meanings from one sentence to the very next).

Some basic but important task information is missing, and makes a huge difference to how decision quantities can be decoded from EEG. For example:<br /> - How do the subjects press the left/right buttons - with different hands or different fingers on the same hand?<br /> - Was the presentation of the Stay/cue and safe/risky options on the left/right sides counterbalanced? If not, decisions can be formed well in advance especially once a policy is in place.<br /> - What were the actual reward distributions ("magnitude X with probability p, magnitude y with probability 1-p") in the risky option? 

The EEG analysis is not sufficiently detailed and motivated. For example,<br /> - why the high lower-filter cutoff of 1 Hz, and shouldn't it be acknowledged that this removes from the EEG any sustained, iteratively updated representation that evolves with learning across trials?<br /> - Since the EEG analysis was done using an array of free-energy-related variables in a regression, was multicollinearity checked between these variables?<br /> - In the initial comparison of the first/second half, why just 5 clusters of electrodes, and why these particular clusters? How many different variables are systematically different in the first vs second half, and how do you rule out less interesting time-on-task effects such as engagement or alertness? In what time windows are these amplitudes being measured? In the comparison of asked and not-asked trials, what trial stage and time window is being measured? Again, how many different variables, of the many estimated per trial in the active inference model, are different in the asked and not-asked trials, and how can you know which of these differences is the one reflected in the EEG effects? The authors choose to interpret that on not-asked trials the subjects are more uncertain because the cue doesn't give them the context, but you could equally argue that they don't ask because they are more certain of the possible hidden states.<br /> - The EEG regressors are not fully explained. For example, an "active learning" regressor is listed as one of the 4 at the beginning of section 3.3, but it is the first mention of this term in the paper and the term does not arise once in the methods.<br /> - In general, it is not clear how one can know that the EEG results reflect that the brain is purposefully encoding these very parameters while implementing this very mechanism, and not other, possibly simpler, factors that correlate with them since there is no engagement with such potential confounds or alternative models. For example, a model-free reinforcement learning model is fit to behaviour for comparison. Why not the EEG?

Reviewer #3 (Public Review):

Summary:<br /> This work uses multiscale molecular dynamics simulations to demonstrate molecular mechanism(s) for phosphatidylinositol regulation of voltage gated sodium channel (Nav1.4) gating. Recent experimental work by Gada et al. JGP 2023 showed altered Nav1.4 gating when Nav1.4 current was recorded with simultaneous application of PI(4,5)P2 dephosphorylate. Here the authors revealed probable molecular mechanism that can explain PI(4,5)P2 modulation of Nav1.4 gating. They found PIP lipids interacting with the gating charges - potentially making it harder to move the voltage sensor domain and altering the channels voltage sensitivity. They also found a stable PIP binding site that reaches the D_IV S4-S5 linker, reducing the mobility of the linker and potentially competing with the C-terminal domain.

Strengths:<br /> Using multiscale simulations with course-grained simulations to capture lipid-protein interactions and the overall protein lipid fingerprint and then all-atom simulations to verify atomistic details for specific lipid-protein interactions is extremely appropriate for the question at hand. Overall, the types of simulation and their length are suitable for the questions the authors pose and a thorough set of analysis was done which illustrates the observed PIP-protein interactions.

Weaknesses:<br /> Although the set of current simulations and analysis supports the conclusions drawn nicely, the course-grained simulations have further utility than that utilized by the authors. With the 4to1 heavy atoms bead mapping in Martini 2 some detailed chemical specificity is averaged out but parameters for different PIP family members do exist - including specific PIP(4,5)P2 vs PIP(3,4)P2, and could have been explored at the course-grained level. However, performing more detailed all-atom simulation, as done in this manuscript, is always advisable to extend and/or confirm course-grained results.

Reviewer #3 (Public Review):

Summary:<br /> The authors of this manuscript identified the fossils of the newly designated species Beretella spinosa and analyzed its phylogenetic position in relation to the extinct described species and extant species. Their analysis placed the newly described species Beretella spinosa and Saccorhytus as an independent clade from the rest of the ecdysozoans. Remarkably, these species are non-vermiform, and the resulting evolutionary scenario assumes non-vermiform as early ecdysozoans.

Strengths:<br /> The study presents outstanding, novel data and provides new insights into the evolution of animal forms especially regarding their morphological diversity after the Cambrian explosion.

Weaknesses:<br /> I, as a paleontology non-expert, experienced several difficulties in reading the manuscript. This should be taken into consideration when assuming a wide range of readers including non-experts.

Reviewer #3 (Public Review):

Summary and strengths:<br /> The manuscript, "Remodelling of skeletal muscle myosin metabolic states in hibernating mammals", by Lewis et al, investigates whether myosin ATP activity may differ between states of hibernation and activity in both large and small mammals. The study interrogates (primarily) permeabilized muscle strips or myofibrils using several state-of-the-art assays, including the mant-ATP assay to investigate ATP utilization of myosin, X-ray diffraction of muscles, proteomics studies, metabolic tests, and computational simulations. The overall data suggests that ATP utilization of myosin during hibernation is different than in active conditions.

A clear strength of this study is the use of multiple animals that utilize two different states of hibernation or torpor. Two large animal hibernators (Eurasian Brown Bear, American Black Bear) represent large animal hibernators that typically undergo prolonged hibernation. Two small animal hibernators (Garden Dormouse, 13 Lined Ground Squirrel) undergo torpor with more substantial reductions in heart rate and body temperature, but whose torpor bouts are interrupted by short arousals that bring the animals back to near-summer-like metabolic conditions.

Especially interesting, the investigators analyze the impact that body temperature may have on myosin ATP utilization by performing assays at two different temperatures (8 and 20 degrees C, in 13 Lined Ground Squirrels).

The multiple assays utilized provide a more comprehensive set of methods with which to test their hypothesis that muscle myosins change their metabolic efficiency during hibernation.

Suggestions and potential weaknesses:<br /> While the samples and assays provide a robust and comprehensive coverage of metabolic needs and testing, the data is less categorical. Some of these may be dependent on sample size or statistical analysis while others may be dependent on interpretation.

(1) Statistical Analysis<br /> (1.a) The results of this study often cannot be assessed properly due to a lack of clarity in the statistical tests.<br /> For example, the results related to the large animal hibernators (Figure 1) do not describe the statistical test (in the text of the results, methods, or figure legends). (Similarly for figure 6 and Supplemental Figure 1). Further, it is not clear whether or when the analysis was performed with paired samples. As the methods described, it appears that the Eurasian Brown Bear data should be paired per animal.

(1.b) The statistical methods state that non-parametric testing was utilized "where data was unevenly distributed". Please clarify when this was used.

(1.c) While there are two different myosin isoforms, the isoform may be considered a factor. It is unclear why a one-way ANOVA is generally used for most of the mant-ATP chase data.

(1.d) While the technical replicates on studies such as the mant-ATP chase assay are well done, the total biological replicates are small. A consideration of the sample power should be included.

(1.e) An analysis of the biological vs statistical significance should be considered, especially for the mant-ATP chase data from the American Black Bear, where there appear to be shifts between the summer and winter data.

(2) Consistency of DRX/SRX data.<br /> (2.a) The investigators performed both mant-ATP chase and x-ray diffraction studies to investigate whether myosin heads are in an "on" or "off" state. The results of these two studies do not appear to be fully consistent with each other, which should not be a surprise. The recent work of Mohran et al (PMID 38103642) suggests that the mant-ATP-predicted SRX:DRX proportions are inconsistent with the position of the myosin heads. The discussion appears to lack a detailed assessment of this prior work and lack a substantive assessment contrasting the differing results of the two assays in the current study. i.e. why the current study's mant-ATP chase and x-ray diffraction results differ.

(2.b) The discussion of the current study's x-ray diffraction data relating to the I_1,1/I_1,0 ratio and how substantially different this is to the M6 results merits discussion. i.e. how can myosin both be more primed to contract during IBA versus torpor (according to intensity ratio), but also have less mass near the thick filament (M6).

(3) Possible interactions with Heat Shock Proteins<br /> Heat Shock Proteins (HSPs), such as HSP70, have been shown to be differential during torpor vs active states. A brief search of HSP and myosin reveals HPSs related to thick filament assembly and Heat Shock Cognate 70 interacting with myosin binding protein C. Especially given the author's discussion of protein stability and the potential interaction with myosin binding protein C and the SRX state, the limitation of not assessing HSPs should be discussed. (While HSP's relation to thick filament assembly might conceivably modify the interpretation of the M3 x-ray diffraction results, this reviewer acknowledges that possibility as a leap.)

Despite the above substantial concerns/weaknesses, this reviewer believes that this manuscript represents a valuable data set.

Other comments related to interpretation:<br /> (4) The authors briefly mention the study by Toepher et al [Ref 25] and that it utilizes cardiac muscles. There would benefit from increased discussion regarding the possible differences in energetics between cardiac and skeletal muscle in these states.

(5) The author's analysis of temperature is somewhat limited.<br /> (5.a) First, the authors use 20 degrees C (room temperature), not 37 degrees C, a more physiologic body temperature for large mammals. While it is true that limbs are likely at a lower temperature, 20 degrees C seems substantially outside of a normal range. Thus, temperature differences may have been minimized by the author's protocol.

(5.b) Second, the authors discuss the possibility of myosin contributing to non-shivering thermogenesis. The magnitude of this impact should be discussed. The suggestion of myosin ATP utilization also implies that there is some basal muscle tone (contraction), as the myosin ATPase utilizes ATP to release from actin, before binding and hydrolyzing again. Evidence of this tone should be discussed.

Reviewer #3 (Public Review):

Summary:<br /> Feng et al. test the hypothesis that human body size constrains the perception of object affordances, whereby only objects that are smaller than the body size will be perceived as useful and manipulable parts of the environment, whereas larger objects will be perceived as "less interesting components."

To test this idea, the study employs a multi-method approach consisting of three parts:

In the first part, human observers classify a set of 24 objects that vary systematically in size (e.g., ball, piano, airplane) based on 14 different affordances (e.g., sit, throw, grasp). Based on the average agreement of ratings across participants, the authors compute the similarity of affordance profiles between all object pairs. They report evidence for two homogenous object clusters that are separated based on their size with the boundary between clusters roughly coinciding with the average human body size. In follow-up experiments, the authors show that this boundary is larger/smaller in separate groups of participants who are instructed to imagine themselves as an elephant/cat.

In the second part, the authors ask different large language models (LLMs) to provide ratings for the same set of objects and affordances and conduct equivalent analyses on the obtained data. Some, but not all, of the models produce patterns of ratings that appear to show similar boundary effects, though less pronounced and at a different boundary size than in humans.

In the third part, the authors conduct an fMRI experiment. Human observers are presented with four different objects of different sizes and asked if these objects afford a small set of specific actions. Affordances are either congruent or incongruent with objects. Contrasting brain activity on incongruent trials against brain activity on congruent trials yields significant effects in regions within the ventral and dorsal visual stream, but only for small objects and not for large objects.

The authors interpret their findings as support for their hypothesis that human body size constrains object perception. They further conclude that this effect is cognitively penetrable, and only partly relies on sensorimotor interaction with the environment (and partly on linguistic abilities).

Strengths:<br /> The authors examine an interesting and relevant question and articulate a plausible (though somewhat underspecified) hypothesis that certainly seems worth testing. Providing more detailed insights into how object affordances shape perception would be highly desirable. Their method of analyzing similarity ratings between sets of objects seems useful and the multi-method approach is original and interesting.

Weaknesses:<br /> The study presents several shortcomings that clearly weaken the link between the obtained evidence and the drawn conclusions. Below I outline my concerns in no particular order:

(1) It is not entirely clear to me what the authors are proposing and to what extent the conducted work actually speaks to this. For example, in the introduction, the authors write that they seek to test if body size serves not merely as a reference for object manipulation but also "plays a pivotal role in shaping the representation of objects." This motivation seems rather vague motivation and it is not clear to me how it could be falsified.

Overall, the lack of theoretical precision makes it difficult to judge the appropriateness of the approaches and the persuasiveness of the obtained results. I would strongly suggest clarifying the theoretical rationale and explaining in more detail how the chosen experiments allow them to test falsifiable predictions.

(2) The authors used only a very small set of objects and affordances in their study and they do not describe in sufficient detail how these stimuli were selected. This renders the results rather exploratory and clearly limits their potential to discover general principles of human perception. Much larger sets of objects and affordances and explicit data-driven approaches for their selection would provide a more convincing approach and allow the authors to rule out that their results are just a consequence of the selected set of objects and actions.

(3) Relatedly, the authors could be more thorough in ruling out potential alternative explanations. Object size likely correlates with other variables that could shape human similarity judgments and the estimated boundary is quite broad (depending on the method, either between 80 and 150 cm or between 105 to 130 cm). More precise estimates of the boundary and more rigorous tests of alternative explanations would add a lot to strengthen the authors' interpretation.

(4) While I appreciate the manipulation of imagined body size, as a clever way to solidify the link between body size and affordance perception, I find it unfortunate that it is implemented in a between-subjects design, as this clearly leaves open the possibility of pre-existing differences between groups. I certainly disagree with the authors' statement that their findings suggest "a causal link between body size and affordance perception."

(5) The use of LLMs in the current study is not clearly motivated and I find it hard to understand what exactly the authors are trying to test through their inclusion. As it currently stands, I find it hard to discern how the presence of perceptual boundaries in LLMs could constitute evidence for affordance-based perception.

(6) Along the same lines, the fMRI study also provides little evidence to support the authors' claims. The use of congruency effects as a way of probing affordance perception is not well motivated. Importantly (and related to comment 2 above), the very small set of objects and affordances in this experiment heavily complicates any conclusions about object size being the crucial variable determining the occurrence of congruency effects.

Overall, I consider the main conclusions of the paper to be far beyond the reported data. Articulating a clearer theoretical framework with more specific hypotheses as well as conducting more principled analyses on more comprehensive data sets could help the authors obtain stronger tests of their ideas.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, the authors use the C. elegans system to explore how already-stressed neurons respond to additional mechanical stress. Exophers are large extracellular vesicles secreted by cells, which can contain protein aggregates and organelles. These can be a way of getting rid of cellular debris, but as they are endocytosed by other cells can also pass protein, lipid, and RNA to recipient cells. The authors find that when the uterus fills with eggs or otherwise expands, a nearby neuron (ALMR) is far more likely to secrete exophers. This paper highlights the importance of the mechanical environment in the behavior of neurons and may be relevant to the response of neurons exposed to traumatic injury.

Strengths:<br /> The paper has a logical flow and a compelling narrative supported by crisp and clear figures.

The evidence that egg accumulation leads to exopher production is strong. The authors use a variety of genetic and pharmacological methods to show that increasing pressure leads to more exopher production, and reducing pressure leads to lower exopher production. For example, egg-laying defective animals, which retain eggs in the uterus, produce many more exophers, and hyperactive egg-laying is accompanied by low exopher production. The authors even inject fluid into the uterus and observe the production of exophers.

Weaknesses:<br /> The main weakness of the paper is that it does not explore the molecular mechanism by which the mechanical signals are received or responded to by the neuron, but this could easily be the subject of a follow-up study.

I was intrigued by this paper, and have many questions. I list a few below, which could be addressed in this paper or which could be the subject of follow-up studies.

- Why do such a low percentage of ALMR neurons produce exophers (5-20%)? Does it have to do with the variability of the proteostress?<br /> - Why does the production of exophers lag the peak in progeny production by 24-48 hours? Especially when the injection method produces exophers right away?<br /> - As mentioned in the discussion, it would be interesting to know if PEZO-1/PIEZO is required for uterine stretching to activate exophergenesis. pezo-1 animals accumulate crushed oocytes in the uterus.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Mazeaud and colleagues pursued a small-scale screen of a targeted RNAi library to identify novel players involved in Zika (ZIKV) and dengue (DENV) virus replication. Loss-of-function of IGF2BP2 resulted in reduced titers for ZIKV of the Asian and African lineages in hepatic Huh7.5 cells, but not for either of the four DENV serotypes nor West Nile virus (WNV). The phenotype was further confirmed in two additional cell lines and using a ZIKV reporter virus. In addition, using immunoprecipitation assays the interaction between IGF2BP2 and ZIKV NS5 protein and RNA genome was detected. The work addressed the role of IGF2BP2 in the infected cell combining confocal microscopy imaging, and proteomic analysis. The approach indicated an altered distribution of IGF2BP2 in infected cells and changes in the protein interactome including disrupted association with partner mRNAs and modulation of the abundance of a specific set of protein partners in IGF2BP2 immunoprecipitated ribonucleoprotein (RNP) complexes. Finally, based on the changes in IGF2BP2 interactome and specifically the increment in the abundance of Atlastin 2, the biogenesis of ZIKV replication organelles (vRO) is investigated using a genetic system that allows virus replication-independent assembly of vRO. Electron microscopy showed that knockdown of IGF2BP2 expression reduced the number of cells with vRO.

Strengths:<br /> The role of IGF2BP2 as a proviral factor for ZIKV replication is novel.

The study follows a logical flow of experiments that altogether support the assembly of a specialized RNP complex containing IGF2BP2 and ZIKV NS5 and RNA genome.

Weaknesses:<br /> The statistical analysis should clearly indicate the number of biological replicates of experiments to support statistical significance.

The claim that IGF2BP2 knockdown impairs de novo viral organelle biogenesis and viral RNA synthesis is built upon data that show a reduction in RNA synthesis <0.5-fold as assessed using a reporter replicon, thus suggesting a limited impact of the knockdown on RNA replication.

Validation of IGF2BP2 partners that are modulated upon ZIKV infection (i.e. virus yield in knocked down cells) can be relevant especially for partners such as Atlastin 2, as the hypothesis of a role for IGF2BP2 RNP in vRO biogenesis is based on the observed increase in the abundance of Atlastin 2 in the RNP complex preciìtated from infected cells.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Rana and colleagues examined the effect of a "low impact" ampakine, an AMPA receptor allosteric modulator, on the voiding function of rats subjected to midline T9 spinal cord contusion injury. Previous studies have shown that the micturition reflex fully depends on AMPA glutaminergic signaling, and, that the glutaminergic circuits are reorganized after spinal cord injury. In chronic paraplegic rats, other circuits (no glutaminergic) become engage in the spinal reflex mechanism controlling micturition. The authors employed continuous flow cystometry and external urethral sphincter electromyography to assess bladder function and bladder-urethral sphincter coordination in naïve rats (control) and rats subjected to spinal cord injury (SCI). In the acute phase after SCI, rats exhibit larger voids with lower frequency than naïve rats. This study shows that CX1739 improves, in a dose-dependent manner, bladder function in rats with SCI. The interval between voids and the voided volume were reduced in rat with SCI when compared to controls. In summary, this is an interesting study that describes a potential treatment for patients with SCI.

Strengths:

The findings described in this manuscript are significant because neurogenic bladder predisposes patients with SCI to urinary tract infections, hydronephrosis and kidney failure. The manuscript is clearly written. The study is technically outstanding, and the conclusions are well justified by the data.

Weaknesses:

The study was conducted 5 days after spinal cord contusion when the bladder is underactive. In rats with chronic SCI, the bladder is overactive. Therefore, the therapeutic approach described here is expected to be effective only in the underactive bladder phase of SCI. The mechanism and site of action of CX1739 is not defined.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Dr. Chen et al. investigates the genes that are differentially methylated and associated with cartilage degeneration in hemophilia patients. The study demonstrates the functional mechanisms of the TNXB gene in chondrocytes and F8-/- mice. The authors first showed significant DNA methylation differences between hemophilic arthritis (HA) and osteoarthritis through genome-wide DNA methylation analysis. Subsequently, they showed a decreased expression of the differentially methylated TNXB gene in cartilage from HA patients and mice. By knocking down TNXB in vivo and in vitro, the results indicated that TNXB regulates extracellular matrix homeostasis and apoptosis by modulating p-AKT. The findings are novel and interesting, and the study presents valuable information in blood-induced arthritis research.

Strengths:<br /> The authors adopted a comprehensive approach by combining genome-wide DNA methylation analysis, in vivo and in vitro experiments using human and mouse samples to illustrate the molecular mechanisms involved in HA progression, which is crucial for developing targeted therapeutic strategies. The study identifies Tenascin XB (TNXB) as a central mediator in cartilage matrix degradation. It provides mechanistic insights into how TNXB influences cartilage matrix degradation by regulating the activation of AKT. It opens avenues for future research and potential therapeutic interventions using AKT agonists for cartilage protection in hemophilic arthropathy. The conclusions drawn from the study are clear and directly tied to the findings.

Weaknesses:<br /> (1) The study utilizes a small sample size (N=5 for both osteoarthritis and hemophilic arthropathy). A larger sample size would enhance the generalizability and statistical power of the findings.<br /> (2) The use of an animal model (F8-/- mouse) to investigate the role of TNXB may not fully capture the complexity of human hemophilic arthropathy. Differences in the biology between species may affect the translatability of the findings to human patients.<br /> (3) The study primarily focuses on TNXB as a central mediator, but it might overlook other potentially relevant factors contributing to cartilage degradation in hemophilic arthropathy. A more holistic exploration of genetic and molecular factors could provide a broader understanding of the condition.

Reviewer #3 (Public Review):

Summary:<br /> In the 'bCFS' paradigm, a monocular target gradually increases in contrast until it breaks interocular suppression by a rich monocular suppressor in the other eye. The present authors extend the bCFS paradigm by allowing the target to reduce back down in contrast until it becomes suppressed again. The main variable of interest is the contrast difference between breaking suppression and (re) entering suppression. The authors find this difference to be constant across a range of target types, even ones that differ substantially in the contrast at which they break interocular suppression (the variable conventionally measured in bCFS). They also measure how the difference changes as a function of other manipulations. Interpretation is in terms of the processing of unconscious visual content, as well as in terms of the mechanism of interocular suppression.

Strengths:<br /> Interpretation of bCFS findings is mired in controversy, and this is an ingenuous effort to move beyond the paradigm's exclusive focus on breaking suppression. The notion of using the contrast difference between breaking and entering suppression as an index of suppression depth is interesting.

Reviewer #3 (Public Review):

Summary: Richter et al. present a comprehensive anatomical analysis of the external sensory organs of the D. melanogaster larva. Extending on their previous study (Rist and Thum 2017) that analyzed the anatomy of the terminal organ, a major external taste organ of fruit fly larva, the authors examined the anatomy of the remaining head sensory organs - the dorsal organ, the ventral organ, and the labial organ-also described the sensory organs of the thoracic and abdominal segments. Using improved electron microscopy, the authors performed a three-dimensional anatomical analysis of the sensilla and adjacent ganglia to construct a complete structural and neuronal map of the external larval sensilla.

Strengths: Though the manuscript is lengthy, it is written clearly, and the presented data supports the conclusion. In addition to the classification and nomenclature of the different types of sensilla throughout the larval body, the wealth of data presented here will be valuable to the scientific community. The study offers fundamental anatomical insights, which will be helpful for future functional studies and to understand the sensory strategies of Drosophila larvae in response to the external environment. By analyzing different larval stages (L1 and L3), this work offers some insights into the developmental aspects of the larval sense organs and their corresponding sensory cells.

Weaknesses: There are no apparent weaknesses. The repetitiveness of some data and prior studies may be avoided for easy readability.

