Reviewer #3 (Public review):

Summary:

In their manuscript, Armand and colleagues investigate the potential of continuing CDK4/6 inhibitors or combining them with CDK2 inhibitors in the treatment of breast cancer that has developed resistance to initial therapy. Utilizing cellular and animal models, the research examines whether maintaining CDK4/6 inhibition or adding CDK2 inhibitors can effectively control tumor growth after resistance has set in. The key findings from the study indicate that the sustained use of CDK4/6 inhibitors can slow down the proliferation of cancer cells that have become resistant, and the combination of CDK2 inhibitors with CDK4/6 inhibitors can further enhance the suppression of tumor growth. Additionally, the study identifies that high levels of Cyclin E play a significant role in resistance to the combined therapy. These results suggest that continuing CDK4/6 inhibitors along with the strategic use of CDK2 inhibitors could be an effective strategy to overcome treatment resistance in hormone receptor-positive breast cancer.

Strengths:

(1) Continuous CDK4/6 Inhibitor Treatment Significantly Suppresses the Growth of Drug-Resistant HR+ Breast Cancer: The study demonstrates that the continued use of CDK4/6 inhibitors, even after disease progression, can significantly inhibit the growth of drug-resistant breast cancer.

(2) Potential of Combined Use of CDK2 Inhibitors with CDK4/6 Inhibitors: The research highlights the potential of combining CDK2 inhibitors with CDK4/6 inhibitors to effectively suppress CDK2 activity and overcome drug resistance.

(3) Discovery of Cyclin E Overexpression as a Key Driver: The study identifies overexpression of cyclin E as a key driver of resistance to the combination of CDK4/6 and CDK2 inhibitors, providing insights for future cancer treatments.

(4) Consistency of In Vitro and In Vivo Experimental Results: The study obtained supportive results from both in vitro cell experiments and in vivo tumor models, enhancing the reliability of the research.

(5) Validation with Multiple Cell Lines: The research utilized multiple HR+/HER2- breast cancer cell lines (such as MCF-7, T47D, CAMA-1) and triple-negative breast cancer cell lines (such as MDA-MB-231), validating the broad applicability of the results.

Weaknesses:

(1) The manuscript presents intriguing findings on the sustained use of CDK4/6 inhibitors and the potential incorporation of CDK2 inhibitors in breast cancer treatment. However, I would appreciate a more detailed discussion of how these findings could be translated into clinical practice, particularly regarding the management of patients with drug-resistant breast cancer.

(2) While the emergence of resistance is acknowledged, the manuscript could benefit from a deeper exploration of the molecular mechanisms underlying resistance development. A more thorough understanding of how CDK2 inhibitors may overcome this resistance would be valuable.

(3) The manuscript supports the continued use of CDK4/6 inhibitors, but it lacks a discussion on the long-term efficacy and safety of this approach. Additional studies or data to support the safety profile of prolonged CDK4/6 inhibitor use would strengthen the manuscript.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors set out to test the "force from lipids" mechanism of mechanosensitive channel gating, which posits that mechanical properties of the membrane are directly responsible for converting membrane tension into useful energy for channel gating. They employ amphiphilic polymers called poloxamers to alter membrane mechanical properties and relate those to the threshold of mechanical activation of the MscL channel of E.coli.

The authors heterologously express the channel, perform electrical recordings, and assess the mechanical properties of vesicles derived from the same membranes. This allows them to directly compare derived mechanical parameters to channel gating in the same environment.

They further repeat experiments in an eukaryotic mechano-channel and show that the same principles apply to gating in this very different protein, providing support for the force from lipids hypothesis.

Strengths:

In this work, characterization of the mechanical properties of the plasma membrane and electrical recordings of channel activity are carried out in membranes derived from the same cells. This is a nice contribution to these experiments since usually these two properties are measured in separate membranes with differing compositions. The experiments are of high quality and the data analysis and interpretation are careful.

Weaknesses:

It is not clear to this reviewer what the relationship is between the mechanical properties the authors measure, the membrane area expansion modulus, and bending rigidity, to what they call "interfacial tension".

Reviewer #3 (Public review):

Summary:

Avila and colleagues investigate the role of glutamate signaling in the dorsomedial striatum in a treadmill-based task where rats learn to turn or stop their walking based on learning cue-associations that allow them to acquire rewards. Phenotypic variation in Pavlovian conditioned sign and goal-tracking behavior was examined, where behavioral differences in stopping and turning were observed. Glutamate signals in the DMS were recording during the treadmill task, and were related to features of cue-controlled movement, with a stronger relationship seen for goal trackers. Finally, chemogenic inhibition of prelimbic neurons projecting to the DMS (the predicted source of those glutamate signals), preferentially affected cued movement in goal trackers. The authors couch these experiments in the context of cognitive control-attentional mechanisms, movement disorders, and individual differences in cue reactivity.

Strengths:

Overall these studies are interesting and are of general relevance to a number of research questions in neurology and psychiatry. The assessment of intersection of individual differences in cue-related learning strategies with movement-related questions - in this case cued turning behavior - is interesting and understudied question. The link between this work and growing notions of corticostriatal control of action selection makes it timely.

Weaknesses:

The clarity of the manuscript could be improved in several places, including in the graphical visualization of data. It is difficult to interpret the glutamate results, as presented, in the context of specific behaviors. It is difficult to assess how many trials/subjects are represented in the data shown, and too much emphasis is placed on representative examples. Averages traces of the glutamate data and other standard analysis approaches would improve the paper and allow for easier interpretation of the data.

Reviewer #3 (Public review):

In this project, Garber and Fiser examined how the structure of incidentally learned regularities influences subsequent learning of regularities, that either have the same structure or a different one. Over a series of six online experiments, it was found that the structure (spatial arrangement) of the first set of regularities affected learning of the second set, indicating that it has indeed been abstracted away from the specific items that have been learned. The effect was found to depend on the explicitness of the original learning: Participants who noticed regularities in the stimuli were better at learning subsequent regularities of the same structure than of a different one. On the other hand, participants whose learning was only implicit had an opposite pattern: they were better in learning regularities of a novel structure than of the same one. However, when an overnight sleep separated the first and second learning phases, this opposite effect was reversed and came to match the pattern of the explicit group, suggesting that the abstraction and transfer in the implicit case were aided by memory consolidation.

In their revision the authors addressed my major comments successfully and I commend them for that.

Reviewer #3 (Public review):

The manuscript by Talbi R et al. generated transgenic mice to assess the reproduction function of MC4R in Kiss1 neurons in vivo and used electrophysiology to test how MC4R activation regulated Kiss1 neuronal firing in ARH and AVPV/PeN. This timely study is highly significant in neuroendocrinology research for the following reasons.

(1) The authors' findings are significant in the field of reproductive research. Despite the known presence of MC4R signaling in Kiss1 neurons, the exact mechanisms of how MC4R signaling regulates different Kiss1 neuronal populations in the context of sex hormone fluctuations are not entirely understood. The authors reported that knocking out Mc4r from Kiss1 neurons replicates the reproductive impairment of MC4RKO mice, and Mc4r expression in Kiss1 neurons in the MC4R null background partially restored the reproductive impairment. MC4R activation excites Kiss1 ARH neurons and inhibits Kiss1 AVPV/PeN neurons (except for elevated estradiol).

(2) Reproduction dysfunction is one of obesity comorbidities. MC4R loss-of-function mutations cause obesity phenotype and impaired reproduction. However, it is hard to determine the causality. The authors carefully measured the body weight of the different mouse models (Figure 1C, Figure 2A, Figure 3B). For example, the Kiss1-MC4RKO females showed no body weight difference at puberty onset. This clearly demonstrated the direct function of MC4R signaling in reproduction but was not a consequence of excessive adiposity.

(3) Gene expression findings in the "KNDy" system align with the reproduction phenotype.

(4) The electrophysiology results reported in this manuscript are innovative and provide more details of MC4R activation and Kiss1 neuronal activation.

Overall, the authors have presented sufficient background in a clear, logical, and organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, and made a justified conclusion.

Comments on revisions:

The authors have addressed my comments.

Reviewer #3 (Public review):

Summary:

The authors aim to explore whether other subunits besides MED1 exert specific functions during the process of terminal erythropoiesis with global gene repression, and finally they demonstrated that MED26-enriched condensates drive erythropoiesis through modulating transcription pausing.

Strengths:

Through both in vitro and in vivo models, the authors showed that while MED1 and MED26 co-occupy a plethora of genes important for cell survival and proliferation at the HSPC stage, MED26 preferentially marks erythroid genes and recruits pausing-related factors for cell fate specification. Gradually, MED26 becomes the dominant factor in shaping the composition of transcription condensates and transforms the chromatin towards a repressive yet permissive state, achieving global transcription repression in erythropoiesis.

Weaknesses:

In the in vitro model, the author only used CD34+ cell-derived erythropoiesis as the validation, which is relatively simple, and more in vitro erythropoiesis models need to be used to strengthen the conclusion.

Reviewer #3 (Public review):

This study was focused on the conserved mechanisms across the Transmembrane Channel/Scramblase superfamily, which includes members of the TMEM16, TMEM63/OSCA, and TMC families. In previous work, the authors have studied the role of the inner activation gate of these proteins. Here, the authors show that the introduction of mutations at the TM4-TM6 interface, which are close to the inactivation gate, can disrupt gating and confer scramblase activity to non-scramblases proteins.

Overall, the confocal imaging experiments, patch clamping experiments, and data analysis are performed well and in line with standard methods. The molecular dynamics simulation work is focused but adds supportive evidence to their findings. Although there could have been more extensive molecular analysis to bolster the authors' arguments on the role of the TM4-TM6 interface (e.g. evaluate effects of size/hydrophobicity, double mutants, cross-linking, more in-depth simulation data), there is adequate evidence to conclude that certain residues at this interface is critical to ion conduction and phospholipid scramblase activity. The data presented only adds incremental depth of knowledge for each individual channel, but together, they show this to be true for conserved TM4 residues across TMEM16F, TMEM16A, OSCA1.2, and TMEM63A proteins. This breadth of data is a major strength of this paper, and provides strong evidence for a coupled pathway for ion conduction and phospholipid transport, though the underlying biophysical mechanism is still speculative and remains to be elucidated.

Reviewer #3 (Public review):

Summary:

The previously described reporter SPOTlight is a fluorescence-based reporter of the integrated stress response, specifically, protein synthesis initiation dynamics. In the current study from the same lab, the authors describe the creation and characterization of a transgenic mouse that expresses SPOTlight.

Strengths:

The previously described reporter has now been made into a Cre-dependent transgene in mice. The authors replicate previous findings from their lab that were acquired using viral vector-mediated delivery of their reporter.

Weaknesses:

There is not a clear advantage to having the Cre-dependent SPOTlight reporter in a transgenic mouse over using a viral vector to deliver the same Cre-dependent SPOTlight based on the experiments presented. There are potential general advantages and disadvantages to virus vs transgenic mouse but no side-by-side comparisons are performed here.

It is not clear whether overexpressing the reporter alters basal ISR/UPR function and gene expression. The CAG is a strong promoter and overexpression of fluorescent proteins (or any protein) can potentially stress protein synthesis and processing mechanisms. The use of the animal as a reporter may be misleading if the presence of the reporter is already altering ISR/UPR.

Reviewer #3 (Public review):

The authors were interested in how Ankle2 regulates nuclear envelope reformation after cell division. They show that Ankle2 can bind in a PP2A complex without other known regulatory subunits of PP2A. The authors also identity a novel interaction with ER protein Vap33 that could be important for localization. This manuscript is a useful finding linking Ankle2 function during nuclear envelope reformation to the PP2A complex. The authors present solid data showing that Ankle2 can form a complex with PP2A-29B and Mts and generate a phosphoproteomic resource that is fundamentally important to understand Ankle2 biology. The caveat should be remembered that most experiments, including subcellular localization, are based on overexpression data. Keeping this in mind, the manuscript is a valuable resource.

Reviewer #3 (Public review):

Summary:

In this study, McAllester and Pool develop a new model to explain the maintenance of balanced inversion polymorphism, based on (sexually) antagonistic alleles and a trade-off between male reproduction and survival (in females or both sexes). Simulations of this model support the plausibility of this mechanism. In addition, the authors use experiments on four naturally occurring inversion polymorphisms in D. melanogaster and find tentative evidence for one aspect of their theoretical model, namely the existence of the above-mentioned trade-off in two out of the four inversions.

Strengths:

(1) The study develops and analyzes a new (Drosophila melanogaster-inspired) model for the maintenance of balanced inversion polymorphism, combining elements of (sexually) antagonistically (pleiotropic) alleles, negative frequency-dependent selection and synergistic epistasis. Simulations of the model suggest that the hypothesized mechanism might be plausible.

(2) The above-mentioned model assumes, as a specific example, a trade-off between male reproductive display and survival; in the second part of their study, the authors perform laboratory experiments on four common D. melanogaster inversions to study whether these polymorphisms may be subject to such a trade-off. The authors observe that two of the four inversions show suggestive evidence that is consistent with a trade-off between male reproduction and survival.

Open issues:

(1) A gap in the current modeling is that, while a diploid situation is being studied, the model does not investigate the effects of varying degrees of dominance. It would thus be important and interesting, as the authors mention, to fill this gap in future work,

(2) It will also be important to further explore and corroborate the potential importance and generality of trade-offs between different fitness components in maintaining inversion polymorphisms in future work.

Reviewer #3 (Public review):

Summary:

The migration of primordial germ cells (PGCs) to the developing gonad is a poorly understood, yet essential step in reproductive development. Here, the authors examine whether there are differences in leading and lagging migratory PGCs using single-cell RNA sequencing of mouse embryos. Cleverly, the authors dissected embryonic trunks along the anterior-to-posterior axis prior to scRNAseq in order to distinguish leading and lagging migratory PGCs. After batch corrections, their analyses revealed several known and novel differences in gene expression within and around leading and lagging PGCs, intercellular signaling networks, as well as number of genes upregulated upon gonad colonization. The authors then compared their datasets with publicly available human datasets to identify common biological themes. Altogether, this rigorous study reveals several differences between leading and lagging migratory PGCs, hints at signatures for different fates among the population of migratory PGCs, and provides new potential markers for post-migratory PGCs in both humans and mice. While many of the interesting hypotheses that arise from this work are not extensively tested, these data provide a rich platform for future investigations.

Strengths:

-The authors have successfully navigated significant technical challenges to obtain a substantial number of mouse migratory primordial germ cells for robust transcriptomic analysis. Here the authors were able to collect quality data on ~13,000 PGCs and ~7,800 surrounding somatic cells, which is ten times more PGCs than previous studies.

- The decision to physically separate leading and lagging primordial germ cells was clever and well-validated based on expected anterior-to-posterior transcriptional signatures.

- Within the PGCs and surrounding tissues, the authors found many gene expression dynamics they would expect to see both along the PGC migratory path as well as across developmental time, increasing confidence in the new differentially expressed genes they found.

- The comparison of their mouse-based migratory PGC datasets with existing human migratory PGC datasets is appreciated.

- The quality control, ambient RNA contamination elimination, batch correction, cell identification and analysis of scRNAseq data were thorough and well-done such that the new hypotheses and markers found through this study are dependable.

- The subsetting of cells in their trajectory analysis is appreciated, further strengthening their cell terminal state predictions.

Weaknesses:

- Although it is useful to compare their mouse-based dataset with human datasets, the authors used two different analysis pipelines for each dataset. While this may have been due to the small number of cells in the human dataset as mentioned, it does make it difficult to compare them.

- There were few validation experiments within this study. For one such experiment, whether there is a difference in pSMAD2/3 along the AP axis is unclear and not quantified as was nicely done for Lefty1/2.

Reviewer #3 (Public review):

Summary:

The paper describes the molecular pathway to regulate germ cell differentiation in zebrafish through ribosomal RNA biogenesis. Meioc sequesters Piwil1, a Piwi homolog, which suppresses the transcription of the 45S pre-rDNA by the formation of heterochromatin, to the perinuclear bodies. The key results are solid and useful to researchers in the field of germ cell/meiosis as well as RNA biosynthesis and chromatin.

Strengths:

The authors nicely provided the molecular evidence on the antagonism of Meioc to Piwil1 in the rRNA synthesis, which supported by the genetic evidence that the inability of the meioc mutant to enter meiosis is suppressed by the piwil1 heterozygosity.

Weaknesses:

(1) Although the paper provides very convincing evidence for the authors' claim, the scientific contents are poorly written and incorrectly described. As a result, it is hard to read the text. Checking by scientific experts would be highly recommended. For example, on line 38, "the global translation activity is generally [inhibited]", is incorrect and, rather, a sentence like "the activity is lowered relative to other cells" is more appropriate here. See minor points for more examples.<br /> (2) In some figures, it is hard for readers outside of zebrafish meiosis to evaluate the results without more explanation and drawing.<br /> (3) Figure 1E, F, cycloheximide experiments: Please mention the toxicity of the concentration of the drug in cell proliferation and viability.

Reviewer #3 (Public review):

Summary:

The authors provide an in-depth analysis of the function of Numb in adult Drosophila midgut. Based on RNAi combinations and double mutant clonal analyses, they propose that Numb has a function in inhibiting Notch pathway to maintain intestinal stem cells, and is a backup mechanism with BMP pathway in maintaining midgut stem cell mediated homeostasis.

Strengths:

Overall, this is a carefully constructed series of experiments, and the results and statistical analyses provides believable evidence that Numb has a role, albeit weak compared to other pathways, in sustaining ISC and in promoting regeneration especially after damage by bleomycin, which may damage enterocytes and therefore disrupt BMP pathway more. The results overall support their claim.

The data are highly coherent, and support a genetic function of Numb, in collaborating with BMP signaling, to maintain the number and proliferative function of ISCs in adult midguts. The authors used appropriate and sophisticated genetic tools of double RNAi, mutant clonal analysis and dual marker stem cell tracing approaches to ensure the results are reproducible and consistent. The statistical analyses provide confidence that the phenotypic changes are reliable albeit weaker than many other mutants previously studied.

Weaknesses:<br /> In the absence of Numb itself, the midgut has a weak reduction of ISC number (Fig. 3 and 5), as well as weak albeit not statistically significant reduction of ISC clone size/proliferation. I think the authors published similar experiments with BMP pathway mutants. The mad1-2 allele used here as stated below may not be very representative of other BMP pathway mutants. Therefore, it could be beneficial to compare the number of ISC number and clone sizes between other BMP experiments to provide the readers with a clearer picture of how these two pathways individually contribute (stronger/weaker effects) to the ISC number and gut homeostasis.

The main weakness of this manuscript is the analysis of the BMP pathway components, especially the mad1-2 allele. The mad RNAi and mad1-2 alleles (P insertion) are supposed to be weak alleles and that might be suitable for genetic enhancement assays here together with numb RNAi. However, the mad1-2 allele, and sometimes the mad RNAi, showed weakly increased ISC clone size. This is kind of counter-intuitive that they should have a similar ISC loss and ISC clone size reduction.

A much stronger phenotype was observed when numb mutants were subject to treatment of tissue damaging agents Bleomycin, which causes damage in different ways than DSS. Bleomycin as previously shown to be causing mainly enterocyte damage, and therefore disrupt BMP signaling from ECs more likely. Therefore, this treatment together with loss of numb led to a highly significant reduction of ISC in clones and reduction of clone size/proliferation. One improvement is that it is not clear whether the authors discussed the nature of the two numb mutant alleles used in this study and the comparison to the strength of the RNAi allele. Because the phenotypes are weak and more variable, the use of specific reagents is important.

Furthermore, the use of possible activating alleles of either or both pathways to test genetic enhancement or synergistic activation will provide strong support for the claims.

Reviewer #3 (Public review):

Summary:<br /> This study advances the field of β cell dysfunction by unveiling an epigenetic mechanism of β cell senescence. By connecting TET2-mediated DNA methylation to histone acetylation and cellular aging, it opens promising new avenues for therapeutic intervention. In particular, the authors aimed at identifying the mechanisms of pancreatic β cell senescence by epigenetic regulation. They conclude that increased TET2 expression in β cells is associated with ageing and metabolic dysfunction in type 2 diabetes by inducing β cell senescence. The authors further propose that TET2-mediated PTEN promoter methylation promotes β cell senescence by regulating H4K16ac. Last, the authors suggest that this could represent new molecular mechanism and therapeutic target against β cell senescence during type 2 diabetes.

Strengths:<br /> The major strengths of the study are the use of both biased and unbiased experimental tools to approach the topic. The authors also provide in vivo and in vitro mechanistic approaches to answer their questions. All of these approaches are valuable and provides robustness to their study. The authors provide solid evidence that TET2 is associated with ageing and that its absence improves glucose metabolism in ageing and β cell senescence. In addition, the mechanistic studies showing that TET2 regulates the PTEN/MOF/H4K16ac signaling pathway in β cell lines is convincing.

Weaknesses:<br /> Although the use of such a variety of tools is a strength, the outcome of each individual tool is somehow superficial. For instance, the authors focus on very specific targets emanating from their omics studies without a clear or logical justification. In addition, the metabolic studies are inaccurate and the authors do not follow an understandable and rational examination of the ageing versus their obesity cohorts. Last, the mechanistic studies using model cell lines are not validated in the available mouse models.

In my opinion, the evidence that TET2 regulates β cell senescence during obesity is not very strong. This is because the effect of deletion of TET2 in senescence markers is the same under 24weeks of age or 52 weeks of age (16 weeks HFD). Both ageing and HFD promoted the same extent of reduction of senescent markers and increase in β cell markers in the absence of TET2. There is no comparison between young glucose tolerant mice and old glucose intolerant mice. There is also no direct comparison of aged matched lean or obese mice. It may seem as if the mechanism by which TET2 regulates senescence in β cells is independent of the diabetic status but it is more related to ageing. Given that there is evidence that TET2 expression in β cells coordinates inflammatory responses in autoimmune diabetes, it would have been interested to check whether this is also the case for T2DM. Also, considering that expression of TET2 in Figure 3 does not seem to be in β cells in db/db mice but rather in the exocrine pancreas. In addition, senescent marker p16 in Figure 5 in the presence of TET2, seems to be localized in alpha cells or immune cells but not in β cells.<br /> Regarding the mechanistic studies, the authors convincingly show that TET2 regulates the PTEN/MOF/H4K16ac signaling pathway in β cell lines and that this is important for β cell senescence. However, there is no validation of whether this holds true in aged, or prediabetic, mice. Given the availability of mice and model samples, this should be possible and meaningful. Last, in the genome-wide bisulfite sequencing (Figure 7), it seems that the authors are cherry picking for PTEN and in the RNAseq, the same applies for MOF. Thus, although the mechanism seems valid, the lack of in vivo validation, and a proper rational for the selected targets in the omics studies, renders the mechanistic studies rather correlative.

In sum, I believe that the study in its current version, unfortunately, does not bear the conceptual advance or the robustness that is required to offer a strong impact on the field. The methods, on the other hand, mainly the omics analyses provided here, could be of potential benefit for the field of epigenetics in β cell biology. However, in the benefit of the current study, the relevance of this data could be more rigorously assessed experimentally. I believe that the study has the potential to provide the required impact, should the authors work on it further to provide more solid functional and mechanistic validation.

Reviewer #3 (Public review):

Summary:

In their paper the authors tackle three things at once in a theoretical model: how can spiking neural networks perform efficient coding, how can such networks limit the energy use at the same time, and how can this be done in a more biologically realistic way than previous work.

They start by working from a long-running theory on how networks operating in a precisely balanced state can perform efficient coding. First, they assume split networks of excitatory (E) and inhibitory (I) neurons. The E neurons have the task to represent some lower dimensional input signal, and the I neurons have the task to represent the signal represented by the E neurons. Additionally, the E and I populations should minimize an energy cost represented by the sum of all spikes. All this results in two loss functions for the E and I populations, and the networks are then derived by assuming E and I neurons should only spike if this improves their respective loss. This results in networks of spiking neurons that live in a balanced state, and can accurately represent the network inputs.

They then investigate in depth different aspects of the resulting networks, such as responses to perturbations, the effect of following Dale's law, spiking statistics, the excitation (E)/inhibition (I) balance, optimal E/I cell ratios, and others. Overall, they expand on previous work by taking a more biological angle on the theory and show the networks can operate in a biologically realistic regime.

Strengths:

* The authors take a much more biological angle on the efficient spiking networks theory than previous work, which is an essential contribution to the field<br /> * They make a very extensive investigation of many aspects of the network in this context, and do so thoroughly<br /> * They put sensible constraints on their networks, while still maintaining the good properties these networks should have

Weaknesses:

* One of the core goals of the paper is to make a more biophysically realistic network than previous work using similar optimization principles. One of the important things they consider is a split into E and I neurons. While this works fine, and they consider the coding consequences of this, it is not clear from an optimization perspective why the split into E and I neurons and following Dale's law would be beneficial. This would be out of scope for the current paper however.<br /> * The theoretical advances in the paper are not all novel by themselves, as most of them (in particular the split into E and I neurons and the use of biophysical constants) had been achieved in previous models. However, the authors discuss these links thoroughly and do more in-depth follow-up experiments with the resulting model.

