Reviewer #2 (Public review):

Summary:

Using in vivo fiber-photometry the authors first establish that DA release when contacting their partner mouse increases with days of cohabitation while this increase is not observed when contacting a stranger mouse. Similar effects are found in D1-MSNs and D2-MSNs with the D1-MSN responses increasing and D2-MSN responses decreasing with days of cohabitation. They then use slice physiology to identify underlying plasticity/adaptation mechanisms that could contribute to the changes in D1/D2-MSN responses. Last, to address causality the authors use chemogenetic tools to selectively inhibit or activate NAc shell D1 or D2 neurons that project to the ventral pallidum. They found that D2 inhibition facilitates bond formation while D2 excitation inhibits bond formation. In contrast, both D1-MSN activation and inhibition inhibits bond formation.

Strengths:

The strength of the manuscript lies in combining in vivo physiology to demonstrate circuit engagement and chemogenetic manipulation studies to address circuit involvement in pair bond formation in a monogamous vole.

Weaknesses:

Weaknesses include that a large set of experiments within the manuscript are dependent on using short promoters for D1 and D2 receptors in viral vectors. As the authors acknowledge this approach can lead to ectopic expression and the presented immunohistochemistry supports this notion. It seems to me that the presented quantification underestimates the degree of ectopic expression that is observed by eye when looking at the presented immunohistochemistry. However, given that Cre transgenic animals are not available for Microtus mandarinus and given the distinct physiological and behavioral outcomes when imaging and manipulating both viral-targeted populations this concern is minor.

The slice physiology experiments provide some interesting outcomes but it is unclear how they can be linked to the in vivo physiological outcomes and some of the outcomes don't match intuitively (e.g. cohabitation enhances excitatory/inhibitory balance in D2-MSNs but the degree of contact-induced inhibition is enhanced in D2-MSN).

One interesting finding is that the relationship between D2-MSN and pair bond formation is quite clear (inhibition facilitates while excitation inhibits pair bond formation). In contrast, the role of D1-MSNs is more complicated since both excitation and inhibition disrupts pair bond formation. This is not convincingly discussed.

It seemed a missed opportunity that physiological read out is limited to males. I understand though that adding females may be beyond the scope of this manuscript.

Comments on revised version:

The authors addressed most of my comments, some would still need to be addressed.

(1) Previous comment: "The authors do not use an isosbestic control wavelength in photometry experiments, although they do use EGFP control mice which show no effects of these interventions, a within-subject control such as an isosbestic excitation wavelength could give more confidence in these data and rule out motion artefacts within subjects."

The authors should include a paragraph in the discussion addressing the limitations of not using an internal control for the fiberphotometric measurements.

(2) Previous Comment: The slice physiology experiments provide some interesting outcomes but it is unclear how they can be linked to the in vivo physiological outcomes and some of the outcomes don't match intuitively (e.g. cohabitation enhances excitatory/inhibitory balance in D2-MSNs but the degree of contact-induced inhibition is enhanced in D2-MSN).

My comment may not have been clear and the response didn't address my comment. What is missing in the discussion is an explanation of why a relative increase in excitation of D2-MSNs in the slice (Fig. 4J) is associated with an increased inhibition in vivo (Fig. 2H)?

(3) Previous Comment: One interesting finding is that the relationship between D2-MSN and pair bond formation is quite clear (inhibition facilitates while excitation inhibits pair bond formation). In contrast, the role of D1-MSNs is more complicated since both excitation and inhibition disrupt pair bond formation. This is not convincingly discussed.

Similarly, here the response provided does not address my question. Please focus on discussing why both excitation and inhibition of D1-MSNs can disrupt pair bond formation (Figure 7).

Reviewer #1 (Public review):

The authors demonstrated that NINJ1 promotes TF-positive MV release during pyroptosis and thereby triggers coagulation. Coagulation is one of the risk factors that can cause secondary complications in various inflammatory diseases, making it a highly important therapeutic target in clinical treatment. This paper effectively explains the connection between pyroptosis and MV release with Ninj1, which is a significant strength. It provides valuable insight into the potential of targeting Ninj1 as a therapeutic strategy.

Although the advances in this paper are valuable, several aspects need to be clarified. Some comments are discussed below.

(1) Since it is not Ninj1 directly regulating coagulation but rather the MV released by Ninj1 playing a role, the title should include that. The current title makes it seem like Ninj1 directly regulates inflammation and coagulation. It would be better to revise the title.

(2) Ninj1 is known to be an induced protein that is barely expressed in normal conditions. As you showed in "Fig1G" data, control samples showed no detection of Ninj1. However, in "Figure S1", all tissues (liver, lung, kidney and spleen) expressed Ninj1 protein. If the authors stimulated the mice with fla injection, it should be mentioned in the figure legend.

(3) In "Fig3A", the Ninj1 protein expression was increased in the control of BMDM +/- cell lysate rather than fla stimulation. However, in MV, Ninj1 was not detected at all in +/- control but was only observed with Fla injection. The authors need to provide an explanation for this observation. Additionally, looking at the MV β-actin lane, the band thicknesses appear to be very different between groups. It seems necessary to equalize the protein amounts. If that is difficult, at least between the +/+ and +/- controls.

(4) Since the authors focused Ninj1-dependent microvesicle (MV) release, they need to show MV characterizations (EM, NTA, Western for MV markers, etc...).

(5) To clarify whether Ninj1-dependent MV induces coagulation, the authors need to determine whether platelet aggregation is reduced with isolated +/- MVs compared to +/+ MVs.

(6) Even with the authors well established experiments with haploid mice, it is a critical limitation of this paper. To improve the quality of this paper, the authors should consider confirming the findings using mouse macrophage cell lines, such as generating Ninj1-/- Raw264.7 cell lines, to examine the homozygous effect.

(7) There was a paper reported in 2023 (Zhou, X. et al., NINJ1 Regulates Platelet Activation and PANoptosis in Septic Disseminated Intravascular Coagulation. Int. J. Mol. Sci. 2023) that revealed the relationship between Ninj1 and coagulation. According to this paper, inhibition of Ninj1 in platelets prevents pyroptosis, leading to reduced platelet activation and, consequently, the suppression of thrombosis. How about the activation of platelets in Ninj1 +/- mice? The author should add this paper in the reference section and discuss the platelet functions in their mice.

Reviewer #2 (Public review):

Summary:

Golamalamdari, van Schaik, Wang, Kumar Zhang, Zhang and colleagues study interactions between the speckle, nucleolus and lamina in multiple cell types (K562, H1, HCT116 and HFF). Their datasets define how interactions between the genome and the different nuclear landmarks relate to each other and change across cell types. They also identify how these relationships change in K562 cells in which LBR and LMNA are knocked out.

Strengths:

Overall, there are a number of datasets that are provided, and several "integrative" analyses performed. This is a major strength of the paper, and I imagine the datasets will be of use to the community to further probed and the relationships elucidated here further studied. An especially interesting result was that specific genomic regions (relative to their association with the speckle, lamina, and other molecular characteristics) segregate relative to the equatorial plane of the cell.

Weaknesses:

The experiments are primarily descriptive, and the cause-and-effect relationships are limited (though the authors do study the role of LMNA/LBR knockdown with their technologies).

Reviewer #1 (Public review):

Summary:

The drug Ivermectin is used to effectively treat a variety of worm parasites in the world, however resistance to Ivermectin poses a rising challenge for this treatment strategy. In this study, the authors found that loss of the E3 ubiquitin ligase UBR-1 in the worm C. elegans results in resistance to Ivermectin. In particular, the authors found that ubr-1 mutants are resistant to the effects of Ivermectin on worm viability, body size, pharyngeal pumping and locomotion. The authors previously showed that loss of UBR-1 disrupts homeostasis of the amino acid and neurotransmitter glutamate resulting in increased levels of glutamate in C. elegans. Here, the authors found that the sensitivity of ubr-1 mutants to Ivermectin can be restored if glutamate levels are reduced using a variety of different methods. Conversely, treating worms with exogenous glutamate to increase glutamate levels also results in resistance to Ivermectin supporting the idea that increased glutamate promotes resistance to Ivermectin. The authors found that the primary known targets of Ivermectin, glutamate-gated chloride channels (GluCls), are downregulated in ubr-1 mutants providing a plausible mechanism for why ubr-1 mutants are resistant to Ivermectin. Although it is clear that loss of GluCls can lead to resistance to Ivermectin, this study suggests that one potential mechanism to decrease GluCl expression is via disruption of glutamate homeostasis that leads to increased glutamate. This study suggests that if parasitic worms become resistant to Ivermectin due to increased glutamate, their sensitivity to Ivermectin could be restored by reducing glutamate levels using drugs such as Ceftriaxone in a combination drug treatment strategy.

Strengths:

- The use of multiple independent assays (i.e., viability, body size, pharyngeal pumping, locomotion and serotonin-stimulated pharyngeal muscle activity) to monitor the effects of Ivermectin<br /> - The use of multiple independent approaches (got-1, eat-4, ceftriaxone drug, exogenous glutamate treatment) to alter glutamate levels to support the conclusion that increased glutamate in ubr-1 mutants contributes to Ivermectin resistance

Weaknesses:

- The primary target of Ivermectin is GluCls so it is not surprising that alteration of GluCl expression or function would lead to Ivermectin resistance<br /> - It remains to be seen what percent of Ivermectin resistant parasites in the wild have disrupted glutamate homeostasis as opposed to mutations that more directly decrease GluCl expression or function.

Comments on revisions: All my concerns have been addressed by the authors.

Reviewer #1 (Public review):

Summary:

Joshua G. Medina-Feliciano et al. investigated the single-cell transcriptomic profile of holoturian regenerating intestine following evisceration, a process used to expel their viscera in response to predation. Using single-cell RNA-Sequencing and standard analysis such as "Find cluster markers", "Enrichment analysis of Gene Ontology" and "RNA velocity", they identify 13 cell clusters and their potential cell identity. Based on bioinformatic analysis they identified potentially proliferating clusters and potential trajectories of cell differentiation. This manuscript represents a useful dataset that can provide candidate cell types and cell markers for more in-depth functional analysis of the holoturian intestine regeneration.

The conclusions of this paper are supported only by bioinformatic analyses since the in vivo validation through HCR is not sufficient to support them.

Strengths:

- The Authors are providing a single-cell dataset obtained from sea cucumbers regenerating their intestines. This represents the first fundamental step to an unbiased approach to better understand this regeneration process and the cellular dynamics taking part in it.

- The Authors run all the standard analyses providing the reader with a well digested set of information about cell clusters, potential cell types, potential functions and potential cell differentiation trajectories.

Weaknesses:

- The Authors frequently report the percentage of cells with a specific feature (either labelled or expressing a certain gene or belonging to a certain cluster). This number can be misleading since that is calculated after cell dissociation and additional procedures (such as staining or sequencing and dataset cleanup) that can heavily bias the ratio between cell types. Similarly, the Authors cannot compare cell percentage between anlage and mesentery samples since that can be affected by technical aspects related to cell dissociation, tissue composition and sequencing depth.

- The Authors did not validate all the clusters.

- There is no validation of the trajectory analysis and there is no validation of the proliferating cluster with H3P or EdU co-labeling.

Joint Public Review:

Páramo et al. used 3D geometric morphometric analyses of the articulated femur, tibia, and fibula of 17 macronarian taxa (known to preserve these three skeletal elements) to investigate morphological changes that occurred in the hind limb through the evolutionary history of this sauropod clade. A principal components analysis was completed to understand the distribution of the morphological variation. A supertree was constructed to place evolutionary trends in morphological variation into phylogenetic context, and hind limb centroid size was used to investigate potential relationships between skeletal anatomy and gigantism. The majority of the results did not yield statistically significant differences, but they did identify interesting shape-change trends, especially within subclades of Titanosauria. Many previous studies have attempted to elucidate a link between wide-gauge posture and gigantism, which in this study Páramo et al. investigate among several titanosaurian subclades. They propose that morphologies associated with wide-gauge posture arose in parallel with increasing body size among basal members of Macronaria and that this connection became less significant once wide-gauge posture was acquired within Titanosauria. The authors also suggest that other biomechanical factors influenced the independent evolution of subclades within Titanosauria and that these influences resulted in instances of convergent evolution. Therefore, they infer that, overall, wide-gauge posture was not significantly correlated with gigantism, though some morphological aspects of hind limb skeletal anatomy appear to have been associated with gigantism. Their work also supports previous findings of a decrease in body size within Titanosauriformes (which they found to be not significant with shape variables but significant with Pagel's lambda). Collectively, their results support and build on previous work to elucidate more specifics on the evolution of this enigmatic clade. Further study will show if their hypotheses stand or if the inclusion of additional specimens and taxa yields alternative results.

[Editors' note: One of the original reviewers, Reviewer 2, reviewed this revised version of the manuscript; they reported satisfaction with the changes made by the authors in response to the original reviewer comments.]

Reviewer #1 (Public review):

In this paper, the authors had 2 aims:

(1) Measure macaques' aversion to sand and see if its' removal is intentional, as it likely in an unpleasurable sensation that causes tooth damage.

(2) Show that or see if monkeys engage in suboptimal behavior by cleaning foods beyond the point of diminishing returns, and see if this was related to individual traits such as sex and rank, and behavioral technique.

They attempted to achieve these aims through a combination of geochemical analysis of sand, field experiments, and comparing predictions to an analytical model.

The authors' conclusions were that they verified a long-standing assumption that monkeys have an aversion to sand as it contains many potentially damaging fine grained silicates, and that removing it via brushing or washing is intentional.

They also concluded that monkeys will clean food for longer than is necessary, i.e. beyond the point of diminishing returns, and that this is rank-dependent.

High and low-ranking monkeys tended not to wash their food, but instead over-brushed it, potentially to minimize handling time and maximize caloric intake, despite the long-term cumulative costs of sand.

This was interpreted through the *disposable soma hypothesis*, where dominants maximize immediate needs to maintain rank and increase reproductive success at the potential expense of long-term health and survival.

# Strengths

The field experiment seemed well designed, and their quantification of the physical and mineral properties of quartz particles (relative to human detection thresholds) seemed good relative to their feret diameter and particle circularity (to a reviewer that is not an expert in sand). The *Rank Determination* and *Measuring Sand* sections were clear.

In achieving Aim 1, the authors validated a commonly interpreted, but unmeasured function, of macaque and primate behavior-- a key study/finding in primate food processing and cultural transmission research.

I commend their approach in trying to develop a quantitative model to generate predictions to compare to empirical data for their second aim.<br /> This is something others should strive for.

I really appreciated the historical context of this paper in the introduction and found it very enjoyable and easy to read.

I do think that interpreting these results in the context of the *disposable soma hypothesis* and the potential implications in the *paleolithic matters* section about interpreting dental wear in the fossil record are worthwhile.

# Weaknesses

Several of my concerns in an earlier review were addressed in revision, which I appreciate. One thing I think could strengthen this paper is a clearer link to social foraging theory to explore heterogeneity in handling times (as the currency they are trying to maximize).

I am satisfied with the improvements in statistics and that I can access the code and data.

I am still struck that there was an analysis of only trials where <3 individuals are present. If rank was important, I would imagine that behavior might be different in social contexts when theft, scrounging, policing, aggression, or other distractions might occur-- where rank would have effects on foraging behavior. Maybe lower rankers prioritize rapid food intake then. If rank should be related to investment in this behavior, we might expect this to be magnified (or different) in social contexts where it would affect foraging. It might just be that the data was too hard to score or process in those settings, or the analysis was limited. Additionally, I think that more robust metrics of rank from more densely sampled focal follow data would be a better measure, but I acknowledge the limitations in getting the ideal . Since rank is central to the interpretation of these results, I think that reduced social contexts in which rank was analyzed and the robustness of the data from which rank was calculated and analyzed are the main weaknesses of the evidence presented in this paper.

While some of the boxes about raccoons and Concorde Fallacy were interesting, they did feel like a bit of a distraction from the main message in the paper.

Reviewer #1 (Public review):

Summary:

In their manuscript, Gomez-Frittelli and colleagues characterize the expression of cadherin6 (and -8) in colonic IPANs of mice. Moreover, they found that these cdh6-expressing IPANs are capable of initiating colonic motor complexes in the distal colon, but not proximal and midcolon. They support their claim by morphological, electrophysiological and optogenetic, and pharmacological experiments.

Strengths:

The work is very impressive and involves several genetic models and state-of-the-art physiological setups including respective controls. It is a very well-written manuscript that truly contributes to our understanding of GI-motility and its anatomical and physiological basis. The authors were able to convincingly answer their research questions with a wide range of methods without overselling their results.

Weaknesses:

The authors put quite some emphasis on stating that cdh6 is a synaptic protein (in the title and throughout the text), which interacts in a homophilic fashion. They deduct that cdh6 might be involved in IPAN-IPAN synapses (line 247ff.). However, Cdh6 does not only interact in synapses and is expressed by non-neuronal cells as well (see e.g., expression in the proximal tubuli of the kidney). Moreover, cdh6 does not only build homodimers, but also heterodimers with Chd9 as well as Cdh7, -10, and -14 (see e.g., Shimoyama et al. 2000, DOI: 10.1042/0264-6021:3490159). It would therefore be interesting to assess the expression pattern of cdh6-proteins using immunostainings in combination with synaptic markers to substantiate the authors' claim or at least add the possibility of cell-cell-interactions other than synapses to the discussion. Additionally, an immunostaining of cdh6 would confirm if the expression of tdTomato in smooth muscle cells of the cdh6-creERT model is valid or a leaky expression (false positive).

Comments on revisions:

The authors have updated their manuscript and have provided insights and discussions to my remarks.

Reviewer #1 (Public review):

Summary:

Asthenospermia, characterized by reduced sperm motility, is one of the major causes of male infertility. The "9 + 2" arranged MTs and over 200 associated proteins constitute the axoneme, the molecular machine for flagellar and ciliary motility. Understanding the physiological functions of axonemal proteins, particularly their links to male infertility, could help uncover the genetic causes of asthenospermia and improve its clinical diagnosis and management. In this study, the authors generated Ankrd5 null mice and found that ANKRD5-/- males exhibited reduced sperm motility and infertility. Using FLAG-tagged ANKRD5 mice, mass spectrometry, and immunoprecipitation (IP) analyses, they confirmed that ANKRD5 is localized within the N-DRC, a critical protein complex for normal flagellar motility. However, transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) of sperm from Ankrd5 null mice did not reveal any structural abnormalities.

Strengths:

The phenotypes observed in ANKRD5-/- mice, including reduced sperm motility and male infertility, are conversing. The authors demonstrated that ANKRD5 is an N-DRC protein that interacts with TCTE1 and DRC4. Most of the experiments are thoughtfully designed and well executed.

Weaknesses:

The cryo-FIB and cryo-ET analyses require further investigation, as detailed below. The molecular mechanism by which the loss of ANKRD5 affects sperm flagellar motility remains unclear. The current conclusion that Ankrd5 knockout reduces axoneme stability is not well-supported. Specifically, are other axonemal proteins diminished in Ankrd5 knockout sperm? Conducting immunofluorescence analyses and revisiting the quantitative proteomics data may help address these questions.

Reviewer #1 (Public review):

Summary:

In this study, the authors examined the role of Afadin, a key adaptor protein associated with cell-adhesion molecules, in retinal development. Using a conditional knockout mouse line (Six3-Cre; AfadinF/F), the authors successfully characterized a disorganized pattern of various neuron types in the mutant retinae. Despite these altered distributions, the retinal neurons maintained normal cell numbers and seemingly preserved some synaptic connections. Notably, tracing results indicated mistargeting of retinal ganglion cell (RGC) axon projections to the superior colliculus, and electroretinography (ERG) analyses suggested deficits in visual functions.

Strengths:

This compelling study provides solid evidence addressing the important question of how cell-adhesion molecules influence neuronal development. Compared to previous research conducted in other parts of the central nervous system (CNS), the clearly defined lamination of cell types in the retina serves as a unique model for studying the aberrant neuronal localizations caused by Afadin knockout. The data suggest that cell-cell interactions are critical for retinal cellular organization and proper axon pathfinding, while aspects of cell fate determination and synaptogenesis remain less understood. This work has broad implications not only for retinal studies but also for developmental biology and regenerative medicine.

Weaknesses:

While the phenotypes observed in the Afadin knockout (cKO) mice are intriguing, I would expect to see evidence confirming that Afadin is indeed knocked out in the retina through immunostaining. Specifically, is Afadin knocked out only in certain retinal regions and not others, as suggested by Figures 4A-B? Are Afadin levels different among distinct neuron types, which could mean that its knockout may have a more pronounced impact on certain cell types, such as rods compared to others?

The authors suggest that synapses may form between canonical synaptic partners, based on the proximity of their processes (Figure 2). However, more solid evidence is needed to verify these synapses through the use of synaptic marker staining or transsynaptic labeling before drawing further conclusions.

Although the Afadin cKO mice displayed dramatic phenotypes, additional experiments are necessary to clarify the details of this process. By manipulating Afadin levels in specific cell types or at different developmental time points, we could gain a better understanding of how Afadin regulates accurate retinal lamination and axonal projection.

Reviewer #1 (Public review):

Summary:

The authors address the role of the centromere histone core in force transduction by the kinetochore.

Strengths:

They use a hybrid DNA sequence that combines CDEII and CDEIII as well as Widom 601 so they can make stable histones for biophysical studies (provided by the Widom sequence) and maintain features of the centromere (CDE II and III).

Weaknesses:

The main results are shown in one figure (Figure 2). Indeed the Centromere core of Widom and CDE II and III contribute to strengthening the binding force for the OA-beads. The data are very nicely done and convincingly demonstrate the point. The weakness is that this is the entire paper. It is certainly of interest to investigators in kinetochore biology, but beyond that, the impact is fairly limited in scope.

Reviewer #2 (Public review):

Summary:

Casas-Tinto et al., provide new insight into glial plasticity using a crush injury paradigm in the ventral nerve cord (VNC) of adult Drosophila. The authors find that both astrocyte-like glia (ALG) and ensheating glia (EG) divide under homeostatic conditions in the adult VNC and identify ALG as the glial population that specifically ramps up proliferation in response to injury, whereas the number of EGs decreases following the insult. Using lineage-tracing tools, the authors interestingly observe interconversion of glial subtypes, especially of EGs into ALGs, which occurs independent of injury and is dependent on the availability of the transcription factor Prospero in EGs, adding to the plasticity observed in the system. Finally, when tracing the progeny of glia, Casas-Tinto and colleagues detect cells of neuronal identity and provide evidence that such glia-derived neurogenesis is favored following ventral nerve cord injury, which puts forward a remarkable way in which glia can respond to neuronal damage.

Strengths:

This study highlights a new facet of adult nervous system plasticity at the level of the ventral nerve cord, supporting the view that proliferative capacity is maintained in the mature CNS and stimulated upon injury.

The injury paradigm is well chosen, as the organization of the neuromeres allows specific targeting of one segment, compared to the remaining intact and with the potential to later link observed plasticity to behavior such as locomotion.

Numerous experiments have been carried out in 7-day old flies, showing that the observed plasticity is not due to residual developmental remodeling or a still immature VNC.

Different techniques are used to observe proliferation in the VNC.

By elegantly combining different methods, the authors show glial divisions including with mitotic-dependent tracing and find that the number of generated glia is refined by apoptosis later on.

The work identifies prospero in glia as important coordinator of glial cell fate, from development to the adult context, which draws further attention to the upstream regulatory mechanisms.

Weaknesses:

The authors do not discuss their results on gliogenesis or neurogenesis in the adult VNC to previous findings made in the context of the injured adult brain.

The authors speculate about the role of glial inter-conversion for tissue homeostasis or regeneration, but no supportive evidence is cited or provided. Further experiments will be required to test the function of the described glial plasticity.

Elav+ cells originating from glia do not express markers for mature neurons at the analysed time-point. If they will eventually differentiate<br /> or what type of structure is formed by them will have to be followed up in future studies.

Context/Discussion

Highlighting some differences in the reactiveness of glia in the VNC compared to the brain could reveal important differences in repair strategies in different areas of the CNS.

Reviewer #1 (Public review):

Summary:

The authors investigated sleep and circadian rhythm disturbances in Fmr1 KO mice. Initially, they monitored daily home cage behaviors to assess sleep and circadian disruptions. Next, they examined the adaptability of circadian rhythms in response to photic suppression and skeleton photic periods. To explore the underlying mechanisms, they traced retino-suprachiasmatic connectivity. The authors further analyzed the social behaviors of Fmr1 KO mice and tested whether a scheduled feeding strategy could mitigate sleep, circadian, and social behavior deficits. Finally, they demonstrated that scheduled feeding corrected cytokine levels in the plasma of mutant mice.

Strengths:

(1) The manuscript addresses an important topic-investigating sleep deficits in an FXS mouse model and proposing a potential therapeutic strategy.

(2) The study includes a comprehensive experimental design with multiple methodologies, which adds depth to the investigation.

Weaknesses:

(1) The first serious issue in the manuscript is the lack of a clear description of how they performed the experiments and the missing definitions of various parameters in the results. Given that monitoring and analyzing sleep behaviors are the key experiments of this manuscript, I use the "Immobility-Based Sleep Behavior" section of Methods as an example to elaborate:

Incomplete or Incorrect Description of Tracking Threshold:<br /> o The phrase "tracked the (40 sec or greater as previously described" is incomplete and does not clarify what is being tracked. This appears to be an error in writing or editing.<br /> Unclear Relationship Between Threshold and EEG Validation:<br /> o The threshold "40 sec or greater" is mentioned without context or explanation of what it represents (e.g., sleep bout duration, inactivity, or another parameter). The reference to Fisher et al. (2016) and "99% correlation with EEG-defined sleep" seems misaligned with the paragraph's content.

Confusing Definition of Sleep Bout:<br /> o The definition of a sleep bout is unclear. Sleep bouts should logically be based on periods of inactivity, not activity. The sentence suggesting sleep is measured by "activity staying above the threshold" is confusing. The phrase "3 counts of sleep per minute for longer than one minute" requires clarification.

Unclear Data Selection for Analysis:<br /> o The phrase "2 days with the best recording quality" is vague and does not specify how "best" was determined or why only two days out of five were analyzed.

Awkward Grammar and Structure:<br /> o Phrases like "Acquiring data were exported in 1-min bins" are grammatically awkward. "Acquiring" should be "Acquired." Some sentences are overly long and lack clarity, making the text harder to follow.<br /> In addition to this section, the authors should review all paragraphs in the Methods section to improve readability.

(2) Although the manuscript has a relatively long Methods section, some essential information is missing. For instance, the definition of sleep bout, as described above, is unclear. Additional missing information includes:

Figure 2: "Rhythmic strength (%)" and "Cycle-to-cycle variability (min)."<br /> Figure 3: "Activity suppression."<br /> Figure 4: "Rhythmic power (V%)" (is this different from rhythmic strength (%)?) and "Subjective day activity (%)."<br /> Figure 5: Clear labeling of the SCN's anatomical features and an explanation for quantifying only the ventral part instead of the entire SCN. Alternatively, the authors should consider quantifying the whole SCN.<br /> Figure 6: Inconsistencies in terms like "Sleep frag. (bout #)" and "Sleep bouts (#)." Consistent terminology throughout the manuscript is essential.

(3) Figure 1A shows higher mouse activity during ZT13-16. It is unclear why the authors scheduled feeding during ZT15-21, as this seems to disturb the rhythm. Consistent with this, the body weights of WT and Fmr1 KO mice decreased after scheduled feeding. The authors should explain the rationale for this design clearly.

