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):

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):

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.

Reviewer #2 (Public review):

Summary:

The authors present the results of molecular phylogenetic analysis with very comprehensive samplings including 471 specimens belonging to 250 species, trying to give a holistic reconstruction of the evolutionary history of freshwater fishes (Nemacheilidae) across Eurasia since the early Eocene.

Strengths:

They provide very vast data and conduct comprehensive analysis. They suggested that Nemacheilidae contain 6 major clades, and the earliest differentiation can be dated to early Eocene.

Weaknesses:

They did not discuss the systematic problems widely existing, did not use the conventional way to discuss the evolutionary process of branches or clades, but just chronically describe the overall history.

Comments on revisions:

As the authors are aware that there are some taxonomic problems, which can not be solved at present. And they have mentioned this in the revised manuscript. I can not provide other suggestions at the moment.

Reviewer #1 (Public review):

Summary:

This study shows that the pro-inflammatory S1P signaling regulates the responses of muller glial cells to damage. The authors describe the expression of S1P signaling components. Using agonist and antagonist of the pathways they also investigate their effect on the de-differentiation and proliferation of Muller glial cells in damaged retina of postnatal chicks. They show that S1PR1 is highly expressed in resting MG and non-neurogenic MGPCs. This receptor suppresses the proliferation and neuronal activity promotes MGPC cell cycle re-entry and enhanced the number of regenerated amacrine-like cells after retinal damage. The formation of MGPCs in damaged retinas is impaired in the absence of microglial cells. This study further shows that ablation of microglial cells from the retina increases the expression of S1P-related genes in MG, whereas inhibition of S1PR1 and SPHK1 partially rescues the formation of MGPCs in damaged retinas depleted of microglia. The studies also show that expression of S1P-related genes is conserved in fish and human retinas.

Strengths:

This is well-conducted study, with convincing images and statistically relevant data

Weaknesses:

In a previous study, the authors have shown that S1P is upstream of NF-κB signaling (Palazzo et al. 2020; 2022, 2023). Although S1P and NF-κB signaling have overlapping effects, the authors here provide evidence for S1P specific effects, adding some new information to the field.

Reviewer #1 (Public review):

Summary:

The manuscript by Anbarcia et al. re-evaluates the function of the enigmatic Rete Ovarii (RO), a structure that forms in close association with the mammalian ovary. The RO has generally been considered a functionless structure in the adult ovary. This manuscript follows up on a previous study from the lab) that analyzed ovarian morphogenesis using high-resolution microscopy (McKey et al., 2022). The present study adds finer details to RO development and possible function by 1) identifying new markers for OR sub-regions (e.g. GFR1a labels the connecting rete) suggesting that the sub-regions are functionally distinct, 2) showing that the OR sub-regions are connected by a luminal system that allows transport of material from the extra-ovarian rete (EOR) to the inter-ovarian rete (IOG), 3) identifies proteins that are secreted into the OR lumen and that may regulate ovarian homeostasis, and finally, 4) better defines how the vasculature, nervous, and immune system integrates with the OR.

Strengths:

The data is beautifully present and convincing. They show that the RO is composed of three distinct domains that have unique gene expression signatures and thus likely are functionally distinct.

Reviewer #1 (Public review):

Summary:

This work by Al-Jezani et al. focused on characterizing clonally derived MSC populations from the synovium of normal and osteoarthritis (OA) patients. This included characterizing the cell surface marker expression in situ (at time of isolation), as well as after in vitro expansion. The group also tried to correlate marker expression with trilineage differential potential. They also tested the ability of the different sub-populations for their efficacy in repairing cartilage in a rat model of OA. The main finding of the study is that CD47hi MSCs may have a greater capacity to repair cartilage than CD47lo MSCs, suggesting that CD47 may be a novel marker of human MSCs that have enhanced chondrogenic potential.

Strengths:

Studies on cell characterization of the different clonal populations isolated indicate that the MSC are heterogenous and traditional cell surface markers for MSCs do not accurately predict the differentiation potential of MSCs. While this has been previously established in the field of MSC therapy, the authors did attempt to characterize clones derived from single cells, as well as evaluate the marker profile at the time of isolation. While the outcome of heterogeneity is not surprising, the methods used to isolate and characterize the cells were well developed. The interesting finding of the study is the identification of CD47 as a potential MSC marker that could be related to chondrogenic potential. The authors suggest that MSCs with high CD47 repaired cartilage more effectively than MSC with low CD47 in a rat OA model.

Weaknesses:

While the identification of CD47 as a novel MSC marker could be important to the field of cell therapy and cartilage regeneration, there was a lack of robust data to support the correlation of CD47 expression to chondrogenesis. The authors indicated that the proteomics suggested that the MSC subtype expressed significantly more CD47 than the non-MSC subtype. However, it was difficult to appreciate where this was shown. It would be helpful to clearly identify where in the figure this is shown, especially since it is the key result of the study. The authors were able to isolate CD47hi and CD47 low cells. While this is exciting, it was unclear how many cells could be isolated and whether they needed to be expanded before being used in vivo. Additional details for the CD47 studies would have strengthened the paper. Furthermore, the CD47hi cells were not thoroughly characterized in vitro, particularly for in vitro chondrogenesis. More importantly, the in vivo study where the CD47hi and CD47lo MSCs were injected into a rat model of OA lacked experimental details regarding how many cells were injected and how they were labeled. No representative histology was presented and there did not seem to be a statistically significant difference between the OARSI score of the saline injected and MSC injected groups. The repair tissue was stained for Sox9 expression, which is an important marker of chondrogenesis but does not show production of cartilage. Expression of Collagen Type II would be needed to more robustly claim that CD47 is a marker of MSCs with enhanced repair potential.

Reviewer #1 (Public review):

Summary

The authors describe a method for gastruloid formation using mouse embryonic stem cells (mESCs) to study YS and AGM-like hematopoietic differentiation. They characterise the gastruloids during nine days of differentiation using a number of techniques including flow cytometry and single-cell RNA sequencing. They compare their findings to a published data set derived from E10-11.5 mouse AGM. At d9, gastruloids were transplanted under the adrenal gland capsule of immunocompromised mice to look for the development of cells capable of engrafting the mouse bone marrow. The authors then applied the gastruloid protocol to study overexpression of Mnx1 which causes infant AML in humans.

In the introduction, the authors define their interpretation of the different waves of hematopoiesis that occur during development. 'The subsequent wave, known as definitive, produces: first, oligopotent erythro-myeloid progenitors (EMPs) in the YS (E8-E8.5); and later myelo-lymphoid progenitors (MLPs - E9.5-E10), multipotent progenitors (MPPs - E10-E11.5), and hematopoietic stem cells (HSCs - E10.5-E11.5), in the aorta-gonad-mesonephros (AGM) region of the embryo proper.' Herein they designate the yolk sac-derived wave of EMP hematopoiesis as definitive, according to convention, although paradoxically it does not develop from intra-embryonic mesoderm or give rise to HSCs.

General comments

The authors make the following claims in the paper:

(1) The development of a protocol for hemogenic gastruloids (hGx) that recapitulates YS and AGM-like waves of blood from HE.

(2) The protocol recapitulates both YS and EMP-MPP embryonic blood development 'with spatial and temporal accuracy'.

(3) The protocol generates HSC precursors capable of short-term engraftment in an adrenal niche.

(4) Overexpression of MNX1 in hGx transforms YS EMP to 'recapitulate patient transcriptional signatures'.

(5) hGx is a model to study normal and leukaemic embryonic hematopoiesis.

There are major concerns with the manuscript. The statements and claims made by the authors are not supported by the data presented, data is overinterpreted, and the conclusions cannot be justified. Furthermore, the data is presented in a way that makes it difficult for the reader to follow the narrative, causing confusion. The authors have not discussed how their hGx compares to the previously published mouse embryoid body protocols used to model early development and hematopoiesis.

Specific points

(1) It is claimed that HGxs capture cellularity and topography of developmental blood formation. The hGx protocol described in the manuscript is a modification of a previously published gastruloid protocol (Rossi et al 2022). The rationale for the protocol modifications is not fully explained or justified. There is a lack of novelty in the presented protocol as the only modifications appear to be the inclusion of Activin A and an extension of the differentiation period from 7 to 9 days of culture. No direct comparison has been made between the two versions of gastruloid differentiation to justify the changes.

The inclusion of Activin A at high concentration at the beginning of differentiation would be expected to pattern endoderm rather than mesoderm. BMP signaling is required to induce Flk1+ mesoderm, even in the presence of Wnt. FACS analysis of the hGx during differentiation is needed to demonstrate the co-expression of Flk1-GFP and lineage markers such as CD34 to indicate patterning of endothelium from Flk1+ mesoderm. The FACS plots in Figure 1 show c-Kit expression but very little VE-cadherin which suggests that CD34 is not induced. Early endoderm expresses c-Kit, CXCR4, and Epcam but not CD34 which could account for the lack of vascular structures within the hGx as shown in Figure 1E.

(2) The protocol has been incompletely characterised, and the authors have not shown how they can distinguish between either wave of Yolk Sac (YS) hematopoiesis (primitive erythroid/macrophage and erythro-myeloid EMP) or between YS and intraembryonic Aorta-Gonad-Mesonephros (AGM) hematopoiesis. No evidence of germ layer specification has been presented to confirm gastruloid formation, organisation, and functional ability to mimic early development. Furthermore, differentiation of YS primitive and YS EMP stages of development in vitro should result in the efficient generation of CD34+ endothelial and hematopoietic cells. There is no flow cytometry analysis showing the kinetics of CD34 cell generation during differentiation. Benchmarking the hGx against developing mouse YS and embryo data sets would be an important verification.

Single-cell RNA sequencing was used to compare hGx with mouse AGM. The authors incorrectly conclude that ' ..specification of endothelial and HE cells in hGx follows with time-dependent developmental progression into putative AGM-like HE..' And, '...HE-projected hGx cells.......expressed Gata2 but not Runx1, Myb, or Gfi1b..' Hemogenic endothelium is defined by the expression of Runx1 and Gfli1b is downstream of Runx1.

(3) The hGx protocol 'generates hematopoietic SC precursors capable of short-term engraftment' is not supported by the data presented. Short-term engraftment would be confirmed by flow cytometric detection of hematopoietic cells within the recipient bone marrow, spleen, thymus, and peripheral blood that expressed the BFP transgene. This analysis was not provided. PCR detection of transcripts, following an unspecified number of amplification cycles, as shown in Figure 3G (incorrectly referred to as Figure 3F in the legend) is not acceptable evidence for engraftment. Transplanted hGx formed teratoma-like structures, with hematopoietic cells present at the site of transplant only analysed histologically. Indeed, the quality of the images provided does not provide convincing validation that donor-derived hematopoietic cells were present in the grafts.

There is no justification for the authors' conclusion that '... the data suggest that 216h hGx generate AGM-like pre-HSC capable of at least short-term multilineage engraftment upon maturation...'. Indeed, this statement is in conflict with previous studies demonstrating that pre-HSCs in the dorsal aorta of the mouse embryo are immature and actually incapable of engraftment.

The statement '...low-level production of engrafting cells recapitulates their rarity in vivo, in agreement with the embryo-like qualities of the gastruloid system....' is incorrect. Firstly, no evidence has been provided to show the hGx has formed a dorsal aorta facsimile capable of generating cells with engrafting capacity. Secondly, although engrafting cells are rare in the AGM, approximately one per embryo, they are capable of robust and extensive engraftment upon transplantation.

(4) Expression MNX1 transcript and protein in hematopoietic cells in MNX1 rearranged acute myeloid leukaemia (AML) is one cause of AML in infants. In the hGX model of this disease, Mnx1 is overexpressed in the mESCs that are used to form gastruloids. Mnx1 overexpression seems to confer an overall growth advantage on the hGx and increase the serial replating capacity of the small number of hematopoietic cells that are generated. The inefficiency with which the hGx model generates hematopoietic cells makes it difficult to model this disease. The poor quality of the cytospin images prevents accurate identification of cells. The statement that the kit-expressing cells represent leukemic blast cells is not sufficiently validated to support this conclusion. What other stem cell genes are expressed? Surface kit expression also marks mast cells, frequently seen in clonogenic assays of blood cells. Flow cytometric and gene expression analyses using known markers would be required.

(5) In human infant MNX1 AML, the mutation is thought to arise at the fetal liver stage of development. There is no evidence that this developmental stage is mimicked in the hGx model.

Reviewer #1 (Public review):

While CRISPR/Cas technology has greatly facilitated the ability to perform precise genome edits in Leishmania spp., the lack of a non-homologous DNA end-joining (NHEJ) pathway in Leishmania has prevented researchers from performing large-scale Cas-based perturbation screens. With the introduction of base editing technology to the Leishmania field, the Beneke lab has begun to address this challenge (Engstler and Beneke, 2023). In this study, the authors build on their previously published protocols and develop a strategy that:

a) allows for very high editing efficiency. The cell editing frequency of 1 edit per 70 cells reported in this study represents a 400-fold improvement over the previously published protocol,<br /> b) reduces the negative effects of high sgRNA levels on parasite growth by using a weaker T7 promoter to drive sgRNA transcription.

The combination of these two improvements should open the door to exciting large-scale screens and thus be of great interest to researchers working with Leishmania and beyond.

The authors did a great job responding to our concerns and we have no doubt that the technology established here, will be very useful for the Leishmania research community and beyond.

Joint Public Reviews:

Summary:

This work used a comprehensive dataset to compare the effects of species diversity and genetic diversity on multiple ecosystem functions within each trophic level and across three trophic levels. The authors found that species diversity had negative effects on ecosystem functions, while genetic diversity had positive effects. These effects were only observed within each trophic level and not across the three trophic levels studied. Although the effects of biodiversity, especially genetic diversity across multi-trophic levels, have been shown to be important, there are still very few empirical studies on this topic due to the complex relationships and difficulties in obtaining data. This study collected an excellent dataset to address this question and improve our understanding of the effects of genetic diversity effects in aquatic ecosystems.

Strengths:

The study collected a large, good and rare observational dataset covering different facets of diversity (species vs. genetic, multi-trophic levels) and multiple ecosystem functions (biomass of focal species and overall communities, and decomposition rates). The authors used appropriate statistical analyses to provide a comprehensive analysis about how different facets of diversity affect different ecosystem functions.

Weaknesses:

The nature of this observational study makes it difficult to get compelling evidence of the causal relationships between biodiversity and ecosystem functions. As the ecosystem functions were measured at both species and community levels in natural ecosystems, particular care needs to be taken when interpreting comparisons between these ecosystem functions measured at different levels.

Reviewer #1 (Public review):

Summary:

The authors examined whether aberrantly-projecting retinal ganglion cell in albino mice innervate a separate population of thalamocortical neurons, as would be predicted for Hebbian learning rules. The authors find support for this hypothesis in CLEM reconstructions of retinal ganglion cell axons and thalamocortical neurons. In a second line of investigation, the authors ask the same question about retinal ganglion cell innervation of local inhibitory neurons of the mouse LGN. The authors conclude that these connections are less specific.

Strengths:

Good use of CLEM to test a circuit-level hypothesis

Interesting difference between TC and LIN neurons found

Weaknesses:

The authors have addressed all concerns in the last round to my satisfaction.

Reviewer #1 (Public review):

Summary:

This paper is an incremental follow-up to the authors' recent paper which showed that Purkinje cells make inhibitory synapses onto brainstem neurons in the parabrachial nucleus which project directly to the forebrain. In that precedent paper, the authors used a mouse line which expresses the presynaptic marker synaptophysin in Purkinje cells to identify Purkinje cell terminals in the brainstem and they observed labeled puncta not only in the vestibular and parabrachial nuclei, as expected, but also in neighboring dorsal brainstem nuclei, prominently the central pontine grey. The present study, motivated by the lack of thorough characterization of PC projections to brainstem, uses the same mouse line to anatomically map the density and a PC-specific channelrhodopsin mouse line to electrophysiologically assess the strength of Purkinje cell synapses in dorsal brainstem nuclei. The main findings are (1) the density of Purkinje cell synapses is highest in vestibular and parabrachial nuclei and correlates with the magnitude of evoked inhibitory synaptic currents, and (2) Purkinje cells also synapse in the central pontine grey nucleus but not in the locus coeruleus or mesencephalic nucleus.