Assessment and context:

Overall, although much of the underlying theory is not necessarily new, the work provides an important addition to the field. The authors succeeded well in their goal of making the networks more biologically realistic, and incorporate aspects of energy efficiency. For computational neuroscientists this paper is a good example of how to build models that link well to experimental knowledge and constraints, while still being computationally and mathematically tractable. For experimental readers the model provides a clearer link of efficient coding spiking networks to known experimental constraints and provides a few predictions.

Reviewer #3 (Public review):

Summary:

Rollenhagen at al. offer a detailed description of layer 1 of the human neocortex. They use electron microscopy to assess the morphological parameters of presynaptic terminals, active zones, vesicle density/distribution, mitochondrial morphology and astrocytic coverage. The data is collected from tissue from four patients undergoing epilepsy surgery. As the epileptic focus was localized in all patients to the hippocampus, the tissue examined in this manuscript is considered non-epileptic (access) tissue.

Strengths:

The quality of the electron microscopic images is very high, and the data is analyzed carefully. Data from human tissue is always precious and the authors here provide a detailed analysis using adequate approaches, and the data is clearly presented.

Weaknesses:

The text connects functional and morphological characteristics in a very direct way. For example, connecting plasticity to any measurement the authors present would be rather difficult without any additional functional experiments. References to various vesicle pools based on the location of the vesicles is also more complex than it is suggested in the manuscript. The text should better reflect the limitations of the conclusions that can be drawn from the authors' data.

Reviewer #3 (Public review):

Summary:

This paper uses a new chemogenetic tool to investigate the role of cerebellar Purkinje cells in postural control. Using a high-throughput behavioral assay, they show that activation or ablation of Purkinje cells affects various aspects of postural control in zebrafish larvae during spontaneous swimming, and that the effects are more pronounced at later developmental time points, where the Purkinje cell number is much greater. Using a sophisticated imaging assay, they record Purkinje cell activity in response to tilt of the fish, and show that some Purkinje cells are tuned to tilt direction, and that the direction can even be decoded from untuned neurons.

Strengths:

Overall the study is nice, using a variety of genetic tools and behavioral analysis to address a fundamental question about the role of the cerebellum in postural control in fish

Reviewer #3 (Public review):

This study is a part of the ongoing series of rigorous work from this group exploring neural coding deficits in the auditory nerve, and dissociating the effects of cochlear synaptopathy from other age-related deficits. They have previously shown no evidence of phase-locking deficits in the remaining auditory nerve fibers in quiet-aged gerbils. Here, they study the effects of aging on the perception and neural coding of temporal fine structure cues in the same Mongolian gerbil model.

They measure TFS coding in the auditory nerve using the TFS1 task which uses a combination of harmonic and tone-shifted inharmonic tones which differ primarily in their TFS cues (and not the envelope). They then follow this up with a behavioral paradigm using the TFS1 task in these gerbils. They test young normal hearing gerbils, aged gerbils, and young gerbils with cochlear synaptopathy induced using the neurotoxin ouabain to mimic synapse losses seen with age.

In the behavioral paradigm, they find that aging is associated with decreased performance compared to the young gerbils, whereas young gerbils with similar levels of synapse loss do not show these deficits. When looking at the auditory nerve responses, they find no differences in neural coding of TFS cues across any of the groups. However, aged gerbils show an increase in the representation of periodicity envelope cues (around f0) compared to young gerbils or those with induced synapse loss. The authors hence conclude that synapse loss by itself doesn't seem to be important for distinguishing TFS cues, and rather the behavioral deficits with age are likely having to do with the misrepresented envelope cues instead.

The manuscript is well written, and the data presented are robust. Some of the points below will need to be considered while interpreting the results of the study, in its current form. These considerations are addressable if deemed necessary, with some additional analysis in future versions of the manuscript.

Spontaneous rates - Figure S2 shows no differences in median spontaneous rates across groups. But taking the median glosses over some of the nuances there. Ouabain (in the Bourien study) famously affects low spont rates first, and at a higher degree than median or high spont rates. It seems to be the case (qualitatively) in Figure S2 as well, with almost no units in the low spont region in the ouabain group, compared to the other groups. Looking at distributions within each spont rate category and comparing differences across the groups might reveal some of the underlying causes for these changes. Given that overall, the study reports that low-SR fibers had a higher ENV/TFS log-z-ratio, the distribution of these fibers across groups may reveal specific effects of TFS coding by group.

Threshold shifts - It is unclear from the current version if the older gerbils have changes in hearing thresholds, and whether those changes may be affecting behavioral thresholds. The behavioral stimuli appear to have been presented at a fixed sound level for both young and aged gerbils, similar to the single unit recordings. Hence, age-related differences in behavior may have been due to changes in relative sensation level. Approaches such as using hearing thresholds as covariates in the analysis will help explore if older gerbils still show behavioral deficits.

Task learning in aged gerbils - It is unclear if the aged gerbils really learn the task well in two of the three TFS1 test conditions. The d' of 1 which is usually used as the criterion for learning was not reached in even the easiest condition for aged gerbils in all but one condition for the aged gerbils (Fig. 5H) and in that condition, there doesn't seem to be any age-related deficits in behavioral performance (Fig. 6B). Hence dissociating the inability to learn the task from the inability to perceive TFS 1 cues in those animals becomes challenging.

Increased representation of periodicity envelope in the AN - the mechanisms for increased representation of periodicity envelope cues is unclear. The authors point to some potential central mechanisms but given that these are recordings from the auditory nerve what central mechanisms these may be is unclear. If the authors are suggesting some form of efferent modulation only at the f0 frequency, no evidence for this is presented. It appears more likely that the enhancement may be due to outer hair cell dysfunction (widened tuning, distorted tonotopy). Given this increased envelope coding, the potential change in sensation level for the behavior (from the comment above), and no change in neural coding of TFS cues across any of the groups, a simpler interpretation may be -TFS coding is not affected in remaining auditory nerve fibers after age-related or ouabain induced synapse loss, but behavioral performance is affected by altered outer hair cell dysfunction with age.

Emerging evidence seems to suggest that cochlear synaptopathy and/or TFS encoding abilities might be reflected in listening effort rather than behavioral performance. Measuring some proxy of listening effort in these gerbils (like reaction time) to see if that has changed with synapse loss, especially in the young animals with induced synaptopathy, would make an interesting addition to explore perceptual deficits of TFS coding with synapse loss.

Reviewer #3 (Public review):

Summary:

The authors employ a series of well-conceived and well-executed experiments involving photometric imaging of the dentate gyrus and raphe nucleus, as well as cell-type specific genetic manipulations of serotonergic receptors that together serve to directly implicate serotonergic regulation of dentate gyrus (DG) granule (GC) and mossy cell (MC) activity in association with an infra slow oscillation (ISO) of neural activity has been previously linked to general cortical regulation during NREM sleep and microarousals.

Strengths:

There are a number of novel and important results, including the modulation of dentage granule cell activity by the infraslow oscillation during NREM sleep, the selective association of different subpopulations of granule cells to microarousals (MA), the anticorrelation of raphe activity with infraslow dentate activity.

The discussion includes a general survey of ISOs and recent work relating to their expression in other brain areas and other potential neuromodulatory system involvement, as well as possible connections with infraslow oscillations, micro arousals, and sensory sensitivity.

Weaknesses:

- The behavioral results showing contextual memory impairment resulting from 5-HT1a knockdown are fine, but are over-interpreted. The term memory consolidation is used several times, as well as references to sleep-dependence. This is not what was tested. The receptor was knocked down, and then 2 weeks later animals were found to have fear conditioning deficits. They can certainly describe this result as indicating a connection between 5-HT1a receptor function and memory performance, but the connection to sleep and consolidation would just be speculation. The fact that 5-HT1a knockdown also impacted DG ISOs does not establish dependency. Some examples of this are:<br /> o The final conclusion asserts "Together, our study highlights the role of neuromodulation in organizing neuronal activity during sleep and sleep-dependent brain functions, such as memory.", but the reported memory effects (impairment of fear conditioning) were not shown to be explicitly sleep-dependent.<br /> o Earlier in the discussion it mentions "Finally, we showed that local genetic ablation of 5-HT1a receptors in GCs impaired the ISO and memory consolidation". The effect shown was on general memory performance - consolidation was not specifically implicated.

- The assertion on page 9 that the results demonstrate "that the 5-HT is directly acting in the DG to gate the oscillations" is a bit strong given the magnitude of effect shown in Fig. 6D, and the absence of demonstration of negative effect on cortical areas that also show ISO activity and could impact DG activity (see requested cortical sigma power analysis).

- Recent work has shown that abnormal DG GC activity can result from the use of the specific Ca indicator being used (GCaMP6s). (Teng, S., Wang, W., Wen, J.J.J. et al. Expression of GCaMP6s in the dentate gyrus induces tonic-clonic seizures. Sci Rep 14, 8104 (2024). https://doi.org/10.1038/s41598-024-58819-9). The authors of that study found that the effect seemed to be specific to GCaMP6s and that GCaMP6f did not lead to abnormal excitability. Note this is of particular concern given similar infraslow variation of cortical excitability in epilepsy (cf Vanhatalo et al. PNAS 2004). While I don't think that the experiments need to be repeated with a different indicator to address this concern, you should be able to use the 2p GCaMP7 experiments that have already been done to provide additional validation by repeating the analyses done for the GCaMP6s photometry experiments. This should be done anyway to allow appropriate comparison of the 2p and photometry results.

- While the discussion mentions previous work that has linked ISOs during sleep with regulation of cortical oscillations in the sigma band, oddly no such analysis is performed in the current work even though it is presumably available and would be highly relevant to the interpretation of a number of primary results including the relationship between the ISOs and MAs observed in the DG and similar results reported in other areas, as well as the selective impact of DG 5-HT1a knockdown on DG ISOs. For example, in the initial results describing the cross correlation of calcium activity and EMG/EEG with MA episodes (paragraph 1, page 4), similar results relating brief arousals to the infraslow fluctuation in sleep spindles (sigma band) have been reported also at .02 Hz associated with variation in sensory arousability (cf. Cardis et al., "Cortico-autonomic local arousals and heightened somatosensory arousability during NREMS of mice in neuropathic pain", eLife 2021). It would be important to know whether the current results show similar cortical sigma band correlations. Also, in the results on ISO attenuation following 5-HT1 knockdown on page 7 (fig. 6), how is cortical EEG affected? is ISO still seen in EEG but attenuated in DG?

- The illustrations of the effect of 5-HT1a knockdown shown in Figure 6 are somewhat misleading. The examples in panels B and C show an effect that is much more dramatic than the overall effect shown in panel D. Panels B and C do not appear to be representative examples. Which of the sample points in panel D are illustrated in panels B, C? it is not appropriate to arbitrarily select two points from different animals for comparison, or worse, to take points from the extremes of the distributions. If the intent is to illustrate what the effect shown in D looks like in the raw data, then you need to select examples that reflect the means shown in panel D. It is also important to show the effect on cortical EEG, particularly in sigma band to see if the effects are restricted to the DG ISOs. It would also be helpful to show that MAs and their correlations as shown in Fig 1 or G as well as broader sleep architecture are not affected.

- On page 9 of the results it states that GCs and MCs are upregulated during NREM and their activity is abruptly terminated by MAs through a 5-HT mediated mechanism. I didn't see anything showing the 5-HT dependence of the MA activity correlation. The results indicate a reduction in ISO modulation of GC activity but not the MA correlated activity. I would like to see the equivalent of Fig 1,2 G panels with the 5-HT1a manipulation.

Reviewer #4 (Public review):

The work by Yingying Chen, Jeong Han Lee, and co-authors summarizes the morphological and functional outcomes of Cldn9 loss in the inner ear, particularly in the organ of Corti. While the study does not provide mechanistic insights into how the developmental loss of Cldn9 leads to ectopic hair cell formation, the phenomenon itself is curious. The work primarily focuses on a detailed characterization of the ectopic hair cells, which is well done. Despite the lack of mechanistic insights, the study will be of interest to the inner ear field if several major issues with the manuscript are addressed.

(1) The title, "Genetic and pharmacologic alterations of claudin9 levels suffice to induce functional and mature inner hair cells," is misleading. First, both manipulations (knockout and knockdown) are genetic, and no pharmacology is involved. Second, both manipulations are carried out during the embryonic and neonatal periods, and there is no evidence of mature hair cell regeneration in this study. The title should be revised to reflect this. A more accurate title could be: "Developmental loss of Cldn9 results in functional ectopic inner hair cells that persist through adulthood."<br /> (2) Contact-mediated lateral inhibition in hair cell fate determination is one of the most well-studied phenomena in the inner ear field, and numerous groups have shown that it is mediated by Notch signaling. This must be added to the introduction.<br /> (3) A large body of literature has demonstrated that Notch inhibition alone is not sufficient to regenerate hair cells in adult mice. Therefore, if the loss of claudins disrupts Notch signaling-the proposed mechanisms in the discussion - it is unlikely to be a viable therapeutic strategy for hair cell regeneration in the adult ear. Furthermore, no hair cell ablation experiments were conducted to demonstrate what could be considered true regeneration. These speculative statements should be removed or revised accordingly.<br /> (4) Cldn9 is a tight junction protein and should localize to the membrane. Yet, the data presented show what appears to be diffuse cytoplasmic staining, which is concerning.

Reviewer #3 (Public review):

In this paper, the authors use a three-phase economic game to examine the tendency to engage in prosocial versus competitive exchanges with three anonymous partners. In particular, they consider individual differences in the tendency to infer about others' tendencies based on one's preferences and to update one's preferences based on observations of others' behavior. The study includes a sample of individuals diagnosed with borderline personality disorder and a matched sample of psychiatrically healthy control participants.

On the whole, the experimental design is well-suited to the questions and the computational model analyses are thorough, including modern model-fitting procedures. I particularly appreciated the clear exposition regarding model parameterization and the descriptive Table 2 for qualitative model comparison. My broad question about the experiment (in terms of its clinical and cognitive process relevance): Does the task encourage competition or give participants a reason to take advantage of others? I don't think it does, so it would be useful to clarify the normative account for prosociality in the introduction (e.g., some of Robin Dunbar's work).

The finding that individuals with BPD do not engage in self-other generalization on this task of social intentions is novel and potentially clinically relevant. The authors find that BPD participants' tendency to be prosocial when splitting points with a partner does not transfer into their expectations of how a partner will treat them in a task where they are the passive recipient of points chosen by the partner. In the discussion, the authors reasonably focus on model differences between groups (Bayesian model comparison), yet I thought this finding -- BPD participants not assuming prosocial tendencies in phase 2 while CON participant did -- merited greater attention. Although the BPD group was close to 0 on the \beta prior in Phase 2, their difference from CON is still in the direction of being more mistrustful (or at least not assuming prosociality). This may line up with broader clinical literature on mistrustfulness and attributions of malevolence in the BPD literature (e.g., a 1992 paper by Nigg et al. in Journal of Abnormal Psychology). My broad point is to consider further the Phase 2 findings in terms of the clinical interpretation of the shift in \beta relative to controls.

On the conceptual level, I had two additional concerns. First, the authors note that they have "proposed a theory with testable predictions" (p. 4 but also elsewhere) but they do not state any clear predictions in the introduction, nor do they consider what sort of patterns will be observed in the BPD group in view of extant clinical and computational literature. Rather, the paper seems to be somewhat exploratory, largely looking at group differences (BPD vs. CON) on all of the shared computational parameters and additional indices such as belief updating and reaction times. Given this, I would suggest that the authors make stronger connections between extant research on intention representation in BPD and their framework (model and paradigm). In particular, the authors do not address related findings from Ereira (2020) and Story (2024) finding that in a false belief task that BPD participants *overgeneralize* from self to other. A critical comparison of this work to the present study, including an examination of the two tasks differ in the processes they measure, is important.

In addition, perhaps it is fairer to note more explicitly the exploratory nature of this work. Although the analyses are thorough, many of them are not argued for a priori (e.g., rate of belief updating in Figure 2C) and the reader amasses many individual findings that need to by synthesized.

Second, in the discussion, the authors are too quick to generalize to broad clinical phenomena in BPD that are not directly connected to the task at hand. For example, on p. 22: "Those with a diagnosis of BPD also show reduced permeability in generalising from other to self. While prior research has predominantly focused on how those with BPD use information to form impressions, it has not typically examined whether these impressions affect the self." Here, it's not self-representation per se (typically, identity or one's view of oneself), but instead cooperation and prosocial tendencies in an economic context. It is important to clarify what clinical phenomena may be closely related to the task and which are more distal and perhaps should not be approached here.

On a more technical level, I had two primary concerns. First, although the authors consider alternative models within a hierarchical Bayesian framework, some challenges arise when one analyzes parameter estimates fit separately to two groups, particularly when the best-fitting model is not shared. In particular, although the authors conduct a model confusion analysis, they do not as far I could tell (and apologies if I missed it) demonstrate that the dynamics of one model are nested within the other. Given that M4 has free parameters governing the expectations on the absolute and relative reward preferences in Phase 2, is it necessarily the case that the shared parameters between M1 and M4 can be interpreted on the same scale? Relatedly, group-specific model fitting has virtues when believes there to be two distinct populations, but there is also a risk of overfitting potentially irrelevant sample characteristics when parameters are fit group by group.

To resolve these issues, I saw one straightforward solution (though in modeling, my experience is that what seems straightforward on first glance may not be so upon further investigation). M1 assumes that participants' own preferences (posterior central tendency) in Phase 1 directly transfer to priors in Phase 2, but presumably the degree of transfer could vary somewhat without meriting an entirely new model (i.e., the authors currently place this question in terms of model selection, not within-model parameter variation). I would suggest that the authors consider a model parameterization fit to the full dataset (both groups) that contains free parameters capturing the *deviations* in the priors relative to the preceding phase's posterior. That is, the free parameters $\bar{\alpha}_{par}^m$ and $\bar{\beta}_{par}^m$ govern the central tendency of the Phase 2 prior parameter distributions directly, but could be reparametrized as deviations from Phase 1 $\theta^m_{ppt}$ parameters in an additive form. This allows for a single model to be fit all participants that encompasses the dynamics of interest such that between-group parameter comparisons are not biased by the strong assumptions imposed by M1 (that phase 1 preferences and phase 2 observations directly transfer to priors). In the case of controls, we would expect these deviation parameters to be centred on 0 insofar as the current M1 fit them best, whereas for BPD participants should have significant deviations from earlier-phase posteriors (e.g., the shift in \beta toward prior neutrality in phase 2 compared to one's own prosociality in phase 1). I think it's still valid for the authors to argue for stronger model constraints for Bayesian model comparison, as they do now, but inferences regarding parameter estimates should ideally be based on a model that can encompass the full dynamics of the entire sample, with simpler dynamics (like posterior -> prior transfer) being captured by near-zero parameter estimates.

My second concern pertains to the psychometric individual difference analyses. These were not clearly justified in the introduction, though I agree that they could offer potentially meaningful insight into which scales may be most related to model parameters of interest. So, perhaps these should be earmarked as exploratory and/or more clearly argued for. Crucially, however, these analyses appear to have been conducted on the full sample without considering the group structure. Indeed, many of the scales on which there are sizable group differences are also those that show correlations with psychometric scales. So, in essence, it is unclear whether most of these analyses are simply recapitulating the between-group tests reported earlier in the paper or offer additional insights. I think it's hard to have one's cake and eat it, too, in this regard and would suggest the authors review Preacher et al. 2005, Psychological Methods for additional detail. One solution might be to always include group as a binary covariate in the symptom dimension-parameter analyses, essentially partialing the correlations for group status. I remain skeptical regarding whether there is additional signal in these analyses, but such controls could convince the reader. Nevertheless, without such adjustments, I would caution against any transdiagnostic interpretations such as this one in the Highlights: "Higher reported childhood trauma, paranoia, and poorer trait mentalizing all diminish other-to-self information transfer irrespective of diagnosis." Since many of these analyses relate to scales on which the groups differ, the transdiagnostic relevance remains to be demonstrated.

Reviewer #3 (Public review):

The manuscript of Kowalewski et al. titled "Machine learning of honey bee olfactory behavior identifies repellent odorants in free flying bees in the field" did machine learning to predict potential candidates for honeybee repellents, which may keep foraging bees from pesticides. This is a pilot research with strong significance in the research of olfactory behavior and in pest control. However, some major issues need to be addressed to enhance the manuscript's clarity, strength, and overall coherence.

(1) Drosophila melanogaster is not considered as a true agricultural pest. The manuscript would be more compelling if using true pests, for example, Drosophila suzukii or others.<br /> (2) For repellency test, the result relies on dosage. An attractant may become a repellent at high concentration. Test a range of concentrations for each chemicals and compare responses between honeybees and pests.<br /> (3) Be more clear about bee behavior data and their scores (as in Page 4 Results "184 training chemicals and later for 203 chemicals" and Page 10 Methods). I suggest that authors add a supplemental table with each chemical and its behavioral score, feature and reference - which ones were used for training, and which ones for testing. Also add your own behavioral test data (second input) to this table.<br /> (4) The AUC in the first validation was 0.88 (Page 4), and in Page 5, "As expected, the computational validation results based on the AUC values, show an improvement." However, there were no other AUC values to show improvement.<br /> (5) Show plots of ROC AUC curves from Round 1 and Round 2.<br /> (6) In the Discussion, the authors mentioned olfactory receptors in honeybees. It would be useful to provide a general review of the current understanding of these receptors and their (potential) functions.<br /> (7) I suggest combining Fig. 1 and Fig. 3A as one pipeline for this work.<br /> (8) Figure 2C, some sample sizes are very small, such as 2-piperidone: 1 first-choice control vs 0 first-choice repellent? Increase sample size and do statistical analysis.<br /> (9) In general, to assist reviewers, include line numbers to the manuscript.

Reviewer #3 (Public review):

Summary:

The authors created and characterized genetic tools that allow for precise manipulation of individual or small subsets of central complex (CX) cell types in the Drosophila brain. They developed split-GAL4 driver lines and integrated this with a detailed survey of neurotransmitter and neuropeptide expression and receptor localization in the central brain. The manuscript also explores the functional relevance of CX cell types by evaluating their roles in sleep regulation and linking circadian clock signals to the CX. This work represents an ambitious and comprehensive effort to provide both molecular and functional insights into the CX, offering tools and data that will serve as a critical resource for researchers.

Strengths:

(1) The extensive collection of split-GAL4 lines targeting specific CX cell types fills a critical gap in the genetic toolkit for the Drosophila neuroscience community.<br /> (2) By combining anatomical, molecular, and functional analyses, the authors provide a holistic view of CX cell types that is both informative and immediately useful for researchers across diverse disciplines.<br /> (3) The identification of CX cell types involved in sleep regulation and their connection to circadian clock mechanisms highlights the functional importance of the CX and its integrative role in regulating behavior and physiological states.<br /> (4) The authors' decision to present this work as a single, comprehensive manuscript rather than fragmenting it into smaller publications each focusing on separate central complex components is commendable. This decision prioritizes accessibility and utility for the broader neuroscience community, which will enable researchers to approach CX-related questions with a ready-made toolkit.

Weaknesses:

While the manuscript is an outstanding resource, it leaves room for more detailed mechanistic exploration in some areas. Nonetheless, this does not diminish the immediate value of the tools and data provided.

Appraisal:

The authors have succeeded in achieving their aims of creating well-characterized genetic tools and providing a detailed survey of neurochemical and functional properties in the CX. The results strongly support their conclusions and open numerous avenues for future research. The work effectively bridges the gap between genetic manipulation, molecular characterization, and functional assessment, enabling a deeper understanding of the CX's diverse roles.

Impact and Utility

This manuscript will have a significant and lasting impact on the field, providing tools and data that facilitate new discoveries in the study of the CX, sleep regulation, circadian biology, and beyond. The genetic tools developed here are likely to become a standard resource for Drosophila researchers, and the comprehensive dataset on neurotransmitter and neuropeptide expression will inspire investigations into the interplay between neuromodulation and classical neurotransmission.

Additional Context

By delivering an integrated dataset that spans anatomy, molecular properties, and functional relevance, the authors have created a resource that will serve the neuroscience community for years to come.

Reviewer #3 (Public review):

The central goal of this paper as I understand it is to extract the "integration hierarchy" of stimulus in the dorsal and ventrolateral visual fields. The segregation of these responses is different from what is thought to occur in bees and flies and was established in the authors' prior work. Showing how the stimuli combine and are prioritized goes beyond the authors' prior conclusions that separated the response into two visual regions. The data presented do indeed support the hierarchy reported in Figure 5 and that is a nice summary of the authors' work. The moths respond to combinations of dorsal and lateral cues in a mixed way but also seem to strongly prioritize avoiding dorsal optic flow which the authors interpret as a closed and potentially dangerous ecological context for these animals. The authors use clever combinations of stimuli to put cues into conflict to reveal the response hierarchy.