Reviewer #3 (Public Review):

Summary:<br /> The authors investigate the hypothesis that neurexins serve a crucial role as regulators of the synaptic strength and timing at the glycinergic synapse between neurons of the medial nucleus of the trapezoid body (MNTB) and the lateral superior olivary complex (LSO). It is worth mentioning that LSO neurons are an integration station of the auditory brainstem circuit displaying high reliability and temporal precision. These features are necessary for computing interaural cues to derive sound source location from comparing the intensities of sounds arriving at the two ears. In this context, the authors' findings build up according to the hypothesis first by displaying that neurexins were expressed in the MNTB at varying levels. They followed this up with the deletion of all neurexins in the MNTB through the employment of a triple knock-out (TKO). Using electrophysiological recordings in acute brainstem slices of these TKO mice, they gathered solid evidence for the role of neurexins in synaptic transmission at this glycinergic synapse primarily by ensuring tight coupling of Ca2+ channels and vesicular release sites. Additionally, the authors uncovered a connection between the deletion of neurexins and a higher number of glycinergic synapses in TKO mice, for which they provided evidence in the form of immunostainings and related it to electrophysiological data on spontaneous release. Consequently, this investigation expands our knowledge on the molecular regulation of synaptic transmission at glycinergic synapses, as well as on the auditory processing at the level of the brainstem.

Strengths:<br /> The authors demonstrate substantial results in support of the hypothesis of a critical role of neurexins for regulating glycinergic transmission in the LSO using various techniques. They provide evidence for the expression of neurexins in the MNTB and consecutively successfully generate and characterize the neurexin TKO. For their study on LSO IPSCs the authors transduced MNTB neurons by co-injection of virus-carrying Cre and ChR2 and subsequently optogenetically evoke release of glycine. As a result, they observed a significant reduction in amplitude and significantly slower rise and decay times of the IPSCs of the TKO in comparison with control mice in which MNTB neurons were only transduced with ChR2. Furthermore, they observed an increased paired pulse ratio (PPR) of LSO IPSCs in the TKO mice, indicating lower release probability. Elaborating on the hypothesis that neurexins are essential for the coupling of synaptic vesicles to Ca2+ channels, the authors show lowered Ca2+ sensitivity in the TKO mice. Additionally, they reveal convincing evidence for the connection between the increased frequency of spontaneous IPSC and the higher number of glycinergic synapses of the LSO in the TKO mice, revealed by immunolabeling against the glycinergic presynaptic markers GlyT2 or VGAT.

Weaknesses:<br /> The major concern is novelty as this work on the effects of pan-neurexin deletion in a glycinergic synapse is quite consistent with the authors' prior work on glutamatergic synapses (Luo et al., 2020). The authors might want to further work out novel aspects and strengthen the comparative perspective. Conceptually, the authors might want to be more clear about interpreting the results on the altered dependence of release on voltage-gated Ca2+ influx (Ca2+ sensitivity, coupling).

Reviewer #3 (Public Review):

This study demonstrated the application of OPM-MEG in neurodevelopment studies of somatosensory beta oscillations and connections with children as young as 2 years old. It provides a new functional neuroimaging method that has a high spatial-temporal resolution as well wearable which makes it a new useful tool for studies in young children. They have constructed a 192-channel wearable OPM-MEG system that includes field compensation coils which allow free head movement scanning with a relatively high ratio of usable trials. Beta band oscillations during somatosensory tasks are well localized and the modulation with age is found in the amplitude, connectivity, and pan-spectral burst probability. It is demonstrated that the wearable OPM-MEG could be used in children as a quite practical and easy-to-deploy neuroimaging method with performance as good as conventional MEG. With both good spatial (several millimeters) and temporal (milliseconds) resolution, it provides a novel and powerful technology for neurodevelopment research and clinical applications not limited to somatosensory areas.

The conclusions of this paper are mostly well supported by data acquired under the proper method. However, some aspects of data analysis need to be improved and extended.

(1) The colour bars selected for the pseudo-T-static pictures of beta modulation in Figures 2 and 3, which are blue/black and red/black, are not easily distinguished from the anatomical images which are grey-scale. A colour bar without black/white would make these figures better. The peak point locations are also suggested to be marked in Figure 2 and averaged locations in Figure 3 with an error bar.

(2) The data points in plots are not constant across figures. In Figures 3 and 5, they are classified into triangles and circles for children and adults, but all are circles in Figures 4 and 6.

(3) Although MEG is much less susceptible to conductivity inhomogeneity of the head than EEG, the forward modulating may still be impacted by the small head profile. Add more information about source localization accuracy and stability across ages or head size.

Reviewer #3 (Public Review):

The authors describe the role, location, and function of the MTA and MTB mating type genes in the multi-mating-type species T. thermophila. The ciliate is an important group of organisms to study the evolution of mating types, as it is one of the few groups in which more than two mating types evolved independently. In the study, the authors use deletion strains of the species to show that both mating types genes located in each allele are required in both mating individuals for successful matings to occur. They show that the proteins are localized in the cell membrane, not the cilia, and that they interact in a complex (MTRC) with a set of 6 associated (non-mating type-allelic) genes. This complex is furthermore likely to interact with a cyclin-dependent kinase complex. It is intriguing that T. thermophila has two genes that are allelic and that are both required for successful mating. This coevolved double recognition has to my knowledge not been described for any other mating-type recognition system. I am not familiar with experimental research on ciliates, but as far as I can judge, the experiments appear well performed and mostly support the interpretation of the authors with appropriate controls and statistical analyses.

The results show clearly that the mating type genes regulate non-self-recognition, however, I am not convinced that self-recognition occurs leading to the suppression of mating. An alternative explanation could be that the MTA and MTB proteins form a complex and that the two extracellular regions together interact with the MTA+MTB proteins from different mating types. This alternative hypothesis fits with the coevolution of MTA and MTB genes observed in the phylogenetic subgroups as described by Yan et al. (2021 iScience). Adding MTAxc and/or MTBxc to the cells can lead to the occupation of the external parts of the full proteins thereby inhibiting the formation of the complex, which in turn reduces non-self interactions. Self-recognition as explained in Figure 2S1 suggests an active response, which should be measurable in expression data for example. This is in my opinion not essential, but a claim of self-recognition through the MTA and MTB should not be made.

The authors discuss that T. thermophila has special mating-type proteins that are large, while those of other groups are generally small (lines 157-160 and discussion). The complex formed is very large and in the discussion, they argue that this might be due to the "highly complex process, given that there are seven mating types in all". There is no argument given why large is more complex, if this is complex, and whether more mating types require more complexity. In basidiomycete fungi, many more mating types than 7 exist, and the homeodomain genes involved in mating types are relatively small but highly diverse (Luo et al. 1994 PMID: 7914671). The mating types associated with GPCR receptors in fungi are arguably larger, but again their function is not that complex, and mating-type specific variations appear to evolve easily (Fowler et al 2004 PMID: 14643262; Seike et al. 2015 PMID: 25831518). The large protein complex formed is reminiscent of the fusion patches that develop in budding or fission yeasts. In these species, the mating type receptors are activated by ligand pheromones from the opposite mating type that induce polarity patch formation (see Sieber et al. 2023 PMID: 35148940 for a recent review). At these patches, growth (shmooing) and fusion occur, which is reminiscent (in a different order) of the tip transformation in T. thermophilia. The fusion of two cells is in all taxa a dangerous and complex event that requires the evolution of very strict regulation and the existence of a system like the MTRC and cyclin-dependent complex to regulate this process is therefore not unexpected. The existence of multiple mating types should not greatly complicate the process, as most of the machinery (except for the MTA and MTB) is identical among all mating types.

The Tetrahymena/ciliate genetics and lifecycle could be better explained. For a general audience, the system is not easy to follow. For example, the ploidy of the somatic nucleus with regards to the mating type is not clear to me. The MAC is generally considered "polyploid", but how does this work for the mating type? I assume only a single copy of the mating type locus is available in the MAC to avoid self-recognition in the cells. Is it known how the diploid origin reduces to a single mating type? This does not become apparent from Cervantes et al. 2013. Also, the explanation of co-stimulation is not completely clear (lines 49-60). Initially, direct cell-cell contact is mentioned, but later it is mentioned that "all cells become fully stimulated", even when unequal ratios are used. Is physical contact necessary? Or is this due to the "secrete mating-essential factors" (line 601)? These details are essential, for interpretation of the results and need to be explained better.

Abstract and introduction: Sexes are not mating types. In general, mating types refer to systems in which there is no obvious asymmetry between the gametes, beyond the compatibility system. When there is a physiological difference such as size or motility, sexes are used. This distinction is of importance because in many species mating types and sexes can occur together, with each sex being able to have either (when two) or multiple mating types. An example are SI in angiosperms as used as an example by the authors or mating types in filamentous fungi. See Billiard et al. 2011 [PMID: 21489122] for a good explanation and argumentation for the importance of making this distinction.

Reviewer #3 (Public Review):

Summary:

The vesicular monoamine transporter is a key component in neuronal signaling and is implicated in diseases such as Parkinson's. Understanding of monoamine processing and our ability to target that process therapeutically has been to date provided by structural modeling and extensive biochemical studies. However, structural data is required to establish these findings more firmly.

Strengths:

Dalton et al resolved a structure of VMAT2 in the presence of an important inhibitor, tetrabenazine, with the protein in detergent micelles, using cryo-EM and with the aid of protein domains fused to its N- and C-terminal ends, including one fluorescent protein that facilitated protein screening and purification. The resolution of the maps allows clear assignment of the amino acids in the core of the protein. The structure is in good agreement with a wealth of experimental and structural prediction data, and provides important insights into the binding site for tetrabenazine and selectivity relative to analogous compounds. The authors provide additional biochemical analyses that further support their findings. The comparison with AlphaFold models is enlightening.

Weaknesses:

The authors follow up their structures with molecular dynamics simulations of the tetrabenazine-bound state, and test several protonation states of acidic residues in the binding pocket, but not all possible combinations; thus, it is not clear the extent to which tetrabenazine rearrangements observed in these simulations are meaningful. Additional simulations of the substrate dopamine docked into this structure were also carried out, although it is unclear whether this "dead-end" occluded state is a relevant state for dopamine binding. The authors report release of dopamine during these simulations, but it is notable that this only occurs when all four acidic binding site residues were protonated and when an enhanced sampling approach was applied.

Reviewer #3 (Public Review):

Summary:<br /> In the present work, Zhang et al investigate the involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance evolution in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term drug resistance evolution that can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by the accumulation of reactive oxygen species and inhibition of DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance). However, although the authors perform several multi-disciplinary experiments, there are several caveats to the authors' proposal that ultimately do not fully support their interpretation that the observed antimicrobial resistance evolution phenotype is due to compromised DNA repair.

Strengths:<br /> The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to comprised DNA repair and oxidative stress. This is primarily supported by their observations that resistance evolution phenotypes only exist for recA deletion mutants and not other genes in the SOS response

Weaknesses:<br /> The authors do not show any direct evidence (1) that these phenotypes exist in strains harboring deletions in other DNA repair genes outside of the SOS response, (2) that DNA damage is increased, (3) that reactive oxygen species accumulate, (4) that accelerated resistance evolution can be reversed by anything other than recA complementation. The authors do not directly test alternative hypotheses. The conclusions drawn are therefore premature.

Reviewer #3 (Public Review):

Summary:<br /> Pooled optical screening has recently emerged as a powerful approach to associate complex phenotypic information from microscope images with specific genetic perturbations at the single-cell level. This is achieved by amplifying and sequencing DNA barcodes within individual cells through in-situ sequencing. This paper leverages these advances in pooled screening technology to examine the effects of gene knockdowns on high-dimensional cell morphological phenotypes beyond binary readouts.

A key challenge is how to effectively distill meaningful phenotypic dimensions from information-rich image data to connect genotype to phenotype. By screening 366 genes using CRISPRi and analyzing tens of thousands of single-cell images, this paper provides insights into genetic regulators of morphology in osteosarcoma cells. In developing this screen and analyzing its readout, the authors make several notable contributions.

First, the authors tested and optimized molecular inversion probes (MIPs) to improve rolling circle amplification and barcode imaging. Through these optimization experiments, they identified a shortened MIP design that yielded 11-fold more visible amplicons, enabling more robust barcode readout from complex images. Second, the authors address several unresolved questions regarding how to work with single-cell images at this scale. A critical aspect of this is the need to develop analysis strategies using single-cell data rather than commonly used current methodologies that condense down to an agglomerated perturbation level cell morphology information. The authors compare morphological profiling using curated feature extraction and an unsupervised deep learning approach called a β-variational autoencoder on single-cell imaging data, suggesting that the latter can capture salient aspects of variation without requiring much human input. Finally, and perhaps more importantly, the authors develop an approach, Visual Interpretation of Embeddings by constrained Walkthrough Sampling (VIEWS), towards sampling cells at the end of distributions such as a principal component dimension in a reduction of curated features or a latent space dimension extracted from an autoencoder. This allows for a rapid and efficient way of understanding extremes of morphological profiles and allows for quick interpretability of extracted morphological signal which in turn assists with downstream functional understandings of groups of genes that similarly alter a cell's morphology.

Strengths and Weaknesses:<br /> This is an interesting and rigorous paper that provides an important advance in conducting large-scale microscopy-based approaches. The methods development and computational analyses described in this paper are strong and innovative. However, the screening conducted in this paper did not identify a large number of modifiers of general U2OS cell morphology. As the authors rightly point out, several factors could contribute to the modest hit rate, including variable CRISPRi knockdown efficiency and limited phenotypic readout from just two imaging channels. Despite these limitations, the paper makes several key methodological contributions and in the opinion of this reviewer merits revision or benchmarking.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Stubbusch and coauthors examine the foraging behavior of a marine species consuming an abundant marine polysaccharide. Laboratory experiments in a microfluidic setup are complemented with transcriptomic analyses aiming at assessing the genetic bases of the observed behavior. Bacterial cells consuming the polysaccharide form cohesive aggregates, while they start dispersing away when the byproduct of the digestion of the polysaccharide starts accumulating. Dispersing cells, tend to be attracted by the polysaccharide. Expression data show that motility genes are enriched during the dispersal phase, as expected. Counterintuitively, in the same phase, genes for transporters and digestions of polysaccharides are also highly expressed.

Strengths:<br /> The manuscript is very well written and easy to follow. The topic is interesting and timely. The genetic analyses provide a new, albeit complex, angle to the study of foraging behaviors in bacteria, adding to previous studies conducted on other species.

Weaknesses:<br /> I find this paper very descriptive and speculative. The results of the genetic analyses are quite counterintuitive; therefore, I understand the difficulty of connecting them to the observations coming from experiments in the microfluidic device. However, they could be better placed in the literature of foraging - dispersal cycles, beyond bacteria. In addition, the interpretation of the results is sometimes confusing.

Reviewer #3 (Public Review):

Summary:

The study adds to the existing data that have established that cortical development in rhesus macaque is known to recapitulate multiple facets cortical development in humans. The authors generate and analyze single cell transcriptomic data from the timecourse of embryonic neurogenesis.

Strengths:

Studies of primate developmental biology are hindered by the limited availability and limit replication. In this regard, a new dataset is useful.

The study analyzes parietal cortex, while previous studies focused on frontal and motor cortex. This may be the first analysis of macaque parietal cortex and, as such, may provide important insights into arealization, which the authors have not addressed

Reviewer #3 (Public Review):

The authors of this study have examined which cation channels specifically confer to ventral tegmental area dopaminergic neurones their autonomic (spontaneous) firing properties. Having brought evidence for the key role played by NALCN and TRPC6 channels therein, the authors aimed at measuring whether these channels play some role in so-called depression-like (but see below) behaviors triggered by chronic exposure to different stressors. Following evidence for a down-regulation of TRPC6 protein expression in ventral tegmental area dopaminergic cells of stressed animals, the authors provide evidence through viral expression protocols for a causal link between such a down-regulation and so-called depression-like behaviors. The main strength of this study lies on a comprehensive bottom-up approach ranging from patch-clamp recordings to behavioral tasks. These tasks mainly address anxiety-like behaviors and so-called depression-like behaviors (sucrose choice, forced swim test, tail suspension test). The results gathered by means of these procedures are clearcut. However, the reviewer believes that the authors should be more cautious when interpreting immobility responses to stress (forced swim, tail suspension) as "depression-like" responses. These stress models have been routinely used (and validated) in the past to detect the antidepressant properties of compounds under investigation, which by no means indicates that these are depression models. For readers interested by this debate, I suggest to read e.g. De Kloet and Molendijk (Biol. Pscyhiatry 2021).

Reviewer #3 (Public Review):

Summary:

This study uses a state-of-the-art artificial skin assay to determine the quantity of P. falciparum sporozoites expelled during feeding using mosquito infection (by standardised membrane feeding assay SMFA) using both cultured gametocytes and natural infection. Sporozoite densities in salivary glands and expelled into the skin are quantified using a well-validated molecular assay. These studies show clear positive correlations between mosquito infection levels (as determined by oocyst numbers), sporozoite numbers in salivary glands, and sporozoites expelled during feeding. This indicates potentially significant heterogeneity in infectiousness between mosquitoes with different infection loads and thus challenges the often-made assumption that all infected mosquitoes are equally infectious.

Strengths:

Very rigorously designed studies using very well validated, state-of-the-art methods for studying malaria infections in the mosquito and quantifying load of expelled sporozoites. This resulted in very high-quality data that was well-analyzed and presented. Both sources of gametocytes (cultures vs. natural infection) show consistent results further strengthening the quality of the results obtained.

Weaknesses:

As is generally the case when using SMFAs, the mosquito infections levels are often relatively high compared to wild-caught mosquitoes (e.g. Bombard et al 2020 IJP: median 3-4 ), and the strength of the observed correlations between oocyst sheet and salivary gland sporozoite load even more so between salivary gland sporozoite load and expelled sporozoite number may be dominated by results from mosquitoes with infection levels rarely observed in wild-caught mosquitoes. This could result in an overestimation of the importance of these well-observed positive relationships under natural transmission conditions.

The results obtained from these excellently designed and executed studies very well supported their conclusion - with a slight caveat regarding their application to natural transmission scenarios

This work very convincingly highlights the potential for significant heterogeneity in the infectiousness between individual P. falciparum-infected mosquitoes. Such heterogeneity needs to be further investigated and if again confirmed taken into account both when modelling malaria transmission and when evaluating the importance of low-density infections in sustaining malaria transmission.

Reviewer #3 (Public Review):

Summary:<br /> This paper by Stribling and colleagues sheds light on a decade-long P. aeruginosa outbreak of the high-risk lineage ST-621 in a US Military hospital. The origins of the outbreak date back to the late 90s and it was mainly caused by two distinct subclones SC1 and SC2. The data of this outbreak showed the emergence of antibiotic resistance to cephalosporin, carbapenems, and colistin over time highlighting the emerging risk of extensively resistant infections due to P. aeruginosa and the need for ongoing surveillance.

Strengths:<br /> This study overall is well constructed and clearly written. Since detailed information on floor plans of the building and transfers between facilities was available, the authors were able to show that these two subclones emerged in two separate buildings of the hospital. The authors support their conclusions with prospective environmental sampling in 2021 and 2022 and link the role of persistent environmental contamination to sustaining nosocomial transmission. Information on resistance genes in repeat isolates for the same patients allowed the authors to detect the emergence of resistance within patients. The conclusions have broader implications for infection control at other facilities. In particular, the paper highlights the value of real-time surveillance and environmental sampling in slowing nosocomial transmission of P. aeruginosa.

Weaknesses:<br /> My major concern is that the authors used fixed thresholds and definitions to classify the origin of an infection. As such, they were not able to give uncertainty measures around transmission routes nor quantify the relative contribution of persistent environmental contamination vs patient-to-patient transmission. The latter would allow the authors to quantify the impact of certain interventions. In addition, these results represent a specific US military facility and the transmission patterns might be specific to that facility. The study also lacked any data on antibiotic use that could have been used to relate to and discuss the temporal trends of antimicrobial resistance.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, Hajra et al have attempted to identify the role of Sirt1 and Sirt3 in regulating metabolic reprogramming and macrophage host defense. They have performed gene knockdown experiments in RAW macrophage cell lines to show that depletion of Sirt1 or Sirt3 enhances the ability of macrophages to eliminate Salmonella Typhimurium. However, in mice, inhibition of Sirt1 resulted in dissemination of the bacteria but the bacterial burden was still reduced in macrophages. They suggest that the effect they have observed is due to increased inflammation and ROS production by macrophages. They also try to establish a weak link with metabolism. They present data to show that the switch in metabolism from glycolysis to fatty acid oxidation is regulated by acetylation of Hif1a, and PDHA1.

Strengths:<br /> The strength of the manuscript is that the role of Sirtuins in host-pathogen interactions has not been previously explored in-depth making the study interesting. It is also interesting to see that depletion of either Sirt1 or Sirt3 results in a similar outcome.

Weaknesses:<br /> The major weakness of the paper is the low quality of data, making it harder to substantiate the claims. Also, there are too many pathways and mechanisms being investigated. It would have been better if the authors had focussed on either Sirt1 or Sirt3 and elucidated how it reprograms metabolism to eventually modulate host response against Salmonella Typhimurium. Experimental evidence is also lacking to prove the proposed mechanisms. For instance, they show correlative data that the knockdown of Sirt1-mediated shift in metabolism is due to HIF1a acetylation but this needs to be proven with further experiments.

Reviewer #3 (Public Review):

This study investigates subtelomeric repetitive sequences in the budding yeast Saccharomyces cerevisiae, known as Y' and X-elements. Taking advantage of yeast strain SY12 that contains only 3 chromosomes and six telomeres (normal yeast strains contain 32 telomeres) the authors are able to generate a strain completely devoid of Y'- and X-elements.

Strengths: They demonstrate that the SY12 delta XY strain displays normal growth, with stable telomeres of normal length that were transcriptionally silenced, a key finding with wide implications for telomere biology. Inactivation of telomerase in the SY12 and SY12 delta XY strains frequently resulted in survivors that had circularized all three chromosomes, hence bypassing the need for telomeres altogether. They show that survivors with fused chromosomes and so-called atypical survivors arise independently of the central recombination protein Rad52. The SY12 and SY12 delta XY yeast strains can become a useful tool for future studies of telomere biology. The conclusions of this manuscript are well supported by the data and are valuable for researchers studying telomeres.

Weaknesses: A weakness of the manuscript is the analysis of telomere transcriptional silencing. They state: "The results demonstrated a significant increase in the expression of the MPH3 and HSP32 upon Sir2 deletion, indicating that telomere silencing remains effective in the absence of X and Y'-elements". However, for the SY12 strain, their analyses indicate that the difference between the WT and sir2 strains is nonsignificant. In addition, a striking observation is that the SY12 strain (with only three chromosomes) express much less of both MPH3 and HSP32 than the parental strain BY4742 (16 chromosomes), both in the presence and absence of Sir2.

Reviewer #3 (Public Review):

Summary:<br /> In the context of the SCOURGE consortium's research, the authors conduct a GWAS meta-analysis on 4,702 hospitalized individuals of admixed American descent suffering from COVID-19. This study identified four significant genetic associations, including two loci initially discovered in Latin American cohorts. Furthermore, a trans-ethnic meta-analysis highlighted an additional novel risk locus in the CREBBP gene, underscoring the critical role of genetic diversity in understanding the pathogenesis of COVID-19.

Strengths:<br /> 1. The study identified two novel severe COVID-19 loci (BAZ2B and DDIAS) by the largest GWAS meta-analysis for COVID-19 hospitalization in admixed Americans.

2. With a trans-ethnic meta-analysis, an additional risk locus near CREBBP was identified.

Weaknesses:<br /> 1. The GWAS power is limited due to the relatively small number of cases.

2. There is no replication study for the novel severe COVID-19 loci, which may lead to false positive findings.

3. Significant differences exist in the ages between cases and controls, which could potentially introduce biased confounders. I'm curious about how the authors treated age as a covariate. For instance, did they use ten-year intervals? This needs clarification for reproducibility.

4."Those in the top PGS decile exhibited a 5.90-fold (95% CI=3.29-10.60, p=2.79x10-9) greater risk compared to individuals in the lowest decile". I would recommend comparing with the 40-60% PGS decile rather than the lowest decile, as the lowest PGS decile does not represent 'normal controls'.

5. In the field of PGS, it's common to require an independent dataset for training and testing the PGS model. Here, there seems to be an overfitting issue due to using the same subjects for both training and testing the variants.

