Reviewer #1 (Public review):

Summary:

The authors address a fundamental question for cell and tissue biology using the skin epidermis as a paradigm and ask how stratifying self-renewing epithelia induce differentiation and upwards migration in basal dividing progenitor cells to generate suprabasal barrier-forming cells that are essential for a functional barrier formed by such an epithelium. The authors show for the first time that an increase in intracellular actomyosin contractility, a hallmark of barrier-forming keratinocytes, is sufficient to trigger terminal differentiation. Hence the data provide in vivo evidence of the more general interdependency of cell mechanics and differentiation. The data appear to be of high quality and the evidences are strengthened through a combination of different genetic mouse models, RNA sequencing and immunofluorescence analysis.

To generate and maintain the multilayered, barrier-forming epidermis, keratinocytes of the basal stem cell layer differentiate and move suprabasally accompanied by stepwise changes not only in gene expression but also in cell morphology, mechanics and cell position. If any of these changes are instructive for differentiation itself, and whether consecutive changes in differentiation are required, remains unclear. Also, there are few comprehensive data sets on the exact changes in gene expression between different states of keratinocyte differentiation. In this study, through genetic fluorescence labeling of cell states at different developmental timepoints the authors were able to analyze gene expression of basal stem cells and suprabasal differentiated cells at two different stages of maturation: E14 (embryonic day 14) when the epidermis comprises mostly two functional compartments (basal stem cells and suprabasal so called intermediate cells) and E16 when the epidermis comprise three (living) compartments where the spinous layer separates basal stem cells from the barrier forming granular layer, as is the case in adult epidermis. Using RNA bulk sequencing, the authors developed useful new markers for suprabasal stages of differentiation like MafB and Cox1. The transcription factor MafB was then shown to inhibit suprabasal proliferation in a MafB transgenic model.

The data indicate that early in development at E14 the suprabasal intermediate cells resemble in terms of RNA expression, the barrier-forming granular layer at E16, suggesting that keratinocyte can undergo either stepwise (E16) or more direct (E14) terminal differentiation.

Previous studies by several groups found an increased actomyosin contractility in the barrier forming granular layer and showed that this increase in tension is important for epidermal barrier formation and function. However, it was not clear whether contractility itself serves as an instructive signal for differentiation. To address this question, the authors use a previously published model to induce premature hypercontractility in the spinous layer by using spastin overexpression (K10-Spastin) to disrupt microtubules (MT) thereby indirectly inducing actomyosin contractility. A second model activates myosin contractility more directly through overexpression of a constitutively active RhoA GEF (K10-Arhgef11CA). Both models induce late differentiation of suprabasal keratinocytes regardless of the suprabasal position in either spinous or granular layer indicating that increased contractility is key to induce late differentiation of granular cells. A potential weakness is the use of the K10-spastin model that disrupts MT and likely has additional roles in altering differentiation next to the induction of hypercontractility. Their previous publications provided some evidence that the effect on differentiation is driven by the increase in contractility (Ning et al. cell stem cell 2021). Moreover, their data are now further supported by a second model activating myosin through RhoA. This manuscript extends their previous findings that indicated a role for contractility in early differentiation, now focussing on the regulation of late differentiation in barrier forming cells. This data set thus help to unravel the interdependencies of cell position, mechanical state and differentiation in the epidermis, and suggest that an increase in cellular contractility within the epidermis can induce terminal differentiation. Importantly the authors show that despite contractility induced nuclear localization of the mechanoresponsive transcription factor YAP in the barrier forming granular layer, YAP nuclear localization is not sufficient to drive premature differentiation when forced to the nucleus in the spinous layer.

Overall, this is a well written manuscript and comprehensive dataset.

Reviewer #1 (Public Review):

Summary:

The study "Impact of Maximal Overexpression of a Non-toxic Protein on Yeast Cell Physiology" by Fujita et al. aims to elucidate the physiological impacts of overexpressing non-toxic proteins in yeast cells. By identifying model proteins with minimal cytotoxicity, the authors claim to provide insights into cellular stress responses and metabolic shifts induced by protein overexpression.

Strengths:

The study introduces a neutrality index to quantify cytotoxicity and investigates the effects of protein burden on yeast cell physiology. The study identifies mox-YG (a non-fluorescent fluorescent protein) and Gpm1-CCmut (an inactive glycolytic enzyme) as proteins with the lowest cytotoxicity, capable of being overexpressed to more than 40% of total cellular protein while maintaining yeast growth. Overexpression of mox-YG leads to a state resembling nitrogen starvation probably due to TORC1 inactivation, increased mitochondrial function, and decreased ribosomal abundance, indicating a metabolic shift towards more energy-efficient respiration and defects in nucleolar formation.

Weaknesses:

While the introduction of the neutrality index seems useful to differentiate between cytotoxicity and protein burden, the biological relevance of the effects of overexpression of the model proteins is unclear.

Reviewer #1 (Public review):

Summary:

The study aimed to develop a liquid biopsy EV miRNA signature associated with radiomics features for early diagnosis of pancreatic cancer. Flawed study design and inadequate description of clinical characteristics of the enrolled samples makes the findings unconvincing.

Strengths:

The concept of developing EV miRNA signature associated with disease relevant radiomics features is a strength.

Weaknesses:

There are many weaknesses in this manuscript, which include drawing association of data derived from unmatched sample sets, selection of low abundance miRNAs for developing the signature with inadequate rationale, incomplete description of experimental methods and confusing statements in the text.

Reviewer #1 (Public review):

Summary:

Zhu et al., investigate the cellular defects in glia as a result of loss in DEGS1/ifc encoding the dihydroceramide desaturase. Using the strength of Drosophila and its vast genetic toolkit, they find that DEGS1/ifc is mainly expressed in glia and it's loss leads to profound neurodegeneration. This supports a role for DEGS1 in the developing larval brain as it safeguards proper CNS development. Loss of DEGS1/ifc leads to dihydroceramide accumulation in the CNS and induces alteration in the morphology of glial subtypes and a reduction in glial number. Cortex and ensheathing glia appeared swollen and accumulated internal membranes. Astrocyte-glia on the other hand displayed small cell bodies, reduced membrane extension and disrupted organization in the dorsal ventral nerve cord. They also found that DEGS1/ifc localizes primarily to the ER. Interestingly, the authors observed that loss of DEGS1/ifc drives ER expansion and reduced TGs and lipid droplet numbers. No effect on PC and PE and a slight increase in PS.

The conclusions of this paper are well supported by the data.

Strengths:

This is an interesting study that provides new insight into the role of ceramide metabolism in neurodegeneration.

The strength of the paper is the generation of LOF lines, the insertion of transgenes and the use of the UAS-GAL4/GAL80 system to assess the cell-autonomous effect of DEGS1/ifc loss in neurons and different glial subtypes during CNS development.

The imaging, immunofluorescence staining and EM of the larval brain and the use of the optical lobe and the nerve cord as a readout are very robust and nicely done.

Drosophila is a difficult model to perform core biochemistry and lipidomics, but the authors used the whole larvae and CNS to uncover global changes in mRNA levels related to lipogenesis and the unfolded protein responses, as well as specific lipid alterations upon DEGS1/ifc loss.

Weaknesses:

No major weaknesses identified.

Minor point: The authors performed lipidomics and RTqPCR on whole larvae and larval CNS which does not inform of any cell type-specific effects. Performing single-cell RNAseq on larval brains to tease apart the cell-type specific effect of DEGS1/ifc loss would be interesting to explore the future, but beyond the scope of the current study.

Reviewer #1 (Public review):

The study by Lotonin et al. investigates correlates of protection against African swine fever virus (ASFV) infection. The study is based on a comprehensive work, including the measurement of immune parameters using complementary methodologies. An important aspect of the work is the temporal analysis of the immune events, allowing for the capture of the dynamics of the immune responses induced after infection. Also, the work compares responses induced in farm and SPF pigs, showing the latter an enhanced capacity to induce a protective immunity. Overall, the results obtained are interesting and relevant for the field. The findings described in the study further validate work from previous studies (critical role of virus-specific T cell responses) and provide new evidence on the importance of a balanced innate immune response during the immunization process. This information increases our knowledge on basic ASF immunology, one of the important gaps in ASF research that needs to be addressed for a more rational design of effective vaccines. Further studies will be required to corroborate that the results obtained based on the immunization of pigs by a not completely attenuated virus strain are also valid in other models, such as immunization using live attenuated vaccines.

While overall the conclusions of the work are well supported by the results, I consider that the following issues should be addressed to improve the interpretation of the results:

(1) An important issue in the study is the characterization of the infection outcome observed upon Estonia 2014 inoculation. Infected pigs show a long period of viremia, which is not linked to clinical signs. Indeed, animals are recovered by 20 days post-infection (dpi), but virus levels in blood remain high until 141 dpi. This is uncommon for ASF acute infections and rather indicates a potential induction of a chronic infection. Have the authors analysed this possibility deeply? Are there lesions indicative of chronic ASF in infected pigs at 17 dpi (when they have sacrificed some animals) or, more importantly, at later time points? Does the virus persist in some tissues at late time points, once clinical signs are not observed? Has all this been tested in previous studies?

(2) Virus loads post-Estonia infection significantly differ from whole blood and serum (Figure 1C), while they are very similar in the same samples post-challenge. Have the authors validated these results using methods to quantify infectious particles, such as Hemadsorption or Immunoperoxidase assays? This is important, since it would determine the duration of virus replication post-Estonia inoculation, which is a very relevant parameter of the model.

(3) Related to the previous points, do the authors consider it expected that the induction of immunosuppressive mechanisms during such a prolonged virus persistence, as described in humans and mouse models? Have the authors analysed the presence of immunosuppressive mechanisms during the virus persistence phase (IL10, myeloid-derived suppressor cells)? Have the authors used T cell exhausting markers to immunophenotype ASFV Estonia-induced T cells?

(4) A broader analysis of inflammatory mediators during the persistence phase would also be very informative. Is the presence of high VLs at late time points linked to a systemic inflammatory response? For instance, levels of IFN are still higher at 11 dpi than at baseline, but they are not analysed at later time points.

(5) The authors observed a correlation between IL1b in serum before challenge and protection. The authors also nicely discuss the potential role of this cytokine in promoting memory CD4 T cell functionality, as demonstrated in mice previously. However, the cells producing IL1b before ASFV challenge are not identified. Might it be linked to virus persistence in some organs? This important issue should be discussed in the manuscript.

(6) The lack of non-immunized controls during the challenge makes the interpretation of the results difficult. Has this challenge dose been previously tested in pigs of the age to demonstrate its 100% lethality? Can the low percentage of protected farm pigs be due to a modulation of memory T and B cell development by the persistence of the virus, or might it be related to the duration of the immunity, which in this model is tested at a very late time point? Related to this, how has the challenge day been selected? Have the authors analysed ASFV Estonia-induced immune responses over time to select it?

(7) Also, non-immunized controls at 0 dpc would help in the interpretation of the results from Figure 2C. Do the authors consider that the pig's age might influence the immune status (cytokine levels) at the time of challenge and thus the infection outcome?

(8) Besides anti-CD2v antibodies, anti-C-type lectin antibodies can also inhibit hemadsorption (DOI: 10.1099/jgv.0.000024). Please correct the corresponding text in the results and discussion sections related to humoral responses as correlates of protection. Also, a more extended discussion on the controversial role of neutralizing antibodies (which have not been analysed in this study), or other functional mechanisms such as ADCC against ASFV would improve the discussion.

Reviewer #1 (Public review):

Summary:

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

This is an interesting manuscript that describes never before seen melanosome structures and explores fossilised feathers through new methods. This paper creates an opening for new work to explore coloration in extinct birds.

Strengths:

A novel set of methods applied to the study of fossil melanosomes.

Comments on revised version:

The authors provided a response to the previous 9 issues, for which additional response is provided here:

(1) I respectfully disagree with the authors justification regarding the crest. They show one specimen of Confuciusornis with short feathers (which appears to be a unique feature of this species, possibly related to the fact it is beaked) but what about the more primitive Eoconfuciusornis, a referred specimen of which superficially has an enormous "crest" (Zheng et al 2017), as does Changchengornis (Ji et al 1999). Regardless, it would make more sense compare this new specimen to other enantiornithines. Although limited by the preservation of body feathers, which is not all that common, the following published enantiornithines also exhibit a "crest": bohaiornithid indet. (Peteya et al 2017); Brevirostruavis (Li et al 2021); Dapingfangornis (Li et al 2006); Eoenantiornis (Zhou et al 2005); Grabauornis (Dalsatt etal 2014); Junornis (Liu et al 2017); Longirostravis (Hou etal 2004); Monoenantiornis (Hu & O'Connor 2016); Neobohaiornis (Shen etal 2024); Orienantiornis (Liu etal 2019); Parabohaironis (Wang 2023); Parapengornis (Hu etal 2015); Paraprotopteryx (Zheng et al 2007); and every specimen of Protopteryx. In fact, every single published enantiornithine that preserves any feathering on the head has the feathers preserved perpendicular to the bone (in fact, the body feathers on all parts of the bed are splayed at a right angle to the bone due to compression), as shown in the confuciuornis specimen image provided by the authors. Since it is highly improbable they all had crests, the authors have no justification for the interpretation that this new specimen was crested. This does not mean that the feathers were not iridescent or take away from the novel methods these authors have used to explore preserved feathers.

(2) Yes, this is possible, but see above for the very strong argument against interpretation of these feathers as forming a crest.

(3) This just further makes the point that the isolated feather is not likely from the head. Since the neck feathers are missing, it is more likely that it is these feathers that have been disarticulated (and sampled) from the neck region rather than from the very complete looking head feathers; this has significant implications with regards to the birds colour pattern.

(4) Thank you for acknowledging taphonomy.

(5) An interesting hypothesis and one I look forward to seeing explored in the future.

(6) Since the compression is in a single direction, in fact it is not reasonable to assume that distortion would be random. One might predict similar distortion, as with the feathers (spread out from the bone at a 90˚ angle) and bone (crushed), which are all compressed in a single direction. However, I agree that such a consistent discovery suggests it is not an artifact of preservation, and only further studies will elucidate this

(7) I still fail to detect this hexagonal pattern - could machine learning be used to quantify this pattern? The random arrangement of white arrows does little to clarify the authors interpretations.

(8) Great to see additional sampling

(9) Thank you for the explanation.

Reviewer #1 (Public review):

Summary:

Palardy and colleagues examine how transcription factors of the SoxB1 family alter patterning within the zebrafish posterior lateral line primordium and subsequent formation of neuromast organs along the body of the developing fish. They describe how expression of soxb genes changes when Wnt and Fgf signaling pathways are altered, and in addition, how outputs of these signalling pathways change when soxb gene expression is disrupted. Together, experiments suggest a model where the expression of SoxB genes counteracts Wnt signaling. Support comes from the combined inhibition of both pathways, partially restoring the pattern of neuromast deposition. Together, the work reveals an additional layer of control over Wnt and Fgf signals that together ensure proper posterior lateral line development.

Strengths:

The authors provide a clear analysis of changes in RNA expression after systematic manipulation of gene expression and signaling pathways to construct a plausible model of how Sox factors regulate primordium patterning.

Weaknesses:

There is little attempt to capture the variation of expression patterns with each manipulation. Photomicrographs are examples, with little quantification.

While the combined loss of soxb functions shows more severe phenotypes, it is not exactly clear what underlies the apparent redundancy. It would be helpful if the soxb gene family member expression was reported after loss of each. Expression of sox1a is shown in sox2 mutants in Figure 4, but other combinations are not reported. This additional analysis would clarify whether there are alterations in expression that influence apparent redundancy.

Reviewer #1 (Public review):

Summary:

This paper introduces a new class of machine learning models for capturing how likely a specific nucleotide in a rearranged IG gene is to undergo somatic hypermutation. These models modestly outperform existing state-of-the-art efforts, despite having fewer free parameters. A surprising finding is that models trained on all mutations from non-functional rearrangements give divergent results from those trained on only silent mutations from functional rearrangements.

Strengths:

* The new model structure is quite clever and will provide a powerful way to explore larger models.<br /> * Careful attention is paid to curating and processing large existing data sets.<br /> * The authors are to be commended for their efforts to communicate with the developers of previous models and use the strongest possible versions of those in their current evaluation.

Weaknesses:

* No significant weaknesses noted

Reviewer #1 (Public review):

Summary:

The authors revisit the specific domains/signals required for redirection of an inner nuclear membrane protein, emerin, to the secretory pathway. They find that epitope tagging influences protein fate, serving as a cautionary tale for how different visualisation methods are used. Multiple tags and lines of evidence are used, providing solid evidence for the altered fate of different constructs.

Strengths:

This is a thorough dissection of domains and properties that confer INM retention vs secretion to the PM/lysosome, and will serve the community well as a caution regarding placement of tags and how this influences protein fate.

Weaknesses:

The specific biogenesis pathway for C-terminally tagged emerin might confound some interpretations. Appending the large GFP to the C-terminus may direct the fusion protein to a different ER insertion pathway than that used by the endogenous protein. How this might influence the fate of the tagged protein remains to be determined. In some ways this is beyond the scope of the current study, but should serve as a warning to epitope-tagging approaches.

Reviewer #1 (Public review):

Summary:

The authors report four cryoEM structures (2.99 to 3.65 Å resolution) of the 180 kDa, full-length, glycosylated, soluble Angiotensin-I converting enzyme (sACE) dimer, with two homologous catalytic domains at the N- and C-terminal ends (ACE-N and ACE-C). ACE is a protease capable of effectively degrading Aβ. The four structures are C2 pseudo-symmetric homodimers and provide insight into sACE dimerization. These structures were obtained using discrete classification in cryoSPARC and show different combinations of open, intermediate, and closed states of the catalytic domains, resulting in varying degrees of solvent accessibility to the active sites.

To deepen the understanding of the gradient of heterogeneity (from closed to open states) observed with discrete classification, the authors performed all-atom MD simulations and continuous conformational analysis of cryo-EM data using cryoSPARC 3DVA, cryoDRGN, and RECOVAR. cryoDRGN and cryoSPARC 3DVA revealed coordinated open-closed transitions across four catalytic domains, whereas RECOVAR revealed independent motion of two ACE-N domains, also observed with cryoSPARC focused classification. The authors suggest that the discrepancy in the results of the different methods for continuous conformational analysis in cryo-EM could results from different approaches used for dimensionality reduction and trajectory generation in these methods.

Strengths:

This is an important study that shows, for the first time, the structure and the snapshots of the dynamics of the full-length sACE dimer. Moreover, the study highlights the importance of combining insights from different cryo-EM methods that address questions difficult or impossible to tackle experimentally, while lacking ground truth for validation.

Weaknesses:

The open, closed, and intermediate states of ACE-N and ACE-C in the four cryo-EM structures from discrete classification were designated quantitatively (based on measured atomic distances on the models fitted into cryo-EM maps). Unfortunately, atomic models were not fitted into cryo-EM maps obtained with cryoSPARC 3DVA, cryoDRGN, and RECOVAR, and the open/closed states in these cases were designated based on a qualitative analysis.

Reviewer #1 (Public review):

Summary:

This study utilizes polarized second-harmonic generation (pSHG) microscopy to investigate myosin conformation in the relaxed state, distinguishing between the disordered, actin-accessible ON state and the ordered, energy-conserving OFF state. By pharmacologically modulating the ON/OFF equilibrium with a myosin activator (2-deoxyATP) and inhibitor (Mavacamten), the authors demonstrate that pSHG can sensitively quantify the ON/OFF ratio in both skeletal and cardiac muscle. Validation with X-ray diffraction supports the accuracy of the method. Applying this approach to a hypertrophic cardiomyopathy model, the study shows that R403Q/MYH7-mutated minipigs exhibit an increased ON state fraction relative to controls. This difference is eliminated under saturating concentrations of myosin modulators, indicating that the ON/OFF balance can be pharmacologically shifted to its extremes. Additionally, ATPase assays reveal elevated resting ATPase activity in R403Q samples, which persists even when the ON state is saturated, suggesting that increased energy consumption in this mutation is driven by both a shift toward the ON state and inherently higher myosin ATPase activity.

Strengths:

This is a well-written and well-conducted study that clearly reveals the power of SHG microscopy. The study clearly establishes the great utility of SHG to study thick filament regulation.

Weaknesses:

(1) Several studies have shown that the ON state of the thick filament is sensitive to both temperature and filament lattice spacing, with a common recommendation to conduct skinned fiber experiments at temperatures above 27{degree sign}C and in the presence of dextran to better preserve physiological conditions. The authors should clarify the experimental temperature used in their skinned fiber studies, indicate whether dextran was included, and discuss whether adherence to these recommended conditions would have impacted their results.

(2) On page 13, the authors report the proportion of disordered heads as approximately 30% in wild-type and 65% in R403Q fibers. They should clarify whether these values represent the percentage of total myosin heads, or rather the percentage of heads that are responsive to Mavacamten and dATP.

(3) In Figure 5, regarding ATPase measurements, the content of contractile material per unit volume of muscle preparation will influence the results. Did the authors account for this variable, and if not, how might it have affected the conclusions?

(4) For readers primarily interested in assessing the ON/OFF state of thick filaments, could the authors list the specific advantages of polarized second harmonic generation (pSHG) microscopy compared to X-ray diffraction?

(5) Given that many data points were derived from the same fiber or myocyte, how did the authors address the risk of type I errors due to non-independence of measurements? Was a nested or hierarchical statistical approach used?

Reviewer #1 (Public review):

Summary:

This manuscript presents findings on the adaptation mechanisms of Saccharomyces cerevisiae under extreme stress conditions. The authors try to generalize this to adaptation to stress tolerance. A major finding is that S. cerevisiae evolves a quiescence-like state with high trehalose to adapt to freeze-thaw tolerance independent of their genetic background. The manuscript is comprehensive, and each of the conclusions is well supported by careful experiments.

Strengths:

This is excellent interdisciplinary work.

Weaknesses: .

I have questions regarding the overall novelty of the proposal, which I would like the authors to explain.

(1) Earlier papers have shown that loss of ribosomal proteins, that slow growth, leads to better stress tolerance in S. cerevisiae. Given this, isn't it expected that any adaptation that slows down growth would, overall, increase stress tolerance? Even for other systems, it has been shown that slowing down growth (by spore formation in yeast or bacteria/or dauer formation in C. elegans) is an effective strategy to combat stress and hence is a likely route to adaptation. The authors stress this as one of the primary findings. I would like the authors to explain their position, detailing how their findings are unexpected in the context of the literature.

(2) Convergent evolution of traits: I find the results unsurprising. When selecting for a trait, if there is a major mode to adapt to that stress, most of the strains would adapt to that mode, independent of the route. According to me, finding out this major route was the objective of many of the previous reports on adaptive evolution. The surprising part in the previous papers (on adaptive evolution of bacteria or yeast) was the resampling of genes that acquired mutations in multiple replicates of an evolution experiments, providing a handle to understand the major genetic route or the molecular mechanism that guides the adaptation (for example in this case it would be - what guides the over-accumulation of trehalose). I fail to understand why the authors find the results surprising, and I would be happy to understand that from the authors. I may have missed something important.

(3) Adaptive evolution would work on phenotype, as all of selective evolution is supposed to. So, given that one of the phenotypes well-known in literature to allow free-tolerance is trehalose accumulation, I think it is not surprising that this trait is selected. For me, this is not a case of "non-genetic" adaptation as the authors point out: it is likely because perturbation of many genes can individually result in the same outcome - upregulation of trehalose accumulation. Thereby, although the adaptation is genetic, it is not homogeneous across the evolving lines - the end result is. Do the authors check that the trait is actually a non-genetic adaptation, i.e., if they regrow the cells for a few generations without the stress, the cells fall back to being similarly only partially fit to freeze-thaw cycles? Additionally, the inability to identify a network that is conserved in the sequencing does not mean that there is no regulatory pathway. A large number of cryptic pathways may exist to alter cellular metabolic states.<br /> This is a point in continuation of point #2, and I would like to understand what I have missed.