My most significant concern is that this hierarchy of stimulus responses might be limited to the specific parameters chosen in this study. Presumably, there are parameters of these stimuli that modulate the response (spatial frequency, different amounts of optic flow, contrast, color, etc). While I agree that the hierarchy in Figure 5 is consistent for the particular stimuli given, this may not extend to other parameter combinations of the same cues. For example, as the contrast of the dorsal stimuli is reduced, the inequality may shift. This does not preclude the authors' conclusions but it does mean that they may not generalize, even within this species. For example, other cue conflict studies have quantified the responses to ranges of the parameters (e.g. frequency) and shown that one cue might be prioritized or up-weighted in one frequency band but not in others. I could imagine ecological signatures of dorsal clutter and translational positioning cues could depend on the dynamic range of the optic flow, or even having spatial-temporal frequency-dependent integration independent of net optic flow.

The second part of this concern is that there seems to be a missed opportunity to quantify the integration, especially when the optic flow magnitude is already calculated. The discussion even highlights that an advantage of the conflict paradigm is that the weights of the integration hierarchy can be compared. But these weights, which I would interpret as stimulus-responses gains, are not reported. What is the ratio of moth response to optic flow in the different regions? When the moth balances responses in the dorsal and ventrolateral region, is it a simple weighted average of the two? When it prioritizes one over the other is the response gain unchanged? This plays into the first concern because such gain responses could strongly depend on the specific stimulus parameters rather than being constant.

The authors do explain the choice of specific stimuli in the context of their very nice natural scene analysis in Fig. 1 and there is an excellent discussion of the ecological context for the behaviors. However, I struggled to directly map the results from the natural scenes to the conclusions of the paper. How do they directly inform the methods and conclusions for the laboratory experiments? Most important is the discussion in the middle paragraph of page 12, which suggests a relationship with Figure 1B, but seems provocative but lacking a quantification with respect to the laboratory stimuli.

The central conclusion of the first section of the results is that there are likely two different pathways mediating the dorsal and the ventrolateral response. This seems reasonable given the data, however, this was also the message that I got from the authors' prior paper (ref 11). There are certainly more comparisons being done here than in that paper and it is perfectly reasonable to reinforce the conclusion from that study but I think what is new about these results needs to be highlighted in this section and differentiated from prior results. Perhaps one way to help would be to be more explicit with the open hypotheses that remain from that prior paper.

Reviewer #3 (Public review):

Summary:

This study by Glica and colleagues utilized EEG (i.e., Beta power, Gamma power, and aperiodic activity) and 7T MRS (i.e., MRS IE ratio, IE balance) to reevaluate the neural noise hypothesis in Dyslexia. Supported by Bayesian statistics, their results show convincing evidence of no differences in EI balance between groups, challenging the neural noise hypothesis.

Strengths:

Combining EEG and 7T MRS, this study utilized both the indirect (i.e., Beta power, Gamma power, and aperiodic activity) and direct (i.e., MRS IE ratio, IE balance) measures to reevaluate the neural noise hypothesis in Dyslexia.

Reviewer #3 (Public review):

The manuscript contains a carefully designed fMRI study, using MVPA pattern analysis to investigate which high-level associate cortices contain target-related information to guide visual search. A special focus is hereby on so-called 'target-associated' information, that has previously been shown to help in guiding attention during visual search. For this purpose the author trained their participants and made them learn specific target-associations, in order to then test which brain regions may contain neural representations of those learnt associations. They found that at least some of the associations tested were encoded in prefrontal cortex during the cue and delay period.

The manuscript is very carefully prepared. As far as I can see, the statistical analyses are all sound and the results integrate well with previous findings.

I have no strong objections against the presented results and their interpretation.

Reviewer #3 (Public review):

Summary:

The authors optogenetically stimulate 5 neurons all preferring the same pure tone frequency (16 or 54 kHz) in the mouse auditory cortex using a holography-based single cell resolution optogenetics during sound presentation. They demonstrate that the response boosting of target neurons leads to a broad suppression of surrounding neurons, which is significantly more pronounced in neurons that have the same pure tone tuning as the target neurons. This effect is immediate and spans several hundred micrometers. This suggests that the auditory cortical network balances its activity in response to excess spikes, a phenomenon already seen in visual cortex.

Strengths:

The study is based on a technologically very solid approach based on single-cell resolution two-photon optogenetics. The authors demonstrate the potency and resolution of this approach. The inhibitory effects observed upon targeted stimulation are clear and the relative specificity to co-tuned neurons is statistically clear although the effect size is moderate.

Weaknesses:

The evaluation of the results is brief and some aspects of the observed homeostatic are not quantified. For example, it is unclear whether stimulation produces a net increase or decrease of population activity, or if the homeostatic phenomenon fully balances activity. A comparison of population activity for all imaged neurons with and without stimulation would be instructive. The selectivity for co-tuned neurons is significant but weak. Although it is difficult to evaluate this issue, this result may be trivial, as co-tuned neurons fire more strongly. Therefore, the net activity decrease is expected to be larger, in particular, for the number of non-co-tuned neurons which actually do not fire to the target sound. The net effect for the latter neurons will be zero just because they do not respond. The authors do not make a very strong case for a specific inhibition model in comparison to a broad and non-specific inhibitory effect. Complementary modeling work would be needed to fully establish this point.

Reviewer #3 (Public review):

In this work, the authors present an open-source system called behaviourMate for acquiring data related to animal behavior. The temporal alignment of recorded parameters across various devices is highlighted as crucial to avoid delays caused by electronics dependencies. This system not only addresses this issue but also offers an adaptable solution for VR setups. Given the significance of well-designed open-source platforms, this paper holds importance.

Advantages of behaviorMate:

The cost-effectiveness of the system provided.<br /> The reliability of PCBs compared to custom-made systems.<br /> Open-source nature for easy setup.<br /> Plug & Play feature requiring no coding experience for optimizing experiment performance (only text based Json files, 'context List' required for editing).

Reviewer #3 (Public review):

Summary:

The study uses structural MRI to identify how the number, degree of experience, and phonemic diversity of language(s) that a speaker knows can influence the thickness of different sub-segments of auditory cortex. In both a primary and replication sample of adult speakers, the authors find key differences in cortical thickness within specific subregions of cortex due to either the age at which languages are acquired (degree of experience) or the diversity of the phoneme inventories carried by that/those language(s) (breadth of experience).

Strengths:

The results are first and foremost quite fascinating and I do think they make a compelling case for the different ways in which linguistic experience shapes auditory cortex.

The study uses a number of different measures to quantify linguistic experience, related to how many languages a person knows (taking into account the age at which each was learned) as well as the diversity of the phoneme inventories contained within those languages. The primary sample is moderately large for a study that focuses on brain-behaviour relationships; a somewhat smaller replication sample is also deployed in order to test the generality of the effects.

Analytic approaches benefit from the careful use of brain segmentation techniques that nicely capture key landmarks and account for vagaries in the structure of STG that can vary across individuals (e.g., the number of transverse temporal gyri varies from 1-4 across individuals).

Weaknesses:

The specificity of these effects is interesting; some effects really do appear to be localized to left hemisphere and specific subregions of auditory cortex e.g., TTG. There is an ancillary analysis that examines regions outside auditory cortex to examine whether these are the only brain regions for which such effects occur. Expanding the search space to a whole-brain analysis, and a more lenient statistical threshold, does reveal only small patches of the brain outside auditory cortex show similar effects. Notably, these could be due to inflated type-1 error, but overall we would need a much larger sample to be certain.

Discussion of potential genetic differences underlying the findings is interesting. It does represent one alternative account that does not have to do with plasticity/experience, as the authors acknowledge.

The replication sample is useful and a great idea. It does however feature roughly half the number of participants. As the authors are careful to point out, that statistical power is weaker and given small effects in some cases we should not be surprised that the results only partially replicated in that sample.

Reviewer #3 (Public review):

Summary:

m6Am is an abundant mRNA modification present on the TSN. Unlike the structurally similar and abundant internal mRNA modification m6A, m6Am's function has been controversial. One way to resolve controversies surrounding mRNA modification functions has been to develop new ways to better profile said mRNA modification. Here, Liu et al. developed a new method (based on GLORI-seq for m6A-sequencing), for antibody-independent sequencing of m6Am (CROWN-seq). Using appropriate spike-in controls and knockout cell lines, Liu et al. clearly demonstrated CROWN-seq's precision and quantitative accuracy for profiling transcriptome-wide m6Am. Subsequently, the authors used CROWN-seq to greatly expand the number of known m6Am sites in various cell lines and also determine m6Am stoichiometry to generally be high for most genes. CROWN-seq identified gene promoter motifs that correlate best with high stoichiometry m6Am sites, thereby identifying new determinants of m6Am stoichiometry. CROWN-seq also helped reveal that m6Am does not regulate mRNA stability or translation (as opposed to past reported functions). Rather, m6Am stoichiometry correlates well with transcription levels. Finally, Liu et al. reaffirmed that FTO mainly demethylates m6Am, not of mRNA but of snRNAs and snoRNAs.

Strengths:

This is a well-written manuscript that describes and validates a new m6Am-sequencing method: CROWN-seq as the first m6Am-sequencing method that can both quantify m6Am stoichiometry and profile m6Am at single-base resolution. These advantages facilitated Liu et al. to uncover new potential findings related to m6Am regulation and function. I am confident that CROWN-seq will likely be the gold standard for m6Am-sequencing henceforth.

Weaknesses:

Though the authors have uncovered a potentially new function for m6Am, they need to be clear that without identifying a mechanism, their data might only be demonstrating a correlation between the presence of m6Am and transcriptional regulation rather than causality.

Reviewer #3 (Public review):

Summary:

In this paper, Chang and Meliala et al. demonstrate that PEBP1 is a modulator of the ISR, specifically through the induction of mitochondrial stress. The authors utilize thermal proteome profiling (TPP) by which they identify PEPB1 as a thermally stabilized protein upon oligomycin treatment, indicating its role in mitochondrial stress. Moreover, RNA-sequencing analysis indicated that PEBP1 may be specifically modulating the mitochondrial stress-induced ISR, as PEBP1 knock-out reduces phosphorylation of eIF2α. They also show that PEBP1 function is independent of ER stress specifically tunicamycin treatment and loss of PEBP1 does affect mitochondrial ISR but in an OMA1, DELE1 independent manner. Thus, the authors hypothesized that PEBP1 interacts directly with eIF2α, functioning as a scaffolding protein. However, direct co-immunoprecipitation failed to demonstrate PEBP1 and eIF2α potential interaction. The authors then used a NanoBiT luminescence complementation assay to show the PEBP1-eIF2a interaction and its disruption by S51 phosphorylation.

Strengths:

Taken together, this work is novel, and the data presented suggests PEBP1 has a role as a modulator of the mitochondrial ISR, enhancing the signal to elicit the necessary response.

Weaknesses:

The one major issue of this work is the lack of a mechanism showing precisely how PEBP1 amplifies the mitochondrial integrated stress response. The work, as it is described, presents data suggesting PEBP1's role in the ISR but fails to present a more conclusive mechanism.

Reviewer #3 (Public Review):

This work addresses an important question aimed at understanding how membrane docking to the distal appendages is regulated during ciliogenesis. In this study, Tomoharu and colleagues identified interactions between CEP89 (important for RAB34-positive membrane localization to the mother centriole) and NCS1 and C3ORF14. Both these CEP89 interacting proteins were characterized as distal appendage localized proteins between CEP89 and RAB34 based on super-resolution microscopy. Ciliogenesis investigations using knockout cells indicated that NCS1 and CEP89 have similar impaired ciliation due to disruption in vesicle recruitment/RAB34 to the mother centriole, while C3ORF14 had less effect on ciliogenesis. The authors refer to the ciliogenesis requirement for CEP89/NCS1 as ciliary vesicles, which has been previously referred to as preciliary vesicle or distal appendage vesicles. NCS1 distal appendage localization was dependent on CEP89 and TTBK2, but it is not clear how TTBK2 affects NCS1. The authors subsequently performed double knockouts with NCS1 and other distal appendage proteins and showed stronger effects on mother centriole RAB34 levels, suggesting efficient membrane docking during ciliogenesis requires several distal appendage proteins. This is consistent with NCS1 knockout mice which do not display typical ciliopathy phenotypes. These mice do display obesity, which is associated with cilia dysfunction, and show reduced ciliary protein levels. As noted by the authors, the in vivo results for NCS1 knockouts could be affected by the mouse background which was not evaluated. The authors demonstrate the NCS1 myristoylation motif is required for RAB34 localization to the mother centrioles, providing a mechanistic explanation for how distal appendage proteins could interact with membranes during ciliogenesis. Overall the authors' findings support an important role for NCS1 in regulating ciliogenesis via myristoylation-dependent interaction with RAB34-positive membranes docked at the mother centriole.

Reviewer #3 (Public review):

The superfamily I 3'-5' DNA helicase Srs2 is well known for its role as an anti-recombinase, stripping Rad51 from ssDNA, as well as an anti-crossover factor, dissociating extended D-loops and favoring non-crossover outcome during recombination. In addition, Srs2 plays a key role in in ribonucleotide excision repair. Besides DNA repair defects, srs2 mutants also show a reduced recovery after DNA damage that is related to its role in downregulating the DNA damage signaling or checkpoint response. Recent work from the Zhao laboratory (PMID: 33602817) identified a role of Srs2 in downregulating the DNA damage signaling response by removing RPA from ssDNA. This manuscript reports further mechanistic insights into the signaling downregulation function of Srs2.

Using the genetic interaction with mutations in RPA1, mainly rfa1-zm2, the authors test a panel of mutations in Srs2 that affect CDK sites (srs2-7AV), potential Mec1 sites (srs2-2SA), known sumoylation sites (srs2-3KR), Rad51 binding (delta 875-902), PCNA interaction (delta 1159-1163), and SUMO interaction (srs2-SIMmut). All mutants were generated by genomic replacement and the expression level of the mutant proteins was found to be unchanged. This alleviates some concern about the use of deletion mutants compared to point mutations. Double mutant analysis identified that PCNA interaction and SUMO sites were required for the Srs2 checkpoint dampening function, at least in the context of the rfa1-zm2 mutant. There was no effect of this mutants in a RFA1 wild type background. This latter result is likely explained by the activity of the parallel pathway of checkpoint dampening mediated by Slx4, and genetic data with an Slx4 point mutation affecting Rtt107 interaction and checkpoint downregulation support this notion. Further analysis of Srs2 sumoylation showed that Srs2 sumoylation depended on PCNA interaction, suggesting sequential events of Srs2 recruitment by PCNA and subsequent sumoylation. Kinetic analysis showed that sumoylation peaks after maximal Mec1 induction by DNA damage (using the Top1 poison camptothecin (CPT)) and depended on Mec1. This data are consistent with a model that Mec1 hyperactivation is ultimately leading to signaling downregulation by Srs2 through Srs2 sumoylation. Mec1-S1964 phosphorylation, a marker for Mec1 hyperactivation and a site found to be needed for checkpoint downregulation after DSB induction, did not appear to be involved in checkpoint downregulation after CPT damage. The data are in support of the model that Mec1 hyperactivation when targeted to RPA-covered ssDNA by its Ddc2 (human ATRIP) targeting factor, favors Srs2 sumoylation after Srs2 recruitment to PCNA to disrupt the RPA-Ddc2-Mec1 signaling complex. Presumably, this allows gap filling and disappearance of long-lived ssDNA as the initiator of checkpoint signaling, although the study does not extend to this step.

Strengths<br /> (1) The manuscript focuses on the novel function of Srs2 to downregulate the DNA damage signaling response and provide new mechanistic insights.<br /> (2) The conclusions that PCNA interaction and ensuing Srs2-sumoylation are involved in checkpoint downregulation are well supported by the data.

Weaknesses<br /> (1) Additional mutants of interest could have been tested, such as the recently reported Pin mutant, srs2-Y775A (PMID: 38065943), and the Rad51 interaction point mutant, srs2-F891A (PMID: 31142613).<br /> (2) The use of deletion mutants for PCNA and RAD51 interaction is inferior to using specific point mutants, as done for the SUMO interaction and the sites for post-translational modifications.<br /> (3) Figure 4D and Figure 5A report data with standard deviations, which is unusual for n=2. Maybe the individual data points could be plotted with a color for each independent experiment to allow the reader to evaluate the reproducibility of the results.

Comments on revisions:

In this revision, the authors adequately addressed my concerns. The only issue I see remaining is the site of Srs2 action. The authors argue in favor of gaps and against R-loops and ssDNA resulting from excessive supercoiling. The authors do not discuss ssDNA resulting from processing of one-sided DSBs, which are expected to result from replication run-off after CPT damage but are not expected to provide the 3'-junction for preferred PCNA loading. Can the authors exclude PCNA at the 5'-junction at a resected DSB?

Reviewer #3 (Public Review):

Summary:

This study characterizes classical and nonclassical osteoblasts as both types were analyzed independently (integrated ATAC-seq and RNAseq). It was found that gene expression in classical and nonclassical osteoblasts is not regulated in the same way. In classical osteoblasts, Dlx family factors seem to play an important role, while Hox family factors are involved in the regulation of spinal ossification by nonclassical osteoblasts. In the second part of the study, the authors focus on the promoter structure of entpd5a. Through the identification of enhancers, they reveal complex modes of regulation of the gene. The authors suggest candidate transcription factors that likely act on the identified enhancer elements. All the results taken together provide comprehensive new insights into the process of bone development, and point to spatio-temporally regulated promoter/enhancer interactions taking place at the entpd5a locus.

Strengths:

The authors have succeeded in justifying a sound and consistent buildup of their experiments, and meaningfully integrating the results into the design of each of their follow-up experiments. The data are solid, insightfully presented, and the conclusion valid. This makes this manuscript of great value and interest to those studying (fundamental) skeletal biology.

Weaknesses:

The study is solidly constructed, the manuscript is clearly written and the discussion is meaningful - I see no real weaknesses.

Reviewer #4 (Public review):

Summary:

The manuscript by Graça et al. explores the role of MftG in the ethanol metabolism of mycobacteria. The authors hypothesise that MftG functions as a mycofactocin dehydrogenase, regenerating mycofactocin by shuttling electrons to the respiratory chain of mycobacteria. Although the study primarily uses M. smegmatis as a model microorganism, the findings have more general implications for understanding mycobacterial metabolism. Identifying the specific partner to which MftG transfers its electrons within the respiratory chain of mycobacteria would be an important next step, as pointed out by the authors.

Strengths

The authors have used a wide range of tools to support their hypothesis, including co-occurrence analyses, gene knockout and complementation experiments, as well as biochemical assays and transcriptomics studies.<br /> An interesting observation that the mftG deletion mutant grown on ethanol as the sole carbon source exhibited a growth defect resembling a starvation phenotype.<br /> MftG was shown to catalyse the electron transfer from mycofactocinol to components of the respiratory chain, highlighting the flexibility and complexity of mycobacterial redox metabolism.

The authors have taken on the majority of recommendations by the reviewers and made changes in the manuscript accordingly. I don't have any further suggestions.

Reviewer #3 (Public review):

Summary:

This is a valuable study providing solid evidence that the putative non-canonical initiation factor eIF2A has little or no role in the translation of any expressed mRNAs in cultured human (primarily HeLa) cells. Previous studies have implicated eIF2A in GTP-independent recruitment of initiator tRNA to the small (40S) ribosomal subunit, a function analogous to canonical initiation factor eIF2, and in supporting initiation on mRNAs that do not require scanning to select the AUG codon or that contain near-cognate start codons, especially upstream ORFs with non-AUG start codons, and may use the cognate elongator tRNA for initiation. Moreover, the detected functions for eIF2A were limited to, or enhanced by, stress conditions where canonical eIF2 is phosphorylated and inactivated, suggesting that eIF2A provides a back-up function for eIF2 in such stress conditions. CRISPR gene editing was used to construct two different knock-out cell lines that were compared to the parental cell line in a large battery of assays for bulk or gene-specific translation in both unstressed conditions and when cells were treated with inhibitors that induce eIF2 phosphorylation. None of these assays identified any effects of eIF2A KO on translation in unstressed or stressed cells, indicating little or no role for eIF2A as a back-up to eIF2 and in translation initiation at near-cognate start codons, in these cultured cells.

The study is very thorough and generally well executed, examining bulk translation by puromycin labeling and polysome analysis and translational efficiencies of all expressed mRNAs by ribosome profiling, with extensive utilization of reporters equipped with the 5'UTRs of many different native transcripts to follow up on the limited number of genes whose transcripts showed significant differences in translational efficiencies (TEs) in the profiling experiments. They also looked for differences in translation of uORFs in the profiling data and examined reporters of uORF-containing mRNAs known to be translationally regulated by their uORFs in response to stress, going so far as to monitor peptide production from a uORF itself. The high precision and reproducibility of the replicate measurements instil strong confidence that the myriad of negative results they obtained reflects the lack of eIF2A function in these cells rather than data that would be too noisy to detect small effects on the eIF2A mutations. They also tested and found no evidence for a recent claim that eIF2A localizes to the cytoplasm in stress and exerts a global inhibition of translation. Given the numerous papers that have been published reporting functions of eIF2A in specific and general translational control, this study is important in providing abundant, high-quality data to the contrary, at least in these cultured cells.

Strengths:

The paper employed two CRISPR knock-out cell lines and subjected them to a combination of high-quality ribosome profiling experiments, interrogating both main coding sequences and uORFs throughout the translatome, which was complemented by extensive reporter analysis, and cell imaging in cells both unstressed and subjected to conditions of eIF2 phosphorylation, all in an effort to test previous conclusions about eIF2A functioning as an alternative to eIF2.

Weaknesses:

There is some question about whether their induction of eIF2 phosphorylation using tunicamycin was extensive enough to state forcefully that eIF2A has little or no role in the translatome when eIF2 function is strongly impaired. Also, similar conclusions regarding the minimal role of eIF2A were reached previously for a different human cell line from a study that also enlisted ribosome profiling under conditions of extensive eIF2 phosphorylation; although that study lacked the extensive use of reporters to confirm or refute the identification by ribosome profiling of a small group of mRNAs regulated by eIF2A during stress.

Reviewer #3 (Public review):

Summary:

Mitochondrial injury activates eiF2α kinases-PERK, GCN2, HRI and PKR-which collectively regulate the Integrated Stress Response (ISR) to preserve mitochondrial function and integrity. Previous work has demonstrated that stress-induced and pharmacologic stress-independent ISR activation promotes adaptive mitochondrial elongation via the PERK and GCN2 kinases, respectively. Here, the authors demonstrate that pharmacologic ISR inducers of HRI and GCN2 enhance mitochondrial elongation and suppress mitochondrial fragmentation in two disease models, illustrating the therapeutic potential of pharmacologic ISR activators. Specifically, the authors first used an innovative ISR translational reporter to screen for nucleoside mimetic compounds that induce ISR signaling, and identified two compounds, 0357 and 3610, that preferentially activate HRI. Using a mitochondrial-targeted GFP MEF cell line, the authors next determined that these compounds (as well as the GCN2 activator, halofuginone) enhance mitochondrial elongation in an ISR-dependent manner. Moreover, pretreatment of MEFs with these ISR kinase activators suppressed pathological mitochondrial fragmentation caused by a calcium ionophore. Finally, pharmacologic HRI and GCN2 activation was found to preserve mitochondrial morphology in human fibroblasts expressing a pathologic variant in MFN2, a defect that leads to mitochondrial fragmentation and is a cause of Charcot Marie Tooth Type 2A disease.

Strengths:

This well-written manuscript has several notable strengths, including the demonstration of the potential therapeutic benefit of ISR modulation. New chemical entities with which to further interrogate this stress response pathway are also reported. In addition, the authors used an elegant screen to isolate compounds that selectively activate the ISR and identify which of the four kinases was responsible for activation. Special attention was also paid to a thorough evaluation of the effect of their compounds on other stress response pathways (i.e. the UPR, and heat and oxidative stress responses), thereby minimizing the potential for off-target effects. The implementation of automated image analysis rather than manual scoring to quantify mitochondrial elongation is not only practical but also adds to the scientific rigor, as does the complementary use of both the calcium ionophore and MFN2 models to enhance confidence and the broad therapeutic potential for pharmacology ISR manipulation.

Weaknesses:

The only minor concerns are with regard to effects on cell health and the timing of pharmacological administration.

Comments on revisions:

In this revised manuscript the authors demonstrate that pharmacological activation of the eiF2α kinases, HRI and GCN2, induce adaptive mitochondrial elongation and suppress mitochondrial fragmentation in two disease models, illustrating the translational potential of pharmacological ISR modulation.