6. The variants selected for the PGS appear arbitrary and may not leverage the GWAS findings without an independent training dataset.

7. The TWAS models were predominantly trained on European samples, and there is no replication study for the findings as well.

Reviewer #3 (Public Review):

Summary:<br /> As SMAD1/5 activities have previously been indistinguishable, these studies provide a new mouse model to finally understand unique downstream activation of SMAD1/5 target genes, a model useful for many scientific fields. Using CUT&RUN analyses with gene overlap comparisons and signaling pathway analyses, specific targets for SMAD1 versus SMAD5 were compared, identified, and interpreted. These data validate previous findings showing strong evidence that SMADs directly govern critical genes required for endometrial receptivity and decidualization, including cell adhesion and vascular development. Further, SMAD targets were overlapped with progesterone receptor binding sites to identify regions of potential synergistic regulation of implantation. The authors report strong correlations between progesterone receptor and SMAD1/5 direct targets to cooperatively promote embryo implantation. Finally, the authors validated SMAD1/5 gene regulation in primary human endometrial stromal cells. These studies provide a data-rich survey of SMAD family transcription, defining its role as a governor of early pregnancy.

Strengths:<br /> This manuscript provides a valuable survey of SMAD1/5 direct transcriptional events at the time of receptivity. As embryo implantation is controlled by extensive epithelial to stromal molecular crosstalk and hormonal regulation in space and time, the authors state a strong, descriptive narrative defining how SMAD1/5 plays a central role at the site of this molecular orchestration. The implementation of cutting-edge techniques and models and simple comparative analyses provide a straightforward, yet elegant manuscript.

Although the progesterone receptor exists as a major regulator of early pregnancy, the authors have demonstrated clear evidence that progesterone receptor with SMAD1/5 work in concert to molecularly regulate targets such as Sox17, Id2, Tgfbr2, Runx1, Foxo1 and more at embryo implantation. Additionally, the authors pinpoint other critical transcription factor motifs that work with SMADs and the progesterone receptor to promote early pregnancy transcriptional paradigms.

Weaknesses:

Although a wonderful new tool to ascertain SMAD1 versus SMAD5 downstream signaling, the importance of these factors in governing early pregnancy is not novel. Furthermore, functional validation studies are needed to confirm interactions at promoter regions. Additionally, the authors presume that all overlapped genes are shared between progesterone receptor and SMAD1/5, yet some peak representations do not overlap. Although, transcriptional activation can occur at the same time, they may not occur in the same complex. Thus, further confirmation of these transcriptional events is warranted.

Since whole murine uterus was used for these studies, the specific functions of SMAD1/5 in the stroma versus the epithelium (versus the myometrium) remain unknown. Further work is needed to delineate binding and transcriptional activation of SMAD1/5 and the progesterone receptor in the uterine compartments.

There are asynchronous gene responses in the SMAD1/5 ablated mouse model compared to the siRNA-treated human endometrial stromal cells. These differences can be confounding. Further investigation is needed to understand the meaning of these differences and as they relate to the entire SMAD transcriptome.

Reviewer #3 (Public Review):

Summary:<br /> This is an interesting paper investigating fMRI changes during sensory (visual, tactile) stimulation and absence seizures in the GAERS model. The results are potentially important for the field and do suggest that sensory stimulation may not activate brain regions normally during absence seizures. But the findings are limited by substantial methodological issues that do not enable fMRI signals related to absence seizures to be fully disentangled from fMRI signals related to the sensory stimuli.

Strengths:

Investigating fMRI brain responses to sensory stimuli during absence seizures in an animal model is a novel approach with potential to yield important insights.

Use of an awake, habituated model is a valid and potentially powerful approach.

Weaknesses:

The major difficulty with interpreting the results of this study is that the duration of the visual and tactile stimuli were 6 seconds, which is very close to the mean seizure duration per Table 1. Therefore the HRF model looking at fMRI responses to visual or auditory stimuli occurring during seizures was simultaneously weighting both seizure activity and the sensory (visual or auditory) stimuli over the same time intervals on average. The resulting maps and time courses claiming to show fMRI changes from visual or auditory stimulation during seizures will therefore in reality contain some mix of both sensory stimulation-related signals and seizure-related signals. The main claim that the sensory stimuli do not elicit the same activations during seizures as they do in the interictal period may still be true. But the attempts to localize these differences in space or time will be contaminated by the seizure related signals.

In their response to this comment the authors state that some seizures had longer than average duration, and that they attempted to model the effects of both seizures and sensory stimulation. However these factors do not mitigate the concern because the mean duration of seizures and sensory stimulation remain nearly identical, and the models used therefore will not be able to effectively separate signals related to seizures and related to sensory stimulation.

The claims that differences were observed for example between visual cortex and superior colliculus signals with visual stim during seizures vs interictal remain unconvincing due to above.

Maps shown in Figure 3 do not show clear changes in the areas claimed to be involved.

In their response the authors enlarged the cross sections. However there are still discrepancies between the images and the way they are described in the text. For example, in the Results text the authors say that comparing the interictal and ictal states revealed less activation in the somatosensory cortex during the ictal than during the interictal state, yet Figure 3 bottom row left shows greater activation in somatosensory cortex in this contrast.

Reviewer #3 (Public Review):

Summary:

This study used prolonged stimulation of a limb to examine possible plasticity in somatosensory evoked potentials induced by the stimulation. They also studied the extent that the blood brain barrier (BBB) was opened by the prolonged stimulation and whether that played a role in the plasticity. They found that there was potentiation of the amplitude and area under the curve of the evoked potential after prolonged stimulation and this was long-lasting (>5 hrs). They also implicated extravasation of serum albumin, caveolae-mediated transcytosis, and TGFb signalling, as well as neuronal activity and upregulation of PSD95. Transcriptomics was done and implicated plasticity related genes in the changes after prolonged stimulation, but not proteins associated with the BBB or inflammation. Next, they address the application to humans using a squeeze ball task. They imaged the brain and suggest that the hand activity led to an increased permeability of the vessels, suggesting modulation of the BBB.

Strengths:

The strengths of the paper are the novelty of the idea that stimulation of the limb can induce cortical plasticity in a normal condition, and it involves opening of the BBB with albumin entry. In addition, there are many datasets and both rat and human data.

Weaknesses:

The conclusions are not compelling however because of a lack of explanation of methods. The explanation of why prolonged stimulation in the rat was considered relevant to normal conditions should be as clear in the paper as it is in the rebuttal. The authors need to ensure other aspects of the rebuttal are as clear in the paper as in the rebuttal too. The only remaining concern that is significant is that it is hard to understand the figures.

Reviewer #3 (Public Review):

A brain region called the retrotrapezoid nucleus (RTN) regulates breathing in response to changes in CO2/H+, a process termed central chemoreception. A transcription factor called PHOX2B is important for RTN development and mutations in the PHOX2B gene result in a severe type of sleep apnea called Congenital Central Hypoventilation Syndrome. PHOX2B is also expressed throughout life, but its postmitotic functions remain unknown. This study shows that knockdown of PHOX2B in the RTN region in adult rats decreased expression of Task2 and Gpr4 in Nmb-expressing RTN chemoreceptors and this corresponded with a diminished ventilatory response to CO2 but did not impact baseline breathing or the hypoxic ventilatory response. These results provide novel insight regarding the postmitotic functions of PHOX2B in RTN neurons.

Main issues:<br /> 1) The experimental approach was not targeted to Nmb+ neurons and since other cells in the area also express Phox2b, conclusions should be tempered to focus on Phox2b expressing parafacial neurons NOT specifically RTN neurons

2) It is not clear whether PHOX2B is important for the transcription of pH sensing machinery, cell health, or both. If knockdown of PHOX2B knockdown results in loss of RTN neurons this is also expected to decrease Task2 and Gpr4 levels, albeit by a transcription-independent mechanism.

Reviewer #3 (Public Review):

Summary:<br /> The study claims to investigate trunk representations in elephant trigeminal nuclei located in the brainstem. The researchers identified large protrusions visible from the ventral surface of the brainstem, which they examined using a range of histological methods. However, this ventral location is usually where the inferior olivary complex is found, which challenges the author's assertions about the nucleus under analysis. They find that this brainstem nucleus of elephants contains repeating modules, with a focus on the anterior and largest unit which they define as the putative nucleus principalis trunk module of the trigeminal. The nucleus exhibits low neuron density, with glia outnumbering neurons significantly. The study also utilizes synchrotron X-ray phase contrast tomography to suggest that myelin-stripe-axons traverse this module. The analysis maps myelin-rich stripes in several specimens and concludes that based on their number and patterning they likely correspond with trunk folds; however, this conclusion is not well supported if the nucleus has been misidentified.

Strengths:<br /> The strength of this research lies in its comprehensive use of various anatomical methods, including Nissl staining, myelin staining, Golgi staining, cytochrome oxidase labeling, and synchrotron X-ray phase contrast tomography. The inclusion of quantitative data on cell numbers and sizes, dendritic orientation and morphology, and blood vessel density across the nucleus adds a quantitative dimension. Furthermore, the research is commendable for its high-quality and abundant images and figures, effectively illustrating the anatomy under investigation.

Weaknesses:<br /> While the research provides potentially valuable insights if revised to focus on the structure that appears to be the inferior olivary nucleus, there are certain additional weaknesses that warrant further consideration. First, the suggestion that myelin stripes solely serve to separate sensory or motor modules rather than functioning as an "axonal supply system" lacks substantial support due to the absence of information about the neuronal origins and the termination targets of the axons. Postmortem fixed brain tissue limits the ability to trace full axon projections. While the study acknowledges these limitations, it is important to exercise caution in drawing conclusions about the precise role of myelin stripes without a more comprehensive understanding of their neural connections.

Second, the quantification presented in the study lacks comparison to other species or other relevant variables within the elephant specimens (i.e., whole brain or brainstem volume). The absence of comparative data for different species limits the ability to fully evaluate the significance of the findings. Comparative analyses could provide a broader context for understanding whether the observed features are unique to elephants or more common across species. This limitation in comparative data hinders a more comprehensive assessment of the implications of the research within the broader field of neuroanatomy. Furthermore, the quantitative comparisons between African and Asian elephant specimens should include some measure of overall brain size as a covariate in the analyses. Addressing these weaknesses would enable a richer interpretation of the study's findings.

Reviewer #3 (Public Review):

Summary: This study investigated the role of mTORC1 and 2 in a mouse model of developmental epilepsy which simulates the epilepsy in cortical malformations. Given activation of genes such as PTEN activate TORC1, and this is considered to be excessive in cortical malformations, the authors asked whether inactivating mTORC1 and 2 would ameliorate the seizures and malformation in the mouse model. The work is highly significant because a new mouse model is used where Raptor and Rictor, which regulate mTORC1 and 2 respectively, were inactivated in one hemisphere of the cortex. The work is also significant because the deletion of both Raptor and Rictor improved the epilepsy and malformation. In the mouse model, the seizures were generalized or there were spike wave discharges (SWD). They also examined the interictal EEG. The malformation was manifested by increased cortical thickness and soma size.

Strengths: The presentation and writing is strong. Quality of data are strong. The data support the conclusions for the most part. The results are significant: Generalized seizures and SWDs were reduced when both Torc1 and 2 were inactivated but not when one was inactivated.

Weaknesses: One of the limitations is a somewhat small sample size. Another is that there was hippocampal expression. A third is that recordings of seizures were not continuous and different for each mouse. Another concern is they only measured layer II/III neurons.

Reviewer #3 (Public Review):

Summary:<br /> This work examined efference copy related to eye movements in healthy adults who have high autistic traits. Efference copies allow the brain to make predictions about sensory outcomes of self-generated actions, and thus serve important roles in motor planning and maintaining visual stability. Consequently, disrupted efference copies have been posited as a potential mechanism underlying motor and sensory symptoms in psychopathology such as Autism Spectrum Disorder (ASD), but so far very few studies have directly investigated this theory. Therefore, this study makes an important contribution as an attempt to fill in this knowledge gap. The authors conducted two eye-tracking experiments examining the accuracy of motor planning and visual perception following a saccade and found that participants with high autistic traits exhibited worse task performance (i.e., less accurate second saccade and biased perception of object displacement), consistent with their hypothesis of less impact of efference copies on motor and visual updating. Moreover, the motor and visual biases are positively correlated, indicative of a common underlying mechanism. These findings are promising and can have important implications for clinical intervention if they can be replicated in a clinical sample.

Strengths:<br /> The authors utilized well-established and rigorously designed experiments and sound analytic methods. This enables easy translations between similar work in non-human primates and humans and readily points to potential candidates for underlying neural circuits that could be further examined in follow-up studies (e.g., superior colliculus, frontal eye fields, mediodorsal thalamus). The finding of no association between initial saccade accuracy and level of autistic trait in both experiments also serves as an important control analysis and increases one's confidence in the conclusion that the observed differences in task performance were indeed due to disrupted efference copies, not confounding factors such as basic visual/motor deficits or issues with working memory. The strong correlation between the observed motor and visual biases further strengthens the claim that the findings from both experiments may be explained by the same underlying mechanism - disrupted efference copies. Lastly, the authors also presented a thoughtful and detailed mechanistic theory of how efference copy impairment may lead to ASD symptomatology, which can serve as a nice framework for more research into the role of efference copies in ASD.

Weaknesses:<br /> Although the paper has a lot of strengths, the main weakness of the paper is that a direct link with ASD symptoms (i.e., sensory overload and motor inflexibility as the authors suggested) cannot be established. First of all, the participants are all healthy adults who do not meet the clinical criteria for an ASD diagnosis. Although they could be considered a part of the broader autism phenotype, the results cannot be easily generalized to the clinical population without further research. Secondly, the measure used to quantify the level of autistic traits, Autistic Quotient (AQ), does not actually capture any sensory or motor symptoms of ASD. Therefore, it is unknown whether those who scored high on AQ in this study experienced high, or even any, sensory or motor difficulties. In other words, more evidence is needed to demonstrate a direct link between disrupted efference copies and sensory/motor symptoms in ASD.

Reviewer #3 (Public Review):

Observers make judgements about expected stimuli faster and more accurately. How expectations facilitate such perceptual decisions remains an ongoing area of investigation, however, as expectations may exert their effects in multiple ways. Expectations may directly influence the encoding of sensory signals. Alternatively (or additionally), expectations may influence later stages of decision-making, such as motor preparation, when they bear on the appropriate behavioral response.

In the present study, Walsh and colleagues directly measured the effect of expectations on sensory and motor signals by making clever use of the encephalogram (EEG) recorded from human observers performing a contrast discrimination task. On each trial, a predictive cue indicated which of two superimposed stimuli would likely be higher contrast and, therefore, whether a left or right button press was likely to yield a correct response. Deft design choices allowed the authors to extract both contrast-dependent sensory signals and motor preparation signals from the EEG. The authors provide compelling evidence that, when predictive cues provide information about both a forthcoming stimulus and the appropriate behavioral response, expectation effects are immediately manifest in motor preparation signals and only emerge in sensory signals after extensive training.

Future work should attempt to reconcile these results with related investigations in the field. As the authors note, several groups have reported expectation-induced modulation of sensory signals (using both fMRI and EEG/MEG) on shorter timescales (e.g. just one or two sessions of a few hundred trials, versus the intensive multi-session study reported here). One interesting possibility is that perceptual expectations are not automatic but demand the deployment of feature-based attention, while motor preparation is comparatively less effortful and so dominates when both sources of information are available, as in the present study. This hypothesis is consistent with the authors' thoughtful analysis showing decreased neural signatures of attention over posterior electrodes following predictive cues. Therefore, observing the timescale of sensory effects using the same design and methods (facilitating direct comparison with the present work), but altering task demands slightly such that cues are no longer predictive of the appropriate behavioral response, could be illuminating.

Reviewer #3 (Public Review):

Summary:<br /> This paper explores the relationships among evolutionary and epidemiological quantities in influenza, using a wide range of datasets and features, and using both correlations and random forests to examine, primarily, what are the drivers of influenza epidemics. It's a strong paper representing a thorough and fascinating exploration of potential drivers, and it makes a trove of relevant data readily available to the community.

Strengths:<br /> This paper makes links between epidemiological and evolutionary data for influenza. Placing each in the context of the other is crucial for understanding influenza dynamics and evolution and this paper does a thorough job of this, with many analyses and nuances. The results on the extent to which evolutionary factors relate to epidemic burden, and on interference among influenza types, are particularly interesting. The github repository associated with the paper is clear, comprehensive, and well-documented.

Weaknesses:<br /> The format of the results section can be hard to follow, and we suggest improving readability by restructuring and simplifying in some areas. There are a range of choices made about data preparation and scaling; the authors could explore sensitivity of the results to some of these.

Reviewer #3 (Public Review):

In this study, Ruan et al. investigate the role of the IQCH gene in spermatogenesis, focusing on its interaction with calmodulin and its regulation of RNA-binding proteins. The authors examined sperm from a male infertility patient with an inherited IQCH mutation as well as Iqch CRISPR knockout mice. The authors found that both human and mouse sperm exhibited structural and morphogenetic defects in multiple structures, leading to reduced fertility in Ichq-knockout male mice. Molecular analyses such as mass spectrometry and immunoprecipitation indicated that RNA-binding proteins are likely targets of IQCH, with the authors focusing on the RNA-binding protein HNRPAB as a critical regulator of testicular mRNAs. The authors used in vitro cell culture models to demonstrate an interaction between IQCH and calmodulin, in addition to showing that this interaction via the IQ motif of IQCH is required for IQCH's function in promoting HNRPAB expression. In sum, the authors concluded that IQCH promotes male fertility by binding to calmodulin and controlling HNRPAB expression to regulate the expression of essential mRNAs for spermatogenesis. These findings provide new insight into molecular mechanisms underlying spermatogenesis and how important factors for sperm morphogenesis and function are regulated.

The strengths of the study include the use of mouse and human samples, which demonstrate a likely relevance of the mouse model to humans; the use of multiple biochemical techniques to address the molecular mechanisms involved; the development of a new CRISPR mouse model; ample controls; and clearly displayed results. There are some minor weaknesses in that more background details could be provided to the reader regarding the proteins involved; some assays could benefit from more rigorous quantification; some of the mouse testis images and analyses could be improved; and larger sample sizes, especially for the male mouse breeding tests, could be increased. Overall, the claims made the authors in this manuscript are well-supported by the data provided, but there some technical issues that, if addressed, could increase the robustness and rigor of the study.

1. More background details are needed regarding the proteins involved, in particular IQ proteins and calmodulin. The authors state that IQ proteins are not well-represented in the literature, but do not state how many IQ proteins are encoded in the genome. They also do not provide specifics regarding which calmodulins are involved, since there are at least 5 family members in mice and humans. This information could help provide more granular details about the mechanism to the reader and help place the findings in context.

2. The mouse fertility tests could be improved with more depth and rigor. There was no data regarding copulatory plug rate; data was unclear regarding how many WT females were used for the male breeding tests and how many litters were generated; the general methodology used for the breeding tests in the Methods section was not very explicitly or clearly described; the sample size of n=3 for the male breeding tests is rather small for that type of assay; and, given that ICHQ appears to be expressed in testicular interstitial cells (Fig. S10) and somewhat in other organs (Fig. S2), another important parameter of male fertility that should be addressed is reproductive hormone levels (e.g., LH, FSH, and testosterone). While normal epididymal size in Fig. S3 suggests that hormone (testosterone) levels are normal, epididymal size and/or weight were not rigorously quantified.

3. The Western blots in Figure 6 should be rigorously quantified from multiple independent experiments so that there is stronger evidence supporting claims based on those assays.

4. Some of the mouse testis images could be improved. For example, the PNA and PLCz images in Figure S7 are difficult to interpret in that the tubules do not appear to be stage-matched, and since the authors claimed that testicular histology is unaffected in knockout testes, it should be feasible to stage-match control and knockout samples. Also, the anti-ICHQ and CaM immunofluorescence in Figure S10 would benefit from some cell-type-specific co-stains to more rigorously define their expression patterns, and they should also be stage-matched.

Reviewer #3 (Public Review):

In this study, the authors investigate the role of the Notch signalling regulator RBP-J on Ly6Clow monocyte biology starting with the observation that RBP-J-deficient mice have increased circulating Ly6low monocytes. Using myeloid specific conditional mouse models, the authors investigate how RBP-J deficiency effects circulating monocytes and lung interstitial macrophages.<br /> A major strength of this study is that it provides compelling evidence that RBP-J is a novel, critical factor regulating Ly6Clow monocyte cell frequency in the blood. The authors demonstrate that RBP-J deficiency leads to increased Ly6Clow monocytes in the blood and lung and CD16.2+ interstitial macrophages in steady state. The authors use a number of different techniques to confirm this finding including bone marrow transplantation experiments and parabiosis.

The main conclusion of the paper is that RBP-J controls the fate of Ly6ClowCCR2hi monocytes in a cell-intrinsic manner. This conclusion is strongly supported by the data provided. However, this paper is predominantly descriptive and further research is required to fully uncover the mechanisms by which RBP-J deficiency leads to Ly6Clo monocyte numbers increasing specifically in the blood and lungs and the consequence of RBP-J deficiency on Ly6C-low monocyte functionality.

The authors have performed RNA-seq and more in-depth analysis of this sequencing may provide clues for uncovering the thus far elusive mechanism.

Reviewer #3 (Public Review):

In this study, the authors utilized mass spectrometry-based quantification of polar metabolites and lipids in normal and cancerous tissue interstitial fluid and plasma. This showed that nutrient availability in tumor interstitial fluid was similar to that of interstitial fluid in adjacent normal kidney tissue, but that nutrients found in both interstitial fluid compartments were different from those found in plasma. This suggests that the nutrients in kidney tissue differ from those found in blood and that nutrients found in kidney tumors are largely dictated by factors shared with normal kidney tissue. Those data could be useful as a resource to support further study and modeling of the local environment of RCC and normal kidney physiology.

In Figures 1D and 1E, there were about 30% of polar metabolites and 25% of lipids significantly different between TIF and KIF, which could be key factors for RCC tumors. This reviewer considers that the authors should make comments on this.

Reviewer #3 (Public Review):

Mohammed et al perform functional follow-up studies on the single nucleotide polymorphism rs6740960 located on chromosome 2p21 that was previously linked to lower jaw and chin shape variation and an increased risk of non-syndromic orofacial clefting. Through a combination of in silico multi-species alignment, in vitro enhancer marks, and finally in vivo data the team could confirm that the SNP is located in an active enhancer element driving transgene expression in the upper and lower jaw. The team tested the human and chimp orthologs in transgenic mice. Interestingly the mouse ought to look did not show any active enhancer activity in the LacZ reporter assay. Next, the authors could show a selective interaction of the enhancer element with the neighboring gene PKDCC in chondrocytes using H3K27ac HiChIP. Deletion of this enhancer in vitro led to an allele specific reduction of PKC expression. Finally, the authors aimed at evaluating the effect of rs6740960 in vivo using a mouse model. Since the enhancer sequence of the mouse did not show any positive reporter activity, the authors decided to use previously described Pkdcc full knockout mouse model (Kinoshita et al. 2009). Using sophisticated imaging technologies the authors were able to show that in mice several facial bones are Pkdcc dose sensitive.

Overall this is an extremely exciting manuscript that addresses one of the key challenges in the post GWAS time: the functional connection of lead SNPs to their target genes and a detailed evaluation of the biological and morphological consequences.<br /> The manuscript is well written, and the conclusions are completely supported by the evidence provided. I really think this is a great paper, however I have several major concerns with the manuscript and its current format.

Major comments:

1: My main concern about the manuscript in its current format is the disconnection between the beautiful work of linking rs6740960 to Pkdcc in the first part of the manuscript and the investigation of dose sensitivity of Pkdcc itself in end of the manuscript. While I realized that this is because the enhancer itself is not conserved between humans and mice, in my opinion it still weakens the novelty of the finding of the second part of the manuscript quite significantly. The Pkdcc knockout has been well described and that the authors now present evidence that also heterozygous knockouts show a minimal phenotype in the facial bones is really not surprising. More importantly it doesn't show how the rs6740960 influences Pkdcc expression in vivo.