(4) To propose the convergent nature, it would be important to check for independently evolved lines and most probably more than 2 lines. It is not clear from their results section if they have multiple lines that have evolved independently.

(5) For the genomic studies, it is not clear if the authors sequenced a pool or a single colony from the evolved strains. This is an important point, since an average sequence will miss out on many mutations and only focus on the mutations inherited from a common ancestral cell. It is also not clear from the section.

Reviewer #1 (Public review):

Summary:

This study builds off prior work that focused on the molecule AA147 and its role as an activator of the ATF6 arm of the unfolded protein response. In prior manuscripts, AA147 was shown to enter the ER, covalently modify a subset of protein disulfide isomerases (PDIs), and improve ER quality control for the disease-associated mutants of AAT and GABAA. Unsuccessful attempts to improve the potency of AA147 have led the authors to characterize a second hit from the screen in this study: the phenylhydrazone compound AA263. The focus of this study on enhancing the biological activity of the AA147 molecule is compelling, and overcomes a hurdle of the prior AA147 drug that proved difficult to modify. The study successfully identifies PDIs as a shared cellular target of AA263 and its analogs. The authors infer, based on the similar target hits previously characterized for AA147, that PDI modification accounts for a mechanism of action for AA263.

Strengths:

The authors are able to establish that, like AA147, AA263 covalently targets ER PDIs. The work establishes the ability to modify the AA263 molecule to create analogs with more potency and efficacy for ATF6 activation. The "next generation" analogs are able to enhance the levels of functional AAT and GABAA receptors in cellular models expressing the Z-variant of AAT or an epilepsy-associated variant of the GABAA receptor, outlining the therapeutic potential for this molecule and laying the foundation for future organism-based studies.

Weaknesses:

Arguably, the work does not fully support the statement provided in the abstract that the study "reveals a molecular mechanism for the activation of ATF6". The identification of targets of AA263 and its analogs is clear. However, it is a presumption that the overlap in PDIs as targets of both AA263 and AA147 means that AA263 works through the PDIs. While a likely mechanism, this conclusion would be bolstered by establishing that knockdown of the PDIs lessens drug impact with respect to ATF6 activation. Alternatively, it has previously been suggested that the cell-type dependent activity of AA263 may be traced to the presence of cell-type specific P450s that allow for the metabolic activation of AA263 or cell-type specific PDIs (Plate et al 2016; Paxman et al 2018). If the PDI target profile is distinct in different cell types, and these target difference correlates with ATF6-induced activity by AA263, that would also bolster the authors' conclusion.

DOI: 10.1038/s44319-025-00511-8

Resource: None

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-ALT-151022-1

What is this?

Reviewer #1 (Public review):

Summary:

This study provided key experimental evidence for the "Solstice-as-Phenology-Switch Hypothesis" through two temperature manipulation experiments.

Strengths:

The research is data-rich, particularly in exploring the effects of pre- and post-solstice cooling, as well as daytime versus nighttime cooling, on bud set timing, showcasing significant innovation. The article is well-written, logically clear, and is likely to attract a wide readership.

Weaknesses:

However, there are several issues that need to be addressed.

(1) In Experiment 1, significant differences were observed in the impact of cooling in July versus August. July cooling induced a delay in bud set dates that was 3.5 times greater in late-leafing trees compared to early-leafing ones, while August cooling induced comparable advances in bud set timing in both early- and late-leafing trees. The study did not explain why the timing (July vs. August) resulted in different mechanisms. Can a link be established between phenology and photosynthetic product accumulation? Additionally, can the study differentiate between the direct warming effect and the developmental effect, and quantify their relative contributions?

(2) The two experimental setups differed in photoperiod: one used a 13-hour photoperiod at approximately 4,300 lux, while the other used an ambient day length of 16 hours with a light intensity of around 6,900 lux. What criteria were used to select these conditions, and do they accurately represent real-world scenarios? Furthermore, as shown in Figure S1, significant differences in soil moisture content existed between treatments - could this have influenced the conclusions?

(3) The authors investigated how changes in air temperature around the summer solstice affected primary growth cessation, but the summer solstice also marks an important transition in photoperiod. How can the influence of photoperiod be distinguished from the temperature effect in this context?

(4) The study utilized potted trees in a controlled environment, which limits the generalization of the results to natural forests. Wild trees are subject to additional variables, such as competition and precipitation. Moreover, climate differences between years (2022 vs. 2023) were not controlled. As such, the conclusions may be overgeneralized to "all temperate tree species", as the experiment only involved potted European beech seedlings. The discussion would benefit from addressing species-specific differences.

Reviewer #1 (Public review):

Summary:

The paper describes the cryoEM structure of RAD51 filament on the recombination intermediate. In the RAD51 filament, the insertion of a DNA-binding loop called the L2 loop stabilizes the separation of the complementary strand for the base-pairing with an incoming ssDNA and the non-complementary strand, which is captured by the second DNA-binding channel called the site II. The molecular structure of the RAD51 filament with a recombination intermediate provides a new insight into the mechanism of homology search and strand exchange between ssDNA and dsDNA.

Strong points:

This is the first human RAD51 filament structure with a recombination intermediate called the D-loop. The work has been done with great care, and the results shown in the paper are compelling based on cryo-EM and biochemical analyses. The paper is really nice and important for researchers in the field of homologous recombination, which gives a new view on the molecular mechanism of RAD51-mediated homology search and strand exchange.

Comments on revisions:

The authors nicely address most of the previous points.

Reviewer #1 (Public review):

Summary:

The authors extended a previous study of selective response to herbivory in Arabidopsis, in order to look specifically for selection on induced epigenetic variation ("Lamarckian evolution"). They found no evidence. In addition, the re-examined result from a previously published study arguing that environmentally induced epigenetic variation was common, and found that these findings were almost certainly artifactual.

Strengths:

The paper is very clearly written, there is no hype, and the methods used are state-of-the-art.

Weaknesses:

The result is negative, so the best you can do is put an upper bound on any effects.

Significance:

Claims about epigenetic inheritance and Lamarckian evolution continue to be made based on very shaky evidence. Convincing negative results are therefore important. In addition, the study presents results that, to this reviewer, suggest that the 2024 paper by Lin et al. [26] should probably be retracted.

Reviewer #1 (Public review):

Summary:

The authors quantified information in gesture and speech, and investigated the neural processing of speech and gestures in pMTG and LIFG, depending on their informational content, in 8 different time-windows, and using three different methods (EEG, HD-tDCS and TMS). They found that there is a time-sensitive and staged progression of neural engagement that is correlated with the informational content of the signal (speech/gesture).

Strengths:

A strength of the paper is that the authors attempted to combine three different methods to investigate speech-gesture processing.

Comments on revisions:

I thank the authors for their careful responses to my comments. However, I remain not convinced by their argumentation regarding the specificity of their spatial targeting and the time-windows that they used.

I do not believe the authors have adequately demonstrated the spatial and temporal specificity required to disentangle the contributions of the IFG and pMTG during the gesture-speech integration process. While the authors have made a sincere effort to address the concerns raised by the reviewers, and have done so with a lot of new analyses, I remain doubtful that the current methodological approach is sufficient to draw conclusions about the causal roles of the IFG and pMTG in gesture-speech integration.

Reviewer #1 (Public review):

Summary:

Kang et al. provide the first experimental insights from holographic stimulation of auditory cortex. Using stimulation of functionally-defined ensembles, they test whether overactivation of a specific subpopulation biases simultaneous and subsequent sensory-evoked network activations.

Strengths:

The investigators use a novel technique to investigate the sensory response properties in functionally defined cell assemblies in auditory cortex. These data provide the first evidence of how acutely perturbing specific frequency-tuned neurons impacts the tuning across a broader population. Their revised manuscript appropriately tempers any claims about specific plasticity mechanisms involved.

Weaknesses:

Although the single cell analyses in this manuscript are comprehensive, questions about how holographic stimulation impacts population coding are left to future manuscripts, or perhaps re-analyses of this unique dataset.

Reviewer #1 (Public review):

Summary:

The authors have developed self-amplifying RNAs (saRNAs) encoding additional genes to suppress dsRNA-related inflammatory responses and cytokine release. Their results demonstrate that saRNA constructs encoding anti-inflammatory genes effectively reduce cytotoxicity and cytokine production, enhancing the potential of saRNAs. This work is significant for advancing saRNA therapeutics by mitigating unintended immune activation.

Strengths:

This study successfully demonstrates the concept of enhancing saRNA applications by encoding immune-suppressive genes. A key challenge for saRNA-based therapeutics, particularly for non-vaccine applications, is the innate immune response triggered by dsRNA recognition. By leveraging viral protein properties to suppress immunity, the authors provide a novel strategy to overcome this limitation. The study presents a well-designed approach with potential implications for improving saRNA stability and minimizing inflammatory side effects.

Comments on revisions:

All comments have been thoroughly addressed, and the manuscript has been significantly improved.

Reviewer #1 (Public review):

Summary:

The study by Wu et al. uses endogenous bruchpilot expression in a cell-type-specific manner to assess synaptic heterogeneity in adult Drosophila melanogaster mushroom body output neurons. The authors performed genomic on locus tagging of the presynaptic scaffold protein bruchpilot (BRP) with one part of splitGFP (GFP11) using the CRISPR/Cas9 methodology and co-expressed the other part of splitGFP (GFP1-10) using the GAL4/UAS system. Upon expression of both parts of splitGFP, fluorescent GFP is assembled at the N-terminus of BRP, exactly where BRP is endogenously expressed in active zones. For manageable analysis, a high-throughput pipeline was developed. This analysis evaluated parameters like location of BRP clusters, volume of clusters, and cluster intensity as a direct measure of the relative amount of BRP expression levels on site, using publicly available 3D analysis tools that are integrated in Fiji. Analysis was conducted for different mushroom body cell types in different mushroom body lobes using various specific GAL4 drivers. To test this new method of synapse assessment, Wu et al. performed an associative learning experiment in which an odor was paired with an aversive stimulus and found that, in a specific time frame after conditioning, the new analysis solidly revealed changes in BRP levels at specific synapses that are associated with aversive learning.

Strengths:

Expression of splitGFP bound to BRP enables intensity analysis of BRP expression levels as exactly one GFP molecule is expressed per BRP. This is a great tool for synapse assessment. This tool can be widely used for any synapse as long as driver lines are available to co-express the other part of splitGFP in a cell-type-specific manner. As neuropils and thus the BRP label can be extremely dense, the analysis pipeline developed here is very useful and important. The authors have chosen an exceptionally dense neuropil - the mushroom bodies - for their analysis and convincingly show that BRP assessment can be achieved with such densely packed active zones. The result that BRP levels change upon associative learning in an experiment with odor presentation paired with punishment is likewise convincing, and strongly suggests that the tool and pipeline developed here can be used in an in vivo context.

Weaknesses:

Although BRP is an important scaffold protein and its expression levels were associated with function and plasticity, I am still somewhat reluctant to accept that synapse structure profiling can be inferred from only assessing BRP expression levels and BRP cluster volume. Also, is it guaranteed that synaptic plasticity is not impaired by the large GFP fluorophore? Could the GFP10 construct that is tagged to BRP in all BRP-expressing cells, independent of GAL4, possibly hamper neuronal function? Is it certain that only active zones are labeled? I do see that plastic changes are made visible in this study after an associative learning experiment with BRP intensity and cluster volume as read-out, but I would be reassured by direct measurement of synaptic plasticity with splitGFP directly connected to BRP, maybe at a different synapse that is more accessible.

Reviewer #1 (Public review):

Summary:

In this study, the authors aim to uncover how the Parkinson's disease-linked LRRK2 G2019S mutation affects synaptic integrity through astrocyte-intrinsic mechanisms. Specifically, they investigate whether LRRK2-driven ERM hyperphosphorylation disrupts astrocyte morphology and excitatory synapse maintenance, with a focus on regional specificity within the cortex.

Strengths:

(1) Novelty and significance: The work provides important insights into non-neuronal contributions to Parkinson's disease (PD) pathology by highlighting a previously underappreciated role of astrocytic ERM signaling in synapse maintenance. This astrocyte-specific mechanism might help explain early cognitive dysfunctions in PD.

(2) Mechanistic depth: The authors present a detailed molecular pathway where the LRRK2 G2019S mutation increases ERM phosphorylation, disrupting Ezrin-Atg7 interactions critical for astrocyte morphology.

(3) Robust methodology: The study uses a powerful combination of tools, including AAV-mediated gene delivery, BioID-based interactome mapping, PALE labeling, and patch-clamp electrophysiology to link molecular, morphological, and functional changes.

(4) Physiological relevance: Parallel findings in both mouse models and human post-mortem brains suggest conservation of the observed phenotypes and strengthen the relevance to PD pathogenesis.

Weaknesses:

(1) Causal directionality: While ERM hyperphosphorylation is clearly shown to correlate with morphological and synaptic changes, the specific causal hierarchy-especially between Ezrin-Atg7 interaction loss and synapse alteration, is inferred but not definitively proven. For example, a rescue experiment directly restoring Atg7 function alongside Ezrin manipulation could strengthen this point.

(2) Brain region specificity: Although regional differences between ACC and MOp are well documented, the underlying cause of this differential vulnerability remains speculative. Examining astrocyte heterogeneity within cortical layers or via transcriptomic/proteomic profiling could clarify these regional effects.

(3) Autophagy function: While Atg7 knockdown leads to clear morphological changes, autophagic flux (e.g., LC3-II turnover or p62 accumulation) is not directly assessed. This would strengthen the mechanistic link to autophagy disruption.

(4) GFAP-based astrogliosis interpretation: The conclusion that no astrogliosis occurs in LRRK2 G2019S mice is based solely on GFAP staining. However, GFAP-negative reactive states have been reported. Including additional markers would help validate this interpretation.

(5) Impact on neuronal populations: The authors conclude that changes in inhibitory synapse density in the MOp are not rescued by astrocytic Ezrin manipulation and suggest developmental effects on interneurons. However, this is speculative without neuronal cell-type-specific data. Including interneuron density or synaptic connectivity analysis would make this claim more robust.

(6) Despite these limitations, the authors substantially achieve their stated aims. Their results provide strong support for a model in which astrocytic ERM signaling downstream of LRRK2 contributes to region-specific synaptic changes, particularly in the anterior cingulate cortex. While certain mechanistic links-such as the role of Ezrin-Atg7 interaction in synaptic maintenance-would benefit from further functional validation, the study offers a well-supported framework for understanding astrocyte-intrinsic contributions to synaptic dysfunction in Parkinson's disease.

This work is likely to contribute meaningfully to ongoing research in neurodegeneration, glial biology, and synaptic regulation. The methodological approaches - especially the combination of in vivo models with proteomics and electrophysiology - will be of interest to others studying astrocyte function and neuron-glia interactions. More broadly, the study highlights the importance of astrocyte heterogeneity and regional specialization in shaping neural circuit vulnerability, providing a valuable foundation for future investigations.

Reviewer #1 (Public review):

Summary:

The authors have investigated the role of GAT3 in the visual system. First, they have developed a CRISPR/Cas9-based approach to locally knock out this transporter in the visual cortex. They then demonstrated electrophysiologically that this manipulation increases inhibitory synaptic input into layer 2/3 pyramidal cells. They further examined the functional consequences by imaging neuronal activity in the visual cortex in vivo. They found that the absence of GAT3 leads to reduced spontaneous neuronal activity and attenuated neuronal responses and reliability to visual stimuli, but without an effect on orientation selectivity. Further analysis of this data suggests that Gat3 removal leads to less coordinated activity between individual neurons and in population activity patterns, thereby impairing information encoding. Overall, this is an elegant and technically advanced study that demonstrates a new and important role of GAT3 in controlling the processing of visual information.

Strengths:

(1) Development of a new approach for a local knockout (GAT3).

(2) Important and novel insights into visual system function and its dependence on GAT3.

(3) Plausible cellular mechanism.

Weaknesses:

No major weaknesses were identified by this reviewer.

Reviewer #1 (Public review):

Summary:

This study aims to investigate the development of infants' responses to music by examining neural activity via EEG and spontaneous body kinematics using video-based analysis. The authors also explore the role of musical pitch in eliciting neural and motor responses, comparing infants at 3, 6, and 12 months of age.

Strengths:

A key strength of the study lies in its analysis of body kinematics and modeling of stimulus-motor coupling, demonstrating how the amplitude envelope of music predicts infant movement, and how higher musical pitch may enhance auditory-motor synchronization.

Weaknesses:

The neural data analysis is currently limited to auditory evoked potentials aligned with beat timing. A more comprehensive approach is needed to robustly support the proposed developmental trajectory of neural responses to music.

Reviewer #1 (Public review):

Summary:

This article investigates the origin of movement slowdown in weightlessness by testing two possible hypotheses: the first is based on a strategic and conservative slowdown, presented as a scaling of the motion kinematics without altering its profile, while the second is based on the hypothesis of a misestimation of effective mass by the brain due to an alteration of gravity-dependent sensory inputs, which alters the kinematics following a controller parameterization error.

Strengths:

The article convincingly demonstrates that trajectories are affected in 0g conditions, as in previous work. It is interesting, and the results appear robust. However, I have two major reservations about the current version of the manuscript that prevent me from endorsing the conclusion in its current form.

Weaknesses:

(1) First, the hypothesis of a strategic and conservative slowdown implicitly assumes a similar cost function, which cannot be guaranteed, tested, or verified. For example, previous work has suggested that changing the ratio between the state and control weight matrices produced an alteration in movement kinematics similar to that presented here, without changing the estimated mass parameter (Crevecoeur et al., 2010, J Neurophysiol, 104 (3), 1301-1313). Thus, the hypothesis of conservative slowing cannot be rejected. Such a strategy could vary with effective mass (thus showing a statistical effect), but the possibility that the data reflect a combination of both mechanisms (strategic slowing and mass misestimation) remains open.

(2) The main strength of the article is the presence of directional effects expected under the hypothesis of mass estimation error. However, the article lacks a clear demonstration of such an effect: indeed, although there appears to be a significant effect of direction, I was not sure that this effect matched the model's predictions. A directional effect is not sufficient because the model makes clear quantitative predictions about how this effect should vary across directions. In the absence of a quantitative match between the model and the data, the authors' claims regarding the role of misestimating the effective mass remain unsupported.

In general, both the hypotheses of slowing motion (out of caution) and misestimating mass have been put forward in the past, and the added value of this article lies in demonstrating that the effect depended on direction. However, (1) a conservative strategy with a different cost function can also explain the data, and (2) the quantitative match between the directional effect and the model's predictions has not been established.

Specific points:

(1) I noted a lack of presentation of raw kinematic traces, which would be necessary to convince me that the directional effect was related to effective mass as stated.

(2) The presentation and justification of the model require substantial improvement; the reason for their presence in the supplementary material is unclear, as there is space to present the modelling work in detail in the main text. Regarding the model, some choices require justification: for example, why did the authors ignore the nonlinear Coriolis and centripetal terms?

(3) The increase in the proportion of trials with subcomponents is interesting, but the explanatory power of this observation is limited, as the initial percentage was already quite high (from 60-70% during the initial study to 70-85% in flight). This suggests that the potential effect of effective mass only explains a small increase in a trend already present in the initial study. A more critical assessment of this result is warranted.

Reviewer #1 (Public review):

Summary:

This manuscript focuses on single-cell RNA sequencing (scRNA-seq) analysis following chronic methamphetamine (METH) treatment in mice. The authors propose two hypotheses:

(1) METH induces neuroinflammation involving T and NKT cells, and (2) METH alters neuronal stem cell differentiation.

Strengths:

The authors provide a substantial dataset with numerous replicates, offering valuable resources to the research community.

Weaknesses:

Concerns persist regarding the interpretation of data and the validation of experiments. First, the presence of T cells, NKT cells, and neutrophils in both the control and METH-treated hippocampi suggests that blood contamination rather than immune cell infiltration is the cause. Since the authors claim that METH disrupts the blood-brain barrier, increasing the infiltration of these immune cells, identifying the source of these immune cells is critical.

Secondly, the pseudotime analysis, which suggests altered neural stem cell (NSC) differentiation, is not conclusively supported by the current data and requires further validation.

Overall, the authors provided comprehensive in vivo data on the impact of methamphetamine on the hippocampus; however, further in vivo and in vitro experimental validation of the key findings is needed.

Reviewer #1 (Public review):

Summary:

This work by Hall et al provides a novel and important new finding about communication between the anterior cingulate cortex (ACC) and the CA1 region of the dorsal hippocampus: there is a clear ability of ACC to predict CA1 activity, and that is modulated by learning/experience. Furthermore, they have some evidence that the modulation differs by whether the CA1 neurons were in the deep versus superficial sub-layer of CA1. The evidence is suggestive of new and exciting findings, but some gaps and weaknesses remain to be addressed before I believe all of the authors' claims can be supported. The figures also need to be slightly better organized, and the discussion is missing a major dimension in my opinion. Overall, this is a strong submission, but with some gaps to fill.

Strengths:

(1) This is a well-written manuscript - the introduction was especially clear, well-cited, and motivating.

(2) The sub-layer specific communication between ACC and CA1 represents the discovery of a novel and functionally impactful piece of neurobiology.

(3) Optogenetics was an important verification of ACC-CA1 communication, as was the analysis of neurons by waveform type.

Weaknesses:

(1) Figure 2: Why are the data separated into two groups from the outset? If all data are combined, is there a general drop in prediction gain from pre to post?

(2) 2b and 2c are important since they are complementary means to show the same thing, and it is important that they cross-validate each other, especially since the non-significant task active neuron difference in 2b appears to be nearly as strong as the significant difference to its left. A more holistic analysis can be done to compare these dimensions.

(3) Sup vs deep neuron definition: Did the authors have any means to validate this anatomical separation using histology or otherwise? I don't believe they described anything like that, and instead use physiology to infer anatomical location. I understand anatomy-based methods may be practically impossible with tetrodes, but this limitation should at least be mentioned, and it should be explained that without something like silicon probes or histological validation, anatomy had to be inferred from physiology.

(4) Superficial vs deep differences in firing rate ratio based on PG: there are many fewer CAdeep neurons, but in 4c, the trends appear to be the same pre-training, top PG lower than others. It seems the lack of difference in CA1deep in 4c may be due to the much lower power/n. This should be discussed or addressed.

(5) In Figure 5, the term "firing rate ratio" is used, and it sounds the same as in previous figures, but this is a different ratio (based on modulation by opto stim, not task).

(6) I would like to learn more about these v-type neurons. I understand we do not yet know about their molecular or morphologic correlate, but more analysis can be done with the current data.

(7) I would like more discussion of ACC-CA1 connectivity.

(8) Some elements may be missing from the discussion, relating baseline functioning versus post-learning function.

Reviewer #1 (Public review):

Summary:

Zhang et al. addressed the question of whether hyperaltruistic preference is modulated by decision context and tested how oxytocin (OXT) may modulate this process. Using an adapted version of a previously well-established moral decision-making task, healthy human participants in this study undergo decisions that gain more (or lose less, termed as context) meanwhile inducing more painful shocks to either themselves or another person (recipient). The alternative choice is always less gain (or more loss) meanwhile less pain. Through a series of regression analyses, the authors reported that hyperaltruistic preference can only be found in the gain context but not in the loss context, however, OXT reestablished the hyperaltruistic preference in the loss context similar to that in the gain context.