In revising their manuscript the authors adequately addressed the concerns. In response to comments about the potential toxicity of their compounds, 0357 and 3610, the authors furnish evidence that neither compound significantly reduced viability of HEK293 cells (Figure S1G). Understandably, the authors focused the present work on the acute effects of their compounds. Several other attributes are noteworthy: First, that injury attributable to chronic ISR activation in cell culture may ultimately be circumvented by altering the in vivo pharmacodynamic and pharmacodynamic properties of the compounds, thereby preserving the translation potential for these (and related) compounds. Second, the authors also reasonably explain that the rapidity of ionomycin-induced injury, necessitating that the inducers are administered prior to treatment. Their assessment of the effects of the compounds on mitochondrial fragmentation in MFN2 mutant fibroblasts-in combination with the preserved viability of HEK293 cells-is sufficient to demonstrate the practical pharmacological potential for these (or related) agents.

Reviewer #3 (Public review):

Summary:

This study characterizes classical and nonclassical osteoblasts as both types were analyzed independently (integrated ATAC-seq and RNAseq). It was found that gene expression in classical and nonclassical osteoblasts is not regulated in the same way. In classical osteoblasts Dlx family factors seem to play an important role, while Hox family factors are involved in the regulation of spinal ossification by nonclassical osteoblasts. In the second part of the study, the authors focus on the promoter structure of entpd5a. Through the identification of enhancers they reveal complex modes of regulation of the gene. The authors suggest candidate transcription factors that likely act on the identified enhancer elements. All the results taken together provide comprehensive new insights into the process of bone development, and point to spatio-temporally regulated promoter/enhancer interactions taking place at the entpd5a locus.

Strengths:

The authors have succeeded in justifying a sound and consistent buildup of their experiments, and meaningfully integrate the results into the design of each of their follow-up experiments. The data are solid, insightfully presented, and the conclusion valid. This makes this manuscript of great value and interest to those studying (fundamental) skeletal biology.

Weaknesses:

The study is solidly constructed, the manuscript is clearly written and the discussion is meaningful - I see no real weaknesses.

Reviewer #3 (Public review):

Summary:

The relationships of proteins and lipids: it's complicated. This paper illustrates how cardiolipins can stabilize membrane protein subunits - and not surprisingly, positively charged residues play an important role here. But more and stronger binding of such structural lipids does not necessarily translate to stabilization of oligomeric states, since many proteins have alternative binding sites for lipids which may be intra- rather than intermolecular. Mutations which abolish primary binding sites can cause redistribution to (weaker) secondary sites which nevertheless stabilize interactions between subunits. This may be at first sight counterintuitive but actually matches expectations from structural data and MD modelling. An analogous cardiolipin binding site between subunits is found in E.coli tetrameric GlpG, with cardiolipin (thermally) stabilizing the protein against aggregation.

Strengths:

The use of the artificial scaffold allows testing of hypothesis about the different roles of cardiolipin binding. It reveals effects which are at first sight counterintuitive and are explained by the existence of a weaker, secondary binding site which unlike the primary one allows easy lipid-mediated interaction between two subunits of the protein. Introducing different mutations either changes the balance between primary and secondary binding sites or introduced a kink in a helix - thus affecting subunit interactions which are experimentally verified by native mass spectrometry.

Weaknesses:

The artificial scaffold is not necessarily reflecting the conformational dynamics and local flexibility of real, functional membrane proteins. The example of GlpG, while also showing interesting cardiolipin dependency, illustrates the case of a binding site across helices further but does not add much to the main story. It should be evident that structural lipids can be stabilizing in more than one way depending on how they bind, leading to different and possibly opposite functional outcomes.

Reviewer #3 (Public review):

Summary:

The authors investigated the role of secretory leukocyte protease inhibitors (SLPI) in developing Lyme disease in mice infected with Borrelia burgdorferi. Using a combination of histological, gene expression, and flow cytometry analyses, they demonstrated significantly higher bacterial burden and elevated neutrophil and macrophage infiltration in SLPI-deficient mouse ankle joints. Furthermore, they also showed direct interaction of SLPI with B. burgdorferi, which likely depletes the local environment of SLPI and causes excessive protease activity. These results overall suggest ankle tissue inflammation in B. burgdorferi-infected mice is driven by unchecked protease activity.

Strengths:

Utilizing a comprehensive suite of techniques, this is the first study showing the importance of anti-protease-protease balance in the development of periarticular joint inflammation in Lyme disease.

Weaknesses:

Due to the limited sample availability, the authors investigated the serum level of SLPI in both in Lyme arthritis patients and patients with earlier disease manifestations.

Reviewer #3 (Public review):

Summary:

In this report, De Franceschi et al. purify components of the Cdv machinery in archaeon M. sedula and probe their interactions with membrane and with one-another in vitro using two main assays - liposome flotation and fluorescent imaging of encapsulated proteins. This has the potential to add to the field by showing how the order of protein recruitment seen in cells is related to the differential capacity of individual proteins to bind membranes when alone or when combined.

Strengths:

Using the floatation assay, they demonstrate that CdvA and CdvB bind liposomes when combined. While CdvB1 also binds liposomes under these conditions, in the floatation assay, CdvB2 lacking its C-terminus is not efficiently recruited to membranes unless CdvAB or CdvB1 are present. The authors then employ a clever liposome assay that generates chained spherical liposomes connected by thin membrane necks, which allows them to accurately control the buffer composition inside and outside of the liposome. With this, they show that all four proteins accumulate in necks of dumbbell-shaped liposomes that mimic the shape of constricting necks in cell division. Taken altogether, these data lead them to propose that Cdv proteins are sequentially recruited to the membrane as has also been suggested by in vivo studies of ESCRT-III dependent cell division in crenarchaea.

Weaknesses:

These experiments provide a good starting point for the in vitro study the interaction of Cdv system components with the membrane and their consecutive recruitment. However, several experimental controls are missing that complicate their ability to draw strong conclusions. Moreover, some results are inconsistent across the two main assays which make the findings difficult to interpret.

(1) Missing controls.

Various protein mixtures are assessed for their membrane-binding properties in different ways. However, it is difficult to interpret the effect of any specific protein combination, when the same experiment is not presented in a way that includes separate tests for all individual components. In this sense, the paper lacks important controls.

For example, Fig 1C is missing the CdvB-only control. The authors remark that CdvB did not polymerise (data not shown) but do not comment on whether it binds membrane in their assays. In the introduction, Samson et al., 2011 is cited as a reference to show that CdvB does not bind membrane. However, here the authors are working with protein from a different organism in a different buffer, using a different membrane composition and a different assay. Given that so many variables are changing, it would be good to present how M. sedula CdvB behaves under these conditions.

Similarly, there is no data showing how CdvB alone or CdvA alone behave in the dumbbell liposome assay. Without these controls, it's impossible to say whether CdvA recruits CdvB or the other way around.

The manuscript would be much stronger if such data could be added.

(2) Some of the discrepancies in the data generated using different assays are not discussed.

The authors show that CdvB2∆C binds membrane and localizes to membrane necks in the dumbbell liposome assay, but no membrane binding is detected in the flotation assay. The discrepancy between these results further highlights the need for CdvB-only and CdvA-only controls.

(3) Validation of the liposome assay.

The experimental setup to create dumbbell-shaped liposomes seems great and is a clever novel approach pioneered by the team. Not only can the authors manipulate liposome shape, they also state that this allows them to accurately control the species present on the inside and outside of the liposome. Interpreting the results of the liposome assay, however, depends on the geometry being correct. To make this clearer, it would seem important to include controls to prove that all the protein imaged at membrane necks lie on the inside of liposomes. In the images in SFig3 there appears to be protein outside of the liposome. It would also be helpful to present data to show test whether the necks are open, as suggested in the paper, by using FRAP or some other related technique.

(4) Quantification of results from the liposome assay.

The paper would be strengthened by the inclusion of more quantitative data relating to the liposome assay. Firstly, only a single field of view is shown for each condition. Because of this, the reader cannot know whether this is a representative image, or an outlier? Can the authors do some quantification of the data to demonstrate this? The line scan profiles in the supplemental figures would be an example of this, but again in these Figures only a single image is analyzed.

We would recommend that the authors present quantitative data to show the extent of co-localization at the necks in each case. They also need a metric to report instances in which protein is not seen at the neck, e.g. CdvB2 but not CdvB1 in Fig2I, which rules out a simple curvature preference for CdvB2 as stated in line 182.

Secondly, the authors state that they see CdvB2∆C recruited to the membrane by CdvB1 (lines 184-187, Fig 2I). However, this simple conclusion is not borne out in the data. Inspecting the CdvB2∆C panels of Fig 2I, Fig3C, and Fig3D, CdvB2∆C signal can be seen at positions which don't colocalize with other proteins. The authors also observe CdvB2∆C localizing to membrane necks by itself (Fig 2E). Therefore, while CdvB1 and CdvB2∆C colocalize in the flotation assay, there is no strong evidence for CdvB2∆C recruitment by CdvB1 in dumbbells. This is further underscored by the observation that in the presented data, all Cdv proteins always appear to localize at dumbbell necks, irrespective of what other components are present inside the liposome. Although one nice control is presented (ZipA), this suggests that more work is required to be sure that the proteins are behaving properly in this assay. For example, if membrane binding surfaces of Cdv proteins are mutated, does this lead to the accumulation of proteins in the bulk of the liposome as expected?

(5) Rings.

The authors should comment on why they never observe large Cdv rings in their experiments. In crenarchaeal cell division, CdvA and CdvB have been observed to form large rings in the middle of the 1 micron cell, before constriction. Only in the later stages of division are the ESCRTs localized to the constricting neck, at a time when CdvA is no longer present in the ring. Therefore, if the in vitro assay used by the authors really recapitulated the biology, one would expect to see large CdvAB rings in Figs 1EF. This is ignored in the model. In the proposed model of ring assembly (line 252), CdvAB ring formation is mentioned, but authors do not discuss the fact that they do not observe CdvAB rings - only foci at membrane necks. The discussion section would benefit from the authors commenting on this.

(6) Stoichiometry

It is not clear why 100% of the visible CdvA and 100% of the the visible CdvB are shifted to the lipid fraction in 1C. Perhaps this is a matter of quantification. Can the authors comment on the stoichiometry here?

(7) Significance of quantification of MBP-tagged filaments.

Authors use tagging and removal of MBP as a convenient, controllable system to trigger polymerisation of various Cdv proteins. However, it is unclear what is the value and significance of reporting the width and length of the short linear filaments that are formed by the MBP-tagged proteins. Presumably they are artefactual assemblies generated by the presence of the tag? Similar Figure 2C doesn't seem a useful addition to the paper.

DOI: 10.1038/s41419-025-07335-3

Resource: (IMSR Cat# JAX_007850,RRID:IMSR_JAX:007850)

Curator: @dhovakimyan1

SciCrunch record: RRID:IMSR_JAX:007850

What is this?

Reviewer #3 (Public review):

Summary:<br /> In this paper, Xu, Dantu and coworkers report a protocol for analyzing coevolutionary and dynamical information to identify a subset of communities that capture functionally relevant sites in beta-lactamases.

Strengths:<br /> The combination of coevolutionary information and metrics from MD simulations is interesting for capturing functionally relevant sites, which can have implications in the fields of drug discovery but also in protein design.

Weaknesses:<br /> The combination of coevolutionary information and metrics from MD simulations is not new as other protocols have been proposed along the years (the current version of the paper neglects some of them, see below), and there are a few parameters of the protocol that, in my opinion, should be better analyzed and discussed.

(1) As mentioned, the introduction of the paper lacks some important publications in the field of using graph theory to represent important interaction networks extracted from MD simulations (DOI: 10.1002/pro.4911), and also combining MD data with MSA to identify functionally relevant sites for enzyme design (doi: 10.1021/acscatal.4c04587, 10.1093/protein/gzae005).<br /> (2) The matrix used to apply graph theory (J_ij) is built from summing the scaled coevolution and degree of correlation values. The alpha and beta weights are defined, and the authors mention that alpha is set to 0.5, thus beta as well to fulfil with the alpha + beta = 1. Why a value of 0.5 has been selected? How this affects the overall results and conclusions extracted? The finding that many catalytically relevant residues are identified in the communities is not surprising given that such sites usually present a high conservation score.<br /> (3) Another important point that needs further explanation is the selection of the relevant descriptor of protein dynamics. In this study two different strategies have been used (one more global the other more local), but more details should be provided regarding their choice. What is the best strategy according to the authors? Why not using the same strategy for both related systems? The obtained results using one methodology or the other will have a large impact on the dynamical score. Another related point is: what is the impact of the MD simulation length, how the MSA is generated and number of sequences used for MSA construction?

Reviewer #3 (Public review):

Summary:

Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that can be amplified in gastric cancer and serves as a potential therapeutic target for this patient population. However, targeting FGFR2 has shown limited efficacy. Thus, this study seeks to identify additional molecules that can be effectively targeted in FGFR2 amplified gastric cancer, with a focus on Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2). The authors first demonstrate that 6% of gastric cancer patients in a cohort of human patient samples exhibit FGFR2 amplification. Furthermore, they demonstrate that FGFR2 mRNA expression is positively correlated with PTPN11 gene expression (which is the gene that encodes the SHP2 protein). Using human gastric cancer cell lines with amplified FGFR2, the authors then test the effects of combining the FGFR inhibitor AZD4547 with the SHP2 inhibitor SHP099 on tumor cell death and signaling molecules. They demonstrate that combining the two inhibitors is more effective at tumor cell killing and reducing activation of downstream signaling pathways than either inhibitor alone. In further studies, the authors obtained gastric cancer cells with FGFR2 amplification from a patient that was treated with FGFR2 inhibitor. While this patient initially showed a partial response, the patient ultimately progressed, demonstrating resistance to FGFR2 inhibition. Following isolation of tumor cells from the patient's ascites, the authors demonstrate that these cells are sensitive to the combination treatment of AZD4547 and SHP099. Further studies were performed using a xenograft model using athymic nude mice in which the combination of SHP099 and AZD4547 were found to reduce tumor growth more significantly than either treatment alone. Finally, the authors demonstrate using an in vitro culture model that this combination treatment enhances T cell mediated cytotoxicity. The authors conclude that targeting FGFR2 and SHP2 represents a potential combination strategy in gastric patients with FGFR2 amplification.

Strengths:

The authors demonstrate that FGFR2 amplification positively correlates with PTPN11 in human gastric cancer samples, providing rationale for combination therapies. Furthermore, convincing data are provided demonstrating that targeting both FGFR and SHP2 is more effective than targeting either pathway alone using in vitro and in vivo models. The use of cells derived from a gastric cancer patient that progressed following treatment with an FGFR inhibitor is also a strength. The findings from this study support the conclusion that SHP2 inhibitors enhance the efficacy of FGFR-targeted therapies in cancer patients. This study also suggests that targeting SHP2 may also be an effective strategy for targeting cancers that are resistant to FGFR-targeted therapies.

Weaknesses:

The main caveat with these studies is the lack of an immune competent model with which to test the finding that this combination therapy enhances T cell cytotoxicity in vivo. Discussing this limitation within the context of these findings and future directions for this work, particularly since the combination therapy appears to work quite well without the presence of T cells in the environment, would be beneficial.

Reviewer #3 (Public review):

Xiaoyu Wu and colleagues examined the potential role in sleep of a Drosophila ribosomal RNA methyltransferase, mettl5. Based on sleep defects reported in CRISPR generated mutants, the authors performed both RNA-seq and Ribo-seq analyses of head tissue from mutants and compared to control animals collected at the same time point. While these data were subjected to a thorough analysis, it was difficult to understand the relative direction of differential expression between the two genotypes. In any case, a major conclusion was that the mutant showed altered expression of circadian clock genes, and that the altered expression of the period gene in particular accounted for the sleep defect reported in the mettl5 mutant. As noted above, a strength of this work is its relevance to a human developmental disorder as well as the transcriptomic and ribosomal profiling of the mutant. However, there are numerous weaknesses in the manuscript, most of which stem from misinterpretation of the findings, some methodological approaches, and also a lack of method detail provided. The authors seemed to have missed a major phenotype associated with the mettl5 mutant, which is that it caused a significant increase in period length, which was apparent even in a light: dark cycle. Thus the effect of the mutant on clock gene expression more likely contributed to this phenotype than any associated with changes in sleep behavior.

Reviewer #3 (Public review):

Summary:

The authors describe important new biochemical elements in the synthesis of a class of critical developmental signaling molecules, BMP4. They also present a highly detailed description of developmental anomalies in mice bearing known human mutations at these specific elements.

Strengths:

Exceptionally detailed descriptions of pathologies occurring in mutant mice. Novel findings regarding the interaction of propeptide phosphorylation and convertase cleavage, both of which will move the field forward. Provocative hypothesis regarding furin access to cleavage sites, supported by Alphafold predictions.

Weaknesses:

Figure 6A presents two testable models for pre-release access of furin to cleavage sites since physical separation of enzyme from substrate only occurs in one model; could immunocytochemistry resolve?

Reviewer #3 (Public review):

Summary:

In this report, the authors tested how manipulating the contiguous set of stimuli on the screen that should be used to guide behavior - that is, the scope of visual spatial attention - impacts the magnitude and profile of well-established attentional enhancements in visual retinotopic cortex. During fMRI scanning, participants attended to a cued section of the screen for blocks of trials and performed a letter vs digit discrimination task at each attended location (and judged whether the majority of characters were letters/digits). Importantly, the visual stimulus was identical across attention conditions, so any observed response modulations are due to top-down task demands rather than visual input. The authors employ population receptive field (pRF) models, which are used to sort voxel activation with respect to the location and scope of spatial attention and fit a Gaussian-like function to the profile of attentional enhancement from each region and condition. The authors find that attending to a broader region of space expands the profile of attentional enhancement across the cortex (with a larger effect in higher visual areas), but does not strongly impact the magnitude of this enhancement, such that each attended stimulus is enhanced to a similar degree. Interestingly, these modulations, overall, mimic changes in response properties caused by changes to the stimulus itself (increase in contrast matching the attended location in the primary experiment). The finding that attentional enhancement primarily broadens, but does not substantially weaken in most regions, is an important addition to our understanding of the impact of distributed attention on neural responses, and will provide meaningful constraints to neural models of attentional enhancement.

Strengths:

- Well-designed manipulations (changing location and scope of spatial attention), and careful retinotopic/pRF mapping, allow for a robust assay of the spatial profile of attentional enhancement, which has not been carefully measured in previous studies<br /> - Results are overall clear, especially concerning width of the spatial region of attentional enhancement, and lack of clear and consistent evidence for reduction in the amplitude of enhancement profile<br /> - Model-fitting to characterize spatial scope of enhancement improves interpretability of findings

Weaknesses:

- Task difficulty seems to vary as a function of spatial scope of attention, with varying ratios of letters/digits across spatial scope conditions, which may complicate interpretations of neural modulation results<br /> - Some aspects of analysis/data sorting are unclear (e.g., how are voxels selected for analyses?)<br /> - While the focus of this report is on modulations of visual cortex responses due to attention, the lack of inclusion of results from other retinotopic areas (e.g. V3AB, hV4, IPS regions like IPS0/1) is a weakness<br /> - Additional analyses comparing model fits across amounts of data analyzed suggest the model fitting procedure is biased, with some parameters (e.g., FWHM, error, gain) scaling with noise.

Reviewer #3 (Public review):

Summary:

This manuscript describes the characterization of mycobacterial cytoskeleton protein Wag31, examining its role in orchestrating protein-lipid and protein-protein interactions essential for mycobacterial survival. The most significant finding is that Wag31, which directs polar elongation and maintains the intracellular membrane domain, was revealed to have membrane tethering capabilities.

Strengths:

The authors provided a detailed analysis of Wag31 domain architecture, revealing distinct functional roles: the N-terminal domain facilitates lipid binding and membrane tethering, while the C-terminal domain mediates protein-protein interactions. Overall, this study offers a robust and new understanding of Wag31 function.

Weaknesses:

The following major concerns should be addressed.

• Authors use 10-N-Nonyl-acridine orange (NAO) as a marker for cardiolipin localization. However, given that NAO is known to bind to various anionic phospholipids, how do the authors know that what they are seeing is specifically visualizing cardiolipin and not a different anionic phospholipid? For example, phosphatidylinositol is another abundant anionic phospholipid in mycobacterial plasma membrane.

• Authors' data show that the N-terminal region of Wag31 is important for membrane tethering. The authors' data also show that the N-terminal region is important for sustaining mycobacterial morphology. However, the authors' statement in Line 256 "These results highlight the importance of tethering for sustaining mycobacterial morphology and survival" requires additional proof. It remains possible that the N-terminal region has another unknown activity, and this yet-unknown activity rather than the membrane tethering activity drives the morphological maintenance. Similarly, the N-terminal region is important for lipid homeostasis, but the statement in Line 270, "the maintenance of lipid homeostasis by Wag31 is a consequence of its tethering activity" requires additional proof. The authors should tone down these overstatements, or provide additional data to support their claims.

• Authors suggest that Wag31 acts as a scaffold for the IMD (Fig. 8). However, Meniche et. al. has shown that MurG as well as GlfT2, two well-characterized IMD proteins, do not colocalize with Wag31 (DivIVA) (https://doi.org/10.1073/pnas.1402158111). IMD proteins are always slightly subpolar while Wag31 is located to the tip of the cell. Therefore, the authors' biochemical data cannot be easily reconciled with microscopic observations in the literature. This raises a question regarding the validity of protein-protein interaction shown in Figure 7. Since this pull-down assay was conducted by mixing E. coli lysate expressing Wag31 and Msm lysate expression Wag31 interactors like MurG, it is possible that the interactions are not direct. Authors should interpret their data more cautiously. If authors cannot provide additional data and sufficient justifications, they should avoid proposing a confusing model like Figure 8 that contradicts published observations.

Reviewer #3 (Public review):

Summary:

In their manuscript titled "Multiplexed Assays of Human Disease‐relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability" the authors aim to investigate the effect of sequence variations in 3'UTR and 5'UTRs on the stability of mRNAs in two different human cell lines.

To do so, the authors use a massively parallel reporter assay (MPRA). They transfect cells with a set of mRNA reporters that contain sequence variants in their 3' or 5' UTRs, which were previously reported in human diseases. They follow their clearance from cells over time relative to the matching non-variant sequence. To analyze their results, they define a set of factors (RBP and miRNA binding sites, sequence features, secondary structure etc.) and test their association with differences in mRNA stability. For features with a significant association, they use clustering to select a subset of factors for LASSO regression and identify factors that affect mRNA stability.<br /> They conclude that the TA dinucleotide content of UTRs is the strongest destabilizing sequence feature. Within that context, elevated GC content and protein binding can protect susceptible mRNAs from degradation. They also show that TA dinucleotide content of UTRs affects native mRNA stability and that it is associated with specific functional groups. Finally, they link disease associated sequence variants with differences in mRNA stability of reporters.

Strengths:

(1) This work introduces a different MPRA approach to analyze the effect of genetic variants. While previous works in tissue culture use DNA transfections that require normalization for transcription efficiency, here the mRNA is directly introduced into cells at fixed amounts, allowing a more direct view of the mRNA regulation.

(2) The authors also introduce a unique analysis approach, which takes into account multiple factors that might affect mRNA stability. This approach allows them to identify general sequence features that affect mRNA stability beyond specific genetic variants, and reach important insights on mRNA stability regulation. Indeed, while the conclusions to genetic variants identified in this work are interesting, the main strength of the work involves general effect of sequence features rather than specific variants.

(3) The authors provide adequate support for their claims and validate their analysis using both their reporter data and native genes. For the main feature identified, TA di-nucleotides, they perform follow-up experiments with modified reporters that further strengthen their claims, and also validate the effect on native cellular transcripts (beyond reporters), demonstrating its validity also within native scenarios.

(4) The work provides a broad analysis of mRNA stability, across two mRNA regulatory segments (3'UTR and 5'UTR) and is performed in two separate cell-types. Comparison between two different cell-types is adequate, and the results demonstrate, as expected, the dependence of mRNA stability on the cellular context. Analysis of 3'UTR and 5'UTR regulatory effects also shows interesting differences and similarities between these two regulatory regions.

Weaknesses:

In their revised manuscripts, the authors successfully address many of the weaknesses raised in the original review, including the effect of possible confounding effects, and additional methodology details. Notably, two of the issues raised in the original report, have only been partially addressed in the revision.