Strengths:

The complementary use of anatomical and electrophysiological methods to survey the distribution and efficacy of Purkinje cell synapses on brainstem neurons in mouse lines that express markers and light-sensitive opsins specifically in Purkinje cells is the major strength of this study. By systematically mapping presynaptic terminals and light-evoked inhibitory postsynaptic currents in dorsal brainstem, the authors provide convincing evidence that Purkinje cells do synapse directly onto pontine central grey and nearby neurons but do not synapse onto trigeminal motor or locus coeruleus neurons. Their results also confirm previously documented heterogeneity of Purkinje cell inputs to vestibular nucleus and parabrachial neurons.

Weaknesses:

Although the study provides strong evidence that Purkinje cells do not make extensive synapses onto LC neurons, which is a helpful caveat given previous reports to the contrary, it falls short of providing the comprehensive characterization of Purkinje cell brainstem synapses which seemed to be the primary motivation of the study. The main information provided is a regional assessment of PC density and efficacy, which seems of limited utility given that we are not informed about the different sources of PC inputs, variations in the sizes of PC terminals, the subcellular location of synaptic terminals, or the anatomical and physiological heterogeneity of postsynaptic cell types. The title of this paper would be more accurate if "characterization" were replaced by "survey".

Several of the study's conclusions are quite general and have already been made for vestibular nuclei, including the suggestions in Abstract, Results, and Discussion that PCs selectively influence brainstem subregions and that PCs target cell types with specific behavioral roles.

Reviewer #1 (Public review):

Summary:

Insulin is crucial for maintaining metabolic homeostasis, and its release is regulated by various pathways, including blood glucose levels and neuromodulatory systems. The authors investigated the role of neuromodulators in regulating the dynamics of the adult Drosophila IPC population. They showed that IPCs express various receptors for monoaminergic and peptidergic neuromodulators, as well as synaptic neurotransmitters with highly heterogeneous profiles across the IPC population. Activating specific modulatory inputs, e.g. dopaminergic, octopaminergic or peptidergic (Leucokinin) using an optogenetic approach coupled with in vivo electrophysiology unveiled heterogeneous responses of individual IPCs resulting in excitatory, inhibitory or no responses. Interestingly, calcium imaging of the entire IPC population with or without simultaneous electrophysiological recording of individual cells showed highly specific and stable responses of individual IPCs suggesting their intrinsic properties are determined by the expressed receptor repertoire. Using the adult fly connectome they further corroborate the synaptic input of excitatory and inhibitory neuronal subsets of IPCs. The authors conclude that the heterogeneous modulation of individual IPC activity is more likely to allow for flexible control of insulin release to adapt to changes in metabolic demand and environmental cues.

Strengths:

This study provides a comprehensive, multi-level analysis of IPC properties utilizing single-nucleus RNA sequencing, anatomical receptor expression mapping, connectomics, electrophysiological recordings, calcium-imaging and an optogenetics-based 'intrinsic pharmacology' approach. It highlights the heterogeneous receptor profiles of IPCs, demonstrating complex and differential modulation within the IPC population. The authors convincingly showed that different neuromodulatory inputs exhibit varied effects on IPC activity and simultaneous occurrence of heterogeneous responses in IPCs with some populations exciting a subset of IPCs while inhibiting others, showcasing the intricate nature of IPC modulation and diverse roles of IPC subgroups. The temporal dynamic of IPC modulation showed that polysynaptic and neuromodulatory connections play a major role in IPC response. The authors demonstrated that certain neuromodulatory inputs, e.g. dopamine, can shift the overall IPC population activity towards either an excited or inhibited state. The study thus provides a fundamental entry point to understanding the complex influence of neuromodulatory inputs on the insulinergic system of Drosophila.

Weakness:

GPCRs are typically expressed at low levels and while the transcriptomic and reporter expression analysis by the authors was comprehensive, challenges remain to fully validate receptor expression and function. It will thus require future studies to elucidate how these modulatory inputs affect insulin release and transcriptional long-term changes using receptor-specific manipulation and readouts for insulin release. Similarly, optogenetically driven excitation of modulatory neuronal subsets limits the interpretation of the results due to the possibly confounding direct or indirect effect of fast synaptic transmission on IPC excitation/inhibition, and the broad expression of some neuromodulatory lines used in this analysis.

Despite these limitations that are beyond the scope of this study, the conclusions made by the authors are balanced and well supported by the data provided. Moreover, their detailed and thorough analysis of IPC modulation will have a significant impact on the field of metabolic regulation to understand the complex regulatory mechanism of insulin release, which can now be studied further to provide insight about metabolic homeostasis and neural control of metabolic processes.

Reviewer #1 (Public review):

Summary:

The work by Fisher et al describes the role of novel RSPO mimetics in the activation of WNT signaling and hepatocyte regeneration. However, the results of the experiments and weaknesses of the methods used do not support the conclusions of the authors that the new therapy can promote liver regeneration in alcohol-induced liver cirrhosis.

Strengths:

Similarly to its precursor, aASGR1-RSPO2-RA-IgG, SZN-043 can upregulate Wnt target genes and promote hepatocyte proliferation in the liver.

Weaknesses:

(1) The authors rely on the expression of a single gene, CYP1A1, as a readout of Wnt/ß-catenin target gene expression. A more systemic evaluation of Wnt/ß-catenin activity should be performed.

(2) The lack of the mRNA upregulation of cell cycle genes is not sufficient to draw a conclusion of the impaired regeneration in cirrhotic livers.

(3) The authors present single-dose pharmacokinetic (PK) profile of SZN-04. It is not clear how that compares to its precursor, to justify better pharmacokinetic properties.

(4) The specificity of Wnt/ß-catenin activation should be evaluated in ß-catenin KO mice to show no target gene induction in the absence of ß-catenin.

(5) The authors demonstrated that the drug promoted hepatocyte proliferation. How it affects liver functional parameters in alcohol-fed mice, hepatocyte differentiation markers, albumin production, and coagulation factor synthesis is not clear.

(6) Female mice only were used for alcohol studies; the effect on the male mice needs to be evaluated as well.

(7) Alcohol feeding did not reduce Wnt/ß-catenin target gene expression in mice suggesting that it is a bad model to study the efficacy of the SZN-043 in alcohol-induced liver cirrhosis.

(8) The authors used CCl4-induced fibrosis as a model of ALD fibrosis. However, this is not a suitable fibrosis model for ALD studies. Adding alcohol to CCl4 treatment could potentially address this issue. Alternatively, the authors should use an ALD model that produces significant fibrosis.

(9) Sex for the CCl4-treated mice is not indicated.

(10) Histology and fibrosis assessment data for alcohol-fed mice should be presented.

(11) The rationale for using 13.5-month-old aging mice for alcohol studies and immunodeficient mice only for CCl4 studies is not clear.

Reviewer #1 (Public review):

Summary:

Participants in this study completed three visits. In the first, participants received experimental thermal stimulations which were calibrated to elicit three specific pain responses (30, 50, 70) on a 0-100 visual analogue scale (VAS). Experimental pressure stimulations were also calibrated at an intensity to the same three pain intensity responses. In the subsequent two visits, participants completed another pre-calibration check (Visit 2 of 3 only). Then, prior to the exercise NALOXONE or a SALINE placebo-control was administered intravenously. Participants then completed 1 of 4 blocks of HIGH (100%) or LOW (55%) intensity cycling which was tailored according to a functional threshold power (FTP) test completed in Visit 1. After each block of cycling lasting 10 minutes, participants entered an MRI scanner and were stimulated with the same thermal and pressure stimulations that corresponded to 30, 50, and 70 pain intensity ratings from the calibration stage. Therefore, this study ultimately sought to investigate whether aerobic exercise does indeed incur a hypoalgesia effect. More specifically, researchers tested the validity of the proposed endogenous pain modulation mechanism. Further investigation into whether the intensity of exercise had an effect on pain and the neurological activation of pain-related brain centres were also explored.

Results show that in the experimental visits (Visit 2 and 3), when participants exercised at two distinct intensities as intended. Power output, heart rate, and perceived effort ratings were higher during the HIGH versus LOW-intensity cycling. In particular. HIGH intensity exercise was perceived as "hard" / ~15 on the Borg (1974, 1998) scale, whereas LOW intensity exercise was perceived as "very light" / ~9 on the same scale.

The fMRI data from Figure 1 indicates that the anterior insula, dorsal posterior insula, and middle cingulate cortex show pronounced activation as stimulation intensity and subsequent pain responses increased, thus linking these brain regions with pain intensity and corroborating what many studies have shown before.

Results also showed that participants rated a higher pain intensity in the NALOXONE condition at all three stimulation intensities compared to the SALINE condition. Therefore, the expected effect of NALOXONE in this study seemed to occur whereby opioid receptors were "blocked" and thus resulted in higher pain ratings compared to a SALINE condition where opioid receptors were "not blocked". When accounting for participant sex, NALOXONE had negligible effects at lower experimental nociceptive stimulations for females compared to males who showed a hyperalgesia effect to NALOXONE at all stimulation intensities (peak effect at 50 VAS). Females did show a hyperalgesia effect at stimulation intensities corresponding to 50 and 70 VAS pain ratings. The fMRI data showed that the periaqueductal gray (PAG) showed increased activation in the NALOXONE versus SALINE condition at higher thermal stimulation intensities. The PAG is well-linked to endogenous pain modulation.

When assessing the effects of NALOXONE and SALINE after exercise, results showed no significant differences in subsequent pain intensity ratings.

When assessing the effect of aerobic exercise intensity on subsequent pain intensity ratings, authors suggested that aerobic exercise in the form of a continuous cycling exercise tailored to an individual's FTP is not effective at eliciting an exercise-induced hypoalgesia response -irrespective of exercise intensity. This is because results showed that pain responses did not differ significantly between HIGH and LOW intensity exercise with (NALOXONE) and without (SALINE) an opioid antagonist. Therefore, authors have also questioned the mechanisms (endogenous opioids) behind this effect.

Strengths:

Altogether, the paper is a great piece of work that has provided some truly useful insight into the neurological and perceptual mechanisms associated with pain and exercise-induced hypoalgesia. The authors have gone to great lengths to delve into their research question(s) and their methodological approach is relatively sound. The study has incorporated effective pseudo-randomisation and conducted a rigorous set of statistical analyses to account for as many confounds as possible. I will particularly credit the authors on their analysis which explores the impact of sex and female participants' stage of menses on the study outcomes. It would be particularly interesting for future work to pursue some of these lines of research which investigate the differences in the endogenous opioid mechanism between sexes and the added interaction of stage of menses or training status.

There are certainly many other areas that this article contributes to the literature due to the depth of methods the research team has used. For example, the authors provide much insight into: the impact of exercise intensity on the exercise-induced hypoalgesia effect; the impact of sex on the endogenous opioid modulation mechanism; and the impact of exercise intensity on the neurological indices associated with endogenous pain modulation and pain processing. All of which, the researchers should be credited for due to the time and effort they have spent completing this study. Indeed, their in-depth analysis of many of these areas provides ample support for the claims they make in relation to these specific questions. As such, I consider their evidence concerning the fMRI data to be very convincing (and interesting).

Weaknesses:

Although the authors have their own view of their results, I do however, have a slightly different take on what the post-exercise pain ratings seem to show and its implications for judging whether an exercise-induced hypoalgesia effect is present or not. From what I have read, I cannot seem to find whether the authors have compared the post-exercise pain ratings against any data that was collected pre-exercise/at rest or as part of the calibration. Instead, I believe the authors have only compared post-exercise pain ratings against one another (i.e., HIGH versus LOW, NALOXONE versus SALINE). In doing so, I think the authors cannot fully assume that there is no exercise-induced hypoalgesia effect as there is no true control comparison (a no-exercise condition).

In more detail, Figure 6A appears to show an average of all pain ratings combined per participant (is this correct?). As participants were exposed to stimulations expected to elicit a 30, 50, or 70 VAS rating based on pre-calibration values, therefore the average rating would be expected to be around 50. What Figure 6A shows is that in the SALINE condition, average pain ratings are in fact ~10-15 units lower (~35) and then in the NALOXONE condition, average pain ratings are ~5 units lower (~45) for both exercise intensities. From this, I would surmise the following:

It appears there is an exercise-induced hypoalgesia effect as average pain ratings are ~30% lower than pre-calibrated/resting pain ratings within the SALINE condition at the same temperature of stimulation (it would also be interesting to see if this effect occurred for the pressure pain).

It appears there is evidence for the endogenous opioid mechanism as the NALOXONE condition demonstrates a minimal hypoalgesia effect after exercise. I.e., NALOXONE indeed blocked the opioid receptors, and such inhibition prevented the endogenous opioid system from taking effect.

It appears there is no effect of exercise intensity on the exercise-induced hypoalgesia effect. That is, participants can cycle at a moderate intensity (55% FTP) and incur the same hypoalgesia benefits as cycling at an intensity that demarcates the boundary between heavy and severe intensity exercise (100%FTP). This is a great finding in my mind as anyone wishing to reduce pain can do so without having to engage in exercise that is too effortful/intense and therefore aversive - great news! This likely has many applications within the field of public health.

I will very slightly caveat my summaries with the fact that a more ideal comparison here would be a control condition whereby participants did the same experimental visit but without any exercise prior to entering the MRI scanner. I consider the overall strength of the evidence to be solid, with the answer to the primary research question still a little ambiguous.

Reviewer #1 (Public review):

Summary:

Lloyd et al employ an evolutionary comparative approach to study how sleep deprivation affects DNA damage repair in Astyanax mexicanus, using the cave vs surface species evolution as a playground. The work shows, convincingly, that the cavefish population has evolved an impaired DNA damage response both following sleep deprivation or a classical paradigm of DNA damage (UV).

Strengths:

The study employs a thorough multidisciplinary approach. The experiments are well conducted and generally well presented.

Weaknesses:

Having a second experimental mean to induce DNA damage would strengthen and generalise the findings.

Overall, the study represents a very important addition to the field. The model employed underlines once more the importance of using an evolutionary approach to study sleep and provides context and caveats to statements that perhaps were taken a bit too much for granted before. At the same time, the paper manages to have an extremely constructive approach, presenting the platform as a clear useful tool to explore the molecular aspects behind sleep and cellular damage in general. The discussion is fair, highlighting the strengths and weaknesses of the work and its implications.

Comments on revisions:

I was pretty happy with the previous version of the manuscript already and the authors have made all the minor corrections I had suggested so I don't have much to add. The main "weakness", if at all, is that the story would benefit from a secondary stressor (other than UV) but I understand the authors see this more as a long term development than just an addition to this particular paper, which is fair enough.

I don't have any further recommendations. I think this model system is really important for the sleep field and offers a completely new and important perspective to its evolution and function.

Reviewer #1 (Public review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings.

Reviewer #2 (Public review):

Summary:

This study reveals that short-term social isolation increases social behavior at a reunion, and a population of hypothalamic preoptic area neurons become active after social interaction following short-term isolation (POAsocial neurons). Effectively utilizing a TRAP activity-dependent labeling method, the authors inhibit or activate the POAsocial neurons and find that these neurons are involved in controlling various social behaviors, including ultrasonic vocalization, investigation, and mounting in both male and female mice. This work suggests a complex role for the POA in regulating multiple aspects of social behavior, beyond solely controlling male sexual behaviors.

Strengths

While a few studies have shown that optogenetic activation of the POA in females promotes vocalization and mounting behavior similar to the effects observed in males, these were results of artificially stimulating POA neurons, and whether POA neurons play a role in naturally occurring female social behaviors was unknown. This paper clearly demonstrates that a population of POA neurons is necessary for naturally evoked female social vocalizations and mounting behaviors.

Weaknesses

The authors used various gain-of-function and loss-of-function methods to identify the function of POAsocial neurons. However, there were inconsistent results among the different methodologies. As the authors describe in the manuscript, these inconsistencies are potentially due to limitations of the TRAP activity-dependent labeling method; however, different approaches will be necessary to clarify these issues.

Overall, this paper is well-written and provides valuable new data on the neural circuit for female social behaviors and the potentially complex role of POA in social behavior control.

Reviewer #1 (Public review):

The authors report on a thorough investigation of the interaction of megakaryocytes (MK) with their associated ECM during maturation. They report convincing evidence to support the existence of a dense cage-like pericellular structure containing laminin γ1 and α4 and collagen IV, which interacts with integrins β1 and β3 on MK and serves to fix the perisinusoidal localization of MK and prevent their premature intravasation. As with everything in nature, the authors support a Goldilocks range of MK-ECM interactions - inability to digest the ECM via inhibition of MMPs leads to insufficient MK maturation and development of smaller MK. This important work sheds light on the role of cell-matrix interactions in MK maturation, and suggests that higher-dimensional analyses are necessary to capture the full scope of cellular biology in the context of their microenvironment.