Strengths:

This is a solid study that directly adapted a previously well-established task and the analytical pipeline to assess hyperaltruistic preference in separate decision contexts. Context-dependent decisions have gained more and more attention in literature in recent years, hence this study is timely. It also links individual traits (via questionnaires) with task performance, to test potential individual differences. The OXT study is done with great methodological rigor, including pre-registration. Both studies have proper power analysis to determine the sample size.

Weaknesses:

Despite the strengths, multiple analytical decisions have to be explained, justified, or clarified. Also, there is scope to enhance the clarity and coherence of the writing - as it stands, readers will have to go back and forth to search for information. Last, it would be helpful to add line numbers in the manuscript during the revision, as this will help all reviewers to locate the parts we are talking about.

Introduction:<br /> (1) The introduction is somewhat unmotivated, with key terms/concepts left unexplained until relatively late in the manuscript. One of the main focuses in this work is "hyperaltruistic", but how is this defined? It seems that the authors take the meaning of "willing to pay more to reduce other's pain than their own pain", but is this what the task is measuring? Did participants ever need to PAY something to reduce the other's pain? Note that some previous studies indeed allow participants to pay something to reduce other's pain. And what makes it "HYPER-altruistic" rather than simply "altruistic"? Plus, in the intro, the authors mentioned that the "boundary conditions" remain unexplored, but this idea is never touched again. What do boundary conditions mean here in this task? How do the results/data help with finding out the boundary conditions? Can this be discussed within wider literature in the Discussion section? Last, what motivated the authors to examine decision context? It comes somewhat out of the blue that the opening paragraph states that "We set out to [...] decision context", but why? Are there other important factors? Why decision context is more important than studying those others?

Experimental design:<br /> (2) The experiment per se is largely solid, as it followed a previously well-established protocol. But I am curious about how the participants got instructed? Did the experimenter ever mention the word "help" or "harm" to the participants? It would be helpful to include the exact instructions in the SI.

(3) Relatedly, the experimental details were not quite comprehensive in the main text. Indeed, Methods come after the main text, but to be able to guide readers to understand what was going on, it would be very helpful if the authors could include some necessary experimental details at the beginning of the Results section.

Statistical analysis<br /> (3) One of the main analyses uses the harm aversion model (Eq1) and the results section keeps referring to one of the key parameters of it (ie, k). However, it is difficult to understand the text without going to the Methods section below. Hence it would be very helpful to repeat the equation also in the main text. A similar idea goes to the delta_m and delta_s terms - it will be very helpful to give a clear meaning of them, as nearly all analyses rely on knowing what they mean.

(4) There is one additional parameter gamma (choice consistency) in the model. Did the authors also examine the task-related difference of gamma? This might be important as some studies have shown that the other-oriented choice consistency may differ in different prosocial contexts.

(5) I am not fully convinced that the authors included two types of models: the harm aversion model and logistic regression models. Indeed, the models look similar, and the authors have acknowledged that. But I wonder if there is a way to combine them? For example:<br /> Choice ~ delta_V * context * recipient (*Oxt_v._placebo)<br /> The calculation of delta_V follows Equation 1.<br /> Or the conceptual question is, if the authors were interested in the specific and independent contribution of dalta_m and dalta_s to behavior, as their logistic model did, why the authors examine the harm aversion first, where a parameter k is controlling for the trade-off? One way to find it out is to properly run different models and run model comparison. In the end, it would be beneficial to only focus on the "winning" model to draw inferences.

(6) The interpretation of the main OXT results needs to be more cautious. According to the operationalization, "hyperaltruistic" is the reduction of pain of others (higher % of choosing the less painful option) relative to the self. But relative to the placebo (as baseline), OXT did not increase the % of choosing the less painful option for others, rather, it decreased the % of choosing the less painful option for themselves. In other words, the degree of reducing other's pain is the same under OXT and placebo, but the degree of benefiting self-interest is reduced under OXT. I think this needs to be unpacked, and some of the wording needs to be changed. I am not very familiar with the OXT literature, but I believe it is very important to differentiate whether OXT is doing something on self-oriented actions vs other-oriented actions. Relatedly, for results such as that in Fig5A, it would be helpful to not only look at the difference, but also the actual magnitude of the sensitivity to the shocks, for self and others, under OXT and placebo.

Comments on revisions:

I did not change my original public review, as I think it can still be helpful for the field to see the reasoning and argument.

For the revision, the authors have done a thorough job of addressing my previous comments and questions.

The only aspect I would like to ask is that, it would still be great to have a clear definition of hyperaltruism. As it stands, hyperaltruism refers to "people's willingness to pay more to reduce other's pain than<br /> their own pain", ie, this means the "hyper" bit is considered with respect to "self". But shouldn't hyperaltruism be classified contrasting "normal" altruism?

It is fine that it follows a previously published work (Crockett et al., 2014), but it would still be necessary to explain/define the construct being tested in a standalone fashion rather than letting readers to go back to the original work.

Reviewer #1 (Public review):

Summary:

Praegel et al. explore the differences in learning an auditory discrimination task between adolescent and adult mice. Using freely-moving (Educage) and head-fixed paradigms, they compare behavioral performance and neuronal responses over the course of learning. The mice were initially trained for seven days on an easy pure frequency tone Go/No-go task (frequency difference of one octave), followed by seven days of a harder version (frequency difference of 0.25 octave). While adolescents and adults showed similar performance on the easy task, adults performed significantly better on the harder task. Quantifying the lick bias of both groups, the authors then argue that the difference in performance is not due to a difference in perception, but rather to a difference in cognitive control. The authors then used neuropixel recordings across 4 auditory cortical regions to quantify the neuronal activity related to the behavior. At the single cell level, the data shows earlier stimulus-related discrimination for adults compared to adolescents in both the easy and hard tasks. At the neuronal population level, adults displayed a higher decoding accuracy and lower onset latency in the hard task as compared to adolescents. Such differences were not only due to learning, but also to age as concluded from recordings in novice mice. After learning, neuronal tuning properties had changed in adults but not in adolescent. Overall, the differences between adolescent and adult neuronal data correlates with the behavior results in showing that learning a difficult task is more challenging for younger mice.

Strengths:

- The behavioral task is well designed, with the comparison of easy and difficult tasks allowing for a refined conclusion regarding learning across age. The experiments with optogenetics and novice mice are completing the research question in a convincing way.<br /> - The analysis, including the systematic comparison of task performance across the two age groups, is most interesting, and reveals differences in learning (or learning strategies?) that are compelling.<br /> - Neuronal recording during both behavioral training and passive sound exposure is particularly powerful, and allows interesting conclusions.

Weaknesses:<br /> - The presentation of the paper must be strengthened. Inconsistencies, missing information or confusing descriptions should be fixed.<br /> - The recording electrodes cover regions in the primary and secondary cortices. It is well known that these two regions process sounds quite differently (for example, one has tonotopy, the other not), and separating recordings from both regions is important to conclude anything about sound representations. The authors show that the conclusions are the same across regions for Figure 4, but is it also the case for the subsequent analysis? Comparing to the original manuscript, the authors have now done the analysis for AuDp and AUDv separately, and say that the differences are similar in both regions. The data however shows that this is not the case (Fig S7). And even if it were the case, how would it compatible with the published literature?

Reviewer #1 (Public review):

Summary:

Both flies and mammals have D1-like and D2-like dopamine receptors, yet the role of D2-like receptors in Drosophila learning and memory remains underexplored. This paper investigates the role of the D2-like dopamine receptor D2R in single pairs of dopaminergic neurons (DANs) during single-odor aversive learning in the Drosophila larva. First, confocal imaging is used to screen GAL4 driver strains that drive GFP expression in just single pairs of dopaminergic neurons. Next, thermogenetic manipulations of one pair of DANs (DAN-c1) suggest that DAN-c1 activity during larval aversive learning is important. Confocal imaging is then used to reveal expression of D2R in the DANs and mushroom body of the larval brain. Finally, optogenetic activation during training phenocopies D2R knockdown in these neurons: aversive learning is impaired when DAN-c1 is targeted, while appetitive and aversive learning are impaired when the mushroom body is manipulated. Finally, a model is proposed in which D2R limits excessive dopamine release to facilitate successful olfactory learning.

Strengths:

The paper convincingly reproduces prior findings that demonstrated D2R knockdown in DL1 DANs or the mushroom body impairs aversive olfactory learning in Drosophila larvae (Qi and Lee, 2014; doi:10.3390/biology3040831). These previous findings were built upon and extended with a comprehensive confocal imaging screen of 57 GAL4 drivers that identified tools driving GFP expression in individual DANs. One of the drivers, R76F02-AD; R55C10-DBD, was consistently shown to label DAN-c1 neurons and no other DANs in the larval brain. Confocal imaging is also used to demonstrate that GFP-tagged D2R is expressed in most DANs and the mushroom body. Behavioral experiments demonstrate that driving D2R knockdown in DAN-c1 neurons impairs aversive learning, as do other loss-of-function manipulations of DAN-c1 neurons.

Limitations:

(1) The single-odor paradigm used to train larvae does not have the advantages of a more conventional balanced or reciprocal training paradigm. The paper describes how the single-odor experimental design could be controlled for non-associative effects, but does not provide an independent validation of the control experiments performed by a different research group using different odors and genotypes 15 to 20 years ago (see Honjo and Furukubo-Tokunaga, 2005; doi:10.1523/jneurosci.2135-05.2005 and Honjo and Furukubo-Tokunaga, 2009; doi:10.1523/jneurosci.1315-08.2009). Whether the involvement of DAN-c1 for aversive learning generalizes to standard paradigms remains unclear (see Eschbach et al., 2020; doi:10.1038/s41593-020-0607-9 and Weber et al., 2023; doi:10.7554/elife.91387.1).

(2) In 11 of 22 larval brains examined in the paper, R76F02-AD; R55C10-DBD appears to drive GFP expression in 1 to 8 additional non-dopaminergic neurons (Figure S1P and Table S3). Of the remaining 11 brains, 4 of their corresponding ventral nerve cords also have expression in 2 to 4 neurons (Table S3). Therefore, experiments involving with the R76F02-AD; R55C10-DBD driver could be manipulating the activity of additional neurons in around 60% of larvae. The conclusions of the paper would be strengthened if key experiments were repeated with other GAL4 drivers that may label DAN-c1 with even greater specificity, such as SS03066 (Truman et al., 2023; doi:10.7554/elife.80594) or MB320C (Hige et al., 2015; doi:10.1016/j.neuron.2015.11.003).

(3) Successful immunostaining with an anti-D2R antibody (Draper et al., 2007; doi:10.1002/dneu.20355 and Love et al., 2023; doi:10.1111/gbb.12836) could validate GFP-tagged D2R expression (Figure 3) in the same way that TH immunostaining was used throughout the paper to determine whether neurons were dopaminergic.

(4) The paper proposes a model in which DAN-c1 activity conveys an aversive teaching signal (Figure 2f) but excessive artificial DAN-c1 activation causes excessive dopamine release that impairs aversive learning (Figures 2i and 5b). According to this model, thermogenetic DAN-c1 activation during training with water or sucrose conveys an aversive teaching signal that reduces performance (Figure 2i) whereas optogenetic DAN-c1 activation does not due to excessive dopamine release (Figures 5c and 5d). The model suggests that optogenetic DAN-c1 activation is strong enough to cause excessive dopamine release by itself whereas thermogenetic DAN-c1 activation can only achieve the same outcome when it occurs in conjunction with natural DAN-c1 activation evoked by quinine. Therefore, an experiment with weaker optogenetic DAN-c1 activation (with lower intensity light or pulsed at a lower frequency) during water or sucrose training would be expected to convey an aversive teaching signal rather than excessive dopamine release, reducing performance. Such an experiment could reconcile the differing thermogenetic and optogenetic results of the paper.

Reviewer #1 (Public review):

Summary:

The topic of nanobody-based PET imaging is important, and holds great potential for real-world applications since nanobodies have many advantages over full sized immunoglobulins and small molecules.

Strengths:

The submitted manuscript contains quite a bit of interesting data from a collaborative team of well-respected researchers. The authors are to be congratulated for presenting results that may not have turned out the way they had hoped, and doing so in a transparent fashion.

Weaknesses:

However, the manuscript could be considered to be a collection of exploratory findings rather than a complete and mature scientific exposition. Most of the sample sizes were 3 per group, which is fine for exploratory work, but insufficient to draw strong, statistically robust conclusions for definitive results.

Overall, the following specific limitations are noted as suggestions for future work:

(1) The authors used DFO, which is well known to leak Zr, rather than the current standard for 89Zr PET which is DFO* (DFO-star)

(2) The brain tissues were not capillary depleted, which limits interpretation. Capillary depletion, with quantitative assessment of the completion of the depletion process, is the standard in the field.

(3) The authors have not experimentally tested the hypothesis that the PEG adduct reduced BBB transcytosis.

(4) The results in Fig. 7 involving the placenta are interesting, but need confirmation using constructs with 18F labeling and without the PEG adduct.

(5) If this line of investigation were to be translated to humans, an important consideration would be the relative safety of 89Zr and 64Cu. It is likely to be quite a bit worse than for 18F, since the 89Zr and 64Cu have longer half-lives, dissociate from their chelators, and lodge in off-target tissues.

(6) A surprising and somewhat disappointing finding was the modest amount of BBB transcytosis. Clearly additional work will be needed before nanobody-based brain PET becomes feasible.

Reviewer #1 (Public review):

Summary:

The authors use analysis of existing data, mathematical modelling and new experiments to explore the relationship between protein expression noise, translation efficiency and transcriptional bursting.

Strengths:

The analysis of the old data and the new data presented is interesting and mostly convincing.

Weaknesses:

My main concern is the analysis presented in Figure 4. This is the core of mechanistic analysis that suggests ribosomal demand can explain the observed phenomenon. Revisions have improved clarity but I am both confused by the assumptions used here in the mathematical modelling of this section. I said before, the authors assumption that the fluctuations of a single gene mRNA levels will significantly affect ribosome demand is puzzling. The author's seem to dismiss this and maybe I am missing something. However, the specific forms used in equations of table S1 seem very phenomenological and I am not sure how these can be taken as good approximations for modelling ribosome demand. Why kc has this specific form, why such a sharp hill number is appropriate. how many total ribosomes per mRNA is assumed here (if this assumption is indeed needed). Again, my intuition is that on average the total level of mRNA across all genes would stay constant and therefore there are not big fluctuations in the ribosome demand due to the burstiness of transcription of individual genes (as this on average is compensated with drop in level of other transcripts). Should not one be considering all transcripts and total ribosomes to be able to model ribosome demand?

Reviewer #1 (Public review):

Summary:

In this study, Floedder et al report that dopamine ramps in both Pavlovian and Instrumental conditions are shaped by reward interval statistics. Dopamine ramps are an interesting phenomenon because at first glance they do not represent the classical reward prediction errors associated with dopamine signaling. Instead, they seem somewhat to bridge the gap between tonic and phasic dopamine, with an intense discussion still being held in the field about what is their actual behavioral role. Here, in tests with head-fixed mice, and dopamine being recorded with a genetically encoded fluorescent sensor in the nucleus accumbens, the authors find that dopamine ramps were only present when intertrial intervals were relatively short and the structure of the task (Pavlovian cue or progression in a VR corridor) contained elements that indicated progression towards the reward (e.g., a dynamic cue). The authors propose that although these findings can be explained by classical theories of dopamine function, they are better explained by their model of Adjusted Net Contingency of Causal Relation (ANCCR). The results of this study provide constraints on future models of dopamine function, and are of high interest to the field.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors recorded activity in the posterior parietal cortex (PPC) of monkeys performing a perceptual decision-making task. The monkeys were first shown two choice dots of two different colors. Then, they saw a random dot motion stimulus. They had to learn to categorize the direction of motion as referring to either the right or left dot. However, the rule was based on the color of the dot and not its location. So, the red dot could either be to the right or left, but the rule itself remained the same. It is known from past work that PPC neurons would code the learned categorization. Here, the authors showed that the categorization signal depended on whether the executed saccade was in the same hemifield as the recorded PPC neuron or in the opposite one. That is, if a neuron categorized the two motion directions such that it responded stronger for one than the other, then this differential motion direction coding effect was amplified if the subsequent choice saccade was in the same hemifield. The authors then built a computational RNN to replicate the results and make further tests by simulated "lesions".

Strengths:

Linking the results to RNN simulations and simulated lesions.

Weaknesses:

Potential interpretational issues due to a lack of explicit evidence on the sizes and locations of the response fields of the neurons. For example, is the contra/ipsi effect explained by the fact that in the contra condition, the response target and the saccade might have infringed on the outer edges of the response fields?

Reviewer #1 (Public review):

Summary:

Early and accurate diagnosis is critical to treating N. fowleri infections, which often lead to death within 2 weeks of exposure. Current methods are based on sampling cerebrospinal fluid, and are invasive, slow, and sometimes unreliable. Therefore, there is a need for a new diagnostic method. Russell et al. address this need by identifying small RNAs secreted by Naegleria fowleri (Fig. 1) that are detectable by RT-qPCR in multiple biological fluids including blood and urine. SmallRNA-1 and smallRNA-2 were detectable in plasma samples of mice experimentally infected with 6 different N. fowleri strains, and were not detected in uninfected mouse or human samples (Fig. 4). Further, smallRNA-1 is detectable in the urine of experimentally infected mice as early as 24 hours post infection (Fig. 5). The study culminates with testing human samples (obtained from the CDC) from patients with confirmed N. fowleri infections; smallRNA-1 was detectable in cerebrospinal fluid in 6 out of 6 samples (Fig. 6B), and in whole blood from 2 out of 2 samples (Fig. 6C). These results suggest that smallRNA-1 could be a valuable diagnostic marker for N. fowleri infection, detectable in cerebrospinal fluid, blood, or potentially urine.

Strengths:

This study investigates an important problem, and comes to a potential solution with a new diagnostic test for N. fowleri infection that is fast, less invasive than current methods, and seems robust to multiple N. fowleri strains. The work in mice is convincing that smallRNA1 is detectable in blood and urine early in infection. Analysis of patient blood samples shows that whole blood could be tested for smallRNA-1 to diagnose N. fowleri infections. The potential for human blood or urine to be tested for N. fowleri could lead to critical early interventions.

Weaknesses:

There are not many N. fowleri cases, so the authors were limited in the human samples available for testing. It is difficult to know how robust this biomarker is in whole blood, serum, or human urine due to little to no sample material being available for testing. This limitation is examined thoroughly in the discussion section, and additional tests are beyond the scope of this work.

Reviewer #1 (Public review):

Summary

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

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

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

Major comments

What do you mean by bias?

In the second paragraph of the introduction, it is claimed that "if no biases were present in the case of peer review, then we should expect the rate with which members of less powerful social groups enjoy successful peer review outcomes to be proportionate to their representation in submission rates." There are a couple of issues with this statement.

First, the authors are implicitly making a normative assumption that manuscript submission and acceptance rates *should* be equalised across groups. This may very well be the case, but there can also be valid reasons - even when women are not intrinsically better at research than men - why a greater fraction of female-authored submissions are accepted relative to male-authored submissions (or vice versa). For example, if men are more likely to submit their less ground-breaking work, then one might reasonably expect that they experience higher rejection rates compared to women, conditional on submission.

Second, I assume by "bias", the authors are taking a broad definition, i.e., they are not only including factors that specifically relate to gender but also factors that are themselves independent of gender but nevertheless disproportionately are associated with one gender or another (e.g., perhaps women are more likely to write on certain topics and those topics are rated more poorly by (more prevalent) male referees; alternatively, referees may be more likely to accept articles by authors they've met before, most referees are men and men are more likely to have met a given author if he's male instead of female). If that is the case, I would define more clearly what you mean by bias. (And if that isn't the case, then I would encourage the authors to consider a broader definition of "bias"!)

Identifying policy interventions is not a major contribution of this paper

I would take out the final sentence in the abstract. In my opinion, your survey evidence isn't really strong enough to support definitive policy interventions to address the issue and, indeed, providing policy advice is not a major - or even minor - contribution of your paper. (Basically, I would hope that someone interested in policy interventions would consult another paper that much more thoughtfully and comprehensively discusses the costs and benefits of various interventions!) While it's fine to briefly discuss them at the end of your paper - as you currently do - I wouldn't highlight that in the abstract as being an important contribution of your paper.

Minor comments

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

Joint Public Review:

Summary:

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

Strengths:

The single cell voltage imaging used in this study is a highly novel method that may allow recordings that were not previously possible using existing methods.

Weaknesses:

The strength of evidence remains incomplete because of the main claim that synchronous events are not associated with ripples. As was mentioned in previous rounds of review, ripples emerge locally and independently in the two hemispheres. Thus, obtaining ripple recordings from the contralateral hemisphere does not provide solid evidence for this claim. The papers the authors are citing to make the claim that "Additionally, we implanted electrodes in the contralateral CA1 region to monitor theta and ripple oscillations, which are known to co-occur across hemispheres (29-31)" do not support this claim. For example, reference 29 contains the following statement: "These findings suggest that ripples emerge locally and independently in the two hemispheres".

Reviewer #1 (Public review):

The authors of this study use electron microscopy and 3D reconstruction techniques to study the morphology of distinct classes of Drosophila sensory neurons *across many neurons of the same class.* This is a comprehensive study attempting to look at nearly all the sensory neurons across multiple sensilla in the same animal to determine a) how much morphological variability exists between and within neurons of different and similar sensory classes and b) identify dendritic features that may have evolved to support particular sensory functions. This study builds upon the authors' previous work which allowed them to identify and distinguish sensory neuron subtypes in the EM volumes without additional staining so that reconstructed neurons could reliably be placed in the appropriate class. This work is unique in looking at a large number of individual neurons of the same class to determine what is consistent and what is variable about their class-specific morphologies.

This means that in addition to providing specific structural information about these particular cells, the authors explore broader questions of how much morphological diversity exists between sensory neurons of the same class. This then informs our conceptualization about how different dendritic morphologies might affect specific sensory and physiological properties of neurons.

The authors found that CO2 sensing neurons have an unusual, sheet-like morphology in contrast to the thin branches of odor-sensing neurons. They show that this morphology greatly increases the surface area to volume ratio above what could be achieved by modest branching of thin dendrites, and posit that this might be important for their sensory function, though this was not directly tested in their study due to technical limitations. The study is mainly descriptive in nature, but thorough, and provides a nice jumping off point for future functional studies. One interesting future analysis could be to examine all four cell types within a single sensilla together to see if there are any general correlations that could reveal insights about how morphology is determined and relative contributions of intrinsic mechanisms vs interactions with neighboring cells. For example, if higher-than-average branching in one cell type correlated with higher-than-average branching in another type when within the same sensilla, it might suggest differential amounts of extracellular growth or branching cues within a given sensillum drive any heterogeneity observed within a class across sensilla. Conversely, if higher branching in one cell type consistently leads to reduced length or branching of the other neurons within its sensillum, this might point to dendrite-dendrite interactions between cells undergoing competitive or repulsive interactions to define territories within each sensillum as a major determinant of the variability.

Strengths:

This work provides a thorough morphometric analysis of the neurons of the *majority of all ab1 sensilla* across a single antenna. The authors use this analysis to 1) characterize the unique dendritic architecture of ab1C neurons relative to other ORNs including ab1D and 2) provide evidence of substantial morphological diversity even within a single subclass of neuron.

Weaknesses:

This is primarily a descriptive paper due to technical limitations since it is not currently technically feasible to determine individual ORN response properties and tie them to identified neurons with detailed EM-based ultrastructural analyses, nor to predictably alter dendritic morphology of these cells to directly test how different morphologies affect sensory function. However, the quantitative descriptive findings presented here will shape these future questions and are necessary for any such future work.