(4) The interpretation of social behavior results in Figure 6 is questionable. The authors claim that Fmr1 KO mice cannot remember the first stranger in a three-chamber test, writing, "The reduced time in exploring and staying in the novel-mouse chamber suggested that the Fmr1 KO mutants were not able to distinguish the second novel mouse from the first now-familiar mouse." However, an alternative explanation is that Fmr1 KO mice do remember the first stranger but prefer to interact with it due to autistic-like tendencies. Data in Table 5 show that Fmr1 KO mice spent more time interacting with the first stranger in the 3-chamber social recognition test, which support this possibility. Similarly, in the five-trial social test, Fmr1 KO mice's preference for familiar mice might explain the reduced interaction with the second stranger.

In Figure 6C (five-trial social test results), only the fifth trial results are shown. Data for trials 1-4 should be provided and compared with the fifth trial. The behavioral features of mice in the 5-trial test can then be shown completely. In addition, the total interaction times for trials 1-4 (154 {plus minus} 15.3 for WT and 150 {plus minus} 20.9 for Fmr1 KO) suggest normal sociability in Fmr1 KO mice (it is different from the results of 3-chamber). Thus, individual data for trials 1-4 are required to draw reliable conclusions.

In Table 6 and Figure 6G-6J, the authors claim that "Sleep duration (Figures 6G, H) and fragmentation (Figures 6I, J) exhibited a moderate-strong correlation with both social recognition and grooming." However, Figure 6I shows a p-value of 0.077, which is not significant. Moreover, Table 6 shows no significant correlation between SNPI of the three-chamber social test and any sleep parameters. These data do not support the authors' conclusions.

(5) Figure 7 demonstrates the effect of scheduled feeding on circadian activity and sleep behaviors, representing another critical set of results in the manuscript. Notably, the WT+ALF and Fmr1 KO+ALF groups in Figure 7 underwent the same handling as the WT and Fmr1 KO groups in Figures 1 and 2, as no special treatments were applied to these mice. However, the daily patterns observed in Figures 7A, 7B, 7F, and 7G differ substantially from those shown in Figures 2B and 1A, respectively. Additionally, it is unclear why the WT+ALF and Fmr1 KO+ALF groups did not exhibit differences in Figures 7I and 7J, especially considering that Fmr1 KO mice displayed more sleep bouts but shorter bout lengths in Figures 1C and 1D.

Furthermore, it is not specified whether the results in Figure 7 were collected after two weeks of scheduled feeding (for how many days?) or if they represent the average data from the two-week treatment period.

The rationale behind analyzing "ZT 0-3 activity" in Figure 7D instead of the parameters shown in Figures 2C and 2D is also unclear.

In Figure 7F, some data points appear to be incorrectly plotted. For instance, the dark blue circle at ZT13 connects to the light blue circle at ZT14 and the dark blue circle at ZT17. This is inconsistent, as the dark blue circle at ZT13 should link to the dark blue circle at ZT14. Similarly, it is perplexing that the dark blue circle at ZT16 connects to both the light blue and dark blue circles at ZT17. Such errors undermine confidence in the data. The authors need to provide a clear explanation of how these data were processed.

Lastly, in the Figure 7 legend, Table 6 is cited; however, this appears to be incorrect. It seems the authors intended to refer to Table 7.

(6) Similar to the issue in Figure 7F, the data for day 12 in Supplemental Figure 2 includes two yellow triangles but lacks a green triangle. It is unclear how the authors constructed this chart, and clarification is needed.

(7) In Figure 8, a 5-trial test was used to assess the effect of scheduled feeding on social behaviors. It is essential to present the results for all trials (1 to 4). Additionally, it is unclear whether the results for familial mice in Figure 8A correspond to trials 1, 2, 3, or 4.<br /> The legend for Figure 8 also appears to be incorrect: "The left panels show the time spent in social interactions when the second novel stranger mouse was introduced to the testing mouse in the 5-trial social interaction test. The significant differences were analyzed by two-way ANOVA followed by Holm-Sidak's multiple comparisons test with feeding treatment and genotype as factors." This description does not align with the content of the left panels. Moreover, two-way ANOVA is not the appropriate statistical analysis for Figure 8A. The authors need to provide accurate details about the analysis and revise the figure legend accordingly.

(8) The circadian activity and sleep behaviors of Fmr1 KO mice have been reported previously, with some findings consistent with the current manuscript, while others contradict it. Although the authors acknowledge this discrepancy, it seems insufficiently thorough to simply state that the reasons for the conflicts are unknown. Did the studies use the same equipment for behavior recording? Were the same parameters used to define locomotor activity and sleep behaviors? The authors are encouraged to investigate these details further, as doing so may uncover something interesting or significant.

(9) Some subtitles in the Results section and the figure legends do not align well with the presented data. For example, in the section titled "Reduced rhythmic strength and nocturnality in the Fmr1 KOs," it is unclear how the authors justify the claim of altered nocturnality in Fmr1 KO mice. How do the authors define changes in nocturnality? Additionally, the tense used in the subtitles and figure legends is incorrect. The authors are encouraged to carefully review all subtitles and figure legends to correct these errors and enhance readability.

Reviewer #1 (Public review):

Summary:

This manuscript presents a comprehensive structure-guided secretome analysis of gall-forming microbes, providing valuable insights into effector diversity and evolution. The authors have employed AlphaFold2 to predict the 3D structures of the secretome from selected pathogens and conducted a thorough comparative analysis to elucidate commonalities and unique features of effectors among these phytopathogens.

Strengths:

The discovery of conserved motifs such as 'CCG' and 'RAYH' and their central role in maintaining the overall fold is an insightful finding. Additionally, the discovery of a nucleoside hydrolase-like fold conserved among various gall-forming microbes is interesting.

Weaknesses:

Important conclusions are not verified by experiments.

Reviewer #1 (Public review):

Summary:

The goal of this study was to overcome the apparent difficulty in constructing structural models of the open state of the CFTR chloride channel. While several CFTR structural models at near-atomic resolution have been published under a variety of conditions, none of them have demonstrated a pore open across the full dimension of the plasma membrane. Instead, these have routinely been referred to as "near-open" models. In the present study, the authors extended their findings from a prior paper from their group that investigated a series of brief MD simulations, a small number of which exhibited permeation events where chloride ions permeated the pore. This study included massively repeated simulations initiated from these aforementioned Cl permeable conformations. Extensive analysis of the data identified a novel penta-helical structure that comprises the channel pore. This comprehensive study attempted to explain several features of conducting CFTR channels, including single-channel conductance, selectivity, and the mechanisms linking the ATP-induced dimerization of the cytosolic nucleotide-binding domains (NBDs) to the opening of the channel pore (a.k.a., "pore-gating".

Strengths:

The major strength of this study is its comprehensive nature. The approaches applied are cutting-edge and beyond, and are used to explain many different aspects of channel function in CFTR. The strength of evidence is very strong. The paper is extremely well-written, and the arguments are well-supported.

Weaknesses:

The major weakness is that none of the novel conclusions (i.e., those arising solely from this study and not previously published (have been supported by experimental confirmation. That is typical of computational studies such as this.

Reviewer #1 (Public review):

Summary:

This paper investigated the dynamic self-assembly of branched actin networks and the relation between the nonequilibrium features of the dynamics with the thermodynamic cost. The authors constructed a chain model to describe the self-assembly process of a branched actin network, including events like nucleation, polymerization, and capping. The forward and backward transition rates associated with the events allowed them to investigate the entropy production rate of the dynamics. They then used the fact that the entropy production rate has to be greater than zero to derive inequalities that set bounds for the maximum force produced by the branched actin network. The idea is similar to estimating the polymerization force of actin filament via the equation F_{max} = dG/delta, which sets a bound on the maximum force by the thermodynamic potential dG which is the chemical energy associated with ATP hydrolysis and delta is the length increment upon monomer insertion. Furthermore, they speculated the dissipative cost beyond what is necessary to move the load may be necessary to maintain an adaptive steady state.

Strengths:

The authors developed a simple model that is capable of qualitatively reproducing some mechanical phenomena for a branched actin network. The model has captured the essential dynamic elements in the branched actin network and built connections between the maximum load and the adaptation behavior with the energetic cost. It is an interesting study that provides a new perspective to look at the mechanical response of the branched actin network.

Weaknesses:

The text needs to be improved, particularly in the model introduction part. It is unclear to me what happens to the state when the reverse reaction in Figure 2 occurs.

Furthermore, what the authors have done is similar to estimate the polymerization force of actin filaments but in a more complicated scenario. Their conclusion that "dissipative cost in the system beyond what is necessary to move the load may be necessary to maintain an adaptive steady state" is skeptical. The branched actin network is a nonequilibrium system driven by active processes like ATP hydrolysis that converts chemical energy into mechanical work. There has to be a gap between the actual E-C_f curve and that when dissipation rate dot{S} = 0. If the authors want to make the claim, they have to decompose the dissipation into different parts and show that a particular part is associated with adaption. Otherwise, the conclusion about the gap is baseless.

Reviewer #1 (Public review):

Summary:

Li et al describe a novel form of melanosome based iridescence in the crest of an Early Cretaceous enantiornithine avialan bird from the Jehol Group.

Strengths:

Novel set of methods applied to the study of fossil melanosomes.

Weaknesses:

(1) Firstly, several studies have argued that these structures are in fact not a crest, but rather the result of compression. Otherwise, it would seem that a large number of Jehol birds have crests that extend not only along the head but the neck and hindlimb. It is more parsimonious to interpret this as compression as has been demonstrated using actuopaleontology (Foth 2011).<br /> (2) The primitive morphology of the feather with their long and possibly not interlocking barbs also questions the ability of such feathers to be erected without geologic compression.<br /> (3) The feather is not in situ and therefore there is no way to demonstrate unequivocally that it is indeed from the head (it could just as easily be a neck feather)<br /> (4) Melanosome density may be taphonomic; in fact, in an important paper that is notably not cited here (Pan et al. 2019) the authors note dense melanosome packing and attribute it to taphonomy. This paper describes densely packed (taphonomic) melanosomes in non-avian avialans, specifically stating, "Notably, we propose that the very dense arrangement of melanosomes in the fossil feathers (Fig. 2 B, C, and G-I, yellow arrows) does not reflect in-life distribution, but is, rather, a taphonomic response to postmortem or postburial compression" and if this paper was taken into account it seems the conclusions would have to change drastically. If in this case the density is not taphonomic, this needs to be justified explicitly (although clearly these Jehol and Yanliao fossils are heavily compressed).<br /> (5) Color in modern birds is affected by the outer keratin cortex thickness which is not preserved but the authors note the barbs are much thicker (10um) than extant birds; this surely would have affected color so how can the authors be sure about the color in this feather?<br /> (6) Authors describe very strange shapes that are not present in extant birds: "...different from all other known feather melanosomes from both extant and extinct taxa in having some extra hooks and an oblique ellipse shape in cross and longitudinal sections of individual melanosome" but again, how can it be determined that this is not the result of taphonomic distortion?<br /> (7) The authors describe the melanosomes as hexagonally packed but this does not appear to be in fact the case, rather appearing quasi-periodic at best, or random. If the authors could provide some figures to justify this hexagonal interpretation?<br /> (8) One way to address these concerns would be to sample some additional fossil feathers to see if this is unique or rather due to taphonomy<br /> (9) On a side, why are the feet absent in the CT scan image?

Reviewer #1 (Public review):

Summary:

Nahas et al. investigated the roles of herpes simplex virus 1 (HSV-1) structural proteins using correlative cryo-light microscopy and soft X-ray tomography. The authors generated nine viral variants with deletions or mutations in genes encoding structural proteins. They employed a chemical fixation-free approach to study native-like events during viral assembly, enabling observation of a wider field of view compared to cryo-ET. The study effectively combined virology, cell biology, and structural biology to investigate the roles of viral proteins in virus assembly and budding.

Strengths:

(1) The study presented a novel approach to studying viral assembly in cellulo.

(2) The authors generated nine mutant viruses to investigate the roles of essential proteins in nuclear egress and cytoplasmic envelopment.

(3) The use of correlative imaging with cryoSIM and cryoSXT allowed for the study of viral assembly in a near-native state and in 3D.

(4) The study identified the roles of VP16, pUL16, pUL21, pUL34, and pUS3 in nuclear egress.

(5) The authors demonstrated that deletion of VP16, pUL11, gE, pUL51, or gK inhibits cytoplasmic envelopment.

(6) The manuscript is well-written, clearly describing findings, methods, and experimental design.

(7) The figures and data presentation are of good quality.

(8) The study effectively correlated light microscopy and X-ray tomography to follow virus assembly, providing a valuable approach for studying other viruses and cellular events.

(9) The research is a valuable starting point for investigating viral assembly using more sophisticated methods like cryo-ET with FIB-milling.

(10) The study proposes a detailed assembly mechanism and tracks the contributions of studied proteins to the assembly process.

(11) The study includes all necessary controls and tests for the influence of fluorescent proteins.

Weaknesses:

Overall, the manuscript does not have any major weaknesses, just a few minor comments:

(1) The gel quality in Figure 1 is inconsistent for different samples, with some bands not well resolved (e.g., for pUL11, GAPDH, or pUL20).

(2) The manuscript would benefit from a summary figure or table to concisely present the findings for each protein. It is a large body of manuscript, and a summary figure showing the discovered function would be great.

(3) Figure 2 lacks clarity on the type of error bars used (range, standard error, or standard deviation). It says, however, range, and just checking if this is what the authors meant.

(4) The manuscript could be improved by including details on how the plasma membrane boundary was estimated from the saturated gM-mCherry signal. An additional supplementary figure with the data showing the saturation used for the boundary definition would be helpful.

(5) Additional information or supplementary figures on the mask used to filter the YFP signal for Figure 4 would be helpful.

(6) The figure legends could include information about which samples are used for comparison for significance calculations. As the color of the brackets is different from the compared values (dUL34), it would be great to have this information in the figure legend.

(7) In Figure 5B, the association between YFP and mCherry signals is difficult to assess due to the abundance of mCherry signal; single-channel and combined images might improve visualization.

(8) In Figure 6D, staining for tubulin could help identify the cytoskeleton structures involved in the observed virus arrays.

(9) It is unclear in Figure 6D if the microtubule-associated capsids are with the gM envelope or not, as the signal from mCherry is quite weak. It could be made clearer with the split signals to assess the presence of both viral components.

(10) The representation of voxel intensity in Figure 8 is somewhat confusing. Reversion of the voxel intensity representation to align brighter values with higher absorption, which would simplify interpretation.

(11) The visualization in panel I of Figure 8 might benefit from a more divergent colormap to better show the variation in X-ray absorbance.

(12) Figure 9 would be enhanced by images showing the different virus sizes measured for the comparative study, which would help assess the size differences between different assembly stages.

Overall, this is an excellent manuscript and an enjoyable read. It would be interesting to see this approach applied to the study of other viruses, providing valuable insights before progressing to high-resolution methods.

Reviewer #2 (Public review):

Summary:

The authors identified ORMDL3 as a negative regulator of the RLR pathway and anti-tumor immunity. Mechanistically, ORMDL3 interacts with MAVS and further promotes RIG-I for proteasome degradation. In addition, the deubiquitinating enzyme USP10 stabilizes RIG-I and ORMDL3 disturbs this process. Moreover, in subcutaneous syngeneic tumor models in C57BL/6 mice, they showed that inhibition of ORMDL3 enhances anti-tumor efficacy by augmenting the proportion of cytotoxic CD8-positive T cells and IFN production in the tumor microenvironment (TME).

Strengths:

The paper has a clearly arranged structure and the English is easy to understand. It is well written. The results clearly support the conclusion.

Comments on revisions:

All questions have been answered.

Reviewer #1 (Public review):

The study investigates light chains (LCs) using three distinct approaches, with a focus on identifying a conformational fingerprint to differentiate amyloidogenic light chains from multiple myeloma light chains. The study's major contribution is the identification of a low-populated "H state," which the authors propose as a unique marker for AL-LCs. While this finding is promising, the review highlights several strengths and weaknesses. Strengths include the valuable contribution of identifying the H state and the use of multiple approaches, which provide a comprehensive understanding of LC structural dynamics. Weaknesses include a lack of physical insights explaining the changes.

Reviewer #1 (Public review):

The study investigates light chains (LCs) using three distinct approaches, with a focus on identifying a conformational fingerprint to differentiate amyloidogenic light chains from multiple myeloma light chains. The study's major contribution is the identification of a low-populated "H state," which the authors propose as a unique marker for AL-LCs. While this finding is promising, the review highlights several strengths and weaknesses. Strengths include the valuable contribution of identifying the H state and the use of multiple approaches, which provide a comprehensive understanding of LC structural dynamics. Weaknesses include a lack of physical insights explaining the changes.

Reviewer #2 (Public review):

Miyazaki et al. established three distinct BMD mouse models by deleting different exon regions of the dystrophin gene, observed in human BMD. The authors demonstrated that these models exhibit pathophysiological changes, including variations in body weight, muscle force, muscle degeneration, and levels of fibrosis, alongside underlying molecular alterations such as changes in dystrophin and nNOS levels. Notably, these molecular and pathological changes progress at different rates depending on the specific exon deletions in dystrophin gene. Additionally, the authors conducted extensive fiber typing, revealing a site-specific decline in type IIa fibers in BMD mice, which they suggest may be due to muscle degeneration and reduced capillary formation around these fibers.

Strengths:

The manuscript introduces three novel BMD mouse models with different dystrophin exon deletions, each demonstrating varying rates of disease progression similar to the human BMD phenotype. The authors also conducted extensive fiber typing across different muscles and regions within the muscles, effectively highlighting a site-specific decline in type IIa muscle fibers in BMD mice.

Comments on revisions:

The authors did an excellent job addressing all or most of the concerns I raised in my previous review and have incorporated the necessary changes into the manuscript.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors reveal that GIF/MT-3 regulates the zinc homeostasis depending on the cellular redox status. The manuscript technically sounds, and their data concretely suggest that the recombinant MTs, not only GIF/MT-3 but also canonical MTs such as MT-1 and MT-2, contain sulfane sulfur atoms for the Zn-binding. The scenario proposed by the authors seems to be reasonable to explain the Zn homeostasis by the cellular redox balance.

Strengths:

The data presented in the manuscript solidly reveal that recombinant GIF/MT-3 contains sulfane sulfur.

Weaknesses:

It remains unclear whether native MTs, in particular induced MTs in vivo contain sulfane sulfur or not.

Comments on revisions:

Although the authors have revealed the sulfane sulfur content in native MT-3, my question, namely, whether canonical MT-1 and MT-2 contained sulfane sulfur after the induction has been left.<br /> The authors argue that the biological significance of sulfane sulfur in MTs lies in its ability to contribute to metal binding affinity, provide a sensing mechanism against oxidative stress, and aid in the regulation of the protein. Due to their biological roles, induced MT-1 and MT-2 could contain sulfane sulfur in their molecules. Thus, I expect the authors to evaluate or explain the sulfane sulfur content in induced MT-1 and MT-2.

Reviewer #1 (Public review):

Summary:

This manuscript uses the eye lens as a model to investigate basic mechanisms in the Fgf signaling pathway. Understanding Fgf signaling is of broad importance to biologists as it is involved in the regulation of various developmental processes in different tissues/organs and is often misregulated in disease states. The Fgf pathway has been studied in embryonic lens development, namely with regards to its involvement in controlling events such as tissue invagination, vesicle formation, epithelium proliferation and cellular differentiation, thus making the lens a good system to uncover the mechanistic basis of how the modulation of this pathway drives specific outcomes. Previous work has suggested that proteins, other than the ones currently known (e.g., the adaptor protein Frs2), are likely involved in Fgfr signaling. The present study focuses on the role of Shp2 and Shc1 proteins in the recruitment of Grb2 in the events downstream of Fgfr activation.

Strengths:

The findings reveal that the juxtamembrane region of the Fgf receptor is necessary for proper control of downstream events such as facilitating key changes in transcription and cytoskeleton during tissue morphogenesis. The authors conditionally deleted all four Fgfrs in the mouse lens that resulted in molecular and morphological lens defects, most importantly, preventing the upregulation of the lens induction markers Sox2 and Foxe3 and the apical localization of F-actin, thus demonstrating the importance of Fgfrs in early lens development, i.e. during lens induction. They also examined the impact of deleting Fgfr1 and 2, on the following stage, i.e. lens vesicle development, which could be rescued by expressing constitutively active KrasG12D. By using specific mutations (e.g. Fgfr1ΔFrs lacking the Frs2 binding domain and Fgfr2LR harboring mutations that prevent binding of Frs2), it is demonstrated that the Frs2 binding site on Fgfr is necessary for specific events such as morphogenesis of lens vesicle. Further, by studying Shp2 mutations and deletions, the authors present a case for Shp2 protein to function in a context-specific manner in the role of an adaptor protein and a phosphatase enzyme. Finally, the key surprising finding from this study is that downstream of Fgfr signaling, Shc1 is an important alternative pathway - in addition to Shp2 - involved in the recruitment of Grb2 and in the subsequent activation of Ras. The methodologies, namely, mouse genetics and state-of-the-art cell/molecular/biochemical assays are appropriately used to collect the data, which are soundly interpreted to reach these important conclusions. Overall, these findings reveal the flexibility of the Fgf signaling pathway and it downstream mediators in regulating cellular events. This work is expected to be of broad interest to molecular and developmental biologists.

Weaknesses:

A weakness that needs to be discussed is that Le-Cre depends on Pax6 activation, and hence its use in specific gene deletion will not allow evaluation of the requirement of Fgfrs in the expression of Pax6 itself. But since this is the earliest Cre available for deletion in the lens, mentioning this in the discussion would make the readers aware of this issue.

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate the effect of mitochondrial transplantation on post-cardiac arrest myocardial dysfunction (PAMD), which is associated with mitochondrial dysfunction. The authors demonstrate that mitochondrial transplantation enhances cardiac function and increases survival rates after the return of spontaneous circulation (ROSC). Mechanistically, they found that myocardial tissues with transplanted mitochondria exhibit increased mitochondrial complex activity, higher ATP levels, reduced cardiomyocyte apoptosis, and lower myocardial oxidative stress post-ROSC.

Strengths:

Previous studies have reported that mitochondrial transplantation can improve myocardial recovery after regional ischemia, but its potential for treating myocardial injury following cardiac arrest has not been tested yet. Therefore, the findings are somewhat novel. Remarkably, the increased survival in mitochondria treated group post ROSC is very promising and highlights its translational potential.

Comments on revisions:

My concerns are adequately addressed.

Reviewer #1 (Public review):

Summary:

This manuscript investigates a mechanism between the histone reader protein YEATS2 and the metabolic enzyme GCDH, particularly in regulating epithelial-to-mesenchymal transition (EMT) in head and neck cancer (HNC).

Strengths:

Great detailing of the mechanistic aspect of the above axis is the primary strength of the manuscript.

Weaknesses:

Several critical points require clarification, including the rationale behind EMT marker selection, the inclusion of metastasis data, the role of key metabolic enzymes like ECHS1, and the molecular mechanisms governing p300 and YEATS2 interactions.

Major Comments:

(1) The title, "Interplay of YEATS2 and GCDH mediates histone crotonylation and drives EMT in head and neck cancer," appears somewhat misleading, as it implies that YEATS2 directly drives histone crotonylation. However, YEATS2 functions as a reader of histone crotonylation rather than a writer or mediator of this modification. It cannot itself mediate the addition of crotonyl groups onto histones. Instead, the enzyme GCDH is the one responsible for generating crotonyl-CoA, which enables histone crotonylation. Therefore, while YEATS2 plays a role in recognizing crotonylation marks and may regulate gene expression through this mechanism, it does not directly catalyse or promote the crotonylation process.

(2) The study suggests a link between YEATS2 and metastasis due to its role in EMT, but the lack of clinical or pre-clinical evidence of metastasis is concerning. Only primary tumor (PT) data is shown, but if the hypothesis is that YEATS2 promotes metastasis via EMT, then evidence from metastatic samples or in vivo models should be included to solidify this claim.

(3) There seems to be some discrepancy in the invasion data with BICR10 control cells (Figure 2C). BICR10 control cells with mock plasmids, specifically shControl and pEGFP-C3 show an unclear distinction between invasion capacities. Normally, we would expect the control cells to invade somewhat similarly, in terms of area covered, within the same time interval (24 hours here). But we clearly see more control cells invading when the invasion is done with KD and fewer control cells invading when the invasion is done with OE. Are these just plasmid-specific significant effects on normal cell invasion? This needs to be addressed.

(4) In Figure 3G, the Western blot shows an unclear band for YEATS2 in shSP1 cells with YEATS2 overexpression condition. The authors need to clearly identify which band corresponds to YEATS2 in this case.

(5) In ChIP assays with SP1, YEATS2 and p300 which promoter regions were selected for the respective genes? Please provide data for all the different promoter regions that must have been analysed, highlighting the region where enrichment/depletion was observed. Including data from negative control regions would improve the validity of the results.

(6) The authors establish a link between H3K27Cr marks and GCDH expression, and this is an already well-known pathway. A critical missing piece is the level of ECSH1 in patient samples. This will clearly delineate if the balance shifted towards crotonylation.

(7) The p300 ChIP data on the SPARC promoter is confusing. The authors report reduced p300 occupancy in YEATS2-silenced cells, on SPARC promoter. However, this is paradoxical, as p300 is a writer, a histone acetyltransferase (HAT). The absence of a reader (YEATS2) shouldn't affect the writer (p300) unless a complex relationship between p300 and YEATS2 is present. The role of p300 should be further clarified in this case. Additionally, transcriptional regulation of SPARC expression in YEATS2 silenced cells could be analysed via downstream events, like Pol-II recruitment. Assays such as Pol-II ChIP-qPCR could help explain this.

(8) The role of GCDH in producing crotonyl-CoA is already well-established in the literature. The authors' hypothesis that GCDH is essential for crotonyl-CoA production has been proven, and it's unclear why this is presented as a novel finding. It has been shown that YEATS2 KD leads to reduced H3K27cr, however, it remains unclear how the reader is affecting crotonylation levels. Are GCDH levels also reduced in the YEATS2 KD condition? Are YEATS2 levels regulating GCDH expression? One possible mechanism is YEATS2 occupancy on GCDH promoter and therefore reduced GCDH levels upon YEATS2 KD. This aspect is crucial to the study's proposed mechanism but is not addressed thoroughly.

(9) The authors should provide IHC analysis of YEATS2, SPARC alongside H3K27cr and GCDH staining in normal vs. tumor tissues from HNC patients.

Reviewer #1 (Public review):

Summary:

It is essential for Mycobacterium tuberculosis (Mtb) to scavenge trace metals from its host to survive. In this study, the authors explore the effects of copper limitation on Mtb. Mtb synthesizes small molecular diisonitrile lipopeptides termed chalkophores, that chelate host copper for import, whereby the copper is incorporated into Mtb metalloproteins. However, the role of chalkophores in Mtb biology and their targeted metalloproteins are unknown. This study investigates Mtb proteins that require chalkophores for copper incorporation and their effect on Mtb virulence. It is known that the nrp operon is induced by copper deprivation and encodes the synthesis of chalkophores. A genetic analysis revealed transcriptional differences for WT and Mtb∆nrp when exposed to the copper chelator tetrathiomolybdate (TTM). The authors found that copper chelation results in upregulation of genes in the chalkophore cluster as well as genes involved in the respiratory chain: specifically, components of the heme-dependent oxidase CytBD and subunits of the bcc:aa3 heme-copper oxidase. Interestingly, treatment of Mtb∆nrp with an inhibitor of the QcrB subunit of the bcc:aa3 oxidase (Q203) resulted in similar transcriptional changes. The bcc:aa3 oxidase and CytBD are functionally redundant, and while both utilize heme as a cofactor, only the first utilizes heme and copper. Utilizing Mtb∆nrp, Mtb∆cydAB and MtbΔnrpΔcydAB along with single gene complementation, the authors showed that copper starvation survival requires diisonitrile chalkophore synthesis and that copper starvation results in dysfunctional bcc:aa3 oxidase. Further genetic analysis combined with inhibitor studies indicate that bcc:aa3 oxidase is the only target impacted by copper starvation. By monitoring oxygen consumption for mutants in combination with inhibitors, the authors show that copper deprivation inhibits respiration through the bcc:aa3 oxidase. Similarly, they show that TTM or Q203 treatment inhibits ATP production in MtbΔnrpΔcydAB, but not in WT, showing that chalkophores maintain oxidative phosphorylation. Lastly, the authors compare the virulence of WT Mtb, Mtb∆nrp and MtbΔnrpΔcydAB strains in mice spleen and lung. The Mtb∆nrp strain showed mild attenuation, but virulence in MtbΔnrpΔcydAB was severely attenuated, and complementation with the chalkophore biosynthetic pathway restored Mtb virulence. These results suggest that chalkophore mediated protection of the respiratory chain is critical to Mtb virulence, and the that redundant respiratory oxidases within Mtb provides respiratory chain flexibility that may promote host adaptation.

Strengths:

Overall, the paper is very clear and well-written, with thorough and well-thought-out experimentation.

The methods are all quite standard, so there are no weaknesses identified with regard to methodology.

Reviewer #2 (Public review):

In this revised manuscript, Klemm et al., build on top of past published findings (Klemm et al., 2021) to characterize caspase activation in distal cells following necrotic tissue damage within the Drosophila wing imaginal disc. Previously in Klemm et al., 2021, the authors describe necrosis-induced-apoptosis (NiA) following the development of a genetic system to study necrosis that is caused by the expression of a constitutive active GluR1 (Glutamate/Ca2+ channel), and they discovered that the appearance of NiA cells were important for promoting regeneration.

In this manuscript, the authors investigate how tissues regenerate following necrotic cell death. They find that:

(1) the cells of the wing pouch are more likely to have non-autonomous caspase activation than other regions within the wing imaginal disc (hinge and notum),

(2) two signaling pathways that are known to be upregulated during regeneration, Wnt (wingless) and JAK/Stat signaling, act to prevent additional NiA in pouch cells, and may partially explain the region specificity,

(3) the presence of NiA (and/or NiCP) cells promotes regenerative proliferation in the late stages of regeneration,

(4) not all caspase-positive cells are cleared from the epithelium (these cells are then referred to as Necrosis-induced Caspase Positive (NiCP) cells), these NiCP cells continue to live and promote proliferation in adjacent cells,

(5) the initiator caspase Dronc is important for creating NiA/NiCP cells and for these cells to promote proliferation. Animals heterozygous for a Dronc null allele show a decrease in regeneration following necrotic tissue damage. In the revised manuscript, the authors provide improvements through additional data quantifications and text changes to better explain NiA/NiCP lineage tracing methods.

The study has the potential to be broadly interesting due to the insights into how tissues differentially respond to necrosis as compared to apoptosis to promote regeneration. The paper raises many interesting questions for future investigation, including what is the nature of the signaling between the damaged tissue and the NiA/NiCP responsive areas (such as the identity of the DAMPs)? What determines if these cells at a distance undergo apoptosis or remain viable in the tissue as caspase-positive cells? And since the authors have data that indicates that the phenomenon is distinct from 'undead cells', what are the mechanisms by which these cells promote local proliferation?

Reviewer #1 (Public review):

Summary:

This is a very creative study using modeling and measurement of neoblast dynamics to gain insight into the mechanism that allows these highly potent cells to undergo fate-switching as part of their differentiation and self-renewal process. The authors estimate growth equation parameters for expanding neoblast clones based on new and prior experimental observations. These results indicate neoblast likely undergo much more symmetric self-amplifying division than loss of the population through symmetric differentiation, in the case of clone expansion assays after sublethal irradiation. Neoblasts take on multiple distinct transcriptional fates related to their terminally differentiated cell types, and prior work indicated neoblasts have a high plasticity to switch fates in a way linked to cell cycle progression and possibly through a random process. Here, the authors explore the impact of inhibition of key transcription factors defining such states (ie "fate specifying transcription factors", FSTFs) plus measurement and modeling in the clone expansion assay, to find that inhibition of factors like zfp1 likely cause otherwise zfp1-fated neoblasts to fail to proliferate and differentiation without causing compensatory gains in other lineages. A mathematical model of this process assuming that neoblasts do not retain a memory of prior states while they proliferate, and transition across specified states can mimic the experimentally determined decreased sizes of clones following inhibition of zfp1. Complementary approaches to inhibit more than one lineage (muscle plus intestine) supports the idea that this is a more general process in planarian stem cells. These results provide an important advance for understanding the fate-switching process and its relationship to neoblast growth.

Overall I find the evidence very well presented and the study compelling. It offers an important new perspective on the key properties of neoblasts. I do have some comments to clarify the presentation and significance of the work.

Reviewer #1 (Public review):

Summary:

The authors aimed to elucidate the molecular mechanisms underlying HIV-1 persistence and host immune dysfunction in CD4+ T cells during early infection (<6 months). Using single-cell multi-omics technologies-including scRNA-seq, scATAC-seq, and single-cell multiome analyses-they characterized the transcriptional and epigenomic landscapes of HIV-1-infected CD4+ T cells. They identified key transcription factors (TFs), signaling pathways, and T cell subtypes involved in HIV-1 persistence, particularly highlighting KLF2 and Th17 cells as critical regulators of immune suppression. The study provides new insights into immune dysregulation during early HIV-1 infection and reveals potential epigenetic regulatory mechanisms in HIV-1-infected T cells.

Strengths:

The study excels through its innovative integration of single-cell multi-omics technologies, enabling detailed analysis of gene regulatory networks in HIV-1-infected cells. Focusing on early infection stages, it fills a crucial knowledge gap in understanding initial immune responses and viral reservoir establishment. The identification of KLF2 as a key transcription factor and Th17 cells as major viral reservoirs, supported by comprehensive bioinformatics analyses, provides robust evidence for the study's conclusions. These findings have immediate clinical relevance by identifying potential therapeutic targets for HIV-1 reservoir eradication.

Weaknesses:

Despite its strengths, the study has several limitations. By focusing exclusively on CD4+ T cells, the study overlooks other relevant immune cells such as CD14+ monocytes, NK cells, and B cells. Additionally, while the authors generated their own single-cell datasets, they need to validate their findings using other publicly available single-cell data from HIV-1-infected PBMCs.

Reviewer #2 (Public review):

Summary:

In the article, the authors describe their software package in R for visualizing metabolite ratio pairs. I think the work would be of interest to the mass spectrometry community.

Strengths:

The authors describe a software that would be of use to those performing MALDI MSI. This software would certainly add to the understanding metabolomics data and enhance the identification of critical metabolites.

Weaknesses:

The figures are difficult to interpret/ analyze in their current state but are significantly better in the revision.

Joint Public Review:

This study comprehensively presents data from single nuclei sequencing of Heigai pig skeletal muscle in response to conjugated linoleic acid supplementation. The authors identify changes in myofiber type and adipocyte subpopulations induced by linoleic acid at depth previously unobserved. The authors show that linoleic acid supplementation decreased the total myofiber count, specifically reducing type II muscle fiber types (IIB), myotendinous junctions, and neuromuscular junctions, whereas type I muscle fibers are increased. Moreover, the authors identify changes in adipocyte pools, specifically in a population marked by SCD1/DGAT2. To validate the skeletal muscle remodeling in response to linoleic acid supplementation, the authors compare transcriptomics data from Laiwu pigs, a model of high intramuscular fat, to Heigai pigs. The results verify changes in adipocyte subpopulations when pigs have higher intramuscular fat, either genetically or diet-induced. Targeted examination using cell-cell communication network analysis revealed associations with high intramuscular fat with fibro-adipogenic progenitors (FAPs). The authors then conclude that conjugated linoleic acid induces FAPs towards adipogenic commitment. Specifically, they show that linoleic acid stimulates FAPs to become SCD1/DGAT2+ adipocytes via JNK signaling. The authors conclude that their findings demonstrate the effects of conjugated linoleic acid on skeletal muscle fat formation in pigs, which could serve as a model for studying human skeletal muscle diseases.

[Editors' note: the authors have responded to the previous rounds of review: https://doi.org/10.7554/eLife.99790.1.sa1 and https://doi.org/10.7554/eLife.99790.2.sa1]

Reviewer #1 (Public review):

Summary:

This study investigates the role of macrophage lipid metabolism in the intracellular growth of Mycobacterium tuberculosis. By using a CRISPR-Cas9 gene-editing approach, the authors knocked out key genes involved in fatty acid import, lipid droplet formation, and fatty acid oxidation in macrophages. Their results show that disrupting various stages of fatty acid metabolism significantly impairs the ability of Mtb to replicate inside macrophages. The mechanisms of growth restriction included increased glycolysis, oxidative stress, pro-inflammatory cytokine production, enhanced autophagy, and nutrient limitation. The study demonstrates that targeting fatty acid homeostasis at different stages of the lipid metabolic process could offer new strategies for host-directed therapies against tuberculosis.

The work is convincing and methodologically strong, combining genetic, metabolic, and transcriptomic analyses to provide deep insights into how host lipid metabolism affects bacterial survival.

Strengths:

The study uses a multifaceted approach, including CRISPR-Cas9 gene knockouts, metabolic assays, and dual RNA sequencing, to assess how various stages of macrophage lipid metabolism affect Mtb growth. The use of CRISPR-Cas9 to selectively knock out key genes involved in fatty acid metabolism enables precise investigation of how each step-lipid import, lipid droplet formation, and fatty acid oxidation-affects Mtb survival. The study offers mechanistic insights into how different impairments in lipid metabolism lead to diverse antimicrobial responses, including glycolysis, oxidative stress, and autophagy. This deepens the understanding of macrophage function in immune defense.<br /> The use of functional assays to validate findings (e.g., metabolic flux analyses, lipid droplet formation assays, and rescue experiments with fatty acid supplementation) strengthens the reliability and applicability of the results.<br /> By highlighting potential targets for HDT that exploit macrophage lipid metabolism to restrict Mtb growth, the work has significant implications for developing new tuberculosis treatments.

Weaknesses:

The experiments were primarily conducted in vitro using CRISPR-modified macrophages. While these provide valuable insights, they may not fully replicate the complexity of the in vivo environment where multiple cell types and factors influence Mtb infection and immune responses. Yet, I agree that the Hoxb8 in vitro model provides a powerful genetic tool to interrogate host-Mtb interactions using primary macrophages that represent the bone marrow-derived macrophage lineage, instead of using cell lines.

Comments on revisions: The authors have addressed my comment satisfactorily.

Reviewer #1 (Public review):

Summary:

This is a very creative study using modeling and measurement of neoblast dynamics to gain insight into the mechanism that allows these highly potent cells to undergo fate-switching as part of their differentiation and self-renewal process. The authors estimate growth equation parameters for expanding neoblast clones based on new and prior experimental observations. These results indicate neoblast likely undergo much more symmetric self-amplifying division than loss of the population through symmetric differentiation, in the case of clone expansion assays after sublethal irradiation. Neoblasts take on multiple distinct transcriptional fates related to their terminally differentiated cell types, and prior work indicated neoblasts have a high plasticity to switch fates in way linked to cell cycle progression and possibly through a random process. Here, the authors explore the impact of inhibition of key transcription factors defining such states (ie "fate specifying transcription factors", FSTFs) plus measurement and modeling in the clone expansion assay, to find that inhibition of factors like zfp1 likely cause otherwise zfp1-fated neoblasts to fail to proliferate and differentiation, without causing compensatory gains in other lineages. A mathematical model of this process assuming that neoblasts do not retain a memory of prior states while they proliferate and transition across specified states can mimic the experimentally determined decreased sizes of clones following inhibition of zfp1. Complementary approaches to inhibit more than one lineage (muscle plus intestine) supports the idea that this is a more general process in planarian stem cells. These results provide an important advance for understanding the fate-switching process and its relationship to neoblast growth.

Overall I find the evidence very well presented and the study compelling, and offers an important new perspective on the key properties of neoblasts. I have some comments to clarify the presentation and significance of the work.

Comments on revisions:

In this revised version, the authors nicely address all of my comments and I find the work makes a strong case for its main conclusions.

Reviewer #1 (Public review):

Summary:

The authors investigated if/how distractor suppression derived from statistical learning may be implemented in early visual cortex. While in a scanner, participants conducted a standard additional singleton task in which one location more frequently contained a salient distractor. The results showed that activity in EVC was suppressed for the location of the salient distractor as well as for neighbouring neutral locations. This suppression was not stimulus specific - meaning it occurred equally for distractors, targets and neutral items - and it was even present in trials in which the search display was omitted. Generally, the paper was clear, the experiment was well-designed, and the data are interesting.

The authors addressed all of my concerns and the revised manuscript will make a beautiful addition to the literature.

Reviewer #1 (Public review):

Summary:

This manuscript describes the role of PRDM16 in modulating BMP response during choroid plexus (ChP) development. The authors combine PRDM16 knockout mice and cultured PRDM16 KO primary neural stem cells (NSCs) to determine the interactions between BMP signaling and PRDM16 in ChP differentiation.

They show PRDM16 KO affects ChP development in vivo and BMP4 response in vitro. They determine genes regulated by BMP and PRDM16 by ChIP-seq or CUT&TAG for PRDM16, pSMAD1/5/8, and SMAD4. They then measure gene activity in primary NSCs through H3K4me3 and find more genes are co-repressed than co-activated by BMP signaling and PRDM16. They focus on the 31 genes found to be co-repressed by BMP and PRDM16. Wnt7b is in this set and the authors then provide evidence that PRDM16 and BMP signaling together repress Wnt activity in the developing choroid plexus.

Strengths:

Understanding context-dependent responses to cell signals during development is an important problem. The authors use a powerful combination of in vivo and in vitro systems to dissect how PRDM16 may modulate BMP response in early brain development.

Main weaknesses of the experimental setup:

(1) Because the authors state that primary NSCs cultured in vitro lose endogenous Prdm16 expression, they drive expression by a constitutive promoter. However, this means the expression levels are very different from endogenous levels (as explicitly shown in Supplementary Figure 2B) and the effect of many transcription factors is strongly dose-dependent, likely creating differences between the PRDM16-dependent transcriptional response in the in vitro system and in vivo.

(2) It seems that the authors compare Prdm16_KO cells to Prdm16 WT cells overexpressing flag_Prdm16. Aside from the possible expression of endogenous Prdm16, other cell differences may have arisen between these cell lines. A properly controlled experiment would compare Prdm16_KO ctrl (possibly infected with a control vector without Prdm16) to Prdm16_KO_E (i.e. the Prdm16_KO cells with and without Prdm16 overexpression.)

Other experimental weaknesses that make the evidence less convincing:

(1) The authors show in Figure 2E that Ttr is not upregulated by BMP4 in PRDM16_KO NSCs. Does this appear inconsistent with the presence of Ttr expression in the PRDM16_KO brain in Figure1C?

(2) Figure 3: The authors use H3K4me3 to measure gene activity. This is however, very indirect, with bulk RNA-seq providing the most direct readout and polymerase binding (ChIP-seq) another more direct readout. Transcription can be regulated without expected changes in histone methylation, see e.g. papers from Josh Brickman. They verify their H3K4me3 predictions with qPCR for a select number of genes, all related to the kinetochore, but it is not clear why these genes were picked, and one could worry whether these are representative.

(3) Line 256: The overlap of 31 genes between 184 BMP-repressed genes and 240 PRDM16-repressed genes seems quite small.

(4) The Wnt7b H3K4me3 track in Fig. 3G is not discussed in the text but it shows H3K4me3 high in _KO and low in _E regardless of BMP4. This seems to contradict the heatmap of H3K4me3 in Figure 3E which shows H3K4me3 high in _E no BMP4 and low in _E BMP4 while omitting _KO no BMP4. Meanwhile CDKN1A, the other gene shown in 3G, is missing from 3E.

(5) The authors use PRDM16 CUT&TAG on dissected dorsal midline tissues to determine if their 31 identified PRDM16-BMP4 co-repressed genes are regulated directly by PRDM16 in vivo. By manual inspection, they find that "most" of these show a PRDM16 peak. How many is most? If using the same parameters for determining peaks, how many genes in an appropriately chosen negative control set of genes would show peaks? Can the authors rigorously establish the statistical significance of this observation? And why wasn't the same experiment performed on the NSCs in which the other experiments are done so one can directly compare the results? Instead, as far as I could tell, there is only ChIP-qPCR for two genes in NSCs in Supplementary Figure 4D.

(6) In comparing RNA in situ between WT and PRDM16 KO in Figure 7, the authors state they use the Wnt2b signal to identify the border between CH and neocortex. However, the Wnt2b signal is shown in grey and it is impossible for this reviewer to see clear Wnt2b expression or where the boundaries are in Figure 7A. The authors also do not show where they placed the boundaries in their analysis. Furthermore, Figure 7B only shows insets for one of the regions being compared making it difficult to see differences from the other region. Finally, the authors do not show an example of their spot segmentation to judge whether their spot counting is reliable. Overall, this makes it difficult to judge whether the quantification in Figure 7C can be trusted.

(7) The correlation between mKi67 and Axin2 in Figure 7 is interesting but does not convincingly show that Wnt downstream of PRDM16 and BMP is responsible for the increased proliferation in PRDM16 mutants.

Weaknesses of the presentation:

Overall, the manuscript is not easy to read. This can cause confusion.

Reviewer #1 (Public review):

Summary:

This research focuses on C. elegans klinotaxis, a chemotactic behavior characterized by gradual turning, aiming to uncover the neural circuit mechanism responsible for the context-dependent reversal of salt concentration preference. The phenomenon observed is that the preferred salt concentration depends on the difference between the pre-assay cultivation conditions and the current environmental salt levels.

The authors propose that a synaptic-reversal plasticity mechanism at the primary sensory neuron, ASER, is critical for this memory- and context-dependent switching of preference. They build on prior findings regarding synaptic reversal between ASER and AIB, as well as the receptor composition of AIY neurons, to hypothesize that similar "plasticity" between ASER and AIY underpins salt preference behavior in klinotaxis. This plasticity differs conceptually from the classical one as it does not rely on any structural changes but rather synaptic transmission is modulated by the basal level of glutamate, and can switch from inhibitory to excitatory.

To test this hypothesis, the study employs a previously established neuroanatomically grounded model [4] and demonstrates that reversing the ASER-AIY synapse sign in the model agent reproduces the observed reversal in salt preference. The model is parameterized using a computational search technique (evolutionary algorithm) to optimize unknown electrophysiological parameters for chemotaxis performance. Experimental validity is ensured by incorporating constraints derived from published findings, confirming the plausibility of the proposed mechanism.

Finally. the circuit mechanism allowing C. elegans to switch behaviour to an exploration run when starved is also investigated. This extension highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.

Strengths and weaknesses:

The authors' approach of integrating prior knowledge of receptor composition and synaptic reversal with the repurposing of a published neuroanatomical model [4] is a significant strength. This methodology not only ensures biological plausibility but also leverages a solid, reproducible modeling foundation to explore and test novel hypotheses effectively.

The evidence produced that the original model has been successfully reproduced is convincing.

The writing of the manuscript needs revision as it makes comprehension difficult.

One major weakness is that the model does not incorporate key findings that have emerged since the original model's publication in 2013, limiting the support for the proposed mechanism. In particular, ablation studies indicate that AIY is not critical for chemotaxis, and other interneurons may play partially overlapping roles in positive versus negative chemotaxis. These findings challenge the centrality of AIY and suggest the model oversimplifies the circuit involved in klinotaxis.

Reference [1] also shows that ASER neurons exhibit complex, memory- and context-dependent responses, which are not accounted for in the model and may have a significant impact on chemotactic model behaviour.

The hypothesis of synaptic reversal between ASER and AIY is not explicitly modeled in terms of receptor-specific dynamics or glutamate basal levels. Instead, the ASER-to-AIY connection is predefined as inhibitory or excitatory in separate models. This approach limits the model's ability to test the full range of mechanisms hypothesized to drive behavioral switching.

While the main results - such as response dependence on step inputs at different phases of the oscillator - are consistent with those observed in chemotaxis models with explicit neural dynamics (e.g., Reference [2]), the lack of richer neural dynamics could overlook critical effects. For example, the authors highlight the influence of gap junctions on turning sensitivity but do not sufficiently analyze the underlying mechanisms driving these effects. The role of gap junctions in the model may be oversimplified because, as in the original model [4], the oscillator dynamics are not intrinsically generated by an oscillator circuit but are instead externally imposed via $z_\text{osc}$. This simplification should be carefully considered when interpreting the contributions of specific connections to network dynamics. Lastly, the complex and context-dependent responses of ASER [1] might interact with circuit dynamics in ways that are not captured by the current simplified implementation. These simplifications could limit the model's ability to account for the interplay between sensory encoding and motor responses in C. elegans chemotaxis.

Appraisal:

The authors show that their model can reproduce memory-dependent reversal of preference in klinotaxis, demonstrating that the ASER-to-AIY synapse plays a key role in switching chemotactic preferences. By switching the ASER-AIY connection from excitatory to inhibitory they indeed show that salt preference reverses. They also show that the curving/turn rate underlying the preference change is gradual and depends on the weight between ASER-AIY. They further support their claim by showing that curving rates also depend on cultivated (set-point).

Thus within the constraints of the hypothesis and the framework, the model operates as expected and aligns with some experimental findings. However, significant omissions of key experimental evidence raise questions on whether the proposed neural mechanisms are sufficient for reversal in salt-preference chemotaxis.

Previous work [1] has shown that individually ablating the AIZ or AIY interneurons has essentially no effect on the Chemotactic Index (CI) toward the set point ([1] Figure 6). Furthermore, in [1] the authors report that different postsynaptic neurons are required for movement above or below the set point. The manuscript should address how this evidence fits with their model by attempting similar ablations. It is possible that the CI is rescued by klinokinesis but this needs to be tested on an extension of this model to provide a more compelling argument.

The investigation of dispersal behaviour in starved individuals is rather limited to testing by imposing inhibition of the SMB neurons. Although a circuit is proposed for how hunger states modulate taxis in the absence of food, this circuit hypothesis is not explicitly modelled to test the theory or provide novel insights.

Impact :

This research underscores the value of an embodied approach to understanding chemotaxis, addressing an important memory mechanism that enables adaptive behavior in the sensorimotor circuits supporting C. elegans chemotaxis. The principle of operation - the dependence of motor responses to sensory inputs on the phase of oscillation - appears to be a convergent solution to taxis. Similar mechanisms have been proposed in Drosophila larvae chemotaxis [2], zebrafish phototaxis [3], and other systems. Consequently, the proposed mechanism has broader implications for understanding how adaptive behaviors are embedded within sensorimotor systems and how experience shapes these circuits across species.

Although the reported reversal of synaptic connection from excitatory to inhibitory is an exciting phenomenon of broad interest, it is not entirely new, as the authors acknowledge similar reversals have been reported in ASER-to-AIB signaling for klinokinesis ( Hiroki et al., 2022). The proposed reversal of the ASER-to-AIY synaptic connection from inhibitory to excitatory is a novel contribution in the specific context of klinotaxis. While the ASER's role in gradient sensing and memory encoding has been previously identified, the current paper mechanistically models these processes, introducing a hypothesis for synaptic plasticity as the basis for bidirectional salt preference in klinotaxis.

The research also highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.

The methodology of parameter search on a neural model of a connectome used here yielded the valuable insight that connectome information alone does not provide enough constraints to reproduce the neural circuits for behaviour. It demonstrates that additional neurophysiological constraints are required.

Additional Context

Oscillators with stimulus-driven perturbations appear to be a convergent solution for taxis and navigation across species. Similar mechanisms have been studied in zebrafish phototaxis [3], Drosophila larvae chemotaxis [2], and have even been proposed to underlie search runs in ants. The modulation of taxis by context and memory is a ubiquitous requirement, with parallels across species. For example, Drosophila larvae modulate taxis based on current food availability and predicted rewards associated with odors, though the underlying mechanism remains elusive. The synaptic reversal mechanism highlighted in this study offers a compelling framework for understanding how taxis circuits integrate context-related memory retrieval more broadly.

As a side note, an interesting difference emerges when comparing C. elegans and Drosophila larvae chemotaxis. In Drosophila larvae, oscillatory mechanisms are hypothesized to underlie all chemotactic reorientations, ranging from large turns to smaller directional biases (weathervaning). By contrast, in C. elegans, weathervaning and pirouettes are treated as distinct strategies, often attributed to separate neural mechanisms. This raises the possibility that their motor execution could share a common oscillator-based framework. Re-examining their overlap might reveal deeper insights into the neural principles underlying these maneuvers.

(1) Luo, L., Wen, Q., Ren, J., Hendricks, M., Gershow, M., Qin, Y., Greenwood, J., Soucy, E.R., Klein, M., Smith-Parker, H.K., & Calvo, A.C. (2014). Dynamic encoding of perception, memory, and movement in a C. elegans chemotaxis circuit. Neuron, 82(5), 1115-1128.

(2) Antoine Wystrach, Konstantinos Lagogiannis, Barbara Webb (2016) Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae eLife 5:e15504.

(3) Wolf, S., Dubreuil, A.M., Bertoni, T. et al. Sensorimotor computation underlying phototaxis in zebrafish. Nat Commun 8, 651 (2017).

(4) Izquierdo, E.J. and Beer, R.D., 2013. Connecting a connectome to behavior: an ensemble of neuroanatomical models of C. elegans klinotaxis. PLoS computational biology, 9(2), p.e1002890.

Reviewer #1 (Public review):

Summary:

The study investigates the role of vascular mural cells, specifically pericytes and vascular smooth muscle cells (vSMCs), in maintaining blood-brain barrier (BBB) integrity and regulating vascular patterning. Analyzing zebrafish pdgfrb mutants that lack brain pericytes and vSMCs, they show that mural cell deficiency does not impair BBB establishment or maintenance during larval and early juvenile stages. However, mural cells seem to be crucial for preventing vascular aneurysms and hemorrhage in adulthood as focal leakage, basement membrane disruption, and increased caveolae formation are observed in adult zebrafish at aneurysm hotspots. The authors challenge the paradigm that mural cells are essential for BBB regulation in early development while highlighting their importance for long-term vascular stability.

Strengths:

Previous studies have established that the zebrafish BBB shares molecular and morphological homology with e.g. the mammalian BBB and therefore represents a suitable model. By examining mural cell roles across different life stages - from larval to adult zebrafish - the study provides an unprecedented comprehensive developmental analysis of brain vascular development and of how mural cells influence BBB integrity and vascular stability over time. The use of live imaging, whole-brain clearing, and electron microscopy offers high-resolution insights into cerebrovascular patterning, aneurysm development, and structural changes in endothelial cells and basement membranes. By analyzing "leakage hotspots" and their association with structural endothelial defects in adults the presented findings add novel insights into how mural cell loss may lead to vascular instability.

Weaknesses:

The study uses quantitative tracer assays with multiple molecular weight dyes to evaluate blood-brain barrier (BBB) permeability. The study normalizes the intensity of tracer signals (e.g., 10 kDa, 70 kDa dextrans) in the brain parenchyma to the vascular signal of a 2000 kDa dextran tracer (assumed to remain within vessels). Intensity normalization is used to control for variations in tracer injection efficiency or vascular density. This method doesn't directly assess the absolute amount of tracer present in the parenchyma, potentially underestimating leakage severity. As the lack of BBB impairment is a "negative" finding, more rigorous controls or other methods might be needed to corroborate it.

Reviewer #1 (Public review):

Summary:

In this study, the authors employed Saturated Transposon Analysis in Yeast (SATAY) in the model yeast Saccharomyces cerevisiae to uncover mutations conferring resistance to 20 different antifungal compounds. These screens revealed novel resistance mechanisms and the modes of action for the antifungal compounds Chitosan and HTI-2307. The authors discovered that Chitosan electrostatically interacts with cell wall mannosylphosphate and identified Hol1 as the transporter of HTI-2307.

Strengths:

The study highlights the power of SATAY in uncovering drug-resistance mechanisms, modes of action, and cellular processes influencing fungal responses to drugs. Identifying novel resistance mechanisms and modes of action for various compounds in this model yeast provides valuable insights for further investigating these compounds in fungal pathogens and developing antifungal strategies. This study thus represents a significant resource for exploring cellular responses to chemical stresses.

The manuscript is well-written and highly clear.

Weaknesses:

As the study was conducted using highly modified non-pathogenic laboratory yeast strains, verification of the findings in fungal pathogens would greatly enhance its relevance and applicability.

Reviewer #1 (Public review):

Summary:

This study investigates whether pupil dilation reflects prediction error signals during associative learning, defined formally by Kullback-Leibler (KL) divergence, an information-theoretic measure of information gain. Two independent tasks with different entropy dynamics (decreasing and increasing uncertainty) were analyzed: the cue-target 2AFC task and the letter-color 2AFC task. Results revealed that pupil responses scaled with KL divergence shortly after feedback onset, but the direction of this relationship depended on whether uncertainty (entropy) increased or decreased across trials. Furthermore, signed prediction errors (interaction between frequency and accuracy) emerged at different time windows across tasks, suggesting task-specific temporal components of model updating. Overall, the findings highlight that pupil dilation reflects information-theoretic processes in a complex, context-dependent manner.

Strengths:

This study provides a novel and convincing contribution by linking pupil dilation to information-theoretic measures, such as KL divergence, supporting Zénon's hypothesis that pupil responses reflect information gained during learning. The robust methodology, including two independent datasets with distinct entropy dynamics, enhances the reliability and generalisability of the findings. By carefully analysing early and late time windows, the authors capture the temporal dynamics of prediction error signals, offering new insights into the timing of model updates. The use of an ideal learner model to quantify prediction errors, surprise, and entropy provides a principled framework for understanding the computational processes underlying pupil responses. Furthermore, the study highlights the critical role of task context - specifically increasing versus decreasing entropy - in shaping the directionality and magnitude of these effects, revealing the adaptability of predictive processing mechanisms.

Weaknesses:

While this study offers important insights, several limitations remain. The two tasks differ significantly in design (e.g., sensory modality and learning type), complicating direct comparisons and limiting the interpretation of differences in pupil dynamics. Importantly, the apparent context-dependent reversal between pupil constriction and dilation in response to feedback raises concerns about how these opposing effects might confound the observed correlations with KL divergence. Finally, subjective factors such as participants' confidence and internal belief states were not measured, despite their potential influence on prediction errors and pupil responses.

Reviewer #1 (Public review):

Summary:

The investigators in this study analyzed the dataset assembly from 540 Salmonella isolates, and those from 45 recent isolates from Zhejiang University of China. The analysis and comparison of the resistome and mobilome of these isolates identified a significantly higher rate of cross-region dissemination compared to localized propagation. This study highlights the key role of the resistome in driving the transition and evolutionary history of S. Gallinarum.

Strengths:

The isolates included in this study were from 16 countries in the past century (1920 to 2023). While the study uses S. Gallinarun as the prototype, the conclusion from this work will likely apply to other Salmonella serotypes and other pathogens.

Reviewer #2 (Public review):

Summary:

Translation of CGG repeats leads to accumulation of poly G, which is associated with neurological disorders. This is an important paper in which the authors sought out proteins that modulate RAN translation. They determined which proteins in Hela cells were enriched on CGG repeats and affected levels of polyG encoded in the 5'UTR of the FMR1 mRNA. They then showed that siRNA depletion of ribosomal protein RPS26 results in less production of FMR1polyG than in control. Experiments were performed in several cell lines and with several reporters with differences in repeats and transfection methods to increase confidence that changes were occurring. New data and details of the methods increase confidence that reporter translation but not global translation is diminished by RPS26 knockdown as concluded. The manuscript has been improved by data showing that new proteins are being synthesized in cells following RPS26 knockdown, and that near-cognate start codon usage is diminished in lines when RPS26 is knocked down, but the mechanism by which RPS26 depletion affects translation is still unclear.

Strengths:

- The authors have proteomics data that show enrichment of a set of proteins on FMR1-polyG RNA but not a related RNA.<br /> - Knockdown of RPS26, which was enriched on the FMR1 RNA, led to decreases in cell growth, but surprisingly did not strongly affect global translation, as assessed by puromycin incorporation<br /> - There is some new evidence that near-cognate start codon selection is affected by RPS26 knockdown

Weaknesses:

- The mechanism for RPS26 knockdown affecting translation of the polyG sequences is unclear, whether knockdown is affecting ribosome levels, extra ribosomal RPS26 or ribosome composition is not known.

Reviewer #2 (Public review):

Summary

The authors use a tree biodiversity experiment to evaluate the effects of tree community and canopy cover on communities of cavity-nesting Hymenoptera and their parasitoids and the interactions between these two guilds. They find that multiple measures of tree diversity influence the hosts, parasitoids, and their interactions. In addition, host-parasitoid interactions show a phylogenetic signal.

Strength

The authors use a massive, long-term data set, meaningful community descriptors, and a solid set of analyses to explore the impacts of tree communities on host-parasitoid networks. It is rare to have such detailed data from multiple different trophic levels.

Weakness

Even though the data expands over several seasons, this is not considered in the analyses, but communities sampled at different years are pooled at the plot level. A more detailed analysis of the variations between years could reveal underlaying patterns as currently the differences in the communities and their structure between the years are ignored (e.g., when estimating the phylogenetic compositions not all the species pooled together actually coexist in time).<br /> Also, the precision of the writing should be improved as it was not always easy to follow the text and the thoughts.

Reviewer #1 (Public review):

Summary:

In this work, Noorman and colleagues test the predictions of the "four-stage model" of consciousness by combining psychophysics and scalp EEG in humans. The study relies on an elegant experimental design to investigate the respective impact of attentional and perceptual blindness on visual processing.

The study is very well summarised, the text is clear and the methods seem sound. Overall, a very solid piece of work. I haven't identified any major weaknesses. Below I raise a few questions of interpretation that may possibly be the subject of a revision of the text.

(1) The perceptual performance on Fig1D appears to show huge variation across participants, with some participants at chance levels and others with performance > 90% in the attentional blink and/or masked conditions. This seems to reveal that the procedure to match performance across participants was not very successful. Could this impact the results? The authors highlight the fact that they did not resort to post-selection or exclusion of participants, but at the same time do not discuss this equally important point.

(2) In the analysis on collinearity and illusion-specific processing, the authors conclude that the absence of a significant effect of training set demonstrates collinearity-only processing. I don't think that this conclusion is warranted: as the illusory and non-illusory share the same shape, so more elaborate object processing could also be occuring. Please discuss.

(3) Discussion, lines 426-429: It is stated that the results align with the notion that processes of perceptual segmentation and organization represent the mechanism of conscious experience. My interpretation of the results is that they show the contrary: for the same visibility level in the attentional blind or masking conditions, these processes can be implicated or not, which suggests a role during unconscious processing instead.

(4) The two paradigms developed here could be used jointly to highlight non-idiosyncratic NCCs, i.e. EEG markers of visibility or confidence that generalise regardless of the method used. Have the authors attempted to train the classifier on one method and apply it to another (e.g. AB to masking and vice versa)? What perceptual level is assumed to transfer?

(4) How can the results be integrated with the attentional literature showing that attentional filters can be applied early in the processing hierarchy?

Comments on revisions:

I'm very pleased with the responses to my previous comments, and congratulate the authors on this excellent piece of work.

Reviewer #1 (Public review):

Summary:

In this study, Kroll et al. conduct an in-depth behavioral analysis of F0 knockouts of 4 genes associated with late-onset Alzheimer's Disease (AD), together with 3 genes associated with early-onset AD. Kroll and colleagues developed a web application (ZOLTAR) to compare sleep-associated traits between genetic mutants with those obtained from a panel of small molecules to promote identification of affected pathways and potential therapeutic interventions. The authors make a set of potentially important findings vis-à-vis the relationship between AD-associated genes and sleep. First, they find that loss-of-function in late-onset AD genes universally result in nighttime sleep loss, consistent with the well-supported hypothesis that sleep disruption contributes to Alzheimer's-related pathologies. psen-1, an early-onset associated AD gene, which the authors find is principally responsible for the generation of AB40 and AB42 in zebrafish, also shows a slight increase in activity at night and slight decreases in nighttime sleep. Conversely, psen-2 mutations increase daytime sleep, while appa/appb mutations have no impact on sleep. Finally, using ZOLTAR, the authors identify serotonin receptor activity as potentially disrupted in sorl1 mutants, while betamethasone is identified as a potential therapeutic to promote reversal of psen2 knockout-associated phenotypes.

This is a highly innovative and thorough study, yet a handful of key questions remain. First, are the nighttime sleep loss phenotypes observed in all knockouts for late-onset AD genes in the larval zebrafish a valid proxy for AD risk? Can 5-HT reuptake inhibitors reverse other AD-related pathologies in zebrafish? Can compounds be identified which have a common behavioral fingerprint across all or multiple AD risk genes? Do these modify sleep phenotypes? Finally, the authors propose but do not test the hypothesis that sorl1 might regulate localization/surface expression of 5-HT2 receptors. This could provide exciting / more convincing mechanistic support for the assertion that serotonin signaling is disrupted upon loss of AD-associated genes. Despite these important considerations, this study provides a valuable platform for high-throughput analysis of sleep phenotypes and correlation with small-molecule induced sleep phenotypes. The platform could also be expanded to facilitate comparison of other behavioral phenotypes, including stimulus-evoked behaviors. Moreover, the new analyses looking for pathways that might be co-regulated by AD risk genes and discussion of cholinergic signaling as a potentially meaningful target downstream of 5/7 knockouts are valuable.

Strengths:<br /> - Provides a useful platform for comparison of sleep phenotypes across genotypes/drug manipulations.<br /> - Presents convincing evidence that nighttime sleep is disrupted in mutants for multiple late-onset AD-related genes.<br /> - Provides potential mechanistic insights for how AD-related genes might impact sleep and identifies a few drugs that modify their identified phenotypes.

Weaknesses:<br /> - Exploration of potential mechanisms for serotonin disruption in sorl1 mutants is limited<br /> - The pipeline developed is only used to examine sleep-related / spontaneous movement phenotypes. Stimulus-evoked behaviors are not examined.

Reviewer #1 (Public review):

Summary:

This study aims to investigate the links between social behaviors observed in free-moving situations and behavioral performances measured in well-controlled, laboratory settings. The authors assessed general social tendencies and dyadic relationships among four monkeys in a group by scoring agonistic (aggression) and affiliative (grooming and proximity) behaviors in each pair. By measuring the saccadic reaction time in a classic social interference task, the authors reported that the monkeys with higher SEIs (i.e., more social individuals) were less distracted by the faces of other monkeys. These effects were enhanced when the distractors were out-group monkey faces rather than in-group ones. Lastly, oxytocin administration increased the impact of the out-group monkey faces in the social interference task, while reducing the magnitude of general social tendencies measured with SEI.

Strengths:

(1) The combination of behavioral data obtained in a colony room and in a laboratory environment is rare and important.<br /> (2) The evaluation of social interactions were successfully performed based on an automated target detection algorithm. The resulting multi-dimensional, complicated social interactions were summarized into simple indices (SEI and IEI). These indices provide a good measure for the social tendencies of each monkey.<br /> (3) Well-designed and robust experiments in the laboratory environment that are linked nicely with the general social tendencies observed in spontaneous behaviors.

Weaknesses:

(1) While the overall results are interesting, I am somewhat left confused about how to interpret the difference in the scores derived from different conditions. For example, the authors stated "Comparing the weights for in-group and out-group distractors, the effect of proximity was larger than that of aggression and grooming" in p.8. Does this mean that the proximity is indeed the type of behavior most affected in the out-group condition compared to the in-group condition? The out-group effects are difficult to examine with actual behavioral data, but some in-group effects such as those involving OT can be tested, which possibly provides good insights into interpreting the differences of the weights observed across the experimental conditions.

(2) I think it is important to provide how variable spontaneous social interactions were across sessions and how impactful the variability of the interactions is on the SEI and IEI, as it helps to understand how meaningful the differences of weights are across the conditions, but such data are missing. In line with this point, although the conclusions still hold as those data were obtained during the same experimental periods, shouldn't the weights in Fig. 3f and Figs. 4g and 4h (saline) be expected to be similar, if not the same?

Comments on revisions: I do not have further comments.

Reviewer #1 (Public review):

Summary:

Shows a new mechanism of GS regulation in the archaean Methanosarcina maze and clarifies the direct activation of GS activity by 2-oxoglutarate, thus featuring an other way, how 2-oxoglutarate acts as a central status reporter of C/N sensing.

Strengths:

mass photometry reveals a a dynamic mode the effect of 2-OG on the oligomerization state of GS. Single particle Cryo-EM reveals the mechanism of 2-OG mediated dodecamer formation.

Weaknesses:

Not entirely clear, how very high 2-OG concentrations activate GS beyond dodecamer formation.

In the revised version, most of my concerns were adequately addressed. In the summary it is stated that glutamine acts as allosteric inhibitor of dodecameric GS. This is not correct: glutamine binds to the active site and is therefore not allosteric. This way of feedback inhibition is a type of product inhibition

Reviewer #1 (Public review):

Summary:

In their manuscript entitled 'The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition', Kavaklıoğlu and colleagues delve into the role of L1TD1, an RNA binding protein (RBP) derived from a LINE1 transposon. L1TD1 proves crucial for maintaining pluripotency in embryonic stem cells and is linked to cancer progression in germ cell tumors, yet its precise molecular function remains elusive. Here, the authors uncover an intriguing interaction between L1TD1 and its ancestral LINE-1 retrotransposon.

The authors delete the DNA methyltransferase DNMT1 in a haploid human cell line (HAP1), inducing widespread DNA hypo-methylation. This hypomethylation prompts abnormal expression of L1TD1. To scrutinize L1TD1's function in a DNMT1 knock-out setting, the authors create DNMT1/L1TD1 double knock-out cell lines (DKO). Curiously, while the loss of global DNA methylation doesn't impede proliferation, additional depletion of L1TD1 leads to DNA damage and apoptosis.

To unravel the molecular mechanism underpinning L1TD1's protective role in the absence of DNA methylation, the authors dissect L1TD1 complexes in terms of protein and RNA composition. They unveil an association with the LINE-1 transposon protein L1-ORF1 and LINE-1 transcripts, among others.

Surprisingly, the authors note fewer LINE-1 retro-transposition events in DKO cells compared to DNMT1 KO alone.

Strengths:

The authors present compelling data suggesting the interplay of a transposon-derived human RNA binding protein with its ancestral transposable element. Their findings spur interesting questions for cancer types, where LINE1 and L1TD1 are aberrantly expressed.

Weaknesses:

The finding that L1TD1/DNMT1 DKO cells exhibit increased apoptosis and DNA damage but decreased L1 retro-transposition is unexpected. Considering the DNA damage associated with retro-transposition and the DNA damage and apoptosis observed in L1TD1/DNMT1 DKO cells, one would anticipate the opposite outcome. Could it be that the observation of fewer transposition-positive colonies stems from the demise of the most transposition-positive colonies? Future studies are bound to further explore this intriguing phenomenon.

Reviewer #1 (Public review):

Wang et al., recorded concurrent EEG-fMRI in 107 participants during nocturnal NREM sleep to investigate brain activity and connectivity related to slow oscillations (SO), sleep spindles, and in particular their co-occurrence. The authors found SO-spindle coupling to be correlated with increased thalamic and hippocampal activity, and with increased functional connectivity from the hippocampus to the thalamus and from the thalamus to the neocortex, especially the medial prefrontal cortex (mPFC). They concluded the brain-wide activation pattern to resemble episodic memory processing, but to be dissociated from task-related processing and suggest that the thalamus plays a crucial role in coordinating the hippocampal-cortical dialogue during sleep.

The paper offers an impressively large and highly valuable dataset that provides the opportunity for gaining important new insights into the network substrate involved in SOs, spindles, and their coupling. However, the paper does unfortunately not exploit the full potential of this dataset with the analyses currently provided, and the interpretation of the results is often not backed up by the results presented.

I have the following specific comments.

(1) The introduction is lacking sufficient review of the already existing literature on EEG-fMRI during sleep and the BOLD-correlates of slow oscillations and spindles in particular (Laufs et al., 2007; Schabus et al., 2007; Horovitz et al., 2008; Laufs, 2008; Czisch et al., 2009; Picchioni et al., 2010; Spoormaker et al., 2010; Caporro et al., 2011; Bergmann et al., 2012; Hale et al., 2016; Fogel et al., 2017; Moehlman et al., 2018; Ilhan-Bayrakci et al., 2022). The few studies mentioned are not discussed in terms of the methods used or insights gained.

(2) The paper falls short in discussing the specific insights gained into the neurobiological substrate of the investigated slow oscillations, spindles, and their interactions. The validity of the inverse inference approach ("Open ended cognitive state decoding"), assuming certain cognitive functions to be related to these oscillations because of the brain regions/networks activated in temporal association with these events, is debatable at best. It is also unclear why eventually only episodic memory processing-like brain-wide activation is discussed further, despite the activity of 16 of 50 feature terms from the NeuroSynth v3 dataset were significant (episodic memory, declarative memory, working memory, task representation, language, learning, faces, visuospatial processing, category recognition, cognitive control, reading, cued attention, inhibition, and action).

(3) Hippocampal activation during SO-spindles is stated as a main hypothesis of the paper - for good reasons - however, other regions (e.g., several cortical as well as thalamic) would be equally expected given the known origin of both oscillations and the existing sleep-EEG-fMRI literature. However, this focus on the hippocampus contrasts with the focus on investigating the key role of the thalamus instead in the Results section.

(4) The study included an impressive number of 107 subjects. It is surprising though that only 31 subjects had to be excluded under these difficult recording conditions, especially since no adaptation night was performed. Since only subjects were excluded who slept less than 10 min (or had excessive head movements) there are likely several datasets included with comparably short durations and only a small number of SOs and spindles and even less combined SO-spindle events. A comprehensive table should be provided (supplement) including for each subject (included and excluded) the duration of included NREM sleep, number of SOs, spindles, and SO+spindle events. Also, some descriptive statistics (mean/SD/range) would be helpful.

(5) Was the 20-channel head coil dedicated for EEG-fMRI measurements? How were the electrode cables guided through/out of the head coil? Usually, the 64-channel head coil is used for EEG-fMRI measurements in a Siemens PRISMA 3T scanner, which has a cable duct at the back that allows to guide the cables straight out of the head coil (to minimize MR-related artifacts). The choice for the 20-channel head coil should be motivated. Photos of the recording setup would also be helpful.

(6) Was the EEG sampling synchronized to the MR scanner (gradient system) clock (the 10 MHz signal; not referring to the volume TTL triggers here)? This is a requirement for stable gradient artifact shape over time and thus accurate gradient noise removal.

(7) The TR is quite long and the voxel size is quite large in comparison to state-of-the-art EPI sequences. What was the rationale behind choosing a sequence with relatively low temporal and spatial resolution?

(8) The anatomically defined ROIs are quite large. It should be elaborated on how this might reduce sensitivity to sleep rhythm-specific activity within sub-regions, especially for the thalamus, which has distinct nuclei involved in sleep functions.

(9) The study reports SO & spindle amplitudes & densities, as well as SO+spindle coupling, to be larger during N2/3 sleep compared to N1 and REM sleep, which is trivial but can be seen as a sanity check of the data. However, the amount of SOs and spindles reported for N1 and REM sleep is concerning, as per definition there should be hardly any (if SOs or spindles occur in N1 it becomes by definition N2, and the interval between spindles has to be considerably large in REM to still be scored as such). Thus, on the one hand, the report of these comparisons takes too much space in the main manuscript as it is trivial, but on the other hand, it raises concerns about the validity of the scoring.

(10) Why was electrode F3 used to quantify the occurrence of SOs and spindles? Why not a midline frontal electrode like Fz (or a number of frontal electrodes for SOs) and Cz (or a number of centroparietal electrodes) for spindles to be closer to their maximum topography?

(11) Functional connectivity (hippocampus -> thalamus -> cortex (mPFC)) is reported to be increased during SO-spindle coupling and interpreted as evidence for coordination of hippocampo-neocortical communication likely by thalamic spindles. However, functional connectivity was only analysed during coupled SO+spindle events, not during isolated SOs or isolated spindles. Without the direct comparison of the connectivity patterns between these three events, it remains unclear whether this is specific for coupled SO+spindle events or rather associated with one or both of the other isolated events. The PPIs need to be conducted for those isolated events as well and compared statistically to the coupled events.

(12) The limited temporal resolution of fMRI does indeed not allow for easily distinguishing between fMRI activation patterns related to SO-up- vs. SO-down-states. For this, one could try to extract the amplitudes of SO-up- and SO-down-states separately for each SO event and model them as two separate parametric modulators (with the risk of collinearity as they are likely correlated).

(13) L327: "It is likely that our findings of diminished DMN activity reflect brain activity during the SO DOWN-state, as this state consistently shows higher amplitude compared to the UP-state within subjects, which is why we modelled the SO trough as its onset in the fMRI analysis." This conclusion is not justified as the fact that SO down-states are larger in amplitude does not mean their impact on the BOLD response is larger.

(14) Line 77: "In the current study, while directly capturing hippocampal ripples with scalp EEG or fMRI is difficult, we expect to observe hippocampal activation in fMRI whenever SOs-spindles coupling is detected by EEG, if SOs- spindles-ripples triple coupling occurs during human NREM sleep". Not all SO-spindle events are associated with ripples (Staresina et al., 2015), but hippocampal activation may also be expected based on the occurrence of spindles alone (Bergmann et al., 2012).

References:

Bergmann TO, Molle M, Diedrichs J, Born J, Siebner HR (2012) Sleep spindle-related reactivation of category-specific cortical regions after learning face-scene associations. Neuroimage 59:2733-2742.<br /> Caporro M, Haneef Z, Yeh HJ, Lenartowicz A, Buttinelli C, Parvizi J, Stern JM (2011) Functional MRI of sleep spindles and K-complexes. Clin Neurophysiol.<br /> Czisch M, Wehrle R, Stiegler A, Peters H, Andrade K, Holsboer F, Samann PG (2009) Acoustic oddball during NREM sleep: a combined EEG/fMRI study. PLoS One 4:e6749.<br /> Fogel S, Albouy G, King BR, Lungu O, Vien C, Bore A, Pinsard B, Benali H, Carrier J, Doyon J (2017) Reactivation or transformation? Motor memory consolidation associated with cerebral activation time-locked to sleep spindles. PLoS One 12:e0174755.<br /> Hale JR, White TP, Mayhew SD, Wilson RS, Rollings DT, Khalsa S, Arvanitis TN, Bagshaw AP (2016) Altered thalamocortical and intra-thalamic functional connectivity during light sleep compared with wake. Neuroimage 125:657-667.<br /> Horovitz SG, Fukunaga M, de Zwart JA, van Gelderen P, Fulton SC, Balkin TJ, Duyn JH (2008) Low frequency BOLD fluctuations during resting wakefulness and light sleep: a simultaneous EEG-fMRI study. Hum Brain Mapp 29:671-682.<br /> Ilhan-Bayrakci M, Cabral-Calderin Y, Bergmann TO, Tuscher O, Stroh A (2022) Individual slow wave events give rise to macroscopic fMRI signatures and drive the strength of the BOLD signal in human resting-state EEG-fMRI recordings. Cereb Cortex 32:4782-4796.<br /> Laufs H (2008) Endogenous brain oscillations and related networks detected by surface EEG-combined fMRI. Hum Brain Mapp 29:762-769.<br /> Laufs H, Walker MC, Lund TE (2007) 'Brain activation and hypothalamic functional connectivity during human non-rapid eye movement sleep: an EEG/fMRI study'--its limitations and an alternative approach. Brain 130:e75; author reply e76.<br /> Moehlman TM, de Zwart JA, Chappel-Farley MG, Liu X, McClain IB, Chang C, Mandelkow H, Ozbay PS, Johnson NL, Bieber RE, Fernandez KA, King KA, Zalewski CK, Brewer CC, van Gelderen P, Duyn JH, Picchioni D (2018) All-Night Functional Magnetic Resonance Imaging Sleep Studies. J Neurosci Methods.<br /> Picchioni D, Horovitz SG, Fukunaga M, Carr WS, Meltzer JA, Balkin TJ, Duyn JH, Braun AR (2010) Infraslow EEG oscillations organize large-scale cortical-subcortical interactions during sleep: A combined EEG/fMRI study. Brain Res.<br /> Schabus M, Dang-Vu TT, Albouy G, Balteau E, Boly M, Carrier J, Darsaud A, Degueldre C, Desseilles M, Gais S, Phillips C, Rauchs G, Schnakers C, Sterpenich V, Vandewalle G, Luxen A, Maquet P (2007) Hemodynamic cerebral correlates of sleep spindles during human non-rapid eye movement sleep. Proc Natl Acad Sci U S A 104:13164-13169.<br /> Spoormaker VI, Schroter MS, Gleiser PM, Andrade KC, Dresler M, Wehrle R, Samann PG, Czisch M (2010) Development of a large-scale functional brain network during human non-rapid eye movement sleep. J Neurosci 30:11379-11387.<br /> Staresina BP, Bergmann TO, Bonnefond M, van der Meij R, Jensen O, Deuker L, Elger CE, Axmacher N, Fell J (2015) Hierarchical nesting of slow oscillations, spindles and ripples in the human hippocampus during sleep. Nat Neurosci 18:1679-1686.

Reviewer #1 (Public review):

Summary:

Howard et al. performed deep mutational scanning on the MC4R gene, using a reporter assay to investigate two distinct downstream pathways across multiple experimental conditions. They validated their findings with ClinVar data and previous studies. Additionally, they provided insights into the application of DMS results for personalized drug therapy and differential ligand responses across variant types.

Strengths:

They captured over 99% of variants with robust signals and investigated subtle functionalities, such as pathway-specific activities and interactions with different ligands, by refining both the experimental design and analytical methods.

They provided additional details regarding the quality of the library, including the even composition of variants, sufficient readout from tested cells, and adequate sequencing depth. Additionally, they clarified the underlying assay mechanisms, effectively demonstrating the robustness of their results.

Reviewer #1 (Public review):

This study presents Jyvaskylavirus, a new member of the Marseilleviridae family, infecting Acanthamoeba castellanii. The study provides a detailed and comprehensive genomic and structural analysis of Jyvaskylavirus. The authors identified ORF142 as the capsid penton protein and additional structural proteins that comprise the virion. Using a combination of imaging techniques the authors provide new insights into the giant virus architecture and lifecycle. The study could be improved by providing atomic coordinates and refinement statistics, comparisons with available giant virus structures could be expanded, and the novelty in terms of the first isolated example of a giant virus from Finland could be expounded upon.

The study contributes new structural and genomic diversity to the Marseilleviridae family, hinting at a broader distribution and ecological significance of giant viruses than previously thought.

Comments on revisions: I'm satisfied with the authors' responses to the review, and request no further changes.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors provide strong evidence that the cell surface E3 ubiquitin ligases RNF43 and ZNRF3, which are well known for their role in regulating cell surface levels of WNT receptors encoded by FZD genes, also target EGFR for degradation. This is newly identified function for these ubiquitin ligases beyond their role in regulating WNT signaling. Loss of RNF43/ZNRF3 expression leads to elevated EGFR levels and signaling, suggesting a potential new axis to drive tumorigenesis, whereas overexpression of RNF43 or ZNRF3 decreases EGFR levels and signaling. Furthermore, RNF43 and ZNRF3 directly interact with EGFR through their extracellular domains.

Strengths:

The data showing that RNF43 and ZNRF3 interact with EGFR and regulate its levels and activity are thorough and convincing, and the conclusions are largely supported.

Weaknesses:

Prior work established a clear role for RNF43 and ZNRF3 in regulating cell surface levels of FZD, a class of WNT receptors. These new findings that these E3 ubiquitin ligases also target EGFR add a new layer of complexity, and it remains unclear to what extent WNT signaling versus EGFR signaling are impacted in cancer settings. The authors acknowledge this gap in our understanding, which will likely be the topic of follow-up studies.

Comments on revisions:

The authors addressed my main concerns in this revised version and in their rebuttal comments. I have no further critiques to add.

Reviewer #1 (Public review):

Summary:

Prior research indicates that NaV1.2 and NaV1.6 have different compartmental distributions, expression timelines in development, and roles in neuron function. The lack of subtype-specific tools to control Nav1.2 and Nav1.6 activity however has hampered efforts to define the role of each channel in neuronal behavior. The authors attempt to address the problem of subtype specificity here by using aryl sulfonamides (ASCs) to stabilize channels in the inactivated state in combination with mice carrying a mutation that renders NaV1.2 and/or NaV1.6 genetically resistant to the drug. Using this innovative approach, the authors find that action potential initiation is controlled by NaV1.6 while both NaV1.2 and NaV1.6 are involved in backpropagation of the action potential to the soma, corroborating previous findings. Additionally, NaV1.2 inhibition paradoxically increases the firing rate, as has also been observed in genetic knockout models. Finally, the potential anticonvulsant properties of ASCs were tested. NaV1.6 inhibition but not NaV1.2 inhibition was found to decrease action potential firing in prefrontal cortex layer 5b pyramidal neurons in response to current injections designed to mimic inputs during seizure. This result is consistent with studies of loss-of-function Nav1.6 models and knockdown studies showing that these animals are resistant to certain seizure types. These results lend further support for the therapeutic promise of activity-dependent, NaV1.6-selective, inhibitors for epilepsy.

Strengths:

(1) The chemogenetic approaches used to achieve selective inhibition of NaV1.2 and NaV1.6 are innovative and help resolve long-standing questions regarding the role of Nav1.2 and Nav1.6 in neuronal electrogenesis.

(2) The experimental design is overall rigorous, with appropriate controls included.

(3) The assays to elucidate the effects of channel inactivation on typical and seizure-like activity were well selected.

Weaknesses:

(1) The potential impact of the YW->SR mutation in the voltage sensor does not appear to have been sufficiently assessed. The activation/inactivation curves in Figure 1E show differences in both activation and inactivation at physiologically relevant membrane voltages, which may be significant even though the V1/2 and slope factors are roughly similar.

(2) Additional discussion of the fact that channels are only partially blocked by the ASC and that ASCs act in a use-dependent manner would improve the manuscript and help readers interpret these results.

(3) NaV1.6 was described as being exclusively responsible for the change in action potential threshold, but when NaV1.6 alone was inactivated, the effect was significantly reduced from the condition in which both channels were inactivated (Figure 4E). Similarly, Figure 6C shows that blockade of both channels causes threshold depolarization prior to the seizure-like event, but selective inactivation of NaV1.6 does not. As NaV1.2 does not appear to be involved in action potential initiation and threshold change, what is the mechanism of this dissimilarity between the NaV1.6 inactivation and combined NaV1.6/ NaV1.2 inactivation?

(4) The idea that use-dependent VGSC-acting drugs may be effective antiseizure medications is well established. Additional discussion or at least acknowledgement of the existing, widely used, use-dependent VGSC drugs should be included (e.g. Carbamazepine, Lamotrigine, Phenytoin). Also, the idea that targeting NaV1.6 may be effective for seizures is established by studies using genetic models, knockdown, and partially selective pharmacology (e.g. NBI-921352). Additional discussion of how the results reported here are consistent with or differ from studies using these alternative approaches would improve the discussion

Reviewer #1 (Public review):

Summary:

Howard-Spink et al. investigated how older chimpanzees changed their behavior regarding stone tool use for nutcracking over a period of 17 years, from late adulthood to old age. This behavior is cognitively demanding, and it is a good target for understanding aging in wild primates. They used several factors to follow the aging process of five individuals, from attendance at the nut-cracking outdoor laboratory site to time to select tools and efficiency in nut-cracking to check if older chimpanzee changed their behavior.

Indeed, older chimpanzees reduced their visits to the outdoor lab, which was not observed in the younger adults. The authors discuss several reasons for that; the main ones being physiological changes, cognitive and physical constraints, and changes in social associations. Much of the discussion is hypothetical, but a good starting point, as there is not much information about senescence in wild chimpanzees.

The efficiency for nut-cracking was variable, with some individuals taking a long time to crack nuts while others showed little variance. As this is not compared with the younger individuals and the sample is small (only five individuals), it is difficult to be sure if this is also partly a normal variance caused by other factors (ecology) or is only related to senescence.

Strengths:

(1) 17 years of longitudinal data in the same setting, following the same individuals.

(2) Using stone tool use, a cognitively demanding behavior, to understand the aging process.

Weaknesses:

A lack of comparison of the stone tool use behavior with younger individuals in the same period, to check if the changes observed are only related to age or if it is an overall variance. The comparison with younger chimpanzees was only done for one of the variables (attendance).

Reviewer #1 (Public review):

Ejdrup, Gether, and colleagues present a sophisticated simulation of dopamine (DA) dynamics based on a substantial volume of striatum with many DA release sites. The key observation is that a reduced DA uptake rate in the ventral striatum (VS) compared to the dorsal striatum (DS) can produce an appreciable "tonic" level of DA in VS and not DS. In both areas they find that a large proportion of D2 receptors are occupied at "baseline"; this proportion increases with simulated DA cell phasic bursts but has little sensitivity to simulated DA cell pauses. They also examine, in a separate model, the effects of clustering dopamine transporters (DAT) into nanoclusters and say this may be a way of regulating tonic DA levels in VS. I found this work of interest and I think it will be useful to the community. At the same time, there are a number of weaknesses that should be addressed, and the authors need to more carefully explain how their conclusions are distinct from those based on prior models.

(1) The conclusion that even an unrealistically long (1s) and complete pause in DA firing has little effect on DA receptor occupancy is potentially important. The ability to respond to DA pauses has been thought to be a key reason why D2 receptors (may) have high affinity. This simulation instead finds evidence that DA pauses may be useless. This result should be highlighted in the abstract and discussed more.

(2) The claim of "DAT nanoclustering as a way to shape tonic levels of DA" is not very well supported at present. None of the panels in Figure 4 simply show mean steady-state extracellular DA as a function of clustering. Perhaps mean DA is not the relevant measure, but then the authors need to better define what is and why. This issue may be linked to the fact that DAT clustering is modeled separately (Figure 4) to the main model of DA dynamics (Figures 1-3) which per the Methods assumes even distribution of uptake. Presumably, this is because the spatial resolution of the main model is too coarse to incorporate DAT nanoclusters, but it is still a limitation. As it stands it is convincing (but too obvious) that DAT clustering will increase DA away from clusters, while decreasing it near clusters. I.e. clustering increases heterogeneity, but how this could be relevant to striatal function is not made clear, especially given the different spatial scales of the models.

(3) I question how reasonable the "12/40" simulated burst firing condition is, since to my knowledge this is well outside the range of firing patterns actually observed for dopamine cells. It would be better to base key results on more realistic values (in particular, fewer action potentials than 12).

(4) There is a need to better explain why "focality" is important, and justify the measure used.

(5) Line 191: " D1 receptors (-Rs) were assumed to have a half maximal effective concentration (EC50) of 1000 nM"<br /> The assumptions about receptor EC50s are critical to this work and need to be better justified. It would also be good to show what happens if these EC50 numbers are changed by an order of magnitude up or down.

(6) Line 459: "we based our receptor kinetics on newer pharmacological experiments in live cells (Agren et al., 2021) and properties of the recently developed DA receptor-based biosensors (Labouesse & Patriarchi, 2021). Indeed, these sensors are mutated receptors but only on the intracellular domains with no changes of the binding site (Labouesse & Patriarchi, 2021)"<br /> This argument is diminished by the observation that different sensors based on the same binding site have different affinities (e.g. in Patriarchi et al. 2018, dLight1.1 has Kd of 330nM while dlight1.3b has Kd of 1600nM).

(7) Estimates of Vmax for DA uptake are entirely based on prior fast-scan voltammetry studies (Table S2). But FSCV likely produces distorted measures of uptake rate due to the kinetics of DA adsorption and release on the carbon fiber surface.

(8) It is assumed that tortuosity is the same in DS and VS - is this a safe assumption?

(9) More discussion is needed about how the conclusions derived from this more elaborate model of DA dynamics are the same, and different, to conclusions drawn from prior relevant models (including those cited, e.g. from Hunger et al. 2020, etc).

Reviewer #1 (Public review):

Summary:

This study by Xu et al. focuses on the impact of clathrin-independent endocytosis in cancer cells on T cell activation. In particular, by using a combination of biochemical approaches and imaging, the authors identify ICAM1, the ligand for T cell-expressed integrin LFA-1, as a novel cargo for EndoA3-mediated endocytosis. Subsequently, the authors aim to identify functional implications for T cell activation, using a combination of cytokine assays and imaging experiments.

They find that the absence of EndoA3 leads to a reduction in T cell-produced cytokine levels. Additionally, they observe slightly reduced levels of ICAM1 at the immunological synapse and an enlarged contact area between T cells and cancer cells. Taken together, the authors propose a mechanism where EndoA3-mediated endocytosis of ICAM1, followed by retrograde transport, supplies the immunological synapse with ICAM1. In the absence of EndoA3, T cells attempt to compensate for suboptimal ICAM1 levels at the synapse by enlarging their contact area, which proves insufficient and leads to lower levels of T cell activation.

Strengths:

The authors utilize a rigorous and innovative experimental approach that convincingly identifies ICAM1 as a novel cargo for Endo3A-mediated endocytosis.

Weaknesses:

The characterization of the effects of Endo3A absence on T cell activation appears incomplete. Key aspects, such as surface marker upregulation, T cell proliferation, integrin signalling and most importantly, the killing of cancer cells, are not comprehensively investigated.

As Endo- and exocytosis are intricately linked with the biophysical properties of the cellular membrane (e.g. membrane tension), which can significantly impact T-cell activation and cytotoxicity, the authors should address this possibility and ideally address it experimentally to some degree.

Crucially, key literature relevant to this research, addressing the role of ICAM1 endocytosis in antigen-presenting cells, has not been taken into consideration.

Reviewer #1 (Public review):

Summary:

In this study, Ana Lapao et al. investigated the roles of Rab27 effector SYTL5 in cellular membrane trafficking pathways. The authors found that SYTL5 localizes to mitochondria in a Rab27A-dependent manner. They demonstrated that SYTL5-Rab27A positive vesicles containing mitochondrial material are formed under hypoxic conditions, thus they speculate that SYTL5 and Rab27A play roles in mitophagy. They also found that both SYTL5 and Rab27A are important for normal mitochondrial respiration. Cells lacking SYTL5 undergo a shift from mitochondrial oxygen consumption to glycolysis which is a common process known as the Warburg effect in cancer cells. Based on the cancer patient database, the author noticed that low SYTL5 expression is related to reduced survival for adrenocortical carcinoma patients, indicating SYTL5 could be a negative regulator of the Warburg effect and potentially tumorigenesis.

Strengths:

The authors take advantage of multiple techniques and novel methods to perform the experiments.

(1) Live-cell imaging revealed that stably inducible expression of SYTL5 co-localized with filamentous structures positive for mitochondria. This result was further confirmed by using correlative light and EM (CLEM) analysis and western blotting from purified mitochondrial fraction.

(2) In order to investigate whether SYTL5 and RAB27A are required for mitophagy in hypoxic conditions, two established mitophagy reporter U2OS cell lines were used to analyze the autophagic flux.

Weaknesses:

This study revealed a potential function of SYTL5 in mitophagy and mitochondrial metabolism. However, the mechanistic evidence that establishes the relationship between SYTL5/Rab27A and mitophagy is insufficient. The involvement of SYTL5 in ACC needs more investigation. Furthermore, images and results supporting the major conclusions need to be improved.

Reviewer #1 (Public review):

Summary:

Amaral et al. presents a study investigating the mesoscale modelling and dynamics of bolalipids.

Strengths:

The figures in this paper are exceptional. Both those to outline and introduce the lipid types, but also the quality and resolution of the plots. The data held within also appears to be outstanding and of significant (hopefully) general interest.

Weaknesses:

In the introduction, I would like to have read more specifics on the biological role of bolalipids. Archaea are mentioned, but this kingdom is huge - there must be specific species that can be discussed where bolalipids are integral to archaeal life. The authors should go beyond 'extremophiles'. In short, they should unpack why the general audience should be interested in these lipids, within a subset of organisms that are often forgotten about.

Reviewer #1 (Public review):

G. Squiers et al. analyzed a previously reported CRISPR genetic screening dataset of engineered GLUT4 cell-surface presentation and identified the Commander complex subunit COMMD3 as being required for endosomal recycling of specific cargo proteins, such as transferrin receptor (TfR), to the cell surface. Through comparison of COMMD3-KO and other Commander subunit-KO cells, they demonstrated that the role of COMMD3 in mediating TfR recycling is independent of the Commander complex. Structural analysis and co-immunoprecipitation followed by mass spectrometry revealed that TfR recycling by COMMD3 relies on ARF1. COMMD3 interacts with ARF1 through its N-terminal domain (NTD) to stabilize ARF1. A mutation in the NTD of COMMD3, which disrupts the NTD-ARF1 interaction, failed to rescue cell surface TfR in COMMD3-KO cells. In conclusion, the authors assert that COMMD3 stabilizes ARF1 in a Commander complex-independent manner, which is essential for recycling specific cargo proteins from endosomes to the plasma membrane.

The conclusions of this paper are generally supported by data, but some validation experiments and control conditions should be included to strengthen the study.

(1) Commander-Independent Role of COMMD3:<br /> While the authors provided evidence to support the Commander-independent role of COMMD3-such as the absence of other Commander subunits in the CRISPR screen and not decreased COMMD3 levels in other subunit-KO cells-direct evidence is lacking. The mutation that specifically disrupts the COMMD3-ARF1 interaction could serve as a valuable tool to directly address this question.

(2) Role of ARF1 in Cargo Selection:<br /> The Commander-independent function of COMMD3 appears cargo-dependent and relies on ARF1's role in cargo selection. The authors should investigate whether KO/KD of ARF1 reduces cell surface levels of ITGA6 and TfR.

(3) Impact on TfR Stability:<br /> Figure 7D suggests that TfR protein levels are reduced in COMMD3-KO cells, potentially due to degradation caused by disrupted recycling. This raises the question of whether the observed reduction in cell surface TfR is due to impaired endosomal recycling or decreased total protein levels. The authors should quantify the ratio of cell surface protein to total protein for TfR, GLUT-SPR, and ITGA6 in COMMD3-KO cells.

Reviewer #1 (Public review):

In this study, Marocco and colleagues perform a deep characterization of the complex molecular mechanism guiding the recognition of a particular CELLmotif previously identified in hepatocytes in another publication. Having miR-155-3p with or without this CELLmotif as the initial focus, the authors identify 21 proteins differentially binding to these two miRNA versions. From there, they decided to focus on PCBP2. They elegantly demonstrate PCBP2 binding to the miR-155-3p WT version but not to the CELLmotif-mutated version. miR-155-3p contains a hEXOmotif identified in a different report, whose recognition is largely mediated by another RNA-binding protein called SYNCRIP. Interestingly, mutation of the hEXOmotif contained in miR-155-3p did not only blunt SYNCRIP binding but also PCBP2 binding despite the maintenance of the CELLmotif. This indicates that somehow SYNCRIP binding is a pre-requisite for PCBP2 binding. EMSA assay confirms that SYNCRIP is necessary for PCBP2 binding to miR-155-3p, while PCBP2 is not needed for SYNCRIP binding. The authors aim to extend these findings to other miRNAs containing both motifs. For that, they perform a small-RNA-Seq of EVs released from cells knockdown for PCBP2 versus control cells, identifying a subset of miRNAs whose expression either increases or decreases. The assumption is that those miRNAs containing PCBP2-binding CELLmotif should now be less retained in the cell and go more to extracellular vesicles, thus reflecting a higher EV expression. The specific subset of miRNAs having both the CELLmotif and hEXOmotif (9 miRNAs) whose expressions increase in EVs due to PCBP2 reduction is also affected by knocking-down SYNCRIP in the sense that reduction of SYNCRIP leads to lower EV sorting. Further experiments confirm that PCBP2 and SYNCRIP bind to these 9 miRNAs and that knocking down SYNCRIP impairs their EV sorting.

While the process studied in this work is novel and interesting, there are several aspects of this manuscript that should be improved:

(1) First of all, the nature of the CELLmotif and the hEXOmotif they are studying is extremely confusing. For the CELLmotif, the authors seem to focus on the Core CELLmotif AUU A/G in some experiments and the extended 7-nucleotide version in others. The fact that these CELLmotif and hEXOmotif are not shown anywhere in the figures (I mean with the full nucleotide variability described in the original publications) but only referred to in the text further complicates the identification of the motifs and the understanding of the experiments. Moreover, I am not convinced that the sequences they highlight in grey correspond to the original CELLmotif in all cases. For instance, in the miR-155-3p sequence, GCAUU is highlighted in grey. However, the original CELLmotif is basically 7-nucleotide long: C, A/U, G/A/C, U, U/A, C/G/A, A/U/C or CAGUUCA in its more abundant version. I can only see clearly the presence of the Core CELLmotif AUUA in miR-155-3p; however, the last A is not highlighted in grey. It is true that there is some nucleotide variability in each position in the originally reported CELLmotif by the authors in ref. 5 and the hEXOmotifs in ref. 7; however, not all nucleotides are equally likely to be found in each position. This fact seems to be not to be taken into account by the authors as they took basically any sequence with any length and almost sequence combination as valid CELLmotif. This means that I cannot identify the CELLmotif in many cases among the ones they highlight in grey. Instead, they should really focus on the most predominant CELLmotif sequence or, instead, take a reduced subset of "more abundant" CELLmotif versions from the ones that could fit in the originally described CELLmotif. Altogether, the authors need to explain much better what they have considered as the CELLmotif, what is the Core CELLmotif and what is hEXOmotif in each case and restrict to the most likely versions of the CELLmotif and hEXOmotif.

(2) Validation of EV isolation method: first, a large part of Supplementary Figure 2 is not readable. EV markers seem to be enriched in EV isolates; however, more EV and cell markers should be assayed to fulfill MISEV guidelines.

(3) A key variable is missing in Supplementary Figure 2, which is whether PCBP2 or SYNCRIP knockdowns impair EV secretion rates. A quantification of the nr vesicles released per cell upon knocking down each of these factors would be essential to rule out that any of the effects seen throughout the paper are not due to reduced or enhanced EV production rather than miRNA sorting/retention.

(4) The EMSA experiment is important to support their claims. Given the weak bands that are shown, the authors need to show all their replicates to convince the readers that it is reproducible.

(5) Although the bindings of SYNCRIP and PCBP2 to miR-155-3p and other miRNAs having both hEXOmotif and CELLmotif seem clear, the need for SYNCRIP binding to allow for PCBP2-mediated cellular retention is counterintuitive. What happens to those miRNAs that only contain a CELLmotif in terms of cellular retention and SYNCRIP dependence for cellular retention? In this regard, a representative miRNA (miR-31-3p) is analyzed in several experiments, showing that PCBP2 does not bind to it unless a hEXOmotif is introduced (Figure 3). However, this type of experiment should definitely be extended to other miRNAs containing only CELLmotif without hEXOmotif.

(6) Along the same line, I am missing another important experiment: the artificial incorporation of CELLmotif. For example, miR-365-2-5p lacks a CELLmotif but has a hEXOmotif. Does PCBP2 bind to this miRNA upon incorporation of CELLmotif? Does this lead now to enhanced cellular retention of this miRNA?

(7) What would be the net effect of knocking down both SYNCRIP and PCBP2 at the same time? Would this neutralize each other's effect or would the lack of one impose on the other? That could help in understanding the complex interplay between these two factors for mediating cellular retention and EV sorting.

(8) The authors have here a great opportunity to shed some light on an unclear aspect of miRNA EV sorting and cellular retention: whether the RBPs go together with the miRNA to the EVs or not. While the original paper describing hEXOmotif found SYNCRIP in EVs, another publication (Jeppesen et al, Cell 2019; PMID: 30951670) later found this RBP being very scarce in small EVs compared to cellular bodies or large EVs (Supplementary Tables 3 and 4 in that publication). Can the authors find SYNCRIP and PCBP2 in the EVs? Another important question would be the colocalization of these RBPs in the place where the miRNA selection is supposed to take place: in multivesicular bodies (MVB). Is there a colocalization of these RBPs with MVBs in the cell?

(9) In Figure 4C, the authors state in the text that CELLmotif and hEXOmotif are present in extra-seed region; however, for miR-181d-5p and miR-122-3p this is not true as their CELLmotifs fall within the seed sequence.

(10) The authors need to describe how they calculate the EV/cell ratio in gene expression in some experiments (for instance, Figures 1H, 4D, etc). Did they use any housekeeping gene for EV RNA content, the same RNA load, or some other alternative method to normalize EV vs cell RNA content?

(11) I would suggest that the authors speculate a bit in the discussion section on how the interaction between PCBP2 and SYNCRIP takes place. Do they contain any potential interacting domain? The binding of one to the miRNA would impose a topological interference on the binding of the other?

Joint Public Review:

In this manuscript, Weiguang Kong et al. investigate the role of immunoglobulin M (IgM) in antiviral defense in the teleost largemouth bass (Micropterus salmoides). The study employs an IgM depletion model, viral infection experiments, and complementary in vitro assays to explore the role of IgM in systemic and mucosal immunity. The authors conclude that IgM is crucial for both systemic and mucosal antiviral defense, highlighting its role in viral neutralization through direct interactions with viral particles. The study's findings have theoretical implications for understanding immunoglobulin function across vertebrates and practical relevance for aquaculture immunology.

Strengths:

The manuscript applies multiple complementary approaches, including IgM depletion, viral infection models, and histological and gene expression analyses, to address an important immunological question. The study challenges established views that IgT is primarily responsible for mucosal immunity, presenting evidence for a dual role of IgM at both systemic and mucosal levels. If validated, the findings have evolutionary significance, suggesting the conserved role of IgM as an antiviral effector across jawed vertebrates for over 500 million years. The practical implications for vaccine strategies targeting mucosal immunity in fish are noteworthy, addressing a key challenge in aquaculture.

Weaknesses:

Several conceptual and technical issues undermine the strength of the evidence:

Monoclonal Antibody (MoAb) Validation: The study relies heavily on a monoclonal antibody to deplete IgM, but its specificity and functionality are not adequately validated. The epitope recognized by the antibody is not identified, and there is no evidence excluding cross-reactivity with other isotypes. Mass spectrometry, immunoprecipitation, or Western blot analysis using tissue lysates with varying immunoglobulin expression levels would strengthen the claim of IgM-specific depletion.

IgM Depletion Kinetics: The rapid depletion of IgM from serum and mucus (within one day) is unexpected and inconsistent with prior literature. Additional evidence, such as Western blot analyses comparing treated and control fish, is necessary to confirm this finding.

Novelty of Claims: The manuscript claims a novel role for IgM in viral neutralization, despite extensive prior literature demonstrating this role in fish. This overstatement detracts from the contribution of the study and requires a more accurate contextualization of the findings.

Support for IgM's Crucial Role: The mortality data following IgM depletion do not fully support the claim that IgM is indispensable for antiviral defense. The survival of IgM-depleted fish remains high (75%) compared to non-primed controls (~50%), suggesting that other immune components may compensate for IgM loss.

Presentation of IgM Depletion Model: The study describes the IgM depletion model as novel, although similar models have been previously published (e.g., Ding et al., 2023). This should be clarified to avoid overstating its novelty.

While the manuscript attempts to address an important question in teleost immunology, the current evidence is insufficient to fully support the authors' conclusions. Addressing the validation of the monoclonal antibody, re-evaluating depletion kinetics, and tempering claims of novelty would strengthen the study's impact. The findings, if rigorously validated, have important implications for understanding the evolution of vertebrate immunity and practical applications in fish health management.

This work is of interest to immunologists, evolutionary biologists, and aquaculture researchers. The methodological framework, once validated, could be valuable for studying immunoglobulin function in other non-model organisms and for developing targeted vaccine strategies. However, the current weaknesses limit its broader applicability and impact.

Reviewer #1 (Public review):

Summary:

PRMT1 overexpression is linked to poor survival in cancers, including acute megakaryocytic leukemia (AMKL). This manuscript describes the important role of PRMT1 in the metabolic reprograming in AMKL. In a PRMT1-driven AMKL model, only cells with high PRMT1 expression induced leukemia, which was effectively treated with the PRMT1 inhibitor MS023. PRMT1 increased glycolysis, leading to elevated glucose consumption, lactic acid accumulation, and lipid buildup while downregulating CPT1A, a key regulator of fatty acid oxidation. Treatment with 2-deoxy-glucose (2-DG) delayed leukemia progression and induced cell differentiation, while CPT1A overexpression rescued cell proliferation under glucose deprivation. Thus, PRMT1 enhances AMKL cell proliferation by promoting glycolysis and suppressing fatty acid oxidation.

Strengths:

This study highlights the clinical relevance of PRMT1 overexpression with AMKL, identifying it as a promising therapeutic target. A key novel finding is the discovery that only AMKL cells with high PRMT1 expression drive leukemogenesis, and this PRMT1-driven leukemia can be effectively treated with the PRMT1 inhibitor MS023. The work provides significant metabolic insights, showing that PRMT1 enhances glycolysis, suppresses fatty acid oxidation, downregulates CPT1A, and promotes lipid accumulation, which collectively drive leukemia cell proliferation. The successful use of the glucose analogue 2-deoxy-glucose (2-DG) to delay AMKL progression and induce cell differentiation underscores the therapeutic potential of targeting PRMT1-related metabolic pathways. Furthermore, the rescue experiment with ectopic Cpt1a expression strengthens the mechanistic link between PRMT1 and metabolic reprogramming. The study employs robust methodologies, including Seahorse analysis, metabolomics, FACS analysis, and in vivo transplantation models, providing comprehensive and well-supported findings. Overall, this work not only deepens our understanding of PRMT1's role in leukemia progression but also opens new avenues for targeting metabolic pathways in cancer therapy.

Weaknesses:

This study, while significant, has some limitations.

(1) The findings rely heavily on a single AMKL cell line, with no validation in patient-derived samples to confirm clinical relevance or even another type of leukemia line. Adding the discussion of PRMT1's role in other leukemia types will increase the impact of this work.

(2) The observed heterogeneity in Prmt1 expression is noted but not further investigated, leaving gaps in understanding its broader implications.

(3) Some figures and figure legends didn't include important details or had not matching information. For example,<br /> • Figure 2D, E, F, I (wrong label with D), p-value was not shown. Panel I figure legend is missing.<br /> • Figure 6E, F, p value was not shown.<br /> • Line 272-278, figures should be Figures 7 D-F.

(4) Some wording is not accurate, such as line 80 "the elevated level of PRMT1 maintains the leukemic stem cells", the study is using the cell line, not leukemia stem cells.

(5) In the disease model, histopathology of blood, spleen, and BM should be shown.

(6) Can MS023 treatment reverse the metabolic changes in PRMT1 overexpression AMKL cells?

(7) It would be helpful if a summary graph is provided at the end of the manuscript.

for book - Pedagogies of Collapse - Ginie Servant-Miklos - Chapter 1 - Collapse, Climate and Capitalism - 2024

Reviewer #1 (Public review):

Summary:

The authors used high-density probe recordings in the medial prefrontal cortex (PFC) and hippocampus during a rodent spatial memory task to examine functional sub-populations of PFC neurons that are modulated vs. unmodulated by hippocampal sharp-wave ripples (SWRs), an important physiological biomarker that is thought to have a role in mediating information transfer across hippocampal-cortical networks for memory processes. SWRs are associated with the reactivation of representations of previous experiences, and associated reactivation in hippocampal and cortical regions has been proposed to have a role in memory formation, retrieval, planning, and memory-guided behavior. This study focuses on awake SWRs that are prevalent during immobility periods during pauses in behavior. Previous studies have reported strong modulation of a subset of prefrontal neurons during hippocampal SWRs, with some studies reporting prefrontal reactivation during SWRs that have a role in spatial memory processes. The study seeks to extend these findings by examining the activity of SWR-modulated vs. unmodulated neurons across PFC sub-regions, and whether there is a functional distinction between these two kinds of neuronal populations with respect to representing spatial information and supporting memory-guided decision-making.

Strengths:

The major strength of the study is the use of Neuropixels 1.0 probes to monitor activity throughout the dorsal-ventral extent of the rodent medial prefrontal cortex, permitting an investigation of functional distinction in neuronal populations across PFC sub-regions. They are able to show that SWR-unmodulated neurons, in addition to having stronger spatial tuning than SWR-modulated neurons as previously reported, also show stronger directional selectivity and theta-cycle skipping properties.

Weaknesses:

(1) While the study is able to extend previous findings that SWR-modulated PFC neurons have significantly lower spatial tuning that SWR-unmodulated neurons, the evidence presented does not support the main conclusion of the paper that only the unmodulated neurons are involved in spatial tuning and signaling upcoming choice, implying that SWR-modulated neurons are not involved in predicting upcoming choice, as stated in the abstract. This conclusion makes a categorical distinction between two neuronal populations, that SWR-modulated neurons are involved and SWR-unmodulated are not involved in predicting upcoming choice, which requires evidence that clearly shows this absolute distinction. However, in the analyses showing non-local population decoding in PFC for predicting upcoming choice, the results show that SWR-unmodulated neurons have higher firing rates than SWR-modulated neurons, which is not a categorical distinction. Higher firing rates do not imply that only SWR-unmodulated neurons are contributing to the non-local decoding. They may contribute more than SWR-modulated neurons, but there are no follow-up analyses to assess the contribution of the two sub-populations to non-local decoding.

(2) Further, the results show that during non-local representations of the hippocampus of the upcoming options, SWR-excited PFC neurons were more active during hippocampal representations of the upcoming choice, and SWR-inhibited PFC neurons were less active during hippocampal representations of the alternative choice. This clearly suggests that SWR-modulated neurons are involved in signaling upcoming choice, at least during hippocampal non-local representations, which contradicts the main conclusion of the paper.

(3) Similarly, one of the analyses shows that PFC nonlocal representations show no preference for hippocampal SWRs or hippocampal theta phase. However, the examples shown for non-local representations clearly show that these decodes occur prior to the start of the trajectory, or during running on the central zone or start arm. The time period of immobility prior to the start arm running will have a higher prevalence of SWRs and that during running will have a higher prevalence of theta oscillations and theta sequences, so non-local decoded representations have to sub-divided according to these known local-field potential phenomena for this analysis, which is not followed.

(4) The primary phenomenon that the manuscript relies on is the modulation of PFC neurons by hippocampal SWRs, so it is necessary to perform the PFC population decoding analyses during SWRs (or examine non-local decoding that occurs specifically during SWRs), as reported in previous studies of PFC reactivation during SWRs, to see if there is any distinction between modulated and unmodulated neurons in this reactivation. Even in the case of independent PFC reactivation as reported by one study, this PFC reactivation was still reported to occur during hippocampal SWRs, therefore decoding during SWRs has to be examined. Similarly, the phenomenon of theta cycle skipping is related to theta sequence representations, so decoding during PFC and hippocampal theta sequences has to be examined before coming to any conclusions.

Reviewer #1 (Public review):

This study presents evidence that a special group of place cells, those tuned to fast-gamma oscillations, play a key role in theta sequence development. How theta sequences are formed and developed during experience is an important question, because these sequences have been implicated in several cognitive functions of place cells, including memory-guided spatial navigation. The revised version of this paper has been significantly improved. Major concerns in the previous round of review on technical and conceptual aspects of the relationship between gamma oscillations and theta sequences are addressed. The main conclusion is supported by the data presented.

Reviewer #1 (Public review):

Summary:

The manuscript by Hall et al reports a genome-wide map of supercoiling in yeast using psoralen as a probe that intercalates more effectively into underwound DNA and can then be fixed in place by UV-cross-linking. Sites of cross-linking are revealed by exonuclease digestion and sequencing. Cross-linking is compared with samples that are first fixed with formaldehyde, permeabilized, digested with Dpn II to release unrestrained torsion, and then crosslinked. The authors promote this "zero-torsion" approach as an improvement that corrects for nucleosomes (or binding by other macromolecules) that mask psoralen binding. The investigators then examine patterns of psoralen binding (and hence supercoiling) that are associated with promoter strength, promoter type (sequence-specific transcription factor dependent, insulator associated, or general TFs only) and gene length.

Strengths:

This is an interesting paper that reports an approach that reveals some new information about the relationship between torsional stress and gene activity in the yeast genome. The method is logical and interesting and provides evidence that spread of torsional stress through the genome is regulated.

Weaknesses:

The analysis is not entirely novel, and I believe that more valuable information can be culled from these datasets than is reported here.

Reviewer #1 (Public review):

Summary:

This paper concerns mechanisms of foraging behavior in C. elegans. Upon removal from food, C. elegans first executes a stereotypical local search behavior in which it explores a small area by executing many random, undirected reversals and turns called "reorientations." If the worm fails to find food, it transitions to a global search in which it explores larger areas by suppressing reorientations and executing long forward runs (Hills et al., 2004). At the population level, the reorientation rate declines gradually. Nevertheless, about 50% of individual worms appear to exhibit an abrupt transition between local and global search, which is evident as a discrete transition from high to low reorientation rate (Lopez-Cruz et al., 2019). This observation has given rise to the hypothesis that local and global search correspond to separate internal states with the possibility of sudden transitions between them (Calhoun et al., 2014). The main conclusion of the paper is that it is not necessary to posit distinct internal states to account for discrete transitions from high to low reorientation rates. On the contrary, discrete transitions can occur simply because of the stochastic nature of the reorientation behavior itself.

Strengths:

The strength of the paper is the demonstration that a more parsimonious model explains abrupt transitions in the reorientation rate.

Weaknesses:

(1) Use of the Gillespie algorithm is not well justified. A conventional model with a fixed dt and an exponentially decaying reorientation rate would be adequate and far easier to explain. It would also be sufficiently accurate - given the appropriate choice of dt - to support the main claims of the paper, which are merely qualitative. In some respects, the whole point of the paper - that discrete transitions are an epiphenomenon of stochastic behavior - can be made with the authors' version of the model having a constant reorientation rate (Figure 2f).

(2) In the manuscript, the Gillespie algorithm is very poorly explained, even for readers who already understand the algorithm; for those who do not it will be essentially impossible to comprehend. To take just a few examples: in Equation (1), omega is defined as reorientations instead of cumulative reorientations; it is unclear how (4) follows from (2) and (3); notation in (5), line 133, and (7) is idiosyncratic. Figure 1a does not help, partly because the notation is unexplained. For example, what do the arrows mean, what does "*" mean?

(3) In the model, the reorientation rate dΩ⁄dt declines to zero but the empirical rate clearly does not. This is a major flaw. It would have been easy to fix by adding a constant to the exponentially declining rate in (1). Perhaps fixing this obvious problem would mitigate the discrepancies between the data and the model in Figure 2d.

(4) Evidence that the model fits the data (Figure 2d) is unconvincing. I would like to have seen the proportion of runs in which the model generated one as opposed to multiple or no transitions in reorientation rate; in the real data, the proportion is 50% (Lopez). It is claimed that the "model demonstrated a continuum of switching to non-switching behavior" as seen in the experimental data but no evidence is provided.

(5) The explanation for the poor fit between the model and data (lines 166-174) is unclear. Why would externally triggered collisions cause a shift in the transition distribution?

(6) The discussion of Levy walks and the accompanying figure are off-topic and should be deleted.

Reviewer #2 (Public review):

The authors identified new target elements for prostaglandin E2 (PGE2) through which insulin release can be regulated in pancreatic beta cells under physiological conditions. In vitro extracellular exposure to PGE2 could directly and dose-dependently inhibit the potassium channel Kv2.2. In vitro pharmacology revealed that this inhibition occurs through the EP2/4 receptors, which activate protein kinase A (PKA). By screening specific sites of the Kv2.2 channel, the target phosphorylation site (S448) for PKA regulation was found. The physiological relevance of the described signaling cascade was investigated and confirmed in vivo, using a Kv2.2 knockdown mouse model.

The strength of this manuscript is the novelty of the (EP2/4-PKA-Kv2.2 channel) molecular pathway described and the comprehensive methodological toolkit the authors have relied upon.

The introduction is detailed and contains all the information necessary to place the claims in context. Although the dataset is comprehensive and a logical lead is consistently built, there is one important point to consider: to clarify that the described signaling pathway is characteristic of normal physiological conditions and thus differs from pathological changes. It would be useful to carry out basic experiments in a diabetes model (regardless of in mouse or rat even).

Comments on revisions:

The authors addressed my comments sufficiently. I have no additional questions to clarify.

Reviewer #1 (Public review):

O'Neill et al. have developed a software analysis application, miniML, that enables the quantification of electrophysiological events. They utilize a supervised deep learned-based method to optimize the software. miniML is able to quantify and standardize the analyses of miniature events, using both voltage and current clamp electrophysiology, as well as optically driven events using iGluSnFR3, in a variety of preparations, including in the cerebellum, calyx of held, golgi cell, human iPSC cultures, zebrafish, and Drosophila. The software appears to be flexible, in that users are able to hone and adapt the software to new preparations and events. Importantly, miniML is an open source software free for researchers to use and enables users to adapt new features using Python.

Overall this new software has the potential to become widely used in the field and an asset to researchers. Importantly, a new graphical user interface has been generated that enables more user control and a more user-friendly experience. Further, the authors demonstrate how miniML performs relative to other platforms that have been developed, and highlight areas where miniML works optimally. With these revisions, miniML should now be of considerable benefit and utility to a variety of researchers.

Reviewer #1 (Public review):

Summary:

This is a new and important system that can efficiently train mice to perform a variety of cognitive tasks in a flexible manner. It is innovative and opens the door to important experiments in the neurobiology of learning and memory.

Strengths:

Strengths include: high n's, a robust system, task flexibility, comparison of manual-like training vs constant training, circadian analysis, comparison of varying cue types, long-term measurement, and machine teaching.

Weaknesses:

I find no major problems with this report.

Minor weaknesses:

(1) Line 219: Water consumption per day remained the same, but number of trails triggered was more as training continued. First, is this related to manual-type training? Also, I'm trying to understand this result quantitatively, since it seems counter-intuitive: I would assume that with more trials, more water would be consumed since accuracy should go up over training (so more water per average trial). Am I understanding this right? Can the authors give more detail or understanding to how more trials can be triggered but no more water is consumed despite training?

(2) Figure 2J: The X-axis should have some label: at least "training type". Ideally, a legend with colors can be included, although I see the colors elsewhere in the figure. If a legend cannot be added, then the color scheme should be explained in the caption.

(3) Figure 2K: What is the purple line? I encourage a legend here. The same legend could apply to 2J.

(4) Supplementary Figure S2 D: I do not think the phrase "relying on" is correct. Instead, I think "predicted by" or "correlating with" might be better.

Reviewer #1 (Public review):

Summary:

The authors aim to predict ecological suitability for the transmission of highly pathogenic avian influenza (HPAI) using ecological niche models. This class of models identify correlations between the locations of species or disease detections and the environment. These correlations are then used to predict habitat suitability (in this work, ecological suitability for disease transmission) in locations where surveillance of the species or disease has not been conducted. The authors fit separate models for HPAI detections in wild birds and farmed birds, for two strains of HPAI (H5N1 and H5Nx) and for two time periods, pre- and post-2020. The authors also validate models fitted to disease occurrence data from pre-2020 using post-2020 occurrence data.

Strengths:

The authors follow the established methods of Dhingra et al., 2016 to provide an updated spatial assessment of HPAI transmission suitability for two time periods, pre- and post-2020. They explore further methods of model cross-validation and consider the diversity of the bird species that HPAI has been detected in.

Weaknesses:

The precise ecological niche that the authors are modelling here is ambiguous: if we treat the transmission of HPAI in the wild bird population and in poultry populations as separate transmission cycles, linked by spillover events, then these transmission cycles are likely to have fundamentally different ecological niches. While an "index case" in farmed poultry is relevant to the wildlife transmission cycle, further within-farm and farm-to-farm transmission is likely to be contingent on anthropogenic factors, rather than the environment. Similarly, we would expect "index cases" in outbreaks of HPAI in mammals to be relevant to transmission risk in wild birds - this data is not included in this manuscript. Such "index cases" in farmed poultry occur under separate ecological conditions to subsequent transmission in farmed poultry, so should be separated if possible. Some careful editing of the language used in the manuscript may elucidate some of my questions related to model conceptualisation.

The authors' handling of sampling bias in disease detection data in poultry is possibly inappropriate: one would expect the true spatial distribution of disease surveillance in poultry to be more closely correlated with poultry farming density, in contrast to human population density. This shortcoming in the modelling workflow possibly dilutes a key finding of the Results, that the transmission risk of HPAI in poultry is greatest in areas where poultry farming density is high.

Reviewer #1 (Public review):

Wang et al. investigated how sexual failure influences sweet taste perception in male Drosophila. The study revealed that courtship failure leads to decreased sweet sensitivity and feeding behavior via dopaminergic signaling. Specifically, the authors identified a group of dopaminergic neurons projecting to the subesophageal zone that interacts with sweet-sensing Gr5a+ neurons. These dopaminergic neurons positively regulate the sweet sensitivity of Gr5a+ neurons via DopR1 and Dop2R receptors. Sexual failure diminishes the activity of these dopaminergic neurons, leading to reduced sweet-taste sensitivity and sugar-feeding behavior in male flies. These findings highlight the role of dopaminergic neurons in integrating reproductive experiences to modulate appetitive sensory responses.

Previous studies have explored the dopaminergic-to-Gr5a+ neuronal pathways in regulating sugar feeding under hunger conditions. Starvation has been shown to increase dopamine release from a subset of TH-GAL4 labeled neurons, known as TH-VUM, in the subesophageal zone. This enhanced dopamine release activates dopamine receptors in Gr5a+ neurons, heightening their sensitivity to sugar and promoting sucrose acceptance in flies. Since the function of the dopaminergic-to-Gr5a+ circuit motif has been well established, the primary contribution of Wang et al. is to show that mating failure in male flies can also engage this circuit to modulate sugar-feeding behavior. This contribution is valuable because it highlights the role of dopaminergic neurons in integrating diverse internal state signals to inform behavioral decisions.

An intriguing discrepancy between Wang et al. and earlier studies lies in the involvement of dopamine receptors in Gr5a+ neurons. Prior research has shown that Dop2R and DopEcR, but not DopR1, mediate starvation-induced enhancement of sugar sensitivity in Gr5a+ neurons. In contrast, Wang et al. found that DopR1 and Dop2R, but not DopEcR, are involved in the sexual failure-induced decrease in sugar sensitivity in these neurons. I wish the authors had further explored or discussed this discrepancy, as it is unclear how dopamine release selectively engages different receptors to modulate neuronal sensitivity in a context-dependent manner.

The data presented by Wang et al. are solid and effectively support their conclusions. However, certain aspects of their experimental design, data analysis, and interpretation warrant further review, as outlined below.

(1) The authors did not explicitly indicate the feeding status of the flies, but it appears they were not starved. However, the naive and satisfied flies in this study displayed high feeding and PER baselines, similar to those observed in starved flies in other studies. This raises the concern that sexually failed flies may have consumed additional food during the 4.5-hour conditioning period, potentially lowering their baseline hunger levels and subsequently reducing PER responses. This alternative explanation is worth considering, as an earlier study demonstrated that sexually deprived males consumed more alcohol, and both alcohol and food are known rewards for flies. To address this concern, the authors could remove food during the conditioning phase to rule out its influence on the results.

(2) Figure 1B reveals that approximately half of the males in the Failed group did not consume sucrose, yet Figure 1-S1A suggests that the total volume consumed remained unchanged. Were the flies that did not consume sucrose omitted from the dataset presented in Figure 1-S1A? If so, does this imply that only half of the male flies experience sexual failure, or that sexual failure affects only half of males while the others remain unaffected? The authors should clarify this point.

(3) The evidence linking TH-GAL4 labeled dopaminergic neurons to reduced sugar sensitivity in Gr5a+ neurons in sexually failed males could be further strengthened. Ideally, the authors would have activated TH-GAL4 neurons and observed whether this restored GCaMP responses in Gr5a+ neurons in sexually failed males. Instead, the authors performed a less direct experiment, shown in Figures 3-S1C and D. The manuscript does not describe the condition of the flies used in this experiment, but it appears that they were not sexually conditioned. I have two concerns with this experiment. First, no statistical analysis was provided to support the enhancement of sucrose responses following activation of TH-GAL4 neurons. Second, without performing this experiment in sexually failed males, the authors lack direct evidence to confirm that the dampened response of Gr5a+ neurons to sucrose results from decreased activity in TH-GAL4 neurons.

(4) The statistical methods used in this study are poorly described, making it unclear which method was used for each experiment. I suggest that the authors include a clear description of the statistical methods used for each experiment in the figure legends. Furthermore, as I have pointed out, there is a lack of statistical comparisons in Figures 3-S1C and D, a similar problem exists for Figures 6E and F.

(5) The experiments in Figure 5 lack specificity. The target neurons in this study are Gr5a+ neurons, which are directly involved in sugar sensing. However, the authors used the less specific Dop1R1- and Dop2R-GAL4 lines for their manipulations. Using Gr5a-GAL4 to specifically target Gr5a+ neurons would provide greater precision and ensure that the observed effects are directly attributable to the modulation of Gr5a+ neurons, rather than being influenced by potential off-target effects from other neuronal populations expressing these dopamine receptors.

(6) I found the results presented in Fig. 6F puzzling. The knockdown of Dop2R in Gr5a+ neurons would be expected to decrease sucrose responses in naive and satisfied flies, given the role of Dop2R in enhancing sweet sensitivity. However, the figure shows an apparent increase in responses across all three groups, which contradicts this expectation. The authors may want to provide an explanation for this unexpected result.

(7) In several instances in the manuscript, the authors described the effects of silencing dopamine signaling pathways or knocking down dopamine receptors in Gr5a neurons with phrases such as 'no longer exhibited reduced sweet sensitivity' (e.g., L269 and L288), 'prevent the reduction of sweet sensitivity' (e.g., L292), or 'this suppression was reversed' (e.g. L299). I found these descriptions misleading, as they suggest that sweet sensitivity in naive and satisfied groups remains normal while the reduction in failed flies is specifically prevented or reversed. However, this is not the case. The data indicate that these manipulations result in an overall decrease in sweet sensitivity across all groups, such that a further reduction in failed flies is not observed. I recommend revising these descriptions to accurately reflect the observed phenotypes and avoid any confusion regarding the effects of these manipulations.

Reviewer #1 (Public review):

The authors set out to develop a contextual fear learning (CFC) paradigm in head-fixed mice that would produce freezing as the conditioned response. Typically, lick suppression is the conditioned response in such designs, but this (1) introduces a potential confounding influence of reward learning on neural assessments of aversion learning and (2) does not easily allow comparison of head-fixed studies with extensive previous work in freely moving animals, which use freezing as the primary conditioned response.

The first part of this study is a report on the development and outcomes of 3 variations of the CFC paradigm in a virtual reality environment. The fundamental design is strong, with head-fixed mice required to run down a linear virtual track to obtain a water reward. Once trained, the water reward is no longer necessary and mice will navigate virtual reality environments. There are rigorous performance criteria to ensure that mice that make it to the experimental stage show very low levels of inactivity prior to fear conditioning. These criteria do result in only 40% of the mice making it to the experimental stage, but high rates of activity in the VR environment are crucial for detecting learning-related freezing. It is possible that further adjustments to the procedure could improve attrition rates.

Paradigm versions 1 and 2 vary the familiarity of the control context while paradigm versions 2 and 3 vary the inter-shock interval. Paradigm version 1 is the most promising, showing the greatest increase in conditioned freezing (~40%) and good discrimination between contexts (delta ~15-20%). Paradigm version 2 showed no clear evidence of learning - average freezing at recall day 1 was not different than pre-shock freezing. First-lap freezing showed a difference, but this single-lap effect is not useful for many of the neural circuit questions for which this paradigm is meant to facilitate. Also, the claim that mice extinguished first-lap freezing after 1 day is weak. Extinction is determined here by the loss of context discrimination, but this was not strong to begin with. First-lap freezing does not appear to be different between Recall Day 1 and 2, but this analysis was not done. Paradigm version 3 has some promise, but the magnitude of the context discrimination is modest (~10% difference in freezing). Thus, further optimization of the VR CFC will be needed to achieve robust learning and extinction. This could include factors not thoroughly tested in this study, including context pre-exposure timing and duration and shock intensity and frequency.

The second part of the study is a validation of the head-fixed CFC VR protocol through the demonstration that fear conditioning leads to the remapping of dorsal CA1 place fields, similar to that observed in freely moving subjects. The results support this aim and largely replicate previous findings in freely moving subjects. One difference from previous work of note is that VR CFC led to the remapping of the control environment, not just the conditioning context. The authors present several possible explanations for this lack of specificity to the shock context, further underscoring the need for further refinement of the CFC protocol before it can be widely applied. While this experiment examined place cell remapping after fear conditioning, it did not attempt to link neural activity to the learned association or freezing behavior.

In summary, this is an important study that sets the initial parameters and neuronal validation needed to establish a head-fixed CFC paradigm that produces freezing behaviors. In the discussion, the authors note the limitations of this study, suggest the next steps in refinement, and point to several future directions using this protocol to significantly advance our understanding of the neural circuits of threat-related learning and behavior.

Reviewer #1 (Public review):

Summary:

In this remarkable study, the authors use some of their recently-developed oxytocin receptor knockout voles (Oxtr1-/- KOs) to re-examine how oxytocin might influence partner preference. They show that shorter cohabitation times lead to decreased huddling time and partner preference in the KO voles, but with longer periods preference is still established, i.e., the KO animals have a slower rate of forming preference or are less sensitive to whatever cues or experiences lead to the formation of the pair bond as measured by this assay. This helps relate the authors' recent study to the rest of the literature on oxytocin and partner preference in prairie voles. To better understand what might lead to slower partner preference, they quantified changes to the durations and frequency of huddling. In separate assays, they also found that Oxtr1-/- KOs interacted more with stranger males than wild-type females. In a partner choice assay, they found that wild-type males prefer wild-type females more than Oxtr1-/- KO females. They then performed bulk RNA-Seq profiling of nucleus accumbens of both wild-type and Oxtr1-/- KO males and females, either housed with animals of the same sex or paired with a wild-type of the opposite sex. 13 differentially expressed genes were identified, mostly due to downregulation in wild-type females. These genes were also identified in a module lost in the Oxtr1-/- voles by correlated expression profiling. They also compared results of transcriptional profiling in female and male wild-type vs Oxtr1-/- voles (independently of bonding state) and found hundreds of differentially expressed genes in nucleus accumbens, mostly in females and often with some relation to neural development and/or autism. Some of the reduction in the transcript was confirmed with in-situs, as well as compared to changes in transcription in the lateral septum and paraventricular nucleus (PVN) of the hypothalamus. Finally, they find fewer oxytocin+ and AVP+ neurons in the anterior PVN.

Strengths:

This is an important study helping to reveal the effects of oxytocin receptor knockout on behavior and gene expression. The experiments are thorough and reveal a surprising number of genetic and anatomical differences, with some sexual dimorphism as well, and the authors have more carefully examined the behavioral changes after shorter and longer periods of partner preference formation.

Weaknesses:

It is surprising that given all the genetic changes identified by the authors, the behavioral phenotypes are fairly mild. The extent of gene changes also might be under-reported given the variability in the behavior and relatively low number of animals profiled.

Reviewer #1 (Public review):

Summary:

The authors attempted to identify whether a new deep-learning model could be applied to both resting and task state fMRI data to predict cognition and dopaminergic signaling. They found that resting state and moving watching conditions best predict episodic memory, but only movie watching predicts both episodic and working memory. A negative 'brain gap' (where the model trained on brain connectivity predicts worse performance than what is actually observed) was associated with less physical activity, poorer cardiovascular function, and lower D1R availability.

Strengths:

The paper should be of broad interest to the journal's readership, with implications for cognitive neuroscience, psychiatry, and psychology fields. The paper is very well-written and clear. The authors use two independent datasets to validate their findings, including two of the largest databases of dopamine receptor availability to link brain functional connectivity/activity with neurochemical signaling.

Weaknesses:

The deep learning findings represent a relatively small extension/enhancement of knowledge in a very crowded field.

It's unclear from these results how much utility the brain gaps provide above and beyond observed performance. It would be helpful to take a median split of the dataset on observed performance and plot aside the current Figure 3 results to see how the cardiovascular and physical activity measures differ based on actual performance. Could the authors perform additional analyses describing how much additional variance is explained in these measures by including brain gaps?

Some of the imaging findings require deeper analysis. For Figure 1f - Which default mode regions have high salience? DMN is a huge network with subregions having differing functions.

Along the same lines, were the striatal D1R findings regionally specific at all? It would be informative to test whether the three nuclei (Accumbens, Caudate, Putamen) and/or voxelwise models would show something above and beyond what is achieved from averaging D1R across the striatum. What about cortical D1R, which is highly abundant, strongly associated with cognitive (especially WM) performance, and has much unique variance beyond striatal D1R? https://www.science.org/doi/full/10.1126/sciadv.1501672. The PET findings are one of the unique strengths of this paper and are underexplored. It's also unclear if the measure of brain entropy should simply be averaged across all regions.

It is not clear from the text that the authors met the preconditions for mediation analysis (that is, demonstrating significant correlations between D1R and entropy, in addition to the correlation with brain gap. The authors should report this as well.

Was age controlled for in the mediation analysis? I would not consider this result valid unless that is the case.

The discussion section is long, but the authors would do better to replace some less helpful sections (e.g., the paragraph on methodological tweaks to parcellations and model alignment) with a couple of other important points, including:

(1) Discuss the 'sweet-spot' of movie watching for behavior prediction in the context of studies showing that task states 'quench' neural variability: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1007983. This may not be mutually exclusive of the discussion on dopamine and signal-to-noise ratio, but it would be helpful for the authors to discuss their potential overlap vs. unique contributions to the observed findings.

(2) The argument that dopamine signaling increases signal-to-noise ratio is based on some preclinical data as well as correlational data using fMRI with pharmacological challenges. It is less clear how PET-derived estimates of D1R and D2R availability equate to 'dopamine signaling' as it is thought of in this context. Presumably, based on these data, higher D1R or D2R availability would be related to greater levels of tonic dopaminergic signaling. However, in the case of the COBRA dataset with D2R estimates, those are based on raclopride -- which competes with endogenous dopamine for the D2 receptor. Therefore, someone with higher levels of endogenous dopamine signaling should theoretically have lower raclopride binding and lower D2R estimates. I'm not arguing that the authors' logic is flawed or that D1R and D2R are not good measures of dopamine signaling, but I'd ask the authors to dig into the literature and describe more direct potential links for how greater receptor availability might be associated with greater dopamine signaling (and hence lower entropy). Adding this to the discussion would be very valuable for PET research.

Reviewer #1 (Public review):

Summary:

This is a very well-written paper presenting interesting findings related to the recovery following the end-Permian event in continental settings, from N China. The finding is timely as the topic is actively discussed in the scientific community. The data provides additional insights into the faunal, and partly, floral global recovery following the EPE, adding to the global picture.

Strengths:

The conclusions are supported by an impressive amount of sedimentological and paleontological data (mainly trace fossils) and illustrations.

Weaknesses:

The occurrence of MISS (Microbially Induced Sedimentary Structures) could be discussed more in detail as these provide interesting information directly linked to the delayed recovery of the biota.

Reviewer #1 (Public review):

Summary:

The authors investigate the relationship between 3D chromatin architecture and innate immune gene regulation in monocytes from patients with alcohol-associated hepatitis (AH). Using Hi-C technology, they attempt to identify structural changes in the genome that correlate with altered gene expression. Their central claim is that genome restructuring contributes to the hyper-inflammatory phenotype associated with AH.

Strengths:

(1) The manuscript employs Hi-C technology, which, in principle, is a powerful approach for studying genome organization.

(2) The focus on disease-relevant genes, particularly innate immune loci, provides a contextually important angle for understanding AH.

Weaknesses:

(1) Sample Size: The study relies on an exceptionally small cohort (4 AH patients and 4 healthy controls), rendering the results statistically underpowered and highly susceptible to variability.

(2) Hi-C Resolution unpaired to RNA seq: The data are presented at a resolution of 100kb, which is insufficient to uncover meaningful chromatin interactions at the level of individual genes. This data is unpaired.

(3) Functional Validation: The manuscript lacks experiments to directly link changes in chromatin architecture with gene expression or monocyte function, leaving the claims speculative.

(4) Data Integration: The lack of Hi-C with ATAC and RNA-seq data handicaps the analysis and really makes it superficial. In short, it does not convincingly demonstrate a functional relationship.

(5) Confounding Factors: The manuscript neglects critical confounding variables such as comorbidities, medications, and lifestyle factors, which could influence chromatin structure and gene expression independently of AH.

Appraisal of the Aims and Results:

The manuscript sets out to establish a connection between chromatin architecture and AH pathology. However, the study fails to achieve its stated aims due to inadequate methods and insufficient data. The conclusions drawn from the Hi-C analyses alone are poorly supported, and the lack of functional validation undermines the credibility of the proposed mechanisms. Overall, the results do not provide compelling evidence to substantiate the authors' claims.

Impact on the Field and Utility to the Community:

The work, in its current form, is unlikely to have a meaningful impact on the field. The limited scope, methodological shortcomings, and lack of robust data significantly diminish its potential utility. Without addressing these critical gaps, the study does not offer new insights into the role of genome architecture in AH or provide useful methodologies or datasets for the community.

Additional Context:

The manuscript would benefit from a more comprehensive analysis of potential mechanisms underlying the observed changes, including the interplay between chromatin architecture and epigenetic modifications. Furthermore, longitudinal studies or therapeutic interventions could provide insights into the dynamic aspects of genome restructuring in AH. These considerations are entirely absent from the current study.

Conclusion:

The manuscript does not achieve its stated goals and does not present sufficient evidence to support its conclusions. The limitations in sample size, resolution, and experimental rigor severely hinder its contribution to the field. Addressing these fundamental flaws will be essential for the work to be considered a meaningful addition to the literature.

Reviewer #1 (Public review):

Summary:

Winkler et al. present brain activity patterns related to complex motor behaviour by combining whole-head magnetoencephalography (MEG) with subthalamic local field potential (LFP) recordings from people with Parkinson's disease. The motor task involved repetitive circular movements with stops or reversals associated with either predictable or unpredictable cues. Beta and gamma frequency oscillations are described, and the authors found complex interactions between recording sites and task conditions. For example, they observed stronger modulation of connectivity in unpredictable conditions. Moreover, STN power varied across patients during reversals, which differed from stopping movements. The authors conclude that cortex-STN beta modulation is sensitive to movement context, with potential relevance for movement redirection.

Strengths:

This study employs a unique methodology, leveraging the rare opportunity to simultaneously record both invasive and non-invasive brain activity to explore oscillatory networks.

Weaknesses:

It is difficult to interpret the role of the STN in context of reversals, because no consistent activity pattern emerged.

Comments on revisions: The authors have adequately addressed my comments.

Reviewer #1 (Public review):

Summary:

This study is an important follow-up to their prior work - Wong et al. (2019), starting with clear questions and hypotheses, followed by a series of thoughtful and organized experiments. The method and results are convincing. Experiment 1 demonstrated the sensory preconditioned fear with few (8) or many (32) sound-light pairings. Experiments 2A and 2B showed the role of PRh NMDA receptors during conditioning for online integration, revealing that this contribution is present only after few sound-light pairings, not after many sound-light pairings. Experiments 3A and 3B showed the contribution of PRh-BLA communication to online integration, again only after few but not after many. Contrary to Experiments 3A and 3B, Experiments 4A and 4B showed the contribution of PRh-BLA communication to integration at test only after many but not few sound-light pairings.

Strengths:

Throughout the manuscript, the methods and results are clearly organized and described, and the use of statistics is solid, all contributing to the overall clarity of the research. The discussion section was also well written, effectively comparing the current research with the prior work and offering insightful interpretations and potential future directions for this line of research.

All my previous concerns have been well addressed in this revised version. I do not have further concerns about the current version of this manuscript.

Joint Public Review:

Summary:

The behavioral switch between foraging and mating is important for resource allocation in insects. This study characterizes the role of sulfakinin and the sulfakinin receptor 1 in changes in olfactory responses associated with foraging versus mating behavior in the oriental fruit fly (Bactrocera dorsalis), a significant agricultural pest. This pathway regulates food consumption and mating receptivity in other species; here the authors use genetic disruption of sulfakinin and sulfakinin receptor 1 to provide strong evidence that changes in sulfakinin signaling modulate antennal responses to food versus pheromonal cues and alter the expression of ORs that detect relevant stimuli.

Strengths:

The authors utilize multiple complementary approaches including CRISPR/Cas9 mutagenesis, behavioral characterization, electroantennograms, RNA sequencing and heterologous expression to convincingly demonstrate the involvement of the sulfakinin pathway in the switch between foraging and mating behaviors. The use of both sulfakinin peptide and receptor mutants is a strength of the study and implicates specific signaling actors.

Weaknesses:

The authors demonstrate that SKR is expressed in olfactory neurons, however there are additional potential sites of action that may contribute to these results.

Reviewer #1 (Public review):

Summary:

This manuscript by Ishii et al utilize a classical, but extremely understudied, female self-paced assay to directly address aspects of female sxual motivation independent from the male's behavior. This allowed for clear separation of appetitive and consummatory events, of which whole brain unbiased activity was mapped. Mating completion in females was then focused to the medial preoptic nucleus where the authors performed a rigorous set of single-cell GCaMP recordings in populations marked by Vglut2 and Vgat, finding the latter display stronger and prolonged activity after the onset of mating completion. Finally, they demonstrate function to these Fos-TRAPPED completion cells demonstrating their capacity to suppress female sexual behavior.

Strengths:

This manuscript sought to explicitly explore female mating drive as dictated by the female, a very rare angle for those studying mating behavior which almost always is controlled by the male's behavior. To achieve this, the authors went back to old literature and modified a classical paradigm in which measurable approach and avoidance of male conspecifics can be measured in female mice using a self-paced mating assay. Strengths include a detailed quantification of female behaviors demonstrating a robust attenuated sexual motivation in females after mating completion. To determine the neural basis behind this, a brain wide analysis of cells responding to mating completion in the female brain was conducted which revealed numerous anatomical regions displaying increased Fos activity, including the MPOA, of which the authors concentrated the remaining of their study. Employing microendoscopic imaging, the authors discovered that this mating completion signal was strongly represented in the MPOA. The single cell data analyses are of very high quality as is the number of individual cells resolved. While they identified both excitatory and inhibitory cell types that were activated by mating completion, they found the latter exhibited stronger and more persistent activity. Segmentation into individual mating behaviors reinforced the importance of GABAergic completion cells, which display prolonged activity late after the onset of mating completion. This information provides a potential mechanism for how female mice suppress further mating activity following completion. The authors then definitively demonstrate this function by TRAP'ping completion cells with chemogenetic actuators and show that CNO-induced activation of these cells specifically and strongly suppresses female sexul behavior. All experiments were extremely well-designed and performed carefully and expertly with the necessary controls solidifying the conclusions.

Weaknesses:

While there are no glaring weaknesses in this study, it should be noted that a great deal of literature has pinpointed the MPOA (and specifically inhibitory cells in this area) as being critical to sexual behavior, including female mating. However, no study to my knowledge has explored self-paced female mating with such fine control over manipulating and monitoring cellular activity in this region. In addition, this study may act to inspire others to further explore the additional brain regions found to show upregulation of neural activity (Fos) during mating completion in the female using the data sets generated here.

Comments on revisions: The data has been provided in a public database.

Reviewer #3 (Public review):

Human and simian immunodeficiency viruses (HIV and SIV, respectively) evolved numerous mechanisms to compromise effective immune responses but the underlying mechanisms remain incompletely understood. Here, Yamamoto and Matano examined the humoral immune response in a large number of rhesus macaques infected with the difficult-to-neutralize SIVmac239 strain and identified a subgroup of animals showing significant neutralizing Ab responses. Sequence analyses revealed that in most of these animals (7/9) but only a minority in the control group (2/19) SIVmac variants containing a CD8+ T-cell escape mutation of G63E/R in the viral Nef gene emerged. Functional analyses revealed that this change attenuates the ability of Nef to stimulate PI3K/Akt/mTORC2 signalling. The authors propose that this improved induction of SIVmac239 nAb is reciprocal to antibody dysregulation caused by a previously identified human PI3K gain-of-function mutation associated with impaired anti-viral B-cell responses. Altogether, the results suggest that PI3K signalling plays a role in B-cell maturation and generation of effective nAb responses. Preliminary data indicate that Nef might be transferred from infected T cells to B cells by direct contact. However, the exact mechanism and the relevance for vaccine development requires further studies

The strengths of the study are that the authors analyzed a large number of SIVmac-infected macaques to unravel the biological significance of the known effect of the interaction of Nef with PI3K/Akt/mTORC2 signaling. This is interesting and may provide a novel means to improve humoral immune responses to HIV. In the revised version the authors made an effort to address previous concerns. Especially, they provide data supporting that Nef might be transferred to B cells by direct cell-cell contact. In addition, they provide some evidence that G63R that also emerged in most animals does not share the disruptive effect of G63G although experimental examination and discussion why G63R might emerge remains poor. A weakness that remains is that some effects of the G63E mutation are modest and effects were not compared to SIVmac constructs lacking Nef entirely. The evidence for a role of Nef G63E mutation on PI3K and the association with improved nAb responses is convincing and it is appreciated that the authors provide additional evidence for a potential impact of "soluble" Nef on neighboring B cells. The presentation of the experimental set-up and the results has been improved but is in part still challenging to comprehend. It seems that direct cell-cell contact is required and membranes are exchanged. Since Nef is associated with cellular membranes this might lead to some transfer of Nef to B cells. However, the immunological and functional consequences of this largely remain to be determined. Alternatively, Nef-mediated manipulation of helper CD4 T cells might also impact B cell function and effective humoral immune responses. Additional editing of the manuscript has been performed to make the results accessible to a broad readership.

Reviewer #1 (Public review):

Summary:

Wang et al. generate XAP5 and XAP5L knockout mice and find that they are male infertile due to spermatogonial/meiotic arrest and reduced sperm motility, respectively. CUT & Tag data were added in this revision in order to support that XAP5 and XAP5L are antagonistic transcription factors of cilliogenesis.

Strengths:

Knockout mouse models provided strong evidence to indicate that XAP5 and XAP5L are critical for spermatogenesis. RNA-seq and CUT & Tag are valuable sources to further explore their molecular mechanisms.

Weaknesses:

The authors claim that XAP5 and XAP5L transcriptionally regulate sperm flagella development; however, expression, physiological role, and molecular evidence do not well support this concept. This reviewer still thinks the physiological roles of XAP5 and XAP5l are different. (i) XAP5 is expressed at spermatogonia within testes while XAP5l is localized at round/elongating spermatids (their expressions are different). (ii) Spermatogenesis was arrested at spermatogonia/early spermatocyte stage in Xap5-KO mice while sperm abnormalities were observed in Xap5l-KO mice (their roles are different). This reviewer still can't get the authors' viewpoint that XAP5 and XAP5l are 'antagonistic relationship' to regulate sperm flagella development. RNA-seq and CUT & Tag data are valuable source; however, this reviewer suggests exploring how XAP5 regulates spermatogonia differentiation and how XAP5l regulates sperm flagella motility.

Reviewer #1 (Public review):

Summary:

In this manuscript, Corso-Diaz et al, focus on the NRL transcription factor (TF), which is critical for retinal rod photoreceptor development and function. The authors profile NRL's protein interactome, revealing several RNA-binding proteins (RBPs) among its components. Notably, many of these RBPs are associated with R-loop biology, including DHX9 helicase, which is the primary focus of this study. R-loops are three-stranded nucleic acid structures that frequently form during transcription. The authors demonstrate that R-loop levels increase during photoreceptor maturation and establish an interaction between NRL TF and DHX9 helicase. The association between NRL and RBPs like DHX9 suggests a cooperative regulation of gene expression in a cell-type-specific manner, an intriguing discovery relevant to photoreceptor health. Since DHX9 is a key regulator of R-loop homeostasis, the study proposes a potential mechanism where a cell-type-specific TF controls the expression of certain genes by modulating R-loop homeostasis. The authors also identify another R-loop resolvase, DDX5 as having weaker interaction with NRL, perhaps due to indirect mechanism.

This is a very interesting study providing genome-wide mapping of R-loops in mammalian retina, which shows an enrichment of R-loops over intergenic regions as well as genes encoding neuronal function factors. The R-loop-enriched genes are longer than genes without R-loops, which supports previous findings from studies in neuronal cells. This is a very relevant study highlighting the possible mechanism of gene expression regulation via interactions between TFs, RNA binding proteins, and R-loops. In that regard, it would be very interesting to uncover the biological relevance of such gene regulation. The authors provide adequate evidence of interaction between R-loops and NRL TF via in vitro IP assay and genomic co-localization, however, this interaction can be mediated via multiple R-loop or RNA-binding proteins. Thus, follow-up studies would be appropriate to characterize this interaction in more detail.

Reviewer #1 (Public review):

The work by Kim et al. shows that a parameter generator for biophysical HH-like models can be trained through a GAN-based approach, to reproduce experimentally measured voltage responses and IV curves.<br /> A particularly interesting aspects of this generator is that, once it has been learned, it can be applied to new recordings to generate appropriate parameter sets at a low computational cost, a feature missing from more commonplace evolutionary approaches.

I appreciate the changes the authors have made to the manuscript. The authors have clarified their inverse gradient method. They also provide a better validation and a rich set of ablations. However, I still have major concerns that should be addressed.

Major concerns:

(1) The bad equilibria of the model still remain a concern, as well as other features like the transient overshoots that do not match with the data. I think they could achieve more accuracy here by assigning more weight to such specific features, through adding these as separate objectives for the generator explicitly. The traces contain a five-second current steps, and one second before and one second after the training step. This means that in the RMSE, the current step amplitude will dominate as a feature, as this is simply the state for which the data trace contains most time-points. Note that this is further exacerbated by using the IV curve as an auxiliary objective. I believe a better exploration of specific response features, incorporated as independently weighted loss terms for the generator, could improve the fit. E.g. an auxiliary term could be the equilibrium before and after the current step, another term could penalise response traces that do not converge back to their initial equilibrium, etc.

(2) The explanation of what the authors mean with 'inverse gradient operation' is clear now. However, this term is mathematically imprecise, as the inverse gradient does not exist because the gradient operator is not injective. The method is simply forward integration under the assumption that the derivate of the voltage is known at the grid time-points, and should be described as such.

(3) I appreciate that the authors' method provides parameters of models at a minimal computational cost compared to running an evolutionary optimization for every new recording. I also believe that with some tweaking of the objective, the method could improve in accuracy. However, I share reviewer 2's concerns that the evolutionary baseline methods are not sufficiently explored, as these methods have been used to successfully fit considerably more complex response patterns. One way out of the dilemma is to show that the EP-GAN estimated parameters provide an initial guess that considerably narrows the search space for the evolutionary algorithm. In this context, the authors should also discuss the recent gradient based methods such as Deistler et al. (https://doi.org/10.1101/2024.08.21.608979) or Jones et al (https://doi.org/10.48550/arXiv.2407.04025).

Reviewer #1 (Public review):

Koesters and colleagues investigated the role of the small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cortical cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed no significant changes in GluA2 puncta size, intensity, and integral after TTX treatment in control and Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which neuronal Rab3A is required for homeostatic scaling of synaptic transmission, potentially through GluA2-independent mechanisms.

The major finding - impaired homeostatic up-scaling after TTX treatment in Rab3A KO and Rab3 earlybird mutant neurons - is supported by data of high quality. However, the paper falls short of providing any evidence or direction regarding potential mechanisms. The data on GluA2 modulation after TTX incubation are likely statistically underpowered, and do not allow drawing solid conclusions, such as GluA2-independent mechanisms of up-scaling.

The study should be of interest to the field because it implicates a presynaptic molecule in homeostatic scaling, which is generally thought to involve postsynaptic neurotransmitter receptor modulation. However, it remains unclear how Rab3A participates in homeostatic plasticity.

Major (remaining) point:

(1) Direct quantitative comparison between electrophysiology and GluA2 imaging data is complicated by many factors, such as different signal-to-noise ratios. Hence, comparing the variability of the increase in mini amplitude vs. GluA2 fluorescence area is not valid. Thus, I recommend removing the sentence "We found that the increase in postsynaptic AMPAR levels was more variable than that of mEPSC amplitudes, suggesting other factors may contribute to the homeostatic increase in synaptic strength." from the abstract.<br /> Similarly, the data do not directly support the conclusion of GluA2-independent mechanisms of homeostatic scaling. Statements like "We conclude that these data support the idea that there is another contributor to the TTX- induced increase in quantal size." should be thus revised or removed.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the full-length BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

Strengths:

The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

Weaknesses:

The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice.

Reviewer #1 (Public review):

Summary:

A central function of glial cells is the ensheathment of axons. Wrapping of larger-diameter axons involves myelin-forming glial classes (such as oligodendrocytes), whereas smaller axons are covered by non-myelin-forming glial processes (such as olfactory ensheathing glia). While we have some insights into the underlying molecular mechanisms orchestrating myelination, our understanding of the signaling pathways at work in non-myelinating glia remains limited. As non-myelinating glial ensheathment of axons is highly conserved in both vertebrates and invertebrates, the nervous system of Drosophila melanogaster, and in particular the larval peripheral nerves, have emerged as a powerful model to elucidate the regulation of axon ensheathment by a class of glia called wrapping glia. Using this model, this study seeks to specifically address the question, as to which molecular mechanisms contribute to the regulation of the extent of glial ensheathment focusing on the interaction of wrapping glia with axons.

Strengths and Weaknesses:

For this purpose, the study combines state-of-the-art genetic approaches with high-resolution imaging, including classic electron microscopy. The genetic methods involve RNAi-mediated knockdown, acute Crispr-Cas9 knock-outs, and genetic epistasis approaches to manipulate gene function with the help of cell-type specific drivers. The successful use of acute Crispr-Cas9 mediated knockout tools (which required the generation of new genetic reagents for this study) will be of general interest to the Drosophila community.

The authors set out to identify new molecular determinants mediating the extent of axon wrapping in the peripheral nerves of third-instar wandering Drosophila larvae. They could show that over-expressing a constitutive-active version of the Fibroblast growth factor receptor Heartless (Htl) causes an increase in wrapping glial branching, leading to the formation of swellings in nerves close to the cell body (named bulges). To identify new determinants involved in axon wrapping acting downstream of Htl, the authors next conducted an impressive large-scale genetic interaction screen (which has become rare, but remains a very powerful approach), and identified Uninflatable (Uif) in this way. Uif is a large single-pass transmembrane protein that contains a whole series of extracellular domains, including Epidermal growth factor-like domains. Linking this protein to glial branch formation is novel, as it has so far been mostly studied in the context of tracheal maturation and growth. Intriguingly, a knock-down or knock-out of uif reduces branch complexity and also suppresses htl over-expression defects. Importantly, uif over-expression causes the formation of excessive membrane stacks. Together these observations are in in line with the notion that htl may act upstream of uif.

Further epistasis experiments using this model implicated also the Notch signaling pathway as a crucial regulator of glial wrapping: reduction in Notch signaling reduces wrapping, whereas over-activation of the pathway increases axonal wrapping (but does not cause the formation of bulges). Importantly, defects caused by the over-expression of uif can be suppressed by activated Notch signaling. Knock-down experiments in neurons suggest further that neither Delta nor Serrate act as neuronal ligands to activate Notch signaling in wrapping glia, whereas knock-down of Contactin, a GPI anchored Immunoglobulin domain-containing protein led to reduced axon wrapping by glia, and thus could act as an activating ligand in this context.

Based on these results the authors put forward a model proposing that Uif normally suppresses Notch signaling, and that activation of Notch by Contactin leads to suppression of Htl, to trigger the ensheathment of axons. While these are intriguing propositions, future experiments would need to conclusively address whether and how Uif could "stabilize" a specific membrane domain capable of interacting with specific axons. Moreover, to obtain evidence for Uif suppression by Notch to inhibit "precocious" axon wrapping and for a "gradual increase" of Notch signaling that silences uif and htl, (1) reporters for N and Htl signaling in larvae, (2) monitoring of different stages at a time point when branch extension begins, and (3) a reagent enabling to visualize Uif expression could be important next tools/approaches. Considering the qualitatively different phenotypes of reduced branching, compared to excessive membrane stacks close to cell bodies, it would perhaps be worthwhile to explore more deeply how membrane formation in wrapping glia is orchestrated at the subcellular level by Uif.

Finally, in light of the importance of correct ensheathment of axons by glia for neuronal function, this study will be of general interest to the glial biology community.

Reviewer #2 (Public review):

Summary:

Previously, the authors developed a zebrafish model for cerebral cavernous malformations (CCMs) via CRISPR/Cas9-based mosaic inactivation of the ccm2 gene. This model yields CCM-like lesions in the caudal venous plexus of 2 days post-fertilization embryos and classical CNS cavernomas in 8-week fish that depend, like the mouse model, on the upregulation of the KLF2 transcription factor. Remarkably, the morpholino-based knockdown of the gene encoding the Beta1 adrenergic receptor or B1AR (adrb1; a hemodynamic regulator) in fish and treatment with the anti-adrenergic S enantiomer of propranolol in both fish and mice reduce the frequency and size of CMM lesions.

In the present study, the authors aim to test the model that adrb1 is required for CCM lesion development using adrb1 mutant fish (rather than morpholino-mediated knockdown and pharmacological treatments with the anti-adrenergic S enantiomer of propranolol or a racemic mix of metoprolol (a selective B1AR antagonist).

Strengths:

The goal of the work is important, and the findings are potentially highly relevant to cardiovascular medicine.

Comments on latest version:

This reviewer is largely satisfied and congratulates the authors on their updated work. However, the comments regarding the caveats of morpholino use and lack of validation that the morphants phenocopy the mutants using the readouts that they employ still stand (for instance, the tnnt2a MO has been extensively validated for phenocopying lack of cardiac contractility, not for the phenotypes under study). Finally, while using the cytosolic red line to mask a nuclear green readout is suboptimal (not for FRET reasons), this is now a minor issue given that all comparisons are made using this method and the increase in sample size.

Reviewer #1 (Public review):

Summary:

This impressive study presents a comprehensive scRNAseq atlas of the cranial region during neural induction, patterning, and morphogenesis. The authors collected a robust scRNAseq dataset covering six distinct developmental stages. The analysis focused on the neural tissue, resulting in a highly detailed temporal map of neural plate development. The findings demonstrate how different cell fates are organized in specific spatial patterns along the anterior-posterior and medial-lateral axes within the developing neural tissue. Additionally, the research utilized high-density single-cell RNA sequencing (scRNAseq) to reveal intricate spatial and temporal patterns independent of traditional spatial techniques.

The investigation utilized diffusion component analysis to spatially order cells based on their positioning along the anterior-posterior axis, corresponding to the forebrain, midbrain, hindbrain, and medial-lateral axis. By cross-referencing with MGI expression data, the identification of cell types was validated, affirming the expression patterns of numerous known genes and implicating others as differentially expressed along these axes. These findings significantly advance our understanding of the spatially regulated genes in neural tissues during early developmental stages. The emphasis on transcription factors, cell surface, and secreted proteins provides valuable insights into the intricate gene regulatory networks underpinning neural tissue patterning. Analysis of a second scRNAseq dataset where Shh signaling was inhibited by culturing embryos in SAG identified known and previously unknown transcripts regulated by Shh, including the Wnt pathway.

The data includes the neural plate and captures all major cell types in the head, including the mesoderm, endoderm, non-neural ectoderm, neural crest, notochord, and blood. With further analyses, this high-quality data promises to significantly advance our understanding of how these tissues develop in conjunction with the neural tissue, paving the way for future breakthroughs in developmental biology and genomics.

Strengths:

The data is well presented in the figures and thoroughly described in the text. The quality of the scRNAseq data and bioinformatic analysis is exceptional.

Weaknesses:

None

Reviewer #2 (Public review):

Summary:

This paper derives the first three functional gradients in the left and right hippocampus across two datasets. These gradient maps are then compared to dopamine receptor maps obtained with PET, associated with age, and linked to memory. Results reveal links between dopamine maps and gradient 2, age with gradients 1 and 2, and memory performance.

Strengths:

This paper investigates how hippocampal gradients relate to aging, memory, and dopamine receptors, which are interesting and important questions. A strength of the paper is that some of the findings were replicated in a separate sample.

Assessment after revision:

The authors addressed concerns about unclear multiple comparison correction in the revision. The replication sample was primarily used to replicate the topographic organization of functional hippocampal-neocortical connectivity within the hippocampus across the adult lifespan, which was the central goal of this paper. Not all other analyses replicated, which the authors nicely clarified in the revised manuscript. Overall, this work is a thorough and valuable contribution to the literature.

Reviewer #1 (Public review):

Based on the reviewers' comments, the authors conducted additional analyses to enhance their study. By utilizing publicly available datasets (Guo et al., 2017) capable of distinguishing the sex of embryos, they examined DNA methylation in male embryos and identified minor de novo DNA methylation events initiating at the 8-cell stage, predominantly on the X chromosome. However, this finding introduces confusion, as the authors had previously suggested that such minor de novo DNA methylation regulates imprinted X chromosome inactivation, a process specific to female embryos.

The key unresolved issue is whether minor de novo DNA methylation in female embryos occurs exclusively on the "inactive" X chromosome or on both the active and inactive X chromosomes. The authors did not provide direct evidence supporting de novo DNA methylation specifically at the inactive X chromosome. Furthermore, it remains unclear whether this methylation influences embryonic development independent of sex or is specific to female embryos undergoing imprinted X chromosome inactivation. While the authors present data on decreased live birth rates in Figure 2F, they did not address whether there is a sex bias among the live pups, such as male-biased survival. Clarifying this point would strengthen their conclusions.<br /> In summary, the critical issue with the revised manuscript is that it does not adequately resolve whether minor de novo DNA methylation regulates embryonic development irrespective of sex or specifically impacts female embryos where imprinted X chromosome inactivation occurs. This distinction is essential for understanding the broader implications of their findings.

Joint Public Review:

Summary:

This interesting study applies the PSMC model to a set of new genome sequences for migratory and nonmigratory thrushes and seeks to describe differences in the population size history among these groups. The authors create a set of summary statistics describing the PSMC traces - mean and standard deviation of Ne, plus a set of metrics describing the shape of the oldest Ne peak - and use these to compare across migratory and resident species (taking single samples sequenced here as representative of the species). The analyses are framed as supporting or refuting aspects of a biogeographic model describing colonization dynamics from tropical to temperate North and South America.

Strengths:

* This is a creative use of PSMC to test explicit a priori hypotheses about season migration and Ne. The PSMC analyses seem well done and the authors acknowledge much of the complexity of interpretation in the discussion.

* We appreciate the test-of-hypothesis design of the study and the explicit formulation of three main expectations to test. The data analysis has been done with appropriate available tools.

Key weaknesses from the original round of review:

* Short of developing some novel theory deep in the PSMC model, I think readers would need to see simulations showing that the analyses employed in this paper are capable of supporting or refuting their biogeographic hypothesis before viewing them as strongly supporting a specific biogeographic model. Tools like msprime and stdpopsim can be used to simulate genome-scale data with fairly complex biogeographic models. Running simulations of a thrush-like population under different biogeographic scenarios and then using PSMC to differentiate those patterns would be a more convincing argument for the biogeographic aspects of this paper. The other benefit of this approach would be to nail down a specific quantitative version of the taxon cycles model referenced in the abstract, and it would allow the authors to better study and explain the motivation behind the specific summary statistics they develop for PSMC posthoc analysis.

* The authors hypothesized that the wider realized breeding and ecological range characterising migrants versus resident lineages could be a major drive for increased effective population size and population expansion in migrants versus residents. I understand that this pattern (wider range in migrants) is a common characteristic across bird lineages and that it is viewed as a result of adapting to migration. A problem that I see in their dataset is that the breeding grounds range of the two groups are located in very different geographic areas (mainly South versus North America). The authors could have expanded their dataset to include species whose breeding grounds are from the two areas, regardless of their migratory behaviour, as a comparison to disentangle whether ecological differences of these two areas can affect the population sizes or growth rates.

* As I understand from previous literature, the time-scale to population growth and estimates of effective population sizes considered in the present paper for the resident versus migratory clades seem to widely predate the times to speciation for the same lineages, which were reported in previous work of the same authors (Everson et al 2019) and others (Termignoni-Garcia et al 2022). This piece of information makes the calculation of species-specific population size changes difficult to interpret in the light of lineages' comparison. It is unclear what the authors consider to be lineage-specific in these estimates, as the clades were likely undergoing substantial admixture during the time predating full isolation.

* Regarding the methodological difficulties in interpreting the impact of population structure on the estimates of effective population sizes with the PSMC approach, I would think that performing simulations to compare different scenarios of different degrees of structured populations would have helped substantially understand some of the outcomes.

* The authors use an average generation time for all taxa, but the citations imply generation time is known for at least some of them. Are there differences in generation time associated with migration? I am not a bird biologist, but quick googling suggests maybe this is the case? (https://doi.org/10.1111/1365-2656.13983). I think it important the authors address this, as differences in generation time I believe should affect estimates of Ne and growth.

[Editors' note: the original reviews in full are here: https://elifesciences.org/reviewed-preprints/90848/reviews. The reviewers were not available to comment on the latest version of the submission.]

Reviewer #1 (Public review):

Summary:

Even though this is not the first report that the mutation in the DNAH12 gene causes asthenoteratozoospermia, the current study explores the sperm phenotype in-depth. The authors show experimentally that the said mutation disrupts the proper axonemal arrangement and recruitment of DNALI1 and DNAH1 - proteins of inner dynein arms. Based on these results, the authors propose a functional model of DNAH12 in proper axonemal development. Lastly, the authors demonstrate that the male infertility caused by the studies mutation can be rescued by ICSI treatment at least in the mouse. This study furthers our understanding of male infertility caused by a mutation of axonemal protein DNAH12, and how this type of infertility can be overcome using assisted reproductive therapy.

Strengths:

This is an in-depth functional study, employing multiple, complementary methodologies to support the proposed working model.

Weaknesses:

The structure and interaction model between DNAH12, DNALI1, and DNAH1 relies on in silico methodologies, and further studies are required to validate these predictions.

Reviewer #1 (Public review):

Summary:

The paper demonstrated through a comprehensive multi-omics study of the oviduct that the transcriptomic and proteomic landscape of the oviduct at 4 different preimplantation periods was dynamic during natural fertilization, pseudopregnancy, and superovulation using three independent cell/tissue isolation and analytical techniques. This work is very important for understanding oviductal biology and physiology. In addition, the authors have made all the results available in a web search format, which will maximize the public's access and foster and accelerate research in the field.

Strengths:

(1) The manuscript addresses an important and interesting question in the field of reproduction: how does the oviduct at different regions adapt to the sperm and embryos for facilitating fertilization and preimplantation embryo development and transport?<br /> (2) Authors used cutting-edge techniques: Integrated multi-modal datasets followed with in vivo confirmation and machine learning prediction.<br /> (3) RNA-seq, scRNA-seq and proteomic results are immediately available to the scientific community in a web search format<br /> (4) Substantiated results indicate the source of inflammatory responses was the secretory cell population in the IU region when compared to other cell types; sperm modulate inflammatory responses in the oviduct; the oviduct displays immuno-dynamism.

In addition, the revised version has addressed weaknesses adequately.<br /> (1) The revised version provided a clear explanation and the rationale for using the superovulation model.<br /> (2) The revised version generated a graphic abstract/summary of their major findings.

Reviewer #1 (Public review):

Summary:

In this study, the authors showed that enalapril was able to reduce cellular senescence and improve health status in aged mice. The authors further showed that phosphorylated Smad1/5/9 was significantly elevated and blocking this pathway attenuated the protection of cells from senescence. When middle-aged mice were treated with enalapril, the physiological performance in several tissues, including memory capacity, renal function, and muscle strength, exhibited significant improvement.

Strengths:

The strength of the study lies in the identification of the pSMAD1/5/9 pathway as the underlying mechanism mediating the anti-senescence effects of enalapril with comprehensive evaluation both in vitro and in vivo.

Weaknesses:

The major weakness of the study is the in vivo data. Despite the evidence shown in the in vitro study, there is no data to show that blocking the pSmad1/5/9 pathway is able to attenuate the anti-aging effects of enalapril in the mice. In addition, the aging phenotypes mitigation by enalapril is not evidenced by the extension of lifespan. If it is necessary to show that NAC is able to attenuate enalapril effects in the aging mice. In addition, it would be beneficial to test if enalapril is able to achieve similar rescue in a premature aging mouse model.

Reviewer #1 (Public review):

The manuscript by Lucie Oriol et al. revisits the understanding of interneurons in the ventral tegmental area (VTA). The study challenges the traditional notion that VTA interneurons exclusively form local synapses within the VTA. Key findings of the study indicate that VTA GABA and glutamate projection neurons also make local synapses within the VTA. This evidence suggests that functions previously attributed to VTA interneurons could be mediated by these projection neurons.

The study tested four genetic markers-Parvalbumin (PV), Somatostatin (SST), Mu-opioid receptor (MOR), and Neurotensin (NTS)-to determine if they selectively label VTA interneurons. The findings indicate that these markers label VTA projection neurons rather than selectively identifying interneurons. Using a combination of anatomical tracing and brain slice physiological recordings, the study demonstrates that VTA projection neurons make functional inhibitory or excitatory synapses locally within the VTA. These data challenge the conventional view that VTA GABA neurons are purely interneurons and suggests that inhibitory projection neurons can serve functions previously attributed to VTA interneurons. Thus, some functions traditionally ascribed to interneurons may be carried out by projection neurons with local synapses. This has significant implications for understanding the neural circuits underlying reward, motivation, and addiction.

Joint Public Review:

Summary:

This study used a simulation approach with a large-scale compilation of published meta-analytic data sets to address the generalizability of meta-analyses. The authors used prediction interval/distribution as a central tool to evaluate whether future meta-analysis is likely to generate a non-zero effect.

Strengths:

Although the concept of prediction intervals is commonly taught in statistics courses, its application in meta-analysis remains relatively rare. The authors' creative use of this concept, combined with the decomposition of heterogeneity, provides a new perspective for meta-analysts to evaluate the generalizability of their findings. As such, I consider this to be a timely and practically valuable development.

Weaknesses:

First, in their re-analysis of the compiled meta-analytic data to assess generalizability, the authors used a hierarchical model with only the intercept as a fixed effect. In practice, many meta-analyses include moderators in their models. Ignoring these moderators could result in attributing heterogeneity to unexplained variation at the study or paper level, depending on whether the moderators vary across studies or papers. As a consequence, the prediction interval may be inaccurately wide or narrow, leading to an erroneous assessment of the generalizability of results derived from large meta-analytic data sets. A more accurate approach would be to include the same moderators as in the original meta-analyses and generate prediction intervals that reflect the effects of these moderators.

Second, the authors used a t-distribution to generate the prediction intervals and distributions for the hierarchical meta-analysis model. While the t-distribution is exact for prediction intervals in linear models, it is not strictly appropriate for models with random effects. This discrepancy arises because the variances of random effects must be estimated from the data, and using a t-distribution for prediction intervals does not account for the uncertainty in estimating these variance components. Unless the data is perfectly balanced (i.e., all random effects are nested and sample sizes within each level of the random factor are equal), it is well established that t-distribution (or equivalently, F-distribution) based hypothesis testing and confidence/prediction intervals are typically anti-conservative. As recommended in the linear mixed models literature, bootstrapping methods or some form of degrees-of-freedom correction would be more appropriate for generating prediction intervals in this context.

Finally, the authors define generalizability as the likelihood that a future study will yield a significantly non-zero effect. While this is certainly useful information, it is not necessarily the primary concern for many meta-analyses or individual studies. In fact, many studies aim to understand the mean response or effect within a specific context, rather than focusing on whether a future study will produce a significant result. For many research questions, the concern is not whether a future study will generate a significant finding, but whether the true mean response is different from zero. In this regard, the authors may have overstated the importance of knowing the outcome of a single future study, and framing this as the sole goal of research seems somewhat misguided.

Reviewer #1 (Public review):

Summary:

The manuscript by Senn and colleagues presents a comprehensive study on the developing synthetic gene circuits targeting mutant RAS-expressing cells. This study aims to exploit these RAS-targeting circuits as cancer cell classifiers, enabling the selective expression of an output protein in correlation with RAS activity. The system is based on the bacterial two-component system NarX/NarL. A RAS-binding domain, the RBDCRD domain of the RAS effector protein CRAF, is fused to the histidine kinase domain, which carries an inactivating amino acid exchange either in its ATP-binding site (N509A) or in its phosphorylation site (H399Q). Dimerization or nanocluster formation of RAS-GTP reconstitutes an active histidine kinase sensor dimer that phosphorylates the response regulator NarL. The phosphorylated DNA-binding protein NarL, fused to the transcription activator domain VP48, binds its responsive element and induces the expression of the output protein. In comparison to mutated RAS, the effect of the RAS activator SOS-1 and the RAS inhibitor NF1 on the sensing ability as well as the tunability of the RAS sensor were examined. A RAS targeting circuit with an AND gate was designed by expressing the RAS sensor proteins under the control of defined MAPK response elements, resulting in a large increase in the dynamic range between mutant and wild-type RAS. Finally, the RAS targeting circuits were evaluated in detail in a set of twelve cancer cell lines expressing endogenous levels of mutant or wild-type RAS or oncogenes affecting RAS signaling upstream or downstream.

Strengths:

This proof-of-concept study convincingly demonstrates the potential of synthetic gene circuits to target oncogenic RAS in tumor cell lines and to function, at least in part, as an RAS mutant cell classifier.

Weaknesses:

The use of an appropriate "therapeutic gene" might revert the oncogenic properties of RAS mutant cell lines. However, a therapeutic strategy based on this four-plasmid-based system might be difficult to implement in RAS-driven solid cancers.

Reviewer #1 (Public review):

The manuscript presents a novel nonlinear mathematical model that addresses a critical gap in our understanding of how cell shape transitions in response to ECM stiffness. The focus on the interplay between actomyosin contractility and ECM stiffening is highly relevant, especially in the context of cancer invasion and tissue morphogenesis. The originality of the proposed trizonal model is commendable, as it offers a comprehensive framework that could significantly advance the field.

More specifically, the paper makes a significant contribution by providing a model that can predict multimodal cell shapes based on motility levels, which is a substantial improvement over current constitutive models. The potential to calibrate the model against experimental cell shape data is a strong point, as it ensures that the model's predictions are grounded in empirical evidence. The methodology appears to be rigorous and should provide reliable results when applied. This advancement could lead to a better understanding of the complex dynamics involved in cell-matrix interactions, particularly at intermediate ranges of collagen density. The potential applications of this research are vast and span across various medical and biological fields. The ability to predict cancer-induced tissue impairment, cachexia, and muscle injury, as well as to assess therapeutic methods, is particularly noteworthy. The mention of specific treatments like Blebbistatin and HAPLN1 treatments further adds depth to the discussion and highlights the practical relevance of the model.

I'm curious if the authors could further elaborate on the use of this model to examine cellular unjamming transition or the cell shape changes during cancer invasion in various scenarios. Some discussions on that aspect will be helpful. It will also be useful to provide some perspectives on how this model could be integrated with others in a multi-scale modeling framework for understanding cell shape transitions during collective cell migration in various physiologically relevant scenarios.

I recommend some minor revisions but overall, this is a very nice paper.

Reviewer #1 (Public review):

Summary:

This manuscript by Kremer et al. characterizes the tissue-specific responses to changes in TFAM levels and mtDNA copy number in prematurely aging mice (polg mutator model). The authors find that overexpression of TFAM can have beneficial or detrimental effects depending on the tissue type. For instance, increased TFAM levels increase mtDNA copy number in the spleen and improve spleen homeostasis but do not elevate mtDNA copy number in the liver and impair mtDNA expression. Similarly, the consequences of reduced TFAM expression are tissue-specific. Reduced TFAM levels improve brown adipocyte tissue function while other tissues are unaffected. The authors conclude that these tissue-specific responses to altered TFAM levels demonstrate that there are tissue-specific endogenous compensatory mechanisms in response to the continuous mutagenesis produced in the prematurely aging mice model, including upregulation of TFAM expression, elevated mtDNA copy number, and altered mtDNA gene expression. Thus, the impact of genetically manipulating global TFAM expression is limited and there must be other determinants of mtDNA copy number under pathological conditions beyond TFAM.

Strengths:

Overall, this is an interesting study. It does a good job of demonstrating that given the multi-functional role of TFAM, the outcome of manipulating its activity is complex.

Weaknesses:

No major weaknesses were noted. We have minor suggestions for improving the clarity of the manuscript that are detailed in the "recommendations for the authors" section.

Reviewer #1 (Public review):

Summary:

In this manuscript submitted by Qi et al., the authors study the RNA methylation mechanism by the METTL3-METTL14 complex. This complex catalyzes the major epitranscriptome methylation mark of nuclear RNA, including mRNA and lncRNAs. They catalyze the transfer of methyl group from SAM to convert the N6 of adenosine in RNA to m6A. Mutations in this complex have been associated with several diseases, such as type 2 diabetes and several types of cancer. The primary focus of this study was to understand the post-catalytic state of the METTL3-14 bound to a structural mimic of a reaction product known as N6-methyladenosine monophosphate (m6A) using X-ray crystallography. The authors show that the m6A occupies a novel pocket at the interface of the METTL3-14 complex and identified that residues interacting with m6A are mutated in several cancers. Furthermore, the authors demonstrate that the mutations lead to a significant loss in catalytic activity, alter RNA binding, and hinder the proper positioning of the substrate adenine in the active site. Lastly, the authors perform supervised molecular dynamics simulations to understand the effect of the mutations on the interaction network with m6A. The evidence for this study is good, with the combination of X-ray, functional assays, and molecular dynamics justifying their overall conclusions. This structure is significant as it provides new insights into the structural determinants of known cancer-associated mutations of this important class of enzymes. However, some issues need to be addressed.

Strengths:

(1) The X-ray structure is well determined, and the density map has the quality to observe all the interactions of the METTL3-14 complex with m6A.

(2) The structure reveals a novel 'cryptic pocket' in the complex that is 16 Å away from the SAM binding site. It is a functional m6A-sensor, illustrating a mechanism where the complex switches its functionality from an m6A writer to a reader.

(3) The structure illustrates that the residues forming cryptic pockets are found in multiple Cancer-associated mutations and are well conserved across several organisms.

(4) The functional assays (methyl transferase, RNA binding, kinetic, and SPR assays) provide a complete picture of the effect of the mutations on the activity of the METTL3-14 complex.

(5) Molecular dynamics simulations were done to understand the impact of the mutations on the pocket structure and its dynamics and support the X-ray structure findings.

Weaknesses:

(1) Although the X-ray structure is well determined, the statistics are a bit troubling, particularly the Ramachandran, Sidechain and RSRZ outliers. It is well above the average for structures at that resolution. Maybe the use of alternative software such as ISOLDE may be adequate to improve those parameters.

(2) The authors should expand their discussion as to why the affinity for the product is higher than the substrate and the implications on the mechanism.

(3) The SPR profiles of the association kinetics look to have several minor association-dissociation events occurring. Multiple binding sites? Authors should provide an explanation for such behavior. Also, what is the structural explanation of the difference in binding modes between the wt vs. mutant (one vs. two-state binding modes)?

(4) In materials and methods, it shows the data in Figure 2a was fitted to a Michaelis-Menten equation, however, the Y axis shows Normalized methylation and not initial rates. The authors should elaborate on their approach. In addition, more than three initial velocity rate points per protein are needed to fit a Michaelis-Menten curve confidently. Additionally, where can the Michaelis-Menten parameters be found?

Reviewer #1 (Public review):

Summary:

The manuscript by Li and colleagues describes the impact of deficiency on the DKGα and ζ on Treg cells and follicular responses. The experimental approach is based on the characterization of double KO mice that show the emergence of autoimmune manifestations that include the production of autoantibodies. Additionally, there is an increase in Tfh cells, but also Tfr cells in these mice deficient in both DKGα and ζ. Although the observations are interesting, the interpretation of the observations is difficult in the absence of data related to single mutations. While a supplementary figure shows that the autoimmune manifestations are more severe in the DKGα and ζ deficient mice, prior observations show that a single DKGα deficiency has an impact on Treg homeostasis. As such, the contribution of the two chains to the overall phenotype is hard to establish.

Strengths:

Well-conducted experiments with informative mouse models with defined genetic defects.

Weaknesses:

The major weakness is the lack of clarity concerning what can be attributed to simultaneous DKGα and ζ deficiency versus deficiency on DKGα or ζ alone.

Some interpretations are also not conclusively supported by data.

Reviewer #1 (Public review):

Summary:

The authors present a new protocol to assess social dominance in pairs and triads of C57BL/6j mice, based on a competition to access a hidden food pellet. Using this new protocol, the authors have been able to identify stable ranking among male and female pairs, while reporting more fluctuant hierarchies among triads of males. Ranking readouts identified with this new apparatus were compared to the outcomes obtained with the same animals competing in the tube and in the warm spot tests, which have been both commonly used during the last decade to identify social ranks in rodents under laboratory conditions.

Strengths:

FPCT allows for easy and fast identification of a winner and a loser in the context of food competition. The apparatus and the protocol are relatively easy and quick to implement in the lab and free from any complex post-processing/analysis, which qualifies it for wide distribution, particularly within laboratories that do not have the resources to implement more sophisticated protocols. Hierarchical readouts identified through the FPCT correlate with social ranks identified with the tube and the warm spot tests, which have been widely adopted during the last decade and allow for study comparison.

Weaknesses:

While the FPCT is validated by the tube and the warm spot test, this paper would have gained strength by providing a more ethologically based validation. Tube and warm spot tests have been shown to provide conflicting results and might not been a sufficient measurement for social ranking (see Varholik et al, Scientific reports, 2019; Battivelli et al, Biological psychiatry, 2024). Instead, a general consensus pushing toward more ethological approaches for neuroscience studies is emerging.<br /> Other papers already successfully identified social ranks dyadic food competition, using relatively simple scoring protocol (see for example Merlot et al., 2006), within a more naturalistic set-up, allowing the 2 opponents to directly interact while competing for the food. A potential issue with the FPCT, is that the opponents being isolated from each other, the normal inhibition expected to appear in subordinates in the presence of a dominant to access food, could be diminished, and usually avoiding subordinates could be more motivated to push for the access to the food pellet.

There are issues with use of the English language throughout the text. Some sentences are difficult to understand and should be clarified and/or synthesized.

Open question:<br /> Is food restriction mandatory? Palatable food pellet is not sufficient to trigger competition? Food restriction has numerous behavioral and physiological consequences that would be better to prevent to be able to clearly interpret behavioral outcomes in FPCT (see for example Tucci et al., 2006).

Conclusive remarks:<br /> Although this protocol attempts to provide a novel approach to evaluate social ranks in mice, it is not clear how it really brings a significant advance in neuroscience research. The FPCT dynamic is very similar to the one observed in the tube test, where mice compete to navigate forward in a narrow space, constraining the opponent to go backward. The main difference between the FPCT and the tube test is the presence of food between the opponents. In the tube test, a food reward was initially used to increase motivation to cross the tube and push the opponent upon the testing day. This component has been progressively abandoned, precisely because it was not necessary for the mice to compete in the tube.

This paper would really bring a significant contribution to the field by providing a neuronal imaging or manipulation correlate to the behavioral outcome obtained by the application of the FPCT.

Reviewer #1 (Public review):

Summary:

The authors of the study are trying to show that RNAseq can be used for neoantigen prediction and the machine learning approach to the prediction can reveal very useful information for the selection of neoantigens for personalized antitumor vaccination.

Strengths:

The authors demonstrated that RNA expression of a neoantigen is very important factor in the selection of peptides for the creation of personalized vaccines. They proved in vivo that in silico-predicted neoantigens can trigger antitumor response in mice.

Weaknesses:

The authors replied to my previous comment about the selection of the peptides for vaccination in the responses to reviewers, but didn't include that in the revised manuscript. I think all that information should be in the manuscript.<br /> Here is the original comment: "The selection of the peptides for vaccination is not clear. Some peptides were selected before and some after processing. What processing is also not clear. The authors didn't provide the full list of peptides before and after processing, please add those. And it wasn't clear that these peptides were previously published. Looking at the previously published table with peptide from B16 F10 (https://www.nature.com/articles/s41598-021-89927-5/tables/3), there are other genes with high expression, e.g. Tab2, Tm9sf3 that have higher expression than Herc6, please clarify the choice."

Reviewer #1 (Public review):

Hearing and balance rely on specialized ribbon synapses that transmit sensory stimuli between hair cells and afferent neurons. Synaptic adhesion molecules that form and regulate transsynaptic interactions between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) are crucial for maintaining auditory synaptic integrity and, consequently, for auditory signaling. Synaptic adhesion molecules such as neurexin-3 and neuroligin-1 and -3 have recently been shown to play vital roles in establishing and maintaining these synaptic connections ( doi: 10.1242/dev.202723 and DOI: 10.1016/j.isci.2022.104803). However, the full set of molecules required for synapse assembly remains unclear.

Karagulan et al. highlight the critical role of the synaptic adhesion molecule RTN4RL2 in the development and function of auditory afferent synapses between IHCs and SGNs, particularly regarding how RTN4RL2 may influence synaptic integrity and receptor localization. Their study shows that deletion of RTN4RL2 in mice leads to enlarged presynaptic ribbons and smaller postsynaptic densities (PSDs) in SGNs, indicating that RTN4RL2 is vital for synaptic structure. Additionally, the presence of "orphan" PSDs-those not directly associated with IHCs-in RTN4RL2 knockout mice suggests a developmental defect in which some SGN neurites fail to form appropriate synaptic contacts, highlighting potential issues in synaptic pruning or guidance. The study also observed a depolarized shift in the activation of CaV1.3 calcium channels in IHCs, indicating altered presynaptic functionality that may lead to impaired neurotransmitter release. Furthermore, postsynaptic SGNs exhibited a deficiency in GluA2/3 AMPA receptor subunits, despite normal Gria2 mRNA levels, pointing to a disruption in receptor localization that could compromise synaptic transmission. Auditory brainstem responses showed increased sound thresholds in RTN4RL2 knockout mice, indicating impaired hearing related to these synaptic dysfunctions.

The findings reported here significantly enhance our understanding of synaptic organization in the auditory system, particularly concerning the molecular mechanisms underlying IHC-SGN connectivity. The implications are far-reaching, as they not only inform auditory neuroscience but also provide insights into potential therapeutic targets for hearing loss related to synaptic dysfunction.