A rather straightforward and very interesting experimental approach would be to replace the mouse enhancer sequence with the human or chimp enhancer carrying the risk allele or the wild type. In the last figure the authors have nicely shown that the entire experimental setup for the functional analysis of even minor changes to the facial bones caused by the SNP are available to the team. Even if the result was negative this experiment would significantly enhance the scientific impact of the paper.

2: Another option would be to repeat the LacZ reporter essay with the human wild type and the risk allele in direct comparison. A beautiful example of such an experiment was recently shown by Yanchuset et al (A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation, Yanchuset al.,Science378,68-78 2022)

3: It is unclear how the H3K27ac HiChIP signal looks like at the Pkdcc locus in H9 ESC. What is the naïve interaction profile?

Reviewer #3 (Public Review):

This study represents a useful addition to the authors' previous study examining the effects of paternal high-fat diet on offspring metabolism and gene expression in offspring (PMID: 35183795). It differs from the previous study in some of the details of the experimental model (age of sire when exposed to the diet manipulation, mouse substrain, and the nature of the control diet) and the results are largely in line with previous findings. The major finding is that many genes at which sperm H3K4me3 signal is altered also have altered expression in the placenta; some of these genes are paternally imprinted, providing a paternal-specific epigenetic signature. Strengths of the study include establishment of an important dataset correlating the sperm epigenome with gene expression in placental tissue, leading to an interesting and provocative conclusion. Weaknesses include a relatively superficial analysis of the dataset, revealing broad patterns but few specific conclusions, reliance on correlative analysis to draw conclusions, and absence of validation studies. Deconvolution analysis of bulk RNA-seq data helps to account for differences in cell composition between placental datasets, but does not add additional insight toward the central question of how sperm epigenetic state contributes to offspring gene expression. Overall the advance over previous work is relatively small.

Specific points:

1) The analysis as it stands is limited. To compare sperm H3K4me3 and placental expression, numbers of overlapping genes are provided, but no statistical analysis is done to indicate the significance of the overlap.

2) There is little direct connection to biological systems or validation of differential enrichment/expression analysis. Gene ontology enrichments for genes differentially enriched for H3K4me3 in sperm or differentially expressed in placenta (broken up by sex) are performed, but the biological significance of these categories is not clear.

3) The overall effect size is small. In most cases the magnitude of differences is minor, and it is not clear which of these changes are significant over noise. For example, the y-axis for the metagene plots in Figure 2B does not start at zero, so the total range of the difference in H3K4me3 is small. In Figure 6C, DEGs detected in hypoxic placenta after deconvolution analysis do not look very different compared to control.

4) Deconvolution analysis was done on bulk RNA-seq data from placenta, and the numbers of DEGs identified with this analysis compared to the original analysis are shown, but is not clear how the deconvolution analysis changes the specific biological conclusions. In addition, the reference dataset for deconvolution is a published dataset generated in another lab, and it is unclear how comparable the reference sample is to the samples analyzed in this study, or how robust this analysis is when using a dataset generated under different conditions.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Verma et al. set out to visualize cytoplasmic dynein in living cells and describe their behaviour. They first generated heterozygous CRISPR-Cas9 knock-ins of DHC1 and p50 subunit of dynactin and used spinning disk confocal microscopy and TIRF microscopy to visualize these EGFP-tagged molecules. They describe robust localization and movement of DHC and p50 at the plus tips of MTs, which was abrogated using SiR tubulin to visualize the pool of DHC and p50 on the MTs. These DHC and p50 punctae on the MTs showed similar, highly processive movement on MTs. Based on comparison to inducible EGFP-tagged kinesin-1 intensity in Drosophila S2 cells, the authors concluded that the DHC and p50 punctae visualized represented 1 DHC-EGFP dimer+1 untagged DHC dimer and 1 p50-EGFP+3 untagged p50 molecules.

Strengths:<br /> The idea and motivation behind this work are commendable.

Weaknesses:<br /> There are several major issues with the characterization of the knock-in lines generated, the choice of imaging and analysis methods, and inadequate discussion of prior findings.

The specific points are below:

1. CRISPR-edited HeLa clones:<br /> (i) The authors indicate that both the DHC-EGFP and p50-EGFP lines are heterozygous and that the level of DHC-EGFP was not measured due to technical difficulties. However, quantification of the relative amounts of untagged and tagged DHC needs to be performed - either using Western blot, immunofluorescence or qPCR comparing the parent cell line and the cell lines used in this work.<br /> (ii) The localization of DHC predominantly at the plus tips (Fig. 1A) is at odds with other work where endogenous or close-to-endogenous levels of DHC were visualized in HeLa cells and other non-polarized cells like HEK293, A-431 and U-251MG (e.g.: OpenCell (https://opencell.czbiohub.org/target/CID001880), Human Protein Atlas (https://www.proteinatlas.org/ENSG00000197102-DYNC1H1/subcellular#human), https://www.biorxiv.org/content/10.1101/2021.04.05.438428v3). The authors should perform immunofluorescence of DHC in the parental cells and DHC-EGFP cells to confirm there are no expression artifacts in the latter. Additionally, a comparison of the colocalization of DHC with EB1 in the parental and DHC-EGFP and p50-EGFP lines would be good to confirm MT plus-tip localisation of DHC in both lines.<br /> (iii) It would also be useful to see entire fields of view of cells expressing DHC-EGFP and p50-EGFP (e.g. in Spinning Disk microscopy) to understand if there is heterogeneity in expression. Similarly, it would be useful to report the relative levels of expression of EGFP (by measuring the total intensity of EGFP fluorescence per cell) in those cells employed for the analysis in the manuscript.<br /> (iv) Given that the authors suspect there is differential gene regulation in their CRISPR-edited lines, it cannot be concluded that the DHC-EGFP and p50-EGFP punctae tracked are functional and not piggybacking on untagged proteins. The authors could use the FKBP part of the FKBP-EGFP tag to perform knock-sideways of the DHC and p50 to the plasma membrane and confirm abrogation of dynein activity by visualizing known dynein targets such as the Golgi (Golgi should disperse following recruitment of EGFP-tagged DHC-EGFP or p50-EGFP to the PM), or EGF (movement towards the cell center should cease).

2. TIFRM and analysis:<br /> (i) What was the rationale for using TIRFM given its limitation of visualization at/near the plasma membrane? Are the authors confident they are in TIRF mode and not HILO, which would fit with the representative images shown in the manuscript?<br /> (ii) At what depth are the authors imaging DHC-EGFP and p50-EGFP?<br /> (iii) The authors rely on manual inspection of tracks before analyzing them in kymographs - this is not rigorous and is prone to bias. They should instead track the molecules using single particle tracking tools (eg. TrackMate/uTrack), and use these traces to then quantify the displacement, velocity, and run-time.<br /> (iv) It is unclear how the tracks that were eventually used in the quantification were chosen. Are they representative of the kind of movements seen? Kymographs of dynein movement along an entire MT/cell needs to be shown and all punctae that appear on MTs need to be tracked, and their movement quantified.<br /> (v) What is the directionality of the moving punctae?<br /> (vi) Since all the quantification was performed on SiR tubulin-treated cells, it is unclear if the behavior of dynein observed here reflects the behavior of dynein in untreated cells. Analysis of untreated cells is required.

3. Estimation of stoichiometry of DHC and p50<br /> Given that the punctae of DHC-EGFP and p50 seemingly bleach on MT before the end of the movie, the authors should use photobleaching to estimate the number of molecules in their punctae, either by simple counting the number of bleaching steps or by measuring single-step sizes and estimating the number of molecules from the intensity of punctae in the first frame.

4. Discussion of prior literature<br /> Recent work visualizing the behavior of dyneins in HeLa cells (DOI: 10.1101/2021.04.05.438428), which shows results that do not align with observations in this manuscript, has not been discussed. These contradictory findings need to be discussed, and a more objective assessment of the literature in general needs to be undertaken.

Reviewer #3 (Public Review):

Summary:<br /> Using a protein degradation approach, Eaton et al show that INST11 can terminate the sense and anti-sense transcription but higher activity of CDK9 in the sense direction protects it from INS11-dependent termination. They developed sPOINT-seq that detects nascent 5'-capped RNA. The technique allowed them to reveal robust transcription initiation of sense-RNA as compared to anti-sense.

Strengths:<br /> The strength of the paper is the acute degradation of proteins, eliminating the off-target effects. Further, the paper uses elegant approaches such as POINT and sPOINT-seq to measure nascent RNA and 5'-capped short RNA. Together, the combination of these three allowed the authors to make clean interpretations of data.

Weaknesses:<br /> While the manuscript is well written, the details on the panel are not sufficient. The methods could be elaborated to aid understanding. Additional discussion on howthe authors' findings contradict the existing model of anti-sense transcription termination should be added.

Reviewer #3 (Public Review):

Summary:

The manuscript by Flaherty III S.E. et al identified SPAG7 gene in their forward mutagenetic screening and created the germline knockout and inducible knockout mice. The authors reported that the SPAG7 germline knockout mice had lower birth weight likely due to intrauterine growth restriction and placental insufficiency. The SPAG7 KO mice later developed obesity phenotype as result of reduced energy expenditure. However, the inducible SPAG7 knockout mice had normal body weight and composition.

Strengths:

In this reviewer's opinion, this study has high significance in the field of metabolic research for the following reasons.

1) The authors' findings are significant in the field of obesity research, especially from the perspective of maternal-fetal medicine. The authors created and analyzed the SPAG7 KO mice and found that the KO mice had a "thrifty phenotype" and developed obesity.

2) SPAG7 gene function hasn't been thoroughly studied. The reported phenotype will fill the gap of knowledge.

Overall, the authors have presented their results in a clear and logically organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings.

Comments on revised version:

The authors have satisfactorily addressed my previous concerns.

Reviewer #3 (Public Review):

Summary:<br /> Radial muscle growth involves an increase in overall muscle cross-sectional area. For decades this process has been described as the splitting of myofibrils to produce more myofibrils during the growth process. However, a closer look at the original papers shows that the evidence underlying this description was incomplete. In this paper, the authors have developed a novel method using fluorescence microscopy to directly measure myofibril size and number. Using a mouse model of mechanical loading and a human model of resistance exercise they discovered that myofibrillogenesis is playing a key role in the radial growth of muscle fibers.

Strengths:<br /> 1. Well-written and clear description of hypothesis, background, and experiments.<br /> 2. Compelling series of experiments.<br /> 3. Different approaches to test the hypothesis.<br /> 4. Rigorous study design.<br /> 5. Clear interpretation of results.<br /> 6. Novel findings that will be beneficial to the muscle biology field.<br /> 7. Innovative microscopy methods that should be widely available for use in other muscle biology labs.

Reviewer #3 (Public Review):

Summary:<br /> Previous research on the Drosophila mushroom body (MB) has made this structure the best-understood example of an associative memory center in the animal kingdom. This is in no small part due to the generation of cell-type specific driver lines that have allowed consistent and reproducible genetic access to many of the MB's component neurons. The manuscript by Shuai et al. now vastly extends the number of driver lines available to researchers interested in studying learning and memory circuits in the fly. It is an 800-plus collection of new cell-type specific drivers target neurons that either provide input (direct or indirect) to MB neurons or that receive output from them. Many of the new drivers target neurons in sensory pathways that convey conditioned and unconditioned stimuli to the MB. Most drivers are exquisitely selective, and researchers will benefit from the fact that whenever possible, the authors have identified the targeted cell types within the Drosophila connectome. Driver expression patterns are beautifully documented and are publicly available through the Janelia Research Campus's Flylight database where full imaging results can be accessed. Overall, the manuscript significantly augments the number of cell type-specific driver lines available to the Drosophila research community for investigating the cellular mechanisms underlying learning and memory in the fly. Many of the lines will also be useful in dissecting the function of the neural circuits that mediate sensorimotor circuits.

Strengths:<br /> The manuscript represents a huge amount of careful work and leverages numerous important developments from the last several years. These include the thousands of recently generated split-Gal4 lines at Janelia and the computational tools for pairing them to make exquisitely specific targeting reagents. In addition, the manuscript takes full advantage of the recently released Drosophila connectomes. Driver expression patterns are beautifully illustrated side-by-side with corresponding skeletonized neurons reconstructed by EM. A comprehensive table of the new lines, their split-Gal4 components, their neuronal targets, and other valuable information will make this collection eminently useful to end-users. In addition to the anatomical characterization, the manuscript also illustrates the functional utility of the new lines in optogenetic experiments. In one example, the authors identify a specific subset of sugar reward neurons that robustly promotes associative learning.

Weaknesses:<br /> While the manuscript succeeds in making a mass of descriptive detail quite accessible to the reader, the way the collection is initially described - and the new lines categorized - in the text is sometimes confusing. Most of the details can be found elsewhere, but it would be useful to know how many of the lines are being presented for the first time and have not been previously introduced in other publications/contexts. And where can the lines be found at Flylight? Are they listed as one collection or as many? Also, the authors say that some of the lines were included in the collection despite not necessarily targeting the intended type of neuron (presumably one that is involved in learning and memory). What percentage of the collection falls into this category? And what about the lines that the authors say they included in the collection despite a lack of specificity? How many lines does this represent?

Reviewer #3 (Public Review):

Summary:

Te Rietmolen et al., investigated the selectivity of cortical responses to speech and music stimuli using neurosurgical stereo EEG in humans. The authors address two basic questions: 1. Are speech and music responses localized in the brain or distributed; 2. Are these responses selective and domain-specific or rather domain-general and shared? To investigate this, the study proposes a nomenclature of shared responses (speech and music responses are not significantly different), domain selective (one domain is significant from baseline and the other is not), domain preferred (both are significant from baseline but one is larger than the other and significantly different from each other). The authors employ this framework using neural responses across the spectrum (rather than focusing on high gamma), providing evidence for a low level of selectivity across spectral signatures. To investigate the nature of the underlying representations they use encoding models to predict neural responses (low and high frequency) given a feature space of the stimulus envelope or peak rate (by time delay) and find stronger encoding for both in the low-frequency neural responses. The top encoding electrodes are used as seeds for a pair-wise connectivity (coherence) in order to repeat the shared/selective/preferred analysis across the spectra, suggesting low selectivity. Spectral power and connectivity are also analyzed on the level of the regional patient population to rule out (and depict) any effects driven by a select few patients. Across analyses the authors consistently show a paucity of domain selective responses and when evident these selective responses were not represented across the entire cortical region. The authors argue that speech and music mostly rely on shared neural resources.

Strengths:

I found this manuscript to be rigorous providing compelling and clear evidence of shared neural signatures for speech and music. The use of intracranial recordings provides an important spatial and temporal resolution that lends itself to the power, connectivity, and encoding analyses. The statistics and methods employed are rigorous and reliable, estimated based on permutation approaches, and cross-validation/regularization was employed and reported properly. The analysis of measures across the entire spectra in both power, coherence, and encoding models provides a comprehensive view of responses that no doubt will benefit the community as an invaluable resource. Analysis of the level of patient population (feasible with their high N) per region also supports the generalizability of the conclusions across a relatively large cohort of patients. Last but not least, I believe the framework of selective, preferred, and shared is a welcome lens through which to investigate cortical function.

Weaknesses:

I did not find methodological weaknesses in the current version of the manuscript. I do believe that it is important to highlight that the data is limited to passively listening to naturalistic speech and music. The speech and music stimuli are not completely controlled with varying key acoustic features (inherent to the different domains). Overall, I found the differences in stimulus and lack of attentional controls (passive listening) to be minor weaknesses that would not dramatically change the results or conclusions.

Reviewer #3 (Public Review):

Summary:<br /> This paper explores the cost of toxin resistance in snakes that prey on newts defended by highly potent TTX. Two species of garter snakes, T. atratus and T. sirtalis, are examined. Both species have resistant and sensitive populations. Resistance is achieved by substitutions in the voltage-gated sodium channels, which block TTX binding. Resistant T. atratus carry the triple substitutions EPN while resistant T. sirtalis carry the quadruple LVNV. These substitutions occur on the third and fourth intracellular domains of the voltage-gated sodium-channel gene Nav1.4, which is the paralog found in skeletal muscle. EPN and LVNV have been previously attributed to conferring resistance to TXX through target-site insensitivity of the channel. Previous work has also shown that snakes from resistant populations have reduced locomotor capabilities compared to their non-resistant counterparts.

The authors systematically test the hypothesis that the resistance-conferring substitutions affect other phenotypes related to the function of the voltage-gated sodium channel, which is, in turn, responsible for the reduced locomotor capabilities. First, they compare the effects of EPN and LVNV on recombinantly expressed rat Nav1.4 with and without EPN and LVNV (in vitro). They find that both EPN and LVNV significantly reduce the channel's conductance. On top of that, LVNV also causes premature deactivation of the channel, thus reducing the current passing through the membrane. Next, they compare muscle tissue function between resistant and non-resistant populations of T. atratus and T. sirtalis (ex vivo). They find that both resistant populations have reduced twitch force (with T. sirtalis, carrying LVNV, having an even stronger reduction), reduced peak rate of force development, and overall reduced force. In addition, T. sirtalis (LVNV) muscle also has reduced peak tetanic force. Finally, they compare the biophysical effects of EPN and LVNV through homology modeling of Nav1.4 to explain the in vitro and in vivo results (in silico). They found that E1248 (of EPN) has a counteracting effect on the destabilizing effect of N1539, shared by both species. T. sirtalis (LVNV) lacks such a counteracting mutation, which could explain the stronger negative effects observed in LVNV channels and muscles.

Strengths:<br /> A particular strength of this paper is the multi-level approach used to tease apart the negative pleiotropic effects of resistance-conferring substitutions. Each level of experiments informed the next, creating a focused comprehensive analysis of the costs associated with this specialized dietary adaptation in snakes. The results make an important contribution to our understanding of the role of negative pleiotropy in adaptive evolution and would be of broad interest to readers. The paper is well-written, and the data and analyses are clearly presented.

Weaknesses:<br /> The sheer size of the Nav1.4 gene makes it difficult to clone into an expression vector and that's probably why an already cloned rat Nav1.4 was selected for the in vitro experiments. It would be great if the authors could comment on how the level of resistance produced by mutations on the rat Nav1.4 compared to the garter snake Nav1.4s. Are there previous data on tissue-isolated T. sirtalis and T. atratus channels? Is it possible that the snake mutations have slightly different effects on the rat genetic background due to epistatic interactions with sites beyond the 3rd and 4th domains?

Following up on the first comment, sometimes negative pleiotropic effects are mitigated by compensatory mutations in other regions of the protein. This reviewer would recommend that the authors comment on this possibility. Are there substitutions beyond the 3rd and 4th domains that could potentially play a role in this adaptation?

Based on the results, it seems that resistant T. sirtalis got the shorter end of the stick concerning negative pleiotropic effects, despite having similar (the same?) levels of resistance to TTX. Does this difference/disadvantage scale up to locomotor performance as well?

It would be great if the authors could comment on how these resistant populations have persisted despite the locomotor/muscular disadvantages. Are there known differences in predation rates between the populations? The benefit must have outweighed the cost in these cases.

Reviewer #3 (Public Review):

Summary:<br /> The authors of this study aim to develop OpenNucleome, a computational tool designed to simulate the structure and dynamics of the human nucleus. This software models nuclear components like chromosomes and nuclear bodies, leveraging GPU acceleration for improved performance. The key objective is to enhance our understanding of nuclear organization, providing a tool that aligns with experimental data and is accessible to the genome architecture scientific community.

Strengths:<br /> OpenNucleome offers a detailed and dynamic model of the nucleus, a significant step forward in computational biology.

The integration of GPU acceleration with the OpenMM package is a good technical advancement, potentially enhancing performance.

The comparison with experimental data adds credibility to the tool's accuracy and relevance.

Weaknesses:<br /> The lack of comprehensive tutorials and clear documentation on the OpenNucleome GitHub page is a considerable barrier to accessibility and user-friendliness.

The process for generating necessary input files is not adequately explained, which could hinder the tool's practical application.

The paper could benefit from more explicit explanations on the standardization of practices and cross-validation with existing tools like OpenMiChroM.

Reviewer #3 (Public Review):

Summary:<br /> In the manuscript "Overflow metabolism originates from growth optimization and cell heterogeneity" the author Xin Wang investigates the hypothesis that the transition into overflow metabolism at large growth rates actually results from an inhomogeneous cell population, in which every individual cell either performs respiration or fermentation.

Weaknesses:<br /> The paper has several major flaws. First, and most importantly, it repeatedly and wrongly claims that the origins of overflow metabolism are not known. The paper is written as if it is the first to study overflow metabolism and provide a sound explanation for the experimental observations. This is obviously not true and the author actually cites many papers in which explanations of overflow metabolism are suggested (see e.g. Basan et al. 2015, which even has the title "Overflow metabolism in E. coli results from efficient proteome allocation"). The paper should be rewritten in a more modest and scientific style, not attempting to make claims of novelty that are not supported. In fact, all hypotheses in this paper are old. Also the possiblility that cell heterogeneity explains the observed 'smooth' transition into overflow metabolism has been extensively investigated previously (see de Groot et al. 2023, PNAS, "Effective bet-hedging through growth rate dependent stability") and the random drawing of kcat-values is an established technique (Beg et al., 2007, PNAS, "Intracellular crowding defines the mode and sequence of substrate uptake by Escherichia coli and constrains its metabolic activity"). Thus, in terms of novelty, this paper is very limited. It reinvents the wheel and it is written as if decades of literature debating overflow metabolism did not exist.

Moreover, the manuscript is not clearly written and is hard to understand. Variables are not properly introduced (the M-pools need to be discussed, fluxes (J_E), "energy coefficients" (eta_E), etc. need to be more explicitly explained. What is "flux balance at each intermediate node"? How is the "proteome efficiency" of a pathway defined? The paper continues to speak of energy production. This should be avoided. Energy is conserved (1st law of thermodynamics) and can never be produced. A scientific paper should strive for scientific correctness, including precise choice of words.

The statement that the "energy production rate ... is proportional to the growth rate" is, apart from being incorrect - it should be 'ATP consumption rate' or similar (see above), a non-trivial claim. Why should this be the case? Such statements must be supported by references. The observation that the catabolic power indeed appears to increase linearly with growth rate was made, based on chemostat data for E.coli and yeast, in a recent preprint (Ebenhöh et al, 2023, bioRxiv, "Microbial pathway thermodynamics: structural models unveil anabolic and catabolic processes").

All this criticism does not preclude the possibility that cell heterogeneity plays a role in overflow metabolism. However, according to Occam's razor, first the simpler explanations should be explored and refuted before coming up with a more complex solution. Here, it means that the authors first should argue why simpler explanations (e.g. the 'Membrane Real Estate Hypothesis', Szenk et al., 2017, Cell Systems; maximal Gibbs free energy dissipation, Niebel et al., 2019, Nature Metabolism; Saadat et al., 2020, Entropy) are not considered, resp. in what way they are in disagreement with observations, and then provide some evidence of the proposed cell heterogeneity (are there single-cell transcriptomic data supporting the claim?).

Reviewer #3 (Public Review):

The manuscript by Agha et al. explores mechanisms of rhythmicity in V2a neurons in larval zebrafish. Two subpopulations of V2a neurons are distinguishable by anatomy, connectivity, level of GFP, and speed-dependent recruitment properties consistent with V2a neurons involved in rhythm generation and pattern formation. The descending neurons proposed to be consistent with rhythm-generating neurons are active during either slow or fast locomotion, and their firing frequencies during current steps are well matched with the swim frequency they firing during. The bifurcating (patterning neurons) are active during a broader swim frequency range unrelated to their firing during current steps. All of the V2a neurons receive strong inhibitory input but the phasing of this input is based on neuronal type and swim speed when the neuron is active, with prominent in-phase inhibition in slow descending V2a neurons and bifurcating V2a neurons active during fast swimming. Antiphase inhibition is observed in all V2a neurons but it is the main source of rhythmic inhibition in fast descending V2a neurons and bifurcating neurons active during slow swimming. The authors suggest that properties supporting rhythmic bursting are not directly related to locomotor speed but rather to functional neuronal subtypes.