Reviewer #1 (Public Review):

Summary:

Here, Millet et al. consider whether the nematode C. elegans 'discounts' the value of reward due to effort in a manner similar to that shown in other species, including rodents and humans. They designed a T-maze effort choice paradigm inspired by previous literature, but manipulated how effortful the food is to consume. C. elegans worms were sensitive to this novel manipulation, exhibiting effort-discounting-like behaviour that could be shaped by varying the density of food at each alternative in order to calculate an indifference point. This discounting-like behaviour was related to worms' rates of patch leaving, which differed between the low and high effort patches in isolation. The authors also found a potential relationship to dopamine signalling, and also that this discounting behaviour was not specific to lab-based strains of C. elegans.

Strengths:

The question is well-motivated, and the approach taken here is novel. The authors are careful in their approach to altering and testing the properties of the effortful, elongated bacteria. Similarly, they go to some effort to understand what exactly is driving behavioural choices in this context, both through the application of simple standard models of effort discounting and a kinetic analysis of patch leaving. The comparisons to various dopamine mutants further extend the translational potential of their findings. I also appreciate the comparison to natural isolate strains, as the question of whether this behaviour may be driven by some sort of strain-specific adaptation to the environment is not regularly addressed in mammalian counterparts. The manuscript is well-written, and the figures are clear and comprehensible.

Weaknesses:

Discounting is typically defined as the alteration of a subjective value by effort (or time, risk, etc.), which is then used to guide future decision-making. By adapting the standard t-maze task for C. elegans as a patch-leaving paradigm, the authors observe behaviour strongly consistent with discounting models, but that is likely driven by a different process, in particular by an online estimate of the type of food in the current patch, which then influences patch-leaving dynamics (Figure 3). This is fundamentally different from decision-making strategies relating to effort that have been described in the rodent and human literatures. Similarly, the calculation of indifference points at the group instead of at the individual level also suggests a different underlying process and limits the translational potential of their findings. The authors do not discuss the implications of these differences or why they chose not to attempt a more analogous trial-based experiment.

In the case of both the dopamine and natural isolate experiments, the data are very noisy despite large (relative to other C. elegans experiments) sample sizes. In the dopamine experiment, disruption of dop-1, dop-2, and cat-2 had no statistically significant effect. There do not appear to be any corrections for multiple comparisons, and the single significant comparison, for dop-3, had a small effect size. More detailed behavioural analyses on both these and the wild isolate strains, for example by applying their kinetic analysis, would likely give greater insight as to what is driving these inconsistent effects.

Reviewer #1 (Public review):

Summary:

Ritzau-Jost et al. investigate the potential contribution of AP broadening in homeostatic upregulation of neuronal network activity with a specific focus on dissociated neuronal cultures. In cultures obtained from a few brain regions from mice or rats using different culture conditions and examined by different laboratories, AP half-width remained stable despite chronic activity block with TTX. The finding suggests that AP width is not significantly modulated by changes in sodium channel activity.

Strengths:

The collaborative nature of the study amongst the neuronal culture experts and the rigorous electrophysiological assessments provides for a compelling support of the main conclusion.

Weaknesses:

Given the negative nature of the results, a couple of remaining issues (such as the cell density of cultures and the presentation of imaging experiments with a voltage sensor) warrant further consideration. In addition, a discussion of the reasons for the seeming stability of AP half-width to sodium channel modulation might help extend the scope of the study beyond the presentation of a negative conclusion.

Reviewer #1 (Public review):

Summary:

Wu and colleagues aimed to explain previous findings that adolescents, compared to adults, show reduced cooperation following cooperative behaviour from a partner in several social scenarios. The authors analysed behavioural data from adolescents and adults performing a zero-sum Prisoner's Dilemma task and compared a range of social and non-social reinforcement learning models to identify potential algorithmic differences. Their findings suggest that adolescents' lower cooperation is best explained by a reduced learning rate for cooperative outcomes, rather than differences in prior expectations about the cooperativeness of a partner. The authors situate their results within the broader literature, proposing that adolescents' behaviour reflects a stronger preference for self-interest rather than a deficit in mentalising.

Strengths:

The work as a whole suggests that, in line with past work, adolescents prioritise value accumulation, and this can be, in part, explained by algorithmic differences in weighted value learning. The authors situate their work very clearly in past literature, and make it obvious the gap they are testing and trying to explain. The work also includes social contexts that move the field beyond non-social value accumulation in adolescents. The authors compare a series of formal approaches that might explain the results and establish generative and model-comparison procedures to demonstrate the validity of their winning model and individual parameters. The writing was clear, and the presentation of the results was logical and well-structured.

Weaknesses:

I also have some concerns about the methods used to fit and approximate parameters of interest. Namely, the use of maximum likelihood versus hierarchical methods to fit models on an individual level, which may reduce some of the outliers noted in the supplement, and also may improve model identifiability.

There was also little discussion given the structure of the Prisoner's Dilemma, and the strategy of the game (that defection is always dominant), meaning that the preferences of the adolescents cannot necessarily be distinguished from the incentives of the game, i.e. they may seem less cooperative simply because they want to play the dominant strategy, rather than a lower preferences for cooperation if all else was the same.

Appraisal & Discussion:

The authors have partially achieved their aims, but I believe the manuscript would benefit from additional methodological clarification, specifically regarding the use of hierarchical model fitting and the inclusion of Bayes Factors, to more robustly support their conclusions. It would also be important to investigate the source of the model confusion observed in two of their models.

I am unconvinced by the claim that failures in mentalising have been empirically ruled out, even though I am theoretically inclined to believe that adolescents can mentalise using the same procedures as adults. While reinforcement learning models are useful for identifying biases in learning weights, they do not directly capture formal representations of others' mental states. Greater clarity on this point is needed in the discussion, or a toning down of this language.

Additionally, a more detailed discussion of the incentives embedded in the Prisoner's Dilemma task would be valuable. In particular, the authors' interpretation of reduced adolescent cooperativeness might be reconsidered in light of the zero-sum nature of the game, which differs from broader conceptualisations of cooperation in contexts where defection is not structurally incentivised.

Overall, I believe this work has the potential to make a meaningful contribution to the field. Its impact would be strengthened by more rigorous modelling checks and fitting procedures, as well as by framing the findings in terms of the specific game-theoretic context, rather than general cooperation.

Reviewer #1 (Public review):

Summary:

The authors investigated the potential role of IgG N-glycosylation in Haemorrhagic Fever with Renal Syndrome (HFRS), which may offer significant insights for understanding molecular mechanisms and for the development of therapeutic strategies for this infectious disease. However, several issues need to be addressed.

Major Points:

(1) The authors should provide a detailed description of the pathogenesis of Haemorrhagic Fever with Renal Syndrome (HFRS) and elaborate on the crucial role of IgG proteins in the disease's progression (line 65).

(2) An additional discussion on the significance of glycosylation, particularly IgG N-glycosylation, in viral infections should be included in the Introduction section.

(3) In the Abstract section, the authors state that HTNV-specific IgG antibody titers were detected and IgG N-glycosylation was analyzed. However, the analysis of plasma IgG N-glycans is described in the Methods section. Therefore, the authors should clarify the glycome analysis process. Was the specific IgG glycome profile similar to the total IgG N-glycome? Given the biological relevance of specific IgG in immunological diseases, characterizing the specific IgG N-glycome profile would be more significant than analyzing the total plasma IgG.

(4) Further details regarding the N-glycome analysis should be provided, including the quantity of IgG protein used and the methodology employed for analyzing IgG N-glycans (lines 286-287).

(5) Additional statistical analyses should be performed, including multiple comparisons with p-value adjustment, false discovery rate (FDR) control, and Pearson correlation (line 291).

(6) Quality control should be conducted prior to the IgG N-glycome analysis. Additionally, both biological and technical replicates are essential to assess the reproducibility and robustness of the methods.

(7) Multiple regression analysis should be conducted to evaluate the influence of genetic and environmental factors on the IgG N-glycome.

(8) Line 196. Additional discussions should be included, focusing on the underlying correlation between the differential expression of B-cell glycogenes and the dysregulated IgG N-glycome profile, as well as the potential molecular mechanisms of IgG N-glycosylation in the development of HFRS.

Reviewer #1 (Public review):

Building upon their previous work, the authors present an enhanced method for confocal live imaging of leg regeneration in the crustacean Parhyale hawaiensis. Parhyale is an emerging and tractable model system that offers insights into the evolution and mechanisms of development and regeneration. Çevrim et al. demonstrate the ability to image the complete leg regeneration process, spanning several days, with 10-20 minute time intervals and cellular resolution. They have concurrently optimized imaging conditions to enable cell tracking while minimizing phototoxicity. Additionally, they report successfully implementing HCR in situ hybridization in Parhyale, allowing for specific gene transcript staining at the endpoint of live imaging. This opens the possibility of assigning molecular identities to tracked cells.

A key challenge in many regeneration models is achieving continuous imaging throughout the entire regenerative process, as many organisms are difficult to immobilize or cannot tolerate extended imaging without stress. This manuscript's major strength lies in providing practical solutions to these challenges in Parhyale, a compelling and accessible arthropod model for limb regeneration. The authors also employ complementary tools to analyze time-lapse movies and correlate them with endpoint staining. Together, these advances will serve as a useful resource for researchers studying regeneration in Parhyale or in other systems where parts of this workflow can be adapted.

While the data demonstrating the methodological advancement and technical feasibility are solid, much of the benchmarking and regeneration characterization remains qualitative. This does not undermine the validity of the proof-of-principle, but limits the study's broader appeal.

Reviewer #1 (Public review):

Functional lateralization between the right and left hemispheres is reported widely in animal taxa, including humans. However, it remains largely speculative as to whether the lateralized brains have a cognitive gain or a sort of fitness advantage. In the present study, by making use of the advantages of domestic chicks as a model, the authors are successful in revealing that the lateralized brain is advantageous in the number sense, in which numerosity is associated with spatial arrangements of items. Behavioral evidence is strong enough to support their arguments. Brain lateralization was manipulated by light exposure during the terminal phase of incubation, and the left-to-right numerical representation appeared when the distance between items gave a reliable spatial cue. The light-exposure induced lateralization, though quite unique in avian species, together with the lack of intense inter-hemispheric direct connections (such as the corpus callosum in the mammalian cerebrum), was critical for the successful analysis in this study. Specification of the responsible neural substrates in the presumed right hemisphere is expected in future research. Comparable experimental manipulation in the mammalian brain must be developed to address this general question (functional significance of brain laterality) is also expected.

Reviewer #1 (Public review):

Summary:

The image analysis pipeline is tested in analysing microscopy imaging data of gastruloids of varying sizes, for which an optimised protocol for in toto image acquisition is established based on whole mount sample preparation using an optimal refractive index matched mounting media, opposing dual side imaging with two-photon microscopy for enhanced laser penetration, dual view registration, and weighted fusion for improved in toto sample data representation. For enhanced imaging speed in a two-photon microscope, parallel imaging was used, and the authors performed spectral unmixing analysis to avoid issues of signal cross-talk.

In the image analysis pipeline, different pre-treatments are done depending on the analysis to be performed (for nuclear segmentation - contrast enhancement and normalisation; for quantitative analysis of gene expression - corrections for optical artifacts inducing signal intensity variations). Stardist3D was used for the nuclear segmentation. The study analyses into properties of gastruloid nuclear density, patterns of cell division, morphology, deformation, and gene expression.

Strengths:

The methods developed are sound, well described, and well-validated, using a sample challenging for microscopy, gastruloids. Many of the established methods are very useful (e.g. registration, corrections, signal normalisation, lazy loading bioimage visualisation, spectral decomposition analysis), facilitate the development of quantitative research, and would be of interest to the wider scientific community.

Weaknesses:

A recommendation should be added on when or under which conditions to use this pipeline.

Reviewer #1 (Public review):

Summary:

In this manuscript, Rainey et al investigated the effects of transcription factors, ATOH1, POU4F3, GFI1, and SIX1 on the induction of hair cells from human pluripotent stem cells. The authors used a doxycycline-inducible system to control transgene expression and demonstrated significant improvement in the efficiency of MYO7A+ hair cell differentiation compared to the retrovirus-mediated approach. Next, they characterized differentiated cells using single-cell RNA-seq and identified a population of hair cell-like cells with gene expression profiles similar to the fetal human vestibular hair cells. Finally, they revealed the electrophysiological properties of induced cells consistent with those of mechanosensitive hair cells.

Strengths:

A key finding in this study is the rapid induction of cells expressing multiple hair cell markers that takes place within 21 days after overexpression of the four transcription factors. Additionally, the authors demonstrate that doxycycline-mediated gene overexpression outperforms retroviral-mediated gene transfer in terms of both the efficiency and reproducibility of hair cell induction. Furthermore, the authors demonstrate that these induced hair cells can be used to study hair cell protection from cisplatin ototoxicity.

Weaknesses:

The authors conclude that the induced cells lack distinct hair cell subtypes. However, the characterization of generated hair cells in single-cell RNA-seq data is insufficient. Additional vestibular or cochlear hair cell-enriched marker gene and protein expression should be analyzed. Moreover, the morphological features and mechanotransduction channel activity of the induced hair cells have not been analyzed.

Reviewer #1 (Public review):

Summary:

This work integrates two timepoints from the Adolescent Brain Cognitive Development Study to understand how neuroimaging, genetic and environmental data contribute to the predictive power of mental health variables in predicting cognition in a large early adolescent sample. Their multimodal and multivariate prediction framework involves a novel opportunistic stacking model to handle complex types of information to predict variables that are important in understanding mental health-cognitive performance associations.

Strengths:

The authors are commended for incorporating and directly comparing the contribution of multiple imaging modalities (task fMRI, resting state fMRI, diffusion MRI, structural MRI), neurodevelopmental markers, environmental factors and polygenic risk scores in a novel multivariate framework (via opportunistic stacking), as well as interpreting mental health-cognition associations with latent factors derived from Partial Least Squares. The authors also use a large well-characterized and diverse cohort of adolescents from the Adolescent Brain Cognitive Development (ABCD) Study. The paper is also strengthened by commonality analyses to understand the shared and unique contribution of different categories of factors (e.g., neuroimaging vs mental health vs polygenic scores vs sociodemographic and adverse developmental events) in explaining variance in cognitive performance

Weaknesses:

The paper is framed with an over-reliance on the RDoC framework in the introduction, despite deviations from the RDoC framework in the methods. The field is also learning more about RDoC's limitations when mapping cognitive performance to biology. The authors also focus on a single general factor of cognition as the core outcome of interest as opposed to different domains of cognition. The authors could consider predicting mental health rather than cognition. Using mental health as a predictor could be limited by the included 9-11 year age range at baseline (where mental health concerns are likely to be low or not well captured), as well as the nature of how the data was collected, i.e., either by self-report or from parent/caregiver report.

Comments on revisions:

The authors have done an excellent job of addressing my comments. I have no other suggestions to add. Great work!

Reviewer #1 (Public review):

Summary:

Perlee et al. sought to generate a zebrafish line where CRISPR-based gene editing is exclusively limited to the melanocyte lineage, allowing assessment of cell-type restricted gene knockouts. To achieve this, they knocked in Cas9 to the endogenous mitfa locus, as mitfa is a master regulator of melanocyte development. The authors use multiple candidate genes - albino, sox10, tuba1a, ptena/ptenb, tp53 - to demonstrate that their system induces lineage-restricted gene editing. This method allows researchers to bypass embryonic lethal and non-cell autonomous phenotypes emerging from whole body knockout (sox10, tuba1a), drive directed phenotypes, such as depigmentation (albino), and induce lineage-specific tumors, such as melanomas (ptena/ptenb, tp53, when accompanied with expression of BRAFV600E). The main weakness of the manuscript is that the mechanistic explanations proposed to underlie the presented phenotypes are minimally interrogated, but nonetheless interesting and motivating for future experimentation. Overall, there is a clear use for this genetic methodology, and its implementation will be of value to many in vivo researchers.

Strengths:

The strongest component of this manuscript is the genetic control offered by the mitfa:Cas9 system and the ability to make stable, lineage-specific knockouts in zebrafish. This is exemplified by the studies of tuba1a, where the authors nicely show non-cell autonomous mechanisms have obfuscated the role of this gene in melanocyte development. In addition, the mitfa:Cas9 system is elegantly straightforward and can be easily implemented in many labs. Mostly, the figures are clean, controls are appropriate, and phenotypes are reproducible. The invented method is a welcome addition to the arsenal of genetic tools used in zebrafish. The authors kindly and honestly responded to reviewer criticism, which has led to an improved manuscript and a pleasant review process.

Weaknesses:

The authors argue that the benefit of their system is the maintenance of endogenous regulatory elements. However, no direct comparison is made with other tools that offer similar genetic control, such as MAZERATI. This is a missed opportunity to provide researchers the ability to evaluate these two similar genetic approaches. There is a slight concern that tumor onset with this system is hindered by the heterozygous state it imparts to the lineage master regulator (here, mitfa). The authors do a good job at addressing these issues in the Discussion, but experimentation would have been appreciated. Additionally, the authors claim 86% of mitfa+ cells express Cas9. The image shown in Figure 1C does not do a convincing job at showing this percentage.

Another weakness of the manuscript regards minimally investigated mechanistic explanations for each biological vignette. Detailed mechanistic information is indeed out-of-scope for this manuscript, which intends to prove the efficacy of a genetic tool. Readers are cautioned to use the mechanistic insights from these vignettes as inspiration rather than bona fide truth.

The authors performed the necessary experiments to address each of the reviewers' concerns and thereby quell any substantial issues raised during the first review. They have additionally edited their language appropriately to make their claims more accurate. Their efforts during the review process are appreciated.

Conclusion:

The authors were highly receptive to reviewer comments and improved their manuscript from the first submission. The authors were successful in their goal of creating a rapid genetic approach to study cell-type specific genetic insults in vivo. They have presented multiple interesting and convincing stories to support the power of their invented methodology. The refined mechanisms underlying their observed phenotypes may be lacking but this does not take away from the methodological benefit this manuscript provides to the large field of in vivo researchers.

Reviewer #1 (Public review):

Summary:

This manuscript by Garbelli et al. investigates the roles of excitatory amino acid transporters (EAATs) in retinal bipolar cells. The group previously identified that EAAT5b and EAAT7 are expressed at the dendritic tips of bipolar cells, where they connect with photoreceptor terminals. The previous study found that the light responses of bipolar cells, measured by electroretinogram (ERG) in response to white light, were reduced in double mutants, though there was little to no reduction in light responses in single mutants of either EAAT5b or EAAT7.

The current study further explores the roles of EAAT5b and EAAT7 in bipolar cells' chromatic responses. The authors found that bipolar cell responses to red light, but not to green or UV-blue light, were reduced in single mutants of both EAAT5b and EAAT7. In contrast, UV-blue light responses were reduced in double mutants. Additionally, the authors observed that EAAT5b, but not EAAT7, is strongly localized in the UV cone-enriched area of the eye, known as the "Strike Zone (SZ)." This led them to investigate the impact of the EAAT5b mutation on prey detection performance, which is mediated by UV cones in the SZ. Surprisingly, contrary to the predicted role of EAAT5b in prey detection, EAAT5b mutants did not show any changes in prey detection performance compared to wild-type fish. Interestingly, EAAT7 mutants exhibited enhanced prey detection performance, though the underlying mechanisms remain unclear.

The distribution of EAAT7 protein in the outer plexiform layer across the eye correlates with the distribution of red cones. Based on this, the authors tested the behavioral performance driven by red light in EAAT5b and EAAT7 mutants. The results here were again somewhat contrary to predictions based on ERG findings and protein localization: the optomotor response was reduced in EAAT5b mutants, but not in EAAT7 mutants.

Strengths:

Although the paper lacks cohesive conclusions, as many results contradict initial predictions as mentioned above, the authors discuss possible mechanisms for these contradictions and suggest future avenues for study. Nevertheless, this paper demonstrates a novel mechanism underlying chromatic information processing.<br /> The manuscript is well-written, the data are well-presented, and the analysis is thorough.

Weaknesses:

I have only a minor comment. The authors present preliminary data on mGluR6b distribution across the eye. Since this result is based on a single fish, I recommend either adding more samples or removing this data, as it does not significantly impact the paper's main conclusions.

Comments on revisions:

The authors addressed all of the concerns that I had in the original manuscript.

Reviewer #1 (Public review):

Summary

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

Strengths

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

Weaknesses

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

Comments on revisions:

We thank the authors for the significant improvement of the English in the revised version and for the replacement of some conceptual terms that now seem more relevant and appropriate. We only noticed that the term "society" remains in use, although it might not be appropriate to describe a mouse colony (see previous review).

Conclusive remarks

Although this protocol aims to provide a novel approach to evaluate social ranks in mice, it is not clear how it really brings a significant advance in neuroscience research. The FPCT dynamic is very similar to the one observed in the tube test, where mice compete to navigate forward in a narrow space, constraining the opponent to go backwards. The main difference between the FPCT and the tube test is the presence of food between the opponents. In the tube test, food reward was initially used to increase motivation to cross the tube and push the opponent upon the testing day. This component has been progressively abandoned, precisely because it was not necessary for the mice to compete in the tube.<br /> This paper would really bring a significant contribution to the field by providing a neuronal imaging or manipulation correlate to the behavioral outcome obtained by the application of the FPCT.

Reviewer #2 (Public review):

Summary:

In this manuscript, Wang et al analyze ~17,000 transcriptomes from 35 human tissues from the GTEx database and address transcriptomic variations due to age and sex. They identified both gene expression changes as well as alternative splicing events that differ among sexes. Using breakpoint analysis, the authors find sex dimorphic shifts begin with declining sex hormone levels with males being affected more than females. This is an important pan-tissue transcriptomic study exploring age and sex-dependent changes although not the first one.

Strengths:

(1) The authors use sophisticated modeling and statistics for differential, correlational and predictive analysis.

(2) The authors consider important variables such as genetic background, ethnicity, sampling bias, sample sizes, detected genes etc.

(3) This is likely the first study to evaluate alternative splicing changes with age and sex at a pan-tissue scale.

(4) Sex dimorphism with age is an important topic and is thoroughly analyzed in this study.

Reviewer #1 (Public review): <br /> The authors survey the ultrastructural organization of glutamatergic synapses by cryo-ET and image processing tools using two complementary experimental approaches. The first approach employs so-called "ultra-fresh" preparations of brain homogenates from a knock-in mouse expressing a GFP-tagged version of PSD-95, allowing Peukes and colleagues to specifically target excitatory glutamatergic synapses. In the second approach, direct in-tissue (using cortical and hippocampal regions) targeting of the glutamatergic synapses employing the same mouse model is presented. In order to ascertain whether the isolation procedure causes any significant changes in the ultrastructural organization (and possibly synaptic macromolecular organization) the authors compare their findings using both of these approaches. The quantitation of the synaptic cleft height reveals an unexpected variability, while the STA analysis of the ionotropic receptors provides insights into their distribution with respect to the synaptic cleft.

The main novelty of this study lies in the continuous claims by the authors that the sample preservation methods developed here are superior to any others previously used. This leads them as well to systematically downplay or directly ignore a substantial body of previous cryo-ET studies of synaptic structure. Without comparisons with the cryo-ET literature, it is very hard to judge the impact of this work in the field. Furthermore, the data does not show any better preservation in the so-called "ultra-fresh" preparation than in the literature, perhaps to the contrary as synapses with strangely elongated vesicles are often seen. Such synapses have been regularly discarded for further analysis in previous synaptosome studies (e.g. Martinez-Sanchez 2021). Whilst the targeting approach using a fluorescent PSD95 marker is novel and seems sufficiently precise, the authors use a somewhat outdated approach (cryo-sectioning) to generate in-tissue tomograms of poor quality. To what extent such tomograms can be interpreted in molecular terms is highly questionable. The authors also don't discuss the physiological influence of 20% dextran used for high-pressure freezing of these "very native" specimens.