There are several outstanding questions that this work does not address.

Major:

The authors postulate a synergistic role for Itgb1 and Itgb3 in the intravasation phenotype, because the single KOs did not replicate the phenotype of the DKO. However, this is not a correct interpretation in the opinion of this reviewer. The roles appear rather to be redundant. Synergistic roles would rather demonstrate a modest effect in the single KO with potentiation in the DKO.

Furthermore, the experiment does not explain how these integrins influence the interaction of the MK with their microenvironment. It is not surprising that attachment will be impacted by the presence or absence of integrins. However, it is unclear how activation of integrins allows the MK to become "architects for their ECM microenvironment" as the authors posit. A transcriptomic analysis of control and DKO MKs may help elucidate these effects.

Integrin DKO have a 50% reduction in platelets counts as reported previously, however laminin α4 deficiency only leads to 20% reduction in counts. This suggests a more nuanced and subtle role of the ECM in platelet growth. To this end, functional assays of the platelets in the KO and wildtype mice may provide more information.

There is insufficient information in the Methods Section to understand the BM isolation approach. Did the authors flush the bone marrow and then image residual bone, or the extruded bone marrow itself as described in PMID: 29104956?

The references in the Methods section were very frustrating. The authors reference Eckly et al 2020 (PMID: 32702204) which provides no more detail but references a previous publication (PMID: 24152908), which also offers no information and references a further paper (PMID: 22008103), which, as far as this reviewer can tell, did not describe the methodology of in situ bone marrow imaging.

Therefore, this reviewer cannot tell how the preparation was performed and, importantly, how can we be sure that the microarchitecture of the tissue did not get distorted in the process?

Reviewer #1 (Public review):

Summary:

This paper contains what could be described as a "classic" approach towards evaluating a novel taste stimuli in an animal model, including standard behavioral tests (some with nerve transections), taste nerve physiology, and immunocytochemistry of taste cells of the tongue. The stimulus being tested is ornithine, from a class of stimuli called "kokumi" (in terms of human taste); these kokumi stimuli appear to enhance other canonical tastes, increasing what are essentially hedonic attributes of other stimuli. The mechanism for ornithine detection is thought to be GPRC6A receptors expressed in taste cells. The authors showed evidence for this in an earlier paper with mice; this paper evaluates ornithine taste in a rat model, and comes to a similar conclusion, albeit with some small differences between the two rodent species.

Strengths:

The data show effects of ornithine on taste/intake in laboratory rats: In two-bottle and briefer intake tests, adding ornithine results in higher intake of most, but all not all stimuli tested. Bilateral chorda tympani (CT) nerve cuts or the addition of GPRC6A antagonists decreased or eliminated these effects. Ornithine also evoked responses by itself in the CT nerve, but mainly at higher concentrations; at lower concentrations it potentiated the response to monosodium glutamate. Finally, immunocytochemistry of taste cell expression indicated that GPRC6A was expressed predominantly in the anterior tongue, and co-localized (to a small extent) with only IP3R3, indicative of expression in a subset of type II taste receptor cells.

Weaknesses:

As the authors are aware, it is difficult to assess a complex human taste with complex attributes, such as kokumi, in an animal model. In these experiments they attempt to uncover mechanistic insights about how ornithine potentiates other stimuli by using a variety of established experimental approaches in rats. They partially succeed by finding evidence that GPRC6A may mediate effects of ornithine when it is used at lower concentrations. In the revision they have scaled back their interpretations accordingly. A supplementary experiment measuring certain aspects of the effects of ornithine added to Miso soup in human subjects is included for the express purpose of establishing that the kokumi sensation of a complex solution is enhanced by ornithine; however, they do not use any such complex solutions in the rat studies. Moreover, the sample size of the human experiment is (still) small - it really doesn't belong in the same manuscript with the rat studies.

Reviewer #1 (Public review):

The Bagnat and Rawls groups' previous published work (Park et al., 2019) described the kinetics and genetic basis of protein absorption in a specialized cell population of young vertebrates termed lysosome-rich enterocytes (LREs). In this study they seek to understand how the presence and composition of the microbiota impacts the protein absorption function of these cells and reciprocally, how diet and intestinal protein absorption function impact the microbiome.

Strengths of the study include the functional assays for protein absorption performed in live larval zebrafish, which provides detailed kinetics on protein uptake and degradation with anatomic precision, and the gnotobiotic manipulations. The authors clearly show that the presence of the microbiota or of certain individual bacterial members slows the uptake and degradation of multiple different tester fluorescent proteins.

To understand the mechanistic basis for these differences, the authors also provide detailed single-cell transcriptomic analyses of cells isolated based on both an intestinal epithelial cell identity (based on a transgenic marker) and their protein uptake activity. The data generated from these analyses, presented in Figures 3-5, are valuable for expanding knowledge about zebrafish intestinal epithelial cell identities, but of more limited interest to a broader readership. Some of the descriptive analysis in this section is circular because the authors define subsets of LREs (termed anterior and posterior) based on their fabp2 expression levels, but then go on to note transcriptional differences between these cells (for example in fabp2) that are a consequence of this initial subsetting.

Inspired by their single-cell profiling and by previous characterization of the genes required for protein uptake and degradation in the LREs, the authors use quantitative hybridization chain reaction RNA-fluorescent in situ hybridization to examine transcript levels of several of these genes along the length of the LRE intestinal region of germ-free versus mono-associated larvae. They provide good evidence for reduced transcript levels of these genes that correlate with the reduced protein uptake in the mono-associated larval groups.

The final part of the study (shown in Figure 7) characterized the microbiomes of 30-day-old zebrafish reared from 6-30 days on defined diets of low and high protein and with or without homozygous loss of the cubn gene required for protein uptake. The analysis of these microbiomes notes some significant differences between fish genotypes by diet treatments, but the discussion of these data does not provide strong support for the hypothesis that "LRE activity has reciprocal effects on the gut microbiome". The most striking feature of the MDS plot of Bray Curtis distance between zebrafish samples shown in Figure 7B is the separation by diet independent of host genotype, which is not discussed in the associated text. Additionally, the high protein diet microbiomes have a greater spread than those of the low protein treatment groups, with the high protein diet cubn mutant samples being the most dispersed. This pattern is consistent with the intestinal microbiota under a high protein diet regimen and in the absence of protein absorption machinery being most perturbed in stochastic ways than in hosts competent for protein uptake, consistent with greater beta dispersal associated with more dysbiotic microbiomes (described as the Anna Karenina principle here: https://pubmed.ncbi.nlm.nih.gov/28836573/). It would be useful for the authors to provide statistics on the beta dispersal of each treatment group.

Overall, this study provides strong evidence that specific members of the microbiota differentially impact gene expression and cellular activities of enterocyte protein uptake and degradation, findings that have a significant impact on the field of gastrointestinal physiology. The work refines our understanding of intestinal cell types that contribute to protein uptake and their respective transcriptomes. The work also provides some evidence that microbiomes are modulated by enterocyte protein uptake capacity in a diet-dependent manner. These latter findings provide valuable datasets for future related studies.

Reviewer #1 (Public review):

IKK is the key signaling node for inflammatory signaling. Despite the availability of molecular structures, how the kinase achieves its specificity remains unclear. This paper describes a dynamic sequence of events in which autophosphorylation of a tyrosine near the activate site facilitates phosphorylation of the serine on the substrate via a phosphor-transfer reaction. The proposed mechanism is conceptually novel in several ways, suggesting that the kinase is dual specificity (tyrosine and serine) and that it mediates a phospho-transfer reaction. While bacteria contain phosphorylation-transfer enzymes, this is unheard of for mammalian kinases. However, what the functional significance of this enzymatic activity might remain unaddressed.

The revised manuscript adequately addresses all the points I suggested in the review of the first submission.

Reviewer #1 (Public review):

Summary:

In this work, Harpring et al. investigated divisome assembly in Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that lacks FtsZ, the canonical master regulator of bacterial cell division. They find that divisome assembly is initiated by the protein FtsK in Ct by showing that it forms discrete foci at the septum and future division sites. Additionally, knocking down ftsK prevents divisome assembly and inhibits cell division, further supporting their hypothesis that FtsK regulates divisome assembly. Finally, they show that MreB is one of the last chlamydial divisome proteins to arrive at the site of division and is necessary for the formation of septal peptidoglycan rings but does not act as a scaffold for division assembly as previously proposed.

Strengths:

The authors use microscopy to clearly show that FtsK forms foci both at the septum as well as at the base of the progenitor cell where the next septum will form. They also show that the Ct proteins PBP2, PBP3, MreC, and MreB localize to these same sites suggesting they are involved in the divisome complex.

Using CRISPRi the authors knockdown ftsK and find that most cells are no longer able to divide and that PBP2 and PBP3 no longer localized to sites of division suggesting that FtsK is responsible for initiating divisome assembly. They also performed a knockdown of pbp2 using the same approach and found that this also mostly inhibited cell division. Additionally, FtsK was still able to localize in this strain however PBP3 did not suggest that FtsK acts upstream of PBP2 in the divisome assembly process while PBP2 is responsible for the localization of PBP3.

The authors also find that performing a knockdown of ftsK also prevents new PG synthesis further supporting the idea that FtsK regulates divisome assembly. They also find that inhibiting MreB filament formation using A22 results in diffuse PG, suggesting that MreB filament formation is necessary for proper PG synthesis to drive cell division.

Overall the authors propose a new hypothesis for divisome assembly in an organism that lacks FtsZ and use a combination of microscopy and genetics to support their model that is rigorous and convincing. The finding that FtsK, rather than a cytoskeletal or "scaffolding" protein is the first division protein to localize to the incipient division site is unexpected and opens up a host of questions about its regulation. The findings will progress our understanding of how cell division is accomplished in bacteria with non-canonical cell wall structure and/or that lack FtsZ.

Weaknesses:

No major weaknesses were noted in the data supporting the main conclusions.

Reviewer #1 (Public review):

The Bagnat and Rawls groups' previous published work (Park et al., 2019) described the kinetics and genetic basis of protein absorption in a specialized cell population of young vertebrates termed lysosome rich enterocytes (LREs). In this study they seek to understand how the presence and composition of the microbiota impacts the protein absorption function of these cells and reciprocally, how diet and intestinal protein absorption function impact the microbiome.

Strengths of the study include the functional assays for protein absorption performed in live larval zebrafish, which provides detailed kinetics on protein uptake and degradation with anatomic precision, and the gnotobiotic manipulations. The authors clearly show that the presence of the microbiota or of certain individual bacterial members slows the uptake and degradation of multiple different tester fluorescent proteins.

To understand the mechanistic basis for these differences, the authors also provide detailed single-cell transcriptomic analyses of cells isolated based on both an intestinal epithelial cell identity (based on a transgenic marker) and their protein uptake activity. The data generated from these analyses, presented in Figures 3-5, are valuable for expanding knowledge about zebrafish intestinal epithelial cell identities, but of more limited interest to a broader readership. Some of the descriptive analysis in this section is circular because the authors define subsets of LREs (termed anterior and posterior) based on their fabp6 expression levels, but then go on to note transcriptional differences between these cells (for example in fabp6) that are a consequence of this initial subsetting.

Inspired by their single-cell profiling and by previous characterization of the genes required for protein uptake and degradation in the LREs, the authors use quantitative hybridization chain reaction RNA-fluorescent in situ hybridization to examine transcript levels of several of these genes along the length of the LRE intestinal region of germ-free versus mono-associated larvae. They provide good evidence for reduced transcript levels of these genes that correlate with the reduced protein uptake in the mono-associated larval groups.

The final part of the study (shown in Figure 7) characterized the microbiomes of 30-day-old zebrafish reared from 6-30 days on defined diets of low and high protein and with or without homozygous loss of the cubn gene required for protein uptake. The analysis of these microbiomes notes some significant differences between fish genotypes by diet treatments, but the discussion of these data does not provide strong support for the hypothesis that "LRE activity has reciprocal effects on the gut microbiome". The most striking feature of the MDS plot of Bray Curtis distance between zebrafish samples shown in Figure 7B is the separation by diet independent of host genotype, which is not discussed in the associated text. Additionally, the high protein diet microbiomes have a greater spread than those of the low protein treatment groups, with the high protein diet cubn mutant samples being the most dispersed. This pattern is consistent with the intestinal microbiota under a high protein diet regimen and in the absence of protein absorption machinery being most perturbed in stochastic ways than in hosts competent for protein uptake, consistent with greater beta dispersal associated with more dysbiotic microbiomes (described as the Anna Karenina principle here: https://pubmed.ncbi.nlm.nih.gov/28836573/). It would be useful for the authors to provide statistics on the beta dispersal of each treatment group.

Overall, this study provides strong evidence that specific members of the microbiota differentially impact gene expression and cellular activities of enterocyte protein uptake and degradation, findings that have a significant impact on the field of gastrointestinal physiology. The work refines our understanding of intestinal cell types that contribute to protein uptake and their respective transcriptomes. The work also provides some evidence that microbiomes are modulated by enterocyte protein uptake capacity in a diet-dependent manner. These latter findings provide valuable datasets for future related studies.

Comments on revisions:

I suggest that the authors clarify the level of protein in the standard fish food and how this relates to the protein levels in the high protein and low protein diets used in their microbiome study.

Reviewer #1 (Public review):

Summary:

In this study, Basha and colleagues aim to test whether the thalamic nucleus reuniens can facilitate the hippocampus/prefrontal cortex coupling during sleep. Considering the importance of sleep in memory consolidation, this study is important to understand the functional interaction between these three majorly involved regions. This work suggests that the thalamic nucleus reuniens has a functional role in synchronizing the hippocampus and prefrontal cortex. Therefore, it paves the way to new perspectives in order to decipher the neuronal and circuit mechanisms underlying such processes.

Strengths:

The authors have used an interdisciplinary approach to determine how the thalamic nucleus reuniens can impact on the cortico-hippocampal dialogue during sleep. They performed recording in naturally sleeping cats, and analysed the correlation between the main slow wave sleep oscillatory hallmarks: slow waves, spindles, and hippocampal ripples, and with reuniens' neurons firing. They also associated intracellular recordings to assess the reuniens-prefrontal connectivity, and computational models of large networks in which they determine that the coupling of oscillations is modulated by the strength of hippocampal-thalamic connections.<br /> Since the literature regarding fundamentals in nucleus reuniens anatomy in cats is much thinner as compared to what is available in rodents, the authors have performed complementary functional anatomy experiments in anesthetised cats in order to support the functional results (aka excitatory links between reuniens and its targets).

Weaknesses:

The authors have used cats as animal models to study the hippocampo-cortical dialogue. Whereas this is a very interesting and well-done study, it will address a more limited audience, as the majority of research is conducted on rodents, which have a different functional anatomy. Hence, the mechanisms of triggering of thalamic spindles would therefore be far less relevant in rodents, unless shown otherwise in the future.

Reviewer #1 (Public review):

Summary:

This manuscript presents a comprehensive exploration of the role of liver-specific Survival Motor Neuron (SMN) depletion in peripheral and central nervous system tissue pathology through a well-constructed mouse model. This study is pioneering in its approach, focusing on the broader physiological implications of SMN, which has traditionally been associated predominantly with spinal muscular atrophy (SMA).

Strengths:

(1) Novelty and Relevance: The study addresses a significant gap in understanding the role of liver-specific SMN depletion in the context of SMA. This is a novel approach that adds valuable insights into the multi-organ impact of SMN deficiency.

(2) Comprehensive Methodology: The use of a well-characterized mouse model with liver-specific SMN depletion is a strength. The study employs a robust set of techniques, including genetic engineering, histological analysis, and various biochemical assays.

(3) Detailed Analysis: The manuscript provides a thorough analysis of liver pathology and its potential systemic effects, particularly on the pancreas and glucose metabolism.

(4) Clear Presentation: The manuscript is well written. The results are presented clearly with well-designed figures and detailed legends.

Weaknesses:

(1) Limited Time Points: The study primarily focuses on a single time point (P19). This limits the understanding of the temporal progression of liver and pancreatic pathology in the context of SMN depletion. Longitudinal studies would provide a better understanding of disease progression.