(1) The analysis and regression models built in this work are not thoroughly investigated relative to native genes within cells.<br /> While using MPRAs indeed allows to isolate regulatory effects that are less influential in-vivo, the resulting effects still provide some regulatory function in-vivo. The goal of such an analysis would not be to demonstrate the predictive power of the models, or to make any claims regarding using these models to fully explain or predict the stability of native transcripts. Clearly, additional more prominent factors could function in controlling endogenous RNA stability.<br /> Instead, the goal of such an investigation is to simply assess the fraction of in-vivo regulation that the factors identified in this work contribute in native contexts, and what is the relative contribution of the phenomena captured by the well-controlled MPRA study.<br /> This reviewer believes that even if the effects identified by the current MPRA study only contribute a small fraction of in-vivo variation, an analysis that aim to estimate what this fraction is, will be very relevant to this study for several reasons. First, in order to appreciate the results of this study within their in-vivo context. Second, in light of the questions raised as motivation for this study, and particularly the need to identify the effect of disease-associated 3'UTR variants, which clearly have an in-vivo effect.

(2) Methodology validation can be performed with simulated data (generated in-silico by the authors) to provide an independent support for the ability of the current methodology to correctly extract regulatory effects from the data.

Reviewer #3 (Public review):

Summary:

Li et al. describe an audiovisual temporal recalibration experiment in which participants perform baseline sessions of ternary order judgments about audiovisual stimulus pairs with various stimulus-onset asynchronies (SOAs). These are followed by adaptation at several adapting SOAs (each on a different day), followed by post-adaptation sessions to assess changes in psychometric functions. The key novelty is the formal specification and application/fit of a causal-inference model for the perception of relative timing, providing simulated predictions for the complete set of psychometric functions both pre and post adaptation.

Strengths:

(1) Formal models are preferable to vague theoretical statements about a process, and prior to this work, certain accounts of temporal recalibration (specifically those that do not rely on a population code) had only qualitative theoretical statements to explain how/why the magnitude of recalibration changes non-linearly with the stimulus-onset asynchrony of the adaptor.<br /> (2) The experiment is appropriate, the methods are well described, and the average model prediction is a good match to the average data (Figure 4). Conclusions are supported by the data and modelling.<br /> (3) The work should be impactful. There seems a good chance that this will become the go-to modelling framework for those exploring non population-code accounts of temporal recalibration (or comparing them with population-code accounts).<br /> (4) Key issues for the generality of the model, such as recalibration asymmetries reported by other authors that are inconsistent with those reported here, are thoughtfully discussed.

Weaknesses:

(1) Models are not compared using a gold-standard measure such as leave-one-out cross validation. However, this is legitimate given lengthy model fitting times, and a sensible approximation is presented.<br /> (2) The model misses in a systematic way for the psychometric functions of some participants/conditions. In addition to misses relating to occasional failures to estimate the magnitude of recalibration, some of the misses are because all functions are only permitted to shift in central tendency (whereas some participants show changes better characterized at one or both decision criteria). Given the fact that the modelling in general embraces individual differences, it might have been worth allowing different kinds of change for different participants. However, this is not really critical for the central concern (changes in the magnitude of recalibration for different adaptors) and there is a limit to how much can be done along these lines without making the model too flexible to test.<br /> (3) As a minor point, the model relies on simulation, which may limit its take-up/application by others in the field (although open access code will be provided).

Reviewer #3 (Public review):

Summary:

In this manuscript, Bimbard and colleagues describe a new implant apparatus called "Apollo Implant", which should facilitate recording in freely moving rodents (both mice and rats) using Neuropixels probes. The authors collected data from both mice and rats, they used 3 different versions of Neuropixels, multiple labs have already adopted this method, which is impressive. They openly share their CAD designs and surgery protocol to further facilitate the adaptation of their method.

Strengths:

Overall, the "Apollo Implant" is easy to use and adapt, as it has been used in other laboratories successfully and custom modifications are already available. The device is reproducible using common 3D printing services and can be easily modified thanks to its CAD design (the video explaining this is extremely helpful). The weight and price are amazing compared to other systems for rigid silicon probes allowing a wide range of use of the "Apollo Implant".

Weaknesses:

The "Apollo Implant" can only handle Neuropixels probes. It cannot hold other widely used and commercially available silicon probes. Certain angles and distances may be better served by 2 implants.

Reviewer #3 (Public review):

Summary:

This study is focused on testing whether statistical learning (a mechanism for parsing the speech signal into smaller chunks) preferentially operates over certain features of the speech at birth in humans. The features under investigation are phonetic content and speaker identity. Newborns are tested in an EEG paradigm in which they are exposed to a long stream of syllables. In Experiment 1, newborns are familiarized with a sound stream that comprises regularities (transitional probabilities) over syllables (e.g., "pe" followed by "tu" in "petu" with 1.0 probability) while the voices uttering the syllables remain random. In Experiment 2, newborns are familiarized with the same sound stream but, this time, the regularities are built over voices (e.g., "green voice" followed by "red voice" with 1.0 probability) while the concatenation of syllables stays random. At the test, all newborns listened to duplets (individual chunks) that either matched or violated the structure of the familiarization. In both experiments, newborns showed neural entrainment to the regularities implemented in the stream, but only the duplets defined by transitional probabilities over syllables (aka word forms) elicited a N400 ERP component. These results suggest that statistical learning operates in parallel and independently on different dimensions of the speech already at birth and that there seems to be an advantage for processing statistics defining word forms rather than voice patterns.

Strengths:

This paper presents an original experimental design that combines two types of statistical regularities in a speech input. The design is robust and appropriate for EEG with newborns. I appreciated the clarity of the Methods section. There is also a behavioral experiment with adults that acts like a control study for newborns. The research question is interesting, and the results add new information about how statistical learning works at the beginning of postnatal life, and on which features of the speech. The figures are clear and helpful in understanding the methods, especially the stimuli and how the regularities were implemented.

Weaknesses:

I appreciated how the authors addressed my previous comments and concerns. I am satisfied with the changes made by the authors. I believe the paper reads much better. Also, the adjustment to the theoretical framework suits well.

Reviewer #3 (Public Review):

Distal appendages are multiprotein complexes that are only present on the mother centriole as a 9-fold symmetric structure that functions in ciliogenesis. How distal appendage proteins are organized and assembled still remains poorly understood. In this manuscript, Kanie et al. comprehensively analyzed the localizations of known and newly described distal appendage proteins using super-resolution microscopy. They investigated mechanisms associated with distal appendage assembly and their roles in the early stages of ciliogenesis in CRISPR-Cas9 knockout cells, which enabled a clearer investigation of these structures compared to previous RNAi depletion studies. These studies confirm previous findings for distal appendage protein ciliogenesis function and demonstrate the CEP83-SCLT1-CEP164-TTBK2 module is critical for both distal appendage assembly and the initiation of ciliogenesis. Notably, they find that CEP89 is dispensable for distal appendage assembly, but is needed for the recruitment of RAB34-positive ciliary vesicles to the mother centriole for ciliogenesis. Finally, this work introduces the application of single-molecule 3D super-resolution microscopy as a tool for interrogating the relationship between membranes and distal appendages. Overall this work extends our fundamental understanding of distal appendage structure/function in ciliogenesis.

An interesting observation from this work is that CEP83 is found localized both at the innermost region and the outermost region of the distal appendages when detected by antibodies that recognize a different epitope of CEP83 (Figure 1A), suggesting a helical structure that could serve as a platform for distal appendage assembly. A previous study using STORM imaging also showed that another distal appendage protein CEP164 occupies a wider region of the distal appendages when using an antibody recognizing the N-terminal residues of Cep164 (M Bowler et al. 2019). Together these studies show the importance of evaluating the structure of distal appendage proteins and the challenges of using antibody detection to reveal distal appendage hierarchy.

This work also highlights the potential differences in functional conclusions that can be drawn when comparing RNAi and CRISPR knockout depletion approaches. The latter which expectedly can lead to a more precise functional analysis of these small distal appendage structures, albeit with the potential for knockout cells to display compensatory regulation. Although not directly addressed in the text, the authors find that RPE-1 MYO5A knockout cells could ciliate which differs from a report by Wu et al. (2018). Furthermore, in the case of RAB34 knockout cells, the authors find CP110 removal from the mother centriole, while in previously published RAB34 KO studies this was not observed. In the case of the. RAB34 data a plausible explanation for the results given by the authors is that different assay conditions were used as was noted by the authors.

Reviewer #3 (Public review):

Summary:

The authors show that ELS induces a number of brain and behavioral changes in the adult lateral amygdala. These changes include enduring astrocytic dysfunction, and inducing astrocytic dysfunction via genetic interventions is sufficient to phenocopy the behavioral and neural phenotypes suggesting astrocyte dysfunction may play a causal role in ELS-associated pathologies.

Strengths:

A strength is the shift in focus to astrocytes to understand how ELS alters adult behavior.

Weaknesses:

The mechanistic links between some of the correlates - altered astrocytic function, changes in neural excitability and synaptic plasticity in the lateral amygdala and behavior - are underdeveloped.

Comments on revisions:

The authors have significantly improved the paper with the addition of new experimental data, analyses, and textual changes.

Reviewer #3 (Public review):

Summary:

The paper studies learning rules in a simple sigmoidal recurrent neural network setting. The recurrent network has a single layer of 10 to 40 units. It is first confirmed that feedback alignment (FA) can learn a value function in this setting. Then so-called bio-plausible constraints are added: (1) when value weights (readout) is non-negative, (2) when the activity is non-negative (normal sigmoid rather than downscaled between -0.5 and 0.5), (3) when the feedback weights are non-negative, (4) when the learning rule is revised to be monotic: the weights are not downregulated. In the simple task considered all four biological features do not appear to impair totally the learning.

Strengths:

(1) The learning rules are implemented in a low-level fashion of the form: (pre-synaptic-activity) x (post-synaptic-activity) x feedback x RPE. Which is therefore interpretable in terms of measurable quantities in the wet-lab.

(2) I find that non-negative FA (FA with non negative c and w) is the most valuable theoretical insight of this paper: I understand why the alignment between w and c is automatically better at initialization.

(3) The task choice is relevant since it connects with experimental settings of reward conditioning with possible plasticity measurements.

Weaknesses:

(4) The task is rather easy, so it's not clear that it really captures the computational gap that exists with FA (gradient-like learning) and simpler learning rule like a delta rule: RPE x (pre-synpatic) x (post-synaptic). To control if the task is not too trivial, I suggest adding a control where the vector c is constant c_i=1.

(5) Related to point 3), the main strength of this paper is to draw potential connection with experimental data. It would be good to highlight more concretely the prediction of the theory for experimental findings. (Ideally, what should be observed with non-negative FA that is not expected with FA or a delta rule (constant global feedback) ?).

(6a) Random feedback with RNN in RL have been studied in the past, so it is maybe worth giving some insights how the results and the analyzes compare to this previous line of work (for instance in this paper [1]). For instance, I am not very surprised that FA also works for value prediction with TD error. It is also expected from the literature that the RL + RNN + FA setting would scale to tasks that are more complex than the conditioning problem proposed here, so is there a more specific take-home message about non-negative FA? or benefits from this simpler toy task?<br /> (6b) Related to task complexity, it is not clear to me if non-negative value and feedback weights would generally scale to harder tasks. If the task in so simple that a global RPE signal is sufficient to learn (see 4 and 5), then it could be good to extend the task to find a substantial gap between: global RPE, non-negative FA, FA, BP. For a well chosen task, I expect to see a performance gap between any pair of these four learning rules. In the context of the present paper, this would be particularly interesting to study the failure mode of non-negative FA and the cases where it does perform as well as FA.

(7) I find that the writing could be improved, it mostly feels more technical and difficult than it should. Here are some recommendations:<br /> (7a) for instance the technical description of the task (CSC) is not fully described and requires background knowledge from other paper which is not desirable.<br /> (7b) Also the rationale for the added difficulty with the stochastic reward and new state is not well explained.<br /> (7c) In the technical description of the results I find that the text dives into descriptive comments of the figures but high-level take home messages would be helpful to guide the reader. I got a bit lost, although I feel that there is probably a lot of depth in these paragraphs.

(8) Related to the writing issue and 5), I wished that "bio-plausibility" was not the only reason to study positive feedback and value weights. Is it possible to develop a bit more specifically what and why this positivity is interesting? Is there an expected finding with non-negative FA both in the model capability? or maybe there is a simpler and crisp take-home message to communicate the experimental predictions to the community would be useful?

(1) https://www.nature.com/articles/s41467-020-17236-y

DOI: 10.1016/j.celrep.2024.115195

Resource: (ZFIN Cat# ZDB-ALT-070612-3,RRID:ZFIN_ZDB-ALT-070612-3)

Curator: @sjvitug

SciCrunch record: RRID:ZFIN_ZDB-ALT-070612-3

What is this?

DOI: 10.1007/s10565-024-09981-3

Resource: KEGG (RRID:SCR_012773)

Curator: @dhovakimyan1

SciCrunch record: RRID:SCR_012773

What is this?

Reviewer #3 (Public review):

Summary:

This study reveals that sound exposure enhances drug delivery to the cochlea through the non-selective action of outer hair cells. The efficiency of sound-facilitated drug delivery is reduced when outer hair cell motility is inhibited. Additionally, low-frequency tones were found to be more effective than broadband noise for targeting substances to the cochlear apex. Computational model simulations support these findings.

Strengths:

The study provides compelling evidence that the broad action of outer hair cells is crucial for cochlear fluid circulation, offering a novel perspective on their function beyond frequency-selective amplification. Furthermore, these results could offer potential strategies for targeting and optimizing drug delivery throughout the cochlear spiral.

Reviewer #3 (Public review):

Summary:

The article provides the most comprehensive overview of primate MHC class I and class II genes to date, combining published data with an exploration of the available genome assemblies in a coherent phylogenetic framework and formulating new hypotheses about the evolution of the primate MHC genomic region.

Strengths:

I think this is a solid piece of work that will be the reference for years to come, at least until population-scale haplotype-resolved whole-genome resequencing of any mammalian species becomes standard. The work is timely because there is an obvious need to move beyond short amplicon-based polymorphism surveys and classical comparative genomic studies. The paper is data-rich and the approach taken by the authors, i.e. an integrative phylogeny of all MHC genes within a given class across species and the inclusion of often ignored pseudogenes, makes a lot of sense. The focus on primates is a good idea because of the wealth of genomic and, in some cases, functional data, and the relatively densely populated phylogenetic tree facilitates the reconstruction of rapid evolutionary events, providing insights into the mechanisms of MHC evolution. Appendices 1-2 may seem unusual at first glance, but I found them helpful in distilling the information that the authors consider essential, thus reducing the need for the reader to wade through a vast amount of literature. Appendix 3 is an extremely valuable companion in navigating the maze of primate MHC genes and associated terminology.

Weaknesses:

I have not identified major weaknesses and my comments are mostly requests for clarification and justification of some methodological choices.

for - insight - good for digital nomads to be rooted somewhere in the physical word - they are like a cosmo guild of knowledge workers - localities tend to repeat the same things - digital nomads as outsiders can inject new patterns - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - shadow side - of root-less digital nomads - when the sh*t hits the fan, working class will target digital nomads - as they will be seen as a parasitical force - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - cosmolocal strategy - locals - permaculture, bioregional regeneration - cosmo - digital nomads - share collective protocols with locals to create cosmolocal networks - Austin Wade Smith - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - stats - 2025 - digital nomads - 34 million - with crypto - 10 million - rootless - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - quote - Web3 is mainly an exit (escape) strategy for privileged layers of society - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - open space for perspectival knowing - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - investigate - quadratic voting - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - paraphrase - history of labor - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2 - to - stats - Gallup Chairman's Blog - world poll 2024 - 15% of employees worldwide are engaged - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

paraphrase - history of labor - Michel gives a nice succinct summary of the broad strokes of the history of labor over the last few millennia: - Civilizations have begun as slave-based societies first - Then when the Christian revolution occurred after the fall of the Roman Empire, "Ora et Labora (Pray and Work)" was adopted to transform work into a spiritually meaningful endeavor - Then in the 16th century, this philosophy was replaced by turning labor into a commodity, where it has remained ever since, - resulting in a world where 85% of those surveyed say they are not engaged with their job

to - stats - Gallup Chairman's Blog - world poll 2024 - 15% of employees worldwide are engaged - https://hyp.is/iOlXbNBOEe-t6hdOWtvTYw/news.gallup.com/opinion/chairman/212045/world-broken-workplace.aspx

for - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

// - COMMENTS - This is a very insightful interview with Michel that provides a lot of historical contexts for the many challenges faced by contemporary society - Within these historical contexts, we can glimpse how today's problems are part of a repeating pattern, albeit with many new elements that have emerged - He offers the possibility of a commons approach of mutualization, - in particular cosmolocalism - as a powerful leverage point to evolve a future wellbeing civilization - Contexualizing modernity in the alternate growth and downfall periods of human civilizations, he points out how we are in a transition period in which the current system is fraying - He outlines the many seed forms that exist now which, just like those that appeared in past cycles of downfall, combined to emerge the next growth cycle - crypto and blockchain - which can provide a global way of coordinating planetary health - the internet in general, which can bring mutualization of knowledge for locailzed production - There are some strong exemplars of promising seed forms but to scale, - the cosmo processes have to integrate with - local, place-anchored processes such as permacutlure and bioregion-based regeneration.

//

for - event - Funding the Commons - Bangkok conference 2024 - Michel Bauwens - guest - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2 - to - Funding the Commons - Bangkok conference 2024

to - Funding the Commons - Bangkok conference 2024 - https://hyp.is/fF-mVNBJEe-OWvM5g4ZLOQ/www.fundingthecommons.io/bangkok-2024

for - bottom up mobilization - leverage the strength of the commons - create global coalition of local projects within in a common area - IE. Land trust - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - work to find way to use web 3 / crypto to fund currently underfunded regenerative work done by millions of people - the missing link - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - adjacency - trans-financial capital - political polarization - powerlessness of two party politics - culture wars distraction - Yanis Varoufakis - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - cosmolicalism - nice articulation - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - mutualise at scale - add much in the SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - example - efficacy of mutualisation - transportation - cars - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2 - stats - mutualisation - transportation - cars - 1 car can replace 13 - car is parked most of the time - 10% of existing cars doubles our requirement - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

for - definition - Civium - Jordan Hall - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2 - adjacency - Civium - Tipping Point Festival - SOURCE - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2

adjacency - between - civium - Tipping Point Festival - Civiums are the terminology that applies for the vision of the TIpping Point Festival, where twice a year, - solstice - equinox - People gather and converge at a central temporary, cosmolocal event to mutually exchange ideas, network, seed new projects and review the past years successes and failures - This is an event also used to operationalize a planetary framework for restoration and regeneration that is syncrhonized to earth system boundaries, but contextualized to each locality, - but needs to be done at the scale of thousands of cities to have planetary-scale impact - It is, by design, a cosmolocal event

for - stats - job satisfaction - Gallup poll 2024 - engaged employees worldwide - 15% - Gallup CEO - Jim Clifton - from - Youtube Ma Earth channel interview - Devcon 2024 - Cosmo Local Commoning with Web 3 - Michel Bauwens - 2025, Jan 2 - https://hyp.is/FiFXpNBMEe-n_Jc4-PJ5_A/www.youtube.com/watch?v=UCkLHj6r7y8

Reviewer #3 (Public review):

Summary:

In this paper, Alonso-Caraballo et al. investigate sex-specific differences in oxycodone self-administration, withdrawal, and relapse behaviors in rats, as well as associated synaptic plasticity in the paraventricular thalamus to nucleus accumbens shell (PVT-NAcSh) circuit. The authors employ a combination of behavioral paradigms and ex vivo electrophysiology to examine how acute (1-day) and prolonged (14-day) abstinence from oxycodone self-administration affect cue-induced drug-seeking and synaptic transmission in male and female rats. Their findings reveal that while both sexes show similar oxycodone self-administration and acute withdrawal symptoms, females exhibit enhanced cue-induced relapse after prolonged abstinence. Furthermore, they show that prolonged abstinence is associated with increased synaptic strength in the PVT-NAcSh circuit (reduced paired-pulse ratio) and enhanced intrinsic excitability of NAcSh medium spiny neurons in both sexes. This study provides important insights into the sex-specific neural adaptations that may underlie vulnerability to opioid relapse and highlights the PVT-NAcSh circuit as a potential target for therapeutic interventions. However, although this study is well designed, no sex differences were observed in the synaptic activity within this pathway that could explain increased oxycodone seeking in females versus male rats. Additional experiments could strengthen the results and help clarify synaptic mechanisms underpinning behavioral sex differences.

Strengths:

The study exhibits several strengths. It provides a comprehensive behavioral analysis of oxycodone self-administration, withdrawal, and cue-induced relapse in both male and female rats at different time points (acute vs. protracted withdrawal) offering valuable insights into sex-specific differences (i.e., increased oxycodone seeking in females over time but not males). The authors examine synaptic plasticity in the PVT-NAcSh circuit at different abstinence time points, integrating behavioral and electrophysiological data to link circuit adaptations with relapse behaviors, although no sex differences in the electrophysiological parameters examined were evident. The investigation of intrinsic excitability changes in NAcSh medium spiny neurons further enhances the study's depth. Overall, the well-designed experiments provide important insights into the neural adaptations that may underlie vulnerability to opioid relapse, highlighting the PVT-NAcSh circuit as a potential target for therapeutic interventions in opioid use disorder.

Weaknesses:

Despite its strengths, the study has several notable limitations. A key weakness is the lack of observed sex differences in synaptic activity within the PVT-NAcSh pathway that could explain the behavioral results. The authors' failure to differentiate between D1 and D2 medium spiny neurons (MSNs) in the nucleus accumbens represents a missed opportunity to identify potential sex-specific differences at the cellular level, although they do discuss reasons for this omission. The only significant synaptic change observed - reduced paired-pulse ratio indicating increased synaptic strength - occurs in both males and females, failing to explain the sex-specific behavioral differences. Furthermore, the investigation of intrinsic excitability in NAc MSNs adds complexity to data interpretation, as the authors neither differentiate between D1 and D2 MSNs nor confirm that recorded neurons receive direct inputs from the PVT. This assumption potentially confounds the results. Overall, while the study provides valuable insights, additional experiments targeting specific cell populations and more detailed synaptic analyses are needed to elucidate the mechanisms underlying the observed behavioral sex differences in opioid relapse vulnerability.

Reviewer #3 (Public review):

Summary:

Jin, Briggs et al. made use of light-sheet 3D imaging and data analysis to assess the collective network activity in isolated mouse islets. The major advantage of using whole islet imaging, despite compromising on a speed of acquisition, is that it provides a complete description of the network, while 2D networks are only an approximation of the islet network. In static-incubation conditions, excluding the effects of perfusion, they assessed two subpopulations of beta cells and their spatial consistency and metabolic dependence.

Strengths:

The authors confirmed that coordinated Ca2+ oscillations are important for glycemic control. In addition, they definitively disproved the role of individual privileged cells, which were suggested to lead or coordinate Ca²⁺ oscillations. They provided evidence for differential regional stability, confirming the previously described stochastic nature of the beta cells that act as strongly connected hubs as well as beta cells in initiating regions (doi.org/10.1103/PhysRevLett.127.168101). This has not been a surprise to the reviewer.

The fact that islet cores contain beta cells that are more active and more coordinated has also been readily observed in high-frequency 2D recordings (e.g. DOI: 10.2337/db22-0952), suggesting that the high-speed capture of fast activity can partially compensate for incomplete topological information.

They also found an increased metabolic sensitivity of mantle regions of an islet with subpopulation of beta cells with a high probability of leading the islet activity and which can be entrained by fuel input. They discuss a potential role of alpha/delta cell interaction, however relative lack of beta cells in the islet border region could also be a factor contributing to less connectivity and higher excitability.

The Methods section contains a useful series of direct instructions on how to approach fast 3D imaging with currently available hardware and software.

The Discussion is clear and includes most of the issues regarding the interpretation of the presented results.

Taken together it is a strong technical paper to demonstrate the stochasticity regarding the functions subpopulations of beta cells in the islets may have and how less well-resolved approaches (both missing spatial resolution as well as missing temporal resolution) led us to jump to unjustified conclusions regarding the fixed roles of individual beta cells within an islet.

Weaknesses:

There are a few relevant issues that need to be addressed.

(1) The study is not internally consistent regarding the Results section. In the text the authors discuss changes in membrane potential (not been measured in this study), while in the figures they exclusively describe Ca2+ oscillations (which were measured). Examples are on lines 149, 150, 153, 154, 263... It is recommended that the silent and active phase in the Results section describe processes actually measured in this study as shown 6A.

(2) There are in fact no radially oriented networks in the core of an islet (l. 130, Fig. 4) apart from the fact that every hub has somewhat radially oriented edges. For radiality to have some general meaning, the normalized distance from the geometric center would need to be lower than 0.4. The networks are centrally located, which does not change the major conclusions of the study.

(3) The study would profit from acknowledging that Ca2+ influx is not a sole mechanism to drive insulin secretion and that KATP channels are not the sole target sensitive to changes in the cytosolic (global or local) ADP and ATP concentration or that there is an absolute concentration-dependence of these ligands on KATP channels. The relatively small conductance changes that have been found associated to active and silent phases (closing and opening of the KATP channels as interpreted by the authors, respectively, doi: 10.1152/ajpendo.00046.2013) and should be due to metabolic factors, could be also associated to desensitization of KATP channels to ATP due to the increase in cytosolic Ca2+ changes after intracellular Ca2+ flux (DOI: 10.1210/endo.143.2.8625) as they have been found to operate also at time scales, significantly faster (DOI: 10.2337/db22-0952) than reported before (refs. 21,22). Metabolic changes influence intracellular Ca2+ flux as well.

(4) There is no explanation for why KL divergence is so different between the pre-test regional consistency of the islets used to test the vehicle compared to those where GKa and PKa have been tested.

Reviewer #3 (Public review):

Summary:

The authors were trying to validate SARS-CoV-2 Mac1 as a drug discovery target and by extension other viral macrodomains.

Strengths:

The medicinal chemistry and structure based optimization is exemplary. Macrodomains and ADPribosyl hydrolases have a reputation for being undruggable, yet the authors managed to optimize hits from a fragment screen using structure based approaches and fragment linking to make a 20nM inhibitor as a tool compound to validate the target.<br /> In addition, the in vivo work is also a strength. The ability to reduce the viral count at a rate comparable to nirmatrelvir is impressive. Tracking the cytokine expression levels also supports much of the genetic data and mechanism of action for macrodomains.

Weaknesses:

The main compound AVI-4206, while being very potent and selective is not appreciably orally bioavailable. The fact that they have to use high doses of the compound IP to see in vivo effects may lead to questions regarding off target effects.

The cellular models are not as predictive of antiviral activity as one would expect. However, the authors had enough chutzpah to test the compound in vivo knowing that cellular models might not be an accurate representation of a living system with a fully functional immune system all of which is most likely needed in an antiviral response to test the importance of Mac1 as a target.

Reviewer #3 (Public review):

Summary:

The authors identified RBM20 expression in neural tissues using cell type-specific transcriptomic analysis. This discovery was further validated through in vitro and in vivo approaches, including RNA fluorescent in situ hybridization (FISH), open-source datasets, immunostaining, western blotting, and gene-edited RBM20 knockout (KO) mice. CLIP-seq and RiboTRAP data demonstrated that RBM20 regulates common targets in both neural and cardiac tissues, while also modulating tissue-specific targets. Furthermore, the study revealed that neuronal RBM20 governs long pre-mRNAs encoding synaptic proteins.

Strengths:

• Utilization of a large dataset combined with experimental evidence to identify and validate RBM20 expression in neural tissues.<br /> • Global and tissue-specific RBM20 KO mouse models provide robust support for RBM20 localization and expression.<br /> • Employing heart tissue as a control highlights the unique findings in neural tissues.

Weaknesses:

• Lack of physiological functional studies to explore RBM20's role in neural tissues.<br /> • Data quality requires improvement for stronger conclusions.<br /> • Western blot sample size should be increased for enhanced reliability.

Reviewer #3 (Public review):

Summary:

This study aims to explore the role of phosphorylated ubiquitin (pUb) in proteostasis and its impact on neurodegeneration. By employing a combination of molecular, cellular, and in vivo approaches, the authors demonstrate that elevated pUb levels contribute to both protective and neurotoxic effects, depending on the context. The research integrates proteasomal inhibition, mitochondrial dysfunction, and protein aggregation, providing new insights into the pathology of neurodegenerative diseases.

Strengths:

- The integration of proteomics, molecular biology, and animal models provides comprehensive insights.<br /> - The use of phospho-null and phospho-mimetic ubiquitin mutants elegantly demonstrates the dual effects of pUb.<br /> - Data on behavioral changes and cognitive impairments establish a clear link between cellular mechanisms and functional outcomes.

Weaknesses:

- While the study discusses the reciprocal relationship between proteasomal inhibition and pUb elevation, causality remains partially inferred.<br /> - The role of alternative pathways, such as autophagy, in compensating for proteasomal dysfunction is underexplored.<br /> - The immunofluorescence images in Figure 1A-D lack clarity and transparency. It is not clear whether the images represent human brain tissue, mouse brain tissue, or cultured cells. Additionally, the DAPI staining is not well-defined, making it difficult to discern cell nuclei or staging. To address these issues, lower-magnification images that clearly show the brain region should be provided, along with improved DAPI staining for better visualization. Furthermore, the Results section and Figure legends should explicitly indicate which brain region is being presented. These concerns raise questions about the reliability of the reported pUb levels in AD, which is a critical aspect of the study's findings.<br /> - Figure 4B should also indicate which brain region is being presented.

Reviewer #3 (Public review):

Summary:

The report by Shin, Lee, Kim, and Lee entitled "Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex" describes electrophysiological experiments of short-term synaptic plasticity during repetitive presynaptic stimulation at synapses between layer 2/3 pyramidal neurons and nearby target neurons. Manipulations include pharmacological inhibition of PLC and actin polymerization, activation of DAG receptors, and shRNA knockdown of Syt7. The results are interpreted as support for the hypothesis that synaptic vesicle release sites are vacant most of the time at resting synapses (i.e., p_occ is low) and that facilitation (and augmentation) components of short-term enhancement are caused by an increase in occupancy, presumably because of acceleration of the transition from not-occupied to occupied. The report additionally describes behavioural experiments where trace fear conditioning is degraded by knocking down syt7 in the same synapses.

Strengths:

The strength of the study is in the new information about short-term plasticity at local synapses in layer 2/3, and the major disruption of a memory task after eliminating short-term enhancement at only 15% of excitatory synapses in a single layer of a small brain region. The local synapses in layer 2/3 were previously difficult to study, but the authors have overcome a number of challenges by combining channel rhodopsins with in vitro electroporation, which is an impressive technical advance.

Weaknesses:

The question of whether or not short-term enhancement causes an increase in p_occ (i.e., "readily releasable pool overfilling") is important because it cuts to the heart of the ongoing debate about how to model short term synaptic plasticity in general. However, my opinion is that, in their current form, the results do not constitute strong support for an increase in p_occ, even though this is presented as the main conclusion. Instead, there are at least two alternative explanations for the results that both seem more likely. Neither alternative is acknowledged in the present version of the report.

The evidence presented to support overfilling is essentially two-fold. The first is strong paired pulse depression of synaptic strength when the interval between action potentials is 20 or 25 ms, but not when the interval is 50 ms. Subsequent stimuli at frequencies between 5 and 40 Hz then drive enhancement. The second is the observation that a slow component of recovery from depression after trains of action potentials is unveiled after eliminating enhancement by knocking down syt7. Of the two, the second is predicted by essentially all models where enhancement mechanisms operate independently of release site depletion - i.e., transient increases in p_occ, p_v, or even N - so isn't the sort of support that would distinguish the hypothesis from alternatives (Garcia-Perez and Wesseling, 2008, https://doi.org/10.1152/jn.01348.2007).

Regarding the paired pulse depression: The authors ascribe this to depletion of a homogeneous population of release sites, all with similar p_v. However, the details fit better with the alternative hypothesis that the depression is instead caused by quickly reversing inactivation of Ca2+ channels near release sites, as proposed by Dobrunz and Stevens to explain a similar phenomenon at a different type of synapse (1997, PNAS,<br /> https://doi.org/10.1073/pnas.94.26.14843). The details that fit better with Ca2+ channel inactivation include the combination of the sigmoid time course of the recovery from depression (plotted backwards in Fig1G,I) and observations that EGTA (Fig2B) increases the paired-pulse depression seen after 25 ms intervals. That is, the authors ascribe the sigmoid recovery to a delay in the activation of the facilitation mechanism, but the increased paired pulse depression after loading EGTA indicates, instead, that the facilitation mechanism has already caused p_r to double within the first 25 ms (relative to the value if the facilitation mechanism was not active). Meanwhile, Ca2+ channel inactivation would be expected to cause a sigmoidal recovery of synaptic strength because of the sigmoidal relationship between Ca2+-influx and exocytosis (Dodge and Rahamimoff, 1967, https://doi.org/10.1113/jphysiol.1967.sp008367).

The Ca2+-channel inactivation hypothesis could probably be ruled in or out with experiments analogous to the 1997 Dobrunz study, except after lowering extracellular Ca2+ to the point where synaptic transmission failures are frequent. However, a possible complication might be a large increase in facilitation in low Ca2+ (Fig2B of Stevens and Wesseling, 1999, https://doi.org/10.1016/s0896-6273(00)80685-6).

On the other hand, even if the paired pulse depression is caused by depletion of release sites rather than Ca2+-channel inactivation, there does not seem to be any support for the critical assumption that all of the release sites have similar p_v. And indeed, there seems to be substantial emerging evidence from other studies for multiple types of release sites with 5 to 20-fold differences in p_v at a wide variety of synapse types (Maschi and Klyachko, eLife, 2020, https://doi.org/10.7554/elife.55210; Rodriguez Gotor et al, eLife, 2024, https://doi.org/10.7554/elife.88212 and refs. therein). If so, the paired pulse depression could be caused by depletion of release sites with high p_v, whereas the facilitation could occur at sites with much lower p_v that are still occupied. It might be possible to address this by eliminating assumptions about the distribution of p_v across release sites from the variance-mean analysis, but this seems difficult; simply showing how a few selected distributions wouldn't work - such as in standard multiple probability fluctuation analyses - wouldn't add much.

In any case, the large increase - often 10-fold or more - in enhancement seen after lowering Ca2+ below 0.25 mM at a broad range of synapses and neuro-muscular junctions noted above is a potent reason to be cautious about the LS/TS model. There is morphological evidence that the transitions from a loose to tight docking state (LS to TS) occur, and even that the timing is accelerated by activity. However, 10-fold enhancement would imply that at least 90 % of vesicles start off in the LS state, and this has not been reported. In addition, my understanding is that the reverse transition (TS to LS) is thought to occur within 10s of ms of the action potential, which is 10-fold too fast to account for the reversal of facilitation seen at the same synapses (Kusick et al, 2020, https://doi.org/10.1038/s41593-020-00716-1).

Individual points:

(1) An additional problem with the overfilling hypothesis is that syt7 knockdown increases the estimate of p_occ extracted from the variance-mean analysis, which would imply a faster transition from unoccupied to occupied, and would consequently predict faster recovery from depression. However, recovery from depression seen in experiments was slower, not faster. Meanwhile, the apparent decrease in the estimate of N extracted from the mean-variance analysis is not anticipated by the authors' model, but fits well with alternatives where p_v varies extensively among release sites because release sites with low p_v would essentially be silent in the absence of facilitation.

(2) Figure S4A: I like the TTX part of this control, but the 4-AP part needs a positive control to be meaningful (e.g., absence of TTX).

(3) Line 251: At least some of the previous studies that concluded these drugs affect vesicle dynamics used logic that was based on some of the same assumptions that are problematic for the present study, so the reasoning is a bit circular.

(4) Line 329 and Line 461: A similar problem with circularity for interpreting earlier syt7 studies.

Reviewer #3 (Public review):

Summary:

Mirkovic et al explore the cause underlying development of aneuploidy during aging. This paper provides a compelling insight into the basis of chromosome missegregation in aged cells, tying this phenomenon to the established Nuclear Pore Complex architecture remodeling that occurs with aging across a large span of diverse organisms. The authors first establish that aged mother cells exhibit aberrant error correction during mitosis. As extrachromosomal rDNA circles (ERCs) are known to increase with age and lead to NPC dysfunction that can result in leakage of unspliced pre-mRNAs, Mirkovic et al search for intron-containing genes in yeast that may be underlying chromosome missegregation, identifying three genes in the aurora B-dependent error correction pathway: MCM21, NBL1, and GLC7. Interestingly, intron-less mutants in these genes suppress chromosome loss in aged cells, with a significant impact observed when all three introns were deleted (3x∆i). The 3x∆i mutant also suppresses the increased chromosome loss resulting from nuclear basket destabilization in a mlp1∆ mutant. The authors then directly test if aged cells do exhibit aberrant mRNA export, using RNA FISH to identify that old cells indeed leak intron-containing pre-mRNA into the cytoplasm, as well as a reporter assay to demonstrate translation of leaked pre-mRNA, and that this is suppressed in cells producing less ERCs. Mutants causing increased pre-mRNA leakage are sufficient to induce chromosome missegregation, which is suppressed by the 3x∆i.

Strengths:

The finding that deleting the introns of 3 genes in the Aurora B pathway can suppress age-related chromosome missegregation is highly compelling. Additionally, the rationale behind the various experiments in this paper is well-reasoned and clearly explained.

Weaknesses:

In some cases, controls for experiments were not presented or were depicted in other figures. High variability was seen in chromosome loss data, leading to large error bars. The text could have been more polished.

Reviewer #3 (Public review):

Summary:

The authors of this work are trying to understand the role dopaminergic terminals coming from VTA have on hippocampal mechanisms of memory consolidation, with emphasis on the replay of hippocampal patterns of activity during periods of consummatory behavior in reward locations. Previous work suggested that replay of relevant spatial trajectories supports reward localization and influences behavior.

The authors then tried to separate two conditions that were known to cause an increase in replay activity - spatial novelty encoding and variation of reward magnitude - and evaluate how these changed when VTA dopamine neurons were inactivated by a chemogenetic tool. They found that the rate of reverse replay (trajectory going away from the goal location) is increased with reward only in novel, but not in familiar environments. Overall this suggests that the VTA dopamine signal is critical during learning of novel locations, but not during explorations of already familiar environments.

Strengths:

The inactivation of VTA projections during goal-oriented behavior and in-vivo analysis of patterns of hippocampal activity during both novelty and reward variability. This work adds to the body of evidence that reverse replay constitutes an important mechanism in learning spatial goal locations. Furthermore, this work also points to the role of VTA in reward prediction error with consequences for spatial navigation and consolidation of spatial memories.

The authors addressed very carefully all the points raised during the revision and I am very pleased with the revised manuscript.

Reviewer #3 (Public review):

Summary:

Combining electrophysiological recording, circuit tracing, single cell RNAseq, and optogenetic and chemogenetic manipulation, Howe and colleagues have identified a graded division between anterior and posterior plCoA and determined the molecular characteristics that distinguish the neurons in this part of the amygdala. They demonstrate that the expression of slc17a6 is mostly restricted to the anterior plCoA whereas slc17a7 is more broadly expressed. Through both anterograde and retrograde tracing experiments, they demonstrate that the anterior plCoA neurons preferentially projected to the MEA whereas those in the posterior plCoA preferentially innervated the nucleus accumbens. Interestingly, optogenetic activation of the aplCoA drives avoidance in a spatial preference assay whereas activating the pplCoA leads to preference. The data support a model that spatially segregated and molecularly defined populations of neurons and their projection targets carry valence specific information for the odors. The discoveries represent a conceptual advance in understanding plCoA function and innate valence coding in the olfactory system.

Strengths:

The strongest evidence supporting the model comes from single cell RNASeq, genetically facilitated anterograde and retrograde circuit tracing, and optogenetic stimulation. The evidence clear demonstrates two molecularly defined cell populations with differential projection targets. Stimulating the two populations produced opposite behavioral responses.

Weaknesses:

There are a couple of inconsistencies that may be addressed by additional experiments and careful interpretation of the data.

Stimulating aplCoA or slc17a6 neurons results in spatial avoidance, and stimulating pplCoA or slc17a7 neurons drives approach behaviors. On the other hand, the authors and others in the field also show that there is no apparent spatial bias in odor-driven responses associated with odor valence. This discrepancy may be addressed better. A possibility is that odor-evoked responses are recorded from populations outside of those defined by slc17a6/a7. This may be addressed by marking activated cells and identifying their molecular markers. A second possibility is that optogenetic stimulation activates a broad set of neurons that and does not recapitulate the sparseness of odor responses. It is not known whether sparsely activation by optogenetic stimulation can still drive approach of avoidance behaviors.

The authors show that inhibiting slc17a7 neurons blocks approaching behaviors toward 2-PE. Consistent with this result, inhibiting NAc projection neurons also inhibits approach responses. However, inhibiting aplCOA or slc17a6 neurons does not reduce aversive response to TMT, but blocking MEA projection neurons does. The latter two pieces of evidence are not consistent with each other. One possibility is that the MEA projecting neurons may not be expressing slc17a6. It is not clear that the retrogradely labeling experiments what percentage of MEA- and NAC-projecting neurons express slc17a6 and slc17a7. It is possible that neurons expressing neither VGluT1 nor VGluT2 could drive aversive or appetitive responses. This possibility may also explain that silencing slc17a6 neurons does not block avoidance.

Reviewer #3 (Public review):

Summary

The study uses publicly available sequences of classical and non-classical genes from a number of primate species to assess the extent and depth of TSP across the primate phylogeny. The analyses were carried out in a coherent and, in my opinion, robust inferential framework and provided evidence for ancient (even > 30 million years) TSP at several classical class I and class II genes. The authors also characterise evolutionary rates at individual codons, map these rates onto MHC protein structures, and find that the fastest evolving codons are extremely enriched for autoimmune and infectious disease associations.

Strengths

The study is comprehensive, relying on a large data set, state-of-the-art phylogenetic analyses and elegant tests of TSP. The results are not entirely novel, but a synthesis and re-analysis of previous findings is extremely valuable and timely.

Weaknesses

I've identified weaknesses in several areas (details follow in the next section):<br /> - Inadequate description and presentation of the data used<br /> - Large parts of the results read like extended figure captions, which breaks the flow.<br /> - Older literature on the subject is duly cited, but the authors don't really discuss their findings in the context of this literature.<br /> - The potential impact of mechanisms other than long-term maintenance of allelic lineages by balancing selection, such as interspecific introgression and incorrect orthology assessment, needs to be discussed.

for - report - climate crisis - food system transformation - Public climate finance for food systems transformation - Global Alliance for the Future of Food - 2024, Nov - from - post - LinkedIn - Jonathan Foley - This is very, very important - stats - 2.5% climate funding for food system that contributes 30% of global climate emissions - 2025, Jan 3 - https://hyp.is/zKE7vsqkEe-RFB8co7Pdqw/www.linkedin.com/posts/jonathan-foley-182808b9_foodsystemeconomicscommission-cop29-climatefinance-activity-7281009061003706369-P1b0/ - TPC network - motivation

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Reviewer #3 (Public review):

Summary:

This work provides insights into predictive coding models of visual cortex processing. These models predict that visual cortex neurons will show elevated responses when there are unexpected changes to learned sequential stimulus patterns. This model is currently controversial, with recent publications providing conflicting evidence. In this work, the authors test two types of unexpected pattern variations in layer 2/3 of the mouse visual cortex. They show that pattern omission evokes elevated responses, in favor of a predictive coding model, but find no evidence for prediction errors with substituted patterns, which conflicts with both prior results in L4, and with the expectations of a predictive coding model. They also report that with sequence training, responses sparsify and decorrelate, but surprisingly find no changes in the ability of an ideal observer to decode stimulus identity or timing.

These results are an important contribution to the understanding of how temporal sequences and expectations are encoded in the primary visual cortex

Comments on revisions:

In this revision, the authors address several of the concerns in the original manuscript. However, the primary issue, raised by all three reviewers, was the block design of the experiments. This design makes disentangling the effects of any rapid (within block) plasticity from any longer term (across days) plasticity-which nominally is the subject of the paper-extremely difficult.

Although it may be the case that re-running the experiments with an interleaved design is beyond the scope of this paper, unfortunately, the revised manuscript still does not adequately discuss this potential confound. The authors note that stimulus A in ABCD, ABBD, and ACBD could be distinguished on day 0, indicating that within block changes do occur. In both the original and revised manuscript this finding is discussed in terms of representational drift, but the authors fail to discuss how such within block plasticity may impact their primary findings of prediction error effects.

This remains a significant concern with the revised manuscript.

Many of the other issues in the original manuscript have been addressed, and in these areas the revised manuscript is both clearer and more accurately reflects the presented data. The additional analyses and controls shown in the supplemental figures aid in the interpretation of the findings.

Reviewer #3 (Public review):

Yonk and colleagues investigate the role of the thalamostriatal pathway. Specifically, they studied the interaction of the posterior thalamic nucleus (PO) and the dorsolateral striatum in the mouse. First, they characterize connectivity by recording DLS neurons in in vitro slices and optogenetically activating PO terminals. PO is observed to establish depressing synapses onto D1 and D2 spiny neurons as well as PV neurons. Second, the image PO axons are imaged by fiber photometry in mice trained to discriminate textures. Initially, no trial-locked activity is observed, but as the mice learn PO develops responses timed to the audio cue that marks the start of the trial and precedes touch. PO does appear to encode the tactile stimulus type or outcome. Optogenetic suppression of PO terminals in striatum slow task acquisition. The authors conclude that PO provides a "behaviorally relevant arousal-related signal" and that this signal "primes" striatal circuitry for sensory processing.

A great strength of this paper is its timeliness. Thalamostriatal processing has received almost no attention in the past, and the field has become very interested in the possible functions of PO. Additionally, the experiments exploit multiple cutting-edge techniques.

There seem to be some technical/analytical weaknesses. The in vitro experiments appear to have some contamination of nearby thalamic nuclei by the virus delivering the opsin, which could change the interpretation. Some of the statistical analysis of these data also appear inappropriate. The correlative analysis of Pom activity in vivo, licking, and pupil could be more convincingly done.

The bigger weakness is conceptual - why should striatal circuitry need "priming" by thalamus in order to process sensory stimuli? Why would such circuitry even be necessary? Why is a sensory signal from cortex insufficient? Why should the animal more slowly learn the task? How does this fit with existing ideas of striatal plasticity? It is unclear from the experiments that the thalamostriatal pathway exists for priming sensory processing. In fact the optogenetic suppression of the thalamostriatal pathway seems to speak against that idea.

Comments on revisions:

The authors have only tweaked the Discussion and not necessarily in ways that addressed our previous comments. They could have fairly easily analyzed the effect of distance of recording from injection site and compared subsets of data depending on contamination beyond PO (my comments 1 and 2) or effects of movements (3 and 4). Minimally, they could have given caveats in the Results and Discussion about these, and I would strongly encourage them to be explicit about the caveats. The analyses would probably be better.

The suggestion that the effects have something to do with priming (5), seems a grasp for function of the circuit.

Reviewer #3 (Public review):

Summary:

The authors find that Sst-EPN neurons, which project to the lateral habenula, encode information about response directionality (left vs right) and outcome (rewarded vs unrewarded). Surprisingly, chronic impairment of vesicular signaling in these neurons onto their LHb targets did not impair probabilistic choice behavior.

Strengths:

Strengths of the current work include extremely detailed and thorough analysis of data at all levels, not only of the physiological data, but also an uncommonly thorough analysis of behavioral response patterns.

Weaknesses:

In this revised manuscript, the authors have addressed my earlier critiques.

Reviewer #3 (Public review):

Summary:

This manuscript introduces a differentiable variant of the Gillespie algorithm (DGA) that allows gradient calculation using backpropagation. The most significant contribution of this work is the development of the DGA itself, a novel approach to making stochastic simulations differentiable. This is achieved by replacing discontinuous operations in the traditional Gillespie algorithm with smooth, differentiable approximations using sigmoid and Gaussian functions. This conceptual advance opens up new avenues for applying powerful gradient-based optimization techniques, prevalent in machine learning, to studying stochastic biological systems.

The method was tested on a simple two-state promoter model of gene expression. The authors found that the DGA accurately captured the moments of the steady-state distribution and other major qualitative features. However, it was less accurate at capturing information about the distribution's tails, potentially because rare events result from frequent low-probability reaction events where the approximations made by the DGA have a greater impact. The authors could further use the DGA to design a four-state promoter model of gene regulation that exhibited a desired input-output relationship. The DGA could learn parameters that produced a sharper response curve, which was achieved by consuming more energy.

The authors conclude that the DGA is a powerful tool for analyzing and designing stochastic systems.

Strengths:

The DGA allows gradient-based optimization techniques to estimate parameters and design networks with desired properties.

The DGA efficacy in estimating kinetic parameters from both synthetic and experimental data. This capability highlights the DGA's potential to extract meaningful biophysical parameters from noisy biological data.

The DGA's ability to design a four-state promoter architecture exhibiting a desired input-output relationship. This success indicates the potential of the DGA as a valuable tool for synthetic biology, enabling researchers to engineer biological circuits with predefined behaviours.

Weaknesses:

The study primarily focuses on analysing the steady-state properties of stochastic systems. It is unclear how and if this framework can be used beyond the steady-state data presented in the case studies, where it is already quite computationally heavy.<br /> A more in-depth exploration of the DGA's performance in analysing dynamic trajectories, which capture the system's evolution over time, would provide a more comprehensive view of the algorithm's capabilities.<br /> Gradient computations in the DGA can be susceptible to numerical instability, particularly when the sharpness parameters of the sigmoid and Gaussian approximations are set to high values. This issue could lead to challenges in convergence during the optimization process.

Reviewer #3 (Public review):

Summary:

The study demonstrates the effectiveness of a cost-effective closed-loop feedback system for modulating brain activity and behavior in head-fixed mice. Authors have tested real-time closed-loop feedback system in head-fixed mice two types of graded feedback: 1) Closed-loop neurofeedback (CLNF), where feedback is derived from neuronal activity (calcium imaging), and 2) Closed-loop movement feedback (CLMF), where feedback is based on observed body movement. It is a python based opensource system, and authors call it CLoPy. The authors also claim to provide all software, hardware schematics, and protocols to adapt it to various experimental scenarios. This system is capable and can be adapted for a wide use case scenario.

Authors have shown that their system can control both positive (water drop) and negative reinforcement (buzzer-vibrator). This study also shows that using the close loop system mice have shown better performance, learnt arbitrary task and can adapt to change in the rule as well. By integrating real-time feedback based on cortical GCaMP imaging and behavior tracking authors have provided strong evidence that such closed-loop systems can be instrumental in exploring the dynamic interplay between brain activity and behavior.

Strengths:

Simplicity of feedback systems designed. Simplicity of implementation and potential adoption.

Weaknesses:

Long latencies, due to slow Ca2+ dynamics and slow imaging (15 FPS), may limit the application of the system.

Major comments:

(1) Page 5 paragraph 1: "We tested our CLNF system on Raspberry Pi for its compactness, general-purpose input/output (GPIO) programmability, and wide community support, while the CLMF system was tested on an Nvidia Jetson GPU device." Can these programs and hardware be integrated with windows-based system and a microcontroller (Arduino/ Tency). As for the broad adaptability that's what a lot of labs would already have (please comment/discuss)?

(2) Hardware Constraints: The reliance on Raspberry Pi and Nvidia Jetson (is expensive) for real-time processing could introduce latency issues (~63 ms for CLNF and ~67 ms for CLMF). This latency might limit precision for faster or more complex behaviors, which authors should discuss in the discussion section.

(3) Neurofeedback Specificity: The task focuses on mesoscale imaging and ignores finer spatiotemporal details. Sub-second events might be significant in more nuanced behaviors. Can this be discussed in the discussion section?

(4) The activity over 6s is being averaged to determine if the threshold is being crossed before the reward is delivered. This is a rather long duration of time during which the mice may be exhibiting stereotyped behaviors that may result in the changes in DFF that are being observed. It would be interesting for the authors to compare (if data is available) the behavior of the mice in trials where they successfully crossed the threshold for reward delivery and in those trials where the threshold was not breached. How is this different from spontaneous behavior and behaviors exhibited when they are performing the test with CLNF?

Reviewer #3 (Public review):

Summary:

In this study, the authors aimed to investigate if hemodynamic occlusion contributes to fluorescent signals measured with two-photon microscopy. For this, they image the activity-independent fluorophore GFP in 2 different cortical areas, at different cortical depths and in different behavioral conditions. They compare the evoked fluorescent signals with those obtained with calcium sensors and neuromodulator sensors and evaluate their relationship to vessel diameter as a readout of blood flow.<br /> They find that GFP fluorescence transients are comparable to GCaMP6f stimuli-evoked signals in amplitude, although they are generally smaller. Yet, they are significant even at the single neuronal level. They show that GFP fluorescence transients resemble those measured with the dopamine sensor GRAB-DA1m and the serotonin sensor GRAB-5HT1.0 in amplitude an nature, suggesting that signals with these sensors are dominated by hemodynamic occlusion. 
Moreover, the authors perform similar experiments with wide-field microscopy which reveals the similarity between the two methods in generating the hemodynamic signals. Together the evidence presented calls for the development and use of high dynamic range sensors to avoid measuring signals that have another origin from the one intended to measure. In the meantime, the evidence highlights the need to control for those artifacts such as with the parallel use of activity independent fluorophores.

Strengths:

- Comprehensive study comparing different cortical regions in diverse behavioral settings in controlled conditions.<br /> - Comparison to the state-of-the-art, i.e. what has been demonstrated with wide-field microscopy.<br /> - Comparison to diverse activity-dependent sensors, including the widely used GCaMP.

Weaknesses:

- The kinetics of GCaMP is stereotypic. An analysis/comment on if and how the kinetics of the signals could be used to distinguish the hemodynamic occlusion artefacts from calcium signals would be useful.<br /> - Is it possible that motion is affecting the signals in a certain degree? This issue is not made clear.<br /> - The causal relationship with blood flow remains open. Hemodynamic occlusion seems a good candidate causing changes in GFP fluorescence, but this remains to be well addressed in further research.

Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides compelling evidence that glia-derived Shriveled (Shv) modulates activity-dependent synaptic plasticity at the Drosophila neuromuscular junction (NMJ). This mechanism differs from the previously reported function of neuronally released Shv, which activates integrin signaling. They further show that this requirement of Shv is acute and that glial Shv supports synaptic plasticity by modulating neuronal Shv release and the ambient glutamate levels. However, there are a number of conceptual and technical issues that need to be addressed.

Major comments

(1) From the images provided for Fig 2B +RU486, the bouton size appears to be bigger in shv RNAi + stimulation, especially judging from the outline of GluR clusters.<br /> (2) The shv result needs to be replicated with a separate RNAi.<br /> (3) The phenotype of shv mutant resembles that of neuronal shv RNAi - no increased GluR baseline. Any insights why that is the case?<br /> (4) In Fig 3B, SPG shv RNAi has elevated GluR baseline, while PG shv RNAi has a lower baseline. In both cases, there is no activity induced GluR increase. What could explain the different phenotypes?<br /> (5) In Fig 4C, the rescue of PTP is only partial. Does that suggest neuronal shv is also needed to fully rescue the deficit of PTP in shv mutants?<br /> (6) The observation in Fig 5D is interesting. While there is a reduction in Shv release from glia after stimulation, it is unclear what the mechanism could be. Is there a change in glial shv transcription, translation or the releasing machinery? It will be helpful to look at the full shv pool vs the released ones.<br /> (7) In Fig 5E, what will happen after stimulation? Will the elevated glial Shv after neuronal shv RNAi be retained in the glia?<br /> (8) It would be interesting to see if the localization of shv differs based on if it is released by neuron or glia, which might be able to explain the difference in GluR baseline. For example, by using glia-Gal4>UAS-shv-HA and neuronal-QF>QUAS-shv-FLAG. It seems important to determine if they mix together after release? It is unclear if the two shv pools are processed differently.<br /> (9) Alternatively, do neurons and glia express and release different Shv isoforms, which would bind different receptors?<br /> (10) It is claimed that Sup Fig 2 shows no observable change in gross glial morphology, further bolstering support that glial Shv does not activate integrin. This seems quite an overinterpretation. There is only one image for each condition without quantification. It is hard to judge if glia, which is labeled by GFP (presumably by UAS-eGFP?), is altered or not.<br /> (11) The hypothesis that glutamate regulates GluR level as a homeostatic mechanism makes sense. What is the explanation of the increased bouton size in the control after glutamate application in Fig 6?<br /> (12) What could be a mechanism that prevents elevated glial released Shv to activate integrin signaling after neuronal shv RNAi, as seen in Fig 5E?<br /> (13) Any speculation on how the released Shv pool is sensed?

Reviewer #3 (Public review):

Summary:

In this work, Ryan et al. have performed a state-of-the-art full genome CRISP-based screen of iNEurons expressing a teggd version of TDP-43 in order to determine expression modifiers of this protein. Unexpectedly, using this approach the authors have uncovered a previously undescribed role of the BORC complex in affecting the levels of TDP-43 protein, but not mRNA expression. Taken together, these findings represent a very solid piece of work that will certainly be important for the field.

Strengths:

- BORC is a novel TDP-43 expression modifier that has never been described before and it seemingly acts on regulating protein half life rather than transcriptome level. It has been long known that different labs have reported different half-lives for TDP-43 depending on the experimental system but no work has ever explained these discrepancies. Now, the work of Ryan et al. has for the time identified one of these factors which could account for these differences and play an important role in disease (although this is left to be determined in future studies).<br /> - The genome wide CRISPR screening has demonstrated to yield novel results with high reproducibility and could eventually be used to search for expression modifiers of many other proteins involved in neurodegeneration or other diseases

Weaknesses:

- The fact that TDP-43 mRNA does not change following BORCS6 KD is based on a single qRT-PCR that does not really cover all possibilities. For example, the mRNA total levels may not change but the polyA sites may have switched from the highly efficient pA1 to the less efficient and nuclear retained pA4. There are therefore a few other experiments that could have been performed to make this conclusion more compelling, maybe also performing RNAscope experiments to make sure that no change occurred in TDP-43 mRNA localisation in cells.<br /> - Even assuming that the mRNA does not change, no explanation for the change in TDP-43 protein half life has been proposed by the authors. This will presumably be addressed in future studies: for example, are mutants that lack different domains of TDP-43 equally affected in their half-lives by BORC KD?. Alternatively, can a mass-spec be attempted to see whether TDP-43 PTMs change following BORCS6 KD?

Reviewer #3 (Public review):

Summary:

This manuscript by Cui et al., studies the mechanisms for the generation of sighing, an essential breathing pattern. This is an important and interesting topic, as sighing maintains normal pulmonary function and is associated with various emotional conditions. However, the mechanisms of its generation remain not fully understood. The authors employed different approaches, including optogenetics, chemogenetics, intersectional genetic approach, and slice electrophysiology and calcium imaging, to address the question, and found several neuronal populations are sufficient to induce sighing when activated. Furthermore, ectopic sighs can be triggered without the involvement of neuromedin B (NMB) or gastrin releasing peptide (GRP) or their receptors in the preBötzinger Complex (preBötC) region of the brainstem. Additionally, activating SST neurons in the preBötC region induces sighing, even when other receptors are blocked. Based on these results, the authors concluded that increased excitability in certain neurons (NMBR or GRPR neurons) activates pathways leading to sigh generation, with SST neurons serving as a downstream component in converting regular breaths into sighs.

Strengths:

The authors employed a combination of various sophisticated approaches, including optogenetics, chemogenetics, intersectional genetic approach, and slice electrophysiology and calcium imaging, to precisely pinpoint the mechanism responsible for sigh generation. They utilized multiple genetically modified mouse lines, enabling them to selectively manipulate and observe specific neuronal populations involved in sighing.<br /> Using genetics and calcium imaging, the authors record the neuronal activity of NMBR and GRPR neurons, respectively, and identified their difference in activity pattern. Furthermore, by applying the intersectional approach, the authors were able to genetically target and manipulate several distinct neuronal populations, such as NMBR+, GRPR- neurons and GRPR+, NMBR- neurons, and conducted a detailed characterization of their functions in influencing sighing.

Weaknesses:

(1) The authors employed two conditions for optogenetic activation: long pulse photostimulation (LPP) and short pulse photostimulation (SPP), with durations ranging from 4-10s for LPP and 100-500 ms for SPP. These could generate huge variability in the experiments. The rationale behind the selection of these conditions in each experiment remains unclear in the manuscript. Additionally, it is not explained why these specific durations were chosen. Furthermore, the interpretation for the varied responses observed under these conditions is not provided. Clarification on the rationale and interpretation of these experimental parameters would enhance the understanding of the results. The description of the experiment conditions should be consistent throughout the manuscript.

(2) Regarding the fiber optics, my understanding is that they are placed outside of the brainstem from the ventral side. Given the locations of the pF and preBötC neurons, could the differences in responses be attributed to the varying distances of each population from the ventral surface? In fact, in Figure 8, NMBR is illustrated as being closer to the ventral surface. Does it represent the actual location of these neurons?

(3) The results of recording on NMBR neurons in Figure 4 were compelling. However, I'm curious why the recording of GRPR neurons and their response to the neuropeptide were not presented or examined. Additionally, considering the known cross-reaction between peptides and their receptors, it might be worthwhile to investigate how GRP modulates NMBR neurons and how NMB modulates GRPR neurons.

(4) The authors found that activation of several preBötC populations, including NMBR, GRPR, and SST neurons, despite pharmacological inhibition of NMBR and GRPR, can still induce sighing, and concluded that "activation of preBötC NMBRs and/or GRPRs is not necessary for sigh production". I disagree with this conclusion. Even when the receptors are silenced, artificial (optogenetic or chemogenetic) activation could still activate the same downstream pathways. This cannot be used as evidence to claim that the receptors are not required for sighing in vivo, because it is possible that the receptors are still necessary for the activation of these neurons under natural conditions. For instance, while diaphragm activation induces breathing, it does not negate the crucial role of the nervous system in regulating this process in physiological conditions.

(5) The authors noted varied responses upon activating specific subpopulations of the preBötC neurons, namely NMBR, GRPR, and SST neurons. Could these differences be attributed to variations in viral labeling efficiency among different mouse genetic lines? Are there discrepancies in the number of labeled neurons across the lines? Additionally, the authors did not thoroughly characterize the specificities of AAV targeting in their Cre and Flp lines. It's uncertain whether the AAV-labeled neurons are strictly restricted to the designated population without notable leakage into other populations. This is particularly crucial for the experiments manipulating SST neurons. If there's substantial labeling of NMBR or GRPR neurons, it could undermine the conclusions drawn. Further examination of the precision and selectivity of the labeling techniques is necessary to ensure the accurate interpretation of the experimental findings.

(6) The authors have addressed some of the reviewers' concerns in the revision; however, many important issues remain unaddressed.

Reviewer #3 (Public review):

In this study, Dong and colleagues set to dissect the role of Rab10 small GTPase on the intracellular trafficking and exocytosis of dense core vesicles (DCVs). While the authors have already shown that Rab3 plays a central role in the exocytosis of DVC in mammalian neurons, the roles of several other Rab-members have been identified genetically, but their precise mechanism of action in mammalian neurons remains unclear. In this study, the authors use a carefully designed and thoroughly executed series of experiments, including live-cell imaging, functional calcium-imaging, proteomics, and electron microscopy, to identify that DCV secretion upon Rab10 depletion in adult neurons is primarily a result of dysregulated protein synthesis and, to a lesser extent, disrupted intracellular calcium buffering. Given that the full deletion of Rab10 has deleterious effect on neurons and that Rab10 has a major role in axonal development, the authors cautiously employed the knock-down strategy from 7 DIV, to focus on the functional impact of Rab10 in mature neurons. The experiments in this study were meticulously conducted, incorporating essential controls and thoughtful considerations, ensuring rigorous and comprehensive results that fully support the conclusions.

Comments on revisions:

The authors have addressed all the comments and suggestions raised by reviewers, making this an excellent and timely study.

Reviewer #3 (Public review):

Summary:

The manuscript by Hallam et al describes the analysis of various biomarkers in patients undergoing complement factor I supplementation treatment (PPY988 gene therapy) as part of the FOCUS Phase I/II clinical trial. The authors used validated methods (multiplexed assays and OLINK proteomics) for measuring multiple soluble complement proteins in the aqueous humour (AH) and vitreous humour (VH) of 28 patients over a series of timepoints, up to and including 96 weeks. Based on biomarker comparisons, the levels of FI synthesised by PPY988 were believed to be insufficient to achieve the desired level of complement inhibition. Subsequent comparative experiments showed that PPY988-delievred FI was much less efficacious than Pegceptacoplan (FDA approved complement inhibitor under the name SYFORVE) when tested in an artificial VH matrix.

Strengths:

The manuscript is well written with data clearly presented and appropriate statistics used for the analysis itself. It's great to see data from real clinical samples that can help support future studies and therapeutic design. The identification that complement biomarker levels present in the AH do not represent the levels found in the VH is an important finding for the field, given the number of complement-targeting therapies in development and the desperate need for good biomarkers for target engagement. This study also provides a wealth of baseline complement protein measurements in both human AH and VH (and companion measurements in plasma) that will prove useful for future studies.

Weaknesses:

No real weaknesses in the manuscript itself. It is only a shame that it would appear that FI supplementation is not a viable way forward for treating GA secondary to AMD.

Comments on revisions:

I think the authors have done all that they can to present this study in the most robust manner possible.

Reviewer #3 (Public review):

Summary:

"Decoding Phase Separation of Prion-Like Domains through Data-Driven Scaling Laws" by Maristany et al. offers a significant contribution to the understanding of phase separation in prion-like domains (PLDs). The study investigates the phase separation behavior of PLDs, which are intrinsically disordered regions within proteins that have the propensity to undergo liquid-liquid phase separation (LLPS). This phenomenon is crucial in forming biomolecular condensates, which play essential roles in cellular organization and function. The authors employ a data-driven approach to establish predictive scaling laws that describe the phase behavior of these domains.

Strengths:

The study benefits from a robust dataset encompassing a wide range of PLDs, which enhances the generalizability of the findings. The authors' meticulous curation and analysis of this data add to the study's robustness. The scaling laws derived from the data provide predictive insights into the phase behavior of PLDs, which can be useful in the future for the design of synthetic biomolecular condensates.

Reviewer #3 (Public review):

This manuscript examines the impact of congenital visual deprivation on the excitatory/inhibitory (E/I) ratio in the visual cortex using Magnetic Resonance Spectroscopy (MRS) and electroencephalography (EEG) in individuals whose sight was restored. Ten individuals with reversed congenital cataracts were compared to age-matched, normally sighted controls, assessing the cortical E/I balance and its interrelationship and to visual acuity. The study reveals that the Glx/GABA ratio in the visual cortex and the intercept and aperiodic signal are significantly altered in those with a history of early visual deprivation, suggesting persistent neurophysiological changes despite visual restoration.

First of all, I would like to disclose that I am not an expert in congenital visual deprivation, nor in MRS. My expertise is in EEG (particularly in the decomposition of periodic and aperiodic activity) and statistical methods. Although the authors addressed some of the concerns of the previous version, major concerns and flaws remain in terms of methodological and statistical approaches along with the (over)interpretation of the results. Specific concerns include:

(1 3.1) Response to Variability in Visual Deprivation<br /> Rather than listing the advantages and disadvantages of visual deprivation, I recommend providing at least a descriptive analysis of how the duration of visual deprivation influenced the measures of interest. This would enhance the depth and relevance of the discussion.

(2 3.2) Small Sample Size<br /> The issue of small sample size remains problematic. The justification that previous studies employed similar sample sizes does not adequately address the limitation in the current study. I strongly suggest that the correlation analyses should not feature prominently in the main manuscript or the abstract, especially if the discussion does not substantially rely on these correlations. Please also revisit the recommendations made in the section on statistical concerns.

(3 3.3) Statistical Concerns<br /> While I appreciate the effort of conducting an independent statistical check, it merely validates whether the reported statistical parameters, degrees of freedom (df), and p-values are consistent. However, this does not address the appropriateness of the chosen statistical methods.

Several points require clarification or improvement:<br /> (4) Correlation Methods: The manuscript does not specify whether the reported correlation analyses are based on Pearson or Spearman correlation.<br /> (5) Confidence Intervals: Include confidence intervals for correlations to represent the uncertainty associated with these estimates.<br /> (6) Permutation Statistics: Given the small sample size, I recommend using permutation statistics, as these are exact tests and more appropriate for small datasets.<br /> (7) Adjusted P-Values: Ensure that reported Bonferroni corrected p-values (e.g., p > 0.999) are clearly labeled as adjusted p-values where applicable.<br /> (8) Figure 2C<br /> Figure 2C still lacks crucial information that the correlation between Glx/GABA ratio and visual acuity was computed solely in the control group (as described in the rebuttal letter). Why was this analysis restricted to the control group? Please provide a rationale.<br /> (9 3.4) Interpretation of Aperiodic Signal<br /> Relying on previous studies to interpret the aperiodic slope as a proxy for excitation/inhibition (E/I) does not make the interpretation more robust.<br /> (10) Additionally, the authors state:<br /> "We cannot think of how any of the exploratory correlations between neurophysiological measures and MRS measures could be accounted for by a difference e.g. in skull thickness."<br /> (11) This could be addressed directly by including skull thickness as a covariate or visualizing it in scatterplots, for instance, by representing skull thickness as the size of the dots.<br /> (12 3.5) Problems with EEG Preprocessing and Analysis<br /> Downsampling: The decision to downsample the data to 60 Hz "to match the stimulation rate" is problematic. This choice conflates subsequent spectral analyses due to aliasing issues, as explained by the Nyquist theorem. While the authors cite prior studies (Schwenk et al., 2020; VanRullen & MacDonald, 2012) to justify this decision, these studies focused on alpha (8-12 Hz), where aliasing is less of a concern compared of analyzing aperiodic signal. Furthermore, in contrast, the current study analyzes the frequency range from 1-20 Hz, which is too narrow for interpreting the aperiodic signal as E/I. Typically, this analysis should include higher frequencies, spanning at least 1-30 Hz or even 1-45 Hz (not 20-40 Hz).<br /> (13) Baseline Removal: Subtracting the mean activity across an epoch as a baseline removal step is inappropriate for resting-state EEG data. This preprocessing step undermines the validity of the analysis. The EEG dataset has fundamental flaws, many of which were pointed out in the previous review round but remain unaddressed. In its current form, the manuscript falls short of standards for robust EEG analysis. If I were reviewing for another journal, I would recommend rejection based on these flaws.<br /> (14) The authors mention:<br /> "The EEG data sets reported here were part of data published earlier (Ossandón et al., 2023; Pant et al., 2023)." Thus, the statement "The group differences for the EEG assessments corresponded to those of a larger sample of CC individuals (n=38) " is a circular argument and should be avoided."<br /> The authors addressed this comment and adjusted the statement. However, I do not understand, why not the full sample published earlier (Ossandón et al., 2023) was used in the current study?

Reviewer #3 (Public review):

Summary:

In this work, Casu et al. have reported the characterization of a previously uncharacterized membrane protein CisA encoded in a non-canonical contractile injection system of Streptomyces coelicolor, CISSc, which is a cytosolic CISs significantly distinct from both intracellular membrane-anchored T6SSs and extracellular CISs. The authors have presented the first high-resolution structure of extended CISSc structure. It revealed important structural insights in this conformational state. To further explore how CISSc interacted with cytoplasmic membrane, they further set out to investigate CisA that was previously hypothesized to be the membrane adaptor. However, the structure revealed that it was not associated with CISSc. Using fluorescence microscope and cell fractionation assay, the authors verified that CisA is indeed a membrane-associated protein. They further determined experimentally that CisA had a cytosolic N-terminal domain and a periplasmic C-terminus. The functional analysis of cisA mutant revealed that it is not required for CISSc assembly but is essential for the contraction, as a result, the deletion significantly affects CISSc-mediated cell death upon stress, timely differentiation, as well as secondary metabolite production. Although the work did not resolve the mechanistic detail how CisA interacts with CISSc structure, it provides solid data and a strong foundation for future investigation toward understanding the mechanism of CISSc contraction, and potentially, the relation between the membrane association of CISSc, the sheath contraction and the cell death.

Strengths:

The paper is well-structured, and the conclusion of the study is supported by solid data and careful data interpretation was presented. The authors provided strong evidence on (1) the high-resolution structure of extended CISSc determined by cryo-EM, and the subsequent comparison with known eCIS structures, which sheds light on both its similarity and different features from other subtypes of eCISs in detail; (2) the topological features of CisA using fluorescence microscopic analysis, cell fractionation and PhoA-LacZα reporter assays, (3) functions of CisA in CISSc-mediated cell death and secondary metabolite production, likely via the regulation of sheath contraction.

Weaknesses:

The data presented are not sufficient to provide mechanistic details of CisA-mediated CISSc contraction, as authors are not able to experimentally demonstrate the direct interaction between CisA with baseplate complex of CISSc (hypothesized to be via Cis11 by structural modeling), since they could not express cisA in E. coli due to its potential toxicity. Therefore, there is a lack of biochemical analysis of direct interaction between CisA and baseplate wedge. In addition, there is no direct evidence showing that CisA is responsible for tethering CISSc to the membrane upon stress, and the spatial and temporal relation between membrane association and contraction remains unclear. Further investigation will be needed to address these questions in future.

Discussion:

Overall, the work provides a valuable contribution to our understanding on the structure of a much less understood subtype of CISs, which is unique compared to both membrane-anchored T6SSs and host-membrane targeting eCISs. Importantly, the work serves as a good foundation to further investigate how the sheath contraction works here. The work contributes to expanding our understanding of the diverse CIS superfamilies.

Reviewer #3 (Public review):

In this manuscript, Natarajan and colleagues report on the role of a prophage, termed SfPat, in the regulation of motility and biofilm formation by the marine bacterium Shewanella fidelis. The authors investigate the in vivo relevance of prophage carriage by studying the gut occupation patterns of Shewanella fidelis wild-type and an isogenic SfPat- mutant derivative in a model organism, juveniles of the marine tunicate Ciona robusta. The role of bacterial prophages in regulating bacterial lifestyle adaptation and niche occupation is a relatively underexplored field, and efforts in this direction are appreciated.

While the research question is interesting, the work presented lacks clarity in its support for several major claims, and, at times, the authors do not adequately explain their data.

Major concerns:

(1) Prophage deletion renders the SfPat- mutant derivative substantially less motile and with a higher biofilm formation capacity than the WT (Fig. 2a-b). The authors claim the mutant is otherwise isogenic to the WT strain upon sequence comparison of draft genome sequences (I'll take the opportunity to comment here that GenBank accessions are preferable to BioSample accessions in Table 1). Even in the absence of secondary mutations, complementation is needed to validate functional associations (i.e., phenotype restoration). A strategy for this could be phage reintegration into the mutant strain (PMID: 19005496).

(2) The authors claim that the downshift in motility (concomitant with an upshift in biofilm formation) is likely mediated by the activity of c-di-GMP turnover proteins. Specifically, the authors point to the c-di-GMP-specific phosphodiesterase PdeB as a key mediator, after finding lower transcript levels for its coding gene in vivo (lines 148-151, Fig. 2c), and suggesting higher activity of this protein in live animals (!)(line 229). I have several concerns here:<br /> (2.1) Findings shown in Fig. 2a-b are in vitro, yet no altered transcript levels for pdeB were recorded (Fig. 2c). Why do the authors base their inferences only on in vivo data?<br /> (2.2) Somewhat altered transcript levels alone are insufficient for making associations, let alone solid statements. Often, the activity of c-di-GMP turnover proteins is local and/or depends on the activation of specific sensory modules - in the case of PdeB, a PAS domain and a periplasmic sensor domain (PMID: 35501424). This has not been explored in the manuscript, i.e., specific activation vs. global alterations of cellular c-di-GMP pools (or involvement of other proteins, please see below). Additional experiments are needed to confirm the involvement of PdeB. Gaining such mechanistic insights would greatly enhance the impact of this study.<br /> (2.3) What is the rationale behind selecting only four genes to probe the influence of the prophage on Ciona gut colonization by determining their transcript levels in vitro and in vivo? If the authors attribute the distinct behavior of the mutant to altered c-di-GMP homeostasis, as may be plausible, why did the authors choose those four genes specifically and not, for example, the many other c-di-GMP turnover protein-coding genes or c-di-GMP effectors present in the S. fidelis genome? This methodological approach seems inadequate to me, and the conclusions on the potential implication of PdeB are premature.

(3) The behavior of the WT strain and the prophage deletion mutant is insufficiently characterized. For instance, how do the authors know that the higher retention capacity reported for the WT strain with respect to the mutant (Fig. 3b) is not merely a consequence of, e.g., a higher growth rate? It would be worth investigating this further, ideally under conditions reflecting the host environment.

(4) Related to the above, sometimes the authors refer to "retention" (e.g., line 162) and at other instances to "colonization" (e.g., line 161), or even adhesion (line 225). These are distinct processes. The authors have only tracked the presence of bacteria by fluorescence labeling; adhesion or colonization has not been assessed or demonstrated in vivo. Please revise.

(5) The higher CFU numbers for the WT after 24 h (line 161) might also indicate a role of motility for successful niche occupation or dissemination in vivo. The authors could test this hypothesis by examining the behavior of, e.g., flagellar mutants in their in vivo model.

(6) The endpoint of experiments with a mixed WT-mutant inoculum (assumedly 1:1? Please specify) was set to 1 h, I assume because of the differences observed in CFU counts after 24 h. In vivo findings shown in Fig. 3c-e are, prima facie, somewhat contradictory. The authors report preferential occupation of the esophagus by the WT (line 223), which seems proficient from evidence shown in Fig. S3. Yet, there is marginal presence of the WT in the esophagus in experiments with a mixed inoculum (Fig. 3d) or none at all (Fig. 3e). Likewise, the authors claim preferential "adhesion to stomach folds" by the mutant strain (line 225), but this is not evident from Fig. 3e. In fact, the occupation patterns by the WT and mutant strain in the stomach in panel 3e appear to differ from what is shown in panel 3d. The same holds true for the claimed "preferential localization of the WT in the pyloric cecum," with Fig. 3d showing a yellow signal that indicates the coexistence of WT and mutant.

(7) In general, and especially for in vivo data, there is considerable variability that precludes drawing conclusions beyond mere trends. One could attribute such variability in vivo to the employed model organism (which is not germ-free), differences between individuals, and other factors. This should be discussed more openly in the main text and presented as a limitation of the study. Even with such intrinsic factors affecting in vivo measurements, certain in vitro experiments, which are expected, in principle, to yield more reproducible results, also show high variability (e.g., Fig. 5). What do the authors attribute this variability to?

(8) Line 198-199: Why not look for potential prophage excision directly rather than relying on indirect, presumptive evidence based on qPCR?

Reviewer #3 (Public review):

This manuscript by Sarkar et al. examines the infection of the liver and hepatocytes during M. tuberculosis infection. They demonstrate that aerosol infection of mice and guinea pigs leads to appreciable infection of the liver as well as the lung. Transcriptomic analysis of HepG2 cells showed differential regulation of metabolic pathways including fatty acid metabolic processing. Hepatocyte infection is assisted by fatty acid synthesis in the liver and inhibiting this caused reduced Mtb growth. The nuclear receptor PPARg was upregulated by Mtb infection and inhibition or agonism of its activity caused a reduction or increase in Mtb growth, respectively, supporting data published elsewhere about the role of PPARg in lung macrophage Mtb infection. Finally, the authors show that Mtb infection of hepatocytes can cause upregulation of enzymes that metabolize antibiotics, resulting in increased tolerance of these drugs by Mtb in the liver.

Overall, this is an interesting paper on an area of TB research where we lack understanding. However, some additions to the experiments and figures are needed to improve the rigor of the paper and further support the findings. Most importantly, although the authors show that Mtb can infect hepatocytes in vitro, they fail to describe how bacteria get from the lungs to the liver in an aerosolized infection. They also claim that "PPARg activation resulting in lipid droplets formation by Mtb might be a mechanism of prolonging survival within hepatocytes" but do not show a direct interaction between PPARg activation and lipid droplet formation and lipid metabolism, only that PPARg promotes Mtb growth. Thus, the correlations with PPARg appear to be there but causation, implied in the abstract and discussion, is not proven.

The human photomicrographs are important and overall well done (lung and liver from the same individuals is excellent). However, in lines 120-121, the authors comment on the absence of studies on the precise involvement of different cells in the liver. In this study there is no attempt to immunophenotype the nature of the cells harboring Mtb in these samples (esp. hepatocytes). Proving that hepatocytes specifically harbor the bacteria in these human samples would add significant rigor to the conclusions made.

Reviewer #3 (Public review):

Summary:

The manuscript demonstrates that starvation induces persister formation in M. abscesses. They also utilized Tn-Seq for the identification of genes involved in persistence. They identified the role of catalase-peroxidase KatG in preventing death from translation inhibitors Tigecycline and Linezolid. They further demonstrated that a combination of these translation inhibitors leads to the generation of ROS in PBS-starved cells.

Strengths:

The authors used high-throughput genomics-based methods for identification of genes playing a role in persistence.

Weaknesses:

The findings could not be validated in clinical strains.

Reviewer #3 (Public review):

The manuscript describes the use of CRISPR gene editing coupled with phenotyping mosaic zebrafish larvae to characterize functions of genes implicated in heritable fragile bone disorders (FBDs). Authors targeted six high-confident candidate genes implicated in severe recessive forms of FBDs and four Osteoporosis GWAS-implicated genes and observe varied developmental phenotypes across all crispants, in addition to adult skeletal phenotypes. While the study lacks insight on underlying mechanisms that contribute to disease phenotypes, a major strength of the paper is the streamlined method that produced significant phenotypes for all candidate genes tested. It also represents a significant increase in number of candidate genes tested using their crispant approach beyond the single mutant that was previously published.

One weakness was the variability of developmental phenotypes, addressed by authors in the Discussion. This might be a product of maternal transcripts not targeted by CRISPR or genetic compensation, which authors have not fully explored. Overall, the paper was well-written and easy to read.

Comments on latest version:

The authors have addressed many concerns in this revision. Figure 1 and Table 2 are much improved.

While details of orthologous gene expression profiles of target genes is a welcome addition, other features of target genes remain unaddressed. For example, do genes with maternally deposited transcript exhibit dampened phenotypes? Or does genetic compensation impact certain genes more than others? Since authors state that the study represents a methods paper, it will be important for users to understand the caveats of gene selection to effectively implement and interpret results of the approach.

Reviewer #3 (Public review):

Summary:

This work is a valuable study that aims to decipher the molecular mechanisms underlying the translation process in Japanese encephalitis virus (JEV), a relevant member of the genus Flavivirus. The authors provide evidence that cap-independent translation, which has already been demonstrated for other flaviviruses, could also account in JEV. This process depends on the genomic 3' UTR, as previously demonstrated in other flaviviruses. Further, the authors find that cellular proteins such as DDX3 or PABP1 could contribute to JEV translation in a cap-independent way. Both DDX3 and PABP1 had previously been described to have a role in cellular protein synthesis and also in the translation step of other flaviviruses distinct from JEV; therefore, this work would expand the cap-independent translation in flaviviruses as a general mechanism to bypass the translation repression exerted by the host cell during viral infection. Further, the findings can be relevant for the development of specific drugs that could interfere with flaviviral translation in the future. Nevertheless, the conclusions are not fully supported by the provided results.

Strengths:

The results provide a good starting point to investigate the molecular mechanism underlying the translation in flaviviruses, which even today is an area of knowledge with many limitations.

Weaknesses:

The main limit of the work is related to the fact that the role of the 3' UTR structural elements and DDX3 is not only circumscribed to translation, but also to replication and encapsidation. In fact, some of the provided results suggest this idea. Particularly, it is intriguing why the virus titer can be completely abrogated while the viral protein levels are only partially affected by the knockdown of DDX3. This points to the fact that many of the drawn conclusions could be overestimated or, at least, all the observed effect cannot be attributed only to the DDX3 effect on translation. Finally, it is noteworthy that the use of uncapped transcripts could be misleading, since this is not the natural molecular context of the viral genome.

Reviewer #3 (Public review):

Summary:

Retroviruses have been endogenized into the genome of all vertebrate animals. The envelope protein of the virus is not well conserved and acquires many mutations hence can be used to monitor viral evolution. Since they are incorporated into the host genome, they also reflect the evolution of the hosts. In this manuscript the authors have focused their analyses on the env genes of endogenous retroviruses in primates. Important observations made include the extensive recombination events between these retroviruses that were previously unknown and the discovery of HML species in genomes prior to the splitting of old and new world monkeys.

Strengths:

They explored a number of databases and made phylogenetic trees to look at the distribution of retroviral species in primates. The authors provide a strong rationale for their study design, they provide a clear description of the techniques and the bioinformatics tools used.

Weaknesses:

The manuscript is based on bioinformatics analyses only. The reference genomes do not reflect the polymorphisms in humans or other primate species. The analyses thus likely under estimates the amount of diversity in the retroviruses. Further experimental verification will be needed to confirm the observations.<br /> Not sure which databases were used, but if not already analyzed, ERVmap.com and repeatmesker are ones that have many ERVs that are not present in the reference genomes. Also, long range sequencing of the human genome has recently become available which may also be worth studying for this purpose.

Reviewer #3 (Public review):

The manuscript by Lu et al. explores the role of the Arp2/3 complex and the actin nucleators N-WASP and WAVE in myoblast fusion during muscle regeneration. The results are clear and compelling, effectively supporting the main claims of the study. However, the manuscript could benefit from a more detailed molecular and cellular analysis of the fusion synapse. Additionally, while the description of macrophage extravasation from ghost fibers is intriguing, it seems somewhat disconnected from the primary focus of the work.

Despite this, the data are robust, and the major conclusions are well supported. Understanding muscle fusion mechanism is still a widely unexplored topic in the field and the authors make important progress in this domain.

I have a few suggestions that might strengthen the manuscript as outlined below.

(1) Could the authors provide more detail on how they defined cells with "invasive protrusions" in Figure 4C? Membrane blebs are commonly observed in contacting cells, so it would be important to clarify the criteria used for counting this specific event.

(2) Along the same line, please clarify what each individual dot represents in Figure 4C. The authors mention quantifying approximately 83 SCMs from 20 fibers. I assume each dot corresponds to data from individual fibers, but if that's the case, does this imply that only around four SCMs were quantified per fiber? A more detailed explanation would be helpful.

(3) Localizing ArpC2 at the invasive protrusions would be a strong addition to this study. Furthermore, have the authors examined the localization of Myomaker and Myomixer in ArpC2 mutant cells? This could provide insights into potential disruptions in the fusion machinery.

(4) As a minor curiosity, can ArpC2 WT and mutant cells fuse with each other?

(5) The authors report a strong reduction in CSA at 14 dpi and 28 dpi, attributing this defect primarily to failed myoblast fusion. Although this claim is supported by observations at early time points, I wonder whether the Arp2/3 complex might also play roles in myofibers after fusion. For instance, Arp2/3 could be required for the growth or maintenance of healthy myofibers, which could also contribute to the reduced CSA observed, since regenerated myofibers inherit the ArpC2 knockout from the stem cells. Could the authors address or exclude this possibility? This is rather a broader criticism of how things are being interpreted in general beyond this paper.

Reviewer #3 (Public review):

Summary:

The article presents a meticulous and quantitative anatomical reconstruction of the compound eye of a tiny wasp at the level of subcellular structures, and cellular and optical organization of the ommatidia and reveals the ectopic photoreceptors, which are decoupled from the eye's dioptrical apparatus.

Strengths:

The graphic material is of very high quality, beautifully organized, and presented in a logical order. The anatomical analysis is fully supported by quantitative numerical data at all scales, from organelles to cells and ommatidia, which should be a valuable source for future studies in cellular biology and visual physiology. The 3D renders are highly informative and a real eye candy.

Weaknesses:

The claim that the large dorsal part of the eye is the dorsal rim area (DRA), supported by anatomical data on rhabdomere geometry and connectomics in authors' earlier work, would eventually greatly benefit from additional evidence, obtained by immunocytochemical staining, that could also reveal a putative substrate for colour vision. The cell nuclei that are located in the optical path in the DRA crystalline cone have only a putative optical function, which may be either similar to pore canals in hymenopteran DRA cornea (scattering) or to photoreceptor nuclei in camera-type eyes (focussing), both explanations being mutually exclusive.

Reviewer #3 (Public review):

Summary:

The authors suggest a mechanism that explains the preference of viral protein 35 (VP35) homologs to bind the backbone of double-stranded RNA versus blunt ends. These preferences have a biological impact in terms of the ability of different viruses to escape the immune response of the host.<br /> The proposed mechanism involves the existence of a cryptic pocket, where VP35 binds the blunt ends of dsRNA when the cryptic pocket is closed and preferentially binds the RNA double-stranded backbone when the pocket is open.<br /> The authors performed MD simulation results, thiol labelling experiments, fluorescence polarization assays, as well as point mutations to support their hypothesis.

Strengths:

This is a genuinely interesting scientific question, which is approached through multiple complementary experiments as well as extensive MD simulations. Moreover, structural biology studies focused on RNA-protein interactions are particularly rare, highlighting the importance of further research in this area.

Weaknesses:

- Sequence similarity between Ebola-Zaire (94% similarity) explains their similar behaviour in simulations and experimental assays. Marburg instead is a more distant homolog (~80% similarity relative to Ebola/Zaire). This difference is sequence and structure can explain the propensities, without the need to involve the existence of a cryptic pocket.<br /> - No real evidence for the presence of a cryptic pocket is presented, but rather a distance probability distribution between two residues obtained from extensive MD simulations. It would be interesting to characterise the modelled RNA-protein interface in more detail.

Reviewer #3 (Public review):

Summary:

Nestor and colleagues identify genes escaping X chromosome inactivation (XCI) in rare individuals with non-mosaic XCI (nmXCI) whose tissue-specific RNA-seq datasets were obtained from the GTEX database. Because XCI is non-mosaic, read counts representing a second allele are tested for statistically significant escape, in this case > 2.5% of active X expression. Whereas a prior GTEX analysis found only one nmXCI female, this study finds two additional donors in GTEX, therefore expanding the number of assessed X-linked genes to 380. Although this is fewer than half of X-linked genes, the study demonstrates that although rare, nmXCI females are represented in RNA-seq databases such as GTEX. Therefore this analytical approach is worthwhile pursuing in other (larger) databases as well, to provide deeper insight into escape from XCI which is relevant to X-linked diseases and sex differences.

Strengths:

The analysis is well-documented, straightforward, and valuable. The supplementary tables are useful, and the claims in the main text well-supported.

Weaknesses:

There are very few, except that this escape catalogue is limited to 3 donors, based on a single (representative) tissue screen in 285 female donors, mostly using muscle samples. However, if only pituitary samples had been screened, nmXCI-1 would have been missed. Additional donors in the 285 representative samples cross a lower threshold of AE = 0.4. It would be worthwhile to query all tissues of the 285 donors to discover more nmXCI cases, as currently fewer than half of X-linked genes received a call using this very worthwhile approach.

Reviewer #3 (Public review):

Summary:

The authors performed time-resolved proteomics and phospho-proteomics in Xenopus oocytes from prophase I through the MII arrest of the unfertilized egg. The data contains protein abundance and phosphorylation sites of a large number set of proteins at different stages of oocyte maturation. The large sets of the data are of high quality. In addition, the authors discussed several key pathways critical for the maturation. The data is very useful for the researchers not only researchers in Xenopus oocytes but also those in oocyte biology in other organisms.

Strengths:

The data of proteomics and phospho-proteomics in Xenopus oocyte maturation is very useful for future studies to understand molecular networks in oocyte maturation.

Weaknesses:

Although the authors offered molecular pathways of the phosphorylation in the translation, protein degradation, cell cycle regulation, and chromosome segregation. The author did not check the validity of the molecular pathways based ontheir proteomic data by the experimentation.

Reviewer #3 (Public review):

Summary:

The present work by Suzuki et al seeks to develop a new embryonic olfactory epithelium organoid culture model, to study OR gene expression and mechanisms involved in epithelium-to-bulb targeting. They characterize an organoid culture derived from E13 mouse olfactory tissue, using RT-qPCR, immunostaining, limited calcium imaging, and single-cell RNA-seq. Main findings show that the cultures produce major olfactory cell types; many olfactory neurons express a single OR; scSeq analysis identifies transcriptional programs associated with specific OR class expressions that may help define mechanisms involved in projection to specific bulb sites (glomeruli).

Strengths:

The organoid model is generally well-characterized and may be a useful approach for studying this question and other problems, such as basal cell lineage choice or damage and repair mechanisms. Overall, the paper is well-written, and the figures are of high quality.

The cultures, produced from E13 mice, appear to produce HBCs, GBCs, neurons, and non-neural cells, providing an important tool. I think a really interesting question is: when do HBCs first appear in these cultures? Developmentally, in rodents, HBCs do not arise until near the end of gestation, and the OE cell populations are instead made from a more GBC-like cell (keratin negative, p63 negative) that proliferates as an apical or basal progenitor. The cell type and architectural descriptions used here repeatedly are really descriptions of the adult OE, yet the cultures are made from E13 mouse olfactory epithelium. Perhaps an important question could be addressed by this model - how this specific adult reserve epithelial stem cell (the HBC) is generated remains unclear. HBCs are a reserve multipotential cell that reconstitutes the entire olfactory epithelium in adults following severe injury, yet is not present during embryonic development until after the epithelium has been largely generated.

Weaknesses:

The paper should discuss the transcriptional programs identified here that correlate with OR class expression in the context of findings from Tsukahara et al, Cell 2021. Tsukahara identified from in vivo olfactory neuron scSeq fixed gene expression programs defining olfactory neuron position in AP or DV axes correlating highly with OR expression.

While the current findings do define the expression of putative targeting, guidance or adhesion molecules in specific OR-expressing neurons in culture, the current results do not provide any experimental evidence that glomerulus targeting is actually mediated by these factors. Further discussion of this limitation may be helpful, along with a discussion of additional approaches to explore these questions.

Calcium imaging: it is not clear why isovaleric acid was chosen as a stimulus for Ca imaging. Is it's known receptor expressed widely in these cultures? Why not use a cocktail of odorants, to activate a broader range of ORs, as has been widely used in in vitro calcium imaging studies of olfactory neurons? Can you show positive control activation (i.e. high potassium)?

How many unique ORs are identified as expressed in the cultures? Figure 5 indicates only 78 genes. Since mice express about 1200 ORs, is this a limitation? How many replicates (individual cells) are found to express each of the ORs? Again, Figure 5 suggests only 202 cells are OR+? Is this enough to define the gene expression programs reliably associated with a given OR or OR class? More detail on this analysis would be helpful.

Reviewer #3 (Public review):

Summary:

The authours present a variant of a previously described fluorescence lifetime sensor for calcium. Much of the manuscript describes the process of developing appropriate assays for screening sensor variants, and thorough characterization of those variants (inherent fluorescence characteristics, response to calcium and pH, comparisons to other calcium sensors). The final two figures show how the sensor performs in cultured cells and in vivo drosophila brains.

Strengths:

The work is presented clearly and the conclusion (this is a new calcium sensor that could be useful in some circumstances) is supported by the data.

Weaknesses:

There are probably few circumstances where this sensor would facilitate experiments (calcium measurements) that other sensors would prove insufficient.

Reviewer #3 (Public review):

Summary:<br /> In this work, the authors present a chromatin polymer model with some specific pattern of transcription units (TUs) and diffusing TFs; they simulate the model and study TFclustering, mixing, gene expression activity, and their correlations. First, the authors designed a toy polymer with colored beads of a random type, placed periodically (every 30 beads, or 90kb). These colored beads are considered a transcription unit (TU). Same-colored TUs attract with each other mediated by similarly colored diffusing beads considered as TFs. This led to clustering (condensation of beads) and correlated (or anti-correlation) "gene expression" patterns. Beyond the toy model, when authors introduce TUs in a specific pattern, it leads to emergence of specialized and mixed cluster of different TFs. Human chromatin models with realistic distribution of TUs also lead to the mixing of TFs when cluster size is large.

Strengths:<br /> This is a valuable polymer model for chromatin with a specific pattern of TUs and diffusing TF-like beads. Simulation of the model tests many interesting ideas. The simulation study is convincing and the results provide solid evidence showing the emergence of mixed and demixed TF clusters within the assumptions of the model.

Weaknesses:<br /> Weakness of the work: The model has many assumptions. Some of the assumptions are a bit too simplistic. Concerns about the work are detailed below:

The authors assume that when the diffusing beads (TFs) are near a TU, the gene expression starts. However, mammalian gene expression requires activation by enhancer-promoter looping and other related events. It is not a simple diffusion-limited event. Since many of the conclusions are derived from expression activity, will the results be affected by the lack of looping details?

Authors neglect protein-protein interactions. Without protein-protein interactions, condensate formation in natural systems is unlikely to happen.

What is described in this paper is a generic phenomenon; many kinds of multivalent chromatin-binding proteins can form condensates/clusters as described here. For example, if we replace different color TUs with different histone modifications and different TFs with Hp1, PRC1/2, etc, the results would remain the same, wouldn't they? What is specific about transcription factor or transcription here in this model?<br /> What is the logic of considering 3kb chromatin as having a size of 30 nm? See Kadam et al. (Nature Communications 2023). Also, DNA paint experimental measurement of 5kb chromatin is greater than 100 nm (see work by Boettiger et al.).

Reviewer #3 (Public review):

Summary:

This work investigates computational consequences of assemblies containing both excitatory and inhibitory neurons (E/I assembly) in a model with parameters constrained by experimental data from the telencephalic area Dp of zebrafish. The authors show how this precise E/I balance shapes the geometry of neuronal dynamics in comparison to unstructured networks and networks with more global inhibitory balance. Specifically, E/I assemblies lead to the activity being locally restricted onto manifolds - a dynamical structure in-between high-dimensional representations in unstructured networks and discrete attractors in networks with global inhibitory balance. Furthermore, E/I assemblies lead to smoother representations of mixtures of stimuli while those stimuli can still be reliably classified, and allows for more robust learning of additional stimuli.

Strengths:

Since experimental studies do suggest that E/I balance is very precise and E/I assemblies exist, it is important to study the consequences of those connectivity structures on network dynamics. The authors convincingly show that E/I assemblies lead to different geometries of stimulus representation compared to unstructured networks and networks with global inhibition. This finding might open the door for future studies for exploring the functional advantage of these locally defined manifolds, and how other network properties allow to shape those manifolds.

The authors also make sure that their spiking model is well-constrained by experimental data from the zebrafish pDp. Both, spontaneous and odor stimulus triggered spiking activity is within the range of experimental measurements. But the model is also general enough to be potentially applied to findings in other animal models and brain regions.

Weaknesses:

All my previous points have been addressed.