Lastly, a large part of the paper is devoted to image analysis of the PSD which is not convincing (including a somewhat forced comparison with the fixed and heavy-metal staining room temperature approach). Despite being a technically challenging study, the results fall short of expectations.

Reviewer #1 (Public review):

Summary:

This paper is a relevant overview of the currently published literature on low-intensity focused ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems nevertheless relevant to summarize the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data-free permutation of the parameter space.

The meta-analysis concludes that there are, at best, weak trends toward specific parameters predicting the direction of the stimulation effects. The data have been incorporated into an open database that will ideally continue to be populated by the community and thereby become a helpful resource as the field moves forward.

Strengths:

The current state of human TUS is concisely and well summarized. The methods of the meta-analysis are appropriate. The database is a valuable resource.

Suggestions:<br /> - The paper remains lengthy and somewhat unfocused, to the detriment of readability. One can understand that the authors wish to include as much information as possible, but this reviewer is sceptical that this will aid the use of the databank, or help broaden the readership. For one, there is a good chunk of repetition throughout. The intro is also somewhat oscillating between TMS, tDCS and TUS. While the former two help contextualizing the issue, it doesn't seem necessary. In the section on clinical applications of TUs and possible outcomes of TUS, there's an imbalance of the content across examples. That's in part because of the difference in knowledge base but some sections could probably be shortened, eg stroke. In any case, the authors may want to consider whether it is worth making some additional effort in pruning the paper

- The terms or concept of enhancement and suppression warrant a clearer definition and usage. In most cases, the authors refer to E/S of neural activity. Perhaps using terms such as "neural enhancement" etc helps distinguish these from eg behavioural or clinical effects. Crucially, how one maps onto the other is not clear. But in any case, a clear statement that the changes outlined on lines 277ff do not

- Re tb-TUS (lines 382ff), it is worth acknowledging here that independent replication is very limited (eg Bao et al 2024; Fong et al bioRxiv 2024) and seems to indicate rather different effects

- The comparison with TPS is troublesome. For one, that original study was incredibly poorly controlled and designed. Cherry-picking individual (badly conducted) proof-of-principle studies doesn't seem a great way to go about as one can find a match for any desired use or outcome.

Moreover, other than the concept of "pulsed" stimulation, it is not clear why that original study would motivate the use of TUS in the way the authors propose; both types of stimulation act in very different ways (if TPS "acts" at all). But surely the cited TPS study does not "demonstrate the capability for TUS for pre-operative cognitive mapping". As an aside, why the authors feel the need to state the "potential for TPS... to enhance cognitive function" is unclear, but it is certainly a non-sequitur. This review feels quite strongly that simplistic analogies such as the one here are unnecessary and misleading, and don't reflect the thoughtful discussion of the rest of the paper. In the other clinical examples, the authors build their suggestions on other TUS studies, which seems more sensible.

Reviewer #1 (Public review):

Summary:

This manuscript presents a compelling and innovative approach that combines Track2p neuronal tracking with advanced analytical methods to investigate early postnatal brain development. The work provides a powerful framework for exploring complex developmental processes such as the emergence of sensory representations, cognitive functions, and activity-dependent circuit formation. By enabling the tracking of the same neurons over extended developmental periods, this methodology sets the stage for mechanistic insights that were previously inaccessible.

Strengths:

(1) Innovative Methodology:<br /> The integration of Track2p with longitudinal calcium imaging offers a unique capability to follow individual neurons across critical developmental windows.

(2) High Conceptual Impact:<br /> The manuscript outlines a clear path for using this approach to study foundational developmental questions, such as how early neuronal activity shapes later functional properties and network assembly.

(3) Future Experimental Potential:<br /> The authors convincingly argue for the feasibility of extending this tracking into adulthood and combining it with targeted manipulations, which could significantly advance our understanding of causality in developmental processes.

(4) Broad Applicability:<br /> The proposed framework can be adapted to a wide range of experimental designs and questions, making it a valuable resource for the field.

Weaknesses:

No major weaknesses were identified by this reviewer. The manuscript is conceptually strong and methodologically sound. Future studies will need to address potential technical limitations of long-term tracking, but this does not detract from the current work's significance and clarity of vision.

Reviewer #1 (Public review):

Summary:

In this manuscript, Mack and colleagues investigate the role of posttranslational modifications, including lysine acetylation and ubiquitination, in methyltransferase activity of SETD2 and show that this enzyme functions as a tumor suppressor in a KRASG12C-driven lung adenocarcinoma. In contrast to H3K36me2-specific oncogenic methyltransferases, the deletion of SETD2, which is capable of H3K36 trimethylation, increases lethality in a KRASG12C-driven lung adenocarcinoma mouse tumor model. In vitro, the authors demonstrate that polyacetylation of histone H3, particularly of H3K27, H3K14, and H3K23, promotes the catalytic activity of SETD2, whereas ubiquitination of H2A and H2B has no effect.

Strengths:

Overall, this is a well-designed study that addresses an important biological question regarding the functioning of the essential chromatin component. The manuscript contains excellent quality data, and the conclusions are convincing and justified. This work will be of interest to many biochemists working in the field of chromatin biology and epigenetics.

Weaknesses:

A minor comment: labels should be added in the Figures and should be uniform across all Figures (some are distorted).

Reviewer #1 (Public review):

The small conductance calcium-activated potassium channel 2 (SK2) is an important drug target for treating neurological and cardiovascular diseases. However, structural information on this subtype of SK channels has been lacking, and it has been difficult to draw conclusions about activator and inhibitor binding and action in the absence of structural information.

Here the authors set out to (1) determine the structure of the transmembrane regions of a mammalian SK2 channel, (2) determine the binding site of apamin, a historically important SK2 inhibitor whose mode of action is unclear, and (3) use the structural information to generate a novel set of activators/inhibitors that selectively target SK2.

The authors largely achieved all the proposed goals, and they present their data clearly.

Unable to solve the structure of the human SK2 due to excessive heterogeneity in its cytoplasmic regions, the authors create a chimeric construct using SK4, whose structure was previously solved, and use it for structural studies. The data reveal a unique extracellular structure formed by the S2-S3 loop, which appears to directly interact with the selectivity filter and modulate its conductivity. Structures of SK2 in the absence and presence of the activating Ca2+ ions both possess non-K+-selective/conductive selectivity filters, where only sites 3 and 4 are preserved. The S6 gates are captured in closed and open states, respectively. Apamine binds to the S2-S3 loop, and unexpectedly, induces a K+ selective/conductive conformation of the selectivity filter while closing the S6 gate.

Through high-throughput screening of small compound libraries and compound optimization, the group identified a reasonably selective inhibitor and a related compound that acts as an activator. The characterization shows that these compounds bind in a novel binding site. Interestingly, the inhibitor, despite binding in a site different from that of apamine, also induces a K+ selective/conductive conformation of the selectivity filter while the activator induces a non-K+ selective/conductive conformation and an open S6 gate.

The data suggest that the selectivity filter and the S6 gate are rarely open at the same time, and the authors hypothesize that this might be the underlying reason for the small conductance of SK2. The data will be valuable for understanding the mechanism of SK2 channel (and other SK subtypes).

Overall, the data is of good quality and supports the claims made by the authors. However, a deeper analysis of the cryo-EM data sets might yield some important insights, i.e., about the relationship between the conformation of the selectivity filter and the opening of the S6 gate.

Some insight and discussion about the allosteric networks between the SF and the S6 gate would also be a valuable addition.

Reviewer #1 (Public Review):

Summary:

In this paper, Li and colleagues overcome solubility problems to determine the structure of FtsEX bound to EnvC from E. coli.

Strengths:

The structural work is well done, and the work is consistent with previous work on the structure of this complex from P. aerugionsa.

Weaknesses:

The model does not take into account all the information that the authors obtained, as well as known in vivo data.

The work lacks a clear comparison to the Pseudomonas structure highlighting new information that was obtained so that it is readily available to the reader.

The authors set out to obtain the structure of FtsEX-EnvC complex from E. coli. Previously, they were unable to do so but were able to determine the structure of the complex from P. aeruginosa. Here they persisted in attacking the E. coli complex since more is known about its involvement in cell division and there is a wealth of mutants in E. coli. The structural work is well done and recapitulates the results this lab obtained with this complex from P. aeruginosa. It would be helpful to compare more directly the results obtained here with the E. coli complex with the previously reported P. aeruginosa complex - are they largely the same or has some insight been obtained from the work that was not present in the previous complex from P. aeruginosa. This is particularly the case in discussing the symmetrical FtsX dimer binding to the asymmetrical EnvC, since this is emphasized in the paper. However, Figures 3C & D of this paper appear similar to Figures 2D & E of the P. aeruginosa structure. Presumably, the additional information obtained and presented in Figure 4 is due to the higher resolution, but this needs to be highlighted and discussed to make it clear to a general audience.

The main issue is the model (Figure 6). In the model ATP is shown to bind to FtsEX before EnvC, however, in Figure 1c, it is shown that ADP is sufficient to promote binding of FtsEX to EnvC.

The work here is all done in vitro, however, information from in vivo needs to be considered. In vivo results reveal that the ATP-binding mutant FtsE(D162N)X promotes the recruitment of EnvC (Proc Natl Acad Sci U S A 2011 108:E1052-60). Thus, even FtsEX in vivo can bind EnvC without ATP (not sure if this mutant can bind ADP).

Perhaps the FtsE protein from E. coli has to have bound nucleotides to maintain its 3D structure.

Comments after revision:

The most interesting aspect of this complex is that it has yet to be determined the order of events in the ATPase cycle as the authors acknowledge. Although the authors have responded quite well to the comments, I am still worried about the significance of the in vitro results compared to the in vivo results reported by others. In vivo ATP binding does not appear required for complex formation (of course it is possible that ADP is responsible in vivo). Have the authors tried to solve the complex with ADP since they suggested that it is sufficient to hold the complex together). If possible, it would confirm the role of ATP binding by comparing the structures. Also, it is not clear if ADP binds to any of the mutants made by the Bernhardt lab (D162N, K41M). If they do not bind ADP then FtsEX without nucleotide is able to bind EnvC as the authors indicate is the case in Pseudomonas. It is also unclear the significance of the ATPase activity of FtsEX in vitro with or without EnvC. Could the activity be some basal activity that is not relevant to the in vivo situation. If EnvC caused FtsEX to hydrolyze ATP it would be a futile cycle as FtsEX and EnvC are localized to the septum long before they are involved septal hydrolysis.

Reviewer #1 (Public review):

Summary:

Melin et al. developed a quantitative assay to measure the fate of paternal mitochondria after fertilization. They combine this assay with C. elegans genetics to show that multiple genes contribute to paternal mitochondrial elimination. However, despite their claims, they unconvincingly place these genes into distinct pathways and fail to determine whether additional unknown genes are involved in the process.

Strengths:

Melin et al. develop a new assay to quantify the fate of paternal mitochondria during embryonic development in C. elegans. They use complex C. elegans genetics to disrupt 5 different genes and nicely measure their contributions to paternal mitochondrial elimination. In an attempt to place these genes into pathways, the authors interrupt genes in various combinations and measure paternal mitochondrial persistence. The authors discovered that disrupting 4 of the genes known to contribute to paternal mitochondrial elimination still resulted in paternal mitochondrial elimination, suggesting that more genes also contribute to this process. Finally, the authors discovered that pink-1, which had previously been discounted, indeed contributes to paternal mitochondrial elimination when the major pathway involving allo-1 is also disrupted.

Weaknesses:

In the introduction, the authors describe the importance of studying the maternal inheritance of mitochondrial DNA. However, the authors mostly study the inheritance of paternally-derived mitochondrial proteins (HSP6::GFP). While the authors do use a PCR approach to measure paternal mitochondrial DNA, their results are not as quantitative and thorough (applied to multiple mutant combinations) as their microscopy assay. Using their microscopy assay, the authors did not combine mutants for all 5 genes. Therefore, they cannot support or discount the possibility that undiscovered paternal mitochondrial elimination mechanisms exist. The author's genetic epistasis experiments are incomplete and occasionally improperly interpreted (as described below). Finally, the authors were unable to achieve paternal mitochondrial transmission to the F2 generation (which admittedly has not been achieved in any experimental system).

Reviewer #1 (Public review):

Summary:

This study provides new insight into the non-canonicial voltage-gating mechanism of BK channels through prolonged (10 us) MD simulations of the Slo1 transmembrane domain conformation and K+ conduction in response to high imposed voltages (300, 750 mV). The results support previous conclusions based on functional and structural data and MD simulations that the voltage-sensor domain (VSD) of Slo1 undergoes limited conformational changes compared to Kv channels, and predicts gating charge movement comparable in magnitude to experimental results. The gating charge calculations further indicate that R213 and R210 in S4 are the main contributors owing to their large side chain movements and the presence of a locally focused electric field, consistent with recent experimental and MD simulation results by Carrasquel-Ursulaez et al.,2022. Most interestingly, changes in pore conformation and K+ conduction driven by VSD activation are resolved, providing information regarding changes in VSD/pore interaction through S4/S5/S6 segments proposed to underly electromechanical coupling.

Strengths:

Include that the prolonged timescale and high voltage of the simulation allow apparent equilibration in the voltage-sensor domain (VSD) conformational changes and at least partial opening of the pore. The study extends the results of previous MD simulations of VSD activation by providing quantitative estimates of gating charge movement, showing how the electric field distribution across the VSD is altered in resting and activated states, and testing the hypothesis that R213 and R210 are the primary gating charges by steered MD simulations. The ability to estimate gating charge contributions of individual residues in the WT channel is useful as a comparison to experimental studies based on mutagenesis which have yielded conflicting results that could reflect perturbations in structure. Use of dynamic community analysis to identify coupling pathways and information flow for VSD-pore (electromechanical) coupling as well as analysis of state-dependent S4/S5/S6 interactions that could mediate coupling provide useful predictions extending beyond what has been experimentally tested.

Weaknesses:

Weaknesses include that a truncated channel (lacking the C-terminal gating ring) was used for simulations, which is known to have reduced single channel conductance and electromechanical coupling compared to the full-length channel. In addition, as VSD activation in BK channels is much faster than opening, the timescale of simulations was likely insufficient to achieve a fully open state as supported by differences in the degree of pore expansion in replicate simulations, which are also smaller than observed in Ca-bound open structures of the full-length channel. Taken together, these limitations suggest that inferences regarding coupling pathways and interactions in the fully open voltage-activated channel may be only partially supported and therefore incomplete. That said, adequate discussion regarding these limitations are provided together with dynamic community analysis based on the Ca-bound open structure. The latter supports the main conclusions based on simulations, while providing an indication of potential interaction differences between simulated and fully open conformations. Another limitation is that while the simulations convincingly demonstrate voltage-dependent channel opening as evidenced by pore expansion and conduction of K+ and water through the pore, single channel conductance is underestimated by at least an order of magnitude, as in previous studies of other K+ channels. These quantitative discrepancies suggest that MD simulations may not yet be sufficiently advanced to provide insight into mechanisms underlying the extraordinarily large conductance of BK channels.

Comments on revisions:

My previous questions and concerns have been adequately addressed.

Reviewer #1 (Public review):

Summary:

This study provides comprehensive instructions for using the chromatophore tracking software, Chromas, to track and analyse the dynamics of large numbers of cephalopod chromatophores across various spatiotemporal scales. This software addresses a long-standing challenge faced by many researchers who study these soft-bodied creatures, known for their remarkable ability to change colour rapidly. The updated software features a user-friendly interface that can be applied to a wide range of applications, making it an essential tool for biologists focused on animal dynamic signalling. It will also be of interest to professionals in the fields of computer vision and image analysis.

Strengths:

This work provides detailed instructions for this tool kit along with examples for potential users to try. The Gitlab inventory hosts the software package, installation documentation, and tutorials, further helping potential users with a less steep learning curve.

Weaknesses:

The evidence supporting the authors' claims is solid, particularly demonstrated through the use of cuttlefish and squid. However, it may not be applicable to all coleoid cephalopods yet, such as octopuses, which have an incredibly versatile ability to change their body forms.

Comments on revisions:

I am pleased to see the more detailed version of this useful tool along with tutorials designed for diverse users who are interested in animal dynamic colouration. This study provides detailed instructions for using the chromatophore tracking software Chromas to track and analyse the dynamics of large numbers of cephalopod chromatophores across various spatiotemporal scales. The software features a user-friendly interface that is highly compelling and can be applied to a wide range of applications.

Reviewer #2 (Public review):

Summary:

The authors show that a combination of arginine methyltransferase inhibitors synergize with PARP inhibitors to kill ovarian and triple negative cancer cell lines in vitro and in vivo using preclinical mouse models.

Strengths and weaknesses

The experiments are well-performed, convincing and have the appropriate controls (using inhibitors and genetic deletions) and use statistics.

They identify the DNA damage protein ERCC1 to be reduced in expression with PRMT inhibitors. As ERCC1 is known to be synthetic lethal with PARPi, this provides a mechanism for the synergy. They use cell lines only for their study in 2D as well as xenograph models.

Joint Public Review:

Summary:

How mechanical forces transmitted by blood flow contribute to cardiac development remains incompletely understood. Using the unique advantages of the zebrafish model, Chen et al make the fundamental discovery that endocardial expression of the transcriptional repressor, Id2b, is maintained in endocardial cells by blood flow. Id1b zebrafish mutants fail to form the valve in the atrioventricular canal (AVC) and show reduced myocardial contractility that they suggest is due to impaired calcium transients. Id2b mutants are largely viable during the first 6 months of life until ~20% display cardiomyopathy characterized by visible edema, structural abnormalities, retrograde blood flow, and reduced systolic function and calcium handling. Mechanistically, the authors suggest that flow-mediated expression of Id2b leads to neuregulin 1 (nrg1) upregulation by physically interacting with and sequestering the Tcf3b transcriptional repressor from conserved tcf3b binding sites upstream of nrg1. Overall, this study advances our understanding of flow-mediated endocardial-myocardial crosstalk during heart development.

Strengths:

The strengths of the study are the significance of the biological question being addressed, use of the zebrafish model, data quality, and use of genetic tools. The text is generally well-written and easy to understand.

Weaknesses:

The main weakness that remains is the lack of rigor surrounding the molecular mechanism where the authors suggest that blood flow induces endocardial expression of Id2b, which binds to Tcf3b and sequesters it from binding the Nrg1 promoter to repress transcription. Although good faith efforts were made to bolster their model, the physical interaction between Id2b and Tcf3b is limited to overexpression of tagged proteins in HEK293 cells. Moreover, no mutagenesis was performed on the tcf3b binding sites identified in the nrg1 promoter to learn their importance in vivo.

Reviewer #1 (Public review):

Summary:

This study uses a cell-based computational model to simulate and study T cell development in the thymus. They initially applied this model to assess the effect of the thymic epithelial cells (TECs) network on thymocyte proliferation and demonstrated that increasing TEC size, density, or protrusions increased the number of thymocytes. They postulated and confirmed that this was due to changes in IL7 signalling and then expanded this work to encompass various environmental and cell-based parameters, including Notch signalling, cell cycle duration, and cell motility. Critical outcomes from the computational model were tested in vivo using medaka fish, such as the role of IL-7 signalling and minimal effect of Notch signalling.

Strengths:

The strength of the paper is the use of computational modelling to obtain unique insights into the niche parameters that control T cell development, such as the role of TEC architecture, while anchoring those findings with in vivo experiments. I can't comment on the model itself, as I am not an expert in modelling, however, the conclusions of the paper seem to be well-supported by the model.

Reviewer #1 (Public review):

Petrovic et al. investigate CCR5 endocytosis via arrestin2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data are particularly compelling, clearly demonstrating chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants.

To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization.

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays.

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fits-all model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.

Reviewer #1 (Public review):

Summary:

The study shows, perhaps surprisingly, that human fecal homogenates enhance the invasiveness of Salmonella typhimurium into cells of a swine colonic explant. This effect is only seen with chemotactic cells that express the chemoreceptor Tsr. However, two molecules sensed by Tsr that are present at significant concentrations in the fecal homogenates, the repellent indole and the attractant serine, do not, either by themselves or together at the concentrations in which they are present in the fecal homogenates, show this same effect. The authors then go on to study the conflicting repellent response to indole and attractant response to serine in a number of different in vitro assays.

Strengths:

The demonstration that homogenates of human feces enhance the invasiveness of chemotactic Salmonella Typhimurium in a colonic explant is unexpected and interesting. The authors then go on to document the conflicting responses to the repellent indole and the attractant serine, both sensed by the Tsr chemoreceptor, as a function of their relative concentration and the spatial distribution of gradients.

Weaknesses:

The authors do not identify what is the critical compound or combination of compounds in the fecal homogenate that gives the reported response of increased invasiveness. They show it is not indole alone, serine alone, or both in combination that have this effect, although both are sensed by Tsr and both are present in the fecal homogenates. Some of the responses to conflicting stimuli by indole and serine in the in vitro experiments yield interesting results, but they do little to explain the initial interesting observation that fecal homogenates enhance invasiveness.

Reviewer #1 (Public review):

Summary:

The authors developed a tool for simulating multiplexed single-cell RNA-seq and ATAC-seq data with various adjustable settings like ambient RNA/DNA rate and sequencing depth. They used the simulated data with different settings to evaluate the performance of many demultiplexing methods. They also proposed a new metric at single-cell level that correlates with the RNA/DNA contamination level.

Strengths:

The simulation tool has a straightforward design and provides adjustability in multiple parameters that have practical relevance, such as sequencing depth and ambient contamination rate. With the growing use of multiplexing in single-cell RNAseq and ATACseq experiments, the tools and results in this paper can guide the experimental design and tool selection for many researchers. The simulation tool also provides a platform for benchmarking newly developed demultiplexing tools.

Weaknesses:

The usefulness of the proposed new metric of "variant consistency" and how it can guide users in selecting demultiplexing methods seems a little unclear. It correlates with the level of ambient RNA/DNA contamination, which makes it look like a metric on data quality. However, it does depend on the exact demultiplexing method, yet it's not clear how it directly connects to the "accuracy" of each demultiplexing method, which is the most important property that users of these methods care about. Since the simulated data has ground truth of donor identities available, I would suggest using the simulated data to show whether "variant consistency" directly indicates the accuracy of each method, especially the accuracy within those "C2" reads.

I also think the tool and analyses presented in this paper need some further clarification and documentation on the details, such as how the cell-type gene and peak probabilities are determined in the simulation, and how doublets from different cell types are handled in the simulation and analysis. A few analyses and figures also need a more detailed description of the exact methods used.

Reviewer #1 (Public review):

Summary:

The authors had previously found that a brief social isolation could increase the activity of these neurons, and that manipulation of these neurons could alter social behavior in a social rank dependent fashion. This manuscript explored which of the outputs were responsible for this, identifying the central nucleus of the amygdala as the key output region. The authors identified some discrete behavior changes associated with these outputs, and found that during photostimulation of these outputs, neuronal activity appeared altered in 'social response' neurons. In the revised manuscript, the authors address the comments in a rigorous fashion.

Strengths:

Rigorous analysis of the anatomy. Careful examination of the hetergenous effects on cell activity due to stimulation, linking the physiology with the behavior via photostimulation during recording in vivo.

Weaknesses:

The authors have responded to all of my comments.