(2) Incomplete Recombination: The mosaic pattern of Cre-mediated excision leads to variability in SMN depletion, which complicates the interpretation of some results. Ensuring more consistent recombination across samples would strengthen the conclusions.

After the revision, the authors addressed the reviewers' questions by extending their analyses to include P60 mice, conducting both liver and pancreatic analyses, and adding a comprehensive panel of metabolic hormones related to glucose metabolism in animals at P19 and P60. They also corrected all errors identified during the initial review process and expanded the discussion to clarify raised issues. All my questions have now been addressed.

Reviewer #3 (Public review):

Summary:

This paper uses 2D pose estimation and quantitative behavioral analyses to compare patterns of prey capture behavior used by six species of freshwater larval fish, including zebrafish, medaka, and four cichlids. The convincing comparison of tail and eye kinematics during hunts reveals that cichlids and zebrafish use binocular vision and similar hunting strategies, but that cichlids make use of an expanded set of action types. The authors also provide convincing evidence that medaka instead use monocular vision during hunts. This finding has important implications for the evolution of distinct distance estimation algorithms used by larval teleost fish species during prey capture.

Strengths:

The quality of the behavioral data is solid and the high frame rate allowed for careful quantification and comparison of eye and tail dynamics during hunts. The statistical approach to assess eye vergence states (Figure 2B) is elegant, the cross-species comparison of prey location throughout each hunt phase is well done (Figure 3B-D), and the demonstration that swim bout tail kinematics from diverse species can be embedded in a shared "canonical" principal component space to explain most of the variance in 2D postural dynamics for each species (Figure 4A-C) provides a simple and powerful framework for future studies of behavioral diversification across fish species.

Reviewer #1 (Public review):

Summary:

Dorrego-Rivas et al. investigated two different DA neurons and their neurotransmitter release properties in the main olfactory bulb. They found that the two different DA neurons in mostly glomerular layers have different morphologies as well as electrophysiological properties. The anaxonic DA neurons are able to self-inhibit but the axon-bearing ones are not. The findings are interesting and important to increase the understanding both of the synaptic transmissions in the main olfactory bulb and the DA neuron diversity. However, there are some major questions that the authors need to address to support their conclusions.

(1) It is known that there are two types of DA neurons in the glomerular layer with different diameters and capacitances (Kosaka and Kosaka, 2008; Pignatelli et al., 2005; Angela Pignatelli and Ottorino Belluzzi, 2017). In this manuscript, the authors need to articulate better which layer the imaging and ephys recordings took place, all glomerular layers or with an exception. Meanwhile, they have to report the electrophysiological properties of their recordings, including capacitances, input resistance, etc.

(2) It is understandable that recording the DA neurons in the glomerular layer is not easy. However, the authors still need to increase their n's and repeat the experiments at least three times to make their conclusion more solid. For example (but not limited to), Fig 3B, n=2 cells from 1 mouse. Fig.4G, the recording only has 3 cells.

(3) The statistics also use pseudoreplicates. It might be better to present the biology replicates, too.

(4) In Figure 4D, the authors report the values in the manuscript. It is recommended to make a bar graph to be more intuitive.

(5) In Figure 4F and G, although the data with three cells suggest no phenotype, the kinetics looked different. So, the authors might need to explore that aside from increasing the n.

(6) Similarly, for Figure 4I and J, L and M, it is better to present and analyze it like F and G, instead of showing only the after-antagonist effect.

Reviewer #1 (Public review):

The present study by Mikati et al describes an improved method for in-vivo detection of enkephalin release and examines the impact of stress on activation of enkephalin neurons and enkephalin release in the nucleus accumbens (NAc). The authors refine their pipeline to measure met and leu enkephalin using liquid chromatography and mass spectrometry. The authors subsequently measure met and leu enkephalin in the NAc during stress induced by handling and fox urine, in addition to calcium activity of enkephalinergic cells using fiber photometry. The authors conclude that this improved tool for measuring enkephalin reveals experimenter handling stress-induced enkephalin release in the NAc that habituates and is dissociable from calcium activity of these cells, whose activity doesn't habituate. The authors subsequently show that NAc enkephalin neuron calcium activity does habituate to fox urine exposure, is activated by a novel weigh boat, and that fox urine acutely causes increases in met-enk levels, in some animals, as assessed by microdialysis. This study highlights a new approach to monitor two distinct enkephalins and more robust analytical approach for more sensitive detection of neuropeptides. The authors also provide a pipeline that potentially could aid in detection of other neuropeptides and increase our understanding of endogenous opioid neuropeptidergic control in health and disease.

Reviewer #1 (Public review):

The propagation of electrical signals within neuronal circuits is tightly regulated by the physical and molecular properties of neurons. Since neurons vary in size across species, the question arises whether propagation speed also varies to compensate for it. The present article compares numerous speed-related properties in human and rat neurons. They found that the larger size of human neurons seems to be compensated by a faster propagation within dendrites but not axons of these neurons. The faster dendritic signal propagation was found to arise from wider dendritic diameters and greater conductance load in human neurons. In addition, the article provides a careful characterization of human dendrites and axons, as the field has only recently begun to characterize post-operative human cells. There are only a few studies reporting dendritic properties and these are not all consistent, hence there is added value of reporting these findings, particularly given that the characterization is condensed in a compartmental model.

Strengths

The study was performed with great care using standard techniques in slice electrophysiology (pharmacological manipulation with somatic patch-clamp) as well as some challenging ones (axonal and dendritic patch-clamp). Modeling was used to parse out the role of different features in regulating dendritic propagation speed. The finding that propagation speed varies across species is novel as previous studies did not find a large change in membrane time constant nor axonal diameters (a significant parameter affecting speed). A number of possible, yet less likely factors were carefully tested (Ih, membrane capacitance). The main features outlined here are well known to regulate speed in neuronal processes. The modeling was also carefully done to verify that the magnitude of the effects is consistent with the difference in biophysical properties. Hence, the findings appear very solid to me.

Weaknesses

The role of diameter in regulating propagation speed is well known in the axon literature.

Comment on the revised version: the authors have now made clearer that the role of diameter was well known in the manuscript.

Reviewer #1 (Public review):

The results of these experiments support a modest but important conclusion: If sub-optimal methods are used to collect retrospective reports, such as simple yes/no questions, inattentional blindness (IB) rates may be overestimated by up to ~8%.

(1) In experiment 1, data from 374 subjects were included in the analysis. As shown in figure 2b, 267 subjects reported noticing the critical stimulus and 107 subjects reported not noticing it. This translates to a 29% IB rate if we were to only consider the "did you notice anything unusual Y/N" question. As reported in the results text (and figure 2c), when asked to report the location of the critical stimulus (left/right), 63.6% of the "non-noticer" group answered correctly. In other words, 68 subjects were correct about the location while 39 subjects were incorrect. Importantly, because the location judgment was a 2-alternative-forced-choice, the assumption was that if 50% (or at least not statistically different than 50%) of the subjects answered the location question correctly, everyone was purely guessing. Therefore, we can estimate that ~39 of the subjects who answered correctly were simply guessing (because 39 guessed incorrectly), leaving 29 subjects from the non-noticer group who were correct on the 2AFC above and beyond the pure guess rate. If these 29 subjects are moved from the non-noticer to the noticer group, the corrected rate of IB for Experiment 1 is 20.86% instead of the original 28.61% rate that would have been obtained if only the Y/N question was used. In other words, relying only on the "Y/N did you notice anything" question led to an overestimate of IB rates by 7.75% in Experiment 1.

In the revised version of their manuscript, the authors provided the data that was missing from the original submission, which allows this same exercise to be carried out on the other 4 experiments. Using the same logic as above, i.e., calculating the pure-guess rate on the 2AFC, moving the number of subjects above this pure-guess rate to the non-noticer group, and then re-calculating a "corrected IB rate", the other experiments demonstrate the following:

Experiment 2: IB rates were overestimated by 4.74% (original IB rate based only on Y/N question = 27.73%; corrected IB rate that includes the 2AFC = 22.99%)

Experiment 3: IB rates were overestimated by 3.58% (original IB rate = 30.85%; corrected IB rate = 27.27%)

Experiment 4: IB rates were overestimated by ~8.19% (original IB rate = 57.32%; corrected IB rate for color* = 39.71%, corrected IB rate for shape = 52.61%, corrected IB rate for location = 55.07%)

Experiment 5: IB rates were overestimated by ~1.44% (original IB rate = 28.99%; corrected IB rate for color = 27.56%, corrected IB rate for shape = 26.43%, corrected IB rate for location = 28.65%)

*note: the highest overestimate of IB rates was from Experiment 4, color condition, but the authors admitted that there was a problem with 2AFC color guessing bias in this version of the experiment which was a main motivation for running experiment 5 which corrected for this bias.

Taken as a whole, this data clearly demonstrates that even with a conservative approach to analyzing the combination of Y/N and 2AFC data, inattentional blindness was evident in a sizeable portion of the subject populations. An important (albeit modest) overestimate of IB rates was demonstrated by incorporating these improved methods.

(2) One of the strongest pieces of evidence presented in this paper was the single data point in Figure 3e showing that in Experiment 3, even the super subject group that rated their non-noticing as "highly confident" had a d' score significantly above zero. Asking for confidence ratings is certainly an improvement over simple Y/N questions about noticing, and if this result were to hold, it could provide a key challenge to IB. However, this result can most likely be explained by measurement error.

In their revised paper, the authors reported data that was missing from their original submission: the confidence ratings on the 2AFC judgments that followed the initial Y/N question. The most striking indication that this data is likely due to measurement error comes from the number of subjects who indicated that they were highly confident that they didn't notice anything on the critical trial, but then when asked to guess the location of the stimulus, indicated that they were highly confident that the stimulus was on the left (or right). There were 18 subjects (8.82% of the high-confidence non-noticer group) who responded this way. To most readers, this combination of responses (high confidence in correctly judging a stimulus feature that one is highly confident in having not seen at all) indicates that a portion of subjects misunderstood the confidence scales (or just didn't read the questions carefully or made mistakes in their responses, which is common for experiments conducted online).

In the authors' rebuttal to the first round of peer review, they wrote, "it is perfectly rationally coherent to be very confident that one didn't see anything but also very confident that if there was anything to be seen, it was on the left." I respectfully disagree that such a combination of responses is rationally coherent. The more parsimonious interpretation is that a measurement error occurred, and it's questionable whether we should trust any responses from these 18 subjects.

In their rebuttal, the authors go on to note that 14 of the 18 subjects who rated their 2AFC with high confidence were correct in their location judgment. If these 14 subjects were removed from analysis (which seems like a reasonable analysis choice, given their contradictory responses), d' for the high-confidence non-noticer group would most likely fall to chance levels. In other words, we would see a data pattern similar to that plotted in Figure 3e, but with the first data point on the left moving down to zero d'. This corrected Figure 3e would then provide a very nice evidence-based justification for including confidence ratings along with Y/N questions in future inattentional blindness studies.

(3) In most (if not all) IB experiments in the literature, a partial attention and/or full attention trial is administered after the critical trial. These control trials are very important for validating IB on the critical trial, as they must show that, when attended, the critical stimuli are very easy to see. If a subject cannot detect the critical stimulus on the control trial, one cannot conclude that they were inattentionally blind on the critical trial, e.g., perhaps the stimulus was just too difficult to see (e.g., too weak, too brief, too far in the periphery, too crowded by distractor stimuli, etc.), or perhaps they weren't paying enough attention overall or failed to follow instructions. In the aggregate data, rates of noticing the stimuli should increase substantially from the critical trial to the control trials. If noticing rates are equivalent on the critical and control trials, one cannot conclude that attention was manipulated in the first place.

In their rebuttal to the first round of peer review, the authors provided weak justification for not including such a control condition. They cite one paper that argues such control conditions are often used to exclude subjects from analysis (those who fail to notice the stimulus on the control trial are either removed from analysis or replaced with new subjects) and such exclusions/replacements can lead to underestimations of inattentional blindness rates. However, the inclusion of a partial or full attention condition as a control does not necessitate the extra step of excluding or replacing subjects. In the broadest sense, such a control condition simply validates the attention manipulation, i.e., one can easily compare the percent of subjects who answered "yes" or who got the 2AFC judgment correct during the critical trial versus the control trial. The subsequent choice about exclusion/replacement is separate, and researchers can always report the data with and without such exclusions/replacements to remain more neutral on this practice.

If anyone were to follow-up on this study, I highly recommend including a partial or full attention control condition, especially given the online nature of data collection. It's important to know the percent of online subjects who answer yes and who get the 2AFC question correct when the critical stimulus is attended, because that is the baseline (in this case, the "ceiling level" of performance) to which the IB rates on the critical trial can be compared.

Reviewer #1 (Public review):

Summary:

This manuscript seeks to estimate the causal effect of genes on disease. To do so, they introduce a novel algorithm, termed the Root Causal Strength using Perturbations (RCSP) algorithm. RCSP uses perturb-seq to first estimate the gene regulatory network structure among genes, and then uses bulk RNA-seq with phenotype data on the samples to estimate causal effects of genes on the phenotype conditional on the learned network structure. The authors assess the performance of RCSP in comparison to other methods via simulation. Next, they apply RCSP to two real human datasets: 513 individuals age-related macular degeneration and 137 individuals with multiple sclerosis.

Strengths:

The authors tackle an important and ambitious problem - the identification of causal contributors to disease in the context of a causal inference framework. As the authors point out, observational RNA-seq data is insufficient for this kind of causal discovery, since it is very challenging to recover the true underlying graph from observational data; interventional data are needed. However, little perturb-seq data has been generated with annotated phenotype data, and much bulk RNA-seq data has already been generated, so it is useful to propose an algorithm to integrate the two as the authors have done.

The authors also offer substantial theoretical exposition for their work, bringing to bear both the literature on causal discovery as well as literature on the genetic architecture of complex traits. They also benchmark RCSP under multiple challenging simulation settings, including an analysis of RCSP when the underlying graph is not a DAG.

Weaknesses:

The notion of a "root" causal gene - which the authors define based on a graph theoretic notion of topologically sorting graphs - requires a graph that is directed and acyclic. It is the latter that constitutes an important weakness here - it simply is a large simplification of human biology to draw out a DAG including hundreds of genes and a phenotype Y and to claim that the true graph contains no cycles. For example - consider the authors' analysis of T cell infiltration in multiple sclerosis (MS). CD4+ effector T cells have the interesting property that they are stimulated by IL2 as a growth factor; yet IL2 also stimulates the activation of (suppressive) regulatory T cells. What does it mean to analyze CD4+ regulation in disease with a graph that does not consider IL2 (or other cytokine) mediated feedback loops/cycles? To the authors' credit, in the supplementary materials they do consider a simulated example with a cyclic underling causal graph, finding that RCSP performed well comparison to an implementation of the additive noise model (ANM), LiNGAM, CausalCell, and two simpler approaches based on linear regression.

I also encourage the authors to consider more carefully when graph structure learned from perturb-seq can be ported over to bulk RNA-seq. Consider again the MS CD4+ example - the authors first start with a large perturb-seq experiment (Replogle et al., 2022) performed in K562 cells. To what extent are K562 cells, which are derived from a leukemia cell line, suitable for learning the regulatory structure of CD4+ cells from individuals with an MS diagnosis? Presumably this structure is not exactly correct - to what extent is the RCSP algorithm sensitive to false edges in this graph? The authors perform an analysis of this scenario in Supplementary Figure 4, which shows that RCSP is robust to some degree of departure from the underlying true structure. And although challenging - it would be ideal for the RCSP to model or reflect the challenges in correctly identifying the regulatory structure.

It should also be noted that in most perturb-seq experiments, the entire genome is not perturbed, and frequently important TFs (that presumably are very far "upstream" and thus candidate "root" causal genes) are not expressed highly enough to be detected with scRNA-seq. In that context - perhaps slightly modifying the language regarding RCSP's capabilities might be helpful for the manuscript - perhaps it would be better to describe it has an algorithm for causal discovery among a set of genes that were perturbed and measured, rather than a truly complete search for causal factors. Perhaps more broadly - it would also benefit the manuscript to devote slightly more text to describing the kinds of scenarios where RCSP (and similar ideas) would be most appropriately applied - perhaps a well-powered, phenotype annotated perturb-seq dataset performed in a disease relevant primary cell.

Joint public review:

Summary

In this manuscript, Dong et al. study the directed cell migration of tracheal stem cells in Drosophila pupae. The authors study how the directionality of these cells is regulated along the dorsal trunk. They show that inter-organ communication between the tracheal stem cells and the nearby fat body plays a role in posterior migration. They provide compelling evidence that Upd2 production in the fat body and JAK/STAT activation in the tracheal stem cells play a role. Moreover, they show that JAK/STAT signalling might induce the expression of apicobasal and planar cell polarity genes in the tracheal stem cells which appear to be needed to ensure unidirectional migration. Finally, the authors suggest that trafficking and vesicular transport of Upd2 from the fat body towards the tracheal cells might be important.

Strengths

The manuscript is well written and presents extensive and varied experimental data to show a link between Upd2-JAK/STAT signaling from the fat body and tracheal progenitor cell migration. The authors provide convincing evidence that the fat body, located near the trachea, secretes vesicles containing the Upd2 cytokine and that affecting JAK-STAT signaling results in aberrant migration of some of the tracheal stem cells towards the anterior. Using ChIP-seq as well as analysis of GFP-protein trap lines of planar cell polarity genes in combination with RNAi experiments, the authors show that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells which appear to be needed for unidirectional migration. The work presented here provides some novel insights into the mechanism that ensures polarized migration of tracheal stem cells, preventing bidirectional migration. This might have important implications for other types of directed cell migration in invertebrates or vertebrates including cancer cell migration. Overall, the authors have substantially improved their manuscript since the first submission but there are still some weaknesses.

Weaknesses

Overall, the manuscript lacks insights into the potential significance of the observed phenotypes and of the proposed new signaling model. Most of our concerns could be dealt with by adjusting the text (explaining some parts better and toning down some statements).

(1) Directional migration of tracheal progenitors is only partially compromised, with some cells migrating anteriorly and others maintaining their posterior migration, a quite discrete phenotype. The strongest migration defects quantified in graphs (e.g. 100 μm) are not shown in images, since they would be out of frame, it would be beneficial to see them. In addition, the consequence of defects in polarized migration on tracheal development is not clear and data showing phenotypes on the final trachea morphology in pupae are not explained nor linked to the previous phenotypes.

(2) Some important information is lacking, such as the origin of mutant and UAS-RNAi lines, which are not reported in the material and methods. For instance, mutants for components of the JAK-STAT pathway are used but not described. Are they all viable at the pupal stage? Otherwise, pupae would not be homozygous mutants. From the figure legend, it seems that the Stat92EF allele has been used, which is a point mutation, thus not leading to an absence of protein. If the hopTUM allele has been used, as mentioned in the legend, it is a gain-of-function allele. Thus, the authors should not conclude that "The aberrant anterior migration of tracheal progenitors in the absence of JAK/STAT components led to impairment of tracheal integrity and caused melanization in the trachea (Figure 3-figure supplement 1E-I)".

(3) The authors observe that tracheal progenitors display a polarized distribution of Fat that is controlled by JAK-STAT signaling. However, this conclusion is made from a single experiment using only 3 individuals with no statistics. This is insufficient to support the claim that "JAK/STAT signaling promotes the expression of genes involved in planar cell polarity leading to asymmetric localization of Fat in progenitor cells", as mentioned in the abstract, or that "the activated tracheal progenitors establish a disciplined migration through the asymmetrical distribution of polarity proteins which is directed by an Upd2-JAK/STAT signaling stemming from the remote organ of fat body."

(4) The authors demonstrate that Upd2 is transported through vesicles from the fat body to the tracheal progenitors. It remains somewhat unclear in the proposed model how Upd2 activates JAK-STAT signaling. Are vesicles internalized, as it seems to be proposed, and thus how does Upd2 activate JAK-STAT signaling intracellularly? Or is Upd2 released from vesicles to bind Dome extracellularly to activate the JAK-STAT pathway? Moreover, it is not clear nor discussed what would be the advantage of transporting the ligand in vesicles compared to classical ligand diffusion.

Joint Public Review:

Summary:

In this manuscript, the model's capacity to capture epistatic interactions through multi-point mutations and its success in finding the global optimum within the protein fitness landscape highlights the strength of deep learning methods over traditional approaches.

Strengths:

It is impressive that the authors used AI combined with limited experimental validation to achieve such significant enhancements in protein performance. Besides, the successful application of the designed antibody in industrial settings demonstrates the practical and economic relevance of the study. Overall, this work has broad implications for future AI-guided protein engineering efforts.

Reviewing Editor's comments on revised version:

The authors extensively addressed conceptual and methodological points raised by reviewers, as well as constructive comments to clarify the narrative. Consequently, the manuscript experienced a qualitative jump on clarity and appeal for the eLife readership.

Reviewer #1 (Public review):

Summary:

This study investigates the impact of Pink1 loss on glial function and neuronal health in a Drosophila model, highlighting the role of mitochondria-organelle contacts and key genes such as Ccz1, Vps13, Mon1, and Rab7. The work provides insights into cellular processes underlying neurodegenerative diseases, with a focus on glia-neuron interactions. While the findings are promising, the study lacks critical controls, detailed mechanistic evidence, and explanatory figures to strengthen its claims.

Strengths:

(1) The study addresses an important topic in neuroscience, exploring the mechanisms of Pink1 loss, which has implications for Parkinson's disease and neurodegeneration.

(2) The focus on mitochondria-organelle contacts and their regulation by Rab7-mediated pathways is novel and provides a potential mechanism for neuronal dysfunction.

(3) The identification of key genes (Ccz1, Vps13, Mon1, Rab7) and their potential roles in Pink1-related pathways adds valuable knowledge to the field.

(4) The manuscript uses a combination of genetic tools, Drosophila models, and functional assays to approach the problem from multiple angles.

Weaknesses:

(1) Specificity of Mz-Gal4: The study lacks validation of Mz-Gal4 specificity, as it may also drive expression in a few neurons or other types of glia. Additional control experiments using nls-GFP with Elav, Repo, or Draper antibody staining or alternative glial drivers would be helpful.

(2) DLG staining is central to the story but is not well-supported by high-resolution Z-stack imaging, which should be included in the supplementary figures.

(3) The manuscript does not confirm whether the candidate RNAi (Ccz1, Vps13, Mon1, Rab7) directly influence Rab7-mediated membrane trafficking or mitochondria-lysosome contacts in Pink1 mutants.

(4) Using ERG as a readout for EG effects in the antenna is not a direct or appropriate assay. Alternative functional assays relevant to antenna glia should be considered.

(5) A graphical explanation of the interactions and functions of the candidate genes in Pink1 KO mutants is missing. This would greatly enhance the manuscript's clarity.

(6) The study lacks details on sample sizes, effect sizes, and reproducibility, which are necessary for robust conclusions.

(7) There are repeated words on page 3 ("olfactory Olfactory Receptor Neurons") and a lack of explanation in Figure 3C regarding the most up-regulated and down-regulated genes and the significance of large red dots.

Reviewer #1 (Public review):

The authors assess the effectiveness of electroporating mRNA into male germ cells to rescue the expression of proteins required for spermatogenesis progression in individuals where these proteins are mutated or depleted. To set up the methodology, they first evaluated the expression of reporter proteins in wild-type mice, which showed expression in germ cells for over two weeks. Then, they attempted to recover fertility in a model of late spermatogenesis arrest that produces immotile sperm. By electroporating the mutated protein, the authors recovered the motility of ~5% of the sperm; although the sperm regenerated was not able to produce offspring using IVF, the embryos reached the 2-cell state (in contrast to controls that did not progress past the zygote state).

This is a comprehensive evaluation of the mRNA methodology with multiple strengths. First, the authors show that naked synthetic RNA, purchased from a commercial source or generated in the laboratory with simple methods, is enough to express exogenous proteins in testicular germ cells. The authors compared RNA to DNA electroporation and found that germ cells are efficiently electroporated with RNA, but not DNA. The differences between these constructs were evaluated using in vivo imaging to track the reporter signal in individual animals through time. To understand how the reporter proteins affect the results of the experiments, the authors used different reporters: two fluorescent (eGFP and mCherry) and one bioluminescent (Luciferase). Although they observed differences among reporters, in every case expression lasted for at least two weeks.

The authors used a relevant system to study the therapeutic potential of RNA electroporation. The ARMC2-deficient animals have impaired sperm motility phenotype that affects only the later stages of spermatogenesis. The authors showed that sperm motility was recovered to ~5%, which is remarkable due to the small fraction of germ cells electroporated with RNA with the current protocol. The sperm motility parameters were thoroughly assessed by CASA. The 3D reconstruction of an electroporated testis using state-of-the-art methods to show the electroporated regions is compelling.

The main weakness of the manuscript is that although the authors manage to recover motility in a small fraction of the sperm population, it is unclear whether the increased sperm quality is substantial to improve assisted reproduction outcomes. The authors found that the rescued sperm could be used to obtain 2-cell embryos via IVF, but no evidence for more advanced stages of embryo differentiation was provided. The motile rescued sperm was also successfully used to generate blastocyst by ICSI, but the statistical significance of the rate of blastocyst production compared to non-rescued sperm remains unclear. The title is thus an overstatement since fertility was never restored for IVF, and the mutant sperm was already able to produce blastocysts without the electroporation intervention.

Overall, the authors clearly show that electroporating mRNA can improve spermatogenesis as demonstrated by the generation of motile sperm in the ARMC2 KO mouse model.

Reviewer #1 (Public review):

Summary:

This work presents an Interpretable protein-DNA Energy Associative (IDEA) model for predicting binding sites and affinities of DNA-binding proteins. Experimental results demonstrate that such an energy model can predict DNA recognition sites and their binding strengths across various protein families and can capture the absolute protein-DNA binding free energies.

Strengths:

(1) The IDEA model integrates both structural and sequence information, although such an integration is not completely original.

(2) The IDEA predictions seem to have agreement with experimental data such as ChIP-seq measurements.

Weaknesses:

(1) The authors claim that the binding free energy calculated by IDEA, trained using one MAX-DNA complex, correlates well with experimentally measured MAX-DNA binding free energy (Figure 2) based on the reported Pearson Correlation of 0.67. However, the scatter plot in Figure 2A exhibits distinct clustering of the points and thus the linear fit to the data (red line) may not be ideal. As such. the use of the Pearson correlation coefficient that measures linear correlation between two sets of data may not be appropriate and may provide misleading results for non-linear relationships.

(2) In the same vein, the linear Pearson Correlation analysis performed in Figure 5A and the conclusion drawn may be misleading.

(3) The authors included the sequences of the protein and DNA residues that form close contacts in the structure in the training dataset, whereas a series of synthetic decoy sequences were generated by randomizing the contacting residues in both the protein and DNA sequences. In particular, synthetic decoy binders were generated by randomizing either the DNA (1000 sequences) or protein sequences (10,000 sequences) from the strong binders. However, the justification for such randomization and how it might impact the model's generalizability and transferability remain unclear.

(4) The authors performed Receiver Operating Characteristic (ROC) analysis and reported the Area Under the Curve (AUC) scores in order to quantitate the successful identification of the strong binders by IDEA. It would be beneficial to analyze the precision-recall (PR) curve and report the PRAUC metric which could be more robust.

Reviewer #1 (Public review):

Summary:

This study examined the functional organization of the mouse posterior parietal cortex (PPC) using meso-scale two-photon calcium imaging during visually-guided and history-guided tasks. The researchers found distinct functional modules within the medial PPC: area A, which integrates somatosensory and choice information, and area AM, which integrates visual and choice information. Area A also showed a robust representation of choice history and posture. The study further revealed distinct patterns of inter-area correlations for A and AM, suggesting different roles in cortical communication. These findings shed light on the functional architecture of the mouse PPC and its involvement in various sensorimotor and cognitive functions.

Strengths:

Overall, I find this manuscript excellent. It is very clearly written and built up logically. The subject is important, and the data supports the conclusions without overstating implications. Where the manuscript shines the most is the exceptionally thorough analysis of the data. The authors set a high bar for identifying the boundaries of the PPC subareas, where they combine both somatosensory and visual intrinsic imaging. There are many things to compliment the authors on, but one thing that should be applauded in particular is the analysis of the body movements of the mice in the tube. Anyone working with head-fixed mice knows that mice don't sit still but that almost invariable remains unanalyzed. Here the authors show that this indeed explained some of the variance in the data.

Weaknesses:

I see no major weaknesses and I only have minor comments.

Reviewer #1 (Public review):

Summary:

Kohno et al. examined whether the anti-inflammatory cytokine IL-4 attenuates neuropathic pain by promoting the emergence of antinociceptive microglia in the dorsal horn of the spinal cord. In two models of neuropathic pain following peripheral nerve injury, intrathecal administration of IL-4 once a day for 3 days from day 14 to day 17 after injury, attenuates hypersensitivity to mechanical stimuli in the hind paw ipsilateral to nerve injury. Such an antinociceptive effect correlates with a higher number of CD11c+microglia in the dorsal horn of the spinal cord which is the termination area for primary afferent fibres injured in the periphery. Interestingly, CD11c+ microglia emerge spontaneously in the dorsal horn in concomitance with the resolution of pain in the spinal nerve model of pain, but not in the spared nerve injury model where pain does not resolve, confirming that this cluster of microglia is involved in resolution pain.

Based on existing evidence that the receptor for IL-4, namely IL-4R, is expressed by microglia, the authors suggest that IL-4R mediates IL-4 effect in microglia including up-regulation of Igf1 mRNA. They have previously reported that IGF-1 can attenuate pain neuron activity in the spinal cord.

Strengths:

This study includes cutting-edge techniques such as flow cytometry analysis of microglia and transgenic mouse models.

Weaknesses:

The conclusion of this paper is supported by data, but the interpretation of some data requires clarification.

Reviewer #1 (Public review):

Summary:

This manuscript by Tesmer and colleagues uses fiber photometry recordings, sophisticated analysis of movement, and deep learning algorithms to provide compelling evidence that activity in hypothalamic hypocretin/orexin neurons (HONs) correlates with net body movement over multiple behaviors. By examining projection targets, the authors show that hypocretin/orexin release differs in projection targets to the locus coeruleus and substantia nigra, pars compacta. Ablation of HONs does not cause differences in the power spectra of movements. The movement-tracking ability of HONs is independent of HON activity that correlates with blood glucose levels. Finally, the authors show that body movement is not encoded to the same extent in other neural populations.

Strengths:

The major strengths of the study are the combination of fiber photometry recordings, analysis of movement in head-fixed mice, and sophisticated classification of movement using deep learning algorithms. The experiments seem to be well performed, and the data are well presented, visually. The data support the main conclusions of the manuscript.

Weaknesses:

The weaknesses are minor, mostly consisting of writing and data visualization throughout the manuscript. To some degree, it is already known that hypocretin/orexin neurons correlate with movement and arousal, although this manuscript studies this correlation with unprecedented sophistication and scale. It is also unfortunate that most of the experiments throughout the study were only performed in male mice.

Taken together, this study is likely to be impactful to the field and our understanding of HONs across behavioral states.

Reviewer #1 (Public review):

Summary, and Strengths:

The authors and their team have investigated the role of Vimentin Cysteine 328 in epithelial-mesenchymal transition (EMT) and tumorigenesis. Vimentin is a type III intermediate filament, and cysteine 328 is a crucial site for interactions between vimentin and actin. These interactions can significantly influence cell movement, proliferation, and invasion. The team has specifically examined how Vimentin Cysteine 328 affects cancer cell proliferation, the acquisition of stemness markers, and the upregulation of the non-coding RNA XIST. Additionally, functional assays were conducted using both wild-type (WT) and Vimentin Cysteine 328 mutant cells to demonstrate their effects on invasion, EMT, and cancer progression. Overall, the data supports the essential role of Vimentin Cysteine 328 in regulating EMT, cancer stemness, and tumor progression. Overall, the data and its interpretation are on point and support the hypothesis. I believe the manuscript has great potential.

Weaknesses:

Minor issues are related to the visibility and data representation in Figures 2E and 3 A-F.

Reviewer #1 (Public review):

Summary:

Hahn et al use bystander BRET, NanoBiT assays and APEX2 proteomics to investigate endosomal signaling of CCR7 by two agonists, CCL19 and CCL21. The authors suggest that CCR7 signals from early endosomes following internalisation. They use spatial proteomics to try to identify novel interacting partners that may facilitate this signaling and use this data to specifically enhance a Rac1 signaling pathway. The most novel findings are the APEX2 proteomics studies that provide new mechanisms.