This is a well-written paper with many strengths including the rigorous approach. Many parameters, including projection pattern, intracellular properties, inhibition received, and activity during slow/fast swimming were obtained from the same neuron. This links up very well with prior data from the lab on cell position, birth order, morphology/projections, and control of MN recruitment to provide a comprehensive overview of the functioning of V2a interneuronal populations in the larval zebrafish. The overall conclusions are well supported by the data. Weaknesses are relatively minor and were largely related to terminology for some of the secondary conclusions.

1. The assumption is made that all in-phase inhibition is recurrent and out-of-phase inhibition is reciprocal. The latter is likely true but the definition of recurrent may be a bit loose as could be multisegmental feed-forward inhibition as well.

2. In a few places, it is mentioned that the properties of the V2a-D neurons are consistent with pacemakers. This could be true of both the V2a-D and -B neurons that burst in response to depolarizing steps but the properties of the remaining (fast) V2a-D neurons do not seem to be consistent with pacemakers, based on the properties shown. Tonic firing at a frequency related to the locomotor speed the neuron is active during and strong antiphase inhibition may instead suggest a stronger network component driving the rhythmicity.

Reviewer #3 (Public Review):

This manuscript presents a comprehensive investigation into the mechanisms that explain the presence of TADs (P-TADs) in cells where cohesin has been removed. In particular, to study TADs in wildtype and cohesin depleted cells, the authors use a combination of polymer simulations to predict whole chromosome structures de novo and from Hi-C data. Interestingly, they find that those TADs that survive cohesin removal contain a switch in epigenetic marks (from compartment A to B or B to A) at the boundary. Additionally, they find that the P-TADs are needed to retain enhancer-promoter and promoter-promoter interactions.

Overall, the study is well-executed, and the evidence found provides interesting insights into genome folding and interpretations of conflicting results on the role of cohesin on TAD formation.

Reviewer #3 (Public Review):

Summary: The manuscript by Erice et al describes let-7 miRNA promotes Tc17 differentiation and emphysema by repressing the transcription factor RORgt. The authors found that overall expression of the let-7 miRNA clusters, let-7b/let-7c2 and let-7a1/let-30 7f1/let-7d are reduced in the lungs and T cells of mice with cigarette smoke-induced emphysema. They also found that the loss of the let-7b/let-7c2-cluster in T cells exaggerated cigarette smoke-induced emphysema. It appears that deletion of the let-7b/let-7c2-cluster lead to enhancement of IL-17-secreting CD8+ T cells (Tc17) in mice with emphysema. The opposite phenotype was observed when let-7 was overexpressed in T cells. They found a potential let-7 binding site in the 3' UTR of RORgt. They demonstrated a direct effect of let-7 on RORgt expression using let-7 mimic in a RORgt luciferase reporter assay. They have done an outstanding job of translating the finding of reduced let-7 expression in emphysema patients to a thorough delineation of its mechanism in a mouse model. Together, this study suggests an important role for let-7 miRNA in Tc17 cells in emphysema which appears to be mediated via repression of RORgt.

Strengths: This well written manuscript flows logically and the data supports the overall claim let-7 miRNA promotes Tc17 differentiation during emphysema. There are several strengths to this study including the use of conditional let-7 knock out animals to decipher the role of this miRNA in Tc17 cells in emphysema.

Weaknesses: There are no major weaknesses in this study. It would be interesting to see if knockdown RORgt could rescue enhanced Tc17 differentiation seen in let-7b/let-7c2-cluster-deficient T cells. The authors show no change in frequencies of Treg cells in let-7bc2LOF mice exposed to nCB. Do these Treg cells also express higher levels of RORgt and IL-17? The major question that was not addressed in this study is how let-7 expression is regulated in emphysema. The other recommendation is that the authors include the sequences of the let-7 mimic oligos used in the luciferase assay.

Reviewer #3 (Public Review):

Summary:<br /> Adamic and colleagues present fMRI data from ADE patients and a healthy control group acquired during two interoceptive tasks (attention and perturbation) from the same session. They report convergent activity within the granular and dysgranular insular cortex during both tasks, with a patient group-specific lateralisation effect. Furthermore, insular functional connectivity was found to be linked to disease severity.

Strengths:<br /> The study is well-designed and - despite some limitations noted by the authors - provides much-needed insight into the functional pathways of interoceptive processing in health and disease. The manuscript is clear, concise, and well-written so that I only have a few comments I would mostly regard as minor points.

Weaknesses:<br /> There are a few instances where it is not entirely clear whether the authors' claims are fully supported by the underlying statistics.

Reviewer #3 (Public Review):

Summary:<br /> Here the authors show global synchronization of cerebral blood flow (CBF) induced by oscillating visual stimuli in the mouse brain. The study validates the use of endogenous autofluorescence to quantify the vessel "shadow" to assess the magnitude of frequency-locked cerebral blood flow changes. This approach enables straightforward estimation of artery diameter fluctuations in wild-type mice, employing either low magnification wide-field microscopy or deep-brain fibre photometry. For the visual stimuli, awake mice were exposed to vertically oscillating stripes at a low temporal frequency (0.25 Hz), resulting in oscillatory changes in artery diameter synchronized to the visual stimulation frequency. This phenomenon occurred not only in the primary visual cortex but also across a broad cortical and cerebellar surface. The induced CBF changes adapted to various stimulation parameters, and interestingly, repeated trials led to plastic entrainment. The authors control for different artefacts that may have confounded the measurements such as light contamination and eye movements but found no influence of these variables. The study also tested horizontally oscillating visual stimuli, which induce the horizontal optokinetic response (HOKR). The amplitude of eye movement, known to increase with repeated training sessions, showed a strong correlation with CBF entrainment magnitude in the cerebellar flocculus. The authors suggest that parallel plasticity in CBF and neuronal circuits is occurring. Overall, the study proposes that entrained "vasomotion" contributes to meeting the increased energy demand associated with coordinated neuronal activity and subsequent neuronal circuit reorganization.

Strengths:<br /> -The paper describes a simple and useful method for tracking vasomotion in awake mice through an intact skull.<br /> -The work controls for artefacts in their primary measurements.<br /> -There are some interesting observations, including the nearly brain-wide synchronization of cerebral blood flow oscillations to visual stimuli and that this process only occurs after mice are trained in a visual task.<br /> -This topic is interesting to many in the CBF, functional imaging, and dementia fields.

Weaknesses:<br /> -I have concerns with the main concepts put forward, regarding whether the authors are actually studying vasomotion as they state, as opposed to functional hyperemia which is sensory-induced changes in blood flow, which is what they are actually doing. I recommend several additional experiments/analyses for them to explore. This is mostly further characterizing their effect which will benefit the interpretations.

-Neuronal calcium imaging would also benefit the study and improve the interpretations.

-The plastic effects in vasomotion synchronization that occur with training are interesting but they could use an additional control for stress. Is this really a plastic effect, or is it caused by progressively decreasing stress as trials and progress? I recommend a habituation control experiment.

Appraisal<br /> I think the authors have an interesting effect that requires further characterization and controls. Their interpretations are likely sound and additional experiments will continue to support the main hypothesis. If brain-wide synchrony of blood flow can be trained and entrained by external stimuli, this may have interesting therapeutic potential to help clear out toxic proteins from the brain as seen in several neurodegenerative diseases.

Reviewer #3 (Public Review):

Summary:<br /> The study provides a detailed analysis of the chromosomal rearrangements related to the deletions of histidine-rich protein 2 (pfhrp2) and pfhrp3 genes in P. falciparum that have clinical significance since malaria rapid diagnostic tests detect these parasite proteins. A large number of publicly available short sequence reads for the whole genome of the parasite were analyzed, and data on coverage and discordant mapping allowed the authors to identify deletions, duplications, and chromosomal rearrangements related to pfhrp3 deletions. Long-read sequences showed support for the presence of a normal chromosome 11 and a hybrid 13-11 chromosome lacking pfhrp3 in some of the pfhrp3-deleted parasites. The findings support that these translocations have repeatedly occurred in natural populations. The authors discuss the implications of these findings and how they do or do not support previous hypotheses on the emergence of these deletions and the possible selective pressures involved.

Strengths:<br /> The genomic regions where these genes are located are challenging to study since they are highly repetitive and paralogous and the use of long-read sequencing allowed to span the duplicated regions, giving support to the identification of the hybrid 13-11 chromosome.

All publicly available whole-genome sequences of the malaria parasite from around the world were analysed which allowed an overview of the worldwide variability, even though this analysis is biased by the availability of sequences, as the authors recognize.

Despite the reduced sample size, the detailed analysis of haplotypes and identification of the location of breakpoints gives support to a single origin event for the 13-5++ parasites.

The analysis of haplotype variation across the duplicated chromosome-11 segment identified breakpoints at varied locations that support multiple translocation events in natural populations. The authors suggest these translocations may be occurring at high frequency in meiosis in natural populations but are strongly selected against in most circumstances, which remains to be tested.

Weaknesses:<br /> Relying on sequence data publicly available, that were collected based on diagnostic test positivity and that are limited by sequencing availability, limits the interpretation of the occurrence and relative frequency of the deletions. In the discussion, caution is needed when identifying the least common and most common mechanisms and their geographical associations. The identification of only one type of deletion pattern for Pfhrp2 may be related to these biases.

The specific objectives of the study are not stated clearly, and it is sometimes difficult to know which findings are new to this study. Is it the first study analyzing all the worldwide available sequences? Is it the first one to do long-read sequencing to span the entire duplicated region?

Another aspect that should be explained in the introduction is that there was previous information about the association of the deletions to patterns found in chromosomes 5 and 11. In the short-read sequences results, it is not clear if these chromosomes were analysed because of the associations found in this study (and no associations were found to putative duplications or deletions in other chromosomes), or if they were specifically included in the analysis because of the previous information (and the other chromosomes were not analysed).

An interesting statement in the discussion is that existing pfhrp3 deletions in a low-transmission environment may provide a genetic background on which less frequent pfhrp2 deletion events can occur. Does it mean that the occurrence of pfhrp3 deletions would favor the pfhrp2 deletion events? How, and is there any evidence for that?

Reviewer #3 (Public Review):

This neuroimaging study investigated how brain activity related to visual pattern-based reasoning changes over the adult lifespan, addressing the topic of functional compensation in older age. To this end, the authors employed a version of the Cattell task, which probes visual pattern recognition for identifying commonalities and differences within sets of abstract objects in order to infer the odd object among a given set. Using a state-of-the-art univariate analysis approach on fMRI data from a large lifespan sample, the authors identified brain regions in which the activation contrast between hard and easy Cattell task conditions was modulated by both age and performance. Regions identified comprised prefrontal areas and bilateral cuneus. Applying a multivariate decoding approach to activity in these regions, the authors went on to show that only in older adults, the cuneus, but not the prefrontal regions, carried information about the task condition (hard vs. easy) beyond that already provided by activity patterns of voxels that showed a univariate main effect of task difficulty. This was taken as compelling evidence for task-specific compensatory activity in the cuneus in advanced age.

The study is well-motivated and well-written. The authors used appropriate, rigorous methods that allowed them to control for a range of possible confounds or alternative explanations. Laudable aspects include the large sample with a wide and even age distribution, the validation of the in-scanner task performance against previous results obtained with a more standard version outside the scanner, and the control for vascular age-related differences in hemodynamic activity via a BOLD signal amplitude measure obtained from a separate resting-state fMRI scan. Overall, the conclusions are well-supported by the data.

In the following, I list some points of discussion that I would like to see addressed by the authors in a revision:

1) I don't quite follow the argumentation that compensatory recruitment would need to show via non-redundant information carried by any given non-MDN region (cf. p14). Wouldn't the fact that a non-MDN region carries task-related information be sufficient to infer that it is involved in the task and, if activated increasingly with increasing age, that its stronger recruitment reflects compensation, rather than inefficiency or dedifferentiation? Put differently, wouldn't "more of the same" in an additional region suffice to qualify as compensation, as compared to the "additional information in an additional region" requirement set by the authors? As a consequence, in my honest opinion, showing that decoding task difficulty from non-MDN ROIs works better with higher age would already count as evidence for compensation, rather than asking for age-related increases in decoding boosts obtained from adding such ROIs. It would be interesting to see whether the arguably redundant frontal ROI would satisfy this less demanding criterion. At any rate, it seems useful to show whether the difference in log evidence for the real vs. shuffled models is also related to age.

2) Relatedly, does the observed boost in decoding by adding the cuneal ROI (in older adults) really reflect "additional, non-redundant" information carried by this ROI? Or could it be that this boost is just a statistical phenomenon that is obtained because the cuneus just happens to show a more clear-cut, less noisy difference in hard vs. easy task activation patterns than does the MDN (which itself may suffer from increased neural inefficiency in older age), and thus the cuneaus improves decoding performance without containing additional (novel) pieces of information (but just more reliable ones)? If so, the compensation account could still be maintained by reference to the less demanding rationale for what constitutes compensation laid out above.

3) On page 21, the authors state that "...traditional univariate criteria alone are not sufficient for identifying functional compensation." To me, this conclusion is quite bold as I'd think that this depends on the unvariate criterion used. For instance, it could be argued that compensation should be more clearly indicated by an over additive interaction as observed for the relationship of cuneal activity with age and performance (i.e., the activity increase with better performance becomes stronger with age), rather than by an additive effect of age and performance as observed for the prefrontal ROI (see Fig. 2C). In any case, I'd appreciate it if the authors discussed this issue and the relationship between univariate and multivariate results in more detail (e.g. how many differences in sensitivity between the two approaches have contributed), in particular since the sophisticated multivariate approach used here is not widely established in the field yet.

4) As to the exclusion of poorly performing participants (see p24): If only based on the absolute number of errors, wouldn't you miss those who worked (overly) slowly but made few errors (possibly because of adjusting their speed-accuracy tradeoff)? Wouldn't it be reasonable to define a criterion based on the same performance measure (correct - incorrect) as used in the main behavioural analyses?

5) Did the authors consider testing for negative relationships between performance and brain activity, given that there is some literature arguing that neural efficiency (i.e. less activation) is the hallmark of high intelligence (i.e. high performance levels in the Cattell task)? If that were true, at least for some regions, the set of ROIs putatively carrying task-related information could be expanded beyond that examined here. If no such regions were found, it would provide some evidence bearing on the neural efficiency hypothesis.

Reviewer #3 (Public Review):

In this manuscript, Sperry and colleagues identify SNC80 as a compound that can slow metabolism and mimic hibernation, thereby prolonging tissue viability in organ transplantation and cardiovascular disease settings. Overall, the use of varied and relevant model systems is a strength of this study.

The authors perform a literature search to identify SNC80 as a promising hit. However, the details of the literature search, a list of other potential hits, and the criteria for identification of SNC80 are not described. The hypometabolic effect of SNC80 exposure is well-characterized in the Xenopus model. Furthermore, the authors show that SNC80 localises to the brain, but do not discuss several studies that have pointed to convulsions induced by exposure to high doses of SCN80, and whether this would be apparent in the Xenopus studies. The authors have promising data on the WB3 morpholino that retains or even improves on the hypometabolism phenotype of SCN80 while likely not retaining delta opioid activity. However, this is not functionally demonstrated. Moreover, WB3 is not used in any of the other assays and models used in the study. In the setting of cardiac transplant surgery, co-administration of SNC80 reduces metabolic activity and inflammation, although it is unclear if there is an improvement in recovery of organ function due to SCN80. The reversible induction of hypometabolic status is also demonstrated in two different organ chips. These models could identify the differential response of epithelial cells and vascular cells to drug perfusion, but the authors have mostly focused on the former. Finally, the authors identify specific targets for the hypometabolic effect of SNC80, which is a valuable resource for other screening studies and can form the basis for future work.

Reviewer #3 (Public Review):

Summary:

The authors are trying to find a vaccine solution for invasive candidiasis.

Strengths:

The testing of the antifungal activity of EDTA on Candida is not new as many other papers have examined this effect. The novelty here is the use of this EDTA-treated strain as a vaccine to protect against a secondary challenge with wild-type Candida.

Weaknesses:

However, data presented in Figure 5 and Figure 6 are not convincing and need further experimental controls and analysis as the authors do not show a time-dependent effect on the CFU of their vaccine formulation.

The methodology used is also an issue. As it stands, the impact is minor.

Reviewer #3 (Public Review):

Summary:<br /> These studies focus on a very interesting, understudied phenomenon in vascular development - the formation of pial collaterals between cerebral arteries. Understanding the mechanism(s) that regulates this process during normal development could provide important insights for the treatment of adult stroke patients, for which repair is highly dependent on collateral formation. Insights may also be relevant to other collateral-dependent diseases, such as heart disease and chronic peripheral ischemia.

Strengths:<br /> The investigators use lineage tracing and 3D imaging to show that, in mouse embryos, endothelial cells (ECs) predominantly from Bmx+ arteries and some from the Vegfr3+ microvasculature, invade pre-existing pre-collateral vascular structures in a process they termed "mosaic colonization", and arterialization of the vessel segments is said to occur concurrently with colonization, although details about EC phenotypes are lacking. Growth of the collaterals in response to ischemic injury relies on local replication of the ECs within the collaterals and not further recruitment from veins and the microvasculature. Although detailed molecular mechanisms are not provided, demonstration of the "cellular mechanism" of pial collateral vascularization is novel.

Weaknesses:<br /> Nonetheless, there are some issues that should be addressed, particularly to clarify the phenotype of the ECs forming the collaterals and expanding in response to injury; only their "origin" was traced and not their identity/growth after labeling in Bmx+ vessels.

Reviewer #3 (Public Review):

Summary:

Machhua et al. in their work focused on unravelling the molecular mechanism of daptomycin binding and interaction with bacterial cell membranes. Daptomycin (Dap) is an acidic, cyclic lipopeptide composed of 13 amino acids, known for preferential binding to anionic lipids, particularly phosphatidylglycerol (PG), which are prevalent components in the membranes of Gram-positive bacteria. The process of binding and antimicrobial efficacy of Dap is significantly influenced by the ionic composition of the surrounding environment, especially the presence of Ca2+ ions. The authors underscore the presence of significant knowledge gaps in our understanding of daptomycin's mode of action. Several critical questions remain unanswered, including the basis for selective recognition and accumulation in membranes of Gram-positive strains, the specific role of Ca2+ ions in this process, and the mechanisms by which daptomycin binds to and inserts into the cell membrane.

Dap is intrinsically fluorescent due to its kynurenine residue (Kyn-13) and this property allows direct imaging of Dap binding to model cell membranes without the need for additional labeling. Taking advantage of this Dap autofluorescence, authors monitored the emission intensity of micelles, composed of varying DMPG content upon their exposure to Dap and compared it with the kinetics of fluorescence observed for zwitterionic DMPC and other negatively charged lipids such as cardiolipin (CA), POPA and POPS. The authors noted that the linear relationship between DMPG content and Dap fluorescence is strongly lipid-specific, as it was not observed for other anionic lipids. The manuscript sheds light on the specificity of Dap's interaction with CA and DMPG lipids. Through Ca2+ sequestration with EGTA, the authors demonstrated that the binding of Dap with CA is reversible, while its interaction with DMPG results in the irreversible insertion of Dap into the lipid membrane structure, caused by the significant conformational change of this lipopeptide. The formation of a stable DMPG-Dap complex was also verified in bacterial cells isolated from Gram-positive bacteria B. subtilis, where Dap exhibited a permanent binding to PG lipids.

Altogether, the authors endeavored to illuminate novel insights into the molecular basis of Dap binding, interaction, and the mechanism of insertion into bacterial cell membranes. Such understanding holds promise for the development of innovative strategies in combating drug resistance and the emergence of the so-called superbugs.

Strengths:

- The manuscript by Machhua et al. provides a comprehensive analysis of the Dap mechanism of binding and interaction with the membrane. It discusses various aspects of this, only apparently trivial interactions such as the importance of PG presence in the membrane, the impact of Ca2+ ions, and different mechanisms of Dap binding with other negatively charged lipids.

- The authors focused not only on model membranes (micelles) but also extended their research to bacterial cell membranes obtained from B. subtilis.

- The research is not only a report of the experimental findings but tries to give potential hypotheses explaining the molecular mechanisms behind the observed results.

Weaknesses:

- The authors overestimate their findings, stating that they propose a novel mechanism of Dap interaction with bacterial cell membranes. In fact, they rather extend the already reported hypotheses.

- The literature study was not done as thoroughly as it should be. Many publications discussing the importance and mechanism of action of Ca2+ ions or conformational changes of daptomycin were not cited.

Reviewer #3 (Public Review):

Summary:

Yamada et al. build on classic and more recent studies (Chen et al., 2023; Lemmon et al., 1992; Nichol et al., 2016; Zheng et al., 1994; Schense and Hubbell, 2000) to better understand the relationship between substrate adhesion and neurite outgrowth.

Strengths:

The primary strength of the manuscript lies in developing a method for investigating the role of adhesion in axon outgrowth and traction force generation using a femtosecond laser technique. The most exciting finding is that both outgrowth and traction force generation have a biphasic relationship with laminin concentration.

Weaknesses:

The primary weaknesses are a lack of discussion of prior studies that have directly measured the strength of growth cone adhesions to the substrate (Zheng et al., 1994) and traction forces (Koch et al., 2012), the inverse correlation between retrograde flow rate and outgrowth (Nichol et al., 2016), and prior studies noting a biphasic effect of substrate concentration of neurite outgrowth (Schense and Hubbell, 2000).

Overall, the claims and conclusions are well justified by the data. The main exception is that the data is more relevant to how the rate of neurite outgrowth is controlled rather than axonal guidance.

This manuscript will help foster interest in the interrelationship between neurite outgrowth, traction forces, and substrate adhesion, and the use of a novel method to study this problem.

Reviewer #3 (Public Review):

Summary:<br /> This work addresses an important question of how Gtr1/2 small GTPases and Pib2, two major regulators of the TORC1 cell growth controller, differentially operate in yeast. They found not all the TORC1 downstream targets respond to Gtr1/2 and Pib2 equally. In fact, they demonstrate that TORC1-dependent phosphorylation of Ser33, a 3-phosphoglycerate dehydrogenase, is responsive to only Pib2. They attributed this specificity to the physical interaction between Ser33 and Pib2. This part is novel and important, revising the canonical view in the field that Gtr1/2 and Pib2 branches act towards the same TORC1 downstream targets. Of note, this claim largely agrees with a recent independent study (PMID: 38127619).

Moving on, the authors describe different behaviors of TORC1 downstream readouts in intermediate nutrient conditions with a poor nitrogen source, with some readouts still active while others inactive. They argue that selective activation of certain TORC1 downstream targets reflects the "Gtr1/2 off, Pib2 on" state. However, this claim is not sufficiently supported by the presented data.

Strengths:<br /> The data presented in this paper has high value to the TOR community. In particular, a rigorous and comprehensive phospho-proteomic dataset that compares the Gtr1/2- and Pib2-dependency of diverse TORC1 downstream targets is very informative, potentially stimulating follow-up studies on each target.

Identification of Ser33 as a Pib2-specific TORC1 downstream is important and convincing (although whether Ser33 is a direct substrate of TORC1 was not addressed in this work). Physical interaction between Ser33 and Pib2 could represent a novel layer of TORC1 signaling regulation, in line with the mammalian Rag-TFEB interaction model, as discussed by the authors.

Weaknesses:<br /> The authors' three-state model, particularly the claim that cells are in the "Gtr1/2 off, Pib2 on" state in a poor nitrogen condition (e.g., proline medium), is not convincing enough because of the following reasons.

1) The "Pib2 on" claim contradicts with the observation that Ser33, Pib2-specific readout, is hypo-phosphorylated in proline medium (Fig 5F).

2) In the genetic experiments (Figure 8), the authors compare pib2D with Gtr1/2OFF. This is not appropriate, because GTR1/2OFF (GTR1-GDP and Gtr2-GTP) actively inhibits TORC1, differing from the null nature of pib2D. pib2D should be compared with gtr1/2D instead.

3) In general, diverse behaviors of TORC1 targets are not unexpected because their phosphorylation levels should have different dynamic ranges depending on how "good" they are as TORC1 substrates, with some requiring a higher TORC1 activity than others to be detectably phosphorylated. Although this aspect can be physiologically meaningful, and it is indeed important to look at multiple substrates as the authors suggest, this approach does not inform whether the signal is coming from Gtr1/2 or Pib2. An informative way in this context would be to look at the Gtr1/2- or Pib2-specific targets, but the former has not been identified, and observations on the latter, Ser33, do not support the "Pib2 on" claim as mentioned in the above 1).

4) In addition, comparisons made between direct TORC1 substrates (e.g., Sch9) and indirect downstream targets (e.g., Rps6 and Par32) are not very informative, because indirect targets can be impacted by TORC1-independent regulation of the mediating factors (e.g., Ypk3 for Rps6 and Npr1 for Par32).

In summary, the presented data do not tell us which of the two branches (Gtr1/2 or Pib2) is "more active" in the poor nitrogen condition. Their observations do not necessarily prefer their 3-state on/off model (Figure 8) over the more natural assumption that both branches have the gradation of activity depending on the nutrient status.

Reviewer #3 (Public Review):

Bing et al. attempt to address fundamental mechanisms of TAD formation in Drosophila by analyzing gene expression and 3D conformation within the vicinity of the eve TAD after insertion of a transgene harboring a Homie insulator sequence 142 kb away in different orientations. These transgenes along with spatial gene expression analysis were previously published in Fujioka et al. 2016, and the underlying interpretations regarding resulting DNA configuration in this genomic region were also previously published. This manuscript repeats the expression analysis using smFISH probes in order to achieve more quantitative analysis, but the main results are the same as previously published. The only new data are the Micro-C and an additional modeling/analysis of what they refer to as the 'Z3' orientation of the transgenes. The rest of the manuscript merely synthesizes further interpretation with the goal of addressing whether loop extrusion may be occurring or if boundary:boundary pairing without loop extrusion is responsible for TAD formation. The authors conclude that their results are more consistent with boundary:boundary pairing and not loop extrusion; however, most of this imaging data seems to support both loop extrusion and the boundary:boundary models. This manuscript lacks support, especially new data, for its conclusions. Furthermore, there are many parts of the manuscript that are difficult to follow. There are some minor errors in the labelling of the figures that if fixed would help elevate understanding. Lastly, there are several major points that if elaborated on, would potentially be helpful for the clarity of the manuscript.

Major Points:<br /> 1. The authors suggest and attempt to visualize in the supplemental figures, that loop extrusion mechanisms would appear during crosslinking and show as vertical stripes in the micro-C data. In order to see stripes, a majority of the nuclei would need to undergo loop extrusion at the same rate, starting from exactly the same spots, and the loops would also have to be released and restarted at the same rate. If these patterns truly result from loop extrusion, the authors should provide experimental evidence from another organism undergoing loop extrusion.<br /> 2. On lines 311-314, the authors discuss that stem-loops generated by cohesin extrusion would possibly be expected to have more next-next-door neighbor contacts than next-door neighbor contacts and site their models in Figure 1. Based on the boundary:boundary pairing models in the same figure would the stem-loops created by head-to-tail pairing also have the same phenotype? Making possible enrichment of next-next-door neighbor contacts possible in both situations? The concepts in the text are not clear, and the diagrams are not well-labeled relative to the two models.<br /> 3. The authors appear to cite Chen et al., 2018 as a reference for the location of these transgenes being 700nM away in a majority of the nuclei. However, the exact transgenes in this manuscript do not appear to have been measured for distance. The authors could do this experiment and include expression measurements.<br /> 4. The authors discuss the possible importance of CTCF orientation in forming the roadblock to cohesin extrusion and discuss that Homie orientation in the transgene may impact Homie function as an effective roadblock. However, the Homie region inserted in the transgene does not contain the CTCF motif. Can the authors elaborate on why they feel the orientation of Homie is important in its ability to function as a roadblock if the CTCF motif is not present? Trans-acting factors responsible for Homie function have not been identified and this point is not discussed in the manuscript.<br /> 5. The imaging results seem to be consistent with both boundary:boundary interaction and loop extrusion stem looping.<br /> 6. The authors suggest that the eveMa TAD could only be formed by extrusion after the breakthrough of Nhomie and several other roadblocks. Additionally, the overall long-range interactions with Nhomie appear to be less than the interactions with endogenous Homie (Figures 7, 8, and supplemental 5). Is it possible that in some cases boundary:boundary pairing is occurring between only the transgenic Homie and endogenous Homie and not including Nhomie?<br /> 7. In Figure 4E, the GFP hebe expression shown in the LhomieG Z5 transgenic embryo does not appear in the same locations as the LlambdaG Z5 control. Is this actually hebe expression or just a background signal?<br /> 8. Figure 6- The LhomieG Z3 late-stage embryo appears to be showing the ventral orientation of the embryo rather than the lateral side of the embryo as was shown in the previous figure. Is this for a reason? Additionally, there are no statistics shown for the Z3 transgenic images. Were these images analyzed in the same way as the Z5 line images?<br /> 9. Do the Micro-C data align with the developmental time points used in the smFISH probe assays?

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Wang et. al. described the crystal structures of the N-terminal fragments of Scavenger receptor class F member 1 (SCARF1) ectodomains. SCARF1 recognizes modified LDLs, including acetylated LDL and oxidized LDL, and it plays an important role in both innate and adaptive immune responses. They characterized the dimerization of SCARF1 and the interaction of SCARF1 with modified lipoproteins by mutational and biochemical studies. The authors identified the critical residues for dimerization and demonstrated that SCARF1 may function as homodimers. They further characterized the interaction between SCARF1 and LDLs and identified the lipoprotein ligand recognition sites, the highly positively charged areas. Their data suggested that the teichoic acid inhibitors may interact with SCARF1 in the same areas as LDLs.

Strengths:<br /> The crystal structures of SCARF1 were high quality. The authors performed extensive site-specific mutagenesis studies using soluble proteins for ELISA assays and surface-expressed proteins for flow cytometry.

Weaknesses:<br /> 1. The schematic drawing of human SCARF1 and SCARF2 in Fig 1A did not show the differences between them. It would be useful to have a sequence alignment showing the polymorphic regions.<br /> 2. The description of structure determination was confusing. The f1 crystal structure was determined by SAD with Pt derivatives. Why did they need molecular replacement with a native data set? The f2 crystal structure was solved by molecular replacement using the structure of the f1 fragment. Why did they need to use EGF-like fragments predicted by AlphaFold as search models?<br /> 3. It's interesting to observe that SCARA1 binds modified LDLs in a Ca2+-independent manner. The authors performed the binding assays between SCARF1 and modified LDLs in the presence of Ca2+ or EDTA on Page 9. However, EDTA is not an efficient Ca2+ chelator. The authors should have performed the binding assays in the presence of EGTA instead.<br /> 4. The authors claimed that SCARF1Δ353-415, the deletion of a C-terminal region of the ectodomain, might change the conformation of the molecule and generate hinderance for the C-terminal regions. Why didn't SCARF1Δ222-353 have a similar effect? Could the deletion change the interaction between SCARF1 and the membrane? Is SCARF1Δ353-415 region hydrophobic?<br /> 5. What was the point of having Figure 8? Showing the SCARF1 homodimers could form two types of dimers on the membrane surface proposed? The authors didn't have any data to support that.

Reviewer #3 (Public Review):

Cheng et al. studied if and how blood flow regulates the differentiation of vascular smooth muscle cells (VSMC) in the Circle of Willis (CW) in zebrafish embryos. They show that CW vessels gradually acquire an arterial identity. VSMCs also undergo gradual differentiation, which correlates with blood flow velocity. Using cell culture they show that pulsatile blood flow promotes pericyte differentiation into smooth muscle cells. They further identify transcription factor klf2a as differentially regulated by blood flow, and show that klf2a inhibition results in VSMC differentiation. The authors conclude that pulsatile flow promotes VSMC differentiation through klf2a activation.

Overall this is an important study, because VSMC differentiation in CW has not been previously studied, although analogous observations regarding the role of blood flow and klf2 involvement have been previously made in other systems and other vascular beds, for example, mouse klf2 mutants, which have deficient VSMC coverage of the dorsal aorta (Wu et al., 2008, JBC 283: 3942-50). The results convincingly show that VSMC differentiation in CW depends on the blood flow and that klf2a flow-dependent function regulates VSMC differentiation.

Reviewer #3 (Public Review):

Summary:<br /> It has been proposed in the literature, that the ATP release channel Panx1 can be activated in various ways, including by tyrosine phosphorylation of the Panx1 protein. The present study reexamines the commercial antibodies used previously in support of the phosphorylation hypothesis and the presented data indicate that the antibodies may recognize proteins unrelated to Panx1. Consequently, the authors caution about the use and interpretation of results obtained with these antibodies.

Strengths:<br /> The manuscript by Ruan et al. addresses an important issue in Panx1 research, i.e. the activation of the channel formed by Panx1 via protein phosphorylation. If the authors' conclusions are correct, the previous claims for Panx1 phosphorylation on the basis of the commercial anti-phospho-Panx1 antibodies would be in question.

This is a very detailed and comprehensive analysis making use of state-of-the-art techniques, including mass spectrometry and phos-tag gel electrophoresis.

In general, the study is well-controlled as relating to negative controls.

The value of this manuscript is, that it could spawn new, more function-oriented studies on the activation of Panx1 channels.

Weaknesses:<br /> Although the manuscript addresses an important issue, the activation of the ATP-release channel Panx1 by protein phosphorylation, the data provided do not support the firm conclusion that such activation does not exist. The failure to reproduce published data obtained with commercial anti-phospho Panx1 antibodies can only be of limited interest for a subfield.

1. The title claiming that "Panx1 is NOT phosphorylated..." is not justified by the failure to reproduce previously published data obtained with these antibodies. If, as claimed, the antibodies do not recognize Panx1, their failure cannot be used to exclude tyrosine phosphorylation of the Panx1 protein. There is no positive control for the antibodies.

2. The authors claim that exogenous SRC expression does not phosphorylate Y198. DeLalio et al. 2019 show that Panx1 is constitutively phosphorylated at Y198, so an effect of exogenous SRC expression is not necessarily expected.

3. The authors argue that the GFP tag of Panx1at the COOH terminus does not interfere with folding since the COOH modified (thrombin cleavage site) Panx1 folds properly, forming an amorphous glob in the cryo-EM structure. However, they do not show that the COOH-modified Panx1 folds properly. It may not, because functional data strongly suggest that the terminal cysteine dives deep into the pore. For example, the terminal cysteine, C426, can form a disulfide bond with an engineered cysteine at position F54 (Sandilos et al. 2012).

4. The authors dismiss the additional arguments for tyrosine phosphorylation of Panx1 given by the various previous studies on Panx1 phosphorylation. These studies did not, as implied, solely rely on the commercial anti-phospho-Panx1 antibodies, but also presented a wealth of independent supporting data. Contrary to the authors' assertion, in the previous papers the pY198 and pY308 antibodies recognized two protein bands in the size range of glycosylated and partial glycosylated Panx1.

5. A phosphorylation step triggering channel activity of Panx1 would be expected to occur exclusively on proteins embedded in the plasma membrane. The membrane-bound fraction is small in relation to the total protein, which is particularly true for exogenously expressed proteins. Thus, any phosphorylated protein may escape detection when total protein is analyzed. Furthermore, to be of functional consequence, only a small fraction of the channels present in the plasma membrane need to be in the open state. Consequently, only a fraction of the Panx1 protein in the plasma membrane may need to be phosphorylated. Even the high resolution of mass spectroscopy may not be sufficient to detect phosphorylated Panx1 in the absence of enrichment processes.

6. In the electrophysiology experiments described in Figure 7, there is no evidence that the GFP-tagged Panx1 is in the plasma membrane. Instead, the image in Figure 7a shows prominent fluorescence in the cytoplasm. In addition, there is no evidence that the CBX-sensitive currents in 7b are mediated by Panx1-GFP and are not endogenous Panx1. Previous literature suggests that the hPanx1 protein needs to be cleaved (Chiu et al. 2014) or mutated at the amino terminus (Michalski et al 2018) to see voltage-activated currents, so it is not clear that the currents represent hPANX1 voltage-activated currents.

Reviewer #3 (Public Review):

This is a manuscript that attempts to validate Plasmodium M1 alanyl aminopeptidase as a target for antimalarial drug development. The authors provide evidence that MIPS2673 inhibits recombinant enzymes from both Pf and Pv and is selective over other proteases. There is in vitro antimalarial activity. Chemoproteomic experiments demonstrate selective targeting of the PfA-M1 protease.

This is a continuation of previous work focused on designing inhibitors for aminopeptidases by a subset of these authors. Medicinal chemistry explorations resulted in the synthesis of MIPS2673 which has improved properties including potent inhibition of PfA-M1 and PvA-M1 with selectivity over a closed related peptidase. The compound also demonstrated selectivity over several human aminopeptidases and was not toxic to HEK293 cells at 40 uM. The activity against P. falciparum blood-stage parasites was about 300 nM.

Thermal stability studies confirmed that PfA-M1 was a binding target, however, there were other proteins consistently identified in the thermal stability studies. This raises the question as to their potential role as additional targets of this inhibitor. The authors dismiss these because they are not metalloproteases, but further analysis is warranted. This is particularly important as the authors were not able to generate mutants using in vitro evolution of resistance strategies. This often indicates that the inhibitor has more than one target.

The next set of experiments focused on a limited proteolysis approach. Again several proteins were identified as interacting with MIPS2673 including metalloproteases. The authors go on to analyze the LiP-MS data to identify the peptide from PfA-M1 which putatively interacts with MIPS2673. The authors are clearly focused on PfA-M1 as the target, but a further analysis of the other proteins identified by this method would be warranted and would provide evidence to either support or refute the authors' conclusions.

The final set of experiments was an untargeted metabolomics analysis. They identified 97 peptides as significantly dysregulated after MIPS2673 treatment of infected cells and most of these peptides were derived from one of the hemoglobin chains. The accumulation of peptides was consistent with a block in hemoglobin digestion. This experiment does reveal a potential functional confirmation, but questions remain as to specificity.

Overall, this is an interesting series of experiments that have identified a putative inhibitor of PfA-M1 and PvA-M1. The work would be significantly strengthened by structure-aided analysis. It is unclear why putative binding sites cannot be analyzed via specific mutagenesis of the recombinant enzyme. In the thermal stability and LiP -MS analysis, other proteins were consistently identified in addition to PfA-M1 and yet no additional analysis was undertaken to explore these as potential targets. The metabolomics experiments were potentially interesting, but without significant additional work including different lengths of treatment and different stages of the parasite, the conclusions drawn are overstated. Many treatments disrupt hemoglobin digestion - either directly or indirectly and from the data presented here it is premature to conclude that treatment with MIPS2673 directly inhibits hemoglobin digestion. Finally, the potency of this compound on parasites grown in vitro is 300 nM - this would need improvements in potency and demonstration of in vivo efficacy in the SCID mouse model to consider this a candidate for a drug.

Summary:<br /> Overall, this is an interesting series of experiments that have identified a putative inhibitor of the Plasmodium M1 alanyl aminopeptidases, PfA-M1 and PvA-M1.

Strengths:<br /> The main strengths include the synthesis of MIPS2673 which is selectively active against the enzymes and in whole-cell assay.

Weaknesses:<br /> The weaknesses include the lack of additional analysis of additional targets identified in the chemoproteomic approaches.

Reviewer #4 (Public Review):

I am a new reviewer for this manuscript, which has been reviewed before. The authors provide a variational autoencoder that has three objectives in the loss: linear reconstruction of behavior from embeddings, reconstruction of neural data, and KL divergence term related to the variational model elements. They take the output of the VAE as the "behaviorally relevant" part of neural data and call the residual "behaviorally irrelevant". Results aim to inspect the linear versus nonlinear behavior decoding using the original raw neural data versus the inferred behaviorally relevant and irrelevant parts of the signal.

Overall, studying neural computations that are behaviorally relevant or not is an important problem, which several previous studies have explored (for example PSID in (Sani et al. 2021), TNDM in (Hurwitz et al. 2021), TAME-GP in (Balzani et al. 2023), pi-VAE in (Zhou and Wei 2020), and dPCA in (Kobak et al. 2016), etc). However, this manuscript does not properly put their work in the context of such prior works. For example, the abstract states "One solution is to accurately separate behaviorally-relevant and irrelevant signals, but this approach remains elusive", which is not the case given that these prior works have done that. The same is true for various claims in the main text, for example "Furthermore, we found that the dimensionality of primary subspace of raw signals (26, 64, and 45 for datasets A, B, and C) is significantly higher than that of behaviorally-relevant signals (7, 13, and 9), indicating that using raw signals to estimate the neural dimensionality of behaviors leads to an overestimation" (line 321). This finding was presented in (Sani et al. 2021) and (Hurwitz et al. 2021), which is not clarified here. This issue of putting the work in context has been brought up by other reviewers previously but seems to remain largely unaddressed. The introduction is inaccurate also in that it mixes up methods that were designed for separation of behaviorally relevant information with those that are unsupervised and do not aim to do so (e.g., LFADS). The introduction should be significantly revised to explicitly discuss prior models/works that specifically formulated this behavior separation and what these prior studies found, and how this study differs.

Beyond the above, some of the main claims/conclusions made by the manuscript are not properly supported by the analyses and results, which has also been brought up by other reviewers but not fully addressed. First, the analyses here do not support the linear readout from the motor cortex because i) by construction, the VAE here is trained to have a linear readout from its embedding in its loss, which can bias its outputs toward doing well with a linear decoder/readout, and ii) the overall mapping from neural data to behavior includes both the VAE and the linear readout and thus is always nonlinear (even when a linear Kalman filter is used for decoding). This claim is also vague as there is no definition of readout from "motor cortex" or what it means. Why is the readout from the bottleneck of this particular VAE the readout of motor cortex? Second, other claims about properties of individual neurons are also confounded because the VAE is a population-level model that extracts the bottleneck from all neurons. Thus, information can leak from any set of neurons to other sets of neurons during the inference of behaviorally relevant parts of signals. Overall, the results do not convincingly support the claims, and thus the claims should be carefully revised and significantly tempered to avoid misinterpretation by readers.

Below I briefly expand on these as well as other issues, and provide suggestions:

1) Claims about linearity of "motor cortex" readout are not supported by results yet stated even in the abstract. Instead, what the results support is that for decoding behavior from the output of the dVAE model -- that is trained specifically to have a linear behavior readout from its embedding -- a nonlinear readout does not help. This result can be biased by the very construction of the dVAE's loss that encourages a linear readout/decoding from embeddings, and thus does not imply a finding about motor cortex.

2) Related to the above, it is unclear what the manuscript means by readout from motor cortex. A clearer definition of "readout" (a mapping from what to what?) in general is needed. The mapping that the linearity/nonlinearity claims refer to is from the *inferred* behaviorally relevant neural signals, which themselves are inferred nonlinearly using the VAE. This should be explicitly clarified in all claims, i.e., that only the mapping from distilled signals to behavior is linear, not the whole mapping from neural data to behavior. Again, to say the readout from motor cortex is linear is not supported, including in the abstract.

3) Claims about individual neurons are also confounded. The d-VAE distilling processing is a population level embedding so the individual distilled neurons are not obtainable on their own without using the population data. This population level approach also raises the possibility that information can leak from one neuron to another during distillation, which is indeed what the authors hope would recover true information about individual neurons that wasn't there in the recording (the pixel denoising example). The authors acknowledge the possibility that information could leak to a neuron that didn't truly have that information and try to rule it out to some extent with some simulations and by comparing the distilled behaviorally relevant signals to the original neural signals. But ultimately, the distilled signals are different enough from the original signals to substantially improve decoding of low information neurons, and one cannot be sure if all of the information in distilled signals from any individual neuron truly belongs to that neuron. It is still quite likely that some of the improved behavior prediction of the distilled version of low-information neurons is due to leakage of behaviorally relevant information from other neurons, not the former's inherent behavioral information. This should be explicitly acknowledged in the manuscript.

4) Given the nuances involved in appropriate comparisons across methods and since two of the datasets are public, the authors should provide their complete code (not just the dVAE method code), including the code for data loading, data preprocessing, model fitting and model evaluation for all methods and public datasets. This will alleviate concerns and allow readers to confirm conclusions (e.g., figure 2) for themselves down the line.

5) Related to 1) above, the authors should explore the results if the affine network h(.) (from embedding to behavior) was replaced with a nonlinear ANN. Perhaps linear decoders would no longer be as close to nonlinear decoders. Regardless, the claim of linearity should be revised as described in 1) and 2) above, and all caveats should be discussed.

6) The beginning of the section on the "smaller R2 neurons" should clearly define what R2 is being discussed. Based on the response to previous reviewers, this R2 "signifies the proportion of neuronal activity variance explained by the linear encoding model, calculated using raw signals". This should be mentioned and made clear in the main text whenever this R2 is referred to.

7) Various terms require clear definitions. The authors sometimes use vague terminology (e.g., "useless") without a clear definition. Similarly, discussions regarding dimensionality could benefit from more precise definitions. How is neural dimensionality defined? For example, how is "neural dimensionality of specific behaviors" (line 590) defined? Related to this, I agree with Reviewer 2 that a clear definition of irrelevant should be mentioned that clarifies that relevance is roughly taken as "correlated or predictive with a fixed time lag". The analyses do not explore relevance with arbitrary time lags between neural and behavior data.

8) CEBRA itself doesn't provide a neural reconstruction from its embeddings, but one could obtain one via a regression from extracted CEBRA embeddings to neural data. In addition to decoding results of CEBRA (figure S3), the neural reconstruction of CEBRA should be computed and CEBRA should be added to Figure 2 to see how the behaviorally relevant and irrelevant signals from CEBRA compare to other methods.

References:

Kobak, Dmitry, Wieland Brendel, Christos Constantinidis, Claudia E Feierstein, Adam Kepecs, Zachary F Mainen, Xue-Lian Qi, Ranulfo Romo, Naoshige Uchida, and Christian K Machens. 2016. "Demixed Principal Component Analysis of Neural Population Data." Edited by Mark CW van Rossum. eLife 5 (April): e10989. https://doi.org/10.7554/eLife.10989.

Sani, Omid G., Hamidreza Abbaspourazad, Yan T. Wong, Bijan Pesaran, and Maryam M. Shanechi. 2021. "Modeling Behaviorally Relevant Neural Dynamics Enabled by Preferential Subspace Identification." Nature Neuroscience 24 (1): 140-49. https://doi.org/10.1038/s41593-020-00733-0.

Zhou, Ding, and Xue-Xin Wei. 2020. "Learning Identifiable and Interpretable Latent Models of High-Dimensional Neural Activity Using Pi-VAE." In Advances in Neural Information Processing Systems, 33:7234-47. Curran Associates, Inc. https://proceedings.neurips.cc/paper/2020/hash/510f2318f324cf07fce24c3a4b89c771-Abstract.html.

Hurwitz, Cole, Akash Srivastava, Kai Xu, Justin Jude, Matthew Perich, Lee Miller, and Matthias Hennig. 2021. "Targeted Neural Dynamical Modeling." In Advances in Neural Information Processing Systems. Vol. 34. https://proceedings.neurips.cc/paper/2021/hash/f5cfbc876972bd0d031c8abc37344c28-Abstract.html.

Balzani, Edoardo, Jean-Paul G. Noel, Pedro Herrero-Vidal, Dora E. Angelaki, and Cristina Savin. 2023. "A Probabilistic Framework for Task-Aligned Intra- and Inter-Area Neural Manifold Estimation." In . https://openreview.net/forum?id=kt-dcBQcSA.

Reviewer #3 (Public Review):

Summary:<br /> Object classification serves as a vital normative principle in both the study of the primate ventral visual stream and deep learning. Different models exhibit varying classification performances and organize information differently. Consequently, a thriving research area in computational neuroscience involves identifying meaningful properties of neural representations that act as bridges connecting performance and neural implementation. In the work of Lindsey and Issa, the concept of factorization is explored, which has strong connections with emerging concepts like disentanglement [1,2,3] and abstraction [4,5]. Their primary contributions encompass two facets: (1) The proposition of a straightforward method for quantifying the degree of factorization in visual representations. (2) A comprehensive examination of this quantification through correlation analysis across deep learning models.

To elaborate, their methodology, inspired by prior studies [6], employs visual inputs featuring a foreground object superimposed onto natural backgrounds. Four types of scene variables, such as object pose, are manipulated to induce variations. To assess the level of factorization within a model, they systematically alter one of the scene variables of interest and estimate the proportion of encoding variances attributable to the parameter under consideration.

The central assertion of this research is that factorization represents a normative principle governing biological visual representation. The authors substantiate this claim by demonstrating an increase in factorization from macaque V4 to IT, supported by evidence from correlated analyses revealing a positive correlation between factorization and decoding performance. Furthermore, they advocate for the inclusion of factorization as part of the objective function for training artificial neural networks. To validate this proposal, the authors systematically conduct correlation analyses across a wide spectrum of deep neural networks and datasets sourced from human and monkey subjects. Specifically, their findings indicate that the degree of factorization in a deep model positively correlates with its predictability concerning neural data (i.e., goodness of fit).

Strengths:<br /> The primary strength of this paper is the authors' efforts in systematically conducting analysis across different organisms and recording methods. Also, the definition of factorization is simple and intuitive to understand.

Weaknesses:<br /> This work exhibits two primary weaknesses that warrant attention: (i) the definition of factorization and its comparison to previous, relevant definitions, and (ii) the chosen analysis method.

Firstly, the definition of factorization presented in this paper is founded upon the variances of representations under different stimuli variations. However, this definition can be seen as a structural assumption rather than capturing the effective geometric properties pertinent to computation. More precisely, the definition here is primarily statistical in nature, whereas previous methodologies incorporate computational aspects such as deviation from ideal regressors [1], symmetry transformations [3], generalization [5], among others. It would greatly enhance the paper's depth and clarity if the authors devoted a section to comparing their approach with previous methodologies [1,2,3,4,5], elucidating any novel insights and advantages stemming from this new definition.

Secondly, in order to establish a meaningful connection between factorization and computation, the authors rely on a straightforward synthetic model (Figure 1c) and employ multiple correlation analyses to investigate relationships between the degree of factorization, decoding performance, and goodness of fit. Nevertheless, the results derived from the synthetic model are limited to the low training-sample regime. It remains unclear whether the biological datasets under consideration fall within this low training-sample regime or not.

[1] Eastwood, Cian, and Christopher KI Williams. "A framework for the quantitative evaluation of disentangled representations." International conference on learning representations. 2018.<br /> [2] Kim, Hyunjik, and Andriy Mnih. "Disentangling by factorising." International Conference on Machine Learning. PMLR, 2018.<br /> [3] Higgins, Irina, et al. "Towards a definition of disentangled representations." arXiv preprint arXiv:1812.02230 (2018).<br /> [4] Bernardi, Silvia, et al. "The geometry of abstraction in the hippocampus and prefrontal cortex." Cell 183.4 (2020): 954-967.<br /> [5] Johnston, W. Jeffrey, and Stefano Fusi. "Abstract representations emerge naturally in neural networks trained to perform multiple tasks." Nature Communications 14.1 (2023): 1040.<br /> [6] Majaj, Najib J., et al. "Simple learned weighted sums of inferior temporal neuronal firing rates accurately predict human core object recognition performance." Journal of Neuroscience 35.39 (2015): 13402-13418.

Reviewer #3 (Public Review):

Summary:<br /> Ai et al. studied texture, color, and disparity selectivity in the human visual cortex at the mesoscale level using high-resolution fMRI. They reproduced earlier monkey and human studies showing interdigitated color-selective and disparity-selective sub-compartments within area V2, likely corresponding to thin and thick stripes, respectively. At least with the stimuli used, no clear evidence for texture-selective mesoscale activations was observed in area V2. The most interesting and novel part of this study focused on cortical-depth-dependent connectivity analyses across areas. The data suggest feedback and feedforward functional connectivity between V1 and V3A for disparity signals and feedback from V4 to the deep layers of V2 for textures.

Strengths:<br /> High-resolution fMRI and highly interesting layer-specific informational connectivity analyses.

Weaknesses:<br /> The authors tend to overclaim their results.

Reviewer #3 (Public Review):

Summary:<br /> In this work, Duan and Curtis addressed an important issue related to the nature of working memory representations. This work is motivated by findings illustrating that orientation decoding performance for perceptual representations can be biased by the stimulus aperture (modulator). Here, the authors examined whether the decoding performance for working memory representations is similarly influenced by these aperture biases. The results provide convincing evidence that working memory representations have a different representational structure, as the decoding performance was not influenced by the type of stimulus aperture.

Strengths:<br /> The strength of this work lies in the direct comparison of decoding performance for perceptual representations with working memory representations. The authors take a well-motivated approach and illustrate that perceptual and working memory representations do not share a similar representational structure. The authors test a clear question, with a rigorous approach and provide convincing evidence. First, the presented oriented stimuli are carefully manipulated to create orthogonal biases introduced by the stimulus aperture (radial or angular modulator), regardless of the stimulus carrier orientation. Second, the authors implement advanced methods to decode the orientation information present, in visual and parietal cortical regions, when directly perceiving or holding an oriented stimulus in memory. The data illustrates that working memory decoding is not influenced by the type of aperture, while this is the case in perception. In sum, the main claims are important and shed light on the nature of working memory representations.

Weaknesses:<br /> I have a few minor concerns that, although they don't affect the main conclusion of the paper, should still be addressed.

1. Theoretical framing in the introduction: Recent work has shown that decoding of orientation during perception does reflect orientation selectivity, and it is not only driven by the stimulus aperture (Roth, Kay & Merriam, 2022).

2. Figure 1C illustrates the principle of how the radial and angular modulators bias the contrast energy extracted by the V1 model, which in turn would influence orientation decoding. It would be informative if the carrier orientations used in the experiment were shown in this figure, or at a minimum it would be mentioned in the legend that the experiment used 3 carrier orientations (15{degree sign}, 75{degree sign}, 135{degree sign}) clockwise from vertical. Related, when trying to find more information regarding the carrier orientation, the 'Stimuli' section of the Methods incorrectly mentions that 180 orientations are used as the carrier orientation.

3. The description of the image computable V1 model in the Methods is incomplete, and at times inaccurate. i) The model implements 6 orientation channels, which is inaccurately referred to as a bandwidth of 60{degree sign} (should be 180/6=30). ii) The steerable pyramid combines information across phase pairs to obtain a measure of contrast energy for a given stimulus.<br /> Here, it is only mentioned that the model contains different orientation and spatial scale channels. I assume there were also 2 phase pairs, and they were combined in some manner (squared and summed to create contrast energy). Currently, it is unclear what the model output represents. iii) The spatial scale channel with the maximal response differences between the 2 modulators was chosen as the final model output. What spatial frequency does this channel refer to, and how does this spatial frequency relate to the stimulus?

4. It is not clear from the Methods how the difficulty in the perceptual control task was controlled. How were the levels of task difficulty created?

Reviewer #3 (Public Review):

Summary:<br /> The study conducted by Pisanski et al investigates the role of the lateral parafacial area (pFL) in controlling active expiration. Stereotactic injections of bicuculline were utilized to map various pFL sites and their impact on respiration. The results indicate that injections at more rostral pFL locations induce the most robust changes in tidal volume, minute ventilation, and combined respiratory responses. The study indicates that the rostrocaudal organization of the pFL and its influence on breathing is not simple and uniform.

Strengths:<br /> The data provide novel insights into the importance of rostral locations in controlling active expiration. The authors use innovative analytic methods to characterize the respiratory effects of bicuculline injections into various areas of the pFL.

Weaknesses:<br /> Bicuculline injections increase the excitability of neurons. Aside from blocking GABA receptors, bicuculline also inhibits calcium-activated potassium currents and potentiates NMDA current, thus insights into the role of GABAergic inhibition are limited.

Increasing the excitability of neurons provides little insights into the activity pattern and function of the activated neurons. Without recording from the activated neurons, it is impossible to know whether an effect on active expiration or any other respiratory phase is caused by bicuculline acting on rhythmogenic neurons or tonic neurons that modulate respiration. While this approach is inappropriate to study the functional extent of the conditional "oscillator" for active expiration, it provides valuable insights into this region's complex role in controlling breathing.

Reviewer #3 (Public Review)

Summary<br /> This is an important study that tests the hypothesis that Cav1.4 calcium channels do more than provide a voltage-dependent influx of Ca2+ into photoreceptors. The relevant background can be divided into two tranches. First, deletion of Cav1.4 channels (Cav1.4 knock-out) disrupts rod and cone photoreceptors and their synapses in the outer plexiform layer. Second, knock-in of a non-conducting Cav1.4 channel (Cav1.4 knock-in) partially spares the organization of the outer plexiform layer and photoreceptor synapses (Maddox et al., eLife 2020), which is remarkable considering the disruption of the outer plexiform layer in the Cav1.4 knock-out. In addition, phototransduction, assessed by scotopic and phototopic electroretinography (a-wave amplitude) in the Cav1.4 knock-in retina was partially spared for rods and only slightly impaired for cones. However, the non-conducting Cav1.4 channel of the Cav1.4 knock-in failed to rescue synaptic transmission across the outer retina (electroretinography: b-wave amplitude, Maddox et al., eLife 2020). The 2020 Maddox et al. (eLife) focused more on the rod pathway, while the current work addressed the cone pathway.

Strengths<br /> The study addresses the important question of how disruption of Cav1.4 function in both rod and cone photoreceptors leads to impairment primarily of the rod pathway for scotopic vision. This is clinically relevant as human mutations lead to stationary night blindness rather than blindness. The work relevance provides excellent single-cell electrophysiological recordings of Ca2+ currents from cones of wild-type, Cav1.4 knock-out, and Cav1.4 knock-in mice and, in addition, from ground squirrel and monkey cones. To make these recordings successfully in the various species and the compromised retinas (Cav1.4 knock-out and Cav1.4 knock-in) is very impressive. The findings clearly advance our understanding of Ca2+ channel function in cones. In addition, the study presents high-quality electron microscopy reconstructions of cones and further physiological and behavioral data related to the cone pathway.

Weaknesses<br /> The major critiques are related to the description of the Cav1.4 knock-in mouse as "sparing" function, which can be remedied in part by a simple rewrite, and in certain places, the data may need to be examined more critically. In particular, the authors should address features in the data presented in Figures 6 and 7 that seem to indicate that the retina of the Cav1.4 knock-in is not intact, but the interpretation given by the authors as "intact" is not appropriate and made without rigorous statistical testing.

Reviewer #3 (Public Review):

Summary:<br /> The authors propose to invert a mechanistic model of phototransduction in mouse and rod photoreceptors to derive stimuli that compensate for nonlinearities in these cells. They fit the model to a large set of photoreceptor recordings and show in additional data that the compensation works. This can allow the exclusion of photoreceptors as a source of nonlinear computation in the retina, as desired to pinpoint nonlinearities in retinal computation. Overall, the recordings made by the authors are impressive and I appreciate the simplicity and elegance of the idea. The data support the authors' conclusions but the presentation can be improved.

Strengths:<br /> - The authors collected an impressive set of recordings from mouse and primate photoreceptors, which is very challenging to obtain.<br /> - The authors propose to exploit mechanistic mathematical models of well-understood phototransduction to design light stimuli that compensate for nonlinearities.<br /> - The authors demonstrate through additional experiments that their proposed approach works.

Weaknesses:<br /> - The authors use numerical optimization for fitting the parameters of the photoreceptor model to the data. Recently, the field of simulation-based inference has developed methods to do so, including quantification of the uncertainty of the resulting estimates. Since the authors state that two different procedures were used due to the different amounts of data collected from different cells, it may be worthwhile to rather test these methods, as implemented e.g. in the SBI toolbox (https://joss.theoj.org/papers/10.21105/joss.02505). This would also allow them to directly identify dependencies between parameters, and obtain associated uncertainty estimates. This would also make the discussion of how well constrained the parameters are by the data or how much they vary more principled because the SBI uncertainty estimates could be used.

- In several places, the authors refer the reader to look up specific values e.g. of parameters in the associated MATLAB code. I don't think this is appropriate, important values/findings/facts should be in the paper (lines 142, 114, 168). I would even find the precise values that the authors measure interesting, so I think the authors should show them in a figure/table. In general, I would like to see also the average variance explained by different models summarized in a table and precise mean/median values for all important quantities (like the response amplitude ratios in Figures 6/9).

- If the proposed model is supposed to model photoreceptor adaptation on a longer time scale, I fail to see why this can be an invertible model. Could the authors explain this better? I suspect that the model is mainly about nonlinearities as the authors also discuss in lines 360ff.

- The important Figures 6-8 are very hard to read, as it is not easy to see what the stimulus is, the modified stimulus, the response with and without modification, what the desired output looks like, and what is measured for part B. Reworking these figures would be highly recommended.

- If I understand Figure 6 correctly, part B is about quantifying the relative size of the response to the little first flash to the little second flash. While clearly, the response amplitude of the second flash is only 50% for the second flash compared to the first flash in primate rod and cones in the original condition, the modified stimulus seems to overcompensate and result in 130% response for the second flash. How do the authors explain this? A similar effect occurs in Figure 9, which the authors should also discuss.

Reviewer #3 (Public Review):

Summary:<br /> Aubert et al investigated the role of PENK in regulatory T cells. Through the mining of publicly available transcriptome data, the authors confirmed that PENK expression is selectively enriched in regulatory but not conventional T cells. Further data mining suggested that OX40, 4-1BB as well as BATF, can regulate PENK expression in Tregs. The authors generated fate-mapping mice to confirm selective PENK expression in Tregs and activated effector T cells in the colon and spleen. Interestingly, transgenic mice with conditional deletion of PENK in Tregs resulted in hypersensitivity to heat, which the authors attributed to heat hyperalgesia.

Strengths:<br /> The generation of transgenic mice with conditional deletion of PENK in foxp3 and PENK fate-mapping is novel and can potentially yield significant findings. The identification of upstream signals that regulate PENK is interesting but unlikely to be the main reason why PENK is predominantly expressed in Tregs as both BATF and TNFR are expressed in effector T cells.

Weaknesses:<br /> There is a lack of direct evidence and detailed analysis of Tregs in the control and transgenic mice to support the authors' hypothesis. PENK was previously reported to be expressed in skin Tregs and play a significant role in regulating skin homeostasis: this should be considered as an alternative mechanism that may explain the changed sensitivity to heat observed in the paper.

Reviewer #3 (Public Review):

The introduction/background is excellent. It reviews evidence showing that extinction of conditioned responding is regulated by noradrenaline and suggesting that the locus coeruleus (LC) may be a critical locus of this regulation. This naturally leads to the aim of the study: to determine whether the locus coeruleus is involved in extinction of an appetitive conditioned response. Overall, the study is well designed, nicely conducted and the results advance our understanding of the role of the LC in extinction of conditioned behaviour. Future studies may provide more fine-grained analyses of behavioral data to clarify the impact of the LC manipulations (stimulation and inhibition) on performance in the task.

Reviewer #3 (Public Review):

Summary:<br /> Medina and colleagues explored transcriptional kinetics during SARS-CoV-2 between non-hospitalized and hospitalized cohorts and identified that early NK signaling may be responsible for less severe disease.

Strengths:<br /> The paper includes extremely detailed analyses and makes an interesting attempt to link innate and adaptive responses. The analyses are appropriate for the data and described in clear language. The inclusion of late time points is interesting and potentially relevant to long COVID studies. Most findings were compatible with other detailed immune mapping during severe COVID-19.

Weaknesses:<br /> 1. The authors claim to be looking at the earliest stages of infection but this is not true as all patients enrolled are already symptomatic. The time points selected are unlikely to be useful clinically for biomarker selection as they are too late, and are likely beyond the point when the immune responses between severe and mild infection start to diverge.<br /> 2. The comparator timepoints between mild and severe cases do not match. The most comparison would be between day 7 of mild versus day 0 of severe which is already fairly late during infection.<br /> 3. The authors mention viral clearance but I see no evidence of viral loads measured in these individuals.<br /> 4. The cohort is quite small to draw definitive conclusions.<br /> 5. It is uncertain whether the results are applicable to current conditions as most infected people are immune experienced.<br /> 6. I found the discussion to be a bit too detailed and dense. I would suggest editing to make it more streamlined.

Reviewer #3 (Public Review):

Summary:<br /> The authors have initiated studies to understand the molecular mechanisms underlying the devolvement of multi-drug resistance in clinical Mtb strains. They demonstrate the association of isoniazid-resistant isolates by rifampicin treatment supporting the idea that selection of MDR is a microenvironment phenomenon and involves a group of isolates.

Strengths:<br /> The methods used in this study are robust and the results support the authors' claims to a major extent.

Weaknesses:<br /> The manuscript needs a thorough vetting of the language. At present, the language makes it very difficult to comprehend the methodology and results.

Reviewer #3 (Public Review):

Summary:<br /> Chen et al. are addressing a fundamental question in mammalian gut biology, namely how the host controls a mutualistic host-microbiota symbiosis. The authors focus on a protein called Chitinase 3-like protein 1 (Ch3l1) and its interaction with the protective colonic mucus layer. The rationale for the study comes from previous work showing that microbial-associated molecular patterns (MAMPs) can induce Ch3l1 in vitro, but its biological functions in the colon are unknown. In this study, the authors provide evidence supporting the claim that the gut microbiota induces the expression of Ch3l1 in vivo, mainly in mucus-producing goblet cells. Insightfully, the authors note that Ch3l1, although it lacks enzyme (chitinase) activity, still binds Chitin, a glycan that has structural similarity to bacterial cell wall peptidoglycan. This leads the authors to hypothesize that Ch3l1 binds microbial cell walls, particularly those of peptidoglycan-rich Gram-positive probiotic bacteria within the mucus, to promote their retention in the colon. Using a combination of in vivo work with mice conditionally lacking Ch3l1 in gut epithelium (IEC Ch3l1 KO); microbiota profiling; imaging of host-microbiota interactions with labeled microbes; and fecal transplants, the authors provide compelling evidence that Ch3l1 is secreted into the gut mucus layer and that the presence of Ch3l1 is associated with increased levels of beneficial Gram+ bacteria, including Lactobacillus spp. In turn, using a well-characterized colitis model, the authors show that Ch3l1 is associated with protection from intestinal injury caused by Dextran Sodium Sulfate. While these studies are novel and informative, there are several issues that undermine the authors' conclusions.

Strengths:<br /> The authors nicely link microbial induction of Ch3l1 to mucosal protection from intestinal injury. This is done through the use of germ-free and ex-germ-free studies and by comparing Ch3l1 expression in situ between them; microbial sequencing between Control and IEC Ch3l1 KO mice, and clinical and histological injury metrics between these strains. The authors convincingly demonstrate the presence of Ch3l1 in the gut mucus through imaging, and that the deletion of this protein in mice alters the microbiota by reducing the relative abundance of Gram-positive species.

The study employs a technically diverse set of analyses to address their hypothesis, including fluorescent labelling of microbial species for add-back studies, fecal transplants to distinguish the role(s) of the microbiota vs. host in the IEC Ch3l1 KO phenotypes in the intestinal challenge models.

Weaknesses:<br /> The claim that mucus-associated Ch3l1 controls colonization of beneficial Gram-positive species within the mucus is not conclusive. The study should take into account recent discoveries on the nature of mucus in the colon, namely its mobile fecal association and complex structure based on two distinct mucus barrier layers coming from proximal and distal parts of the colon (PMID: ). This impacts the interpretation of how and where Ch3l1 is expressed and gets into the mucus to promote colonization. It also impacts their conclusions because the authors compare fecal vs. tissue mucus, but most of the mucus would be attached to the feces. Of the mucus that was claimed to be isolated from the WT and IEC Ch3l1 KO, this was not biochemically verified. Such verification (e.g. through Western blot) would increase confidence in the data presented. Further, the study relies upon relative microbial profiling, which can mask absolute numbers, making the claim of reduced overall Gram-positive species in mice lacking Ch3l1 unproven. It would be beneficial to show more quantitative approaches (e.g. Quantitative Microbial Profiling, QMP) to provide more definitive conclusions on the impact of Ch3l1 loss on Gram+ microbes.

Other weaknesses lie in the execution of the aims, leaving many claims incompletely substantiated. For example, much of the imaging data is challenging for the reader to interpret due to it being unfocused, too low of magnification, not including the correct control, and not comparing the same regions of tissues among different in vivo study groups. Statistical rigor could be better demonstrated, particularly when making claims based on imaging data. These are often presented as single images without any statistics (i.e. analysis of multiple images and biological replicates). These images include the LTA signal differences, FISH images, Enterococcus colonization, and mucus thickness.

Reviewer #3 (Public Review):

This is an interesting manuscript that addresses a longstanding debate in evolutionary biology - whether social or ecological factors are primarily responsible for the evolution of the large human brain. To address this, the authors examine the relationship between the size of two prefrontal regions involved in metacognition and working memory (DLPFC and FP) and socioecological variables across 16 primate species. I recommend major revisions to this manuscript due to: 1) a lack of clarity surrounding model construction; and 2) an inappropriate treatment of the relative importance of different predictors (due to a lack of scaling/normalization of predictor variables prior to analysis).

Reviewer #3 (Public Review):

The authors have made simultaneous recordings of the responses of large numbers of neurons from the primary visual cortex using optical two-photon imaging of calcium signals from the superficial layers of the cortex. Recordings were made to compare the responses of the cortical neurons under normal binocular viewing of a flat screen with both eyes open and monocular viewing of the same screen with one eye's view blocked by a translucent filter. The screen displayed visual stimuli comprising small contrast patches of Gabor function distributions of luminance, a stimulus that is known to excite cortical neurons.

This is an important data set, given the large numbers of neurons recorded. The authors present a simple model to explain the binocular combination of neuronal signals from the right and left eyes.

The limitations of the paper as written are as follows. These points can be addressed with some additional analysis and rewriting of sections of the paper. No new experimental data need to be collected.

1) The authors should acknowledge the fact that these recordings arise from neurons in the superficial layers of the cortex. This limitation arises from the usual constraints on optical imaging in the macaque cortex. This means that the sample of neurons forming this data set is not fully representative of the population of binocular neurons within the visual cortex. This limitation is important in comparing the outcome of these experiments with the results from other studies of binocular combination, which have used single-electrode recording. Electrode recording will result in a sample of neurons that is drawn from many layers of the cortex, rather than just the superficial layers.

2) Single-neuron recording of binocular neurons in the primary visual cortex has shown that these neurons often have some spontaneous activity. Assessment of this spontaneous level of firing is important for accurate model fitting [1]. The paper here should discuss the level of spontaneous neuronal firing and its potential significance.