Reviewer #1 (Public review):

Summary:

This paper reports an intracranial SEEG study of speech coordination, where participants synchronize their speech output with a virtual partner that is designed to vary its synchronization behavior. This allows the authors to identify electrodes throughout the left hemisphere of the brain that have activity (both power and phase) that correlates with the degree of synchronization behavior. They find that high-frequency activity in secondary auditory cortex (superior temporal gyrus) is correlated to synchronization, in contrast to primary auditory regions. Furthermore, activity in inferior frontal gyrus shows a significant phase-amplitude coupling relationship that is interpreted as compensation for deviation from synchronized behavior with the virtual partner.

Strengths:

(1) The development of a virtual partner model trained for each individual participant, which can dynamically vary its synchronization to the participant's behavior in real time, is novel and exciting.

(2) Understanding real-time temporal coordination for behaviors like speech is a critical and understudied area.

(3) The use of SEEG provides the spatial and temporal resolution necessary to address the complex dynamics associated with the behavior.

(4) The paper provides some results that suggest a role for regions like IFG and STG in the dynamic temporal coordination of behavior both within an individual speaker and across speakers performing a coordination task.

Reviewer #1 (Public review):

Summary:

The authors developed a novel theoretical/computational procedure to count bacterial populations without introducing artificial randomness effects due to dilution. Surprisingly, this very important aspect of studies of bacterial systems has been overlooked. The proposed method provides a simple and transparent approach to eliminate the randomness of bacterial accounting procedures, allowing now to fully concentrate on the intrinsic effects of the studied systems.

Strengths:

A very simple and clear procedure is introduced and explained in full detail. This elegant approach finds an excellent compromise between mathematical rigor and computational efficiency, which is important for practical applications. The provided examples are convincing beyond a doubt, clearly indicating the potential strong impact of the proposed framework. Various complications and possible issues are also discussed and analyzed. This seems to be a very powerful novel method that should significantly advance the analysis of complex biological systems.

Weaknesses:

The only minor weakness that I found is the assumption of independence of bacterial species, which is expressed as the well-stirred approximation. One could imagine that bacterial species might cooperate, leading to non-uniform distributions that are real. How to distinguish such situations?

I believe that this method can be extended to determine if this is the case or not before the application. For example, if the bacteria species are independent of each other and one can use the binomial distributions, then the Fano factor would be proportional to the overall relative fraction of bacterial species. Maybe a simple test can be added to test it before the application of REPOP. However, I believe that this is a minor issue.

Reviewer #1 (Public review):

The authors conducted an fMRI study to investigate the neural effects of sustaining attention to areas of different sizes. Participants were instructed to attend to alphanumeric characters arranged in a circular array. The size of attention field was manipulated in four levels, ranging from small (18 deg) to large (162 deg). They used a model-based method to visualize attentional modulation in early visual cortex V1 to V3, and found spatially congruent modulations of the BOLD response, i.e., as the attended area increased in size, the neural modulation also increased in size in the visual cortex. They suggest that this result is a neural manifestation of the zoom-lens model of attention and that the model-based method can effectively reconstruct the neural modulation in the cortical space.

The study is well-designed with sophisticated and comprehensive data analysis. The results are robust and show strong support for a well-known model of spatial attention, the zoom-lens model. Overall, I find the results interesting and useful for the field of visual attention research.

Comments on revisions:

The authors have addressed my previous comments satisfactorily. I would encourage the authors to make data and code publicly available, which appears to be the custom in this era.

Reviewer #1 (Public review):

Henshall et al. delete the highly abundant merozoite surface protein PfMSP2 from two Plasmodium falciparum laboratory lines (3D7 and Dd2) using CRISPR-Cas9. Parasites lacking MSP2 replicate and invade red cells normally, opposing the experimental history that suggests MSP2 is essential. Unexpectedly, the knock-outs become more susceptible to several inhibitory antibodies - most strikingly those that target the apical antigen AMA1-while antibodies to other surface or secreted proteins are largely unaffected. Recombinant MSP2 added in vitro can dampen AMA1-antibody binding, supporting a "conformational masking" model. The reported data suggest that MSP2 helps shield key invasion ligands from host antibodies and may itself be a double-edged vaccine target.

Reviewer #3 (Public review):

The central issue for evaluating the overfilling hypothesis is the identity of the mechanism that causes the very potent (>80% when inter pulse is 20 ms), but very quickly reverting (< 50 ms) paired pulse depression (Fig 1G, I). To summarize: the logic for overfilling at local cortical L2/3 synapses depends critically on the premise that probability of release (pv) for docked and fully primed vesicles is already close to 100%. If so, the reasoning goes, the only way to account for the potent short-term enhancement seen when stimulation is extended beyond 2 pulses would be by concluding that the readily releasable pool overfills. However, the conclusion that pv is close to 100% depends on the premise that the quickly reverting depression is caused by exocytosis dependent depletion of release sites, and the evidence for this is not strong in my opinion. Caution is especially reasonable given that similarly quickly reverting depression at Schaffer collateral synapses, which are morphologically similar, was previously shown to NOT depend on exocytosis (Dobrunz and Stevens 1997). Note that the authors of the 1997 study speculated that Ca2+-channel inactivation might be the cause, but did not rule out a wide variety of other types of mechanisms that have been discovered since, including the transient vesicle undocking/re-docking (and subsequent re-priming) reported by Kusick et al (2020), which seems to have the correct timing.

In an earlier round of review, I suggested raising extracellular Ca2+, to see if this would increase synaptic strength. This is a strong test of the authors' model because there is essentially no room for an increase in synaptic strength. The authors have now done experiments along these lines, but the result is not clear cut. On one hand, the new results suggest an increase in synaptic strength that is not compatible with the authors' model; technically the increase does not reach statistical significance, but, likely, this is only because the data set is small and the variation between experiments is large. Moreover, a more granular analysis of the individual experiments seems to raise more serious problems, even supporting the depletion-independent counter hypothesis to some extent. On the other hand, the increase in synaptic strength that is seen in the newly added experiments does seem to be less at local L2/3 cortical synapses compared to other types of synapses, measured by other groups, which goes in the general direction of supporting the critical premise that pv is unusually high at L2/3 cortical synapses. Overall, I am left wishing that the new data set were larger, and that reversal experiments had been included as explained in the specific points below.

Specific Points:

(1) One of the standard methods for distinguishing between depletion-dependent and depletion-independent depression mechanisms is by analyzing failures during paired pulses of minimal stimulation. The current study includes experiments along these lines showing that pv would have to be extremely close to 1 when Ca2+ is 1.25 mM to preserve the authors' model (Section "High double failure rate ..."). Lower values for pv are not compatible with their model because the k1 parameter already had to be pushed a bit beyond boundaries established by other types of experiments. The authors now report a mean increase in synaptic strength of 23% after raising Ca to 2.5 mM. The mean increase is not quite statistically significant, but this is likely because of the small sample size. I extracted a 95% confidence interval of [-4%, +60%] from their numbers, with a 92% probability that the mean value of the increase in the full population is > 5%. I used the 5% value as the greatest increase that the model could bear because 5% implies pv < 0.9 using the equation from Dodge and Rahamimoff referenced in the rebuttal. My conclusion from this is that the mean result, rather than supporting the model, actually undermines it to some extent. It would have likely taken 1 or 2 more experiments to get above the 95% confidence threshold for statistical significance, but this is ultimately an arbitrary cut off.

(2) The variation between experiments seems to be even more problematic, at least as currently reported. The plot in Figure 3-figure supplement 3 (left) suggests that the variation reflects true variation between synapses, not measurement error. And yet, synaptic strength increased almost 2-fold in 2 of the 8 experiments, which back extrapolates to pv < 0.2. If all of the depression is caused by depletion as assumed, these individuals would exhibit paired pulse facilitation, not depression. And yet, from what I can tell, the individuals depressed, possibly as much as the synapses with low sensitivity to Ca2+, arguing against the critical premise that depression equals depletion, and even arguing - to some extent - for the counter hypothesis that a component of the depression is caused by a mechanism that is independent of depletion. I would strongly recommend adding an additional plot that documents the relationship between the amount of increase in synaptic strength after increasing extracellular Ca2+ and the paired pulse ratio as this seems central.

(3) Decrease in PPR. The authors recognize that the decrease in the paired-pulse ratio after increasing Ca2+ seems problematic for the overfilling hypothesis by stating: "Although a reduction in PPR is often interpreted as an increase in pv, under conditions where pv is already high, it more likely reflects a slight increase in pocc or in the number of TS vesicles, consistent with the previous estimates (Lin et al., 2025)." I looked quickly, but did not immediately find an explanation in Lin et al 2025 involving an increase in pocc or number of TS vesicles, much less a reason to prefer this over the standard explanation that reduced PPR indicates an increase in pv. The authors should explain why the most straightforward interpretation is not the correct one in this particular case to avoid the appearance of cherry picking explanations to fit the hypothesis.

(4) The authors concede in the rebuttal that mean pv must be < 0.7, but I couldn't find any mention of this within the manuscript itself, nor any explanation for how the new estimate could be compatible with the value of > 0.99 in the section about failures.

(5) Although not the main point, comparisons to synapses in other brain regions reported in other studies might not be accurate without directly matching experiments. As it is, 2 of 8 synapses got weaker instead of stronger, hinting at possible rundown, but this cannot be assessed because reversibility was not evaluated. In addition, comparing axons with and without channel rhodopsins might be problematic because the channel rhodopsins might widen action potentials.

(6) Perhaps authors could double check with Schotten et al about whether PDBu does/does not decrease the latency between osmotic shock and transmitter release. This might be an interesting discrepancy, but my understanding is that Schotten et al didn't acquire information about latency because of how the experiments were designed.

(7) The authors state: "These data are difficult to reconcile with a model in which facilitation is mediated by Ca2+-dependent increases in pv." However, I believe that discarding the premise that depression is always caused by depletion would open up wide range of viable possibilities.

Reviewer #1 (Public review):

Summary:

Authors benchmarked five IBD detection methods (hmmIBD, isoRelate, hap-IBD, phasedIBD, and Refined IBD) in Plasmodium falciparum using simulated and empirical data. Plasmodium falciparum has a mutation rate similar to that of humans but a much higher recombination rate and lower SNP density. Thus, the authors evaluated how recombination rate and marker density affect IBD segment detection. Next, they performed parameter optimization for Plasmodium falciparum and benchmarked the robustness of downstream analyses (selection detection and Ne inference) using IBD segments detected by each method. They also tracked the computational efficiency of these methods. The authors' work is valuable for the tested species, and the analyses presented support their claim that users should be cautious when calling IBD in contexts of low SNP density and high recombination rate.

Strengths:

The study design is convincing and well-structured. The authors chose to use P. falciparum, which presents an interesting case due to its high recombination rate and a mutation rate similar to that of humans. The authors note that despite the widespread use of IBD for genomic surveillance, comprehensive evaluation of these methods in high-recombination, low-marker-density contexts has been lacking. Furthermore, they also examined the performance of IBD detection methods developed specifically for P. falciparum, and evaluated it with phased data which broadened the applicability of the work.

Weaknesses:

The authors thoughtfully addressed our prior concerns by 1) expanding the simulations; 2) updating figures and methods for clarity; and 3) more clearly framing the broader utility of their benchmarking effort. These updates strengthen the manuscript and make the relevance of their findings beyond Plasmodium falciparum more apparent.

More specifically:

The authors added three full replicates per simulation scenario and updated figures to reflect uncertainty at relevant levels, which addresses earlier concerns about reproducibility. The limited number of replicates is due to computational intensity. In the future, broader generalizability and deeper exploration of variance in benchmarking accuracy across parameter space would further strengthen the conclusions/generalizability. The author's also emphasized that, while the study is centered on Plasmodium falciparum, the benchmarking framework, not the parameters, are broadly applicable to other sexually recombining species. Lastly, they extensively updated multiple figures to include simulation models, results from simulation replicates, and improved the figures from the previous version of the manuscript.

Reviewer #1 (Public review):

Summary:

Gekko, Nomura et al., show that Drp1 elimination in zygotes using the Trim-Away technique leads to mitochondrial clustering and uneven mitochondrial partitioning during the first embryonic cleavage, resulting in embryonic arrest. They monitor organellar localization and partitioning using specific targeted fluorophores. They also describe the effects of mitochondrial clustering in spindle formation and the detrimental effect of uneven mitochondrial partitioning to daughter cells.

Strengths:

The authors have gathered solid evidence for the uneven segregation of mitochondria upon Drp1 depletion through different means: mitochondrial labelling, ATP labelling and mtDNA copy number assessment in each daughter cell. Authors have also characterised the defects in cleavage mitotic spindles upon Drp1 loss

Weaknesses:

This study convincingly describes the phenotype seen upon Drp1 loss. Further studies should be conducted to elucidate the mechanism by which Drp1 ensures even mitochondrial partitioning during the first embryonic cleavage.

Reviewer #1 (Public review):

Summary:

In this manuscript, Chua, Daugherty, and Smith analyze a new set of archaeal 20S proteasomes obtained by cryo-EM that illustrate how the occupancy of the HbYX binding pocket induces gate opening. They do so primarily through a V24Y mutation in the α-subunit. These results are supported by a limited set of mutations in K66 in the α subunit, bringing new emphasis to this unit.

Strengths:

The new structure's analysis is comprehensive, occupying the entire manuscript. As such, the scope of this manuscript is very narrow, but the strength of the data are solid, and they offer an interesting and important new piece to the gate-opening literature.

Weaknesses:

Extrapolating from the core HbYX activating motif shared by Archae and Eukaryotes to the specific operations of gate opening, which is more elaborate in eukaryotes, may prove challenging.

Reviewer #1 (Public review):

N'Guessan et al have improved the report of their study of expression QTL (eQTL) mapping in yeast using single cells. The authors make use of advances in single cell RNAseq (scRNAseq) in yeast to increase the efficiency with which this type of analysis can be undertaken. Building on prior research led by the senior author that entailed genotyping and fitness profiling of almost 100,000 cells derived from a cross between two yeast strains (BY and RM) they performed scRNAseq on a subset of ~5% (n = 4,489) individual cells. To address the sparsity of genotype data in the expression profiling they used a Hidden Markov Model (HMM) to infer genotypes and then identify the most likely known lineage genotype from the original dataset. To address the relationship between variance in fitness and gene expression the authors partition the variance to investigate the sources of variation. They then perform eQTL mapping and study the relationship between eQTL and fitness QTL identified in the earlier study.

This paper seeks to address the question of how quantitative trait variation and expression variation are related. scRNAseq represents an appealing approach to eQTL mapping as it is possible to simultaneously genotype individual cells and measure expression in the same cell. As eQTL mapping requires large sample sizes to identify statistical relationships, the use of scRNAseq is likely to dramatically increase the statistical power of such studies. However, there are several technical challenges associated with scRNAseq and the authors' study is focused on addressing those challenges. The authors have successfully demonstrated their stated goal of developing, and illustrating the benefit of, a one-pot scRNA-seq experiment and analysis for eQTL mapping.

Reviewer #1 (Public review):

Summary:

This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

Strengths/weaknesses:

The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of the neural symptons in ciliopathy patients.

Comments on revisions:

The authors addressed most of the points I raised in my original review. However, I am not convinced by the figures the authors present on Shh protein expression. The "bright tiny dots" of Shh protein in the cortex are not visible on the images in Figure 7. I wonder whether the authors could present higher magnification and/or black and white images with increased contrast.

Reviewer #1 (Public review):

Summary:

In this study by Li et al., the authors re-investigated the role of cDC1 for atherosclerosis progression using the ApoE model. First, the authors confirmed the accumulation of cDC1 in atherosclerotic lesions in mice and humans. Then, in order to examine the functional relevance of this cell type, the authors developed a new mouse model to selectively target cDC1. Specifically, they inserted the Cre recombinase directly after the start codon of the endogenous XCR1 gene, thereby avoiding off-target activity. Following validation of this model, the authors crossed it with ApoE-deficient mice and found a striking reduction of aortic lesions (numbers and size) following a high-fat diet. The authors further characterized the impact of cDC1 depletion on lesional T cells and their activation state. Also, they provide in-depth transcriptomic analyses of lesional in comparison to splenic and nodal cDC1. These results imply cellular interactions between lesion T cells and cDC1. Finally, the authors show that the chemokine XCL1, which is produced by activated CD8 T cells (and NK cells), plays a key role in the interaction with XCR1-expressing cDC1 and particularly in the atherosclerotic disease progression.

Strengths:

The surprising results on XCL1 represent a very important gain in knowledge. The role of cDC1 is clarified with a new genetic mouse model.

Weaknesses:

My criticism is limited to the analysis of the scRNAseq data of the cDC1. I think it would be important to match these data with published data sets on cDC1. In particular, the data set by Sophie Janssen's group on splenic cDC1 might be helpful here (PMID: 37172103; https://www.single-cell.be/spleen_cDC_homeostatic_maturation/datasets/cdc1). It would be good to assign a cluster based on the categories used there (early/late, immature/mature, at least for splenic DC).

Reviewer #1 (Public review):

Summary:

This study presents a compelling strategy for ultrasound-mediated immunomodulation in HCC, supported by robust scRNA-Seq data. While the mechanistic depth and translational validation require further refinement, the work significantly advances the field of noninvasive cancer immunotherapy. Addressing the major concerns, particularly regarding calcium signaling specificity and STNDs@Ca²⁺ safety, will strengthen the manuscript's impact.

Strengths:

(1) Innovative Approach:<br /> The integration of noninvasive ultrasound with calcium-targeted nanotechnology (STNDs@Ca²⁺) represents a significant advancement in cancer immunotherapy. The dual mechanism - direct immunomodulation via FUS and calcium delivery via nanoparticles - is both novel and promising.

(2) Comprehensive Mechanistic Insights:<br /> The use of scRNA-seq and flow cytometry provides a detailed map of immune cell dynamics, highlighting key pathways (TNF, NFκB, MAPK) and cellular transitions (e.g., MDSC suppression, CD8⁺/NK cell activation).

(3) Robust Preclinical Validation:<br /> The study validates findings in two distinct HCC models (H22 and Hepal-6), demonstrating consistent tumor suppression (>70-90%) and prolonged survival, which strengthens translational relevance.

Weaknesses:

Major Issues:

(1) Mechanistic Specificity of Calcium Influx:<br /> While the study attributes immunomodulation to ultrasound-induced calcium influx, the exact mechanism (e.g., involvement of mechanosensitive channels like Piezo1 or TRP families) remains underdiscussed. The qRT-PCR data shows no changes in TRP channels, but the upregulation of Piezo1 warrants deeper exploration.

Suggestion: The authors should include experiments to inhibit Piezo1 or other calcium channels to confirm their role in FUS-mediated effects.

(2) STNDs@Ca²⁺ Biodistribution and Safety:<br /> Although biodistribution data show splenic accumulation, potential off-target effects (e.g., liver/lung uptake) and long-term toxicity are not fully addressed. The serum biochemical analysis (Table 2) lacks critical markers like inflammatory cytokines or immune cell counts.

Suggestion: The authors should provide longitudinal toxicity data (e.g., histopathology beyond 3 hours) and assess systemic immune activation/inflammation.

(3) Statistical and Technical Clarifications:<br /> The statistical methods for multi-group comparisons (e.g., ANOVA vs. t-test) are inconsistently described. For instance, Figure 1 labels significance without specifying correction for multiple comparisons.

Suggestion: the authors should clarify statistical methods in figure legends and the Methods section; apply Bonferroni or FDR correction where appropriate.

(4) Interpretation of scRNA-seq Data:<br /> The clustering of MDSCs using surface markers (Itgam/Ly6c2/Ly6g) overlaps with conventional myeloid populations (Supplementary Figure 16), raising questions about subset specificity.

Suggestion: The authors should validate MDSC identity using functional assays (e.g., T cell suppression) or additional markers (e.g., Arg1, iNOS).

Reviewer #1 (Public review):

Summary:

In this fMRI study, the authors wished to assess neural mechanisms supporting flexible "temporal construals". For this, human participants learned a story consisting of fifteen events. During fMRI, events were shown to them and they were instructed to consider the event from "an internal" or from "an external" perspective. The authors found opposite patterns of brain activity in the posterior parietal cortex and the anterior hippocampus for the internal and the external viewpoint. They conclude that allocentric sequences are stored in the hippocampus, whereas egocentric sequences are used in the parietal cortex. The claims align with previous fMRI work addressing this question.

Strengths:

The research topic is fascinating, and very few labs in the world are asking the question of how time is represented in the human brain. Working hypotheses have been recently formulated, and this work seems to want to tackle some of them.

Weaknesses:

The current writing is fuzzy both conceptually and experimentally. I cannot provide a sufficiently well-informed assessment of the quality of the experimental work because there is a paucity of details provided in the report. Any future revisions will likely improve transparency.

(1) Improving writing and presentation:

The abstract and the introduction make use of loaded terms such as "construals", "mental timeline", "panoramic views" in very metaphoric and unexplained ways. The authors do not provide a comprehensive and scholarly overview of these terms, which results in verbiage and keywords/name-dropping without a clear general framework being presented. Some of these terms are not metaphors. They do refer to computational concepts that the authors should didactically explain to their readership. This is all the more important that some statements in the Introduction are misattributed or factually incorrect; some statements lack attributions (uncited published work).

Once the theory, the question, and the working hypothesis are clarified, the authors should carefully explain the task.

(2) The experimental approach lacks sufficient details to be comprehensible to a general audience. In my opinion, the results are thus currently uninterpretable. I highlight only a couple of specific points (out of many). I recommend revision and clarification.

a) No explanation of the narrative is being provided. The authors report a distribution of durations with no clear description of the actual sequence of events. The authors should provide the text that was used, how they controlled for low-level and high-level linguistic confounds.

b) The authors state, "we randomly assigned 15 phrases to the events twice". It is impossible to comprehend what this means. Were these considered stimuli? Controls? IT is also not clear which event or stimulus is part of the "learning set" and whether these were indicated to be such to participants.

c) The left/right counterbalancing is not being clearly explained. The authors state that there is counterbalancing, but do not sufficiently explain what it means concretely in the experiment. If a weak correlation exists between sequential position and distance, it also means that the position and the distance have not been equated within. How do the authors control for these?

d) The authors used two tasks. In the "external perspective" one, the authors asked participants to report whether events were part of the same or a different part of the day. In the "internal perspective one", the authors asked participants to project themselves to the reference event and to determine whether the target event occurred before or after the projected viewpoint. The first task is a same/different recognition task. The second task is a temporal order task (e.g., Arzy et al. 2009). These two asks are radically different and do not require the same operationalization. The authors should minimally provide a comprehensive comparison of task requirements, their operationalization, and, more importantly, assess the behavioral biases inherent to each of these tasks that may confound brain activity observed with fMRI.

e) The authors systematically report interpreted results, not factual data. For instance, while not showing the results on behavioral outcomes, the authors directly interpret them as symbolic distance effects.

Crucially, the authors do not comment on the obvious differences in task difficulty in these two tasks, which demonstrates a substantial lack of control in the experimental design. The same/different task (task 1 called "external perspective") comes with known biases in psychophysics that are not present in the temporal order task (task 2 called " internal perspective"). The authors also did not discuss or try to match the performance level in these two tasks. Accordingly, the authors claim that participants had greater accuracy in the external (same/different) task than in the internal task, although no data are shown and provided to support this report. Further, the behavioral effect is trivialized by the report of a performance accuracy trade-off that further illustrates that there is a difference in the task requirements, preventing accurate comparison of the two tasks.

All fMRI contrasts are also confounded by this experimental shortcoming, seeing as they are all reported at the interaction level across a task. For instance, in Figure 4, the authors report a significant beta difference between internal and external tasks. It is impossible to disentangle whether this effect is simply due to task difference or to an actual processing of the duration that differs across tasks, or to the nature of the representation (the most difficult to tackle, and the one chosen by the authors).