Strengths:

(1) The APEX2 resource will be valuable to the GPCR and immunology community. It offers many opportunities to follow up on findings and discover new biology. The authors have used the resource to validate earlier findings in the current manuscript and in previous manuscripts.

(2) The results section is well written and can be followed very easily by the reader.

(3) Some findings verify previous studies (e.g. endomembrane signalling).

Weaknesses:

(1) The findings are interesting although the studies are almost all performed in HEK293 cells. I understand that these are commonly used in GPCR biology and current tools need to be improved in order to perform similar analyses in more relevant cell-lines. Future studies should focus on validating the findings of the current study in physiologically-relevant cell-lines.

Reviewer #2 (Public review):

The authors investigate the gene expression variation in a rice diversity panel under normal and saline growth conditions to gain insight into the underlying molecular adaptive response to salinity. They present a convincing case to demonstrate that environment stress can induce selective pressure on gene expression, which is in agreement with their earlier study (Groen et al, 2020). The data seems to be a good fit for their study and overall the analytic approach is robust.

(1) The work started by investigating the effect of genotype and their interaction at each transcript level using 3'-end-biased mRNA sequencing, and detect a wide-spread GXE effect. Later, using the total filled grain number as a proxy of fitness, they estimated the strength of selection on each transcript and reported stronger selective pressure in saline environment. However, this current framework rely on precise estimation of fitness and, therefore can be sensitive to the choice of fitness proxy.

(2) Furthermore, the authors decomposed the genetic architecture of expression variation into cis- and trans-eQTL in each environment separately and reported more unique environment specific trans-eQTLs than cis-. The relative contribution of cis- and trans-eQTL depends on both the abundance and effect size. I wonder why the latter was not reported while comparing these two different genetic architectures. If the authors were to compare the variation explained by these two categories of eQTL instead of their frequency, would the inference that trans-eQTLs are primarily associated with expression variation still hold?

(3) Next, the authors investigated the relationship between cis- and trans-eQTLs at transcript level and revealed an excess of reinforcement over compensation pattern. Here, I struggle to understand the motivation for testing the relationship by comparing the effect of cis-QTL with the mean effect of all trans-eQTLs of a given transcript. My concern is that taking the mean can diminish the effect of small trans-eQTLs potentially biasing the relationship towards the large-effect eQTLs.

Comments on latest version:

After the revision, the article has improved substantially. The authors have addressed most of my concerns and suggestions, except for testing the eQTL reinforcement/compensation relationship in the context of genetic architecture. I understand the motivation for testing this relationship at the gene level to determine whether it arises from directional or stabilizing selection, rather than examining it in a cis-trans pairwise fashion. However, I find the definition of this relationship unclear. The authors state in line 824 that "Genes were defined as compensating and reinforcing if they had at least 60% of individuals with opposite and same cis-trans allelic configuration, respectively." In contrast, if I understood correctly, the response to reviewers describes the relationship as reinforcing if the cis-eQTL effect is in the same direction as the mean effect of all the detected trans-eQTLs. I would request that the authors clarify their method of defining this relationship. Also, one should be aware of the fact that this relationship can evolve neutrally. Since there was no formal test performed to say it is otherwise, the authors might need to interpret the relationship carefully.

While the authors explain the possible factors that could lead to the trend of observing widespread genotype-dependent plastic responsse without significant genotype-dependent plasticity for fitness (L142), it is also important to consider the time axis. While filled grain serves as a proxy for fitness over time, gene expression profiles provide only a snapshot at a given time point. Therefore, temporal GxE dynamics may also play a role here.

Also, I am a little surprised by not mentioning anything about the code availability in this manuscript. I would request the authors to incorporate that in the revised version.

Reviewer #1 (Public review):

The conserved AAA-ATPase PCH-2 has been shown in several organisms including C. elegans to remodel classes of HORMAD proteins that act in meiotic pairing and recombination. In some organisms the impact of PCH-2 mutations is subtle but becomes more apparent when other aspects of recombination are perturbed. Patel et al. performed a set of elegant experiments in C. elegans aimed at identifying conserved functions of PCH-2. Their work provides such an opportunity because in C. elegans meiotically expressed HORMADs localize to meiotic chromosomes independently of PCH-2. Work in C. elegans also allows the authors to focus on nuclear PCH-2 functions as opposed to cytoplasmic functions also seen for PCH-2 in other organisms.

The authors performed the following experiments:

(1) They constructed C. elegans animals with SNPs that enabled them to measure crossing over in intervals that cover most of four of the six chromosomes. They then showed that double-crossovers, which were common on most of the four chromosomes in wild-type, were absent in pch-2. They also noted shifts in crossover distribution in the four chromosomes.

(2) Based on the crossover analysis and previous studies they hypothesized that PCH-2 plays a role at an early stage in meiotic prophase to regulate how SPO-11 induced double-strand breaks are utilized to form crossovers. They tested their hypothesis by performing ionizing irradiation and depleting SPO-11 at different stages in meiotic prophase in wild-type and pch-2 mutant animals. The authors observed that irradiation of meiotic nuclei in zygotene resulted in pch-2 nuclei having a larger number of nuclei with 6 or greater crossovers (as measured by COSA-1 foci) compared to wildtype. Consistent with this observation, SPO11 depletion, starting roughly in zygotene, also resulted in pch-2 nuclei having an increase in 6 or more COSA-1 foci compared to wildtype. The increased number at this time point appeared beneficial because a significant decrease in univalents was observed.

(3) They then asked if the above phenotypes correlated with the localization of MSH-5, a factor that stabilizes crossover-specific DNA recombination intermediates. They observed that pch-2 mutants displayed an increase in MSH-5 foci at early times in meiotic prophase and an unexpectedly higher number at later times. They conclude based on the differences in early MSH-5 localization and the SPO-11 and irradiation studies that PCH-2 prevents early DSBs from becoming crossovers and early loading of MSH-5. By analyzing different HORMAD proteins that are defective in forming the closed conformation acted upon by PCH-2, they present evidence that MSH-5 loading was regulated by the HIM-3 HORMAD.

(4) They performed a crossover homeostasis experiment in which DSB levels were reduced. The goal of this experiment was to test if PCH-2 acts in crossover assurance. Interestingly, in this background PCH-2 negative nuclei displayed higher levels of COSA-1 foci compared to PCH-2 positive nuclei. This observation and a further test of the model suggested that "PCH-2's presence on the SC prevents crossover designation."

(5) Based on their observations indicating that early DSBS are prevented from becoming crossovers by PCH-2, the authors hypothesized that the DNA damage kinase CHK-2 and PCH-2 act to control how DSBs enter the crossover pathway. This hypothesis was developed based on their finding that PCH-2 prevents early DSBs from becoming crossovers and previous work showing that CHK-2 activity is modulated during meiotic recombination progression. They tested their hypothesis using a mutant synaptonemal complex component that maintains high CHK-2 activity that cannot be turned off to enable crossover designation. Their finding that the pch-2 mutation suppressed the crossover defect (as measured by COSA-1 foci) supports their hypothesis.

Based on these studies the authors provide convincing evidence that PCH-2 prevents early DSBs from becoming crossovers and controls the number and distribution of crossovers to promote a regulated mechanism that ensures the formation of obligate crossovers and crossover homeostasis. As the authors note, such a mechanism is consistent with earlier studies suggesting that early DSBs could serve as "scouts" to facilitate homolog pairing or to coordinate the DNA damage response with repair events that lead to crossing over. The detailed mechanistic insights provided in this work will certainly be used to better understand functions for PCH-2 in meiosis in other organisms.

Comments on revisions:

The authors responded very carefully to all of my concerns expressed in the first review, which were primarily aimed at improving the clarity of the manuscript.

Reviewer #1 (Public review):

Summary:

This interesting manuscript first shows that human, murine, and feline sperm penetrate the zona pellucida (ZP) of bovine oocytes recovered directly from the ovary, although first cleavage rates are reduced. Similarly, bovine sperm can penetrate superovulated murine oocytes recovered directly from the ovary. However, bovine oocytes incubated with oviduct fluid (30 min) are generally impenetrable by human sperm.

Thereafter, the cytoplasm was aspirated from murine oocytes - obtained from the ovary or oviduct. Binding and penetration by bovine and human sperm was reduced in both groups relative to homologous (murine) sperm. However, heterologous (bovine and human) sperm penetration was further reduced in oviduct vs. ovary derived empty ZP. These data show that outer (ZP) not inner (cytoplasmic) oocyte alterations reduce heterologous sperm penetration as well as homologous sperm binding.

This was repeated using empty bovine ZP incubated, or not, with bovine oviduct fluid. Prior oviduct fluid exposure reduced non-homologous (human and murine) empty ZP penetration, polyspermy, and sperm binding. This demonstrates that species-specific oviduct fluid factors regulate ZP penetrability.

To test the hypothesis that OVGP1 is responsible, the authors obtained histidiine-tagged bovine and murine OVGP1 and DDK-tagged human OVGP1 proteins. Tagging was to enable purification following over-expression in BHK-21 or HEK293T cells. The authors confirm these recombinant OVGP1 proteins bound to both murine and bovine oocytes. Moreover, previous data using oviduct fluid was mirrored using bovine oocytes supplemented with homologous (bovine) recombinant OVGP1, or not. This confirms the hypothesis, at least in cattle.

Next, the authors exposed bovine and murine empty ZP to bovine, murine, and human recombinant OVGP1, in addition to bovine, murine, or human sperm. Interestingly, both species-specific ZP and OVGP1 seem to be required for optimal sperm binding and penetration.

Lastly, empty bovine and murine ZP were treated with neuraminidase, or not, with or without pre-treatment with homologous OVGP1. In each case, neuraminidase reduced sperm binding and penetration. This further demonstrates that both ZP and OVGP1 are required for optimal sperm binding and penetration.

In summary, the authors demonstrate that two mechanisms seem to underpin mammalian sperm recognition and penetration, the first being specific (ZP-mediated) and the second non-specific (OVGP1 mediated).

Reviewer #1 (Public Review):

Summary:

In this study, a chromosome-level genome of the rose-grain aphid M. dirhodum was assembled with high quality, and A-to-I RNA-editing sites were systematically identified. The authors then demonstrated that: 1) Wing dimorphism induced by crowding in M. dirhodum is regulated by 20E (ecdysone signaling pathway); 2) an A-to-I RNA editing prevents the binding of miR-3036-5p to CYP18A1 (the enzyme required for 20E degradation), thus elevating CYP18A1 expression, decreasing 20E titer, and finally regulating the wing dimorphism of offspring.

Strengths:

The authors present both genome and A-to-I RNA editing data. An interesting finding is that a A-to-I RNA editing site in CYP18A1 ruin the miRNA binding site of miR-3036-5p. And loss of miR-3036-5p regulation lead to less 20E and winged offspring.

Reviewer #1 (Public review):

This paper focuses on secondary structure and homodimers in the HIV genome. The authors introduce a new method called HiCapR which reveals secondary structure, homodimer, and long-range interactions in the HIV genome. The experimental design and data analysis are well-documented and statistically sound.

Comments on revisions:

The authors have addressed key questions and highlighted the advantages of HiCapR.

Reviewer #1 (Public Review):

Summary:

The authors identified nanobodies that were specific for the trypanosomal enzyme pyruvate kinase in previous work seeking diagnostic tools. They have shown that a site involved in the allosteric regulation of the enzyme is targeted by the nanobody and using elegant structural approaches to pinpoint where binding occurs, opening the way to the design of small molecules that could also target this site.

Strengths:

The structural work shows the binding of a nanobody to a specific site on Trypanosoma congolense pyruvate kinase and provides a good explanation as to how binding inhibits enzyme activity. The authors go on to show that by expressing the nanobodies within the parasites they can get some inhibition of growth, which albeit rather weak, they provide a case on how this could point to targeting the same site with small molecules as potential trypanocidal drugs.

Weaknesses:

The impact on growth is rather marginal. Although explanations are offered on the reasons for that, including the high turnover rate of the expressed nanobody and the difficulty in achieving the high levels of inhibition of pyruvate kinase required to impact energy production sufficiently to kill parasites, this aspect of the work doesn't offer great support to developing small molecule inhibitors of the same site.

Reviewer #1 (Public review):

Although the use of antimony has been discontinued in India, the observation that there are Leishmania parasites that are resistant to antimony in circulation has been cited as evidence that these resistant parasites are now a distinct strain with properties that ensure their transmission and persistence. It is of interest to determine what are the properties that favor the retention of their drug resistance phenotype even in the absence of the selective pressure that would otherwise be conferred by the drug. The hypothesis that these authors set out to test is that these parasites have developed a new capacity to acquire and utilize lipids, especially cholesterol which affords them the capacity to grow robustly in infected hosts.

Major issues:

There are several experiments for which they do not provide sufficient details, but proceed to make significant conclusions.

Experiments in section 5 are poorly described. They supposedly isolated PVs from infected cells. No details of their protocol for the isolation of PVs are provided. They reference a protocol for PV isolation that focused on the isolation of PVs after L. amazonensis infection. In the images of infection that they show, by 24 hrs, infected cells harbor a considerable number of parasites. Is it at the 24 hr time point that they recover PVs? What is the purity of PVs? The authors should provide evidence of the success of this protocol in their hands. Earlier, they mentioned that using imaging techniques, the PVs seem to have fused or interconnected somehow. Does this affect the capacity to recover PVs? If more membranes are recovered in the PV fraction, it may explain the higher cholesterol content.

In section 6 they evaluate the mechanism of LDL uptake in macrophages. Several approaches and endocytic pathway inhibitors are employed. The authors must be aware that the role of cytochalasin D in the disruption of fluid phase endocytosis is controversial. Although they reference a study that suggests that cytochalasin D has no effect on fluid-phase endocytosis, other studies have found the opposite (doi: 10.1371/journal.pone.0058054). It wasn't readily evident what concentrations were used in their study. They should consider testing more than 1 concentration of the drug before they make their conclusions on their findings on fluid phase endocytosis.

In Figure 5 they present a blot that shows increased Lamp1 expression from as early as 4 hrs after infection with LD-R and by 12 hrs after infection of both LD-S and LD-R. Increased Lamp1 expression after Leishmania infection has not been reported by others. By what mechanism do they suggest is causing such a rapid increase (at 4hrs post-infection) in Lamp-1 protein? As they report, their RNA seq data did not show an increase in LAMP1 transcription (lines 432 - 434).

In Figure 6, amongst several assays, they reported on studies where SPC-1 is knocked down in PECs. They failed to provide any evidence of the success of the knockdown, but nonetheless showed greater LD-R after NPC-1 was knocked down. They should provide more details of such experiments.

Minor issues

There is an implication that parasite replication occurs well before 24hrs post-infection? Studies on Leishmania parasite replication have reported on the commencement of replication after 24hrs post-infection of macrophages (PMCID: PMC9642900). Is this dramatic increase in parasite numbers that they observed due to early parasite replication?

Several of the fluorescence images in the paper are difficult to see. It would be helpful if a blown-up (higher magnification image of images in Figure 1 (especially D) for example) is presented.

The times at which they choose to evaluate their infections seem arbitrary. It is not clear why they stopped analysis of their KC infections at 24 hrs. As mentioned above, several studies have shown that this is when intracellular amastigotes start replicating. They should consider extending their analyses to 48 or 72 hrs post-infection. Also, they stop in vitro infection of Apoe-/- mice at 11 days. Why? No explanation is given for why only 1 point after infection.

Reviewer #1 (Public review):

Summary:

This work by Ding et al uses agent-based simulations to explore the role of the structure of molecular motor myosin filaments in force generation in cytoskeletal structures. The focus of the study is on disordered actin bundles which can occur in the cell cytoskeleton and have also been investigated with in vitro purified protein experiments.

Strengths:

The key finding is that cooperative effects between multiple myosin filaments can enhance both total force and the efficiency of force generation (force per myosin). These trends were possible to obtain only because the detailed structure of the motor filaments with multiple heads is represented in the model.

Weaknesses:

It is not clearly described what scientific/biological questions about cellular force production the work answers. There should be more discussion of how their simulation results compare with existing experiments or can be tested in future experiments.

The model assumptions and scientific context need to be described better.

The network contractility seems to be a mere appendix to the bundle contractility which is presented in much more detail.

Reviewer #1 (Public review):

Summary:

In this manuscript, Frangos et al. used a transcriptomic and proteomic approach to characterise changes in HER2-driven mammary tumours compared to healthy mammary tissue in mice. They observed that mitochondrial genes, including OXPHOS regulators, were among the most down-regulated genes and proteins in their datasets. Surprisingly, these were associated with higher mitochondrial respiration, in response to a variety of carbon sources. In addition, there seems to be a reduction in mitochondrial fusion and an increase in fission in tumours compared to healthy tissues.

Strengths:

The data are clearly presented and described.

The author reported very similar trends in proteomic and transcriptomic data. Such approaches are essential to have a better understanding of the changes in cancer cell metabolism associated with tumourigenesis

Weaknesses:

This study, despite being a useful resource (assuming all the data will be publicly available and not only upon request) is mainly descriptive and correlative and lacks mechanistic links.

It would be important to determine the cellular composition of the tumour and healthy tissue used. Do the changes described here apply to cancer cells only or do other cell types contribute to this?

Are the changes in metabolic gene expression a consequence of HER2 signalling activation? Ex-vivo experiments could be performed to perturb this pathway and determine cause-effects.

The data of fission/fusion seem quite preliminary and the gene/protein expression changes are not so clear cut to be a convincing explanation that this is the main reason for the increased mitochondria respiration in tumours.

Reviewer #1 (Public review):

De Seze et al. investigated the role of guanine exchange factors (GEFs) in controlling cell protrusion and retraction. In order to causally link protein activities to the switch between the opposing cell phenotypes, they employed optogenetic versions of GEFs which can be recruited to the plasma membrane upon light exposure and activate their downstream effectors. Particularly the RhoGEF PRG could elicit both protruding and retracting phenotypes. Interestingly, the phenotype depended on the basal expression level of the optoPRG. By assessing the activity of RhoA and Cdc42, the downstream effectors of PRG, the mechanism of this switch was elucidated: at low PRG levels, RhoA is predominantly activated and leads to cell retraction, whereas at high PRG levels, both RhoA and Cdc42 are activated but PRG also sequesters the active RhoA, therefore Cdc42 dominates and triggers cell protrusion. Finally, they create a minimal model that captures the key dynamics of this protein interaction network and the switch in cell behavior.

The conclusions of this study are strongly supported by data, harnessing the power of modelling and optogenetic activation. The minimal model captures well the dynamics of RhoA and Cdc42 activation and predicts that by changing the frequency of optogenetic activation one can switch between protruding and retracting behaviour in the same cell of intermediate optoPRG level. The authors are indeed able to demonstrate this experimentally albeit with a very low number of cells. A major caveat of this study is that global changes due to PRG overexpression cannot be ruled out. Also, a quantification of absolute protein concentration, which is notoriously difficult, would be useful to put the level of overexpression here in perspective with endogenous levels. Furthermore, it remains unclear whether in cases of protein overexpression in vivo such as cancer, PRG or other GEFs can activate alternative migratory behaviours.

Previous work has implicated RhoA in both protrusion and retraction depending on the context. The mechanism uncovered here provides a convincing explanation for this conundrum. In addition to PRG, optogenetic versions of two other GEFs, LARG and GEF-H1, were used which produced either only one phenotype or less response than optoPRG, underscoring the functional diversity of RhoGEFs. The authors chose transient transfection to achieve a large range of concentration levels and, to find transfected cells at low cell density, developed a small software solution (Cell finder), which could be of interest for other researchers.

Reviewer #2 (Public review):

The resubmitted version of the paper by Yan et al. titled "Frequent intertrophic transmission of Wolbachia by parasitism but not predation" contains all the major flaws I found in the original submission. As far as I could see, the authors did not address my original concerns.

In short:

(1) A control of Portiera MUST be included in the FISH experiments, if the claim that Wolbachia is not only transferred from a parasitoid to the whitefly, but finds its way to the bacteriocytes. This is especially true for the Q, a biotype for which the pattern of Wolbachia distribution has been documented as scattered in naturally infected populations. The very strong signal in the whitefly bacteriocytes implies Portiera.

(2) In my original review I wrote: "The authors fail to discuss, or even acknowledge, a number of published studies that specifically show no horizontal transmission such as the one claimed to be detected in the study presented." In return the authors wrote in their rebuttal letter: "We have made corresponding modifications to the discussion section (Lines 256-271in the revised manuscript) and have discussed the published studies that report no evidence of horizontal transmission (Lines 260-263 in the revised manuscript)." However, the stated lines are concerned with a different subject. In addition, in their letter the authors write "Additionally, some experiments have found no evidence of horizontal transmission of Wolbachia (39- 42) (Lines 260-263 in the revised manuscript)." Beside the fact that the line numbers are wrong, the papers cited are entirely irrelevant as they do not discuss parasitoids.

(3) My original comment on the origin of sequences used for the phylogenetical analysis still stands. It is hard to claim a data-based search, when most of the data originate in the authors lab. The explanation of the confusion with the Qi et al. (2019) paper should at least be mentioned in the M&M. Apologies if it has been included and I missed it.

Reviewer #1 (Public review):

Summary:

Ghone et al show that HIV-1 Vif causes a pseudo-metaphase arrest rather than a G2 arrest. The metaphase arrest correlates with misregulation of the kinetochore that could be explained by the loss of phosphatase functions that determine chromosome-microtubule interactions.

Strengths:

The single-cell imaging using different reporters of cell cycle progression is very elegant and the quantitation is convincing. The authors clearly show that what others have characterized as a G2 arrest by flow cytometry is somewhat later in metaphase and correlates with kinetocore misregulation.

Weaknesses:

(1) The major problem with the paper is trying to connect what is observed in tumor cell lines with actual infections in primary T cells. While all of the descriptive work in cell lines is convincing, none of these cells are relevant targets and tumor cells have different cell death and cell cycle regulation than primary T cells. Thus, while Vif might well do all of the things described in the manuscript, it is a stretch to connect any of it to what happens in vivo. In the revised version, the authors now acknowledge this caveat.

(2) Line 109 and elsewhere. The ability of Vif to cause cell cycle arrest and bind PP2A subunits is not a completely conserved feature. Rather, it is quite variable in different HIV-1 strains. (e.g. https://doi.org/10.1016/j.bbrc.2020.04.123 and https://elifesciences.org/articles/53036). Therefore, it is necessary for the authors to quite clearly use strain designations in the manuscript rather than a generic "Vif", and to more clearly describe the viruses being used. In the revised version, the authors now make this more clear.

(3) Figure 5: This figure shows disruption of PP2A-B56 at the kinetochores. However, is this specific to the kinetochores? Since Vif has been described to more broadly degrade PP2A-B56, could this not be a result of a more general decrease in PP2A activity throughout the cell? In the revised version, the authors now clarify this point.

Reviewer #1 (Public review):

Summary:

This study examines to what extent this phenomenon varies based on the visibility of the saccade target. Visibility is defined as the contrast level of the target with respect to the noise background, and it is related to the signal-to-noise ratio of the target. A more visible target facilitates the oculomotor behavior planning and execution, however, as speculated by the authors, it can also benefit foveal prediction even if the foveal stimulus visibility is maintained constant. Remarkably, the authors show that presenting a highly visible saccade target is beneficial for foveal vision as detection of stimuli with an orientation similar to that of the saccade target is improved, the lower is the saccade target visibility, the less prominent is this effect.

Strengths:

The results are convincing and the research methodology is technically sound.

Weaknesses:

It is still unclear why the pre-saccadic enhancement would oscillate for targets with higher opacity levels, and what would be the benefit of this oscillatory pattern. The authors do not speculate too much on this and loosely relate it to feedback processes, which are characterized by neural oscillations in a similar range.

Reviewer #1 (Public review):

Summary:

In this work, the authors investigate the functional difference between the most commonly expressed form of PTH, and a novel point mutation in PTH identified in a patient with chronic hypocalcemia and hyperphosphatemia. The value of this mutant form of PTH as a potential anabolic agent for bone is investigated alongside PTH(1-84), which is a current anabolic therapy. The authors have achieved the aims of the study.

Strengths:

The work is novel, as it describes the function of a novel, naturally occurring, variant of PTH in terms of its ability to dimerise, to lead to cAMP activation, to increase serum calcium, and its pharmacological action compared to normal PTH.

[Editors' note: the original reviews are here, https://doi.org/10.7554/eLife.97579.1.sa1, and here, https://doi.org/10.7554/eLife.97579.2.sa1]

Reviewer #1 (Public review):

Summary:

The study describes the migration of epidermal keratinocytes through porous membranes and observes a unique size selection whereby only on 3-micron membrane are keratinocytes able to migrate and reform an intact epidermis. The authors propose that the model replicates three cell states of the intact epidermis, EMT, and MET. They also show that this response depends on the actin cytoskeleton and Piezo1, and the migration could be stimulated with TGFbeta ligands.

Strengths:

Strengths of the study include the establishment of a simple yet robust in vitro model that captures all three cell states, which could be useful for future investigation of wound healing or metastasis. There is also good characterisation of the pore size effects, providing some interesting observations about the physical regulation of keratinocyte migration. The images and presentation are clear.

Weaknesses:

(1) Some of the terminology would benefit from better definition or refinement. Triphasic suggests different physical behaviours (e.g. liquid-liquid phase separation) rather than cellular properties. Perhaps it would be better to refer to these as cell states or to describe the model more specifically as an invasion or EMT model. Likewise, the term 'reciprocating' implies two-way communication, but it is used to describe two-way migration or oscillating migration. Here, perhaps oscillatory would be clearer.

(2) The quantification and statistical analysis of key results could be improved. Notably, quantification of immunostaining in Figures 1 and 2 would strengthen core findings, and greater detail is needed on the sample sizes and number of experiments used for statistical analysis. These details are missing or only appear to N=1 in some places.

(3) There is an attempt to analyse the underlying molecular mechanisms, but these studies lack depth and detail. For example, it is not clear how actin, keratins, and piezo1 communicate to regulate cell migration. Are they acting directly on EMT genes such as SNAI1 or through changes in cell mechanics and cell-cell adhesions? Likewise, is TGF-beta signalling active in the system (e.g. nuclear pSMAD during cell migration)? As a result, the new biological insight is somewhat limited and confirms much of what is known about these pathways in keratinocyte migration.

Reviewer #1 (Public review):

Dixit, Noe, and Weikl apply coarse-grained and all-atom molecular dynamics to determine the response of the mechanosensitive proteins Piezo 1 and Piezo 2 proteins to tension. Cryo-EM structures in micelles show a high curvature of the protein whereas structures in lipid bilayers show lower curvature. Is the zero-stress state of the protein closer to the micelle structure or the bilayer structure? Moreover, while the tension sensitivity of channel function can be inferred from the experiment, molecular details are not clearly available. How much does the protein's height and effective area change in response to tension? With these in hand, a quantitative model of its function follows that can be related to the properties of the membrane and the effect of external forces.

Simulations indicate that in a bilayer the protein relaxes from the highly curved cryo-EM dome (Figure 1).

Under applied tension, the dome flattens (Figure 2) including the underlying lipid bilayer. The shape of the system is a combination of the membrane mechanical and protein conformational energies (Equation 1). The membrane's mechanical energy is well-characterized. It requires only the curvature and bending modulus as inputs. They determine membrane curvature and the local area metric (Equation 4) by averaging the height on a grid and computing second derivatives (Eqsuations 7, 8) consistent with known differential geometric formulas.

The bending energy can be limited to the nano dome but this implies that the noise in the membrane energy is significant. Where there is noise outside the dome there is noise inside the dome. At the least, they could characterize the noisy energy due to inadequate averaging of membrane shape.

My concern for this paper is that they are significantly overestimating the membrane deformation energy based on their numerical scheme, which in turn leads to a much stiffer model of the protein itself. Two things would address this:

(1) Report the membrane energy under different graining schemes (e.g., report schemes up to double the discretization grain).

(2) For a Gaussian bump with sigma=6 nm I obtained a bending energy of 0.6 kappa, so certainly in the ballpark with what they are reporting but significantly lower (compared to 2 kappa, Figure 5 lower left). It would be simpler to use the Gaussian approximation to their curves in Figure 3 - and I would argue more accurate, especially since they have not reported the variation of the membrane energy with respect to the discretization size and so I cannot judge the dependence of the energy on discretization. I view reporting the variation of the membrane energy with respect to discretization as being essential for the analysis if their goal is to provide a quantitative estimate for the force of Piezo. The Helfrich energy computed from an analytical model with a membrane shape closely resembling the simulated shapes would be very helpful. According to my intuition, finite-difference estimates of curvatures will tend to be overestimates of the true membrane deformation energy because white noise tends to lead to high curvature at short-length scales, which is strongly penalized by the bending energy.

The fitting of the system deformation to the inverse time appears to be incredibly ad hoc ... Nor is it clear that the quantified model will be substantially changed without extrapolation. The authors should either justify the extrapolation more clearly (sorry if I missed it!) or also report the unextrapolated numbers alongside the extrapolated ones.

In summary, this paper uses molecular dynamics simulations to quantify the force of the Piezo 1 and Piezo 2 proteins on a lipid bilayer using simulations under controlled tension, observing the membrane deformation, and using that data to infer protein mechanics. While much of the physical mechanism was previously known, the study itself is a valuable quantification. I identified one issue in the membrane deformation energy analysis that has large quantitative repercussions for the extracted model.

Reviewer #1 (Public review):

Summary:

The authors tried to identify the relationships among the gut microbiota, lipid metabolites, and the host in type 2 diabetes (T2DM) by using macaques that spontaneously develop T2DM, considered one of the best models of the human disease.

Strengths:

The authors comprehensively compared the gut microbiota and plasma fatty acids between macaques with spontaneous T2DM and control macaques and verified the results with macaques on a high-fat diet-fed mice model.

Weaknesses:

The observed multi-omics of the macaques can be done on humans, which weakens the impact of the conclusion of the manuscript.

In addition, the age and sex of the control macaque group did not necessarily match those of the T2DM group, leaving the possibility for compromising the analysis.

Regarding the metabolomic analysis, the authors did not include fecal samples which are important, considering the authors' claim about the importance of gut microbiota in the pathogenesis of T2DM.

In the mouse experiments, the control group should be given a FMT from control macaques rather than just untreated SPF mice since the fecal microbiota composition is likely very different between macaques and mice. Additionally, the palmitic acid-containing diets fed to mice to induce a diabetes-like condition do not mimic spontaneous T2DM in macaques.

Reviewer #1 (Public review):

First, the authors confirm the up-regulation of the main genes involved in the three branches of the Unfolded Protein Response (UPR) system in diet-induced obese mice in AT, observations that have been extensively reported before. Not surprisingly, IRE1a inhibition with STF led to an amelioration of the obesity and insulin resistance of the animals. Moreover, non-alcoholic fatty liver disease was also improved by the treatment. More novel are their results in terms of thermogenesis and energy expenditure, where IRE1a seems to act via activation of brown AT. Finally, mice treated with STF exhibited significantly fewer metabolically active and M1-like macrophages in the AT compared to those under vehicle conditions. Overall, the authors conclude that targeting IRE1a has therapeutical potential for treating obesity and insulin resistance.

The study has some strengths, such as the detailed characterization of the effect of STF in different fat depots and a thorough analysis of macrophage populations. However, the lack of novelty in the findings somewhat limits the study´s impact on the field.

Reviewer #2 (Public review):

Summary:

This manuscript reports a comparison of microbial traits and host response traits in a laboratory model of infected granuloma using Mtb strains from different lineages. The authors report increased bacillary growth and granuloma formation, inversely associated with T cell activation that is characterized by CXCL9, granzyme B and TNF expression. They therefore infer that these T cell responses are likely to be host-protective and that the greater virulence of modern Mtb lineages may be driven by their ability to avoid triggering these responses.

Strengths:

The comparison of multiple Mtb lineages in a granuloma model that enables evaluation of the potential role of multiple host cells in Mtb control, offers a valuable experimental approach to study the biological mechanisms that underpin differential virulence of Mtb lineages that has been previously reported in clinical and epidemiological studies.

Weaknesses:

The study is rather limited to descriptive observations, and lacks experiments to test causal relationships between host and pathogen traits. Some of the presentation of the data are difficult to interpret, and some conclusions are not adequately supported by the data.

Comments on revisions:

The authors have addressed my previous comments with appropriate revisions and explanations.

Reviewer #1 (Public review):

Summary:

In this work Ritchie and colleagues explore functional consequences of neuronal over-expression or deletion of the MAP3K DLK that their labs and others have strongly implicated in both axon degeneration, neuronal cell death, and axon regeneration. Their recent work in eLife (Li, 2021) showed that inducible over-expression of DLK (or the related LZK) induces neuronal death in the cerebellum. Here, they extend this work to show that inducible over-expression in Vglut1+ neuron also kills excitatory neurons in hippocampal CA1, but not CA3. They complement this very interesting finding with translatomics to quantify genes whose mRNAs are differentially translated in the context of DLK over-expression or knockout, the latter manipulation having little to no effect on the phenotypes measured. The authors note that several genes and pathways are differentially regulated according to whether DLK is over-expressed or knocked out. They note DLK-dependent changes in genes related to synaptic function and to the cytoskeleton and ultimately relate this in cultured neurons to findings that DLK over-expression negatively impacts synapse number and changes microtubules and neurites, though with a less obvious correlation.

Strengths:

Where this work represents a conceptual advance is in defining DLK-dependent changes in translation. Moreover, the finding that DLK may differentially impact neuronal death will become the basis for future studies exploring whether DLK contributes to differential neuronal susceptibility to death, which is a broadly important topic.

Comments on the latest version:

The addition of the P10 data is an important advance. With this, the authors have satisfactorily addressed the concerns that I raised.

Reviewer #1 (Public review):

The role of enteric glial cells in regulating intestinal mucosal functions at steady state has been a matter of debate in recent years. Enteric glial cell heterogeneity and related methodological differences likely underlie the contrasting findings obtained by different laboratories. Here, Prochera and colleagues used Plp1-CreERT2 driver mice to deplete the vast majority of enteric glia from the gut, and performed an elegant set of transcriptomic, microscopic and biochemical essays to examine the impact of enteric glia loss. It was found that enteric glia depletion has very limited effects on the transcriptome of gut cells 11 days after tamoxifen treatment (used to induce Diphtheria Toxin A expression in the majority of enteric glia including those present in the mucosa), and by extension - more specifically, has only minimal impact on cells of the intestinal mucosa. Interestingly, in the colon (where Paneth cells are not present) they did observe transcriptomic changes related to Paneth cell biology. Although no overt gene expression alterations were found in the small intestine - also not in Paneth cells - morphological, ultrastructural and functional changes were detected in the Paneth cells of enteric glia-depleted mice. In addition, and likely related to impaired Paneth cell secretory activity, enteric glia-depleted mice also show alterations in intestinal microbiota composition. This is an excellent study that convincingly demonstrates a role for enteric glia in supporting Paneth cells of the intestinal mucosa, suggesting that enteric glial cells shape host-microbiome interactions via the regulation of Paneth cell homeostasis.

Reviewer #1 (Public review):

Summary:

The article by Piersma et al. aims to reduce the complex process of NK cell licensing to the action of a single inhibitory receptor for MHC class I. This is achieved using a mouse strain lacking all of the Ly49 receptors expressed by NK cells and inserting the Ly49a gene into the Ncr1 locus, leading to expression on all the majority of NK cells.

Strengths:

The mouse model used represents a precise deletion of all NK-expressed genes within the Ly49 cluster. Re-introduction of the Ly49a gene into the Ncr1 locus allows expression by most NK cells. Convincing effects of Ly49a expression on in vitro activation and in vivo killing assay are shown.

Weaknesses:

The choice of Ly49a provides a clear picture of H-2Dd recognition by this Ly49. It would be valuable to perform additional studies investigating Ly49c and Ly49i receptors for H-2b. This is of interest because there are reports indicating that Ly49c may not be a functional receptor in B6 mice due to strong cis interactions. Investigation of the Ly49c and Ly49i receptors in this model would be the basis of future studies that are beyond the scope of the current report.

This work generates an excellent mouse model for the study of NK cell licensing by inhibitory Ly49s that will be useful for the community. It provides a platform whereby the functional activity of a single Ly49 can be assessed.

Comments on revisions: No additional concerns

Reviewer #1 (Public review):

Summary:

Zanetti et al use biophysical and cellular assays to investigate the interaction of the birnavirus VP3 protein with the early endosome lipid PI3P. The major novel finding is that association of the VP3 protein with an anionic lipid (PI3P) appears to be important for viral replication, as evidenced through a cellular assay on FFUs.

Strengths:

Support previously published claims that VP3 associates with early endosome membrane, potentially through binding to PI3P. The finding that mutating a single residue (R200) critically affects early endosome binding and that the same mutation also inhibits viral replication suggests a very important role for this binding in the viral life cycle.

Weaknesses:

The manuscript is relatively narrowly focused: the specifics of the bi-molecular interaction between the VP3 of an unusual avian virus and a host cell lipid (PIP3). Further, the affinity of this interaction is low and its specificity relative to other PIPs is not tested, leading to questions about whether VP3-PI3P binding is relevant.

Reviewer #1 (Public review):

Summary:

Hua et al show how targeting amino acid metabolism can overcome Trastuzumab resistance in HER2+ breast cancer.

Strengths:

The authors used metabolomics, transcriptomics and epigenomics approaches in vitro and in preclinical models to demonstrate how trastuzumab-resistant cells utilize cysteine metabolism.

Weaknesses:

However, there are some key aspects that needs to be addressed.

Major:

(1) Patient Samples for Transcriptomic Analysis: It is unclear from the text whether tumor tissues or blood samples were used for the transcriptomic analysis. This distinction is crucial, as these two sample types would yield vastly different inferences. The authors should clarify the source of these samples.

(2) The study only tested one trastuzumab-resistant and one trastuzumab-sensitive cell line. It is unclear whether these findings are applicable to other HER2-positive tumor cell lines, such as HCC1954. The authors should validate their results in additional cell lines to strengthen their conclusions.

(3) Relevance to Metastatic Disease: Trastuzumab resistance often arises in patients during disease recurrence, which is frequently associated with metastasis. However, the mouse experiments described in this paper were conducted only in the primary tumors. This article would have more impact if the authors could demonstrate that the combination of Erastin or cysteine starvation with trastuzumab can also improve outcomes in metastasis models.

Minor:

(1) The figures lack information about the specific statistical tests used. Including this information is essential to show the robustness of the results.

(2) Figure 3K Interpretation: The significance asterisks in Figure 3K do not specify the comparison being made. Are they relative to the DMSO control? This should be clarified.

Reviewer #1 (Public review):

In this manuscript, the authors report that GPR55 activation in presynaptic terminals of Purkinje cells decrease GABA release at the PC-DCN synapse. The authors use an impressive array of techniques (including highly challenging presynaptic recordings) to show that GPR55 activation reduces the readily releasable pool of vesicle without affecting presynaptic AP waveform and presynaptic Ca2+ influx. This is an interesting study, which is seemingly well-executed and proposes a novel mechanism for the control of neurotransmitter release. However, the authors' main conclusions are heavily, if not solely, based on pharmacological agents that most often than not demonstrate affinity at multiple targets. Below are points that the authors should consider in a revised version.

Major points:

(1) There is no clear evidence that GPR55 is specifically expressed in presynaptic terminals at the PC-DCN synapse. The authors cited Ryberg 2007 and Wu 2013 in the introduction, mentioning that GPR55 is potentially expressed in PCs. Ryberg (2007) offers no such evidence, and the expression in PC suggested by Wu (2013) does not necessarily correlate with presynaptic expression. The authors should perform additional experiments to demonstrate the presynaptic expression of GPR55 at PC-DCN synapse.

(2) The authors' conclusions rest heavily on pharmacological experiments, with compounds that are sometimes not selective for single targets. Genetic deletion of GPR55 would be a more appropriate control. The authors should also expand their experiments with occlusion experiments, showing if the effects of LPI are absent after AM251 or O-1602 treatment. In addition, the authors may want to consider AM281 as a CB1R antagonist without reported effects at GPR55.

(3) It is not clear how long the different drugs were applied, and at what time the recordings were performed during or following drug application. It appears that GPR55 agonists can have transient effects (Sylantyev, 2013; Rosenberg, 2023), possibly due to receptor internalization. The timeline of drug application should be reported, where IPSC amplitude is shown as a function of time and drug application windows are illustrated.

(4) A previous investigation on the role of GPR55 in the control of neurotransmitter release is not cited nor discussed Sylantyev et al., (2013, PNAS, Cannabinoid- and lysophosphatidylinositol-sensitive receptor GPR55 boosts neurotransmitter release at central synapses). Similarities and differences should be discussed.

Minor point:

(1) What is the source of LPI? What isoform was used? The multiple isoforms of LPI have different affinities for GPR55.

Reviewer #1 (Public review):

In their paper entitled "Combined transcriptomic, connectivity, and activity profiling of the medial amygdala using highly amplified multiplexed in situ hybridization (hamFISH)" Edwards et al. present a new method designated as hamFISH (highly amplified multiplexed in situ hybridization) that enables sequential detection of {less than or equal to}32 genes using multiplexed branched DNA amplification. As proof-of-principle, the authors apply the new technique - in conjunction with connectivity, and activity profiling - to the medial amygdala (MeA) of the mouse, which is a critical nucleus for innate social and defensive behaviors.

As mentioned by Edwards et al., hamFISH could prove beneficial as an affordable alternative to other in situ transcriptomic methods, including commercial platforms, that are resource-intensive and require complex analysis pipelines. Thus, the authors envision that the method they present could democratize in situ cell-type identification in individual laboratories.

The data presented by Edwards et al. is convincing. The authors use the appropriate and validated methodology in line with the current state-of-the-art. The paper makes a strong case for the benefits of hamFISH when combining transcriptomics studies with connectivity tracing and immediate early gene-based activity profiling. Notably, the authors also discuss the caveats and limitations of their study/approach in an open and transparent manner.

In its current state, the manuscript touches upon a number of most intriguing, yet rather preliminary findings. For example, the roles of inhibitory neuron cluster i3 or of the selective and apparently MeA neuron-specific projections (Figure 3 - Figure Supplement 2D) remain elusive. As it is the authors' prime intent to provide "a proof-of-principle example of overlaying transcriptomic types, projection, and activity in a behaviorally relevant manner and demonstrates the usefulness of hamFISH in multiplexed in situ gene expression profiling", such studies might be beyond the scope of the present manuscript. The absence of such more in-depth hypothesis-based analysis, however, prevents an even more enthusiastic overall assessment.

Reviewer #1 (Public review):

Summary:

This study experimentally examined diet-microbe-host interactions through a complex systems framework, centered on dietary oxalate. Multiple, independent molecular, animal, and in vitro experimental models were introduced into this research. The authors found that microbiome composition influenced multiple oxalate-microbe-host interfaces. Oxalobacter formigenes were only effective against a poor oxalate-degrading microbiota background and give critical new insights into why clinical intervention trials with this species exhibit variable outcomes. Data suggest that, while heterogeneity in the microbiome impacts multiple diet-host-microbe interfaces, metabolic redundancy among diverse microorganisms in specific diet-microbe axes is a critical variable that may impact the efficacy of bacteriotherapies, which can help guide patient and probiotic selection criteria in probiotic clinical trials.

Strengths:

The paper has made significant progress in both the depth and breadth of scientific research by systematically comparing multiple experimental methods across multiple dimensions. Particularly through in-depth analysis from the enzymatic perspective, it has not only successfully identified several key strains and redundant genes, which is of great significance for understanding the functions of enzymes, the characteristics of strains, and the mechanisms of genes in microbial communities, but also provided a valuable reference for subsequent experimental design and theoretical research.

More importantly, the establishment of a novel research approach to probiotics and gut microbiota in this paper represents a major contribution to the current research field. The proposal of this new approach not only breaks through the limitations of traditional research but also offers new perspectives and strategies for the screening, optimization of probiotics, and the regulation of gut microbiota balance. This holds potential significant value for improving human health and the prevention and treatment of related diseases.

Weaknesses:

While the study has excellently examined the overall changes in microbial community structure and the functions of individual bacteria, it lacks a focused investigation on the metabolic cross-feeding relationships between oxalate-degrading bacteria and related microorganisms, failing to provide a foundational microbial community or model for future research. Although this paper conducts a detailed study on oxalate metabolism, it would be beneficial to visually present the enrichment of different microbial community structures in metabolic pathways using graphical models.

Furthermore, the authors have done a commendable job in studying the roles of key bacteria. If the interactions and effects of upstream and downstream metabolically related bacteria could be integrated, it would provide readers with even more meaningful information. By illustrating how these bacteria interact within the metabolic network, readers can gain a deeper understanding of the complex ecological and functional relationships within microbial communities. Such an integrated approach would not only enhance the scientific value of the study but also facilitate future research in this area.

Reviewer #1 (Public review):

Summary:

This is a comprehensive study that clearly and deeply investigates the function of GATA6 in human early cardiac development.

Strengths:

This study combines hESC engineering, differentiation, detailed gene expression, genome occupancy, and and pathway modulation to elucidate the role of GATA6 in early cardiac differentiation. The work is carefully executed and the results support the conclusions. The use of publicly available data is well integrated throughout the manuscript. The RIME experiments are excellent.

Weaknesses:

Much has been known about GATA6 in mesendoderm development, and this is acknowledged by the authors.

Comments on revised version:

The authors have addressed my comments appropriately.

Reviewer #1 (Public review):

Summary:

The study by Pudlowski et al. investigates how the intricate structure of centrioles is formed by studying the role of a complex formed by delta- and epsilon-tubulin and the TEDC1 and TEDC2 proteins. For this they employ knockout cell lines, EM and ultrastructure expansion microscopy as well as pull-downs. Previous work has indicated a role of delta- and epsilon-tubulin in triplet microtubule formation. Without triplet microtubules centriolar cylinders can still form, but are unstable, resulting is futile rounds of de novo centriole assembly during S phase and disassembly during mitosis. Here the authors show that all four proteins function as a complex and knockout of any of the four proteins results in the same phenotype. They further find that mutant centrioles lack inner scaffold proteins and contain an extended proximal end including markers such as SAS6 and CEP135, suggesting that triplet microtubule formation is linked to limiting proximal end extension and formation of the central region that contains the inner scaffold. Finally, they show that mutant centrioles seem to undergo elongation during early mitosis before disassembly, although it is not clear if this may also be due to prolonged mitotic duration in mutants.

Strengths:

Overall this is a well-performed study, well presented, with conclusions supported by convincing data based on knockout cell lines, rescue experiments, and detailed quantifications.

Weaknesses:

Most weaknesses have been addressed in the revised version. The precise mapping of TED complex proteins to centrioles remains challenging with the available tools but has been addressed through the use of several complementary super-resolution techniques.

Reviewer #1 (Public review):

Summary:

Gene transfer agent (GTA) from Bartonella is a fascinating chimeric GTA that evolved from the domestication of two phages. Not much is known about how the expression of the BaGTA is regulated. In this manuscript, Korotaev et al noted the structural similarity between BrrG (a protein encoded by the ror locus of BaGTA) to a well-known transcriptional anti-termination factor, 21Q, from phage P21. This sparked the investigation into the possibility that BaGTA cluster is also regulated by anti-termination. Using a suite of cell biology, genetics, and genome-wide techniques (ChIP-seq), Korotaev et al convincingly showed that this is most likely the case. The findings offer the first insight into the regulation of GTA cluster (and GTA-mediated gene transfer) particularly in this pathogen Bartonella. Note that anti-termination is a well-known/studied mechanism of transcriptional control. Anti-termination is a very common mechanism for gene expression control of prophages, phages, bacterial gene clusters, and other GTAs, so in this sense, the impact of the findings in this study here is limited to Bartonella.

Strengths:

Convincing results that overall support the main claim of the manuscript.

Weaknesses:

A few important controls are missing.

Reviewer #1 (Public review):

Summary:

In this work, Huang et al used SMRT sequencing to identify methylated nucleotides (6mA, 4mC, and 5mC) in Pseudomonas syringae genome. They show that the most abundant modification is 6mA and they identify the enzymes required for this modification as when they mutate HsdMSR they observe a decrease of 6mA. Interestingly, the mutant also displays phenotypes of change in pathogenicity, biofilm formation, and translation activity due to a change in gene expression likely linked to the loss of 6mA.

Overall, the paper represents an interesting set of new data that can bring forward the field of DNA modification in bacteria.

Comments on revisions:

Thank you for the additional work. The authors have now addressed all my concerns.