3) The arrangements for visual stimulation and comparison of binocular and monocular responses mean that the stereoscopic disparity of the binocular stimuli is always at zero or close to zero. The animal's fixation point is in the centre of a single display that is viewed binocularly. The fixation point is, by definition, at zero disparity. The other points on the flat display are also at zero disparity or very close to zero because they lie in the same depth plane. There will be some small deviations from exactly zero because the geometry of the viewing arrangements results in the extremities of the display being at a slightly different distance than the centre. Therefore, the visual stimulation used to test the binocular condition is always at zero disparity, with a slight deviation from zero at the edges of the display, and never changes. [There is a detail that can be ignored. The experimenters tested neurons with visual stimulation at different real distances from the eyes, but this is not relevant here. Provided the animals accurately converged their eyes on the provided binocular fixation point, then the disparity of the visual stimuli will always be at or close to zero, regardless of viewing distance in these circumstances.] However, we already know from earlier work that neurons in the visual cortex exhibit a range of selectivity for binocular disparity. Some neurons have their peak response at non-zero disparities, representing binocular depths nearer than the fixation depth or beyond it. The response of other neurons is maximally suppressed by disparities at the depth of the fixation point (so-called Tuned Inhibitory [TI] neurons). The simple model and analysis presented in the paper for the summation of monocular responses to predict binocular responses will perform adequately for neurons that are tuned to zero disparity, so-called tuned excitatory neurons [TE], but is necessarily compromised when applied to neurons that have other, different tuning profiles. Specifically, when neurons are stimulated binocularly with a non-preferred disparity, the binocular response may be lower than the monocular response[2, 3]. This more realistic view of binocular responses needs to be considered by the authors and integrated into their modelling.

4) The data in the paper show some features that have been reported before but are not captured by the model. Notably for neurons with extreme values of ocular dominance, the binocular response is typically less than the larger of the two monocular responses. This is apparent in the row of plots in Figure 2D from individual animals and in the pooled data in Figure 2E. Responses of this type are characteristic of tuned inhibitory [TI] neurons[2]. It is not immediately clear why this feature of the data does not appear in the summary and analysis in Figure 3. The paper text states that the responses were "first normalized by the median of the binocular responses". This will certainly get rid of this characteristic of the data, but this step needs better justification, or an amendment to the main analysis is needed. In the present form, the model and analysis do not appear to fit the data in Figure 2 as accurately as needed. The authors should address the discrepancy between the data as presented in Figures 2D, E, and Figure 3.

Citations<br /> 1. Prince, S.J.D., Pointon, A.D., Cumming, B.G., and Parker, A.J., (2002). Quantitative analysis of the responses of V1 neurons to horizontal disparity in dynamic random-dot stereograms. Journal of Neurophysiology, 87: 191-208.<br /> 2. Prince, S.J.D., Cumming, B.G., and Parker, A.J., (2002). Range and mechanism of encoding of horizontal disparity in macaque V1. Journal of Neurophysiology, 87: 209-221.<br /> 3. Poggio, G.F. and Fischer, B., (1977). Binocular interaction and depth sensitivity in striate and prestriate cortex of behaving rhesus monkey. Journal of Neurophysiology, 40: 1392-1405 doi 10.1152/jn.1977.40.6.1392.

Reviewer #3 (Public Review):

Summary:<br /> This paper presents a new formulation of a computational model of adaptive learning amid environmental volatility. Using a behavioral paradigm and data set made available by the authors of an earlier publication (Gagne et al., 2020), the new model is found to fit the data well. The model's structure consists of three weighted controllers that influence decisions on the basis of (1) expected utility, (2) potential outcome magnitude, and (3) habit. The model offers an interpretation of psychopathology-related individual differences in decision-making behavior in terms of differences in the relative weighting of the three controllers.

Strengths:<br /> The newly proposed "mixture of strategies" (MOS) model is evaluated relative to the model presented in the original paper by Gagne et al., 2020 (here called the "flexible learning rate" or FLR model) and two other models. Appropriate and sophisticated methods are used for developing, parameterizing, fitting, and assessing the MOS model, and the MOS model performs well on multiple goodness-of-fit indices. The parameters of the model show decent recoverability and offer a novel interpretation for psychopathology-related individual differences. Most remarkably, the model seems to be able to account for apparent differences in behavioral learning rates between high-volatility and low-volatility conditions even with no true condition-dependent change in the parameters of its learning/decision processes. This finding calls into question a class of existing models that attribute behavioral adaptation to adaptive learning rates.

Weaknesses:<br /> 1. Some aspects of the paper, especially in the methods section, lacked clarity or seemed to assume context that had not been presented. I found it necessary to set the paper down and read Gagne et al., 2020 in order to understand it properly.

2. There is little examination of why the MOS model does so well in terms of model fit indices. What features of the data is it doing a better job of capturing? One thing that makes this puzzling is that the MOS and FLR models seem to have most of the same qualitative components: the FLR model has parameters for additive weighting of magnitude relative to probability (akin to the MOS model's magnitude-only strategy weight) and for an autocorrelative choice kernel (akin to the MOS model's habit strategy weight). So it's not self-evident where the MOS model's advantage is coming from.

3. One of the paper's potentially most noteworthy findings (Figure 5) is that when the FLR model is fit to synthetic data generated by the expected utility (EU) controller with a fixed learning rate, it recovers a spurious difference in learning rate between the volatile and stable environments. Although this is potentially a significant finding, its interpretation seems uncertain for several reasons:

- According to the relevant methods text, the result is based on a simulation of only 5 task blocks for each strategy. It would be better to repeat the simulation and recovery multiple times so that a confidence interval or error bar can be estimated and added to the figure.

- It makes sense that learning rates recovered for the magnitude-oriented (MO) strategy are near zero, since behavior simulated by that strategy would have no reason to show any evidence of learning. But this makes it perplexing why the MO learning rate in the volatile condition is slightly positive and slightly greater than in the stable condition.

- The pure-EU and pure-MO strategies are interpreted as being analogous to the healthy control group and the patient group, respectively. However, the actual difference in estimated EU/MO weighting between the two participant groups was much more moderate. It's unclear whether the same result would be obtained for a more empirically plausible difference in EU/MO weighting.

- The fits of the FLR model to the simulated data "controlled all parameters except for the learning rate parameters across the two strategies" (line 522). If this means that no parameters except learning rate were allowed to differ between the fits to the pure-EU and pure-MO synthetic data sets, the models would have been prevented from fitting the difference in terms of the relative weighting of probability and magnitude, which better corresponds to the true difference between the two strategies. This could have interfered with the estimation of other parameters, such as learning rate.

- If, after addressing all of the above, the FLR model really does recover a spurious difference in learning rate between stable and volatile blocks, it would be worth more examination of why this is happening. For example, is it because there are more opportunities to observe learning in those blocks?

4. Figure 4C shows that the habit-only strategy is able to learn and adapt to changing contingencies, and some of the interpretive discussion emphasizes this. (For instance, line 651 says the habit strategy brings more rewards than the MO strategy.) However, the habit strategy doesn't seem to have any mechanism for learning from outcome feedback. It seems unlikely it would perform better than chance if it were the sole driver of behavior. Is it succeeding in this example because it is learning from previous decisions made by the EU strategy, or perhaps from decisions in the empirical data?

5. For the model recovery analysis (line 567), the stated purpose is to rule out the possibility that the MOS model always wins (line 552), but the only result presented is one in which the MOS model wins. To assess whether the MOS and FLR models can be differentiated, it seems necessary also to show model recovery results for synthetic data generated by the FLR model.

6. To the best of my understanding, the MOS model seems to implement valence-specific learning rates in a qualitatively different way from how they were implemented in Gagne et al., 2020, and other previous literature. Line 246 says there were separate learning rates for upward and downward updates to the outcome probability. That's different from using two learning rates for "better"- and "worse"-than-expected outcomes, which will depend on both the direction of the update and the valence of the outcome (reward or shock). Might this relate to why no evidence for valence-specific learning rates was found even though the original authors found such evidence in the same data set?

7. The discussion (line 649) foregrounds the finding of greater "magnitude-only" weights with greater "general factor" psychopathology scores, concluding it reflects a shift toward simplifying heuristics. However, the picture might not be so straightforward because "habit" weights, which also reflect a simplifying heuristic, correlated negatively with the psychopathology scores.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript from Tariq and Maurici et al. presents important biochemical and biophysical data linking protein phosphorylation to phase separation behavior in the repressive arm of the Neurospora circadian clock. This is an important topic that contributes to what is likely a conceptual shift in the field.

Reviewer #3 (Public Review):

Summary<br /> Pham, Pahuja, Hagenbeek, et al. have conducted a comprehensive range of assays to biochemically and genetically determine TEAD degradation through RNF146 ubiquitination. Additionally, they designed a PROTAC protein degrader system to regulate the Hippo pathway through TEAD degradation. Overall, the data appears robust. However, the manuscript lacks detailed methodological descriptions, which should be addressed and improved. For instance, the methods used to analyze the K48 ubiquitination site on TEAD and the gene expression analysis of Hippo Signaling are unclear. Furthermore, the multiple proteomics, RNA-seq, and ATAC-seq data must be made publicly available upon publication to ensure reproducibility. Most of the main figures are of low resolution, which needs addressing.

Strengths:<br /> - A broad range of assays was used to robustly determine the role of RNF146 in TEAD degradation.<br /> - Development of novel PROTAC for degrading TEAD.

Weaknesses:<br /> - An orthogonal approach is needed (e.g., PARP1 inhibitor) to demonstrate PARP1's dependency in TEAD ubiquitination.

- The data from Table 2 is unclear in illustrating the association of identified K48 ubiquitination with TEAD4, especially since the experiments were presumably to be conducted on whole cell lysates with KGG enrichment. This raises the possibility that the K48 ubiquitination could originate from other proteins. Alternatively, if the authors performed immunoprecipitation on TEAD followed by mass spectrometry, this should be explicitly described in the text and materials and methods section.

- Figure 2D: The methodology for measuring the Hippo signature is unclear, as is the case for Figures 7E and F regarding the analysis of Hippo target genes.

- Figure S3F requires quantification with additional replicates for validation.

- There is a misleading claim in the discussion stating "TEAD PARylation by PAR-family members (Figure 3)"; however, the demonstration is only for PARP1, which should be corrected.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, Curran et al investigate the role of Ntn, Slit1, and Slit 2 in the axon patterning of DRG neurons. The paper uses mouse genetics to perturb each guidance molecule and its corresponding receptor. Cre-based approaches and immunostaining of DRG neurons are used to assess the phenotypes. Overall, the study uses the strength of mouse genetics and imaging to reveal new genetic modifiers of DRG axons. The conclusions of the experiments match the presented results. The paper is an important contribution to the field, as evidence that dorsal funiculus formation is impacted by Ntn and Slit signaling. However, there are some potential areas of the manuscript that should be edited to better match the results with the conclusions of the work.

Strengths:<br /> The manuscript uses the advantage of mouse genetics to investigate the axon patterning of DRG neurons. The work does a great job of assessing individual phenotypes in single and double mutants. This reveals an intriguing cooperative and independent function of Ntn, Slit1, and Slit2 in DRG axon patterning. The sophisticated triple mutant analysis is lauded and provides important insight.

Weaknesses:<br /> Overall, the manuscript is sound in technique and analysis. However, the majority of the manuscript is about the dorsal funiculus and not the bifurcation of the axons, as the title would make a reader believe. Further, the manuscript would provide a more scholarly discussion of the current knowledge of DRG axon patterning and how their work fits into that knowledge.

Reviewer #3 (Public Review):

In this work, the authors shed light onto the structures of Fusarium oxysporum f.sp. lycopersici proteins involved in the infection of tomato. They unravelled several new secreted effector protein structures and additionally used computational approaches to structurally classify the remaining effectors known from this pathogen. Through this they uncovered a new and unique structural class of proteins which they found to be present and widely distributed in fungal plant pathogens and plant symbiotic fungi. The authors further predicted structural models for the full SIX effector set revealing that genome-proximal effector pairs share similar structural classes. Building on their Avr1 structure, the authors also define the C-terminal domain and specific amino acid residues that are essential to Avr1 detection by its cognate immune receptor.

A major strength of this work is a portfolio of several (Avr1, Avr3, SIX6, SIX8) new structurally resolved proteins which led to the discovery that several of them fall into the same structural class. These findings are supported by strong results.

The experiments addressing the structure-function relationship of Avr1's avirulence activity are highly relevant to our understanding of disease resistance mechanisms against Fusarium. Additional controls would allow for better support of the conclusions to be drawn. An example is FonSIX4's cell death activity in N.benthamiana leaves and whether FonSIX4 cdll death is indeed dependent on the tomato I receptor. Complementary work in Fusarium mutants lacking Avr1 and expressing chimeric versions would document that the observations from transient expressions in Nicotiana benthamiana are relevant in the biological context of a Fusarium/tomato interaction.

The discovered solvent-exposed residues conditioning Avr1 recognition by the I receptor seem to be positioned in an area of the protein which had previously been highlighted as being highly variable in FOLD proteins of symbiotic fungi but it is not clear from the work whether this is indeed the case or whether Avr1 differs significantly in its structure from FOLD proteins found in other fungi.<br /> It remains to be tested whether the residues conditioning avirulence activity are also crucial for virulence activity in Fusarium.

This work uncovered a new structural class of proteins with critical roles in plant-pathogen interactions. Structure-based predictions and genome-wide comparisons have emerged as a new approach enabling the identification of similar proteins with divergent sequences. The work undertaken by the authors adds to a growing body of work in plant-microbe research, predominantly from pathogenic organisms, and more recently in symbiotic fungi.

Reviewer #3 (Public Review):

Summary:<br /> In this study, the authors investigated the potential of nifuroxazide to enhance responsiveness to radiotherapy, employing both an in vitro cell culture system and an in vivo syngeneic mouse tumor model.

Strengths:<br /> The researchers conducted a series of experiments to elucidate the role of nifuroxazide in facilitating the radiotherapy-induced reduction of proliferation, migration, and invasion of HepG2 cells.

Weaknesses:<br /> The evidence supporting the claim that nifuroxazide contributes to the degradation of radiotherapy-induced upregulation of PD-L1 via the ubiquitin-proteasome pathway is still relatively weak.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript introduces a new computational framework for choosing 'the best method' according to the case for getting the best possible structural prediction for the CDR-H3 loop. The authors show their strategy improves on average the accuracy of the predictions on datasets of increasingly difficulty in comparison to several state-of-the-art methods. They also show the benefits of improving the structural predictions of the CDR-H3 in the evaluation of different properties that may be relevant for drug discovery and therapeutics design.

Strengths:<br /> Authors introduce a novel framework, which can be easily adapted and improved. Authors use a well defined dataset to test their new method. A modest average accuracy gain is obtained in comparison to other state-of-the art methods for the same task, while avoiding for testing different prediction approaches. Although the accuracy gain is mainly ascribed to easy cases, the accuracy and precision for moderate to challenging cases is comparable to the best PLM methods (see Fig. 4b and Extended Data Fig. 2), reflecting the present methodological limit in the field.

Weaknesses:<br /> The proposed method lacks of a confidence score or a warning to help guiding the users in moderate to challenging cases.

Reviewer #3 (Public Review):

Summary:<br /> This study examines the impact of CTRP10/C1QL2 absence on obesity and metabolic health in mice. Female mice lacking CTRP10 tend to develop obesity, particularly on a high-fat diet. Surprisingly, they do not display the typical metabolic traits associated with obesity, like fatty liver or glucose intolerance. This indicates a disconnection between weight gain and metabolic issues in these female mice. The research underscores the need to understand sex-specific factors in how obesity influences metabolic health.

Strengths:<br /> The study provides compelling evidence regarding Ctrp10's role in female-specific metabolic regulation in mice, shedding light on its potential significance in metabolically healthy obese (MHO) individuals.

Weaknesses:<br /> -The analysis and description of sex-specific human data require more details to highlight the relevance of Ctrp10 mouse data and the analysis of differentially expressed genes in humans.<br /> -There's a lack of analysis regarding secreted Ctrp10 under various dietary conditions.<br /> -The study didn't assess adipose tissue function to evaluate metabolic health.

Reviewer #3 (Public Review):

Summary:

Bartolome et al. present ProteasomeID, a novel method to identify components, interactors, and (potentially) substrates of the proteasome in cell lines and mouse models. As a major protein degradation machine that is highly conserved across eukaryotes, the proteasome has historically been assumed to be relatively homogeneous across biological scales (with few notable exceptions, e.g., immunoproteasomes and thymoproteasomes). However, a growing body of evidence suggests that there is some degree of heterogeneity in the composition of proteasomes across cell tissues, and can be highly dynamic in response to physiologic and pathologic stimuli. This work provides a methodological framework for investigating such sources of variation. The authors start by adapting the increasingly popular biotin ligation strategy for labelling proteins coming into close proximity with one of three different subunits of the proteasome, before proceeding with PSMA4 for further development and analysis based on their preliminary labelling data. In a series of well-constructed and convincing validation experiments, the authors go on to show that the tagged PSMA4 construct can be incorporated into functional proteasomes, and is able to label a broad set of known proteasome components and interacting proteins in HEK293T cells. They also attempt to identify novel proteasomal degradation substrates with ProteasomeID; while this was convincing for known substrates with particularly short half-lives (exemplified by the transcription factor c-myc), follow-up validation experiments with other substrates were less clear. One of the most compelling results was from a similar experiment to confirm proteasomal degradation induced by a BRD-targeting PROTAC, which I think is likely to be of keen interest to the targeted degradation community. Finally, the authors establish a ProteasomeID mouse model, and demonstrate its utility across several tissues.

Strengths:

1) ProteasomeID itself is an important step forward for researchers with an interest in protein turnover across biological scales (e.g., in sub-cellular compartments, in cells, in tissues, and whole organisms). I especially see interest from two communities: those studying fundamental proteostasis in physiological and pathologic processes (e.g., ageing; tissue-specific protein aggregation diseases), and those developing targeted protein degradation modalities (e.g., PROTACs; molecular glues). All the datasets generated and deposited here are likely to provide a rich resource to both. The HEK293T cell line data are a valuable proof-of-concept to allow expansion into more biologically-relevant cell culture settings; however, I envision the greatest innovation here to be the mouse model. For example, in the targeted protein degradation space, two major hurdles in early-stage pre-clinical development are (i) evaluation of degradation efficacy across disease-relevant tissues, and (ii) toxicity and safety implications caused by off-target degradation, e.g., of newly-identified molecular glues and/or in particularly-sensitive tissues. The ProteasomeID mouse allows early in vivo assessment of both these questions. The results of the BRD PROTAC experiment in 293T cells provides an excellent in vitro proof-of-concept for this approach.

2) The mass spectrometry-based proteomics workflows used and presented throughout the manuscript are robust, rigorous, and convincing. For example, the algorithm the authors use for defining enrichment score cut-offs are logical and based on rational models, rather than on arbitrary cut-offs that are common for similar proteomics studies. The construction (and subsequent validation) of both BirA*- and miniTurbo- tagged PSMA4 variants also increases the utility of the method, allowing researchers to choose the variant with the labelling time-scale required for their particular research question.

3) The optimised BioID and TurboID protocol the authors develop (summarised in Fig. S2A) and validate (Fig. S2B-D) is likely to be of broad interest to cell and molecular biologists beyond the protein degradation field, given that proximity labelling is a current gold-standard in global protein:protein interaction profiling.

Limitations:

I think the authors do an excellent job in highlighting the limitations of ProteasomeID throughout the Results and Discussion. I do have some specific comments that might provide additional context for the reader.

1) The authors do a good job in showing that a substantial proportion of PSMA4-BirA* is incorporated into functional proteasome particles; however, it is not immediately clear to me how much background (false-positive IDs) might be contributed by the ~40 % of PSMA4-BirA* that is not incorporated into the mature core particle (based on the BirA* SEC-MS traces in Fig. 2b and S3b, i.e., the large peak ~ fraction 20). Are there any bands lower down in the native gel shown in Fig. 2c, i.e., corresponding to lower molecular weight complexes or monomeric PSMA4-BirA*? The enrichment of proteasome assembly factors in all the ProteasomeID experiments might suggest the presence of assembly intermediates, which might themselves become substrates for proteasomal degradation (as has been shown for other incompletely-assembled protein complexes, e.g., the ribosome, TRiC/CCT).

2) Although the authors attempt to show that BirA* tagging of PSMA4 does not interfere with proteasome activity (Fig. 2e-f), I think the experimental evidence for this is incomplete. They show that the overall chymotrypsin-like activity (attributable to PSMB5) in cells expressing PSMA4-BirA* is not markedly reduced compared with control BirA*-expressing cells. However, they do not show that the activity of the specific proteasome sub-population that contains PSMA4-BirA* is unaffected (e.g., by purifying this sub-population via the Flag tag). The proteasome activity of the sub-population of wild-type proteasome complexes that do not contain the PSMA4-BirA* (~50%, based on the earlier immunoblots) could account for the entire chymotrypsin-like activity-especially in the context of HEK293T cells, where steady-state proteasome levels are unlikely to be limiting. It would also be useful to assess any changes in tryspin- and caspase- like activities, especially as tagging of PSMA4 could conceivably interfere with the activity of some PSMB subunits, but not others.

3) I was left unsure of the general utility of ProteasomeID for identifying novel proteasomal substrates in homeostatic or stressed conditions. The immunoblots for the two candidates the authors follow up in Fig. 4g was not especially clear; the reduction in the bands are modest, at best. Furthermore, classifying candidates based on enrichment following proteasome inhibition with MG-132 have the potential to lead to a high number of false positives. ProteasomeID's utility in identifying potential substrates in more targeted settings (e.g., molecular glues, off-target PROTAC substrates) is far more apparent.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Zhang et al. analyzes the function of the centrosomal protein CEP44 in centriole duplication and in the formation of the mitotic spindle. The first part addresses the role of CEP44 at centrioles. Using mostly RNAi-mediated depletion in cell lines and in some cases KO cells, the authors find increased centriole numbers in depleted cells and, based on quantification of centrioles stained with various centriole markers as well as live imaging, conclude that this is due to premature centriole disengagement and overduplication. The second part, which is largely independent of the first, focuses on the role of CEP44 in the mitotic spindle. The authors find that CEP44 is phosphorylated in mitosis in an Aurora A-dependent manner and identify the phosphorylation site, which controls CEP44 spindle localization and functions in maintaining spindle integrity.

Strength:<br /> The manuscript makes the interesting observation that reduced expression of CEP44 is observed in breast cancer and correlated with poor survival in patients.<br /> The analysis of mitotic phosphorylation including the identification of the modified site and its role in spindle recruitment is interesting and useful.

Weakness:<br /> The authors seem to largely ignore previously published work that contrasts with the findings presented in the current study. The previous work found a role of CEP44 in centriole formation and centrosome conversion and observed reduced centriole numbers in depleted cells, whereas the current study claims the opposite, a role in centriole engagement that leads to overduplication and increased centriole number in depleted cells. However, the supporting evidence is not strong enough, especially in light of the previous work. Considering that CEP44 depletion also disrupts mitosis, which could affect centriole numbers by failed segregation/division, a more careful analysis in synchronized cultures would be needed. Also, cell cycle analysis would be required to rule out cell cycle effects in CEP44-depleted cells, which could also explain altered centriole numbers. Moreover, the quality of the imaging is often not sufficient to support the claims.<br /> The second part is largely disconnected from the first and reads as if it was a separate study. There is no attempt to integrate both parts. For example, the second part seems to largely focus on normal bipolar spindles, even though the first part reveals multipolarity as a phenotype after CEP44 knockdown. It remains unclear if the spindle defects are due to centriole defects, defective spindle microtubule stability/organization, or both, and whether the centriole-localized or spindle-localized CEP44 is involved.

Another weak aspect is that neither for RNAi nor for KO cells the authors show that CEP44 is depleted at centrioles and to what extent. This is only shown in cell extract.

Reviewer #3 (Public Review):

Summary:<br /> The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:<br /> The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1, and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs.

Weaknesses:<br /> Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. In addition, some conclusions about the role of specific DDC proteins in cell cycle arrest at G2/M need further experimental support. The results with Bfa1/Bub2 are surprising and somewhat unexpected. There is no clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block.