Conclusion:

In conclusion, the current experimental work is confounded and lacks controls. Any behavioral or fMRI contrasts between the two proposed tasks can be parsimoniously accounted for by difficulty or attentional differences, not the claim of representational differences being argued for here.

Reviewer #1 (Public review):

Summary:

This is a rigorous data-driven modeling study, extending the authors' previous model of spinal locomotor central pattern generator (CPG) circuits developed for the mouse spinal cord and adapted here to the rat to explore potential circuit-level changes underlying altered speed-dependent gaits, due to asymmetric (lateral) thoracic spinal hemisection and symmetric midline contusion. The model reproduces key features of the rat speed-dependent gait-related experimental data before injury and after recovery from these two different thoracic spinal cord injuries and suggests injury-specific mechanisms of circuit reorganization underlying functional recovery. There is much interest in the mechanisms of locomotor behavior recovery after spinal cord injury, and data-driven behaviorally relevant circuit modeling is an important approach. This study represents an important advance in the authors' previous experimental and modeling work on locomotor circuitry and in the motor control field.

Strengths:

(1) The authors use an advanced computational model of spinal locomotor circuitry to investigate potential reorganization of neural connectivity underlying locomotor control following recovery from symmetrical midline thoracic contusion and asymmetrical (lateral) hemisection injuries, based on an extensive dataset for the rat model of spinal cord injury.

(2) The rat dataset used is from an in vivo experimental paradigm involving challenging animals to perform overground locomotion across the full range of speeds before and after the two distinct spinal cord injury models, enabling the authors to more completely reveal injury-specific deficits in speed-dependent interlimb coordination and locomotor gaits.

(3) The model reproduces the rat gait-related experimental data before injury and after recovery from these two different thoracic spinal cord injuries, which exhibit roughly comparable functional recovery, and suggests injury-specific, compensatory mechanisms of circuit reorganization underlying recovery.

(4) The model simulations suggest that recovery after lateral hemisection mechanistically involves partial functional restoration of descending drive and long propriospinal pathways. In contrast, recovery following midline contusion relies on reorganization of sublesional lumbar circuitry combined with altered descending control of cervical networks.

(5) These observations suggest that symmetrical (contusion) and asymmetrical (lateral hemisection) injuries induce distinct types of plasticity in different spinal cord regions, suggesting that injury symmetry partly dictates the location and type of neural plasticity supporting recovery.

(6) The authors suggest that therapeutic strategies may be more effective by targeting specific circuits according to injury symmetry.

Weaknesses:

The recovery mechanisms implemented in the model involve circuit connectivity/connection weights adjustment based on assumptions about the structures involved and compensatory responses to the injury. As the authors acknowledge, other factors affecting locomotor patterns and compensation, such as somatosensory afferent feedback, neurochemical modulator influences, and limb/body biomechanics, are not considered in the model.

Reviewer #1 (Public review):

In this manuscript, Saeb et al reported the mechanistic roles of the flexible stalk domain in sTREM2 function using molecular dynamics simulations. They have reported some interesting molecular bases explaining why sTREM2 shows protective effects during AD, such as partial extracellular stalk domain promoting binding preference and stabilities of sTREM2 with its ligand even in the presence of known AD-risk mutation, R47H. Furthermore, they found that the stalk domain itself acts as the site for ligand binding by providing an "expanded surface", known as 'Expanded Surface 2' together with the Ig-like domain. Also, they observed no difference in the binding free energy of phosphatidyl-serine with wild TREM2-Ig and mutant TREM2-Ig, which is a bit inconsistent with the previous report with experiment studies by Journal of Biological Chemistry 293, (2018), Alzheimer's and Dementia 17, 475-488 (2021), Cell 160, 1061-1071 (2015).

Reviewer #1 (Public review):

Summary:

Odor- and taste-sensing are mediated by two different systems, the olfactory and gustatory systems, and have different behavioral roles. In this study, Wei et al. challenge this dichotomy by showing that odors can activate gustatory receptor neurons (GRNs) in Drosophila to promote feeding responses, including the proboscis extension response (PER) that was previously thought to be driven only by taste. While previous studies suggested that odors can promote PER to appetitive tastants, Wei et al. go further to show that odors alone cause PER, this effect is mediated through sweet-sensing GRNs, and sugar receptors are required. The study also shows that odor detection by bitter-sensing GRNs suppresses PER. The authors' conclusions are supported by behavioral assays, calcium imaging, electrophysiological recordings, and genetic manipulations. The observation that both attractive and aversive odors promote PER leaves an open question as to why this effect is adaptive. Overall, the study sheds new light on chemosensation and multimodal integration by showing that odor and taste detection converge at the level of sensory neurons, a finding that is interesting and surprising while also being supported by another recent study (Dweck & Carlson, Sci Advances 2023).

Strengths:

(1) The main finding that odors alone can promote PER by activating sweet-sensing GRNs is interesting and novel.

(2) The study uses video tracking of the proboscis to quantify PER rather than manual scoring, which is typically used in the field. The tracking method is less subjective and provides a higher-resolution readout of the behavior.

(3) The study uses calcium imaging and electrophysiology to show that odors activate GRNs. These represent complementary techniques that measure activity at different parts of the GRN (axons versus dendrites, respectively) and strengthen the evidence for this conclusion.

(4) Genetic manipulations show that odor-evoked PER is primarily driven by sugar GRNs and sugar receptors rather than olfactory neurons. This is a major finding that distinguishes this work from previous studies of odor effects on PER and feeding (e.g., Reisenman & Scott, 2019; Shiraiwa, 2008) that assumed or demonstrated that odors were acting through olfactory neurons.

Weaknesses/Limitations:

(1) Many of the odor effects on behavior or neuronal responses were only observed at very high concentrations. Most effects seemed to require concentrations of at least 10^-2 (0.01 v/v), which is at the high end of the concentration range used in olfactory studies (e.g., Hallem et al., 2004), and most experiments in the paper used a far higher concentration of 0.5 v/v. It is unclear whether these are concentrations that would be naturally encountered by flies. In addition, it is difficult to compare the concentrations used for electrophysiology and behavior given that they are presented in solution versus volatile form.

(2) The timecourse of GRN activation by odors seems quite prolonged (and possibly delayed, depending on the exact timing of odor onset to the fly), and this timecourse is not directly compared with activation by tastes to determine whether it is a property of the calcium sensor or a real difference.

(3) While the overall effect of different conditions is tested using appropriate statistical methods, post-hoc tests are not always used to determine which specific groups are different from each other (e.g., which odors and concentrations elicit significant PER compared to air or mineral oil controls in Fig. 1; which odors show impaired responses without olfactory organs in Fig. 2A).

Discrepancies with previous studies:

These discrepancies are important to note but should not necessarily be considered "weaknesses" of the present study.

(1) It is not entirely clear why PER to odors alone has not been previously reported, especially as this study shows that it is a broad effect evoked by many different odors. Previous studies (Oh et al., 2021; Reisenman & Scott, 2019; Shiraiwa, 2008) tested the effect of odors on PER and only observed enhancement of PER to sugar rather than odor-evoked PER; some of these studies explicitly show no effect of odor alone or odor with low sugar concentration. In the Response to Reviewers, the authors propose that genetic background may explain discrepancies, but this is not discussed much in the paper itself. Differences in behavioral quantification (automated vs. manual scoring, quantification of PER duration versus probability) may also contribute.

(2) The calcium imaging data showing that sugar GRNs respond to a broad set of odors contrasts with results from Dweck & Carlson (Sci Adv, 2023) who recorded sugar neurons with electrophysiology and observed responses to organic acids, but not other odors. This discrepancy is mentioned in the Discussion but the underlying reason is not clear.

Reviewer #1 (Public review):

Summary:

The authors attempted to dissect the function of a long non-coding RNA, lnc-FANCI-2, in cervical cancer. They profiled lnc-FANCI-2 in different cell lines and tissues, generated knockout cell lines, and characterized the gene using multiple assays.

Strengths:

A large body of experimental data has been presented and can serve as a useful resource for the scientific community, including transcriptomics and proteomics datasets. The reported results also span different parts of the regulatory network and open up multiple avenues for future research.

Weaknesses:

The write-up is somewhat unfocused and lacks deep mechanistic insights in some places.

Comments on revisions:

The manuscript is much improved. I am satisfied with the authors' responses.

Reviewer #1 (Public review):

Summary

Detecting unexpected epistatic interactions between multiple mutations requires a robust null expectation-or neutral function-that predicts the combined effects of multiple mutations on phenotype based on the individual effects of single mutations. This study evaluated the relevance of the product neutrality function, where double-mutant fitness is represented as a multiplicative combination of single-mutant fitness in the absence of epistatic interactions. The authors used a recent large dataset on fitness, specifically yeast colony size, to analyze epistatic interactions.

The study confirmed that the product function outperformed other neutral functions in predicting double-mutant fitness, showing no bias between negative and positive epistatic interactions. Additionally, in the theoretical portion of the study, the authors employed a previously established theoretical model of bacterial cell growth to simulate growth rates of both single- and double-mutants under multiple parameters. The simulations similarly demonstrated that the product function was superior to other functions in predicting the fitness of hypothetical double-mutants. Based on these findings, the authors concluded that the product function is a robust tool for analyzing epistatic interactions in growth fitness and effectively reflects how growth rates depend on the combination of multiple biochemical pathways.

Strength

By leveraging a previously published large dataset of yeast colony sizes for single- and double-knockout mutants, this study validated the relevance of the product function, which has frequently been used in genetics to analyze epistatic interactions. The confirmation that the product function provides a more reliable prediction of double-mutant fitness compared to other neutral functions is valuable for researchers analyzing epistatic interactions, particularly those working with the same dataset.<br /> Notably, this dataset has been previously used in studies exploring epistatic interactions with the product neutrality function. This study's findings affirm the validity of using the product function, which could enhance confidence in the conclusions drawn by those earlier studies. Consequently, both researchers utilizing this dataset and readers of prior research will benefit from the confirmation provided by this study.

Weakness

This study contains several serious problems, primarily stemming from the following issues: ignoring the substantial differences in the mechanisms regulating cell growth between prokaryotes and eukaryotes and adopting an overly specific and unrealistic set of assumptions in the mutation model. Below, the details are discussed.

(1) Misapplication of prokaryotic growth models

The mechanistic origin of the multiplicative model observed in yeast colony fitness is explained using a bacterial cell growth model. However, there is no valid justification for linking these two systems. The bacterial growth model, the Scott-Hwa model, heavily rely on specific molecular mechanisms, such as ppGpp-mediated regulation, which adjusts ribosome expression and activity during translation. In particular, this mechanism is critical to ensure growth-dependency of the fraction of ribosome in proteome in the Scott-Hwa model [https://doi.org/10.1111/j.1462-2920.2010.02357.x; https://doi.org/10.1073/pnas.2201585119]. Yeast cells lack this regulatory mechanism, making it inappropriate to directly apply bacterial growth models to yeast.<br /> The Weiße model is based on a larger set of underlying equations and involves more parameters than the Scott-Hwa model. In the original paper by Weiße et al. (PNAS, 2015), however, the model parameters were fitted solely to experimental data from E. coli, and the model's applicability to yeast was never assessed. In summary, for neither the Scott-Hwa model nor the Weiße model has it been demonstrated that the entire model quantitatively fits experimental data from yeast. A positive correlation between growth rate and RNA/protein ratio, often observed in yeast, supports only a limited portion of either model, and does not constitute validation of the models as a whole.

(2) Overly specific assumptions in the theoretical model

The theoretical model assumes that two mutations affect only independent parameters of specific biochemical processes. However, this overly restrictive assumption weakens the model's validity in explaining the general occurrence of the multiplicative model in mutations. Furthermore, experimental evidence suggests limitations of this approach. For example, in most viable yeast deletion mutants with reduced growth rates, the expression of ribosomal proteins remained largely unchanged, contrary to the predictions of the Scott-Hwa model [https://doi.org/10.7554/eLife.28034]. This discrepancy highlights that the Scott-Hwa model and its derivatives cannot reliably explain mutants' growth rates based on current experimental evidence.

(3) Limited reliability of the mechanistic origin of the multiplicative model

The authors seem to regard growth-optimizing feedback as the mechanistic origin of the multiplicative model. However, the importance of growth-optimizing feedback in explaining product neutrality heavily depends on the very specific framework of the Scott-Hwa model. As I pointed out above, the Scott-Hwa model is a bacterial growth model that considers only a narrowly defined set of biochemical reactions. Using such a narrow model to explore the mechanistic origin of product neutrality observed on a genome-wide scale appears to be inappropriate. Arguments based on either the Scott-Hwa model or the Weiße model fail to account for the generality of product neutrality across diverse genetic perturbations. These models, in their current form, do not explain the broader patterns of product neutrality observed experimentally.

Reviewer #1 (Public review):

Summary:

This work provides a new potential tool to manipulate Tregs function for therapeutic use. It focuses on the role of PGAM in Tregs differentiation and function. The authors, interrogating publicly available transcriptomic and proteomic data of human regulatory T cells and CD4 T cells, state that Tregs express higher levels of PGAM (at both message and protein levels) compared to CD4 T cells. They then inhibit PGAM by using a known inhibitor ECGC and show that this inhibition affects Tregs differentiation. This result was also observed when they used antisense oligonucleotides (ASOs) to knockdown PGAM1.

PGAM1 catalyzes the conversion of 3PG to 2PG in the glycolysis cascade. However, the authors focused their attention on the additional role of 3PG: acting as starting material for the de novo synthesis of serine.

They hypothesized that PGAM1 regulates Tregs differentiation by regulating the levels of 3PG that are available for de novo synthesis of serine, which has a negative impact on Tregs differentiation. Indeed, they tested whether the effect on Tregs differentiation observed by reducing PGAM1 levels was reverted by inhibiting the enzyme that catalyzes the synthesis of serine from 3PG.

The authors continued by testing whether both synthesized and exogenous serine affect Tregs differentiation and continued with in vivo experiments to examine the effects of dietary serine restriction on Tregs function.

In order to understand the mechanism by which serine impacts Tregs function, the authors assessed whether this depends on the contribution of serine to one-carbon metabolism and to DNA methylation.

The authors therefore propose that extracellular serine and serine whose synthesis is regulated by PGAM1 induce methylation of genes Tregs associated, downregulating their expression and overall impacting Tregs differentiation and suppressive functions.

Strengths:

The strength of this paper is the number of approaches taken by the authors to verify their hypothesis. Indeed, by using both pharmacological and genetic tools in in vitro and in vivo systems they identified a potential new metabolic regulation of Tregs differentiation and function.

Reviewer #1 (Public review):

Summary:

The authors strived for an inventory of GPCRs and GPCR pathway component genes within the genomes of 23 choanoflagellates and other close relatives of metazoans.

Strengths:

The authors generated a solid phylogenetic overview of the GPCR superfamily in these species. Intriguingly, they discover novel GPCR families, novel assortments of domain combinations, and novel insights into the evolution of those groups within the Opisthokonta clade. A particular focus is laid on adhesion GPCRs, for which the authors discover many hitherto unknown subfamilies based on Hidden Markov Models of the 7TM domain sequences, which were also reflected by combinations of extracellular domains of the homologs. In addition, the authors provide bioinformatic evidence that aGPCRs of choanoflagellates also contain a GAIN domain, which is self-cleavable, thereby reflecting the most remarkable biochemical feat of aGPCRs.

Weaknesses:

The chosen classification scheme for aGPCRs may require reassessment and amendment by the authors in order to prevent confusion with previously issued classification attempts of this family.

Reviewer #1 (Public review):

Summary:

This is an interesting theoretical study examining the viability of Virtual Circular Genome (VCG) model, a recently proposed scenario of prebiotic replication in which a relatively long sequence is stored as a collection of its shorter subsequences (and their compliments). It was previously pointed out that VCG model is prone to so-called sequence scrambling which limits the overall length of such a genome. In the present paper, additional limitations are identified. Specifically, it is shown that VCG is well replicated when the oligomers are elongated by sufficiently short chains from "feedstock" pool. However, ligation of oligomers from VCG itself results in a high error rate. I believe the research is of high quality and well written. However, the presentation could be improved and the key messages could be clarified.

(1) It is not clear from the paper whether the observed error has the same nature as sequence scrambling

(2) The authors introduce two important lengths LS1 and LS2 only in the conclusions and do not explain enough which each of them is important. It would make sense to discuss this early in the manuscript.

(3) It is not entirely clear why specific length distribution for VCG oligomers has to be assumed rather than emerged from simulations.

(4) Furthermore, the problem has another important length, L0 that is never introduced or discussed: a minimal hybridization length with a lifetime longer than the ligation time. From the parameters given, it appears that L0 is sufficiently long (~10 bases). In other words, it appears that the study is done is a somewhat suboptimal regime: most hybridization events do not lead to a ligation. Am I right in this assessment? If that is the case, the authors might want to explore another regime, L0<br /> Strengths:

High-quality theoretical modeling of an important problem is implemented.

Reviewer #1 (Public review):

Summary:

The authors have assembled a cohort of 10 SiNET, 1 SiAdeno, and 1 lung MiNEN samples to explore the biology of neuroendocrine neoplasms. They employ single-cell RNA sequencing to profile 5 samples (siAdeno, SiNETs 1-3, MiNEN) and single-nuclei RNA sequencing to profile seven frozen samples (SiNET 4-10).

They identify two subtypes of siNETs, characterized by either epithelial or neuronal NE cells, through a series of DE analyses. They also report findings of higher proliferation in non-malignant cell types across both subtypes. Additionally, they identify a potential progenitor cell population in a single lung MiNEN samples.

In the revised study, they have addressed my points and I have no further comments.

Reviewer #2 (Public review):

Summary:

In this manuscript by Peto et al., the authors describe the impact of different antimicrobials on gut microbiota in a prospective observational study of 225 participants (healthy volunteers, inpatients and outpatients). Both cross-sectional data (all participants) and longitudinal data (subset of 79 haematopoietic cell transplant patients) were used. Using metagenomic sequencing, they estimated the impact of antibiotic exposure on gut microbiota composition and resistance genes. In their models, the authors aim to correct for potential confounders (e.g. demographics, non-antimicrobial exposures and physiological abnormalities), and for differences in the recency and total duration of antibiotic exposure. I consider these comprehensive models an important strength of this observational study. Yet, the underlying assumptions of such models may have impacted the study findings and residual confounding is likely. Other strengths include the presence of both cross-sectional and longitudinal exposure data and presence of both healthy volunteers and patients. Together, these observational findings expand on previous studies (both observational and RCTs) describing the impact of antimicrobials on gut microbiota.

Weaknesses:

(1) The main weaknesses result from the observational design. This hampers causal interpretation and makes correction for potential confounding necessary. While the authors have used comprehensive models to correct for potential confounders and for differences between participants in duration of antibiotic exposure and time between exposure and sample collection, I believe residual confounding is likely (which is mentioned as a limitation in the discussion).<br /> For their models, the authors found a disruption half-life of 6 days to be the best fit based on Shannon diversity. Yet, the disruption caused by antimicrobials may be longer than represented in this model - as highlighted in the discussion.

(2) Another consequence of the observational design of this study is the relatively small number of participants available for some comparisons (e.g. oral clindamycin was only used by 6 participants). Care should be taken when drawing any conclusions from such small numbers.

Comments on revisions:

The authors have adequately addressed all of my comments.

Reviewer #1 (Public review):

Summary:

The study by Cao et al. provides a compelling investigation into the role of mutational input in the rapid evolution of pesticide resistance, focusing on the two-spotted spider mite's response to the recent introduction of the acaricide cyetpyrafen. This well-documented introduction of the pesticide-and thus a clearly defined history of selection-offers a powerful framework for studying the temporal dynamics of rapid adaptation. The authors combine resistance phenotyping across multiple populations, extensive resequencing to track the frequency of resistance alleles, and genomic analyses of selection in both contemporary and historical samples. These approaches are further complemented by laboratory-based experimental evolution, which serves as a baseline for understanding the genetic architecture of resistance across mite populations in China. Their analyses identify two key resistance-associated genes, sdhB and sdhD, within which they detect 15 mutations in wild-collected samples. Protein modeling reveals that these mutations cluster around the pesticide's binding site, suggesting a direct functional role in resistance. The authors further examine signatures of selective sweeps and their distribution across populations to infer the mechanisms-such as de novo mutation or gene flow-driving the spread of resistance, a crucial consideration for predicting evolutionary responses to extreme selection pressure. Overall, this is a well-rounded, thoughtfully designed and well-written manuscript. It shows significant novelty, as it is relatively rare to integrate broad-scale evolutionary inference from natural populations with experimentally informed bioassays, however, follow up work will be needed to fully resolve haplotype structure and the functional effects of resistance mutations in the system.

Strengths:

One of the most compelling aspects of this study is its integration of genomic time-series data in natural populations with controlled experimental evolution. By coupling genome sequencing of resistant field populations with laboratory selection experiments, the authors tease apart the individual effects of resistance alleles along with regions of the genome where selection is expected to occur, and compare that to the observed frequency in the wild populations over space and time. Their temporal data clearly demonstrates the pace at which evolution can occur in response to extreme selection. This type of approach is a powerful roadmap for the rest of the field of rapid adaptation.

The study effectively links specific genetic changes to resistance phenotypes. The identification of sdhB and sdhD mutations as major drivers of cyetpyrafen resistance is well supported by allele frequency shifts in both field and experimental populations. The scope of their sampling clearly facilitated the remarkable number of observed mutations within these target genes, and the authors provide a careful discussion of the likelihood of these mutations from de novo or standing variation. Furthermore, the discovered cross-resistance that these mutations confer to other mitochondrial complex II inhibitors highlights the potential for broader resistance management and evolution.

Weaknesses:

(1) Pleiotropy without pesticide modes of action (cyflumetofen and cyetpyrafen) may also play a role in the rapid response to the focal pesticide in this study<br /> (2) Other aspects of the environment that might influence selection were not considered in the structure of resistance alleles (i.e. climate, elevation)<br /> (3) Very little data were used for haplotype reconstruction, only 8 SNPs, and this excluded all heterozygous alleles, which could dramatically influence the complexity of these inferred haplotype networks.<br /> (4) Single Mutations and Their Effects:<br /> - Allelic effects were not estimated in isogenic lines, so the effects presented also include heterogeneity from allelic interactions with the genomic background<br /> - The authors see populations that segregate for resistance mutations but that have no survival to pesticides. This suggests either not all of the resistance mutations studied here actually have functional effects or that dominance is playing a role in masking their effects in the heterozygous state.

Reviewer #1 (Public review):

In this manuscript, Azlan et al. identified a novel maternal factor called Sakura that is required for proper oogenesis in Drosophila. They showed that Sakura is specifically expressed in the female germline cells. Consistent with its expression pattern, Sakura functioned autonomously in germline cells to ensure proper oogenesis. In sakura KO flies, germline cells were lost during early oogenesis and often became tumorous before degenerating by apoptosis. In these tumorous germ cells, piRNA production was defective and many transposons were derepressed. Interestingly, Smad signaling, a critical signaling pathway for the GSC maintenance, was abolished in sakura KO germline stem cells, resulting in ectopic expression of Bam in whole germline cells in the tumorous germline. A recent study reported that Bam acts together with the deubiquitinase Otu to stabilize Cyc A. In the absence of sakura, Cyc A was upregulated in tumorous germline cells in the germarium. Furthermore, the authors showed that Sakura co-immunoprecipitated Otu in ovarian extracts. A series of in vitro assays suggested that the Otu (1-339 aa) and Sakura (1-49 aa) are sufficient for their direct interaction. Finally, the authors demonstrated that the loss of otu phenocopies the loss of sakura, supporting their idea that Sakura plays a role in germ cell maintenance and differentiation through interaction with Otu during oogenesis.

Latest comments:

The reviewer acknowledges the importance of sharing the observed defects in Sakura mutant ovaries and the possible physiological significance of the Sakura-Out interaction with the research community, as this information could lay the groundwork for future functional analysis research.

Reviewer #2 (Public review):

Summary:

The authors report the discovery of a population of gingival fibroblasts displaying the expression of cellular senescence markers P21 and P16 in human periodontitis samples and a murine ligature-induced periodontitis (LIP) model. They support this finding in the murine model through bulk RNA-sequencing and show that differentially expressed genes are significantly enriched in the SenMayo cellular senescence in aging dataset. They then show that Ligature-Induced Periodontitis (LIP) mice treated with the senomorphic drug metformin display overall diminished bone damage, reduced histomorphic alterations, and a reduction in P21 and P16 immunostaining signal. To explore the cell types expressing cellular senescence markers in periodontitis, the authors make use of a combination of bioinformatic analyses on publicly available scRNA-seq data, immunostainings on patient samples and their LIP model; as well as in vitro culture of healthy human gingival fibroblasts treated with LPS. They found that fibroblasts are a cell population expressing P16 in periodontitis which are also enriched for SenMayo genes, suggesting they have a senescent phenotype. They then point to a subgroup of fibroblasts expressing CD81+ with the highest enrichment for a SASP geneset in periodontitis. They also show that treatment of LIP mice and human LPS-treated gingival fibroblasts with metformin leads to a reduction of P21 and P16-positive cells, as well as the senescence-associated beta-galactosidase (SA-beta-gal) marker. Finally, they show evidence suggesting that CD81+ senescent fibroblasts are the source of C3 complement protein, which they stipulate signals through the C3AR1 receptor present in neutrophils in periodontitis. The authors observed that both CD81+ fibroblast and C3AR1+ neutrophil populations are expanded in periodontitis, that both populations appear to be in close contact, and that treatment with metformin reduced both C3 and the neutrophil marker MPO in their mouse LIP model.

After a round of revision, the authors have made significant improvements to their manuscript, such as improving the quality of the data/evidence and also included new data from experiments using a well-known senolytic and the senomorphic metformin, which all together provide a solid support to their main claims.

Strengths:

The study implements several different techniques and tools on human samples, mouse models, fibroblast cultures, and publicly available data to support their conclusions. In summary, they provide solid evidence showing that in the context of periodontitis, there is an expansion of cells expressing senescence markers P21, and P16, as well as members of the SASP, and that this includes CD81+ fibroblasts.

Weaknesses:

The fact that in this study the periodontitis samples belonged to patients with a significantly higher median age (all older than 50 years of age) and the healthy samples belonged to young adults (all younger than 35 years of age), raises the need for caution in interpretation due to a possible effect of aging in the accumulation of CD81+ senescent fibroblasts. However, the recruitment of similar age groups in this case is of course difficult due to the higher prevalence of periodontitis in older adults. In this regard it is important to note that the authors still support their findings using a mouse ligature model. Similar studies comparing healthy and periodontitic patients from similar age groups will be of great importance in the future.

Reviewer #1 (Public review):

Summary:

In this paper, Wang et al show that differentiated peridermal cells of the zebrafish epidermis extend cytoneme-like protrusions toward the less differentiated, intermediate layer below. They present evidence that expression of a dominant-negative cdc42, inhibits cytoneme formation and leads to elevated expression of a marker of undifferentiated keratinocytes, krtt1c19e, in the periderm layer. It is demonstrated that Delta-Notch signaling is involved in keratinocyte differentiation and that loss of cytonemes correlates with a loss of Notch signaling. Finally, changes in expression of the inflammatory cytokine IL-17 and its receptors is shown to affect cytoneme number and periderm structure in a manner similar to Notch and cdc42 perturbations.

Strengths:

Overall, the idea that differentiated cells signal to underlying undifferentiated cells via membrane protrusions in skin keratinocytes is interesting and novel, and it is clear that periderm cells send out thin membrane protrusions that contain a Notch ligand. Further, and perturbations that affect cytoneme number, Notch signaling and IL-17 expression clearly lead to changes in periderm structure and gene expression.

Weaknesses:

The mechanisms by which IL-17 affects cytoneme formation requires further investigation.

Reviewer #1 (Public review):

Summary:

This fundamental study presents a practical modification of the orthogonal hybridization chain reaction (HCR) technique, a promising yet underutilized method with broad potential for future applications across various fields. The authors advance this technique by integrating peptide ligation technology and nanobody-based antibody mimetics-cost-effective and scalable alternatives to conventional antibodies-into a DNA-immunoassay framework that merges oligonucleotide-based detection with immunoassay methodologies. They demonstrate this with compelling evidence that this approach facilitates a modified ELISA platform capable of simultaneously quantifying multiple target protein expression levels within a single protein mixture sample.

Strengths:

The hybridization chain reaction (HCR) technique was initially developed to enable the simultaneous detection of multiple mRNA expression levels within the same tissue. This method has since evolved into immuno-HCR, which extends its application to protein detection by utilizing antibodies. A key requirement of immuno-HCR is the coupling of oligonucleotides to antibodies, a process that can be challenging due to the inherent difficulties in expressing and purifying conventional antibodies.<br /> In this study, the authors present an innovative approach that circumvents these limitations by employing nanobody-based antibody mimetics, which recognize antibodies, instead of directly coupling oligonucleotides to conventional antibodies. This strategy facilitates oligonucleotide conjugation-designed to target the initiator hairpin oligonucleotide of HCR-through peptide ligation and click chemistry.

Weaknesses:

The sandwich-format technique presented in this study, which employs a nanobody that recognizes primary IgG antibodies, may have limited scalability compared to existing methods that directly couple oligonucleotides to primary antibodies. This limitation arises because the C-region types of primary antibodies are relatively restricted, meaning that the use of nanobody-based detection may constrain the number of target proteins that can be analyzed simultaneously. In contrast, the conventional approach of directly conjugating oligonucleotides to primary antibodies allows for a broader range of protein targets to be analyzed in parallel.

Additionally, in the context of HCR-based protein detection, the number of proteins that can be analyzed simultaneously is inherently constrained by fluorescence wavelength overlap in microscopy, which limits its multiplexing capability. By comparison, direct coupling oligonucleotides to primary antibodies can facilitate the simultaneous measurement of a significantly greater number of protein targets than the sandwich-based nanobody approach in the barcode-ELISA/NGS-based technique.

Comments on revisions:

The previous suggestions were well incorporated in the revised manuscript.

Reviewer #2 (Public review):

Summary:

This study explores how a simple sensorimotor circuit in the nematode C. elegans enables it to navigate salt gradients based on past experiences. Using computational simulations and previously described neural connections, the study demonstrates how a single neuron, ASER, can change its signaling behavior in response to different salt conditions, with which the worm is able to "remember" prior environments and adjust its navigation toward "preferred" salinity accordingly.

Strengths:

The key novelty and strength of this paper is the explicit demonstration of computational neurobehavioral modeling and evolutionary algorithms to elucidate the synaptic plasticity in a minimal neural circuit that is sufficient to replicate memory-based chemotaxis. In particular, with changes in ASER's glutamate release and sensitivity of downstream neurons, the ASER neuron adjusts its output to be either excitatory or inhibitory depending on ambient salt concentration, enabling the worm to navigate toward or away from salt gradients based on prior exposure to salt concentration.

Weaknesses:

While the model successfully replicates some behaviors observed in previous experiments, some key assumptions of the work still need to be verified by biological validation of further experiments.

Comments on revisions:

Thank you for the authors' response. The revision and their response have substantially addressed my concerns.

Reviewer #1 (Public review):

Summary:

In the present study, Chen et al. investigate the role of Endophilin A1 in regulating GABAergic synapse formation and function. To this end, the authors use constitutive or conditional knockout of Endophilin A1 (EEN1) to assess the consequences on GABAergic synapse composition and function, as well as the outcome for PTZ-induced seizure susceptibility. The authors show that EEN1 KO mice show a higher susceptibility to PTZ-induced seizures, accompanied by a reduction in the GABAergic synaptic scaffolding protein gephyrin as well as specific GABAAR subunits and eIPSCs. The authors then investigate the underlying mechanisms, demonstrating that Endophilin A1 binds directly to gephyrin and GABAAR subunits, and identifying the subdomains of Endophilin A1 that contribute to this effect. Overall, the authors state that their study places Endophilin A1 as a new regulator of GABAergic synapse function.

Strengths:

Overall, the topic of this manuscript is very timely, since there has been substantial recent interest in describing the mechanisms governing inhibitory synaptic transmission at GABAergic synapses. The study will therefore be of interest to a wide audience of neuroscientists studying synaptic transmission and its role in disease. The manuscript is well written and contains a substantial quantity of data. In the revised version of the manuscript, the authors have increased the number of samples analyzed and have significantly improved the statistical analysis, thereby substantially strengthening the conclusions of their study.

Comments on revised version:

The authors have addressed all of my concerns, and the manuscript has been substantially improved.

Reviewer #1 (Public review):

Summary:

The manuscript by Ross, Miscik, and others describes an intriguing series of observations made when investigating the requirement for podxl during hepatic development in zebrafish. Podxl morphants and CRISPants display a reduced number of hepatic stellate cells (HSCs), while mutants are either phenotypically wild type or display an increased number of HSCs.

The absence of observable phenotypes in genetic mutants could indeed be attributed to genetic compensation, as the authors postulate. However, in my opinion, the evidence provided in the manuscript at this point is insufficient to draw a firm conclusion. Furthermore, the opposite phenotype observed in the two deletion mutants is not readily explainable by genetic compensation and invokes additional mechanisms.

Major concerns:

(1) Considering discrepancies in phenotypes, the phenotypes observed in podxl morphants and CRISPants need to be more thoroughly validated. To generate morphants, authors use "well characterized and validated ATG Morpholino" (lines 373-374). However, published morphants, in addition to kidney malformations, display gross developmental defects including pericardial edema, yolk sack extension abnormalities, and body curvature at 2-3 dpf (reference 7 / PMID: 24224085). Were these gross developmental defects observed in the knockdown experiments performed in this paper? If yes, is it possible that the liver phenotype observed at 5 dpf is, to some extent, secondary to these preceding abnormalities? If not, why were they not observed? Did kidney malformations reproduce? On the CRISPant side, were these gross developmental defects also observed in sgRNA#1 and sgRNA#2 CRISPants? Considering that morphants and CRISPants show very similar effects on HSC development and assuming other phenotypes are specific as well, they would be expected to occur at similar frequencies. It would be helpful if full-size images of all relevant morphant and CRISPant embryos were displayed, as is done for tyr CRISPant in Figure S2. Finally, it is very important to thoroughly quantify the efficacy of podxl sgRNA#1 and sgRNA#2 in CRISPants. The HRMA data provided in Figure S1 is not quantitative in terms of the fraction of alleles with indels. Figure S3 indicates a very broad range of efficacies, averaging out at ~62% (line 100). Assuming random distribution of indels among cells and that even in-frame indels result in complete loss of function (possible for sgRNA#1 due to targeting the signal sequence), only ~38% (.62*.62) of all cells will be mutated bi-allelically. That does not seem sufficient to reliably induce loss-of-function phenotypes. My guess is that the capillary electrophoresis method used in Figure S3 underestimates the efficiency of mutagenesis, and that much higher mutagenesis rates would be observed if mutagenesis were assessed by amplicon sequencing (ideally NGS but Sanger followed by deconvolution analysis would suffice). This would strengthen the claim that CRISPant phenotypes are specific.

(2) In addition to confidence in morphant and CRISPant phenotypes, the authors' claim of genetic compensation rests on the observation that podxl (Ex1(p)_Ex7Δ) mutants are resistant to CRISPant effect when injected with sgRNA#1 (Figure 3L). Considering the issues raised in the paragraph above, this is insufficient. There is a very straightforward way to address both concerns, though. The described podxl(-194_Ex7Δ) and podxl(-319_ex1(p)Δ) deletions remove the binding site for the ATG morpholino. Therefore, deletion mutants should be refractive to the Morpholino (specificity assessment recommended in PMID: 29049395, see also PMID: 32958829). Furthermore, both deletion mutants should be refractive to sgRNA#1 CRISPant phenotypes, with the first being refractive to sgRNA#2 as well.

Reviewer #1 (Public review):

This study presents cryoEM-derived structures of the Trypanosome aquaporin AQP2, in complex with its natural ligand, glycerol, as well as two trypanocidal drugs, pentamidine and melarsoprol, which use AQP2 as an uptake route. The structures are high quality, and the density for the drug molecules is convincing, showing a binding site in the centre of the AQP2 pore.

The authors then continue to study this system using molecular dynamics simulations. Their simulations indicate that the drugs can pass through the pore and identify a weak binding site in the centre of the pore, which corresponds with that identified through cryoEM analysis. They also simulate the effect of drug resistance mutations, which suggests that the mutations reduce the affinity for drugs and therefore might reduce the likelihood that the drugs enter into the centre of the pore, reducing the likelihood that they progress through into the cell.

While the cryoEM and MD studies are well conducted, it is a shame that the drug transport hypothesis was not tested experimentally. For example, did they do cryoEM with AQP2 with drug resistance mutations and see if they could see the drugs in these maps? They might not bind, but another possibility is that the binding site shifts, as seen in Chen et al. Do they have an assay for measuring drug binding? I think that some experimental validation of the drug binding hypothesis would strengthen this paper. Without this, I would recommend the authors to soften the statement of their hypothesis (i.e, lines 65-68) as this has not been experimentally validated.

Reviewer #1 (Public review):

The manuscript by Chiu et al describes the modification of the Zwitch strategy to efficiently generate conditional knockouts of zebrafish betapix. They leverage this system to identify a surprising glia-exclusive function of betapix in mediating vascular integrity and angiogenesis. Betapix has been previously associated with vascular integrity and angiogenesis in zebrafish, and betapix function in glia has also been proposed. However, this study identifies glial betapix in vascular stability and angiogenesis for the first time.

The study derives its strength from the modified CRISPR-based Zwitch approach to identify the specific role of glial betapix (and not neuronal, mural, or endothelial). Using RNA-in situ hybridization and analysis of scRNA-Seq data, they also identify delayed maturation of neurons and glia and implicate a reduction in stathmin levels in the glial knockouts in mediating vascular homeostasis and angiogenesis. The study also implicates a betapix-zfhx3/4-vegfa axis in mediating cerebral angiogenesis.

There is both technical (the generation of conditional KOs) and knowledge-related (the exclusive role of glial betapix in vascular stability/angiogenesis) novelty in this work that is going to benefit the community significantly.<br /> While the text is well written, it often elides details of experiments and relies on implicit understanding on the part of the reader. Similarly, the figure legends are laconic and often fail to provide all the relevant details.

Specific comments:

(1) While the evidence from cKO's implicating glial betapix in vascular stability/angiogenesis is exciting, glia-specific rescue of betapix in the global KOs/mutants (like those performed for stathmin) would be necessary to make a water-tight case for glial betapix.

(2) Splice variants of betapix have been shown to have differential roles in haemorrhaging (Liu, 2007). What are the major glial isoforms, and are there specific splice variants in the glial that contribute to the phenotypes described?

(3) Liu et al, 2012 demonstrated reduced proliferation of endothelial cells in bbh fish and linked it to deficits in angiogenesis. Are there proliferation/survival defects in endothelial cells in the glial KOs?

Reviewer #1 (Public review):

Summary:

This study employs spatial transcriptomics to explore the molecular architecture of the adult mouse adrenal gland and the adjacent adipose tissue. The research aimed to identify zonation-specific genetic markers, elucidate cellular differentiation patterns, and investigate inter- and intra-zone communication within the adrenal gland. The findings support the centripetal differentiation model, highlighting the transition of cell populations across different cortical zones. The study also integrates ligand-receptor interaction analysis to uncover the adrenal gland's role in endocrine and neuroendocrine signaling, particularly in stress response. This high-resolution spatial transcriptomic map provides novel insights into adrenal gland biology and is a resource for further investigations.

Strengths:

The study, using the latest technologies and methods such as Visium CytAssist technology, UMAP & Seurat analysis, Gene Ontology (GO) & KEGG pathway enrichment analysis, Monocle3, and CellChat analysis, performed three-dimensional analysis, which has been challenging to achieve using the two-dimensional transcriptomics that have been commonly used up until now.

The unique gene expression patterns were demonstrated for each adrenal zone. Spatial transcriptomics confirmed unique gene expression patterns for each adrenal zone (ZG, ZF, ZX, medulla). The centripetal differentiation model shows the migration of the progenitor cells from the adrenal capsule towards the inner cortex. Key genetic markers were identified in each adrenal zone and adjacent adipose tissues. In addition, CellChat analysis identified major signaling pathways, including Wnt signaling, Hedgehog signaling, IGF2-IGF2R interactions, and Neuropeptide Y (NPY) signaling in the medulla. All these results offer a valuable dataset for future adrenal biology research, with potential applications in disease modeling and therapeutic target identification.

The results, high-resolution mapping of adrenal gland zonation, validation of the centripetal differentiation model, perspective on cell-cell communication, and potential translational impact on human adrenal gland function and disorders, are quite noble.

Weaknesses:

The reviewer requests that the following issues be addressed in the text:

(1) The study focuses only on adult male mice, which limits insights into developmental and sex-specific differences. What do the authors predict about the gender and age difference?

(2) Despite advanced methodologies, single-cell heterogeneity may not be fully captured, as Visium technology has limited spatial resolution.

(3) While the study suggests that ZX might have a role in androgen synthesis, further functional validation is required.

(4) The study is primarily descriptive, lacking in-depth mechanistic experiments to validate cell-cell communication interactions. It is quite interesting to suggest cell-cell communication, but the authors are still required to provide some evidence to support it.

(5) The data supports the conclusions, particularly in validating the centripetal differentiation model using Monocle3 trajectory analysis. However, functional validation experiments (e.g., gene knockout studies) would strengthen the findings, especially regarding ZX function and ligand-receptor interactions.

DOI: 10.1016/j.devcel.2025.06.015

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:ZFIN_ZDB-ALT-081110-1

What is this?

Reviewer #1 (Public review):

Summary:

This study addresses the issue of rapid skill learning and whether individual sequence elements (here: finger presses) are differentially represented in human MEG data. The authors use a decoding approach to classify individual finger elements, and accomplish an accuracy of around 94%. A relevant finding is that the neural representations of individual finger elements dynamically change over the course of learning. This would be highly relevant for any attempts to develop better brain machine interfaces - one now can decode individual elements within a sequence with high precision, but these representations are not static but develop over the course of learning.

Strengths:

The work follows from a large body of work from the same group on the behavioural and neural foundations of sequence learning. The behavioural task is well established a neatly designed to allow for tracking learning and how individual sequence elements contribute. The inclusion of short offline rest periods between learning epochs has been influential because it has revealed that a lot, if not most of the gains in behaviour (ie speed of finger movements) occur in these so-called micro-offline rest periods.

The authors use a range of new decoding techniques, and exhaustively interrogate their data in different ways, using different decoding approaches. Regardless of the approach, impressively high decoding accuracies are observed, but when using a hybrid approach that combines the MEG data in different ways, the authors observe decoding accuracies of individual sequence elements from the MEG data of up to 94%.

Reviewer #1 (Public review):

Summary:

Diarrheal diseases represent an important public health issue. Among the many pathogens that contribute to this problem, Salmonella enterica serovar Typhimurium is an important one. Due to the rise in antimicrobial resistance and the problems associated with widespread antibiotic use, the discovery and development of new strategies to combat bacterial infections is urgently needed. The microbiome field is constantly providing us with various health-related properties elicited by the commensals that inhabit their mammalian hosts. Harnessing the potential of these commensals for knowledge about host-microbe interactions as well as useful properties with therapeutic implications will likely to remain a fruitful field for decades to come. In this manuscript, Wang et al use various methods, encompassing classic microbiology, genomics, chemical biology, and immunology, to identify a potent probiotic strain that protects nematode and murine hosts from S. enterica infection. Additionally, authors identify gut metabolites that are correlated with protection, and show that a single metabolite can recapitulate the effects of probiotic administration.

Strengths:

The utilization of varied methods by the authors, together with the impressive amount of data generated, to support the claims and conclusions made in the manuscript is a major strength of the work. Also, the ability the move beyond simple identification of the active probiotic, also identifying compounds that are at least partially responsible for the protective effects, is commendable.

Weaknesses:

No major weaknesses noted.

Reviewer #1 (Public review):

Summary:

Walton et al. set out to isolate new phages targeting the opportunistic pathogen Pseudomonas aeruginosa. Using a double ∆fliF ∆pilA mutant strain, they were able to isolate 4 new phages, CLEW-1. -3, -6 and -10, that were unable to infected the parental PAO1F Wt strain. Further experiments showed that the 4 phages were only able to infect a ∆fliF strain, indicating a role of the MS-protein in the flagellum complex. Through further mutational analysis of the flagellum apparatus, the authors were able to identify the involvement of c-di-GMP in phage infection. Depletion of c-di-GMP levels by an inducible phosphodiesterase render the bacteria resistant to phage infection, while elevation of c-di-GMP through the Wsp system made the cells sensitive to infection by CLEW-1. Using TnSeq, the authors were able to not only reaffirm the involvement of c-di-GMP in phage infection but also able to identify the exopolysaccharide PSL as a downstream target for CLEW-1. C-di-GMP is a known regulator of PSL biosynthesis. The authors show that CLEW-1 binds directly to PSL on the cell surface and that deletion of the pslC gene resulted in complete phage resistance. The authors also provide evidence that the phage - PSL interaction happens during the biofilm mode of growth and that the addition of the CLEW-1 phage specifically resulted in a significant loss of biofilm biomass. Lastly, the authors set out to test if CLEW-1 could be used to resolve a biofilm infection using a mouse keratitis model. Unfortunately, while the authors noted a reduction in bacterial load assessed by GFP fluorescence, the keratitis did not resolve under the tested parameters.

Strengths:

The experiments carried out in this manuscript are thoughtful and rational, and sufficient explanation is provided for why the authors chose each specific set of experiments. The data presented strongly supports their conclusions and they give present compelling explanations for any deviation. The authors have not only developed a new technique for screening for phages targeting P. aeruginosa, but also highlights the importance of looking for phages during the biofilm mode of growth, as opposed to the more standard techniques involving planktonic cultures.

Weaknesses:

The authors did not include host-range testing or resistance development in this study, which would have strengthened the paper. Additionally, further characterisation of the CLEW-1 interaction with PSL at the molecular level would also have been welcomed. However, this will likely be the subject of future studies.

Reviewer #1 (Public review):

Summary:

In this paper, Behruznia and colleagues use long-read sequencing data for 339 strains of the Mycobacterium tuberculosis complex to study genome evolution in this clonal bacterial pathogen. They use both a "classical" pangenome approach that looks at the presence and absence of genes, and a pangenome graph based on whole genomes in order to investigate structural variants in non-coding regions. The comparison of the two approaches is informative and shows that much is missed when focussing only on genes. The two main biological results of the study are that 1) the MTBC has a small pangenome with few accessory genes, and that 2) pangenome evolution is driven by genome reduction. In the revised article, the description of the data set and the methods is much improved, and the comparison of the two pangenome approaches is more consistent. I still think, however, that the discussion of genome reduction suffers from a basic flaw, namely the failure to distinguish clearly between orthologs and homologs/paralogs.

Strengths: