Reviewer #3 (Public review):

Summary:

The paper offers a systematic and rigorous description of the layer-and sublayer specific outputs of the somatosensory cortex using a modern toolbox for the analysis of brain connectivity which combines: 1) Layer-specific genetic drivers for conditional viral tracing; 2) whole brain analyses of axon tracts using tissue clearing and imaging; 3) Segmentation and quantification of axons with normalization to the number of transduced neurons; 4) registration of connectivity to a widely used anatomical reference atlas; 5) functional validation of the connectivity using optogenetic approaches in vivo.

Strengths:

Although the connectivity of the somatosensory cortex is already known, precise data are dispersed in different accounts (papers, online resources, ) using different methods. So the present account has the merit of condensing this information in one very precisely documented report. It also brings new insights on the connectivity, such as the precise comparison of layer specific outputs, and of the primary and secondary somatosensory areas. It also shows a topographic organization of the circuits linking the somatosensory and motor cortices. The paper also offers a clear description of the methodology and of a rigorous approach to quantitative anatomy.

Weaknesses:

The weakness relates to the intrinsic limitations of the in toto approaches, that currently lack the precision and resolution allowing to identify single axons, axon branching or synaptic connectivity. These limitations are identified and discussed by the authors.

Reviewer #2 (Public review):

Summary:

The authors developed a bioinformatic pipeline to aid the screening and identification of inhibitory receptors suitable as drug targets. The challenge lies in the large search space and lack of tools for assessing the likelihood of their inhibitory function. To make progress, the authors used a consensus protein membrane topology and sequence motif prediction tool (TOPCOS) combined with both a statistical measure assessing their likelihood function and a machine learning protein structural prediction model (AlphaFold) to greatly cut down the search space. After obtaining a manageable set of 398 high confidence known and putative inhibitory receptors through this pipeline, the authors then mapped these receptors to different functional categories across different cell types based on their expression both in the resting and activated state. Additionally, by using publicly available pan cancer scRNA-seq for tumor-infiltrating T cells data, they showed that these receptors are expressed across various cellular subsets.

Strengths:

The authors presented sound arguments motivating the need to efficiently screen inhibitory receptors and to identify those that are functional. Key components of the algorithm were presented along with solid justification for why they addressed challenges faced by existing approaches. To name a few:

• TOPCON algorithm was elected to optimize the prediction of membrane topology<br /> • A statistical measure was used to remove potential false positives<br /> • AlphaFold is used to filter out putative receptors that are low confidence (and likely intrinsically disordered)

To examine receptors screened through this pipeline through a functional lens, the authors proposed to look at their expression of various immune cell subsets to assign functional categories. This is a reasonable and appropriate first step for interpreting and understanding how potential drug targets are differentially expressed in some disease contexts. They also presented an example showing this pipeline can be used to "rediscover" known targets.

Weaknesses:

The paper has strength in the pipeline they presented, but the weakness, in my opinion, lies in the lack of direct experimental validation on putative receptors. That said, the authors presented in the revised manuscript, as a proof-of-concept, an analytic approach for using functional categorization of putative inhibitory receptors to select therapeutic targets based on in vitro RNAseq. Such analysis will benefit from further investigation across different cancer types using in vivo expression.

Reviewer #1 (Public review):

Summary:

The manuscript by Jang et al. describes the application of new methods to measure the localization GTP-binding signaling proteins (G proteins) on different membrane structures in a model mammalian cell line (HEK293). G proteins mediate signaling by receptors found at the cell surface (GPCRs), with evidence from the last 15 years suggesting that GPCRs can induce G-protein mediated signaling from different membrane structures within the cell, with variation in signal localization leading to different cellular outcomes. While it has been clearly shown that different GPCRs efficiently traffic to various intracellular compartments, it is less clear whether G proteins traffic in the same manor, and whether GPCR trafficking facilitates "passenger" G protein trafficking. This question was a blind spot in the burgeoning field of GPCR localized signaling in need of careful study, and the results obtained will serve as an important guide post for further work in this field.<br /> The extent to which G proteins localize to different membranes within the cell is the main experimental question tested in this manuscript. This question is pursued by through two distinct methods, both relying on genetic modification of the G-beta subunit with a tag. In one method, G-beta is modified with a small fragment of the fluorescent protein mNG, which combines with the larger mNG fragment to form a fully functional fluorescent protein to facilitate protein trafficking by fluorescent microscopy. This approach was combined with expression of fluorescent proteins directed to various intracellular compartments (different types of endosomes, lysosome, endoplasmic reticulum, golgi, mitochondria) to look for colocalization of G-beta with these markers. These experiments showed compelling evidence that G-beta co-localizes with markers at the plasma membrane and the lysosome, with weak or absent co-localization for other markers. A second method for measuring localization relied on fusing G-beta with a small fragment from a miniature luciferase (HiBit) that combines with a larger luciferase fragment (LgBit) to form an active luciferase enzyme. Localization of G-beta (and luciferase signal) was measured using a method known as bystander BRET, which relies on expression of a fluorescent protein BRET acceptor in different cellular compartments. Results using bystander BRET supported findings from fluorescence microscopy experiments. These methods for tracking G protein localization were also used to probe other questions. The activation of GPCRs from different classes had virtually no impact on the localization of G-beta, suggesting that GPCR activation does not result in shuttling of G proteins through the endosomal pathway with activated receptors.

In the revised version of this manuscript the authors have performed informative and important new experiments in addition to adding new text to address conceptual questions. These new data and discussions are commendable and address most or all of the weaknesses listed in the initial review.

Strengths:

The question probed in this study is quite important and, in my opinion, understudied by the pharmacology community. The results presented here are an important call to be cognizant of the localization of GPCR coupling partners in different cellular compartments. Abundant reports of endosomal GPCR signaling need to consider how the impact of lower G protein abundance on endosomal membranes will affect the signaling responses under study.

*The work presented is carefully executed, with seemingly high levels of technical rigor. These studies benefit from probing the experimental questions at hand using two different methods of measurement (fluorescent microscopy and bystander BRET). The observation that both methods arrive at the same (or a very similar) answer inspires confidence about the validity of these findings.

Weaknesses:

*As noted by the authors, they do not demonstrate that the tagged G-beta is predominantly found within heterotrimeric G protein complexes. In the revised manuscript the authors have added new discussion text on why it is likely that G-beta is mostly found in complexes. This line of reasoning is convincing, although more robust experimental methods for assessing the assembly status of G-beta could be a valuable target for future experimental developments.

Reviewer #2 (Public review):

This study assess the subcellular distribution of a major G protein subunit (Gβ1) when expressed at an endogenous level in a well-studied model cell system (293 cells). The approach elegantly extends a gene editing strategy described by Leonetti's group and combines it with a FRET-based proximity assay to detect the presence of endogenously tagged Gβ1 on membrane compartments of 293 cells. The authors achieve their goal, and the data are convincing and interesting. The authors do a nice job of integrating their results with previous work in the field. The methods are now sufficiently well-described to enable other investigators to apply or adapt them in future studies.

Reviewer #3 (Public review):

Summary:

This article addresses an important and interesting question concerning intracellular localization and dynamics of endogenous G proteins. The fate and trafficking of G protein-coupled receptors (GPCRs) have been extensively studied but so far little is known about the trafficking routes of their partner G proteins that are known to dissociate from their respective receptors upon activation of the signaling pathway. Authors utilize modern cell biology tools including genome editing and bystander bioluminescence resonance energy transfer (BRET) to probe intracellular localization of G proteins in various membrane compartments in steady state and also upon receptor activation. Data presented in this manuscript shows that while G proteins are mostly present on the plasma membrane, they can be also detected in endosomal compartments, especially in late endosomes and lysosomes. This distribution, according to data presented in this study, seems not to be affected by receptor activation. These findings will have implications in further studies addressing GPCR signaling mechanisms from intracellular compartments.

Strengths:

The methods used in this study are adequate for the question asked. Especially use of genome-edited cells (for addition of the tag on one of the G proteins) is a great choice to prevent effects of overexpression. Moreover, use of bystander BRET allowed authors to probe intracellular localization of G proteins in a very high-throughput fashion. By combining imaging and BRET authors convincingly show that G proteins are very low abundant on early endosomes (also ER, mitochondria, and medial Golgi), however seem to accumulate on membranes of late endosomal compartments. Moreover, authors also looked at the dynamics of G protein trafficking by tracking them over multiple time points in different compartments.

Weaknesses:

While authors provide a novel dataset, many questions regarding G protein trafficking remain open. For example, it is not entirely clear which pathway is utilized to traffic G proteins from the plasma membrane to intracellular compartments. Additionally, future studies should also include more quantitative details considering G-protein distribution in different compartments as well as more detailed dynamic data on G protein internalization as well as intracellular trafficking kinetics.

Reviewer #3 (Public review):

Summary:

This paper provides presents an automated method to track individual mammalian cells as they progress through the cell cycle using the FUCCI system, and applies the method to look at different tumor cell lines that grow in suspension and determine their cell cycle profile and the effect of drugs that directly affect the cell cycles, on progression through the cell cycle for a 72 hour period.

Strengths:

This is a METHODS paper. The one potentially novel finding is that they can identify cells which are at the G1-S transition by the change in color as one protein starts to go up and the other one goes down, similar to change seen as cells enter G2/M. They have provided detailed data in the resubmission, demonstrating how this can be done in different cell lines and that the resolution of the brief time is about (about 1 hr) when the cells are determined to be in the transition from G1 to S. They further showed how one can explore this period (using EDU labeling in conjunction with FUCCI how one can determine whether cells have entered S-phase. This nicely addressed a weakness identified in the previous review.

Reviewer #1 (Public review):

Summary:

In this revised manuscript, Rincon-Torroella et al. developed ME3BP-7, a microencapsulated formulation of 3BP, as a potential agent to target MCT1 overexpressing PDACs. The authors provided compelling experimental evidence demonstrating the specific and rapid killing of MCT1 overexpressing PDAC cells in vitro, along with the safety and significant anti-tumor efficacy of ME3BP-7 in multiple PDAC orthotopic mouse models. Overall, this study is very novel, with well-designed experiments and a clear, organized presentation of data that supports the conclusions. The authors have effectively addressed the questions raised in the primary review and provided a thorough discussion of the study's significance, limitations, and future directions, which enhances the readers' understanding of the potential clinical impact of this research.

Strengths:

* Developed a novel agent.<br /> * Well-designed experiments and an organized presentation of data that support the conclusions.

Weaknesses:

No significant weaknesses are noticed.

Reviewer #2 (Public review):

Summary:

In the manuscript by Rincon-Torroella et al, the authors evaluated the therapeutic potential of ME3BP-7, a microencapsulated formulation of 3BP which specifically target MCT-1 high tumor cells, in pancreatic cancer models. The authors showed that, compared to 3BP, ME3BP-7 exhibited much enhanced stability in serum. In addition, the authors confirmed the specificity of ME3BP-7 toward MCT-1 high tumor cells and demonstrated the in vivo anti-tumor effect of ME3BP-7 in orthotopic xenograft of human PDAC cell line and PDAC PDX model.

Strengths:

(1) The study convincingly demonstrated the superior stability of ME3BP-7 in serum.<br /> (2) the specificity of ME3BP-7 and 3BP toward MCT-1 high PDAC cells was clearly demonstrated with CRISPR-mediated knockout experiments.<br /> (3) The advantage of ME3BP-7 over 3BP under in vivo situation is highlighted in the revised manuscript.

Reviewer #1 (Public Review):

Summary:

The manuscript by Kim et al. describes a role for axonal transport of Wnd (a dual leucine zipper kinase) for its normal degradation by the Hiw ubiquitin ligase pathway. In Hiw mutants, the Wnd protein accumulates dramatically in nerve terminals compared to the cell body of neurons. In the absence of axonal transport, Wnd levels rise and lead to excessive JNK signaling that makes neurons unhappy.

Strengths:

Using GFP-tagged Wnd transgenes and structure-function approaches, the authors show that palmitoylation of the protein at C130 plays a role in this process by promoting golgi trafficking and axonal localization of the protein. In the absence of this transport, Wnd is not degraded by Hiw. The authors also identify a role for Rab11 in the transport of Wnd, and provide some evidence that Rab11 loss-of-function neuronal degenerative phenotypes are due to excessive Wnd signaling. Overall, the paper provides convincing evidence for a preferential site of action for Wnd degradation by the Hiw pathway within axonal and/or synaptic compartments of the neuron. In the absence of Wnd transport and degradation, the JNK pathway becomes hyperactivated. As such, the manuscript provides important new insights into compartmental roles for Hiw-mediated Wnd degradation and JNK signaling control.

Weaknesses:

It is unclear if the requirement for Wnd degradation at axonal terminals is due to restricted localization of HIW there, but it seems other data in the field argues against that model. The mechanistic link between Hiw degradation and compartmentalization is unknown.

Reviewer #2 (Public Review):

Summary:

Utilizing transgene expression of Wnd in sensory neurons in Drosophila, the authors found that Wnd is enriched in axonal terminals. This enrichment could be blocked by preventing palmitoylation or inhibiting Rab1 or Rab11 activity. Indeed, subsequent experiments showed that inhibiting Wnd can prevent toxicity by Rab11 loss of function.

Strengths:

This paper evaluates in detail Wnd location in sensory neurons, and identifies a novel genetic interaction between Rab11 and Wnd that affects Wnd cellular distribution.

Weaknesses:

The authors report low endogenous expression of wnd, and expressing mutant hiw or overexpressing wnd is necessary to see axonal terminal enrichment. It is unclear if this overexpression model (which is known to promote synaptic overgrowth) would be relevant to normal physiology.

Palmitoylation of the Wnd orthologue DLK in sensory neurons has previously been identified as important for DLK trafficking in a cell culture model.

Reviewer #1 (Public review):

Summary:

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

Strengths:

The study collected an extensive dataset that includes species diversity of primary producers (riparian trees), primary consumers (macroinvertebrate shredders), and secondary consumers (fish). It also includes the genetic diversity of the dominant species at each trophic level, biomass production, decomposition rates, and environmental data.

The conclusions of this paper are mostly well supported by the data and the writing is logical and easy to follow.

Weaknesses:

While the dataset is impressive, the authors conducted analyses more akin to a "meta-analysis," leaving out important basic information about the raw data in the manuscript. Given the complexity of the relationships between different trophic levels and ecosystem functions, it would be beneficial for the authors to show the results of each SEM (structural equation model).

The main results presented in the manuscript are derived from a "metadata" analysis of effect sizes. However, the methods used to obtain these effect sizes are not sufficiently clarified. By analyzing the effect sizes of species diversity and genetic diversity on these ecosystem functions, the results showed that species diversity had negative effects, while genetic diversity had positive effects on ecosystem functions. The negative effects of species diversity contradict many studies conducted in biodiversity experiments. The authors argue that their study is more relevant because it is based on a natural system, which is closer to reality, but they also acknowledge that natural systems make it harder to detect underlying mechanisms. Providing more results based on the raw data and offering more explanations of the possible mechanisms in the introduction and discussion might help readers understand why and in what context species diversity could have negative effects.

Environmental variation was included in the analyses to test if the environment would modulate the effects of biodiversity on ecosystem functions. However, the main results and conclusions did not sufficiently address this aspect.

Reviewer #2 (Public review):

Summary:

Fargeot et al. investigated the relative importance of genetic and species diversity on ecosystem function and examined whether this relationship varies within or between trophic-level responses. To do so, they conducted a well-designed field survey measuring species diversity at 3 trophic levels (primary producers [trees], primary consumers [macroinvertebrate shredders], and secondary consumers [fishes]), genetic diversity in a dominant species within each of these 3 trophic levels and 7 ecosystem functions across 52 riverine sites in southern France. They show that the effect of genetic and species diversity on ecosystem functions are similar in magnitude, but when examining within-trophic level responses, operate in different directions: genetic diversity having a positive effect and species diversity a negative one. This data adds to growing evidence from manipulated experiments that both species and genetic diversity can impact ecosystem function and builds upon this by showing these effects can be observed in nature.

Strengths:

The study design has resulted in a robust dataset to ask questions about the relative importance of genetic and species diversity of ecosystem function across and within trophic levels.

Overall, their data supports their conclusions - at least within the system that they are studying - but as mentioned below, it is unclear from this study how general these conclusions would be.

Weaknesses:

(1) While a robust dataset, the authors only show the data output from the SEM (i.e., effect size for each individual diversity type per trophic level (6) on each ecosystem function (7)), instead of showing much of the individual data. Although the summary SEM results are interesting and informative, I find that a weakness of this approach is that it is unclear how environmental factors (which were included but not discussed in the results) nor levels of diversity were correlated across sites. As species and genetic diversity are often correlated but also can have reciprocal feedbacks on each other (e.g., Vellend 2005), there may be constraints that underpin why the authors observed positive effects of one type of diversity (genetic) when negative effects of the other (species). It may have also been informative to run SEM with links between levels of diversity. By focusing only on the summary of SEM data, the authors may be reducing the strength of their field dataset and ability to draw inferences from multiple questions and understand specific study-system responses.

(2) My understanding of SEM is it gives outputs of the strength/significance of each pathway/relationship and if so, it isn't clear why this wasn't used and instead, confidence intervals of Z scores to determine which individual BEFs were significant. In addition, an inclusion of the 7 SEM pathway outputs would have been useful to include in an appendix.

(3) I don't fully agree with the authors calling this a meta-analysis as it is this a single study of multiple sites within a single region and a specific time point, and not a collection of multiple studies or ecosystems conducted by multiple authors. Moreso, the authors are using meta-analysis summary metrics to evaluate their data. The authors tend to focus on these patterns as general trends, but as the data is all from this riverine system this study could have benefited from focusing on what was going on in this system to underpin these patterns. I'd argue more data is needed to know whether across sites and ecosystems, species diversity and genetic diversity have opposite effects on ecosystem function within trophic levels.

Reviewer #3 (Public review):

The manuscript by Fargeot and colleagues assesses the relative effects of species and genetic diversity on ecosystem functioning. This study is very well written and examines the interesting question of whether within-species or among-species diversity correlates with ecosystem functioning, and whether these effects are consistent across trophic levels. The main findings are that genetic diversity appears to have a stronger positive effect on function than species diversity (which appears negative). These results are interesting and have value.

However, I do have some concerns that could influence the interpretation.

(1) Scale: the different measures of diversity and function for the different trophic levels are measured over very different spatial scales, for example, trees along 200 m transects and 15 cm traps. It is not clear whether trees 200 m away are having an effect on small-scale function.

(2) Size of diversity gradients: More information is needed on the actual diversity gradients. One of the issues with surveys of natural systems is that they are of species that have already gone through selection filters from a regional pool, and theoretically, if the environments are similar, you should get similar sets of species, without monocultures. So, if the species diversity gradients range from say, 6 to 8 species, but genetic diversity gradients span an order of magnitude more, you can explain much more variance with genetic diversity. Related to this, species diversity effects on function are often asymptotic at high diversity and so if you are only sampling at the high diversity range, we should expect a strong effect.

(3) Ecosystem functions: The functions are largely biomass estimates (expect decomposition), and I fail to see how the biomass of a single species can be construed as an ecosystem function. Aren't you just estimating a selection effect in this case?

Note that the article claims to be one of the only studies to look at function across trophic levels, but there are several others out there, for example:

Li, F., Altermatt, F., Yang, J., An, S., Li, A., & Zhang, X. (2020). Human activities' fingerprint on multitrophic biodiversity and ecosystem functions across a major river catchment in China. Global change biology, 26(12), 6867-6879.

Luo, Y. H., Cadotte, M. W., Liu, J., Burgess, K. S., Tan, S. L., Ye, L. J., ... & Gao, L. M. (2022). Multitrophic diversity and biotic associations influence subalpine forest ecosystem multifunctionality. Ecology, 103(9), e3745.

Moi, D. A., Romero, G. Q., Antiqueira, P. A., Mormul, R. P., Teixeira de Mello, F., & Bonecker, C. C. (2021). Multitrophic richness enhances ecosystem multifunctionality of tropical shallow lakes. Functional Ecology, 35(4), 942-954.

Wan, B., Liu, T., Gong, X., Zhang, Y., Li, C., Chen, X., ... & Liu, M. (2022). Energy flux across multitrophic levels drives ecosystem multifunctionality: Evidence from nematode food webs. Soil Biology and Biochemistry, 169, 108656.

And the case was made strongly by:

Seibold, S., Cadotte, M. W., MacIvor, J. S., Thorn, S., & Müller, J. (2018). The necessity of multitrophic approaches in community ecology. Trends in ecology & evolution, 33(10), 754-764.

Reviewer #1 (Public Review):

Summary:

In this paper the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:

The experiments are comprehensive and clearly described, both in text and in the figures. Statistical analyses are provided throughout.

Weaknesses:

None.

Reviewer #2 (Public Review):

The focus of this manuscript was to investigate whether Kv1.8 channels, which have previously been suggested to be expressed in type I hair cells of the mammalian vestibular system, are responsible for the potassium conductance gK,L. This is an important study because gK,L is known to be crucial for the function of type I hair cells, but the channel identity has been a matter of debate for the past 20 years. The authors have addressed this research topic by primarily investigating the electrophysiological properties of the vestibular hair cells from Kv1.8 knockout mice. Interestingly, gK,L was completely abolished in Kv1.8-deficient mice, in agreement with the hypothesis put forward by the authors based on the literature. The surprising observation was that in the absence of Kv1.8 potassium channels, the outward potassium current in type II hair cells was also largely reduced. Type II hair cells express the largely inactivating potassium conductance g,K,A, but not gK,L. The authors concluded that heteromultimerization of non-inactivating Kv1.8 and the inactivating Kv1.4 subunits could be responsible for the inactivating gK,A. Overall, the manuscript is very well written and most of the conclusions are supported by the experimental work. The figures are well described, and the statistical analysis is robust.

Reviewer #3 (Public Review):

Summary:

This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts and heterozygotes) are examined here and whole cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

(1) From previous work it is known that GK,L in type I hair cells has unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+ and modulated by cyclic GMP. The question arises-if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells. However, hair cell specific partner interactions with Kv1.8 that result in GK, L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

(2) Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

(3) A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be really interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Reviewer #1 (Public review):

In the revised manuscript Vicario et al. provide new insights on a potential contribution of somatic mutations within the microglia population of the CNS that accelerates microglia activation and disease-associated gene signatures in Alzheimer's disease. Here they especially identified an "enrichment" of pathological SNVs in microglia, but not the peripheral blood, that are associated with clonal proliferative disorders and neurological diseases in a subset of patients with AD. They identified P-SNVs in microglia of AD patients located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in human microglia-like cells in vitro.

Overall, this study provides novel evidence from an AD patient cohort pointing to a potential contribution of microglia-specific somatic mutations to disease onset and/or progression in at least a subset of patients with Alzheimer's disease.

The work within this study is highly relevant and will open new study lines to explore somatic mutations within the microglia compartment and neurodegenerative diseases.

Strengths:

As outlined above, the study identified P-SNVs in microglia of AD patients associated with clonal proliferative disorders, but also give an in depth analysis in re-occurring P-SNVs located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in HEK cells, BV2 cells, MAC cells and human microglia-like cells in vitro. The over-activation of the cells in vitro is convincing.

Great care was taken to identify the limitations of the possible conclusions and to make careful conclusions. For example, they highlight that the pathway proposed to be affected may be an explanation for a subset of AD patients, and emphasize that it is yet unclear whether this accumulation of pathological SNVs is a cause or consequence of disease progression

The study supports an enrichment of P-SNVs in several genes associated clonal proliferative disorders in microglia and nicely separates this from SNVs associated with clonal hematopoiesis in the peripheral blood found in AD patients and controls.

The authors further acknowledged that several age matched control patients were diagnosed with cancer or tumor-associated diseases and carefully dissected the occurring SNVs in these patients are not associated with the P-SNVs identified in the microglial compartment of the AD cohort.

Weaknesses:

The revised study is overall convincing and has improved in the revised version, but some points especially regarding the clear connection of the seen somatic variants in microglia with a potential role in disease progression remain unanswered.

A potential connection between P-SNVs in microglia and disease pathology and symptoms was not further explored by the authors but might be in future work.

Taken this into account, maybe the title is a bit overstated and could be tuned down.

Reviewer #2 (Public review):

Summary:

In this study, Vicaro et al. aimed to quantify and characterize mosaic mutations in human sporadic Alzheimer's disease (AD) brain samples. They focused on three broad classes of brain cells, neurons that express the marker NeuN, microglia that express the marker PU.1, and double-negative cells that presumably comprise all other brain cell types, including astrocytes, oligodendrocytes, oligodendrocyte progenitor cells, and endothelial cells. The authors find an enrichment of potentially pathogenic somatic mutations in AD microglia compared to controls, with MAPK pathway genes being particularly enriched for somatic mutations in those cells. The authors report a striking enrichment for mutations in the gene CBL and use in vitro functional assays to show that these mutations indeed induce MAPK pathway activation.

The current state of the AD and somatic mutation fields puts this work into context. First, AD is a devastating disease whose prevalence is only increasing as the population of the U.S. is aging, necessitating the investigation of novel features of AD to identify new therapeutic opportunities. Second, microglia have recently come into focus as important players in AD pathogenesis. Many AD risk genes are selectively expressed in microglia, and microglia from AD brain samples show a distinct transcriptional profile indicating an inflammatory phenotype. The authors' previous work shows that a genetic mouse model of mosaic BRAF activation in macrophages (including microglia) displays a neurodegenerative phenotype similar to AD (Mass et al., 2017, doi:10.1038/nature23672). Third, new technological developments have allowed for identifying mosaic mutations present in only a small fraction of or even single cells. Together, these data form a rationale for studying mosaic mutations in microglia in AD. In light of the authors' findings regarding MAPK pathway gene somatic mutations, it is also important to note that MAPK has previously been implicated in AD neuroinflammation in the literature.

Strengths:

The study demonstrated several strengths. Firstly, the authors used two methods to identify mosaic mutations: 1) deep (~1,100x) DNA sequencing of a targeted panel of >700 genes they hypothesized might, if mutated somatically, play a role in AD, and 2) deep (400x) whole-exome sequencing (WES) to identify clonal mosaics outside of those genes. A second strength is the agreement between these experiments, where WES found many variants identified in the panel experiment, and both experiments revealed somatic mutations in MAPK pathway genes. Third, the authors demonstrated in several in vitro systems that many mutations they identified in MAPK genes activate MAPK signaling. Finally, the authors showed that in some human brain samples, single-cell gene expression analysis revealed that cells bearing a mosaic MAPK pathway mutation displayed dysregulated inflammatory signaling and dysregulation in other pathways. This single-cell analysis was in agreement with their in vitro analyses.

Weaknesses:

The study also showed some weaknesses. The sample size (45 AD donors and 44 controls) is small, reflected in the relatively modest effect sizes and p-values observed. This weakness is partially ameliorated by the authors' extensive molecular and functional validation of mutation candidates. Secondly, as the authors point out, this study cannot conclude whether microglial mosaic mutations cause AD or are an effect of AD. Future studies may shed more light on this important question.

Conclusions and Impact:

Considering the study's aims, strengths, and weaknesses, I conclude that the authors achieved their goal of characterizing the role of mosaic mutations in human AD. Their data strongly suggest that mosaic MAPK mutations in microglia are associated with AD. The impacts of this study remain to be seen, but they could include attempts to target CBL or other mutated genes in the treatment of AD. This work also suggests a similar approach to identifying potentially causative somatic mutations in other neurodegenerative diseases.

Reviewer #1 (Public review):

Summary:

The work of Muller and colleagues concerns the question where we place our feet when passing uneven terrain, in particular how we trade-off path length against the steepness of each single step. The authors find that paths are chosen that are consistently less steep and deviate from the straight line more than an average random path, suggesting that participants indeed trade off steepness for path length. They show that this might be related to biomechanical properties, specifically the leg length of the walkers. In addition, they show using a neural network model that participants could choose the footholds based on their sensory (visual) information about depth.

Strengths:

The work is a natural continuation of some of the researchers' earlier work that related the immediately following steps to gaze. Methodologically, the work is very impressive and presents a further step forward towards understanding real-world locomotion and its interaction with sampling visual information. While some of the results may seem somewhat trivial in hindsight (as always in this kind of studies), I still think this is a very important approach to understand locomotion in the wild better.

Weaknesses:

The concerns I had regarding the initial version of the manuscript have all been fixed in the current one.

Reviewer #2 (Public review):

This manuscript examines how humans walk over uneven terrain and use vision to decide where to step. There is a huge lack of evidence about this because the vast majority of locomotion studies have focused on steady, well-controlled conditions, and not on decisions made in the real world. The author team has already made great advances in this topic by pioneering gaze recordings during locomotion, but there has been no practical way to map the gaze targets, specifically the 3D terrain features in naturalistic environments. The team has now developed a way to integrate such measurements along with gaze and step tracking. This allows quantitative evaluation of the proposed trade-offs between stepping vertically onto vs. stepping around obstacles, along with how far people look to decide where to step. The team also introduces several new analysis techniques to accompany these measurements. They use machine learning techniques to examine whether retinocentric depth helps predict footholds and develop simulations to assess possible alternative footholds and walking paths. The technical achievement is impressive.

This study addresses several real-world questions not normally examined in the laboratory. First, do humans elect to walk around steeper footholds rather than over them? Second, is there a quantifiable benefit to walking around, such as allowing for a flatter path? Third, does visual depth of terrain contribute to selection of footholds? Fourth, are there scale effects, where for example a tall adult can easily walk over an obstacle that a toddler must walk around. One might superficially answer yes to all of these questions, but it is highly nontrival to answer them quantitatively. As for the conclusions, my feelings are mixed. I find strengths in answers to two of the questions, and weaknesses in the other two.

Strengths:

I consider the evidence strongest for the first of the main questions. The results show subjects walking with more laterally deviating paths, measured by a quantity called "tortuosity," when the direct straight-ahead paths appear to have steeper ups and downs (Fig. 9). The measure of straight-ahead steepness is fairly complicated (discussed below), but is shown to be well correlated with tortuosity, effectively predicting when subjects will not walk straight ahead.

There is also good evidence for the third question, showing that retinocentric depth is predictive of chosen footholds. Retinocentric depth was computed by a series of steps, starting with scene capture to determine a 3D terrain mesh, projecting that mesh into the eye's perspective, and then discarding all but the depth information. This highly involved process is only the beginning, because the depth was then used to train a neural network classifier with chosen footholds. That network was found to predict footholds better than chance, using a test set independent from the training set, each using half the recorded data. The results are strong and are best interpreted along with a previous study (Bonnen et al. 2021) showing that subjects gaze nearer ahead on rougher terrain, and slightly more so when binocular vision was disrupted. Depth information seems important for foothold selection.

As an aside, humans presumably also select footholds and estimate depth from a number of monocular visual cues, such as shading, shadows, color, and self-motion information. Interestingly, the terrain mesh and depth data here were computed from monocular images, suggesting that monocular vision can in principle be predictive of both depth and footholds. Binocular human vision presumably improves on monocular depth estimation, and so it would be interesting to see whether binocular scene cameras would predict footholds better. In an earlier review, I had suggested other avenues for exploration, but these are not weaknesses so much as opportunities not yet taken. I believe much could be learned from deeper analysis of the neural network, and future experiments using variations of this technique.

There is much to be appreciated about this study. I was impressed by the overarching outlook and ambitiousness of the team. They seek to understand human decision-making in real-world locomotion tasks, a topic of obvious relevance to the human condition but not often examined in research. The field has been biased toward well-controlled, laboratory studies, which have undeniable scientific advantages but are also quite different from the real world. The present study discards all of the usual advantages of the laboratory, yet still finds a way to explore real-world behaviors in a quantitative manner. It is an exciting and forward-thinking approach, used to tackle an ecologically relevant question.

I also appreciate the numerous technical challenges of this study. The state of the art in real-world locomotion studies has largely been limited to kinematic motion capture. This team managed to collect and analyze an unprecedented, one-of-a-kind dataset. They applied a number of non-trivial methods to assess retinocentric depth, simulate would-be walking paths and steepness, and predict footholds from neural network. Any of these could and probably will merit individual papers, and to assemble them all at once is quite beyond other studies I am aware of. I hope this study will spur more inquiries of this type, leveraging mobile electronics and modern machine learning techniques to answer questions that were previously only addressable qualitatively.

Weaknesses:<br /> Although I am highly enthusiastic about this study, I was not entirely convinced by the evidence for the second and fourth questions. Some of this is because I was confused by aspects of the analysis, limiting my understanding of the evidence. But I also question some of the basic conclusions, whether the authors indeed proved that (from Abstract, emphasis mine) "[walkers] change direction TO AVOID taking steeper steps that involve large height changes, instead of [sic] choosing more circuitous, RELATIVELY FLAT paths." (I interpret the "of" as a typo that should have been omitted.) I think it is more objective to say, "walkers changed direction more when straight-ahead paths seemed to have steeper height changes."

I say "seemed" because it is unknown whether humans would have experienced greater height changes if they walked straight ahead (the second main question). The comparison shown is between human tortuous paths taken and simulated straight-ahead paths never experienced by human. Ignoring questions about the simulations for now (discussed below), it is not an apples-to-apples comparison, say between the tortuous paths humans preferred and straight-ahead paths they didn't. The authors determined a measure of steepness, "straight path slope" (Fig. 9), that predicts when humans circuitously, but that is the same as the steepness that humans would actually experience if they had walked straight ahead. That could have been measured with an appropriate control condition, for example asking subjects to walk as straight ahead as they can manage. That also would have eliminated the need for simulations, because the slope of each step actually taken could simply have been measured and compared between conditions. Instead, two different kinds of simulations are compared, where steeper paths are fully simulated, and the circuitous paths are partially simulated but partially based on data. It seems that every fifth circuitous step coincides with a human foothold, but the intervening ones are somewhat random. I don't find this especially strong evidence that the chosen paths were indeed relatively flatter. I would prefer to be convinced by hard data than by unequal simulations.

I also have trouble accepting "TO AVOID" because it implies a degree of intent not evident in the data. I suppose conscious intent could be assessed subjectively by questionnaire, but I don't know how unconscious intent could be tested objectively. I believe my suggested interpretation above is better supported by evidence.

My limited acceptance is due in part to confusion about the simulations. I was especially confused about the connection between feasible steps drawn from the distribution in Figure 7, and the histograms of Figure 8. The feasible steps have clear peaks near zero slope, unity step length, and zero step direction (let's call them Flat). If 5-step simulations of Figure 8 draw from that distribution, why is there zero probability for the 0-3 deg bin (which is within {plus minus}3 deg due to absolute values)? It seems to me that Flat steps were eminently available, so why were they completely avoided? It seems that the simulations were probabilistic (and not just figurative) random walks, which implies they should have had about the same mean as Figure 7 but a wider variance, and then passed through absolute value. They look like something else that I cannot understand. This is important because the RELATIVELY FLAT conclusion is based on the chosen walks apparently being skewed flatter than random simulated walks. I have trouble accepting those distributions because Flat steps were unaccountably never taken by either simulation or human. (This issue is less concerning for Figure 9, because one can accept that some simulation measure is predictive of tortuosity even if the measure is hard to understand.)

I was also confused why Figure 7 distances and directions are nearly normally distributed and not more uniform. The methods only mention constraints to eliminate steps, which to me suggests a truncated uniform distribution. It is not clear to me why the terrain should have a high peak at unity step length, which implies that the only feasible footholds were almost exclusively straight ahead and one step length away. It is possible that the "feasible" footholds are themselves drawn from a "likely" normal distribution, perhaps based on level walking data. It could be argued that simulated steps should be performed by drawing from typical step distributions for level ground, eliminating non-viable footholds, and then repeating that across multiple steps. That would explain the normality, but it is not stated in the Methods, and even if they were "feasible and likely" it would not explain the distributions of Figure 8.

I had some misgivings about the fourth question, where Figure 10 suggests that shorter subjects had greater correlation between straight-path slope and tortuosity than taller ones, who tended to walk straighter ahead. I agree with the authors' rebuttal to my previous review that "the data are the data" but I still have doubts. Now supplied as suggested by another reviewer, Figure 18 provides more detail of the underlying data, with considerably lower correlations. I now suspect that Figure 10 benefits from some statistical artifacts due to binning and other operations, and the weaker correlations of Fig. 18A are closer to reality. I am rather suspicious of correlations of correlations (Figure 18B), which lose some statistical grounding because the second correlation treats all data on equal footing, effectively whitewashing the first correlations of their varying significance (p-values 0.008 to 1e-9).

Furthermore, I am also unsure about Figure 10's comparison of tortuosity vs. straight path slope against leg length. Both tortuosity and straight path slope are already effectively dimensionless and therefore already seem to eliminate scale. It is my understanding that the simulated paths were recomputed for each subject's parameters, and the horizontal axis, slope, is already an angular measure that should affect short and tall people similarly. Shouldn't all subjects equally avoid steep angles, regardless of their dimensional height? If there is indeed a scale effect, then I would expect it to be demonstrated with a dimensional measure (vertical axis) that depends on leg length.

I certainly agree with the hypothetical prior that tall adults walk straight over obstacles that shorter adults (or children) walk around. But I feel that simpler tests would better evidence, perhaps in future work. Did shorter subjects walk with greater tortuosity than taller ones on the same terrain? Did shorter subjects take relatively more steps even after normalizing for leg length? A possible comparison would be (number of steps)*(leg length)/(start to end distance). I feel that the evidence from this study is not that strong.

Although it is a strength of this study that so much can be learned from pure observation, that does not mean controlled conditions are not scientifically helpful. As mentioned earlier, a helpful control could have been to ask subjects to walk straighter but less preferred paths on the same terrain, treating human paths as an independent variable. Another would be to treat terrain as an independent variable, by using level ground and intermediate terrain conditions. This would make it easier to test whether taller subjects walk straighter ahead on more uneven terrain than shorter subjects. Indeed, the data set already includes some patches of flatter terrain, not included here. Additional and simpler tests might be possible based on existing data.

Conclusion

This is an ambitious undertaking, presenting a wealth of unprecedented data to quantitatively test basic ecological questions that have long been unanswered. There are a number of considerable strengths that merit appreciation, especially the ability to quantitatively predict when humans will walk more circuitously. The weaknesses are about limitations in the conclusions that can be drawn thus far rather than the correctness of the study. I consider this to be a first step that will hopefully enable and inspire a long line of future work that will address these questions more in depth.

Reviewer #3 (Public review):

Summary:

The systematic way in which path selection is parametrically investigated is the main contribution.

Strengths:

The authors have developed an impressive workflow to study gait and gaze in natural terrain. They are able to determine footholds and gaze points in the 3D world, and explore different path selections in the terrain.

Weaknesses:

The finding that walkers prefer less tortuous, demanding paths is hardly surprising, and from the data it is still not clear what actual visual features are used to choose among alternative routes or what the nature of the decision process is. The authors discuss energetic cost and other "factors" that might influence path selection, but as yet there is no way to express these ideas rigorously in such complex natural settings.

Reviewer #1 (Public review):

This is an important study to characterize cultured neuronal network dynamics, down to the combinations of individual excitatory and inhibitory inputs that result in spiking. The authors effectively combine high-density multi-electrode arrays with patch recordings and a convincing analysis to work out the contributions of multiple simultaneously active input neurons to postsynaptic activity.

In this study the authors develop methods to interrogate cultured neuronal networks to learn about the contributions of multiple simultaneously active input neurons to postsynaptic activity. They then use these methods to ask how excitatory and inhibitory inputs combine to result in postsynaptic neuronal firing in a network context.

The study uses a compelling combination of high-density multi-electrode array recordings with patch recordings. They make effective use of physiology techniques such as shifting the reversal potential of inhibitory inputs, and identifying inhibitory vs. excitatory neurons through their influence on other neurons, to tease apart the key parameters of synaptic connections. The method appears to work on rather low-density cultures so the size of the networks in the current study is in the low tens, and the number of synaptic inputs coming to each neuron is smaller than what would be encountered in vivo.

The authors obtain a number of findings on the conditions in which the dynamics of excitatory and inhibitory inputs permit spiking, and the statistics of connectivity that result in this. This is of considerable interest, and clearly one would like to see how these findings map to larger networks, to non-cortical networks, and ideally to networks in-vivo. The suite of approaches discussed here could potentially serve as a basis for such further development.

One of the challenges in doing such studies in a dish is that the network is simply ticking away without any neural or sensory context to work on, nor any clear idea of what its outputs might mean. Nevertheless, at a single-neuron level one expects that this system might provide a reasonable subset of the kinds of activity an individual cell might have to work on. In their response to earlier comments the authors have made useful comments on features of in-vivo network activity that are seen in culture. This could ideally be incorporated into the discussion.

Reviewer #2 (Public Review):

The authors had two aims in this study. First, to develop a tool that lets them quantify the synaptic strength and sign of upstream neurons in a large network of cultured neurons. Second, they aimed at disentangling the contributions of excitatory and inhibitory inputs to spike generation.

For the quantification of synaptic currents, their methods allows them to quantify excitatory and inhibitory currents simultaneously, as the sign of the current is determined by the neuron identity in the high-density extracellular recording. They further made sure that their method works for nonstationary firing rates, and they did a simulation to characterize what kind of connections their analysis does not capture. They assume that dendritic integration is linear, which is reasonable for synaptic currents measured using voltage-clamp.

As suggested in a previous review, they have partitioned the explained variance into frequency bands and are able to account for most of the variance in the 3-200Hz range of expected synaptic activity.

For the contributions of excitation and inhibition to neuronal spiking, the authors found a clear reduction of inhibitory inputs and increase of excitation associated with spiking when averaging across many spikes. And interestingly, the inhibition shows a reversal right after a spike and the timescale is faster during higher network activity. These findings provide further support that their method is working. In the revised version the authors now also provide an analysis of which synaptic event is associated with postsynaptic spiking. The large datasets from this study are well-suited to examining these points.

For the first part, the authors achieved their goal in developing a tool to study synaptic inputs driving subthreshold activity at the soma and characterizing such connections. For the second part, they found an effect of EPSCs on firing, and in the revision they have quantified its relevance.

With the availability of Neuropixels probes, there is certainly use for their tool in in vivo applications, and their statistical analysis provides a reference for future studies.<br /> The relevance of excitatory and inhibitory currents on spiking has now been examined in the updated version of the manuscript.

In the following, there is a suggestion on improving Figure 6. Many other suggestions for Fig 6 and 7 have been taken up in the revision and it is OK to consider this as future work:

Figure 6B is useful, but could be done better: The autocovariance of a shotnoise process is a convolution of the autocovariance of the underlying point process and the autocovariance of the EPSC kernel. So one would want to separate those to obtain a better temporal resolution. But a shotnoise process has well defined peaks, and the time of these local maxima can be estimated quite precisely. Now if I would do a peak triggered average instead of the full convolution, I would do half of the deconvolution and obtain a temporally asymmetric curve of what is expected to happen around an EPSC. Importantly, one could directly see expected excitation after inhibition or expected inhibition after excitation, and this visualization could be much better and more intuitive compared to panel 6E.

As a suggestion for further analysis, though I am well aware that this is likely beyond the scope of this manuscript, I'd suggest the following analysis:<br /> I would split the data into the high and low activity states. Then I would compute the average of E/(E+I) values for spikes. Assuming that spikes tend to happen for local maxima of E/(E+I) I would find local maxima for periods without spikes such that their average is equal to the value for actual spikes. Finally, I would test for a systematic difference in either excitation or inhibition.<br /> If there is no difference, you can make the claim that synaptic input does not guarantee a spike, and compare it to a global average of E/(E+I).

Reviewer #1 (Public review):

In this manuscript, the authors use a large dataset of neuroscience publications to elucidate the nature of self-citation within the neuroscience literature. The authors initially present descriptive measures of self-citation across time and author characteristics; they then produce an inclusive model to tease apart the potential role of various article and author features in shaping self-citation behavior. This is a valuable area of study, and the authors approach it with a rich dataset and solid methodology.

The revisions made by the authors in this version have greatly improved the validity and clarity of the statistical techniques, and as a result the paper's findings are more convincing.

This paper's primary strengths are: 1) its comprehensive dataset that allows for a snapshot of the dynamics of several related fields; 2) its thorough exploration of how self-citation behavior relates to characteristics of research and researchers.

Its primary weakness is that the study stops short of digging into potential mechanisms in areas where it is potentially feasible to do so - for example, studying international dynamics by identifying and studying researchers who move between countries, or quantifying more or less 'appropriate' self-citations via measures of abstract text similarity.

Yet while these types of questions were not determined to be in scope for this paper, the study is quite effective at laying the important groundwork for further study of mechanisms and motivations, and will be a highly valuable resource for both scientists within the field and those studying it.

Reviewer #2 (Public review):

The study presents valuable findings on self-citation rates in the field of Neuroscience, shedding light on potential strategic manipulation of citation metrics by first authors, regional variations in citation practices across continents, gender differences in early-career self-citation rates, and the influence of research specialization on self-citation rates in different subfields of Neuroscience. While some of the evidence supporting the claims of the authors is solid, some of the analysis seems incomplete and would benefit from more rigorous approaches.

Reviewer #3 (Public review):

This paper analyses self-citation rates in the field of Neuroscience, comprising in this case, Neurology, Neuroscience and Psychiatry. Based on data from Scopus, the authors identify self-citations, that is, whether references from a paper by some authors cite work that is written by one of the same authors. They separately analyse this in terms of first-author self-citations and last-author self-citations. The analysis is well-executed and the analysis and results are written down clearly. The interpretation of some of the results might prove more challenging. That is, it is not always clear what is being estimated.

This issue of interpretability was already raised in my review of the previous revision, where I argued that the authors should take a more explicit causal framework. The authors have now revised some of the language in this revision, in order to downplay causal language. Although this is perfectly fine, this misses the broader point, namely that it is not clear what is being estimated. Perhaps it is best to refer to Lundberg et al. (2021) and ask the authors to clarify "What is your Estimand?" In my view, the theoretical estimands the authors are interested in are causal in nature. Perhaps the authors would argue that their estimands are descriptive. In either case, it would be good if the authors could clarify that theoretical estimand.

Finally, in my previous review, I raised the issue of when self-citations become "problematic". The authors have addressed this issue satisfactorily, I believe, and now formulate their conclusions more carefully.

Lundberg, I., Johnson, R., & Stewart, B. M. (2021). What Is Your Estimand? Defining the Target Quantity Connects Statistical Evidence to Theory. American Sociological Review, 86(3), 532-565. https://doi.org/10.1177/00031224211004187

Reviewer #1 (Public Review):

In this manuscript, the authors investigate whether enhancers use a common regulatory paradigm to modulate transcriptional bursting in both endogenous and ectopic domains using cis-regulatory mutant reporters of the eve transcriptional locus in early Drosophila embryogenesis.

The authors create a series of cis-regulatory BAC mutants of the eve stripe 1 and 2 enhancers by mutating the binding sites for the transcriptional repressor Giant in the stripe 2 minimal response element (MRE) independently or in combination with deletion of the stripe 1 enhancer sequence. With these enhancer mutations, they are able to generate conditions in which eve is ectopically expressed. Next, the authors investigate if nuclei in these "ectopic" regions have similar transcriptional kinetics to the "endogenous"-expressing eve+ nuclei. They show that bursting parameters are unchanged when comparing endogenous and ectopic gene expression regions. Under a scheme of a 2-state model, the eveS1Δ-EveS2Gt- reporter modulates transcription by increasing the active state switching rate (kon) and the initiation rate (r) while maintaining a constant inactive state switching rate.

Based on these results, the authors support a model whereby kinetic regimes are encoded in the cis-regulatory sequences of a gene instead of imposed by an evolving trans-regulatory environment.

The question asked in this manuscript is important and the eve locus represents an ideal paradigm to address it in a quantitative manner. Most of the results are correctly interpreted and well-presented.

Reviewer #2 (Public Review):

The manuscript by Berrocal et al. asks if shared bursting kinetics, as observed for various developmental genes in animals, hint towards a shared molecular mechanism or result from natural selection favoring such a strategy. Transcription happens in bursts. While transcriptional output can be modulated by altering various properties of bursting, certain strategies are observed more widely. As the authors noted, recent experimental studies have found that even-skipped enhancers control transcriptional output by changing burst frequency and amplitude while burst duration remains largely constant. The authors compared the kinetics of transcriptional bursting between endogenous and ectopic gene expression patterns. It is argued that since enhancers act under different regulatory inputs in ectopically expressed genes, adaptation would lead to diverse bursting strategies as compared to endogenous gene expression patterns. To achieve this goal, the authors generated ectopic even-skipped transcription patterns in fruit fly embryos. The key finding is that bursting strategies are similar in endogenous and ectopic even-skipped expression. According to the authors, the findings favor the presence of a unified molecular mechanism shaping even-skipped bursting strategies. This is an important piece of work. Everything has been carried out in a systematic fashion.

Reviewer #3 (Public Review):

In this manuscript by Berrocal and coworkers, the authors do a deep dive into the transcriptional regulation of the eve gene in both an endogenous and ectopic background. The idea is that by looking at eve expression under non-native conditions, one might infer how enhancers control transcriptional bursting. The main conclusion is that eve enhancers have not evolved to have specific behaviors in the eve stripes, but rather the same rates in the telegraph model are utilized as control rates even under ectopic or 'de novo' conditions. For example, they achieve ectopic expression (outside of the canonical eve stripes) through a BAC construct where the binding sites for the TF Giant are disrupted along with one of the eve enhancers. Perhaps the most general conclusion is that burst duration is largely constant throughout at ~ 1 - 2 min. This conclusion is consistent with work in human cell lines that enhancers mostly control frequency and that burst duration is largely conserved across genes, pointing to an underlying mechanistic basis that has yet to be determined.

Reviewer #1 (Public Review):

Revised Public Review

This reviewed preprint is essentially three papers combined into one-one paper focused on the role of CIB2/CIB3 in vestibular hair cells, one on the role of CIB2/CIB3 in zebrafish, and one on structural modeling of a CIB2/3 and TMC1/2 complex. The authors try to combine the three parts with the overarching theme of demonstrating that CIB2/3 play a functionally conserved role across species and hair cell types. It is important to note that many of the basic results from the mouse have already been reported by other groups in Liang et al. (2021) and Wang et al. (2023).

That said, their demonstration of the importance of CIB2 and CIB3 in zebrafish hair cell function is novel. The results largely coincide with what is seen in the mouse-they are both important, with stimulus-dependent Ca2+ entry reduced more in cib2 KOs than in cib3 KOs, and the cib2;cib3 showing the greatest impact. Interestingly, cib2 is uniquely localized in and important for specific hair cell types in the neuromast and crista.

The last part of the manuscript also offers significant new findings. Here structural studies (AlphaFold 2 modeling, NMR structure determination, and molecular dynamics simulations) brings us closer to the structure of the mammalian TMCs, alone and in complex with the CIB proteins. Moreover, the structural work supports the assignment of the TMC pore to alpha helices 4-7.

In summary, while this reviewed preprint has some data that replicate data from publications from other labs, it provides a comprehensive look at the CIB family in hair cells, especially in vestibular hair cells.

Reviewer #2 (Public Review):

The paper by Giese and coworkers is quite an intense reading. The manuscript is packed with data pertaining to very different aspects of MET apparatus function, scales, and events. I have to praise the team that combined molecular genetics, biochemistry, NMR, microscopy, functional physiology, in-vivo tests for vestibulo-ocular reflexes, and other tests for vestibular dysfunction with molecular modeling and simulations. The authors nicely show the way CIBs are associated with TMCs to form functional MET channels. The authors clarify the specificity of associations and elucidate the functional effects of the absence of specific CIBs and their partial redundancy.

Comments on revised version:

I appreciate the author's effort to address my comments. The revised paper 'Complexes of vertebrate TMC1/2 and CIB2/3 proteins 1 form hair-cell mechanotransduction cation channels' by Giese and coworkers is definitely cleaner but remains a compendium of related but very uneven parts. By saying 'uneven,' I mean that the grounding of the experimental and computational parts is different, and the firmness of conclusions, respectively, is not matched.

My conclusion is that this is a great collaborative project. However, in its present form, different components pull the emphasis in several directions with little cross-talk. It is worth splitting into two papers.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

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

Weaknesses:

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

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

Reviewer #2 (Public review):

Summary:

Marylin Alves de Almeida et al. developed a novel mouse cross via conditionally depleting functional SMN protein in the liver (AlbCre/+;Smn2B/F7). This mouse model retains a proportion of SMN in the liver, which better recapitulates SMN deficiency observed in SMA patients and allows further investigation into liver-specific SMN deficiency and its systemic impact. They show that AlbCre/+;Smn2B/F7 mice do not develop an apparent SMA phenotype as mice did not develop motor neuron death, neuromuscular pathology or muscle atrophy, which is observed in the Smn2B/- controls. Nonetheless, at P19, these mice develop mild liver steatosis, and interestingly, this conditional depletion of SMN in the liver impacts cells in the pancreas.

Strengths:

The current model has clearly delineated the apparent metabolic perturbations which involve a significantly increased lipid accumulation in the liver and pancreatic cell defects in AlbCre/+;Smn2B/F7 mice at P19. Standard methods like H&E and Oil Red-O staining show that in AlbCre/+;Smn2B/F7 mice, their livers closely mimic the livers of Smn2B/- mice, which have the full body knockout of SMN protein. Unlike previous work, this liver-specific conditional depletion of SMN is superior in that it is not lethal to the mouse, which allows an opportunity to investigate the long-term effects of liver-specific SMN on the pathology of SMA.

Weaknesses: Given that SMA often involves fatty liver, dyslipidemia and insulin resistance, using the current mouse model, the authors could have explored the long-term effects of liver-specific depletion of SMN on metabolic phenotypes beyond P19, as well as systemic effects like glucose homeostasis. Given that the authors also report pancreatic cell defects, the long-term effect on insulin secretion and resistance could be further explored. The mechanistic link between a liver-specific SMN depletion and apparent pancreatic cell defects is also unclear.

Discussion:

This current work explores a novel mouse cross in order to specifically deplete liver SMN using an Albumin-Cre driver line. This provides insight into the contribution of liver-specific SMN protein to the pathology of SMA, which is relevant for understanding metabolic perturbations in SMA patients. Nonetheless, given that SMA in patients involve a systemic deletion or mutation of the SMN gene, the authors could emphasize the utility of this liver-specific mouse model, as opposed to using in vitro models, which have been recently reported (Leow et al, 2024, JCI). Authors should also discuss why a mild metabolic phenotype is observed in this current mouse model, as opposed to other SMA mouse models described in literature.

Reviewer #1 (Public review):

Summary:

This work starts with the observation that embryo polarization is asynchronous starting at the early 8-cell stage, with early polarizing cells being biased towards producing the trophectoderm (TE) lineage. They further found that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and TE specification, this piece of evidence connects the previous finding that at Carm1 heterogeneity 4-cell stage guide later cell lineages - the higher Carm1-expressing blastomeres are biased towards ICM lineage. Thus, This work provides a link between asymmetries at the 4-cell stage and polarization at the 8-cell stage, providing a cohesive explanation regarding the first lineage allocation in mouse embryos.

Strengths:

In addition to what has been put in the summary, the advanced 3D image-based analysis has found that early polarization is associated with a change in cell geometry in blastomeres, regarding the ratio of the long axis to the short axis. This is considered a new observation that has not been identified.

Weaknesses:

For the microinjection-based method to overexpression/deletion of proteins, although it has been shown to be effective in the early embryo settings and has been widely used, it may not fully represent the in vivo situation in some cases, compared to other strategies such as the use of knock-in mice. This is a minor weakness; it would be good to include some sentences in the discussion on the potential caveats.

Reviewer #2 (Public review):

Summary:

In this study, Lamba and colleagues suggest a molecular mechanism to explain cell heterogeneity in cell specification during pre-implantation development. They show that embryo polarization is asynchronous. They propose that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and trophectoderm specification.

Strengths:

The authors use appropriate and validated methodology to address their scientific questions. They also report excellent live imaging. Most of the data are accompanied by careful quantifications.

Weaknesses:

I think this manuscript requires some more quantification, increased number of embryos in their evaluations and clearly stating the number of embryos evaluated per experiments.

Here are some points:

(1) It should be clearly stated in all figure legends and in the text how many cells from how many embryos were analyzed.

(2) I think that the number of embryos sometimes are too low. These are mouse embryos easily accessible and the methods used are well established in this lab, so the authors should make an effort to have at least 10/15 embryos per experiment. For example "In agreement with this, hybridization chain reaction (HCR) RNA fluorescence in situ hybridization of early 8-cell stage embryos revealed that the number of CDX2 mRNA puncta was higher in polarized blastomeres with a PARD6-positive apical domain than in unpolarized blastomeres, for 5 out of 6 embryos with EP cells (Figure 3A, B)".. or the data for Figure 4, we know how many cells but now how many embryos.

(3) It would be useful to see in Figure 4 an example of asymmetric cell division as done for symmetric cell division in panel 4B. This could really help the reader to understand how the authors assessed this.

(4) Figure 5C there is a big disproportion of the number of EP and LP identified. Could the authors increase the number of embryos quantified and see if they can increase EP numbers?

(5) Could the authors give more details about how they mount the embryos for live imaging? With agarose or another technique? In which dishes? Overlaid with how much medium and oil? This could help other labs that want to replicate the live imaging in their labs. Also, was it a z-stack analysis? If yes, how many um per stack? Ideally, if they also know the laser power used (at least a range) it would be extremely useful.

Reviewer #1 (Public review):

Summary:

This is a valuable study probing the impact of pH and cancer mutations on nucleosome interactions and higher-order chromatin structures.

Strengths:

The study is comprehensive, covering all the titratable residues of nucleosomes and all known cancer mutations. The analysis was rigorously carried out within the feasibility of current computational capabilities. The methods used in this study are also solid. The results of this study can enhance our understanding of higher-order chromatin organizations and their modulation by various genetic and epigenetic changes.

Weaknesses:

The interpretation and illustration of the data need improvement, such as the change of protonation states of titratable residues on the nucleosome-protein interactions and higher-order chromatin structures.

Reviewer #2 (Public review):

Summary:

The paper by Zhang et al. has two parts.

The first one presents a comprehensive study of the nucleosome pKs, including their shifts from reference values in solution. They also explore changes in the protonation states of the histone residue in response to the formation of various nucleosome complexes, including higher-order nucleosome structures. The overall conclusion is that pH-induced changes in histone residue protonation states modulate nucleosome surface electrostatic potentials, and influence nucleosome-partner protein interactions. Proton uptake or release often accompanied by nucleosome-partner protein interactions affects their binding processes.

In the second part, the authors study the effect of 1266 recurrent histone cancer mutations on the nucleosome surface electrostatics: they show a significant subset of these has a major effect on the nucleosome-partner interactions, with the potential to regulate nucleosome self-association, thereby affecting higher-order chromatin structures.

Strengths:

The main strengths of this work are its technical rigor, comprehensive nature, and novelty of several of its aspects. For example, I am not aware of another work that analyzed pK shifts in the nucleosome in such level of detail, and on for so many different structures. The same for pK shifts upon nucleosome-partner binding. The analysis of pK shifts in nucleosome-nucleosome binding is likely completely new. The authors use an established methodology, check it against experiment at least in some instances, and, very importantly, base their conclusions on many different structures. The specific pK-related numbers they report are believable.

Regarding the second part of the work: the specific connection made between a subset of cancer-associated mutations and the major electrostatic changes in the nucleosome is novel and should be of interest to a broad community. The authors conclude that cancer mutations can also regulate nucleosome self-association, modulating the organization and dynamics of higher-order chromatin structures.

The detailed and comprehensive analysis of the cancer-associated mutations, including their partitioning into multiple relevant categories, is of value in its own right.

Weaknesses:

The main weakness of the first (pK-related) part of this work is the lack of relevance to specific conditions in most living cells of higher eukaryotes. The problem is that the nucleosome resides in the nucleus, where the pH is very tightly controlled, and for good reasons. See e.g. Casey, J., Grinstein, S., and Orlowski, J. ``Sensors and regulators of intracellular pH." Nature Rev. Mol. Cell. Biology. (2009). Parker, M. D., and Boron, W. F. ``The divergence, actions, roles, and relatives of sodium-coupled bicarbonate transporters.", Physiol. Rev. (2013). While intracellular pH does deviate from about 7.2, the naturally occurring deviations are only of the order of 0.3 pH units. In that respect, what the authors call "physiological" range of 6.5 to 7.5 is still too broad, let alone the "slightly basic (pH 5 to 6.5) or ``slightly acidic" (pH 7.5 to 9) conditions, as defined by the authors. It is hard to imagine a situation where intra-nuclear pH changes from e.g. "slightly acidic" to neutral in a live cell nucleus.

This said, there is nothing wrong with studying the response of the nucleosome structures to these large variations of pH, which can be reproduced in-vitro. It is the relevance of the findings to in-vivo conditions that are highly questionable.

The second part of the work - the effect of cancer mutations - is free from this major defect. In the opinion of this reviewer, it can (and should) stand on its feet, as a separate work.

However, the lack of specific, testable (preferably quantitative) biologically relevant predictions is a weakness of both parts. For example, in "Discussion" the authors state that "Histone ionizable residues are highly sensitive to cellular pH fluctuations, leading to changes in their protonation states and consequent alterations in nucleosome surface electrostatic potentials and interactions." This statement is certainly true, based on what is already known about the effect of pH on protein-DNA (or protein-protein) association, from previous works. But what are the specific predictions here?

Reviewer #1 (Public review):

Summary:

This manuscript (Baron, Oviedo et al., 2024) builds on a previous study from the Wiseman lab (Perea, Baron et al., 2023) and describes the identification of novel nucleoside mimetics that activate the HRI branch of the ISR and drive mitochondrial elongation. The authors develop an image processing and analysis pipeline to quantify the effects of these compounds on mitochondrial networks and show that these HRI activators mitigate ionomycin-driven mitochondrial fragmentation. They then show that these compounds rescue mitochondrial morphology defects in patient-derived MFN2 mutant cell lines.

Strengths:

The identification of new ISR modulators opens new avenues for biological discovery surrounding the interplay between mitochondrial form/function and the ISR, a topic that is of broad interest. It also reinforces the possibility that such compounds might represent new potential therapeutics for certain mitochondrial disorders. The development of a quantitative image analysis pipeline is valuable and has the potential to extract the subtle effects of various treatments on mitochondrial morphology.

Weaknesses:

I have three main concerns.

First, support for the selectivity of compounds 0357 and 3610 acting downstream of HRI comes from using knockdown ISR kinase cell lines and measuring the fluorescence of ATF4-mApple (Figure 1G and 1H). However, the selectivity of these compounds acting through HRI is not shown for mitochondrial morphology. Is mitochondrial elongation blocked in HRI knockdown cells treated with the compounds? While the ISRIB treatment does block mitochondrial elongation, ISRIB acts downstream of all ISR kinases and doesn't necessarily define selectivity for the HRI branch of the ISR. Additionally, are the effects of these compounds on ATF4 production and mitochondrial elongation blocked in a non-phosphorylatable eIF2alpha mutant? This point of selectivity/specificity of the compounds gets at a semantic stumbling block I encountered in the text where it was often stated "stress-independent activation" of ISR kinases. Nucleoside mimetics are likely a very biologically active class of molecules and are likely driving some level of cell stress independent of a classical ISR, UPR, heat-shock response, or oxidative stress response.

Second, it is difficult for me to interpret the data for the quantification of mitochondrial morphology. In the legend for Figure 2, it is stated that "The number of individual measurements for each condition are shown above." Are the individual measurements the number of total cells quantified? If not, how many total cells were analyzed? If the individual measurements are distinct mitochondrial structures that could be quantified why are the n's for each parameter (bounding box, ellipsoid principal axis, and sphericity) so different? Does this mean that for some mitochondria certain parameters were not included in the analysis? For me, it seems more intuitive that each mitochondrial unit should have all three parameters associated with it, but if this isn't the case it needs to be more carefully described why.

Third, the impact of these compounds on the physiological function of mitochondria in the MFN2.D414V mutants needs to be measured. Sharma et al., 2021 showed a clear deficit in mitochondrial OCR in MFN2.D414V cells which, if rescued by these compounds, would strengthen the argument that pharmacological ISR kinase activation is a strategy for targeting the functional consequences of the dysregulation of mitochondrial form.

Reviewer #2 (Public review):

Summary.

Mitochondrial dysfunction is associated with a wide spectrum of genetic and age-related diseases. Healthy mitochondria form a dynamic reticular network and constantly fuse, divide, and move. In contrast, dysfunctional mitochondria have altered dynamic properties resulting in fragmentation of the network and more static mitochondria. It has recently been reported that different types of mitochondrial stress or dysfunction activate kinases that control the integrated stress response, including HRI, PERK, and GCN2. Kinase activity results in decreased global translation and increased transcription of stress response genes via ATF4, including genes that encode mitochondrial protein chaperones and proteases (HSP70 and LON). In addition, the ISR kinases regulate other mitochondrial functions including mitochondrial morphology, phospholipid composition, inner membrane organization, and respiratory chain activity. Increased mitochondrial connectivity may be a protective mechanism that could be initiated by pharmacological activation of ISR kinases, as was recently demonstrated for GCN2.

A small molecule screening platform was used to identify nucleoside mimetic compounds that activate HRI. These compounds promote mitochondrial elongation and protect against acute mitochondrial fragmentation induced by a calcium ionophore. Mitochondrial connectivity is also increased in patient cells with a dominant mutation in MFN2 by treatment with the compounds.

Strengths:

(1) The screen leverages a well-characterized reporter of the ISR: translation of ATF4-FLuc is activated in response to ER stress or mitochondrial stress. Nucleoside mimetic compounds were screened for activation of the reporter, which resulted in the identification of nine hits. The two most efficacious dose-response tests were chosen for further analysis (0357 and 3610). The authors clearly state that the compounds have low potency. These compounds were specific to the ISR and did not activate the unfolded protein response or the heat shock response. Kinases activated in the ISR were systematically depleted by CRISPRi revealing that the compounds activate HRI.

(2) The status of the mitochondrial network was assessed with an Imaris analysis pipeline and attributes such as length, sphericity, and ellipsoid principal axis length were quantified. The characteristics of the mitochondrial network in cells treated with the compounds were consistent with increased connectivity. Rigorous controls were included. These changes were attenuated with pharmacological inhibition of the ISR.

(3) Treatment of cells with the calcium ionophore results in rapid mitochondrial fragmentation. This was diminished by pre-treatment with 0357 or 3610 and control treatment with thapsigargin and halofuginone

(4) Pathogenic mutations in MFN2 result in the neurodegenerative disease Charcot-Marie-Tooth Syndrome Type 2A (CMT2A). Patient cells that express Mfn2-D414V possess fragmented mitochondrial networks and treatment with 0357 or 3610 increased mitochondrial connectivity in these cells.

Weaknesses:

The weakness is the limited analysis of cellular changes following treatment with the compounds.

(1) Unclear how 0357 or 3610 alter other aspects of cellular physiology. While this would be satisfying to know, it may be that the authors determined that broad, unbiased experiments such as RNAseq or proteomic analysis are not justified due to the limited translational potential of these specific compounds.

(2) There are many changes in Mfn2-D414V patient cells including reduced respiratory capacity, reduced mtDNA copy number, and fewer mitochondrial-ER contact sites. These experiments are relatively narrow in scope and quantifying more than mitochondrial structure would reveal if the compounds improve mitochondrial function, as is predicted by their model.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

The manuscript is well written. This novel work demonstrates a likely link between Upd2-JAK/STAT signalling in the fat body and tracheal stem cells and the control of unidirectional cell migration of tracheal stem cells. The authors show that hid+rpr or Upd2RNAi expression in a fat body or Dome RNAi, Hop RNAi, or STAT92E RNAi expression in tracheal stem cells results in aberrant migration of some of the tracheal stem cells towards the anterior. Using ChIP-seq as well as analysis of GFP-protein trap lines of planar cell polarity genes in combination with RNAi experiments, the authors show that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells which appear to be needed for unidirectional migration. Moreover, the authors hypothesise that extracellular vesicle transport of Upd2 might be involved in this Upd2-JAK/STAT signalling in the fat body and tracheal stem cells, which, if true, would be quite interesting and novel.

Overall, the work presented here provides some novel insights into the mechanism that ensures unidirectional migration of tracheal stem cells that prevents bidirectional migration. This might have important implications for other types of directed cell migration in invertebrates or vertebrates including cancer cell migration.

Weaknesses:

It remains unclear to what extent Upd2-JAK/STAT signalling regulates unidirectional migration. While there seems to be a consistent phenotype upon genetic manipulation of Upd2-JAK/STAT signalling and planar cell polarity genes, as in the aberrant anterior migration of a fraction of the cells, the phenotype seems to be rather mild, with the majority of cells migrating towards the posterior.

While I am not an expert on extracellular vesicle transport, the data presented here regarding Upd2 being transported in extracellular vesicles do not appear to be very convincing.

Major comments:

(1) The graphs showing the quantification of anterior (and in some cases also posterior migration) are quite confusing. E.g. Figure 1F (and 5E and all others): These graphs are difficult to read because the quantification for the different conditions is not shown separately. E.g. what is the migration distance for Fj RNAi anterior at 3h in Fig5E? Around -205micron (green plus all the other colors) or around -70micron (just green, even though the green bar goes to -205micron). If it's -205micron, then the images in C' or D' do not seem to show this strong phenotype. If it's around -70, then the way the graph shows it is misleading, because some readers will interpret the result as -205.

Moreover, it's also not clear what exactly was quantified and how it was quantified. The details are also not described in the methods. It would be useful, to mark with two arrowheads in the image (e.g. 5 A' -D') where the migration distance is measured (anterior margin and point zero).

Overall, it would be better, if the graph showed the different conditions separately. Also, n numbers should be shown in the figure legend for all graphs.

(2) Figure 2-figure supplement 1: C-L and M: From these images and graph it appears that Upd2 RNAi results in no aberrant anterior migration. Why is this result different from Figures 2D-F where it does?

(3) Figure 5F: The data on the localisation of planar cell polarity proteins in the tracheal stem cell group is rather weak. Figure 5G and J should at least be quantified for several animals of the same age for each genotype. Is there overall more Ft-GFP in the cells on the posterior end of the cell group than on the opposite side? Or is there a more classic planar cell polarity in each cell with Ft-GFP facing to the posterior side of the cell in each cell? Maybe it would be more convincing if the authors assessed what the subcellular localisation of Ft is through the expression of Ft-GFP in clones to figure out whether it localises posteriorly or anteriorly in individual cells.

(4) Regarding the trafficking of Upd2 in the fat body, is it known, whether Grasp65, Lbm, Rab5, and 7 are specifically needed for extracellular vesicle trafficking rather than general intracellular trafficking? What is the evidence for this?

(5) Figure 8A-B: The data on the proximity of Rab5 and 7 to the Upd2 blobs are not very convincing.

(6) The authors should clarify whether or not their work has shown that "vesicle-mediated transport of ligands is essential for JAK/STAT signaling". In its current form, this manuscript does not appear to provide enough evidence for extracellular vesicle transport of Upd2.

(7) What is the long-term effect of the various genetic manipulations on migration? The authors don't show what the phenotype at later time points would be, regarding the longer-term migration behaviour (e.g. at 10h APF when the cells should normally reach the posterior end of the pupa). And what is the overall effect of the aberrant bidirectional migration phenotype on tracheal remodelling?

(8) The RNAi experiments in this manuscript are generally done using a single RNAi line. To rule out off-target effects, it would be important to use two non-overlapping RNAi lines for each gene.

Reviewer #2 (Public review):

Summary:

This work by Dong and colleagues investigates the directed migration of tracheal stem cells in Drosophila pupae, essential for tissue homeostasis. These cells, found in two nearby groups, migrate unidirectionally along the dorsal trunk towards the posterior to replenish degenerating branches that disperse the FGF mitogen. The authors show that inter-organ communication between tracheal stem cells and the neighboring fat body controls this directionality. They propose that the fat body-derived cytokine Upd2 induces JAK/STAT signaling in tracheal progenitors, maintaining their directional migration. Disruption of Upd2 production or JAK/STAT signaling results in erratic, bidirectional migration. Additionally, JAK/STAT signaling promotes the expression of planar cell polarity genes, leading to asymmetric localization of Fat in progenitor cells. The study also indicates that Upd2 transport depends on Rab5- and Rab7-mediated endocytic sorting and Lbm-dependent vesicle trafficking. This research addresses inter-organ communication and vesicular transport in the disciplined migration of tracheal progenitors.

Strengths:

This manuscript presents extensive and varied experimental data to show a link between Upd2-JAK/STAT signaling and tracheal progenitor cell migration. The authors provide convincing evidence that the fat body, located near the trachea, secretes vesicles containing the Upd2 cytokine. These vesicles reach tracheal progenitors and activate the JAK-STAT pathway, which is necessary for their polarized migration. Using ChIP-seq, GFP-protein trap lines of planar cell polarity genes, and RNAi experiments, the authors demonstrate that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells, which seem to be necessary for unidirectional migration.

Weaknesses:

Directional migration of tracheal progenitors is only partially compromised, with some cells migrating anteriorly and others maintaining their posterior migration.<br /> Additionally, the authors do not examine the potential phenotypic consequences of this defective migration.

It is not clear whether the number of tracheal progenitors remains unchanged in the different genetic conditions. If there are more cells, this could affect their localization rather than migration and may change the proposed interpretation of the data.

Upd2 transport by vesicles is not convincingly shown.

Data presentation is confusing and incomplete.

Reviewer #3 (Public review):

Summary:

Dong et al tackle the mechanism leading to polarized migration of tracheal progenitors during Drosophila metamorphosis. This work fits in the stem cell research field and its crucial role in growth and regeneration. While it has been previously reported by others that tracheal progenitors migrate in response to FGF and Insulin signals emanating from the fat body in order to regenerate tracheal branches, the authors identified an additional mechanism involved in the communication of the fat body and tracheal progenitors.

Strengths:

The data presented were obtained using a wide range of complementary techniques combining genetics, molecular biology, quantitative, and live imaging techniques. The authors provide convincing evidence that the fat body, found in close proximity to the trachea, secrete vesicles containing the Upd2 cytokine that reach tracheal progenitors leading to JAK-STAT pathway activation, which is required for their polarized migration. In addition, the authors show that genes regulating planar cell polarity are also involved in this inter-organ communication.

Weaknesses:

(1) Affecting this inter-organ communication leads to a quite discrete phenotype where polarized migration of tracheal progenitors is partially compromised. The study lacks data showing the consequences of this phenotype on the final trachea morphology, function, and/or regeneration capacities at later pupal and adult stages. This could potentially increase the significance of the findings.

(2) The conclusions of this paper are mostly well supported by data, but some aspects of data acquisition and analysis need to be clarified and corrected, such as recurrent errors in plotting of tracheal progenitor migration distance that mislead the reader regarding the severity of the phenotype.

(3) The number of tracheal progenitors should be assessed since they seem to be found in excess in some genetic conditions that affect their behavior. A change in progenitor number could lead to crowding, thus affecting their localization rather than migration capacities, thereby changing the proposed interpretation. In addition, the authors show data suggesting a reduced progenitor migration speed when the fat body is affected, which would also be consistent with a crowding of progenitors.

(4) The authors claim that tracheal progenitors display a polarized distribution of PCP proteins that is controlled by JAK-STAT signaling. However, this conclusion is made from a single experiment that is not quantified and for which there is no explanation of how the plot profile measurements were performed. It also seems that this experiment was done only once. Altogether, this is insufficient to support the claim. Finally, a quantification of the number of posterior edges presenting filopodia rather than the number of filopodia at the anterior and posterior leading edges would be more appropriate.

(5) The authors demonstrate that Upd2 is transported through vesicles from the fat body to the tracheal progenitors where they propose they are internalized. Since the Upd2 receptor Dome ligand binding sites are exposed to the extracellular environment, it is difficult to envision in the proposed model how Upd2 would be released from vesicles to bind Dome extracellularly and activate the JAK-STAT pathway. Moreover, data regarding the mechanism of the vesicular transport of Upd2 are not fully convincing since the PLA experiments between Upd2 and Rab5, Rab7, and Lbm are not supported by proper positive and negative controls and co-immunoprecipitation data in the main figure do not always correlate to the raw data.

Reviewer #1 (Public review):

Summary:

Audio et al. measured cerebral blood volume (CBV) across cortical areas and layers using high-resolution MRI with contrast agents in non-human primates. While the non-invasive CBV MRI methodology is often used to enhance fMRI sensitivity in NHPs, its application for baseline CBV measurement is rare due to the complexities of susceptibility contrast mechanisms. The authors determined the number of large vessels and the areal and laminar variations of CBV in NHP and compared those with various other metrics.

Strengths:

Non-invasive mapping of relative cerebral blood volume is novel for non-human primates. A key finding was the observation of variations in CBV across regions; primary sensory cortices had high CBV, whereas other higher areas had low CBV. The measured CBV values correlated with previously reported neuronal and receptor densities.

Weaknesses:

A weakness of this manuscript is that the quantification of CBV with postprocessing approaches to remove susceptibility effects from pial and penetrating vessels, as well as orientation dependency, is not fully validated, especially on a laminar scale. Further specific comments follow.

(1) Baseline CBV indices were determined using contrast agent-enhanced MRI (deltaR2*). Although this approach is suitable for areal comparisons, its application on a laminar scale has not been validated in the literature or in this study. By comparing with histological vascular information of V1, the authors attempted to validate their approach. However, the generalization of their method is questionable. The main issue is whether the large vessel contribution is minimized by processing approaches properly in various cortical areas (such as clusters 1-3 in Figure 5). It would be beneficial to compare deltaR2* with deltaR2 induced by contrast agents in a few selected slices, as deltaR2 is supposed to be sensitive to microvessels, not macrovessels. Please discuss this issue.

(2) High-resolution MRI with a critical sampling frequency estimated from previous studies (Weber 2008, Zheng 1991) was performed to separate penetrating vessels, which is considered one of the major advancements in this study. However, this approach is still insufficient to accurately identify the number of vessels due to the blooming effects of susceptibility and insufficient spatial resolution. There was no detailed description of the detection criteria. More importantly, the number of observable penetrating vessels is dependent on imaging parameters and the dose of the contrast agent. If imaging slices were obtained in parallel to the cortex with higher in-plane resolution, it would likely improve the detection of penetrating vessels. Using higher-field MRI would further enhance the detection of penetrating vessels. Therefore, the reported value is only applicable to the experimental and processing conditions used in this study. Detailed selection criteria should be mentioned, and all potential pitfalls should be discussed.

(3) Attempts to obtain pial vascular structures were made (Figure 2). As mentioned in this manuscript, the blooming effect of susceptibility contrasts is problematic. In the MRI community, T1-based Gd contrast agents have been used for mapping large vasculature, which is a better approach for obtaining pial vascular structures. Alternatively, computer tomography with a blood contrast agent can be used for mapping blood vasculature noninvasively. This issue should be discussed.

(4) Since baseline R2* is related to baseline R2, vascular volume, iron content, and susceptibility gradients, it is difficult to correlate it with physiological parameters. Baseline R2* is also sensitive to imaging parameters; higher spatial resolution tends to result in lower R2* values (closer to the R2 value). Therefore, baseline R2* findings need to be emphasized.

(5) CBV-weighted deltaR2* is correlated with various other metrics (cytoarchitectural parcellation, myelin/receptor density, cortical thickness, CO, cell-type specificity, etc.). While testing the correlation between deltaR2* and these other metrics may be acceptable as an exploratory analysis, it is challenging for readers to discern a causal relationship between them. A critical question is whether CBV-weighted deltaR2* can provide insights into other metrics in diseased or abnormal brain states. If this is the case, then high-resolution deltaR2* will be useful. Please comment on this possibility.

(6) There is no discussion about the deltaR2* difference across subcortical areas (Figure 1). This finding is intriguing and warrants a thorough discussion in the context of the cortical findings.

(7) Figure 3 is missing. Several statements in the manuscript require statistics (e.g., bimodality in Figure 2D, Figure 3F).

Reviewer #2 (Public review):

Summary:

This manuscript presents a new approach for non-invasive, MRI-based measurements of cerebral blood volume (CBV). Here, the authors use ferumoxytol, a high-contrast agent, and apply specific sequences to infer CBV. The authors then move to statistically compare measured regional CBV with the known distribution of different types of neurons, markers of metabolic load, and others. While the presented methodology captures an estimated 30% of the vasculature, the authors corroborated previous findings regarding the lack of vascular compartmentalization around functional neuronal units in the primary visual cortex.

Strengths:

Non-invasive methodology geared to map vascular properties in vivo.

Implementation of a highly sensitive approach for measuring blood volume.

Ability to map vascular structural and functional vascular metrics to other types of published data.

Weaknesses:

The key issue here is the underlying assumption about the appropriate spatial sampling frequency needed to capture the architecture of the brain vasculature. Namely, ~7 penetrating vessels / mm2 as derived from Weber et al 2008 (Cer Cor). The cited work begins by characterizing the spacing of penetrating arteries and ascending veins using a vascular cast of 7 monkeys (Macaca mulatta, same as in the current paper). The ~7 penetrating vessels / mm2 are computed by dividing the total number of identified vessels by the area imaged. The problem here is that all measurements were made in a "non-volumetric" manner and only in V1. Extrapolating from here to the entire brain seems like an over-assumption, particularly given the region-dependent heterogeneity that the current paper reports.

Reviewer #1 (Public review):

Summary:

In this elegant and thorough study, Sánchez-León et al. investigate the effects of tDCS on the firing of single cerebellar neurons in awake and anesthetized mice. They find heterogeneous responses depending on the orientation of the recorded Purkinje cell.

Strengths:

The paper is important in that it may well explain part of the controversial and ambiguous outcomes of various clinical trials. It is a well-written paper on a deeply analyzed dataset.

Weaknesses:

The sample size could be increased for some of the experiments.

Reviewer #2 (Public review):

Summary:

In this study by Sánchez-León and colleagues, the authors attempted to determine the influence of neuronal orientation on the efficacy of cerebellar tDCS in modulating neural activity. To do this, the authors made recordings from Purkinje cells, the primary output neurons of the cerebellar cortex, and determined the inter-dependency between the orientation of these cells and the changes in their firing rate during cerebellar tDCS application.

Strengths:

(1) A major strength is the in vivo nature of this study. Being able to simultaneously record neural activity and apply exogenous electrical current to the brain during both an anesthetized state and during wakefulness in these animals provides important insight into the physiological underpinnings of tDCS.

(2) The authors provide evidence that tDCS can modulate neural activity in multiple cell types. For example, there is a similar pattern of modulation in Purkinje cells and non-Purkinje cells (excitatory and inhibitory interneurons). Together, these data provide wholistic insight into how tDCS can affect activity across different populations of cells, which has important implications for basic neuroscience, but also clinical populations where there may be non-uniform or staged effects of neurological disease on these various cell types.

(3) There is a systematic investigation into the effects of tDCS on neural activity across multiple regions of the cerebellum. The authors demonstrate that the pattern of modulation is dependent on the target region. These findings have important implications for determining the expected neuromodulatory effects of tDCS when applying this technique over different target regions non-invasively in animals and humans.

Weaknesses:

(1) In the introduction, there is a lack of context regarding why neuronal orientation might be a critical factor influencing the responsiveness to tDCS. The authors allude to in vitro studies that have shown neuronal orientation to be relevant for the effects of tDCS on neural activity but do not expand on why this might be the case. These points could be better understood by informing the reader about the uniformity/non-uniformity of the induced electric field by tDCS. In addition, there is a lack of an a priori hypothesis. For example, would the authors have expected that neuronal orientation parallel or perpendicular to the electrical field to be related to the effects of tDCS on neural activity?

(2) It is unclear how specific stimulation parameters were determined. First, how were the tDCS intensities used in the present experiments determined/selected, and how does the relative strength of this induced electric field equate to the intensities used non-invasively during tDCS experiments in humans? Second, there is also a fundamental difference in the pattern of application used here (e.g., 15 s pulses separated by 10 s of no stimulation) compared to human studies (e.g., 10-20 min of constant stimulation).

(3) In their first experiment, the authors measure the electric field strength at increasing depths during increasing stimulation intensities. However, it appears that an alternating current rather than a direct current, which is usually employed in tDCS protocols, was used. There is a lack of rationale regarding why the alternating current was used for this component. Typically, this technique is more commonly used for entraining/boosting neural oscillations compared to studies using tDCS which aim to increase or decrease neural activity in general.

Reviewer #3 (Public review):

Summary:

In this study, Sanchez-Leon et al. combined extracellular recordings of Purkinje cell activity in awake and anesthetized mice with juxtacellular recordings and Purkinje cell staining to link Purkinje cell orientation to their stimulation response. The authors find a relationship between neuron orientation and firing rate, dependent on stimulation type (anodal/cathodal). They also show the effects of stimulation intensity and rebound effects.

Strengths:

Overall, the work is methodologically sound and the manuscript is well written. The authors have taken great care to explain their rationale and methodological choices.

Weaknesses:

My only reservation is the lack of reporting of the precise test statistics, p-values, and multiple comparison corrections. The work would benefit from adding this and other information.

Major Comments:

(1) The authors should report the exact test statistics. These are missing for all comparisons and hinder the reader from understanding what exactly was tested for each of the experiments. For example, having the exact test statistics would help better understand the non-significant differences in Figure 1h where there is at least a numeric difference in CS firing rate during tDCS.

(2) Did the authors apply any corrections for multiple comparisons? Generally, it would be helpful if they could clarify the statistical analysis (which values were subjected to the tests, how many tests were performed for each question, etc.).

(3) The relationship shown in Figure 2g seems to be influenced by the two outliers. Have the authors confirmed the results using a robust linear regression method?

(4) The authors conclude that tDCS modulates vermal PCs more than Crus I/II PCs - but they don't seem to test this statistically. It would be helpful to submit the firing rate change values to an actual statistical test to conclude this directly from the data

Reviewer #1 (Public review):

Summary:

How reconsolidation works - particularly in humans - remains largely unknown. With an elegant, 3-day design, combining fMRI and psychopharmacology, the authors provide evidence for a certain role for noradrenaline in the reconsolidation of memory for neutral stimuli. All memory tasks were performed in the context of fMRI scanning, with additional resting-state acquisitions performed before and after recall testing on Day 2. On Day 1, 3 groups of healthy participants encoded word-picture associates (with pictures being either scenes or objects) and then performed an immediate cued recall task to presentation of the word (answering is the word old or new, and whether it was paired with a scene or an object). On Day 2, the cued recall task was repeated using half of the stimulus set words encoded on Day 1 (only old words were presented, with subjects required to indicate prior scene vs object pairing). This test was immediately preceded by the oral administration of placebo, cortisol, or yohimbine (to raise noradrenaline levels) depending on group assignment. On Day 3, all words presented on Day 1 were presented. As expected, on Day 3, memory was significantly enhanced for associations that were cued and successfully retrieved on Day 2 compared to uncued associations. However, for associative d', there was no Cued × Group interaction nor a main effect of Group, i.e., on the standard measure of memory performance, post-retrieval drug presence on Day 2 did not affect memory reconsolidation. As further evidence for a null result, fMRI univariate analyses showed no Cued × Group interactions in whole-brain or ROI activity.

Strengths:

There are some aspects of this study that I find impressive. The study is well-designed and the fMRI analysis methodology is innovative and sound. The authors have made meticulous and thorough physiological measurements, and assays of mood, throughout the experiment. By doing so, they have overcome, to a considerable extent, the difficulties inherent in the timing of human oral drug delivery in reconsolidation tasks, where it is difficult to have the drug present in the immediate recall period without affecting recall itself. This is beautifully shown in Figure 3. I also think that having some neurobiological assay of memory reactivation when studying reconsolidation in humans is critical, and the authors provide this. While multi-voxel patterns of hemodynamic responses are, in my view, very difficult to equate with an "engram", these patterns do have something to do with memory.

Weaknesses:

I have major issues regarding the behavioral results and the framing of the manuscript.

(1) To arrive at group differences in memory performance, the authors performed median splitting of Day 3 trials by short and long reaction times during memory cueing on Day 2, as they took this as a putative measure of high/low levels of memory reactivation. Associative category hits on Day 3 showed a Group by Day 2 Reaction time (short, long) interaction, with post-hocs showing (according to the text) worse memory for short Day 2 RTs in the Yohimbine group. These post-hocs should be corrected for multiple comparisons, as the result is not what would be predicted (see point 2). My primary issue here is that we are not given RT data for each group, nor is the median splitting procedure described in the methods. Was this across all groups, or within groups? Are short RTs in the yohimbine group any different from short RTs in the other two groups? Unfortunately, we are not given Day 2 picture category memory levels or reaction times for each group. This is relevant because (as given in Supplemental Table S1) memory performance (d´) for the Yohimbine group on Day 1 immediate testing is (roughly speaking) 20% lower than the other 2 groups (independently of whether the pairs will be presented again the following day). I appreciate that this is not significant in a group x performance ANOVA but how does this relate to later memory performance? What were the group-specific RTs on Day 1? So, before the reader goes into the fMRI results, there are questions regarding the supposed drug-induced changes in behavior. Indeed, in the discussion, there is repeated mention of subsequent memory impairment produced by yohimbine but the nature of the impairment is not clear.

(2) The authors should be clearer as to what their original hypotheses were, and why they did the experiment. Despite being a complex literature, I would have thought the hypotheses would be reconsolidation impairment by cortisol and enhancement by yohimbine. Here it is relevant to point out that - only when the reader gets to the Methods section - there is mention of a paper published by this group in 2024. In this publication, the authors used the same study design but administered a stress manipulation after Day 2 cued recall, instead of a pharmacological one. They did not find a difference in associative hit rate between stress and control groups, but - similar to the current manuscript - reported that post-retrieval stress disrupts subsequent remembering (Day 3 performance) depending on neural memory reinstatement during reactivation (specifically driven by the hippocampus and its correlation with neocortical areas).

Instead of using these results, and other human studies, to motivate the current work, reference is made to a recent animal study: Line 169 "Building on recent findings in rodents (Khalaf et al. 2018), we hypothesized that the effects of post-retrieval noradrenergic and glucocorticoid activation would critically depend on the reinstatement of the neural event representation during retrieval". It is difficult to follow that a rodent study using contextual fear conditioning and examining single neuron activity to remote fear recall and extinction would be relevant enough to motivate a hypothesis for a human psychopharmacological study on emotionally neutral paired associates.

Reviewer #2 (Public review):

Summary:

The authors aimed to investigate how noradrenergic and glucocorticoid activity after retrieval influence subsequent memory recall with a 24-hour interval, by using a controlled three-day fMRI study involving pharmacological manipulation. They found that noradrenergic activity after retrieval selectively impairs subsequent memory recall, depending on hippocampal and cortical reactivation during retrieval.

Overall, there are several significant strengths of this well-written manuscript.

Strengths:

(1) The study is methodologically rigorous, employing a well-structured three-day experimental design that includes fMRI imaging, pharmacological interventions, and controlled memory tests.

(2) The use of pharmacological agents (i.e., hydrocortisone and yohimbine) to manipulate glucocorticoid and noradrenergic activity is a significant strength.

(3) The clear distinction between online and offline neural reactivation using MVPA and RSA approaches provides valuable insights into how memory dynamics are influenced by noradrenergic and glucocorticoid activity distinctly.

Weaknesses:

(1) One potential limitation is the reliance on distinct pharmacodynamics of hydrocortisone and yohimbine, which may complicate the interpretation of the results.

(2) Another point related above, individual differences in pharmacological responses, physiological and cortisol measures may contribute to memory recall on Day 3.

(3) Median-splitting approach for reaction times and hippocampal activity should better be justified.

Reviewer #1 (Public review):

Summary:

In this work, the authors investigate the molecular dynamics of MinD, a component of the Bacillus subtilis Min system, in vitro and in vivo. In Escherichia coli the Min system is highly dynamic and displays rapid pole-to-pole oscillation whereby a time average minimum of the Min proteins at mid-cell is established. However, in B. subtilis, this is not the case, and there is no MinE present. MinD in B. subtilis dynamically relocalizes from the poles to division sites and binds to MinC and MinJ, which mediates its interaction with DivIVA. This paper reports the biochemical characterization of B. subtilis MinD in vitro and dynamics of MinD variants in vivo, providing mechanistic insight into the mechanism of dynamic localization.

Strengths:

In the current study, the authors perform a detailed biochemical characterizion of the in vitro ATPase activity of MinD and demonstrate that rapid hydrolysis is elicited by adding phospholipids. They further show using a collection of substitution mutants of MinD that both monomers and dimers bind to the membrane, and ATP occupancy changes the on and off rates. Identification, quantification, and tracking of discrete Halo-MinD populations were nicely done and showed that mutations in MinD alter dynamic localization, correlating with PL binding on and off rates in vitro.

Weaknesses:

While the study shows that MinD in B. subtilis utilizes a different (MinE-independent) activation mechanism, it remains to be determined the extent to which MinJ and/or MinC play a role.

Reviewer #2 (Public review):

Summary:

Feddersen & Bramkamp determined important characteristics of how MinD protein binds/dissociates to/from the membrane, and dimerizes in relation to its ATPase activity. The presented data clearly shows the differences in function of MinD homologs from B. subtilis and E. coli.

Strengths:

The work presents well-executed experiments that lead to interesting conclusions and a new model of how Min system works during B. subtilis mid-cell division. Importantly, this model is supported by in vitro characterization of well-chosen mutants in the functional domains of MinD. Outstandingly, most of the in vitro data are confirmed by single-molecule localization microscopy.

Weaknesses:

The authors immobilized liposomes, for which they used E. coli total lipids, to measure ATPase activity and liposome association and dissociation of B. subtilis MinD. For these experiments would be more suitable to use B. subtilis total lipids as more biologically relevant data could be gained.

Although the work is in detail and nicely compares the function of B. subtilis Min system with E. coli Min system, it lacks the comparison of the Min system function in other rod-shaped Gram-positive bacteria. I would suggest including in the Discussion the complexity of other Min systems. Especially, this complexity is seen in other rod-shaped and spore formers such as Clostridial species in which one of these Min systems or both are present, an oscillating E. coli Min system type and more static as in B. subtilis.

Reviewer #3 (Public review):

Experimentally, this study provides sufficient data to support the authors' conclusion that MinD dimerization but not ATPase activity is both necessary and sufficient for concentrating it and its binding partner, the division inhibitor MinC, at cell poles. Biochemical data appears to be rigorously acquired and includes proper controls. Although cytological data are consistent with the authors' model, quantitative information on MinD localization in a statistically relevant set of cells is missing (e.g. Figure 2B). 

The study's other major conclusion, as outlined in their discussion, that a reaction-diffusion model explains MinD localization in wild-type cells, is unsubstantiated. If they would like to make this a major conclusion of the final manuscript, they will need to include modeling that takes into account biochemical and cytological data. 

From a presentation perspective, the manuscript is challenging to read and will require substantial rewriting and revision prior to publication.

Reviewer #1 (Public Review):

Summary:

Zheng and colleagues assessed the real world efficacy of SARS-CoV-2 vaccination against re-infection following the large omicron wave in Shanghai in April, 2022. The study was performed among previously vaccinated individuals. The study successfully documents a small but real added protective benefit of re-vaccination, though this diminishes in previously boosted individuals. Unsurprisingly, vaccine preventative efficacy was higher if the vaccine was given in the month before the 2nd large wave in Shanghai. The re-infection rate of 24% suggests that long-term anti-COVID immunity is very difficult to achieve. The conclusions are largely supported by the analyses. These results may be useful for planning the timing of subsequent vaccine rollouts.

Strengths:

The strengths of the study are a very large and unique cohort based on synchronously timed single infection among individuals with well documented vaccine histories. Statistical analyses seem appropriate. As with any cohort study, there are potential confounders and the possibility of misclassification and the authors outline limitations nicely in the discussion.

Weaknesses:

The authors have addressed each of my points thoroughly.

Reviewer #2 (Public Review):

Summary:

This paper evaluates the effect of COVID-19 booster vaccination on reinfection in Shanghai, China among individuals who received primary COVID-19 vaccination followed by initial infection, during an Omicron wave.

Strengths:

A large database is collated from electronic vaccination and infection records. Nearly 200,000 individuals are included in the analysis and 24% became reinfected.

Weaknesses:

The authors have revised the manuscript and have provided satisfactory responses to my prior comments.

Reviewer #1 (Public review):

Ellis et al. investigated the functional and topographical organization of visual cortex in infants and toddlers, as evidenced by movie-viewing data. They build directly on prior research that revealed topographic maps in infants who completed a retinotopy task, claiming that even a limited amount of rich, naturalistic movie-viewing data (3-18 minutes) is sufficient to reveal this organization, within and across participants. Generating this evidence required methodological innovations to acquire high-quality fMRI data from awake infants (which have been described by this group, elsewhere) and analytical creativity. The authors provide evidence for structured functional responses in infant visual cortex at multiple levels of analyses; homotopic brain regions (defined based on a retinotopy task) responded more similarly to one another than to other brain regions in visual cortex during movie-viewing; ICA applied to movie-viewing data revealed components that were identifiable as spatial frequency, and to a lesser degree, meridian maps, and shared response modeling analyses suggested that visual cortex responses were similar across infants/toddlers, as well as across infants/toddlers and adults. These results are suggestive of fairly mature functional response profiles in visual cortex in infants/toddlers and highlight the potential of movie-viewing data for studying finer-grained aspects of functional brain responses.

Strengths:

- This study links the authors' prior evidence for retinotopic organization of visual cortex in human infants (Ellis et al., 2021) and research by others using movie-viewing fMRI experiments with adults to reveal retinotopic organization (e.g., Knapen, 2021) to strengthen our understanding of infant vision during naturalistic contexts and further evidence for the usefulness of movie-based experiments.<br /> - This study provides novel evidence that functional alignment approaches (specifically, shared response modeling) can be usefully applied to infant fMRI data. Further, code for reproducing such analyses (and others) will be made publicly available.<br /> - Awake infant fMRI data are rare and time-consuming and expensive to collect; they are therefore of high value to the community. The raw and preprocessed fMRI and anatomical data analyzed will be made publicly available.

Weakness:

- As the authors clearly state, movie-viewing experiments may not work as well as traditional retinotopy tasks; that is, this approach cannot currently be considered a replacement for retinotopy when accurate maps are needed.

Reviewer #2 (Public review):

Summary:

This manuscript reports analyses of fMRI data from infants and toddlers watching naturalistic movies. Visual areas in the infant brain show distinct functions, consistent with previous studies using resting state and awake task-based infant fMRI. The pattern of activity in visual regions contains some features predicted by the regions' retinotopic responses. The revised version of the manuscript provides additional validation of the methodology and clarifies the claims. As a result, the data provide clear support for the claims.

Strengths:

The authors have collected a unique dataset: the same individual infants both watched naturalistic animations and a specific retinotopy task. Using these data positions the authors show that activity evoked by movies, in infants' visual areas, is correlated with the regions' retinopic response. The revised manuscript validates this methodology, using adult data. The revised manuscript also shows that an infant's movie-watching data is not sufficient or optimal to predict their visual areas' retinotopic responses; anatomical alignment with a group of previous participants provides more accurate prediction of a new participant's retinotopic response.

Weaknesses:

A key step in the analysis of the movie-watching data is the selection of independent components of the movie evoked response, by a trained researcher, that resemble retinotopic spatial patterns. While the researcher is unlikely to be biased by this infant's own retinotopy , as the authors argue, the researcher is actively looking for ICs that resemble average patterns of retinotopic response. So, how likely is it that ICs that resemble retinotopic organization arise by chance (i.e. in noise) in infant fMRI data? I do not see an analysis that addresses this question. With apologies if I missed it.

Reviewer #3 (Public review):

The manuscript reports data collected in awake toddlers recording BOLD while watching videos. The authors analyse the BOLD time series using two different statistical approaches, both very complex but that do not require any a priori determination of the movies features or contents to be associated with regressors. The two main messages are that 1) toddlers have occipital visual areas very similar to adults, given that a SRM model derive from adults BOLD is consistent with the infant brains as well; 2) the retinotopic organization and the spatial frequency selectivity of the occipital maps derived by applying correlation analysis are consistent with the maps obtained by standard and conventional mapping.

Comments on revised version:

The authors did a thorough revision of the manuscript which now is very clear. All the missing information has been added and the technical issue clarified. I think that it is a very good and important paper.

Reviewer #1 (Public review):

Summary:

In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and pro-invasive phenotype.

In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10A) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasma membrane localization correlates with high-grade DCIS cells in patient tissue samples.<br /> Specifically in non-invasive MCF10DCIS.com cells, they showed that overcrowding or over-confluence leads to a decrease in cell volume and intracellular calcium levels. This condition also triggers the trafficking of TRPV4 channels from intracellular stores (nucleus and potentially endosomes), to the plasma membrane (PM). When these over-confluent cells are incubated with a TRPV4 activator, there is an acute and substantial influx of calcium, attesting to the fact that there are a high number of TRPV4 channels present on the PM. Long-term incubation of these over-confluent cells with the TRPV4 activator results in the internalization of the PM-localized TRPV4 channels.

In contrast, cells plated at lower confluence primarily have TRPV4 channels localized in the nucleus and cytosol. Long-term incubation of these cells at lower confluence with a TRPV4 inhibitor leads to the relocation of TRPV4 channels to the plasma membrane from intracellular stores and a subsequent reduction in cell volume. Similarly, incubation of these cells at low confluence with PEG 3000 (a hyperosmotic agent) promotes the trafficking of TRPV4 channels from intracellular stores to the plasma membrane.

Strengths:

The study is elegantly designed and the findings are novel. Their findings on this mechano-transduction pathway involving TRPV4 channels, calcium homeostasis, cell volume plasticity, motility, and invasiveness will have a great impact in the cancer field and are potentially applicable to other fields as well. Experiments are well-planned and executed, and the data is convincing. The authors investigated TRVP4 dynamics using multiple different strategies- overcrowding, hyperosmotic stress, and pharmacological means, and showed a good correlation between different phenomena.

Weaknesses:

A major emphasis in the study is on pharmacological means to relate TRPV4 channel function to the phenotype. I believe the use of genetic means would greatly enhance the impact and provide compelling proof for the involvement of TRPV4 channels in the associated phenotype. In this regard, I wonder if siRNA-mediated knockdown of TRPV4 in over-confluent cells (or knockout) would lead to an increase in cell volume and normalize the intracellular calcium levels back to normal, thus ultimately leading to a decrease in cell invasiveness.

Reviewer #2 (Public review):

Summary:

The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.

Strengths:

The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in vitro and in vivo (patient samples). Activation and inhibition of the TRPV4 channel can modulate the cell volume and cell mobility. These results unveil a novel mechanism of mechanical sensing in cancer cells, potentially offering new avenues for therapeutic intervention targeting cancer metastasis by modulating TRPV4 activity. This is a very comprehensive study, and the data presented in the paper are clear and convincing. The study represents a very important advance in our understanding of the mechanical biology of cancer.

Weaknesses:

However, I do think that there are several additional experiments that could strengthen the conclusions of this work. A critical limitation is the absence of genetic ablation of the TRPV4 gene to confirm its essential role in the response to cell crowding.

Reviewer #1 (Public review):

Summary:

The use of antalarmin, a selective CRF1 receptor antagonist, prevents the deficits in sociability in (acutely) morphine-treated males, but not in females. In addition, cell-attached experiments show a rescue to control levels of the morphine-induced increased firing in PVN neurons from morphine-treated males. Similar results are obtained in CRF receptor 1-/- male mice, confirming the involvement of CRF receptor 1-mediated signaling in both sociability deficits and neuronal firing changes in morphine-treated male mice.

Strengths:

The experiments and analyses appear to be performed to a high standard, and the manuscript is well written and the data clearly presented. The main finding, that CRF-receptor plays a role in sociability deficits occurring after acute morphine administration, is an important contribution to the field.

Weaknesses:

The link between the effect of pharmacological and genetic modulation of CRF 1 receptor on sociability and on PVN neuronal firing, is less well supported by the data presented. No evidence of causality is provided.

Major points:

(1) The results of behavioral tests and the neural substrate are purely correlative. To find causality would be important to selectively delete or re-express CRF1 receptor sequence in the VPN. Re-expressing the CRF1 receptor in the VPN of male mice and testing them for social behavior and for neuronal firing would be the easier step in this direction.

(2) It would be interesting to discuss the relationship between morphine dose and CRF1 receptor expression.

(3) It would be important to show the expression levels of CRF1 receptors in PVN neurons in controls and morphine-treated mice, both males and females.

(4) It would be important to discuss the mechanisms by which CRF1 receptor controls the firing frequency of APV+/OXY+ neurons in the VPN of male mice.

Minor points:

(1) The phase of the estrous cycles in which females are analyzed for both behavior and electrophysiology should be stated.

(2) It would be important to show the statistical analysis between sexes.

Reviewer #2 (Public review):

This manuscript reports a series of studies that sought to identify a biological basis for morphine-induced social deficits. This goal has important translational implications and is, at present, incompletely understood in the field. The extant literature points to changes in periventricular CRF and oxytocin neurons as critical substrates for morphine to alter social behavior. The experiments utilize mice, administered morphine prior to a sociability assay. Both male and female mice show reduced sociability in this procedure. Pretreatment with the CRF1 receptor antagonist, antalarmin, clearly abolished the morphine effect in males, and the data are compelling. Consistently, CRF1-/- male mice appeared to be spared of the effect of morphine (while wild-type and het mice had reduced sociability). The same experiment was reported as non-feasible in females due to the effect of dose on exploratory behavior per se. Seeking a neural correlate of the behavioral pharmacology, acute cell-attached recordings of PVN neurons were made in acute slices from mice pretreated with morphine or anatalarmin. Morphine increased firing frequencies, and both antalarmin and CRF1-/- mice were spared of this effect. Increasing confidence that this is a CRF1 mediated effect, there is a gene deletion dose effect where het's had an intermediate response to morphine. In general, these experiments are well-designed and sufficiently powered to support the authors' inferences. A final experiment repeated the cell-attached recordings with later immunohistochemical verification of the recorded cells as oxytocin or vasopressin positive. Here the data are more nuanced. The majority of sampled cells were positive for both oxytocin and vasopressin, in cells obtained from males, morphine pretreatment increased firing in this population and was CRF1 dependent, however in females the effect of morphine was more modest without sensitivity to CRF1. Given that only ~8 cells were only immunoreactive for oxytocin, it may be premature to attribute the changes in behavior and physiology strictly to oxytocinergic neurons. In sum, the data provide convincing behavioral pharmacological evidence and a regional (and possibly cellular) correlation of these effects suggesting that morphine leads to sociality deficits via CRF interacting with oxytocin in the hypothalamus. While this hypothesis remains plausible, the current data do not go so far as directly testing this mechanism in a site or cell-specific way. With regard to the presentation of these data and their interpretation, the manuscript does not sufficiently draw a clear link between mu-opioid receptors, their action on CRF neurons of the PVN, and the synaptic connectivity to oxytocin neurons. Importantly, sex, cell, and site-specific variations in the CRF are well established (see Valentino & Bangasser) yet these are not reviewed nor are hypotheses regarding sex differences articulated at the outset. The manuscript would have more impact on the field if the implications of the sex-specific effects evident here were incorporated into a larger literature.

With regards to the model proposed in the discussion, it seems that there is an assumption that ip morphine or antalarmin have specific effects on the PVN and that these mediate behavior - but this is impossible to assume and there are many meaningful alternatives (for example, both MOR and CRF modulation of the raphe or accumbens are worth exploration). While it is up to the authors to conduct additional studies, a demonstration that the physiology findings are in fact specific to the PVN would greatly increase confidence that the pharmacology is localized here. Similarly, direct infusion of antalarmin to the PVN, or cell-specific manipulation of OT neurons (OT-cre mice with inhibitory dreadds) combined with morphine pre-exposure would really tie the correlative data together for a strong mechanistic interpretation.

Because the work is framed as informing a clinical problem, the discussion might have increased impact if the authors describe how the acute effects of CRF1 antagonists and morphine might change as a result of repeated use or withdrawal.

Reviewer #3 (Public review):

Summary:

In the current manuscript, Piccin et al. identify a role for CRF type 1 receptors in morphine-induced social deficits using a 3-chamber social interaction task in mice. They demonstrate that pre-treatment with a CRFR1 antagonist blocks morphine-induced social deficits in male, but not female, mice, and this is associated with the CRF R1 antagonist blocking morphine-induced increases in PVN neuronal excitability in male but not female mice. They followed up by using a transgenic mouse CRFR1 knockout mouse line. CRFR1 genetic deletion also blocked morphine-induced social deficits, similar to the pharmacological approach, in male mice. This was also associated with morphine-induced increases in PVN neuronal excitability being blocked in CRFR1 knockout mice. Interestingly they found that the pharmacological antagonism of the CRFR1 specifically blocked morphine-induced increases in oxytocin/AVP neurons in the PVN in male mice.

Strengths:

The authors used both male and female mice where possible and the studies were fairly well controlled. The authors provided sufficient methodological detail and detailed statistical information. They also examined measures of locomotion in all of the behavioral tasks to separate changes in sociability from overall changes in locomotion. The experiments were well thought out and well controlled. The use of both the pharmacological and genetic approaches provides converging lines of evidence for the role of CRFR1 in morphine-induced social deficits. Additionally, they have identified the PVN as a potential site of action for these CRFR1 effects.

Weaknesses:

While the authors included both sexes they analyzed them independently. This was done for simplicity's sake as they have multiple measures but there are several measures where the number of factors is reduced and the inclusion of sex as a factor would be possible. Additionally, single doses of both the CRFR1 antagonist and morphine are used within an experiment without justification for the doses. In fact, a lower dose of morphine was needed for the genetic CRFR1 mouse line. This would suggest that the dose of morphine being used is likely causing some aversion that may be more present in the females, as they have lower overall time in the ROI areas of both the object and the mouse following morphine exposure. As for the discussion, the authors do not sufficiently address why CRFR1 has an effect in males but not females and what might be driving that difference, or why male and female mice have different distribution of PVN cell types during the recordings. Additionally, the authors attribute their effect to CRF and CRFR1 within the PVN but do not consider the role of extrahypothalamic CRF and CRFR1. While the PVN does contain the largest density of CRF neurons there are other CRF neurons, notably in the central amygdala and BNST, that have been shown to play important roles in the impact of stress on drug-related behavior. This also holds true for the expression of CRFR1 in other regions of the brain, including the VTA, which is important for drug-related behavior and social behavior. The treatments used in the current manuscript were systemic or brain-wide deletion of CRFR1. Therefore, the authors should consider that the effects could be outside the PVN.

Reviewer #1 (Public Review):

Summary of the Study:

The manuscript delves into the COVID-19 virus membrane protein M1-subtype and its IgM responses in COVID-19 cohorts. The authors conducted an extensive epitope screening and prediction through delta of the normalized accessible surface area (DASA) and validated their findings across multiple cohorts in Europe. The study aims to provide novel insights into the immune responses to COVID-19 and explore potential clinical implications for long COVID prognostics.

Strengths:

(1) Innovative Approach:<br /> The use of DASA for epitope screening is innovative and allows for detailed mapping of immune responses.

(2) Validation Across Cohorts:<br /> The study's validation of findings across multiple European cohorts adds robustness and generalizability to the results.

(3) Comprehensive Analysis:<br /> The manuscript presents a thorough analysis of IgM responses, contributing valuable data to the understanding of immune responses in COVID-19.

Weaknesses:

(1) Lack of Clarity on T-Independent B Cell Reactions:<br /> The rationale and results regarding T-independent B cell reactions are not well-explained, requiring additional bridging sentences or data for better comprehension.

(2) Limited Sample Size for B Cell Stimulation:<br /> The in vitro B cell stimulation experiments involve a very small number of individuals (2 reacted vs 1 unreacted), which weakens the strength of the conclusions drawn from these experiments.

(3) Insufficient Exploration of Comorbidities:<br /> The manuscript could benefit from exploring correlations with other clinical data on comorbidities or sub-grouping the long COVID cohort by specific outcomes.

Appraisal of the Study's Aims and Conclusions :

The authors have partially achieved their aims by providing novel insights into COVID-19 immune responses and highlighting the potential for using IgM responses in long COVID prognostics. However, the conclusions would be more convincing with additional data and clarity on certain aspects, such as the T-independent B cell reactions and the impact of comorbidities.

Impact on the Field and Utility to the Community:

This study has the potential to significantly impact the field of COVID-19 research by advancing the understanding of immune responses to the virus. The novel insights into IgM responses and epitope screening could inform future diagnostic and prognostic tools for COVID-19, particularly in the context of long COVID. Additionally, the methods and data presented could be valuable to researchers exploring similar viral immune responses.

Additional Context:

For readers and researchers, it is essential to note that while the study offers intriguing results, the manuscript would benefit from more comprehensive data and clearer explanations in certain areas. The inclusion of the DASA equation in the manuscript or a figure would improve readability and contextual comprehension. Further exploration of clinical comorbidities and additional external validation data would enhance the study's robustness and applicability.

Reviewer #2 (Public Review):

Summary:

This paper identifies a novel SARS-CoV-2 epitope that measures host-virus interactions that have clinical correlations and can act as a signature of infection. In doing so, the authors present a novel structure-driven epitope profiling pipeline that allows them to rapidly iterate through multiple possible peptide epitope candidates for directly measuring host-virus binding. With this approach, the authors identify an IgM antibody response driven by the N-terminus of the Membrane protein of SARS-CoV-2, and demonstrate that epitope is directly correlative with cell-level measurements of infection, and can even act as a clinical signature of infection. The findings are significant to those interested in epitope identification and present a unique step forward for incorporating structural data in an iterative screening approach. The study itself presents some unique connections between the models presented, the IgM being generated, and clinical outcomes, but the claim that these IgM levels are indicative of anything more than past infection will require further detailed analysis.

Strengths:

(1) The methodological approach presented in this study is incredibly powerful and shows major promise to identify other peptide epitopes of proteins for antibody profiling. The simplicity of the methodological approach to string together established protocols and measurements offers a unique elegant promise that this is a generalizable method to many other systems and disease contexts.

(2) The clever use of a SASA metric to study and identify each of the major components demonstrates how structural information is a powerful way to approach identifying and nominating candidate peptides.

(3) This paper spans an exciting range of structural data to clinical-derived measurements, demonstrating the powerful possibilities that can arise from connecting structural biophysical data to clinical measurements to build generalized pipelines or models

Weaknesses:

(1) While the authors use SASA as a great way to screen peptides based on the presumption that SASA can act as a measure of the stability of protein folding, there are many caveats that may come with this measurement that can reduce generalizability. Assessing SASA per residue is a high variance metric that requires many additional layers of further analysis to make inferences about peptide stability. Further, since proteins are inherently dynamic, alternative configurations may yield fluctuating SASA values that inherently bias and introduce noise into the results. It would be useful to compare these SASA metrics for peptides to other structural measures often associated with protein stability used in the literature, such as Radius of Gyration, Hydrodynamic Radius, Secondary Structure degree, etc.

(2) In Figure 3G, the author put forth that IgM ELISA results and whole spike IgG correlate with one another. While it is clear that IgM for M1 and IgM for spike S1' subunit both correlate similarly to whole spike IgG levels, the correlation in both cases is incredibly weak, with whole spike IgG fluctuating widely across a narrow range of IgM for M1 values. This interpretation is also contradicted by 3G's best-fit lines that would have a large residual value to the data. Lastly, the Pearson correlation values for both correlations are misleading here as Pearson correlation indicates the strength and direction of two linear variables. This means that any dataset will inherently have a Pearson r value of ~0.40 but one may not be predictive of the other. It would be better for the authors to instead use measures such as Spearman R or additional statistical analysis like histogramming to demonstrate this coupling.

(3) It is not clear from the text if the authors are the first to use LASSO models to correlate IgM levels with infection scores in patients. LASSO-based logistic regressions are powerful tools used widely in statistical approaches to measure the association between two variables. However, there is a lack of citations indicating that the authors' approach is based on previous efforts and matches the best practice in generating these models on clinical data. It would be useful to add citations to indicate that this approach is following established statistical best practices in line with the field. If the use of the LASSO approach is novel, it would be key to mention this and highlight why the authors feel a LASSO model is the appropriate approach here.

(4) The authors demonstrate in Figure 5 that their IgM levels are very clearly correlative with a history of SARS-CoV-2 infection, and provides another avenue for the detection of prior infections. However, these claims are extended to compare to direct symptoms such as fatigue, depression, and quality of life. Specifically, the authors claim that IgM persistence is correlated with lower quality of life and stress-indicative symptoms. However, Figure 5D contradicts this, highlighting that both persistent and non-persistent IgM groups have similar trends and patterns in fatigue, depression, and quality of life. The authors should reexamine this interpretation of their data, and revisit if there are alternative analyses that may indicate where persistent and non-persistent IgM groups separate.

(5) One under-discussed component of this paper is the potential for sequence variation impacting IgM generation and detection. With resistance being a consistent issue amongst infectious diseases and immune evasion, it may be useful to discuss the possible sequence variance seen in the M protein sequence of M1, as well as to see if the IgM levels induced upon M1 presentation can be separated out from their existing analyses (it may not be!). Regardless, it would be useful for the authors to consider the potential for sequence variation in the M1 peptide and its downstream effects.

Reviewer #3 (Public Review):

Summary:

Kearns et al. explored a computational approach DASAr to identify stable peptide epitopes on SARS-CoV-2 proteins. They find that the computational approach has a high success rate at identifying stable and soluble peptides that may reserve the native conformation. The approach identified multiple peptides in Spike, Nucleoprotein, Membrane, and Envelope proteins of SARS-CoV-2. Most surprisingly, a high prevalence of IgM response is to recognize a newly exposed Membrane epitope, M1. Anti-M1 IgM titer is associated with a protective anti-Spike titer, severe disease and long COVID. The data also indicate that anti-M1 IgM may arise from T cell-independent B cell activation.

Strengths:

The computational approach can be widely applied to study antibody epitopes in many pathogens. The observations from this study provide clues to further understanding the role of anti-M1 response and the mechanisms of anti-M1 IgM response to SARS-CoV-2 associated diseases.

Weaknesses:

A subset of the conclusions of this paper are well supported by data, but some statements and analyses need to be clarified, revised, and extended.

Reviewer #1 (Public review):

Summary:

This work combines molecular dynamics (MD) simulations along with experimental elucidation of the efficacy of ATP as biological hydrotrope. While ATP is broadly known as the energy currency, it has also been suggested to modulate the stability of biomolecules and their aggregation propensity. In the computational part of the work, the authors demonstrate that ATP increases the population of the more expanded conformations (higher radius of gyration) in both a soluble folded mini-protein Trp-cage and an intrinsically disordered protein (IDP) Aβ40. Furthermore, ATP is shown to destabilise the pre-formed fibrillar structures using both simulation and experimental data (ThT assay and TEM images). They have also suggested that the biological hydrotrope ATP has significantly higher efficacy as compared to the commonly used chemical hydrotrope sodium xylene sulfonate (NaXS).

Strengths:

This work presents a comprehensive and compelling investigation of the effect of ATP on the conformational population of two types of proteins: globular/folded and IDP. The role of ATP as an "aggregate solubilizer" of pre-formed fibrils has been demonstrated using both simulation and experiments. They also elucidate the mechanism of action of ATP as a multi-purpose solubilizer in a protein-specific manner. Depending on the protein, it can interact through electrostatic interactions (for predominantly charged IDPs like Aβ40), or primarily van der Waals' interactions through (for Trp-Cage).

Weaknesses:

The weaknesses and suggestions mentioned in my first review have been adequately addressed by the authors in the revised version of the manuscript.

Reviewer #3 (Public review):

Since its first experimental report in 2017 (Patel et al. Science 2017), there have been several studies on the phenomenon in which ATP functions as a biological hydrotrope of protein aggregates. In this manuscript, by conducting molecular dynamics simulations of three different proteins, Trp-cage, Abeta40 monomer, and Abeta40 dimer at concentrations of ATP (0.1, 0.5 M), which are higher than those at cellular condition (a few mM), Sarkar et al. find that the amphiphilic nature of ATP, arising from its molecular structure consisting of phosphate group (PG), sugar ring, and aromatic base, enables it to interact with proteins in a protein-specific manner and prevents their aggregation and solubilize if they aggregate. The authors also point out that in comparison with NaXS, which is the traditional chemical hydrotrope, ATP is more efficient in solubilizing protein aggregates because of its amphiphilic nature.

Trp-cage, featured with hydrophobic core in its native state, is denatured at high ATP concentration. The authors show that the aromatic base group (purine group) of ATP is responsible for inducing the denaturation of helical motif in the native state.

For Abeta40, which can be classified as an IDP with charged residues, it is shown that ATP disrupts the salt bridge (D23-K28) required for the stability of beta-turn formation.

By showing that ATP can disassemble preformed protein oligomers (Abeta40 dimer), the authors suggest that ATP is "potent enough to disassemble existing protein droplets, maintaining proper cellular homeostasis," and enhancing solubility.

Overall, the message of the paper is clear and straightforward to follow. In addition to the previous studies in the literature on this subject. (J. Am. Chem. Soc. 2021, 143, 31, 11982-11993; J. Phys. Chem. B 2022, 126, 42, 8486-8494; J. Phys. Chem. B 2021, 125, 28, 7717-7731; J. Phys. Chem. B 2020, 124, 1, 210-223), the study, which tested using MD simulations whether ATP is a solubilizer of protein aggregates, deserves some attention from the community and is worth publishing.

Weakness

My only major concern is that the simulations were performed at unusually high ATP concentrations (100 and 500 mM of ATP), whereas the real cellular concentration of ATP is 1-5 mM.

I was wondering if there is any report on a titration curve of protein aggregates against ATP, and what is the transition mid-point of ATP-induced solubility of protein aggregates. For instance, urea or GdmCl have long been known as the non-specific denaturants of proteins, and it has been well experimented that their transition mid-points of protein unfolding are in the range of ~(1 - 6) M depending on the proteins.

The authors responded to my comment on ATP concentration that because of the computational issue in all-atom simulations, they had no option but to employ mM-protein concentrations instead of micromolar concentrations, thus requiring 1000-folds higher ATP concentration, which is at least in accordance with the protein/ATP stoichiometry. However, I believe this is an issue common to all the researchers conducting MD simulations. Even if the system is in the same stoichiometric ratio, it is never clear to me (is it still dilute enough?) whether the mechanism of solubilization of aggregate at 1000 fold higher concentration of ATP remains identical to the actual process.

Reviewer #1 (Public review):

In this manuscript, Ferhat and colleagues describe their study aimed at developing a blood brain barrier (BBB) penetrant agent that could induce hypothermia and provide neuroprotection from the sequelae of status epilepticus (SE) in mice. Hypothermia is used clinically in an attempt to reduce neurological sequelae of injury and disease. Hypothermia can be effective, but physical means used to reduce core body temperature is associated with untoward effects. Pharmacological means to induce hypothermia could be as effective with fewer untoward complications. Intracerebroventricularly applied neurotensin can cause hypothermia; however, neurotensin applied peripherally is degraded and does not cross the BBB. Here the authors develop and characterize a neurotensin conjugate that can reach the brain, induce hypothermia, and reduce seizures, cognitive changes, and inflammatory changes associated with status epilepticus.

Strengths:

(1) In general, the study is well reasoned, well designed, and seemingly well executed.<br /> (2) Strong dose-response assessment of multiple neurotensin conjugates in mice.<br /> (3) Solid assessment of binding affinity, in vitro stability ion blood, and brain uptake of the conjugate.<br /> (4) Appropriate inclusion of controls for SE and for drug injections.<br /> (5) Multifaceted assessment of neurodegeneration, inflammation, and mossy fiber sprouting in the different groups.<br /> (6) Inclusion of behavioral assessments.<br /> (7) Evaluate NSTR1 receptor distribution in multiple ways.<br /> (8) Demonstrate that this conjugate can induce hypothermia and have positive effects on the sequelae of SE. Could have great impact on the application of pharmacologically-induced hypothermia as a neuroprotective measure in patients.

Weaknesses:

(1) The authors make the claim, repeatedly, that the hypothermia caused by the neurotensin conjugate is responsible for the effects they see; however, what they really show is that the conjugate causes hypothermia AND has favorable effects on the sequelae of SE. They have now discussed this limitation in the manuscript.

Reviewer #2 (Public review):

Summary:

The authors generated analogs consisting of modified neurotensin (NT) peptides capable of binding to low density lipoprotein (LDL) and NT receptors. Their lead analog was further evaluated for additional validation as a novel therapeutic. The putative mechanism of action for NT in its antiseizure activity is hypothermia, and as therapeutic hypothermia has been demonstrated in epilepsy, NT analogs may confer antiseizure activity and avoid the negative effects of induced hypothermia.

Strengths:

The authors demonstrate an innovative approach, i.e. using LDLR as a means of transport into the brain, that may extend to other compounds. They systematically validate their approach and its potential through binding, brain penetration, in vivo antiseizure efficacy, and neuroprotection studies.

Weaknesses:

Tolerability studies are warranted, given the mechanism of action and the potential narrow therapeutic index. In vivo studies were used to assess efficacy of the peptide conjugate analogs in the mouse KA model. However, it would be beneficial to have shown tolerability in naïve animals to better understand the therapeutic potential of this approach.

Mice may be particularly sensitive to hypothermia. It would be beneficial to show similar effects in a rat model.

Reviewer #1 (Public review):

Summary

Das and Menon describe an analysis of a large open-source iEEG dataset (UPENN-RAM). From encoding and recall phases of memory tasks, they analyzed power and phase-transfer entropy as a measure of directed information flow in regions across a hypothesized tripartite network system. The anterior insula (AI) was found to have heightened high gamma power during encoding and retrieval, which corresponded to suppression of high gamma power in medial prefrontal cortex (mPFC) and posterior cingulate cortex (PCC) during encoding but not recall. In contrast, directed information flow from (but not to) AI to mPFC and PCC is high during both time periods when PTE is analyzed with broadband but not narrowband activity. They claim that these findings significantly advance an understanding of how network communication facilitates cognitive operations during memory tasks, and that the AI of the salience network (SN) is responsible for influencing both the frontoparietal network (FPN) and default-mode network (DMN) during memory encoding and retrieval.

I find this question interesting and important, and agree with the authors that iEEG presents a unique opportunity to investigate the temporal dynamics within network nodes. Their findings convey intriguing information about the structure and order of communication between network regions during on-task cognition in general (though, perhaps not specific to memory - see Weaknesses), with the AI of the SN ostensibly playing an important role in possibly influencing the DMN and FPN.

Strengths

- The authors present results from an impressively sized iEEG sample. For reader context, this type of invasive human data is difficult and time-consuming to collect and many similar studies in high-level journals include 5-20 participants, typically not all of whom have electrodes in all regions of interest. It is excellent that they have been able to leverage open-source data in this way.<br /> - Preprocessing of iEEG data also seems sensible and appropriate based on field standards.<br /> - The authors tackle the replication issues inherent in much of the literature by replicating findings across task contexts, demonstrating that the principles of network communication evidenced by their results generalize in multiple task memory contexts. Again, the number of iEEG patients who have multiple tasks' worth of data is impressive.<br /> - Though the revised manuscript presents a broader and more novel investigation of the tripartite network's role in memory encoding and retrieval (as opposed to cognitive control of memory) the authors now thoroughly review the literature motivating this investigation of open-source data.

Weaknesses

- As the authors discuss, it is currently unclear if the directed information flow from AI to DMN and FPN nodes truly arises from memory-associated processes as opposed to more general attentional and cognitive demands, especially given that information flow does not relate meaningfully to task performance (whether memory retrieval is successful or not). I also note this is a concern because - though the authors have now demonstrated that information flow is increased compared to an off-task baseline - influences of AI on DMN or FPN were not increased relative to baseline epochs during the task in the original preprint version, again suggesting these effects may not be specific to the memory component of the analyzed tasks. The authors have thoughtfully noted in the Discussion several ways that experimental design can be improved in future studies to address this limitation.

Because phase-transfer entropy is referenced as a "causal" analysis in this investigation (PTE), I believe it is important to highlight for readers recent discussions surrounding the description of "causal mechanisms" in neuroscience (see "Confusion about causation" section from Ross and Bassett, 2024, Nature Neuroscience). A large proportion of neuroscientists (myself included) use "causal" only to refer to a mechanism whose modulation or removal (with direct manipulation, such as by lesion or stimulation) is known to change or control a given outcome (such as a successful behavior). As Ross and Bassett highlight, it is debatable whether such mechanistic causality is captured by Granger "causality" (a.k.a. Granger prediction) or the parametric PTE, and imprecise use of "causation" may be confusing. The authors have defined in the revised Introduction what their definition of "causality" is within the context of this investigation.

Reviewer #2 (Public review):

Based on reviewer feedback, Das and Menon have made several modifications to their manuscript, including a revised Introduction with a reframed motivation (now more oriented around the role of tripartite network in memory operations), new control analyses (as requested by Reviewers, including an updated and more appropriate baseline period and a control region, the IFG), an assessment of narrowband phase synchronization (as requested), as well as updates for clarity throughout the Methods section.

While I believe the authors have been responsive to reviewer feedback, and these modifications do enhance the manuscript, I have a few suggestions for how these new analyses could be made more statistically robust and better contextualized against the main findings of the manuscript. I continue to have some reservations about a tendency for their data to be overinterpreted, and for conclusions to be drawn more strongly than the data actually warrant.

(1) Clarifying the new control analyses. The authors have been responsive to our feedback and implemented several new analyses. The use of a pre-task baseline period and a control brain region (IFG) definitively help to contextualize their results, and the findings shown in the revision do suggest that (1) relative to a pre-task baseline, directed interactions from the AI are stronger and (2) relative to a nearby region, the IFG, the AI exhibits greater outward-directed influence.

However, it is difficult to draw strong quantitative conclusions from the analyses as presented, because they do not directly statistically contrast the effect in question (directed interactions with the FPN and DMN) between two conditions (e.g. during baseline vs. during memory encoding/retrieval). As I understand it, in their main figures the authors ask, "Is there statistically greater influence from the AI to the DMN/FPN in one direction versus another?" And in the AI they show greater "outward" PTE than "inward" PTE from other networks during encoding/retrieval. The balance of directed information favors an outward influence from the AI to DMN/FPN.

But in their new analyses, they simply show that the degree of "outward" PTE is greater during task relative to baseline in (almost) all tasks. I believe a more appropriately matched analysis would be to quantify the inward/outward balance during task states, quantify the inward/outward balance during rest states, and then directly statistically compare the two. It could be that the relative balance of directed information flow is non-significantly changed between task and rest states, which would be important to know.

Likewise, a similar principle applies to their IFG analysis. They show that the IFG tends to have an "inward" balance of influence from the DMN/FPN (the opposite of the AIs effect), but this does not directly answer whether the AI occupies a statistically unique position in terms of the magnitude of its influence on other regions. More appropriate, as I suggest above, would be to quantify the relative balance inward/outward influence, both for the IFG and the AI, and then directly compare those two quantities. (Given the inversion of the direction of effect, this is likely to be a significant result, but I think it deserves a careful approach regardless.)

(2) Consider additional control regions. The authors justify their choice of IFG as a control region very well. In my original comments, I perhaps should have been more clear that the most compelling control analyses here would be to subject every region of the brain outside these networks (with good coverage) to the same analysis, quantify the degree of inward/outward balance, and then see how the magnitude of the AI effect stacks up against all possible other options. If the assertion is that the AI plays a uniquely important role in these memory processes, showing how its influence stacks up against all possible "competitors" would be a very compelling demonstration of their argument.

(3) Reporting of successful vs. unsuccessful memory results. I apologize if I was not clear in my original comment (2.7, pg. 13 of the response document) regarding successful vs. unsuccessful memory. The fact that no significant difference was found in PTE between successful/unsuccessful memory is a very important finding that adds valuable context to the rest of the manuscript. I believe it deserves a figure, at least in the Supplement, so that readers can visualize the extent of the effect in successful/unsuccessful trials. This is especially important now that the manuscript has been reframed to focus more directly on claims regarding episodic memory processing; if that is indeed the focus, and their central analysis does not show a significant effect conditionalized on the success of memory encoding/retrieval, it is important that readers can see these data directly.

(4) Claims regarding causal relationships in the brain. I understand that the authors have defined "causal" in a specific way in the context of their manuscript; I do believe that as a matter of clear and transparent scientific communication, the authors nonetheless bear a responsibility to appreciate how this word may be erroneously interpreted/overinterpreted and I would urge further review of the manuscript to tone down claims of causality. Reflective of this, I was very surprised that even as both reviewers remarked on the need to use the word "causal" with extreme caution, the authors added it to the title in their revised manuscript.

Reviewer #1 (Public Review):

Summary:

This work focuses on the structure and regulation of the Anaphase-Promoting Complex/Cyclosome (APC/C), a large multi-subunit ubiquitin ligase that controls the onset of chromosome segregation in mitosis. Previous high-resolution structural studies have uncovered numerous structural features and regulatory mechanisms of the human APC/C, but it has remained unclear if these mechanisms are conserved in other model eukaryotes. To address this gap in our understanding, the authors employed cryo-electron microscopy to generate structural models of APC/C from the budding yeast S. cerevisiae, a key model organism in cell cycle analysis. In their comparison of the human and yeast complexes, the authors uncover many conserved structural features that are documented here in detail, revealing widespread similarities in the fundamental structural features of the enzyme. Interestingly, the authors also find evidence that two of the key mechanisms of human APC/C regulation are not conserved in the yeast enzyme. Specifically:

(1) The ubiquitin ligase activity of the APC/C depends on its association with a co-activator subunit such as CDH1 or CDC20, which serves both as a substrate-binding adaptor and as an activator of interactions with the E2 co-enzyme. Previous studies of the human APC/C revealed that co-activator binding induces a conformational change that enables E2 binding. In contrast, the current work shows that this E2-binding conformation already exists in the absence of a co-activator in the yeast enzyme, suggesting that the enhancement of E2 binding in yeast depends on other, as yet undiscovered, mechanisms.

(2) APC/C phosphorylation on multiple subunits is known to enhance APC/C activation by the CDC20 co-activator in mitosis. Previous studies showed that phosphorylation acts by promoting the displacement of an autoinhibitory loop that occupies part of the CDC20-binding site. In the yeast enzyme, however, there is no autoinhibitory loop in the CDC20-binding site, and there is no apparent effect of APC/C phosphorylation on co-activator binding sites. Thus, phosphorylation activates the yeast CDC20-APC/C by unknown mechanisms.

Strengths:

The strength of this paper is that it provides a comprehensive analysis of yeast APC/C structure and how it compares to previously determined human structures. The article systematically unwraps the key features of the structure in a subunit-by-subunit fashion, carefully revealing the key features that are the same or different in the two species. These descriptions are based on a thorough overview of past work in the field; indeed, this article serves as a concise review of the key features, conserved or otherwise, of APC/C structure and regulation.

Weaknesses:

No significant weaknesses were identified.

Reviewer #2 (Public Review):

Summary:

This paper from the Barford lab describes medium/high-resolution cryo-EM structures of three versions of the S. cerevisiae anaphase-promoting complex/cyclosome (APC/C):

(1) the recombinant apo complex purified from insect cells,

(2) the apo complex phosphorylated in vitro by cyclin-dependent kinase, and

(3) an active APC/C-Cdh1-substrate ternary complex.

The focus of the paper is on comparing similarities and differences between S. cerevisiae and human APC/C structures, mechanisms of activation by coactivator, and regulation by phosphorylation. The authors find that the overall structures of S. cerevisiae and human APC/C are remarkably similar, including the binding sites and orientation for the substrate-recruiting coactivator, Cdh1. In addition, the mechanism of Cdh1 inhibition by phosphorylation appears conserved across kingdoms. However, key differences were also observed that reveal divergence in APC/C mechanisms that are important for researchers in this field to know. Specifically, the mechanism of APC/C-Cdc20 activation by mitotic phosphorylation appears to be different, due to the absence of the key Apc1 autoinhibition loop in the S. cerevisiae complex. In addition, the conformational activation of human APC/C by coactivator binding was not observed in the S. cerevisiae complex, implying that stimulation of E2 binding must occur via a different mechanism in this species.

Strengths:

Consistent with the numerous prior cryo-EM structures of human APC/C from the Barford lab, the technical quality of the structure models is a major strength of this work. In addition, the detailed comparison of similarities and differences between the two species will be a very valuable resource for the scientific community. The manuscript is written very well and allows readers lacking expertise in cryo-EM to understand the important aspects of the conservation of APC/C structure and mechanism across kingdoms.

Weaknesses:

The lack of experimentation in this work to test some of the putative differences in APC/C mechanism (e.g. stimulation of E2 binding by coactivator and stimulation of activity by mitotic phosphorylation) could be considered a weakness. Nonetheless, the authors do a nice job explaining how the structure interpretations imply these differences likely exist, and this work sets the stage nicely for future studies to understand these differences at a mechanistic level. There is enough value in having the S. cerevisiae structure models and the comparison to the human structures, without any additional experimentation.

The validation of APC/C phosphorylation in the unphosphorylated and hyperphosphorylated states is not very robust. Given the lack of significant effects of phosphorylation on APC/C structure observed here (compared to the human complex), this becomes important. A list of phosphorylation sites identified by mass spec before and after in vitro phosphorylation is provided but lacks quantitative information. This list indicates that a significant number of phosphorylation sites are detected in the purified APC/C prior to reaction with purified kinases. Many more sites are detected after in vitro kinase reaction, but it isn't clear how extensively any of the sites are modified. There is reason for caution then, in accepting the conclusions that structures of unphosphorylated and hyperphosphorylated APC/C from S. cerevisiae are nearly identical.

Reviewer #3 (Public Review):

Vazquez-Fernandez et al. present a comprehensive and detailed analysis of the S. cerevisiae APC/C complex, providing new insights into its structure and function. The authors determined the medium-resolution structures of three recombinant S. cerevisiae APC/C complexes, including unphosphorylated apo-APC/C (4.9 Å), the ternary APC/CCDH1-substrate complex (APC/CCDH1:Hsl1 , 4.0 Å), and phosphorylated apo-APC/C (4.4 Å). Prior structures of human, E. cuniculi, S. cerevisiae, and S. pombe APC/C subunits, as well as AlphaFold2 predictions were used to guide model building. Although the determined structures are not sufficient to fully explain the molecular mechanism of APC/C activation and regulation in S. cerevisiae, they provide valuable insights into the similarities and differences with the human complex, shedding light on the conserved and divergent features of APC/C function.

The manuscript synthesizes the structural analysis of the APC/C complex in S. cerevisiae, with literature into a cohesive and clear picture of the complex's structure and function. It is well-written and clear, making the complex biology of the APC/C complex accessible to a wide range of readers. The complex forms a triangular shape, with a central cavity surrounded by two modules: the TPR lobe and the platform module. The TPR lobe consists of three TPR proteins (APC3, APC6, and APC8), which stack on top of each other to form a quasi-symmetric structure. The platform module is composed of the large APC1 subunit, together with APC4 and APC5. The authors also analyzed the structure of several smaller subunits that are involved in regulating the activity of the APC/C complex and showed their structural similarities to and discrepancies from their human counterparts. These subunits, including CDC26/APC12, SWM1/APC13, APC9, and MND2/APC15, form extended, irregular structures that simultaneously contact multiple large globular APC/C subunits.

While the authors report the similarity between the overall structure of S. cerevisiae and human APC/C complexes, they also found two unexpected differences. First, in the S. cerevisiae apo-complex, the E2 binding site on APC11RING is accessible, whereas, in humans, it requires CDH1 binding. Second, a structural element similar to the human APC1 auto-inhibitory segment is missing in S. cerevisiae. In humans, the phosphorylation-dependent displacement of this segment allows CDC20 binding to APC/C. In S. cerevisiae, the binding requires phosphorylation however the structures reported here are suggestive that this could involve a different (presently unknown) mechanism. These structural insights highlight the importance of understanding the species-specific features of APC/C function.

Strengths:

The manuscript does a great job of revealing new structures.

Opportunity for increasing impact: It would have been nice if some functional differences were demonstrated, for example regarding the mechanism of CDC20 binding, and the comparison between apo-APC/C and ternary APC/CCDH1:Hsl1 does not explain the molecular activation mechanism of S. cerevisiae APC/C. Nonetheless, the authors nicely integrate their data with well-established literature on the similarities and differences between yeast and human systems.

Reviewer #1 (Public Review):

The study identifies the epigenetic reader SntB as a crucial transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus, although the precise molecular mechanisms remain elusive. Using homologous recombination, researchers constructed sntB gene deletion (ΔsntB), complementary (Com-sntB), and HA tag-fused sntB (sntB-HA) strains. Results indicated that deletion of the sntB gene impaired mycelial growth, conidial production, sclerotia formation, aflatoxin synthesis, and host colonization compared to the wild type (WT). The defects in the ΔsntB strain were reversible in the Com-sntB strain.

Further experiments involving ChIP-seq and RNA-seq analyses of sntB-HA and WT, as well as ΔsntB and WT strains, highlighted SntB's significant role in the oxidative stress response. Analysis of the catalase-encoding catC gene, which was upregulated in the ΔsntB strain, and a secretory lipase gene, which was downregulated, underpinned the functional disruptions observed. Under oxidative stress induced by menadione sodium bisulfite (MSB), the deletion of sntB reduced catC expression significantly. Additionally, deleting the catC gene curtailed mycelial growth, conidial production, and sclerotia formation, but elevated reactive oxygen species (ROS) levels and aflatoxin production. The ΔcatC strain also showed reduced susceptibility to MSB and decreased aflatoxin production compared to the WT.

This study outlines a pathway by which SntB regulates fungal morphogenesis, mycotoxin synthesis, and virulence through a sequence of H3K36me3 modification to peroxisomes and lipid hydrolysis, impacting fungal virulence and mycotoxin biosynthesis.

The authors have achieved the majority of their aims at the beginning of the study, finding target genes, which led to catC mediated regulation of development, growth and aflatoxin metabolism. Overall most parts of the study are solid and clear.

Comments on revision:

The authors have thoroughly addressed all the concerns I raised. The current manuscript is robust and effectively presents evidence supporting its claims. The overall quality of the manuscript has significantly improved.

Reviewer #2 (Public Review):

The authors fully addressed my concerns and made appropriate changes in the manuscript. The quality of the manuscript is now significantly improved.

Reviewer #1 (Public review):

Summary:

The study used root tips from semi-hydroponic tea seedlings. The strategy followed sequential steps to draw partial conclusions.

Initially, protoplasts obtained from root tips were processed for scRNA-seq using the 10x Genomics platform. The sequencing data underwent pre-filtering at cell and gene levels, leading to 10,435 cells. These cells were then classified into eight clusters using t-SNE algorithms. The present study scrutinised cell typification through protein sequence similarity analysis of homologs of cell type marker genes. The analysis was conducted to ensure accuracy using validated genes from previous scRNA-seq studies and the model plant Arabidopsis thaliana. The cluster cell annotation was confirmed using in situ RT-PCR analyses. This methodology provided a comprehensive insight into the cellular differentiation of the sample under study. The identified clusters, spanning 1 to 8, have been accurately classified as xylem, epidermal, stem cell niche, cortex/endodermal, root cap, cambium, phloem, and pericycle cells.

Then, the authors performed a pseudo-time analysis to validate the cell cluster annotation by examining the differentiation pathways of the root cells. Lastly, they created a differentiation heatmap from the xylem and epidermal cells and identified the biological functions associated with the highly expressed genes.

Upon thoroughly analysing the scRNA-seq data, the researchers delved into the cell heterogeneity of nitrate and ammonium uptake, transport, and nitrogen assimilation into amino acids. The scRNA-seq data was validated by in situ RT-PCR. It allows the localisation of glutamate and alanine biosynthetic enzymes along the cell clusters and confirms that both constituent the primary amino acid metabolism in the root. Such investigation was deemed necessary due to the paramount importance of these processes in theanine biosynthesis since this molecule is synthesised from glutamate and alanine-derived ethylamine.

Afterwards, the authors analysed the cell-specific expression patterns of the theanine biosynthesis genes, combining the same molecular tools. They concluded that theanine biosynthesis is more enriched in cluster 8 "pericycle cells" than glutamate biosynthesis (Lines 271-272). However, the statement made in Line 250 states that the highest expression levels of genes responsible for glutamate biosynthesis were observed in Clusters 1, 3, 4, 6 and 8, leading to an unclear conclusion.<br /> The regulation of theanine biosynthesis by the MYB transcription factor family is well-established. In particular, CsMYB6, a transcription factor expressed specifically in roots, has been found to promote theanine biosynthesis by binding to the promoter of the TSI gene responsible for theanine synthesis. However, their findings indicate that CsMYB6 expression is present in Cluster 3 (SCN), Cluster 6 (cambium cells), and Cluster 1 (xylem cells) but not in Cluster 8 (pericycle cells), which is known for its high expression of CsTSI. Similarly, their scRNA-seq data indicated that CsMYB40 and CsHHO3, which activate and repress CsAlaDC expression, respectively, did not show high expression in Cluster 1 (the cell cluster with high CsAlaDC expression). Based on these findings, the authors speculated that transcription factors and target genes are not necessarily always highly expressed in the same cells.

Lastly, the authors have discovered a novel transcription factor belonging to the Lateral Organ Boundaries Domain (LBD) family known as CsLBD37 that can co-regulate the synthesis of theanine and the development of lateral roots. The authors observed that CsLBD37 is located within the nucleus and can repress the CsAlaDC promoter's activity. To investigate this mechanism further, the authors conducted experiments to determine whether CsLBD37 can inhibit CsAlaDC expression in vivo. They achieved this by creating transiently CsLBD37-silenced or over-expression tea seedlings through antisense oligonucleotide interference and generation of transgenic hairy roots. Based on their findings, the authors theorise that CsLBD37 regulates CsAlaDC expression to modulate the synthesis of ethylamine and theanine in tea roots. Apologies for the inadvertent mistake concerning glutamate and glutamine.

Strength:

The manuscript showcases significant dedication and hard work, resulting in valuable insights that are fundamental for generating knowledge. The authors skillfully integrated various tools available for this type of study and meticulously presented and illustrated every step involved in the survey. The overall quality of the work is exceptional, and it would be a valuable addition to any academic or professional setting.

Weaknesses:

The authors have effectively addressed the feedback and revised the manuscript, presenting their debatable conclusions as speculative. Consequently, I find the manuscript's current form free of any apparent weaknesses.

Reviewer #2 (Public review):

Summary:

In their manuscript, Lin et al. present a comprehensive single-cell analysis of tea plant roots. They measured the transcriptomes of 10,435 cells from tea plant root tips, leading to the identification and annotation of 8 distinct cell clusters using marker genes. Through this dataset, they delved into the cell-type-specific expression profiles of genes crucial for the biosynthesis, transport, and storage of theanine, revealing potential multicellular compartmentalization in theanine biosynthesis pathways. Furthermore, their findings highlight CsLBD37 as a novel transcription factor with dual regulatory roles in both theanine biosynthesis and lateral root development.

Strengths:

This manuscript provides the first single-cell dataset analysis of roots of the tea plants. It also enables detailed analysis of the specific expression patterns of the gene involved in theanine biosynthesis. Some of these gene expression patterns in roots were further validated through in-situ RT-PCR. Additionally, a novel TF gene CsLBD37's role in regulating theanine biosynthesis was identified through their analysis.

Weaknesses:

The revised manuscript has addressed the concerns raised during the initial review.

Reviewer #3 (Public review):

Summary:

Lin et al., performed a scRNA-seq-based study of tea roots, as an example, to elucidate the biosynthesis and regulatory processes for theanine, a root-specific secondary metabolite, and established the first map of tea roots comprised of 8 cell clusters. Their findings contribute to deepening our understanding of the regulation of the synthesis of important flavor substances in tea plant roots. They have presented some innovative ideas.

Comment on revised version:

The reviewer has addressed all my concerns and I have no further comments.

Reviewer #1 (Public review):

Summary:

This manuscript addresses two main issues: (i) do MAPKs play an important role in SAC regulation in single cell organism such as S pombe? (ii) what is the nature of their involvement and what are their molecular targets?<br /> The authors have extensively used the cold-sensitive β-tubulin mutant to activate or inactivate SAC employing an arrest-release protocol. Localization of Cdc13 (cyclin B) to the SPBs is used as a readout for the SAC activation or inactivation. The roles of two major MAPK pathways i.e. stress activated pathway (SAP) and cell integrity pathway (CIP), have been explored in this context (with CIP more extensively than SAP). Sty1Δ or pmk1Δ mutants were used to inactivate the SAP or CIP pathways and wis1DD or pek1DD expression was utilized to constitutively activate these pathways, respectively. Lowering of Slp1Cdc20 abundance (by phosphorylation of Slp1-Thr 480) is revealed as the main function of MAPK to augment the robustness of spindle assembly checkpoint.

Strengths:

The experiments are generally well-conducted, and the results support the interpretations in various sections. The experimental data clearly support some of the key conclusions:<br /> (i) while inactivation of SAP and CIP compromises SAC-imposed arrest, their constitutive activation delays the release from the SAC-imposed arrest (ii) CIP signaling, but not SAP signaling, attenuates Slp1Cdc20 levels (iii) Pmk1 and Cdc20 physical interact and Pmk1-docking sequences in Slp1 (PDSS) is identifies and confirmed by mutational/substitution experiments (iv) Thr480 (and also S76) is identified as the residue phosphorylated by Pmk1. S28 and T31 are identified as Cdk1 phosphorylation sites. These are confirmed by mutational and other related analyses (v) Functional aspects of the phosphorylation sites have been elucidated to some extent: (a) Phosphorylation of Slp1-T480 by Pmk1 reduces its abundance thereby augmenting the SAC-induced arrest (b) S28, T31 (also S59) are phosphorylated by Cdk1 (v) K472 and K479 residues are involved in ubiquitylation of Slp1

Weaknesses:

(i) Cdc13 localization to SPBs has been used as a readout for SAC activation/inactivation throughout the manuscript. However, the only image showing such localization (Figure 1C) is of poor quality where the Cdc13 localization to SPBs barely visible. This should be replaced by a better image.

(ii) The overlapping error-bars in Cdc13-localization data in some figures (for instance Figure 3E and 4H) makes the effect of various mutations on SAC activation/inactivation rather marginal. In some of these cases, Western-blotting data support the author's conclusions better.

(iii) This specific point is not really a weakness but rather a loose end:<br /> One of the conclusions of this study is that MAPK (PMK1) contributes to the robustness of SAC-induced arrest by lowering the abundance of Slp1Cdc20. The authors have used pmk1Δ or constitutively activating the MAPK pathways (Pek1DD) and documenting their effect on SAC activation/inactivation dynamics. It is not clear if SAC activation also leads to activation of MAPK pathways for them to contribute to the SAC robustness. To tie this loose end, the author could have checked if MAPK pathway is also activated under the conditions when SAC is activated. Unless this is shown, one must assume that the authors are attributing the effect they observe to the basal activity of MAPKs.

(iv) This is also a loose end:<br /> The authors show that activation of stress pathways (by addition of KCL instance) causes phosphorylation-dependent Slp1Cdc20 downregulation (Figure 6) under SAC-activating conditions. Does activation of the stress pathway cause phosphorylation-dependent Slp1Cdc20 downregulation under non-SAC-activation conditions or does it occur only under SAC-activating conditions?

(v) Although the authors have gone to some length to identify S28, T31 (also S59) as phosphorylation sites for Cdk1, their functional significance in the context of MAPK involvement is not yet clear. Perhaps it is outside the scope of this study to dig deeper into this aspect more than the authors have.

(vi) In its current state, the Discussion section is quite disjointed. The first section "Involvement of MAPKs in cell cycle regulation" should be in the Introduction section (very briefly, if at all). It certainly does not belong to the Discussion section. In any case, the Discussion section should be more organized with better flow of arguments/interpretations.

Reviewer #2 (Public review):

Summary:

This study by Sun et al. presents a role for the S. pombe MAP kinase Pmk1 in the activation of the Spindle Assembly Checkpoint (SAC) via controlling the protein levels of APC/C activator Cdc20 (Slp1 in S. pombe). The data presented in the manuscript is thorough and convincing. The authors have shown that Pmk1 binds and phosphorylates Slp1, promoting its ubiquitination and subsequent degradation. Since Cdc20 is an activator of APC/C, which promotes anaphase entry, constitutive Pmk1 activation leads to an increased percentage of metaphase-arrested cells. The authors have used genetic and environmental stress conditions to modulate MAP kinase signalling and demonstrate their effect on APC/C activation. This work provides evidence for the role of MAP kinases in cell cycle regulation in S. pombe and opens avenues for exploration of similar regulation in other eukaryotes.

Strengths:

The authors have done a very comprehensive experimental analysis to support their hypothesis. The data is well represented, and including a model in every figure summarizes the data well.

Weaknesses:

As mentioned in the comments, the manuscript does not establish that MAP kinase activity leads to genome stability when cells are subjected to genotoxic stressors. That would establish the importance of this pathway for checkpoint activation.

Reviewer #1 (Public review):

Summary:

The manuscript characterizes a functional peptidergic system in the echinoderm Apostichopus japonicus that is related to the widely conserved family of calcitonin/diuretic hormone 31 (CT/DH31) peptides in bilaterian animals. In vitro analysis of receptor-ligand interactions, using multiple receptor activation assays, identifies three cognate receptors for two CT-like peptides in the sea cucumber, which stimulate cAMP, calcium, and ERK signaling. Only one of these receptors is closely related to the family of calcitonin and calcitonin-like receptors (CTR/CLR) in bilaterian animals, whereas two other receptors cluster with invertebrate pigment dispersing factor receptors (PDFRs). In addition, this study sheds light on the transcript expression and in vivo functions of CT-like peptides in A. japonicus, by quantitative real-time PCR, in situ hybridization, pharmacological experiments on body wall muscle and intestine preparations, and peptide injection and RNAi knockdown experiments. This reveals a conserved function of CT-like peptides as muscle relaxants and hints at a potential role as growth regulators in A. japonicus.

Strengths:

This work combines both in vitro and in vivo functional assays to identify a CT-like peptidergic system in an economically relevant echinoderm species, the sea cucumber A. japonicus. A major strength of the study is that it identifies three G protein-coupled receptors for AjCT-like peptides, one related to the CTR/CLR family and two related to the PDFR family. A similar finding was previously reported for the CT-related peptide DH31 in Drosophila melanogaster that activates both CT-type and PDF-type receptors. Here, the authors expand this observation to a deuterostomian animal, which suggests that receptor promiscuity is a more general feature of the CT/DH31 peptide family and that CT/DH31-like peptides may activate both CT-type and PDF-type receptors in other animals as well.

Besides the identification of receptor-ligand pairs, the downstream signaling pathways of AjCT receptors have been characterized, highlighting broad effects on cAMP, calcium, and ERK signaling. Functional characterization of the CT-related peptide system in heterologous cells is complemented with ex vivo and in vivo experiments. First, peptide injection and RNAi knockdown experiments establish transcriptional regulation of all three identified receptors in response to changing AjCT peptide levels. Second, ex vivo experiments reveal a conserved role for the two CT-like peptides as muscle relaxants, which have differential effects on body wall muscle and intestine preparations. Finally, peptide injection studies suggest a putative role for one of the two CT-like peptides (AjCT2) in growth regulation.

Weaknesses:

Analysis of transcript expression is limited to the CT-peptide encoding gene, while no gene expression analysis was attempted for the three identified receptors. Differences in the activation of downstream signaling pathways between the three receptors are also questionable due to unclarities in the statistical analysis and variation in the control and experimental data in heterologous assays. Together, this makes it difficult to propose a mechanism underlying differences in the functions of the two CT-like peptides in muscle control and growth regulation.

The authors also suggest a putative orexigenic role for the CT-like peptidergic system in feeding behavior. This effect is not well supported by the experimental data provided, as no detailed analysis of feeding behavior was carried out (only indirect measurements were performed that could be influenced by other peptidergic effects, such as on muscle relaxation) and no statistically significant differences were reported in these assays.

Overall, details regarding statistical analyses are not (clearly) specified in the manuscript, and there are several instances where statements are not supported by literature evidence.

Reviewer #2 (Public review):

Summary:

The authors show that A. japonicus calcitonins (AjCT1 and AjCT2) activate not only the calcitonin/calcitonin-like receptor but also activate the two PDF receptors, ex vivo. They also explore secondary messenger pathways that are recruited following receptor activation. They determine the source of CT1 and CT2 using qPCR and in situ hybridization and finally test the effects of these peptides on tissue contractions, feeding, and growth. This study provides solid evidence that CT1 and CT2 act as ligands for calcitonin receptors; however, evidence supporting cross-talk between CT peptides and PDF receptors is only based on ex vivo experiments.

Strengths:

This is the first study to report the pharmacological characterization of CT receptors in an echinoderm. Multiple lines of evidence in cell culture (receptor internalization and secondary messenger pathways) support this conclusion.

Weaknesses:

The authors claim that A. japonicus CTs activate "PDF" receptors and suggest that this cross-talk is evolutionarily ancient since a similar phenomenon also exists in the fly Drosophila melanogaster. These conclusions are not fully supported for several reasons. The authors perform phylogenetic analysis to show that the two "PDF" receptors form an independent clade. This clade is sister to the clade comprising CT receptors. This phylogenetic analysis suffers from several issues. Firstly, the phylogenies lack bootstrap support. Secondly, the resolution of the phylogeny is poor because representative members from diverse phyla have not been included. For instance, insect or other protostomian PDF receptors have not been included so how can the authors distinguish between "PDF" receptors or another group of CT receptors? Thirdly, no in vivo evidence has been presented to support that CT can activate "PDF" receptors in vivo.

The source of CT which mediates the effects on longitudinal muscles and intestine is unclear. Is it autocrine or paracrine signaling by CT from the same tissue or is it long-range hormonal signaling?

Pharmacology experiments showing the effects of CT1 and CT2 on ACh-induced contractions were performed. Sample traces have been provided but no traces with ACh alone have been included. How long do ACh-induced contractions persist? These controls are necessary to differentiate between the eventual decay of ACh effects and relaxation induced by CT1 and CT2. The traces also do not reflect the results portrayed in dose-response curves. For instance, in Figure 6B, maximum relaxation is reported for 10-6M. Yet, the trace hardly shows any difference before and after the addition of 10-6M peptide. The maximum effect in the trace appears to be after the addition of 10-8M peptide.

I am unsure how differences in wet mass indicate feeding and growth differences since no justification has been provided. Couldn't wet mass also be influenced by differences in osmotic balance, a key function of calcitonin-like peptides in protostomian invertebrates? The statistical comparisons have not been included in Figure 7B.

While the authors succeeded in knocking down CT, the physiological effects of reduced CT signaling were not examined.

Reviewer #1 (Public review):

Summary:

In this study, the authors use ChEC-seq, an MNase-based method to map yeast RNA pol II. Part of the reasoning for this study is that earlier biochemical work suggested pol II initiation and termination should involve slow steps at the UAS/promoter and termination regions that are not well visualized by formaldehyde-based ChIP methods. Here the authors find that pol II ChIP and ChEC give complementary patterns. Pol II ChIP signals are strongest in the coding region (where ChIP signal correlates well with transcription (rho = 0.62)). In contrast, pol II ChEC signals are strongest at promoters (rho = 0.52) and terminator regions. Weaker upstream ChEC signals are also observed at the STM class genes where biochemical studies have suggested a form of Pol (and maybe other general factors) is recruited to UAS sites. ChEC of TFIIA and TFIIE give promoter-specific ChEC signals as expected. Extending this work to elongation factors Ctk1 and Spt5 unexpectedly give strong signals near the PIC location and little signals over the coding region. This, and mapping CTD S2 and S5 phosphorylation by ChEC suggests to me that, for some reason, ChEC isn't optimal for detecting components of the elongation complex over coding regions.

Examples are also presented where perturbations of transcription can be measured by ChEC. Modeling studies are shown where adjustment of kinetic parameters agree well with ChEC data and that these models can be used to estimate which steps in transcription are affected by various perturbations. No tests were performed to see if the predictions could be validated by other means. Finally, the role of nuclear pore binding by Gcn4 is explored, although the results do not seem convincing. Overall, the authors show that pol II ChEC is a valuable and complementary method for investigating transcription mechanisms and slow steps at the initiation and termination regions.

Reviewer #2 (Public review):

Summary:

The study by VanBelzen et. al. compares chromatin immunoprecipitation (ChIP-seq) and chromatin endogenous cleavage sequencing (ChEC-seq2) to examine RNA polymerase II (RNAPII) binding patterns in yeast. While ChIP-seq shows RNAPII enrichment mainly over transcribed regions, ChEC-seq2 highlights RNAPII binding at promoters and upstream activating sequences (UASs), suggesting it captures distinct RNAPII populations that the authors speculate are linked more tightly to active transcription. The authors develop a stochastic model for RNAPII kinetics using ChEC-seq2 data, revealing insights into transcription regulation and the role of the nuclear pore complex in stabilizing promoter-associated RNAPII. The study suggests that ChEC-seq2 identifies regulatory events that ChIP-seq may overlook.

Strengths:

(1) This is a carefully crafted study that adds significantly to existing literature in this area. Transgenic MNase fusions with endogenous Rpb1 and Rpb3 subunits were carefully performed, and complemented by fusions with several additional proteins that help the authors to dissect the transcription cycle. Both the S. cerevisiae lines and the sequencing data are likely to be of significant use to the community.

(2) The validation of ChEC-seq2 and its comparison with ChIP-seq is highly valuable technical information for the community.

(3) The kinetic modeling appears to be thoughtfully done.

Weaknesses:

(1) The term "nascent transcription" is all too often used interchangeably for NET-seq, PRO-seq, 4sU-seq, and other assays that often provide different types of information. The authors should make it clear their use of the term refers to SLAM-seq data.

(2) The authors do not perform any comparison to run-on (PRO-seq) data. My impression is that the distribution of PRO-seq signal in S. cerevisiae agrees better with the distribution the authors observe by ChIP-seq. PRO-seq only captures RNAPII that is engaged and actively transcribing. If PRO-seq does indeed provide a similar profile as ChIP-seq, wouldn't this indicate that the high frequency of association between RNAPII and either the promoter or UAS reflects RNAPII that has not yet started transcription elongation? Perhaps this could help sort out what types of activities are occurring at the UAS (which does not appear to require a full PIC) or at the promoter (which does)?

Reviewer #1 (Public Review):

Summary:

In a previous study, the authors developed a human iPS cell line which expresses Cre under the control of the Lmx1a promoter in order to track, select for, and differentiate human dopamine neurons. In the manuscript under review, the authors are using methods which they have modified to generate astrocytes from the same cell line. The authors are interested in examining astrocytes which are derived from regionalized, floor plate progenitors.

The fundamental weakness of this paper is that the authors are making arguments about regional identity but their work is limited to experiments in vitro. Some of the claims that the authors make should be tested in vivo - ie, in sections, at least. Are floor plate markers or other ventral markers ever expressed in astrocytes or glial progenitors in the mammalian fetus? When do astrocytes emerge in the floor plate? All of the data here are based on an overly simplified in vitro platform.

Lmx1a expression is not limited to the ventral midbrain; it is also expressed in other parts of the developing, ventral CNS and in the roof plate and dorsal CNS (Millonig et al, Nature 2000). Indeed, many of the phenotypes of the Lmx1a mutant mouse (dreher) have little to do with the ventral midbrain. The authors are making an assumption that regional identity is fixed when they begin their astrocyte differentiation protocol - not necessarily true. After astrocytic differentiation is initiated, the authors have done little to demonstrate that floor plate identity is maintained even in selected cells; in fact, the transcriptomic data suggests that the cells are released from a floor plate fate. The authors seem to realize this but do not make any attempt to prove their thesis. If regional identity is not maintained, the authors need a better experiment.

If regional identity is not maintained, so what? Don't we already know that this can happen? The authors acknowledge that this is known in the discussion.

The authors have done transcriptomics studies to follow the changes in these cells but they have not told us very much that is meaningful. It would be useful to validate some of the new astrocytic markers that they have identified - Pax and Irx genes (Welle et al., Glia 2021) come quickly to mind. What about genes related to Shh and Wnt signaling that are prevalent in the floor plate? In particular, a lot of work has been done examining the role of Shh on the properties and lineage of astrocytes (Farmer et al., Science 2016; Hill et al., eLife 2019; Gingrich et al., Neural Dev 2022; Xie et al., Cell Rep 2022). There are a lot of stones which remain unturned, here, and the authors could actually tell us much more without doing an immense amount of work. These suggestions and criticisms are described in far greater detail in the confidential comments to the authors.

Work Cited:

Chizhikov et al., Mamm Genome 2006. https://pubmed.ncbi.nlm.nih.gov/17019651/

Chizhikov et al., Development 2004. https://journals.biologists.com/dev/article/131/11/2693/42269/Control-of-roof-plate-formation-by-Lmx1a-in-the

Chizhikov et al., PNAS 2010. https://pubmed.ncbi.nlm.nih.gov/20498066/

Emsley and Macklis. Neuron Glia Biol 2007. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820889/

Farmer et al., Science 2016. https://pubmed.ncbi.nlm.nih.gov/26912893/

Gross et al., Development 2016. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958331/

Hill et al., eLife 2019. https://pubmed.ncbi.nlm.nih.gov/31194676/

Gingrich et al., Neural Dev 2022. https://pubmed.ncbi.nlm.nih.gov/35027088/

Iskusnykh et al., eLife 2023. https://elifesciences.org/articles/84095

Millonig et al, Nature 2000. https://pubmed.ncbi.nlm.nih.gov/10693804/

Welle et al. Glia 2021. https://pubmed.ncbi.nlm.nih.gov/36342840/

Xie et al., Cell Rep 2022. https://pubmed.ncbi.nlm.nih.gov/35196485/

Reviewer #2 (Public Review):

In the current manuscript Li et al., study the preservation of the regional identity during the process of astrocyte generation from pluripotent stem cells. More precisely, this work investigates if neural progenitor cells patterned for the ventral midbrain give rise to astrocytes with conserved regional specification, which could reflect the astrocytic heterogeneity in the brain. To this end, the authors utilized a previously generated reporter iPSC line in which the expression of introduced blue fluorescence protein (BFP) is subjacent to the activation of LMXA1, a ventral midbrain floor plate marker. The study reports that following a defined patterning protocol based on SHH and FGF8, over 90% of d19 cells, corresponding to a neural progenitor stage, acquired the midbrain floor plate identity. However, during the subsequent astrogenic induction and glial progenitor expansion, this identity is gradually lost, supposedly due to the growth advantage of cells deriving from the residual LMX1A- neural progenitors. Contrariwise, if the LMX1A+ progenitors were purified, regional identity would be maintained throughout the astrocytic generation and incur an early astrogenic switch and maturation of derived astrocytes. By using single-cell RNA sequencing, the authors further identified distinct transcriptomic signatures on the astrocytic progeny of LMX1A- and LMX1A- progenitors.

Strengths and weaknesses:

(1) The main model utilized was engineered from the KOLF2 human iPSC line into an elegant LMX1A-reporter line based on the expression of BFP. This results in an attractive model for studies tracing the fate of LMX1A cells. However, consideration should be given to the fact that the parental line, exhibits a splice disruption in the COL3A1 gene encoding type III collagen (Pantazis 2022, doi:10.1016/j.stem.2022.11.004 ), which has been identified as being enriched in certain ventral astrocytic populations (Bradley 2019, doi:10.1242/dev.170910).

(2) The authors argue that the depletion of BFP seen in the unsorted population immediately after the onset of astrogenic induction is due to the growth advantage of the derivatives of the residual LMX1A- population. However, no objective data supporting this idea is provided, and one could also hypothesize that the residual LMX1A- cells could affect the overall LMX1A expression in the culture through negative paracrine regulation. Therefore, cell cycle or proliferation studies of these cells are needed to prove the authors' assumption. Furthermore, on line 124 it is stated that: "Interestingly, the sorted BFP+ cells exhibited similar population growth rate to that of unsorted cultures...". In the face of the suggested growth disadvantage of those cells, this statement needs clarification.

(3) Regarding the fidelity of the model system, it is not clear to me how the TagBFP expression was detected in the BFP+ population supposedly in d87 and d136 pooled astrocytes (Fig S6C) while no LMX1A expression was observed in the same cells (Fig S6F).

(4) The generated single-cell RNASeq dataset is extremely valuable. However, given the number of conditions included in this study (i.e. early vs late astrocytes, BFP+ vs BFP-, sorted vs unsorted, plus non-patterned and neuronal samples) the resulting analysis lacks detail. For instance, from a developmental perspective and to better grasp the functional significance of astrocytic heterogeneity, it would be interesting to map the identified clusters to early vs late populations and to the BFP status. Moreover, although comprehensive, Figure S7 is complex to understand given that citations rather than the reference populations are depicted.

(5) Do the authors have any consideration regarding the morphology of the astrocytes obtained in this study? None of the late astrocyte images depict a prototypical stellate morphology, which is reported in many other studies involving the generation of iPSC-derived astrocytes and which is associated with the maturity status of the cell.

Reviewer #1 (Public review):

Summary:

García-Vázquez et al. identify GTSE1 as a novel target of the cyclin D1-CDK4/6 kinases. The authors show that GTSE1 is phosphorylated at four distinct serine residues and that this phosphorylation stabilizes GTSE1 protein levels to promote proliferation.

Strengths:

The authors support their findings with several previously published results, including databases. In addition, the authors perform a wide range of experiments to support their findings.

Weaknesses:

I feel that important controls and considerations in the context of the cell cycle are missing. Cyclin D1 overexpression, Palbociclib treatment and apparently also AMBRA1 depletion can lead to major changes in cell cycle distribution, which could strongly influence many of the observed effects on the cell cycle protein GTSE1. It is therefore important that the authors assess such changes and normalize their results accordingly.

Reviewer #2 (Public review):

Summary:

The manuscript by García-Vázquez et al identifies the G2 and S phases expressed protein 1(GTSE1) as a substrate of the CycD-CDK4/6 complex. CycD-CDK4/6 is a key regulator of the G1/S cell cycle restriction point, which commits cells to enter a new cell cycle. This kinase is also an important therapeutic cancer target by approved drugs including Palbocyclib. Identification of substrates of CycD-CDK4/6 can therefore provide insights into cell cycle regulation and the mechanism of action of cancer therapeutics. A previous study identified GTSE1 as a target of CycB-Cdk1 but this appears to be the first study to address the phosphorylation of the protein by Cdk4/6.

The authors identified GTSE1 by mining an existing proteomic dataset that is elevated in AMBRA1 knockout cells. The AMBRA1 complex normally targets D cyclins for degradation. From this list, they then identified proteins that contain a CDK4/6 consensus phosphorylation site and were responsive to treatment with Palbocyclib.

The authors show CycD-CDK4/6 overexpression induces a shift in GTSE1 on phostag gels that can be reversed by Palbocyclib. In vitro kinase assays also showed phosphorylation by CDK4. The phosphorylation sites were then identified by mutagenizing the predicted sites and phostag got to see which eliminated the shift.

The authors go on to show that phosphorylation of GTSE1 affects the steady state level of the protein. Moreover, they show that expression and phosphorylation of GTSE1 confer a growth advantage on tumor cells and correlate with poor prognosis in patients.

Strengths:

The biochemical and mutagenesis evidence presented convincingly show that the GTSE1 protein is indeed a target of the CycD-CDK4 kinase. The follow-up experiments begin to show that the phosphorylation state of the protein affects function and has an impact on patient outcomes.

Weaknesses:

It is not clear at which stage in the cell cycle GTSE1 is being phosphorylated and how this is affecting the cell cycle. Considering that the protein is also phosphorylated during mitosis by CycB-Cdk1, it is unclear which phosphorylation events may be regulating the protein.

Reviewer #3 (Public review):

Summary:

This paper identifies GTSE1 as a potential substrate of cyclin D1-CDK4/6 and shows that GTSE1 correlates with cancer prognosis, probably through an effect on cell proliferation. The main problem is that the phosphorylation analysis relies on the over-expression of cyclin D1. It is unclear if the endogenous cyclin D1 is responsible for any phosphorylation of GTSE1 in vivo, and what, if anything, this moderate amount of GTSE1 phosphorylation does to drive proliferation.

Strengths:

There are few bonafide cyclin D1-Cdk4/6 substrates identified to be important in vivo so GTSE1 represents a potentially important finding for the field. Currently, the only cyclin D1 substrates involved in proliferation are the Rb family proteins.

Weaknesses:

The main weakness is that it is unclear if the endogenous cyclin D1 is responsible for phosphorylating GTSE1 in the G1 phase. For example, in Figure 2G there doesn't seem to be a higher band in the phos-tag gel in the early time points for the parental cells. This experiment could be redone with the addition of palbociclib to the parental to see if there is a reduction in GTSE1 phosphorylation and an increase in the amount in the G1 phase as predicted by the authors' model.

The experiments involving palbociclib do not disentangle cell cycle effects. Adding Cdk4 inhibitors will progressively arrest more and more cells in the G1 phase and so there will be a reduction not just in Cdk4 activity but also in Cdk2 and Cdk1 activity. More experiments, like the serum starvation/release in Figure 2G, with synchronized populations of cells would be needed to disentangle the cell cycle effects of palbociclib treatment.

It is unclear if GTSE1 drives the G1/S transition. Presumably, this is part of the authors' model and should be tested.

The proliferation assays need to be more quantitative. Figure 4B should be plotted on a log scale so that the slope can be used to infer the proliferation rate of an exponentially increasing population of cells. Figure 4c should be done with more replicates and error analysis since the effects shown in the lower right-hand panel are modest.

Reviewer #1 (Public review):

Summary:

In their paper, Hosack and Arce-McShane investigate how the 3D movement direction of the tongue is represented in the orofacial part of the sensory-motor cortex and how this representation changes with the loss of oral sensation. They examine the firing patterns of neurons in the orofacial parts of the primary motor cortex (MIo) and somatosensory cortex (SIo) in non-human primates (NHPs) during drinking and feeding tasks. While recording neural activity, they also tracked the kinematics of tongue movement using biplanar video-radiography of markers implanted in the tongue. Their findings indicate that most units in both MIo and SIo are directionally tuned during the drinking task. However, during the feeding task, directional turning was more frequent in MIo units and less prominent in SIo units. Additionally, in some recording sessions, they blocked sensory feedback using bilateral nerve block injections, which resulted in fewer directionally tuned units and changes in the overall distribution of the preferred direction of the units.

Strengths:

The most significant strength of this paper lies in its unique combination of experimental tools. The author utilized a video-radiography method to capture 3D kinematics of the tongue movement during two behavioral tasks while simultaneously recording activity from two brain areas. Moreover, they employed a nerve-blocking procedure to halt sensory feedback. This specific dataset and experimental setup hold great potential for future research on the understudied orofacial segment of the sensory-motor area.

Weaknesses:

Aside from the last part of the result section, the majority of the analyses in this paper are focused on single units. I understand the need to characterize the number of single units that directly code for external variables like movement direction, especially for less-studied areas like the orofacial part of the sensory-motor cortex. However, as a field, our decade-long experience in the arm region of sensory-motor cortices suggests that many of the idiosyncratic behaviors of single units can be better understood when the neural activity is studied at the level of the state space of the population. By doing so, for the arm region, we were able to explain why units have "mixed selectivity" for external variables, why the tuning of units changes in the planning and execution phase of the movement, why activity in the planning phase does not lead to undesired muscle activity, etc. See (Gallego et al. 2017; Vyas et al. 2020; Churchland and Shenoy 2024) for a review. Therefore, I believe investigating the dynamics of the population activity in orofacial regions can similarly help the reader go beyond the peculiarities of single units and in a broader view, inform us if the same principles found in the arm region can be generalized to other segments of sensory-motor cortex.

Further, for the nerve-blocking experiments, the authors demonstrate that the lack of sensory feedback severely alters how the movement is executed at the level of behavior and neural activity. However, I had a hard time interpreting these results since any change in neural activity after blocking the orofacial nerves could be due to either the lack of the sensory signal or, as the authors suggest, due to the NHPs executing a different movement to compensate for the lack of sensory information or the combination of both of these factors. Hence, it would be helpful to know if the authors have any hint in the data that can tease apart these factors. For example, analyzing a subset of nerve-blocked trials that have similar kinematics to the control.

Reviewer #2 (Public review):

Summary:

This manuscript by Hosack and Arce-McShane examines the directional tuning of neurons in macaque primary motor (MIo) and somatosensory (SIo) cortex. The neural basis of tongue control is far less studied than, for example, forelimb movements, partly because the tongue's kinematics and kinetics are difficult to measure. A major technical advantage of this study is using biplanar video-radiography, processed with modern motion tracking analysis software, to track the movement of the tongue inside the oral cavity. Compared to prior work, the behaviors are more naturalistic behaviors (feeding and licking water from one of three spouts), although the animals were still head-fixed.

The study's main findings are that:

• A majority of neurons in MIo and a (somewhat smaller) percentage of SIo modulated their firing rates during tongue movements, with different modulations depending on the direction of movement (i.e., exhibited directional tuning). Examining the statistics of tuning across neurons, there was anisotropy (e.g., more neurons preferring anterior movement) and a lateral bias in which tongue direction neurons preferred that was consistent with the innervation patterns of tongue control muscles (although with some inconsistency between monkeys).

• Consistent with this encoding, tongue position could be decoded with moderate accuracy even from small ensembles of ~28 neurons.

• There were differences observed in the proportion and extent of directional tuning between the feeding and licking behaviors, with stronger tuning overall during licking. This potentially suggests behavioral context-dependent encoding.

• The authors then went one step further and used a bilateral nerve block to the sensory inputs (trigeminal nerve) from the tongue. This impaired the precision of tongue movements and resulted in an apparent reduction and change in neural tuning in Mio and SIo.

Strengths:

The data are difficult to obtain and appear to have been rigorously measured, and provide a valuable contribution to this under-explored subfield of sensorimotor neuroscience. The analyses adopt well-established methods, especially from the arm motor control literature, and represent a natural starting point for characterizing tongue 3D direction tuning.

Weaknesses:

There are alternative explanations for some of the interpretations, but those interpretations are described in a way that clearly distinguishes results from interpretations, and readers can make their own assessments. Some of these limitations are described in more detail below.

One weakness of the current study is that there is substantial variability in results between monkeys, and that only one session of data per monkey/condition is analyzed (8 sessions total). This raises the concern that the results could be idiosyncratic. The Methods mention that other datasets were collected, but not analyzed because the imaging pre-processing is very labor-intensive. While I recognize that time is precious, I do think in this case the manuscript would be substantially strengthened by showing that the results are similar on other sessions.

This study focuses on describing directional tuning using the preferred direction (PD) / cosine tuning model popularized by Georgopoulous and colleagues for understanding neural control of arm reaching in the 1980s. This is a reasonable starting point and a decent first-order description of neural tuning. However, the arm motor control field has moved far past that viewpoint, and in some ways, an over-fixation on static representational encoding models and PDs held that field back for many years. The manuscript benefits from drawing the readers' attention (perhaps in their Discussion) that PDs are a very simple starting point for characterizing how cortical activity relates to kinematics, but that there is likely much richer population-level dynamical structure and that a more mechanistic, control-focused analytical framework may be fruitful. A good review of this evolution in the arm field can be found in Vyas S, Golub MD, Sussillo D, Shenoy K. 2020. Computation Through Neural Population Dynamics. Annual Review of Neuroscience. 43(1):249-75

Can the authors explain (or at least speculate) why there was such a large difference in behavioral effect due to nerve block between the two monkeys (Figure 7)?

Do the analyses showing a decrease in tuning after nerve block take into account the changes (and sometimes reduction in variability) of the kinematics between these conditions? In other words, if you subsampled trials to have similar distributions of kinematics between Control and Block conditions, does the effect hold true? The extreme scenario to illustrate my concern is that if Block conditions resulted in all identical movements (which of course they don't), the tuning analysis would find no tuned neurons. The lack of change in decoding accuracy is another yellow flag that there may be a methodological explanation for the decreased tuning result.

The manuscript states that "Our results suggest that the somatosensory cortex may be less involved than the motor areas during feeding, possibly because it is a more ingrained and stereotyped behavior as opposed to tongue protrusion or drinking tasks". Could an alternative explanation be more statistical/technical in nature: that during feeding, there will be more variability in exactly what somatosensation afferent signals are being received from trial to trial (because slight differences in kinematics can have large differences in exactly where the tongue is and the where/when/how of what parts of it are touching other parts of the oral cavity)? This variability could "smear out" the apparent tuning using these types of trial-averaged analyses. Given how important proprioception and somatosensation are for not biting the tongue or choking, the speculation that somatosensory cortical activity is suppressed during feedback is very counter-intuitive to this reviewer.

Reviewer #3 (Public review):

Summary:

In this study, the authors aim to uncover how 3D tongue direction is represented in the Motor (M1o) and Somatosensory (S1o) cortex. In non-human primates implanted with chronic electrode arrays, they use X-ray-based imaging to track the kinematics of the tongue and jaw as the animal is either chewing food or licking from a spout. They then correlate the tongue kinematics with the recorded neural activity. Using linear regressions, they characterize the tuning properties and distributions of the recorded population during feeding and licking. Then, they recharacterize the tuning properties after bilateral lidocaine injections in the two sensory branches of the trigeminal nerve. They report that their nerve block causes a reorganization of the tuning properties. Overall, this paper concludes that M1o and S1o both contain representations of the tongue direction, but their numbers, their tuning properties, and susceptibility to perturbed sensory input are different.

Strengths:

The major strengths of this paper are in the state-of-the-art experimental methods employed to collect the electrophysiological and kinematic data.

Weaknesses:

However, this paper has a number of weaknesses in the analysis of this data.

It is unclear how reliable the neural responses are to the stimuli. The trial-by-trial variability of the neural firing rates is not reported. Thus, it is unclear if the methods used for establishing that a neuron is modulated and tuned to a direction are susceptible to spurious correlations. The authors do not use shuffling or bootstrapping tests to determine the robustness of their fits or determining the 'preferred direction' of the neurons. This weakness colors the rest of the paper.

The authors compare the tuning properties during feeding to those during licking but only focus on the tongue-tip. However, the two behaviors are different also in their engagement of the jaw muscles. Thus many of the differences observed between the two 'tasks' might have very little to do with an alternation in the properties of the neural code - and more to do with the differences in the movements involved. Many of the neurons are likely correlated with both Jaw movements and tongue movements - this complicates the interpretations and raises the possibility that the differences in tuning properties across tasks are trivial.

The population analyses for decoding are rudimentary and provide very coarse estimates (left, center, or right), it is also unclear what the major takeaways from the population decoding analyses are. The reduced classification accuracy could very well be a consequence of linear models being unable to account for the complexity of feeding movements, while the licking movements are 'simpler' and thus are better accounted for.

The nature of the nerve block and what sensory pathways are being affected is unclear - the trigeminal nerve contains many different sensory afferents - is there a characterization of how effectively the nerve impulses are being blocked? Have the authors confirmed or characterized the strength of their inactivation or block, I was unable to find any electrophysiological evidence characterizing the perturbation.

Overall, while this paper provides a descriptive account of the observed neural correlations and their alteration by perturbation, a synthesis of the observed changes and some insight into neural processing of tongue kinematics would strengthen this paper.

Reviewer #1 (Public review):

Summary:

This study by Wang et al. identifies a new type of deacetylase, CobQ, in Aeromonas hydrophila. Notably, the identification of this deacetylase reveals a lack of homology with eukaryotic counterparts, thus underscoring its unique evolutionary trajectory within the bacterial domain.

Strengths:

The manuscript convincingly illustrates CobQ's deacetylase activity through robust in vitro experiments, establishing its distinctiveness from known prokaryotic deacetylases. Additionally, the authors elucidate CobQ's potential cooperation with other deacetylases in vivo to regulate bacterial cellular processes. Furthermore, the study highlights CobQ's significance in the regulation of acetylation within prokaryotic cells.

Weaknesses:

The problem I raised has been well resolved. I have no further questions.

Reviewer #2 (Public review):

In recent years, lots of researchers tried to explore the existence of new acetyltransferase and deacetylase by using specific antibody enrichment technologies and high resolution mass spectrometry. Here is an example for this effort. Yuqian Wang et al. studied a novel Zn2+- and NAD+-independent KDAC protein, AhCobQ, in Aeromonas hydrophila. They studied the biological function of AhCobQ by using biochemistry method and MS identification technology to confirm it. These results extended our understanding of the regulatory mechanism of bacterial lysine acetylation modifications. However, I find this conclusion is a little speculative, and unfortunately, it also doesn't totally support the conclusion as the authors provided.

Reviewer #3 (Public review):

Summary:

This study reports on a novel NAD+ and Zn2+-independent protein lysine deacetylase (KDAC) in Aeromonas hydrophila, termed as AhCobQ (AHA_1389). This protein is annotated as a CobQ/CobB/MinD/ParA family protein and does not show similarity with known NAD+-dependent or Zn2+-dependent KDACs. The authors showed that AhCobQ has NAD+ and Zn2+-independent deacetylase activity with acetylated BSA by western blot and MS analyses. They also provided evidence that the 195-245 aa region of AhCobQ is responsible for the deacetylase activity, which is conserved in some marine prokaryotes and has no similarity with eukaryotic proteins. They identified target proteins of AhCobQ deacetylase by proteomic analysis and verified the deacetylase activity using site-specific Kac proteins. Finally, they showed that AhCobQ activates isocitrate dehydrogenase by deacetylation at K388.

Strengths:

The finding of a new type of KDAC has a valuable impact on the field of protein acetylation. The characters (NAD+ and Zn2+-independent deacetylase activity in an unknown domain) shown in this study are very unexpected.

Weaknesses:

(1) The characters (NAD+ and Zn2+-independent deacetylase activity in an unknown domain) shown in this study are very unexpected. To convince readers, MSMS data must be necessary to accurately detect (de)acetylation at the target site in the deacetylase activity assay. The authors showed the MSMS data in assays with acetylated BSA, but other assays only rely on western blot.

(2) They prepared site-specific Kac proteins and used them in deacetylase activity assays. Incorporation of acetyllysine at the target site should be confirmed by MSMS and shown as supplementary data.

(3) The authors imply that the 195-245 aa region of AhCobQ may represent a new domain responsible for deacetylase activity. The feature of the region would be of interest but is not sufficiently described in Figure 5. The amino acid sequence alignments with representative proteins with conserved residues would be informative. It would be also informative if the modeled structure predicted by AlphaFold is shown and the structural similarity with known deacetylases is discussed.

Reviewer #1 (Public review):<br /> <br /> Summary:

Balasubramanian et al. characterized the cell types comprising mouse Schlemm's canal (SC) using bulk and single cell RNA sequencing (scRNA-seq). The results identify expression patterns the delineate the SC inner and outer wall cells and two inner wall 'states'. Further analysis demonstrates expression patterns of glaucoma associated genes and receptor ligand pairs between SEC's and neighboring trabecular meshwork.

Strengths:

While mouse SC has been profiled in previous scRNA-seq studies (van Zyl et al 2020, Thomson et al 2021), these data provide higher resolution of SC cell types, particularly endothelial cell (SEC) populations. SC is an important regulator of anterior chamber outflow and has important consequences for glaucoma.

Comments on the latest version:

The authors have addressed my primary concerns with the first version of the manuscript. This study represents a valuable resource in the molecular characterization of mouse Schlemm's canal cell types.

Reviewer #2 (Public review):

Summary:

This revised article has characterized the mouse Schlemm's canal expression profile using a comprehensive approach based on sorted SEC, LEC, and BEC total RNA-Seq, scRNA-Seq, and snRNA-Seq to enrich the selection of SECs. The revised study has successfully profiled genome-wide gene expression using sorted SECs, demonstrating that SECs have a closer similarity to LECs than BECs. The combined scRNA- and snRNA-Seq data with deep coverage of gene expression led to the successful identification of many novel biomarkers for inner wall SECs, outer wall SECs, collector channel ECs, and pericytes. In addition, the study also identified two novel states of inner wall SECs separated by new markers. The study provides significant novel information about the biology and expression profile of SECs in the inner and outer walls. It is of great significance to have this novel, convincing, and comprehensive study led by leading researchers published in this journal. The revision has improved the clarity and significance of the study with more details.

Strengths:

This is a comprehensive study using various data to support the expression characterization of mouse SECs. First, the study profiled genome-wide expression using sorted SECs, LECs, and BECs from the same tissue/organ to identify the similarities and differences among the three types of cells. Second, snRNA-Seq was applied to enrich the number of SECs from mouse ocular tissues significantly. Increased sampling of SECs and other cells led to more comprehensive coverage and characterization of cells, including pericytes. Third, the combined scRNA- and snRNA-Seq data analyses increase the power to further characterize the subtle differences within SECs, leading to identifying the expression markers of Inner and Outer wall SECs, collector channel ECs, and distal region cells. Fourth, the identified unique markers were validated for RNA and protein expression in mouse ocular tissues. Fifth, the study explored how the IOP- and glaucoma-associated genes are expressed in the ScRNA- and snRNA-Seq data, providing potential connections of these GWAS genes with IOP and glaucoma. Sixth, the initial pathway and network analyses generated exciting hypotheses that could be tested in other independent studies.

Weaknesses:

The authors have addressed most of the previous comments by adding more details about the protocol and additional discussions. Several comments requiring additional experimental data have been addressed as future directions, such as protein validation, RNA expression validation in human samples, and GWAS-identified IOP genes.

Comments on the latest version:

The authors have addressed previous comments responsively. The authors have suggested several experiments to be completed in the future since these could be time-consuming with human samples. The revised article is with better clarity and clearer significance. No additional comments.

Reviewer #1 (Public review):

Summary:

This paper reports the first results on the effects of a novel waveform for weak transcranial magnetic stimulation, which is refered to as "perturbation" (kTMP). The waveform is sinusoidal at kHz frequency with subthreshold intensities of 2V/m, instead of the suprathreshold pulses used in conventional TMS (~100V/m). The effect reported here concerns motor-evoked potentials (MEPs) elicited on the hand with single-pulse TMS. These MEPs are considered a marker of "corotico-spinal excitability". The manuscripts report that kTMP at 3.5kHz enhances MEPs with a medium effect size, with independent replication of this finding on 3 separate cohorts of subjects (N=16, 15, 16). This result is important for the field of non-invasive brain stimulation. The evidence in support of this claim is compelling. Despite the replications, this remains an exploratory study that will require replication with adequately powered planned comparisons.

Strengths:

• This is a novel modality for non-invasive brain stimulation.<br /> • Knowing the history in this field, this is likely to lead to a large number of follow-up studies in basic and clinical research.<br /> • The modality causes practically no sensation, which makes it perfectly suitable for control conditions. Indeed, the study itself used a persuasive double-blinding procedure.<br /> • The replication of the main result in two subsequent experiments is very compelling.<br /> • The effect size of Cohen's d=0.5 is very promising.<br /> • It is nice the E-fields were measured on a phantom, in addition to modeling.

Weakness:

• Statistical analysis combining Experiments 1, 2, 3 after inspecting the data is inappropriate.<br /> • Post-hoc definition of outliers that were removed is unfortunate.<br /> • While sensation has been documented, blinding was not directly assessed.<br /> • Despite the replications, this remains an exploratory study as it lacks power analysis and planned comparisons.

Other comments from an earlier review were adequately addressed.

Reviewer #2 (Public review):

Summary:

kTMP is a novel method of stimulating the brain using electromagnetic fields. It has potential benefits over existing technology because it is a safe and easy technology. It explores a range of brain frequencies that has not been explored in depth before (2-5kHz) and thus offers new opportunities.

Strengths:

This work relied on standard methods and was carefully and conservatively performed.

Weaknesses:

There were few weaknesses. The sham condition was prepared as well as could be done, but sham is always challenging in a treatment with sound and sensation, and with knowledgeable operators. New technology, also, is very exciting to subjects and it is difficult to achieve a natural experiment. These difficulties are related to the technology, however, and not to the execution of these experiments..

Reviewer #1 (Public review):

Summary:

This work by Leclercq and colleagues performed metabolomics on biospecimens collected from 96 patients diagnosed with severe alcohol use disorder (AUD). The authors discovery strong alterations in circulating glycerophospholipids, bile acids, and some gut microbe-derived metabolites in AUD patients compared to controls. An exciting part of this work is that metabolomics was also done in post-mortem samples of the frontal cortex and cerebrospinal fluid of heavy alcohol users, and some of the same metabolites were seen to be altered in the central nervous system. This important study will form the basis for hypothesis generation around diet-microbe-host interactions in alcohol use disorder. The work is done in a highly rigorous manner, and the rigorously collected human samples is an evident strength of this work. Overall, this work will provide many new insights, and it is poised to have a high impact on the field.

Strengths:

(1) The rigorously collected patient-derived samples<br /> (2) There is high rigorous in the metabolomics investigation<br /> (3) Statistical analyses are well-described and strong.<br /> (4) The careful control of taking blood samples at the same time to avoid alterations in meal- and circadian-related fluctuations in metabolites is a clear strength.

Weaknesses:

None remaining

Reviewer #2 (Public review):

The authors carried out the current studies with the justification that the biochemical mechanisms that lead to alcohol addiction are incompletely understood. The topic and question addressed here are impactful and indeed deserve further research. To this end, a metabolomics approach toward investigating the metabolic effects of alcohol use disorder and the effect of alcohol withdrawal in AUD subjects is valuable. However, this work is primarily descriptive in nature, and these data alone do not meet the stated goal of investigating biochemical mechanisms of alcohol addiction. The current work's most significant limitation is the cross-sectional study design, though inadequate description and citation of the underlying methodological approaches also hampers interest.

Most of the data are cross-sectional in study design, i.e., alcohol use disorder vs controls. However, it is well established that there is a high degree of interpersonal variation with metabolism, and further, there is somewhat high intra-personal variation in metabolism over time. This means that the relatively small cohort of subjects is unlikely to just reflect the broader condition of interest (AUD/withdrawal). The authors report a comparison of a later time-point after alcohol withdrawal (T2) vs the AUD condition. Nonetheless, without replicate time points from the control subjects it is difficult to assess how much of these changes are due to withdrawal vs the intra-personal variation described above. Overall, insufficient experimental context exists to interpret these findings into a biological understanding. For example, while several metabolites are linked with AUD and associated with microbiome or host metabolism based on existing literature, it is unclear from the current study what function these changes have concerning AUD, if any. The authors also argue that alcohol withdrawal shifts the AUD plasma metabolic fingerprint towards healthy controls (line 153). However, this is hard to assess based on the provided plots since the direction of change of the orange data subset considers AUD T2 vs. T1. In contrast, AUD T2 vs. Control would represent the claimed shift. To substantiate these claims, the authors would better support their argument by showing this comparison in all experimental groups (including control subjects) in their multi-dimensional model (e.g., PCA). The authors attempt to extend the significance of their findings by assessing post-mortem brain tissues from AUD subjects; however, the finding that many of the metabolites changed in T2/T1 are also found in AUD brain tissues is interesting but does not strongly support the authors' claims that these metabolites are markers of AUD (line 173). Concerning the plasma cohort itself, it is unclear how the authors assessed for compliance with alcohol withdrawal or whether the subjects' blood-alcohol levels were independently verified.

The second area of concern is the lack of description of the analytical methodology, the lack of metabolite identification validation evidence, and related statistical questions. The authors cite reference #59 regarding the general methodology. However, this reference from their group is a tutorial/review/protocol focused resource paper and it needs to be clarified how specific critical steps were actually applied to the current plasma study samples, given the range of descriptions provided in the citations. The authors report a variety of interesting metabolites, including their primary fragment intensities, which is appreciated (Supp Table 3), but no MS2 matching scores are provided for level 2 or 3 hits. Further, level 1 hits under their definition are validated by an in-house standard, but not supporting data are provided other than this categorization. Finally, a common risk in such descriptive studies is finding spurious associations, especially considering the many factors as described in the current work. These include AUD, depression, anxiety, craving, withdrawal, etc. The authors describe the use of BH correction for multiple-hypothesis testing. Still, this approach only accounts for the many possible metabolite association tests within each comparison (such as metabolites vs. depression) and does not account for the multi-variate comparisons to the many behavior/clinical factors described above. The authors should employ one of several common strategies, such as linear mixed effects models for these types of multi-variate assessments.

Revised Review after Resubmission:

I thank the authors for their responses and revisions to the figures and data and their clarifications of their results and study goals. However, based on this updated information, it is now more apparent that the paper falls into the common trap of descriptive studies where insufficient experimental design was considered to test the association in question robustly. Further, follow-up initiatives are lacking to test the findings by other experimental means. Despite the authors' responses, the paper still fails to convert or interpret the metabolomics findings into any new biological understanding or meaningfully testable hypotheses, and the results remain descriptive in nature with significant caveats.

The authors clarify that their study's "goal was not to investigate the biochemical mechanisms of AUD but how metabolomics could contribute to the psychological alterations of AUD." However, the 2nd sentence of the abstract remains as follows: "The biochemical mechanisms that lead to this disorder are not yet fully understood, and in this respect, metabolomics represents a promising approach to decipher metabolic events related to AUD."This leads the reader to conclude that the purpose of the current study is to use metabolomics to address this gap, despite their later clarification. In the revised response, the authors walk back their claims of these goals, yet the manuscript text and data is largely unchanged in the revision. The serious caveats pointed out by several reviewers concerning the study as reported significantly reduces the utility of the described findings for the broader scientific community, and the authors largely downplay these limitations without addressing the underlying issues.

The authors also clarified in their response that the study's key purpose of the study is to assess "correlations between the blood metabolome and psychological symptoms developed in AUD patients." This goal is dubious as the vast majority of metabolites are not psychoactive, and it is implausible that the metabolome would affect mental state or vice versa. More biological frameworks and citations are needed for this paradigm. The soundness of the goal is further questioned by the study's simplistic design and the authors' admission that "In this discovery-based approach, the aim was to discover potential candidates linked with psychological symptoms for subsequent work to evaluate causality." Yet, the authors side-step the point about the risk of finding spurious associations and decline to control this risk using widely-accepted approaches such as multi-variate correction, instead continuing to use only BH correction for multiple hypothesis testing. The reviewers previously pointed out that BH correction only accounts for the many possible metabolite association tests within each comparison (such as metabolites vs depression). However, it does not account for the multi-variate comparisons to the many behavior/clinical factors. This issue is ignored in the response because the study's goal is hypothesis generating. Instead, the authors focused their responses on the issue of causality which was not the central point of the criticism.

Further, the authors employ mainly systemic plasma analyses unlikely to reflect brain biochemistry. The authors deny that the purpose of including the post-mortem brain tissue data was to demonstrate that "metabolites significantly correlated with the psychological symptoms - and present in the central nervous system (frontal cortex or CSF) - are "markers of AUD," yet if this is not the goal, the structure of the experiment, and the value of these data, is unclear. Another reviewer pointed out that it is difficult to control cross-sectional post-mortem tissue due to a lack of suitable controls, and the authors again side-step the question by citing the lack of suitable controls and the impossibility of "healthy controls" in post-mortem samples. This is true, but this lack of technical feasibility and the confounding factor of CVD/lipid metabolism does not justify the weak experimental design in this respect. Therefore, it remains unclear what can be understood from these data, given the limitations.

Finally, the authors acknowledge the limitation in their revision that they did not assess a second-time point in the control cohort of samples which could have been used to tease apart intra-personal variation from AUD-associated changes during alcohol-abstinence. Unfortunately, this is not a small caveat to simply acknowledge in the discussion section; it severely limits the interpretation and utility of the reported data more broadly, and the authors do not address this underlying problem.

Reviewer #3 (Public review):

Summary:

The authors have compared different groups of AUD patients at different levels and have examined metabolomics.

Strengths:

A well-written and comprehensive study.

Reviewer #1 (Public review):

Summary:

The authors study age-related changes in the excitability and firing properties of sympathetic neurons, which they ascribe to age-related changes in the expression of KCNQ (Kv7, "M-type") K+ currents in rodent sympathetic neurons, whose regulation by GPCRs has been most thoroughly studied for over 40 years.

Strengths:

The strengths include the rigor of the current-clamp and voltage-clamp experiments and the lovely, crisp presentation of the data, The separation of neurons into tonic, phasic and adapting classes is also interesting, and informative. The ability to successfully isolate and dissociate peripheral ganglia from such older animals is also quite rare and commendable! There is much useful detail here.

Weaknesses:

Whereas the description of the data are very nice, and useful, the manuscript does not provide much in the way of mechanistic insights. As such, the effect is more of an epi-phenomenon of unclear insight, and the authors cannot ascribe changes in signaling mechanisms, such as that of M1 mAChRs to the phenomena that is supported by data.

Comments on latest version:

I do not have any additional issues to be addressed by the authors.

Reviewer #2 (Public review):

Summary:

This research provides compelling and detailed evidence showing that aging influences intrinsic membrane properties of peripheral sympathetic motor neurons, which become hyperexcitable. The authors found that sympathetic motor neurons from old mice exhibit increased firing rates (spontaneous and evoked), more depolarized membrane resting potential, and increased rheobase. Furthermore, the study investigates cellular mechanisms underlying age-associated hyperexcitability and shows solid evidence supporting that a decreased activity of KCNQ2/3 channels during aging is a major contributor to the increased excitability of sympathetic old neurons. The conclusions of this paper are supported by the data.

Strengths:

Detailed and rigorous analysis of electrical responses of peripheral sympathetic motor neurons using electrophysiology (perforated patch and whole-cell recordings). The study identifies a decreased KCNQ2/3 current as a cellular mechanism behind age-induced hyperexcitability in sympathetic motor neurons.

Weaknesses:

The revised version of the manuscript has addressed all my concerns.

Reviewer #3 (Public review):

This revised study described changes in membrane excitability and Na+ and K+ current amplitudes of sympathetic motor neurons in culture. The findings indicate that neurons isolated from aged animals show increased membrane excitability manifested as increased firing rates in response to electrical stimulation and changes in related membrane properties including depolarized resting membrane potential, increased rheobase, and spontaneous firing. By contrast, neuron cultures from young mice show little to no spontaneous firing and relatively low firing rates in response to current injection. These changes in excitability correlate with reductions in the magnitude of KCNQ currents in neurons cultured from aged mice compared to neurons from cultured from young mice. The authors conclude that aging promotes hyperexcitability of sympathetic motor neurons through changes in KCNQ channels.

Joint Public Review:

This study describes a group of CRH-releasing neurons, located in the paraventricular nucleus of the hypothalamus, which, in mice, affects both the state of sevoflurane anesthesia and a grooming behavior observed after it. PVHCRH neurons showed elevated calcium activity during the post-anesthesia period. Optogenetic activation of these PVHCRH neurons during sevoflurane anesthesia shifts the EEG from burst-suppression to a seemingly activated state (an apparent arousal effect), although without a behavioral correlate. Chemogenetic activation of the PVHCRH neurons delays sevoflurane-induced loss of righting reflex (another apparent arousal effect). On the other hand, chemogenetic inhibition of PVHCRH neurons delays recovery of righting reflex and decreases sevoflurane-induced stress (an apparent decrease in the arousal effect). The authors conclude that PVHCRH neurons "integrate" sevoflurane-induced anesthesia and stress. The authors also claim that their findings show that sevoflurane itself produces a post-anesthesia stress response that is independent of any surgical trauma, such as an incision. In its revised form, the article does not achieve its intended goal and will not have impact on the clinical practice of anesthesiology nor on anesthesiology research.

Strengths:

The manuscript uses targeted manipulation of the PVHCRH neurons with state-of-the-art methods and is technically sound. Also, the number of experiments is substantial.

Weaknesses:

The most significant weaknesses remain: a) overinterpretation of the significance of their findings b) the failure to use another anesthetic as a control, c) a failure to compellingly link their post-sevoflurane measures in mice to anything measured in humans, and d) limitations in the novelty of the findings. These weaknesses are related to the primary concerns described below:

Concerns about the primary conclusion that PVHCRH neurons integrate the anesthetic effects and post-anesthesia stress response of sevoflurane GA:

It is important to compare the effects of sevoflurane with at least one other inhaled ether anesthetic as one step towards elevating the impact of this paper to the level required for a journal such as eLife. Isoflurane, desflurane, and enflurane are ether anesthetics that are very similar to each other, as well as being similar to sevoflurane. For example, one study cited by the authors (Marana et al. 2013) concludes that there is weak evidence for differences in stress-related hormones between sevoflurane and desflurane, with lower levels of cortisol and ACTH observed during the desflurane intraoperative period. It is important to determine whether desflurane activates PVHCRH neurons in the post-anesthesia period, and whether this is accompanied by excess grooming in the mice because this will distinguish whether the effects of sevoflurane generalize to other inhaled anesthestics, or, alternatively, relate to unique idiosyncratic properties of this gas that may not be a part of its anesthetic properties.

Concerns about the clinical relevance of the experiments:

In anesthesiology practice, perioperative stress observed in patients is more commonly related to the trauma of the surgical intervention, with inadequate levels of antinociception or unconsciousness intraoperatively and/or poor post-operative pain control. The authors seem to be suggesting that the sevoflurane itself is causing stress because their mice receive sevoflurane but no invasive procedures, but there is no evidence of sevoflurane inducing stress in human patients. It is important to know whether sevoflurane effectively produces behavioral stress in the recovery room in patients that could be related to the putative stress response (excess grooming) observed in mice. For example, in surgeries or procedures which required only a brief period of unconsciousness that could be achieved by administering sevoflurane alone (comparable to the 30 min administered to the mice), is there clinical evidence of post-operative stress? It is also important to describe a rationale for using a 30 min sevoflurane exposure. What proportion of human surgeries using sevoflurane use exposure times that are comparable to this?

It is the experience of one of the reviewers that human patients who receive sevoflurane as the primary anesthetic do not wake up more stressed than if they had had one of the other GABAergic anesthetics. If there were signs of stress upon emergence (increased heart rate, blood pressure, thrashing movements) from general anesthesia, this would be treated immediately. The most likely cause of post-operative stress behaviors in humans is probably inadequate anti-nociception during the procedure, which translates into inadequate post-op analgesia and likely delirium. It is the case that children receiving sevoflurane do have a higher likelihood of post-operative delirium. Perhaps the authors' studies address a mechanism for delirium associated with sevoflurane, but this is barely mentioned. Delirium seems likely to be the closest clinical phenomenon to what was studied. As noted by the Besnier et al (2017) article cited by the authors, surgery can elevate postoperative glucocorticoid stress hormones, but it generally correlates with the intensity of the surgical procedure. Besnier et al also note the elevation of glucocorticoids is generally considered to be adaptive. Thus, reducing glucocorticoids during surgery with sevoflurane may hamper recovery, especially as it relates to tissue damage, which was not measured or considered here. This paper only considers glucocorticoid release as a negative factor, which causes "immunosuppression", "proteolysis", and "delays postoperative recovery and...leads to increased morbidity".

It is also the case that there are explicit published findings showing that mild and moderate surgical procedures in children receiving sevoflurane (which might be the closest human proxy to the brief 30 minute sevoflurane exposure used here) do not have elevated cortisol (Taylor et al, J Clin Endocrinol Metab, 2013). This again raises the question of whether the enhanced grooming or elevated corticosterone observed in the mice here has any relevance to humans.

Concerns about the novelty of the findings:

The key finding here is that CRH neurons mediate measures of arousal, and arousal modulates sevoflurane anesthesia induction and recovery. However, CRH is associated with arousal in numerous studies. In fact, the authors' own work, published in eLife in 2021, showed that stimulating the hypothalamic CRH cells lead to arousal and their inhibition promoted hypersomnia. In both papers the authors use fos expression in CRH cells during a specific event to implicate the cells, then manipulate them and measure EEG responses. In the previous work, the cells were active during wakefulness; here- they were active in the awake state the follows anesthesia (Figure 1). Thus, the findings in the current work are incremental and not particularly impactful. Claims like "Here, a core hypothalamic ensemble, corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus, is discovered" are overstated. PVHCRH cell populations were discovered in the 1980s. Suggesting that it is novel to identify that hypothalamic CRH cells regulate post-anesthesia stress is unfounded as well: this PVH population has been shown over four decades to regulate a plethora of different responses to stress. Anesthesia stress is no different. Their role in arousal is not being discovered in this paper. Even their role in grooming is not discovered in this paper.

The activation of CRH cells in PVH has already been shown to result in grooming by Jaideep Bains (a paper cited by the authors). Thus, the involvement of these cells in this behavior is not surprising. The authors perform elaborate manipulations of CRH cells and numerous analyses of grooming and related behaviors. For example, they compare grooming and paw licking after anesthesia with those after other stressors such as forced swim, spraying mice with water, physical attack and restraint. The authors have identified a behavioral phenomenon in a rodent model that does not have a clear correlation with a behavior state observed in humans during the use of sevoflurane as part of an anesthetic regimen. The grooming behaviors are not a model of the emergence delirium or the cognitive dysfunction observed commonly in patients receiving sevoflurane for general anesthesia. Emergence delirium is commonly seen in children after sevoflurane is used as part of general anesthesia and cognitive dysfunction is commonly observed in adults-particularly the elderly -- following general anesthesia.

Reviewer #1 (Public review):

Summary:

Li Zhang et al. characterized two new Gram-negative endolysins identified through an AMP-targeted search in bacterial proteomes. These endolysins exhibit broad lytic activity against both Gram-negative and Gram-positive bacteria and retain significant antimicrobial activity even after prolonged exposure to high temperatures (100{degree sign}C for 1 hour). This stability is attributed to a temperature-reversible transition from a dimer to a monomer. The authors suggest several potential applications, such as complementing heat sterilization processes or being used in animal feed premixes that undergo high-temperature pelleting, which I agree with.

Strengths:

The claims are well-supported by relevant and complementary assays, as well as extensive bioinformatic analyses.

Weaknesses:

My last comments are minor and nearly all aim to improve the use of English language in the manuscript. However, a section describing the statistical analysis is still lacking. I believe that the presented manuscript can benefit from language editing, but I leave this decision with the editor.

Reviewer #2 (Public review):

Summary:

The study explores a new strategy of lysin-derived antimicrobial peptide-primed screening to find peptidoglycan hydrolases from bacterial proteomes. Using this strategy authors identified five peptidoglycan hydrolases from A. baumannii. They further tested their antimicrobial activities on various Gram positive and Gram-negative pathogens.

Strengths:

Overall, the study is good and adds new members to the peptidoglycan hydrolases family. The authors also show that these lysins have bactericidal activities against both Gram-positive and Gram-negative bacteria. The crystal structure data is good, reveals different thermostablility to the peptidoglycan hydrolases. Structural data also reveals that PhAb10 and PHAb11 form thermostable dimer and data is corroborated by generating variant protein defective in supporting intermolecular bond pairs. The mice bacterial infection shows promise for the use of these hydrolases as antimicrobial agents.

Weaknesses:

While the authors have employed various mechanisms to justify their findings, some aspects are still unclear. Only CFU has been used to test bactericidal activity. This should also be corroborated by live/dead assay. Moreover, SEM or TEM analysis would reveal the effect of these peptidoglycan hydrolases on Gram-negative /Gram-positive cell envelopes. The authors claim that these hydrolases are similar to T4 lysozyme, but they have not correlated their findings with already published findings on T4 lysozyme. T4 lysozyme has C-terminal amphipathic helix with antimicrobial properties. Moreover, heat, denatured lysozyme also shows enhanced bactericidal activity due to the formation of hydrophobic dimeric forms, which are inserted in the membrane. Authors also observe that heat denatured PHAb10 and PHAb11 have bactericidal activity but no enzymatic activity. These findings should be corroborated by studying the effect of these holoenzymes/ truncated peptides on bacterial cell membranes. Also, a quantitative peptidoglycan cleavage assay should be performed in addition to halo assay. Including these details would make the work more comprehensive.

Revised version: The authors have addressed most of the questions in the revised version of the paper.

Reviewer #1 (Public review):

Summary:

The work seeks to investigate the efficacy of linalool as a natural alternative for combating Saprolegnia parasitica infections, which would provide great benefit to aquaculture. This paper shows the effect of linalool in vitro using a variety of techniques including changes in S. parasitica membrane integrity following linalool exposure and alterations in cell metabolism and ribosome function. Additionally, this work goes on to show that prophylactic and concurrent treatment of linalool at the time of S. parasitica infection can improve survival and tissue damage in vivo in their grass carp infection model. The conclusions of the paper are partially supported by the data, cleaning up, clarifying, and elaborating on some aspects of this work is necessary.

(1) Adding microscopy of the untreated group to compare Figure 2A with would further strengthen the findings here.

(2) Quantification of immune infiltration and histological scoring of kidney, liver, and spleen in the various treatment groups would increase the impact of Figure 4.

(3) The data in Figure 6 I is not sufficiently convincing as being significant.

(4) Comparisons of the global transcriptomic analysis of the untreated group to the PC, LP, and LT groups would strengthen the author's claims about the immunological and transcriptomic changes caused by linalool and provide a true baseline.

Reviewer #2 (Public review):

Summary:

In this study, the authors aimed to delineate the antimicrobial activity of linalool and tried to investigate the mode of action of linalool against S. parasitica infection. One of the main focuses of this work was to identify the in vitro and in vivo mechanisms associated with the protective role of linalool against S. parasitica infection.

Strengths:

(1) The authors have used a variety of techniques to prove their hypothesis.

(2) An adequate number of replicates were used in their studies.

(3) Their findings showed a protective role of linalool against oomycetes and makes it an attractive future antibiotic in the aquaculture industry.

Weaknesses:

There are several weaknesses in this manuscript.

(1) The authors have taken for granted that the readers already know the experiments/assays used in the manuscript. There was not enough explanation for the figures as well as figure legends.

(2) The authors missed adding the serial numbers to the references.

(3) The introduction section does not provide adequate rationale for their work, rather it is focused more on the assays done.

(4) Full forms are missing in many places (both in the text and figure legends), also the resolution of the figures is not good. In some figures, the font size is too small.

(5) There is much mislabeling of the figure panels in the main text. A detailed explanation of why and how they did the experiments and how the results were interpreted is missing.

(6) There is not enough experimental data to support their hypothesis on the mechanism of action of linalool. Most of the data comes from pathway analysis, and experimental validation is missing.

Overall, the conclusions drawn by the authors are partially justified by the data. Importantly, this paper has discovered the novelty of the compound linalool as a potent antimicrobial agent and might open up future possibilities to use this compound in the aquaculture industry.

Reviewer #1 (Public review):

Summary:

Here, the authors attempt to show that CCL5 is increased after stroke, possibly due to decreased miR-324, and that this is a modifiable system to decrease stroke damage. By bidirectionally manipulating CCL5 levels through direct injection of CCL5; a CCL5 blocking antibody; miR324; miR324 antagomir; or CCR5-blocking Maraviroc, they broadly show improvement with lower CCL5 levels. This includes infarct size, behavioral analysis, and immunohistochemical analysis of astrocytes, microglia, and neurons. They further try to mechanistically tie miR324 and CCL5 in astrocytes specifically to stroke-induced changes using a neuronal/astrocytic coculture system. They argue that decreasing CCL5 leads to increased ERK and CREB phosphorylation as a potential neuroprotective mechanism. CCL5 is one potential ligand for CCR5, and recent work identified CCR5 as a targetable mechanism by clinically-approved drug Maraviroc to enhance stroke recovery. Particularly given the high level of interest in CCR5 in stroke recovery, the focus on CCL5 - one of CCR5's potential ligands - and its miR regulation is an exciting expansion of this area of stroke biology.

Strengths:

The authors' findings that decreasing CCL5 acutely after stroke shows behavioral improvement appear robust. This broadly replicates work from other groups, although the finding that miR324 manipulation can phenocopy direct CCL5 manipulation is novel and intriguing. However, many of their other claims are difficult to evaluate based on a combination of missing methodological information, inappropriate statistical testing, and a flawed culture system.

Weaknesses:

Broadly speaking, the manuscript takes a zoomed-out view of what is fundamentally highly localized biology.

(1) miRNA-based regulation, by definition, has to include miR and mRNA in the same cell type; as the authors note, CCL5 is expressed in many cells. It is therefore impossible to propose any interaction on the basis of the tissue-level changes described; any evidence of in vivo cell-type specificity would dramatically improve the claims.

(2) The authors treat an extensive area of ipsilesional cortex uniformly as "IP". Astrocytic and microglial responses to localized injuries such as stroke are highly location-dependent and undoubtedly change dramatically within this area. The presented data cannot be interpreted without confirmation that these were taken at identical distances from the injury, and what that distance was. These do not appear to be adjacent to the injury, where the responses would presumably be the most informative. Similarly, it is difficult to interpret the neuronal Sholl and spine data without more information on where within the large IP region these neurons were found.

The authors attempt to narrow in on cell-type specificity via culture. However, astrocytes are notoriously prone to a dramatic change in culture and require careful methods (immunopanning; see eg doi: 10.1016/j.neuron.2011.07.022) to maintain much resemblance to their in vivo counterpart. It is difficult to conclude much about the role of astrocytes in the CCL5 pathway based on the use of this shaking-based culture system, particularly in the absence of cell-type specific validation in vivo.

There is missing methodological information, including infarct size measurements, TUNEL staining, and statistical testing. The TTC figures look very odd, like a collection of overlapping stars have been placed on the images rather than the natural relatively smooth infarct edges one would expect. It is unclear if the infarct volume measurements accounted for edema, as is standard; there is no description of the protocol used for quantification. It is also unclear if the infarct volume measurement comparisons were also done with t-tests vs ANOVA, as the statistical test used is not listed in the figure legends. In numerous cases where statistical testing is listed, repeated t-tests between subgroups are used vs the more appropriate ANOVA (assuming normality; nonparametric testing as appropriate), making it difficult to have confidence in the results.

Reviewer #2 (Public review):

The authors presented evidence from various in vivo and in vitro experiments demonstrating the mutual interaction between CCL5 and astrocytic miR-342-5p in the ipsilateral core of cerebral ischemia. However, miR-342-5p was downregulated only late after MCAO (D3-7). Additionally, this downregulation was observed not only in the ipsilateral core but also in the ipsilateral penumbra and contralateral sides. Therefore, it is not convincing that the upregulation of CCL5 in the ipsilateral core at later time points (D3 and D7) is attributable to the decreased expression of miR-342-5p. In particular, infarct injury was already evident within a short time period (say 24 h) following MCAO.

(1) The temporal and spatial expression patterns of miR-324-5p do not match those of CCL-5, especially at D1 and D3 (see Figure 1C, 1D). Despite the inverse relationship between miR-324-5p and CCL-5 expression at D7 after MCAO, what was the purpose of administering miR-324-5p agomir (or antagomir) at D1 post-MCAO? If the connection cannot be clearly established, the conclusion reached at the end will be difficult to accept.

(2) Would administering miR-342-5p or anti-CCL5 at later time points (e.g., after D3) reduce infarct size or improve functional recovery? If this is not the case, the effect of CCL5 on neuronal cell damage (infarct size formation) must occur within a very short time after MCAO. Additionally, if the increased CCL5 expression is due to the downregulation of miR-342-5p, its impact would likely be less significant.

(3) While the study offers valuable insights into the roles of CCL5 and its connection with the regulation of miR-342-5p (though this connection is somewhat weak), it is recommended that the authors explore potential translational applications of these findings.

Overall, given the experimental designs and results, it is difficult to support the conclusions drawn in the manuscript.

Reviewer #1 (Public review):

Summary:

The authors of this study use an optimization algorithm approach, based on the established Nelder-Mead method, to infer polymer models that best match input bulk Hi-C contact data. The procedure infers the best parameters of a generic polymer model that combines loop-extrusion (LE) dynamics and compartmentalisation of chromatin types driven by weak biochemical affinities. Using this and DNA FISH, the authors investigate the chromatin structure of the MYC locus in leukaemia cells, showing that loop extrusion alone cannot explain local pathogenic chromatin rearrangements. Finally, they study the locus single-cell heterogeneity and time dynamics.

In the revised manuscript the authors have adequately addressed my questions and comments. The exception concerns point #5 of my original review:

(5) Besides cumulative probability distributions, I asked the authors to show the TAD2-TAD4 (model vs. exp) distances in Fig. 3c as relative frequency histograms. This allows readers to more accurately evaluate whether model and experimental distributions have same shape and variance.

Reviewer #1 (Public Review):

The individual roles of both cosolvents and intrinsically disordered proteins (IDPs) in desiccation have been well established, but few studies have tried to elucidate how these two factors may contribute synergistically. The authors quantify the synergy for the model and true IDPs involved with desiccation and find that only the true IDPs have strong desiccation tolerance and synergy with cosolvents. Using these as model systems, they quantify the local (secondary structure vis-a-vi CD spectroscopy) and global dimensions (vis-a-vi the Rg of SAXS experiments) and find no obvious changes with the co-solvents. Instead, they focus on the gelation of one of the IDPs and, using theory and experiments, suggest that the co-solvents may enable desiccation tolerance, an interesting hypothesis to guide future in vivo desiccation studies. A few minor points that remained unclear to this reviewer and that were noted previously have been reasonably addressed in this revision.

Strengths:

This paper is quite extensive and has significant strengths worth highlighting. Notably, the number and type of methods employed to study IDPs are quite unusual, employing CD spectroscopy, SAXS measurements, and DSC. The use of the TFE is an exciting integration of the physical chemistry of cosolvents into the desiccation field is a nice approach and a clever way of addressing the gap of the lack of conformational changes depending on the cosolvents. Furthermore, I think this is a major point and strength of the paper; the underlying synergy of cosolvents and IDPs may lie in the thermodynamics of the dehydration process.

Figure S6A is very useful. I encourage readers who are confused about the DSC analysis, interpretation, and calculation to refer to it.

Weaknesses:

All minor weaknesses were addressed in this revision.

Reviewer #2 (Public Review):

Summary:

The paper aims to investigate the synergies between desiccation chaperones and small molecule cosolutes, and describe its mechanistic basis. The paper reports that IDP chaperones have stronger synergies with the cosolutes they coexist with, and in one case suggests that this is related to oligomerization propensity of the IDP.

Strengths:

The authors have done a good job improving the paper. The study uses a lot of orthogonal methods and the experiments are technically well done. They are addressing a new question that has not really been addressed previously.

Weaknesses:

The conclusions are still based on a few examples and only partial correlations. However, this is now acknowledged by the authors and the conclusions are presented with appropriate caveats.

Reviewer #1 (Public review):

Summary:

The manuscript by Sztangierska et al explores how the Hsp70 chaperone together with its JDP-NEF cofactors and Hsp104 disentangle aggregated proteins. Specifically, the study provides mechanistic findings that explain what role the NEF class Hsp110 has in protein disaggregation. The results explain several previous observations related to Hsp110 in protein disaggregation. Importantly, the study provides compelling evidence that Hsp110 acts early in the disaggregation process.

Strengths:

(1) This is a very well performed study with multiple in vitro experiments that provide convincing support for the claims.

(2) An important finding is that the study places Hsp110 function early in the disaggregation process.

(3) The study has an important value in that it picks up on a number of observations in the field that have not been explored or directly tested by experiment. The presented results settle questions and controversy regarding Hsp110 function in disaggregation.

Weaknesses:

(1) While the key finding of this manuscript is that it places Hsp110 early in the disaggregation process, the other findings are advancing the field less.

Reviewer #2 (Public review):

Sztangierska et al. have investigated the impact of the nucleotide exchange (NEF) factor Hsp110 on the Hsp70-dependent dissolution of amorphous aggregates in the presence of representative members of two classes of J-domain protein.<br /> The authors find that the nucleotide exchange factor of the Hsp110 family, sse1, stimulates the disaggregation activity of yeast Hsp70, ssa1, in particular in the presence of the J-domain protein sis1. Linking chaperone-substrate interactions as determined by biolayer interferometry (BLI) to activity assays, they show that sse1 facilitates the loading of more ssa1 onto the aggregate substrate and propose that this is due to active remodelling of the protein aggregate which exposes more chaperone binding sites and thus facilitates reactivation. This study highlights two important facets of Hsp70 biology: different Hsp70 functions rely on the functional cooperation of specific co-chaperone combinations and the stoichiometry of the different players of the Hsp70 system is an important parameter in tuning Hsp70 chaperone activity.

Strengths:

The manuscript presents a systematic analysis of the functional cooperation of sse1 with a class B J-domain protein sis1 in the disaggregation of two different model aggregate substrates, allowing the authors to draw more general conclusions about Hsp70 disaggregation activity.

The authors can pinpoint the role of sse1 to the initial remodeling of aggregates, rather than the later stages of refolding, highlighting the functional specificity of Hsp70 co-chaperones.

They demonstrate the competitive nature of binding to ssa1 between sse1 and sis1 which can explain the poisoning of Hsp70 chaperone activities observed at high NEF concentrations.

Weaknesses:

While structural requirements have been identified that allow sse1, in cooperation with sis1, to facilitates the loading of Hsp70 on the amorphous aggregate substrate, how this is achieved on a mechanistic level remains an open question.

Reviewer #3 (Public review):

Summary:

The authors studied the function of Hsp110 co-chaperones (e.g. yeast Sse1) in Hsp70-dependent protein disaggregation reactions. The study builds on former work by the authors (Wyszkowski et al., 2021, PNAS), analyzing the binding of Hsp70 and J-domain protein (JDP) cochaperones to protein aggregates using bio-layer interferometry (BLI). It was shown before by other groups that Hsp110 enhances Hsp70 disaggregation activity. The mechanism of Hsp110-stimulated disaggregation activity, however, remained poorly defined. Here, the authors show that yeast Hsp110 increases Hsp70 recruitment to the surface of protein aggregates. The effect is largely dependent on J-domain protein (JDP) identity and particularly pronounced for class B JDPs (e.g. yeast Sis1), which are also more effective in disaggregation reactions. The authors also confirm former results, showing inhibition by increased Hsp110 levels and provide novel evidence that the inhibitory effect is caused by competition between Hsp110 and JDPs for Hsp70 binding.

Strengths:

The work represents a very thoroughly executed study, which provides novel insights into the mechanism of Hsp70-mediated protein disaggregation. Key findings established for yeast chaperones are also documented for human counterparts. The observation that Hsp110 might displace JDPs from Hsp70 during the disaggregation reaction is very appealing. It will now become important to validate this initial finding and dissect how it propels the disaggregation reaction.

Weaknesses:

How exactly the interplay between JDPs and Hsp110 orchestrates protein disaggregation remains largely speculative and further analysis is required for a deeper mechanistic understanding.

Reviewer #1 (Public review):

Summary:

Matsui et al. present an experimental pipeline for visualizing molecular machinery of synapses in the brain, which includes numerous techniques, starting with generating labeled antibodies and recombinant mice, continuing with HPF and FIB milling and finishing with tilt series collection and 3D image processing. This pipeline represents a breakthrough in preparation of brain tissue for high resolution imaging and can be used in future tomographic research to reconstruct molecular details of synaptic complexes as well as pre- and post-synaptic assemblies. This methodology can also be adapted for a broader range of tissue preparations and signifies the next step towards better structural understanding of how molecular machineries operate in natural conditions.

Strengths:

The manuscript is very well written, contains a detailed description of methodology, provides nice illustrations and will be an outstanding guide for future research.

Weaknesses:

None noted.

Reviewer #2 (Public review):

Summary

The authors present a method that allows for the identification and localization of molecular machinery at chemical synapses in unstained, unfixed native brain tissue slices. They believe that this approach will provide a 3D structural basis for understanding different mechanisms of synaptic transmission, plasticity, and development. To achieve this, the group used genetically engineered mouse lines and generated thin brain slices that underwent high-pressure freezing (HPF) and focused ion beam (FIB) milling. Utilizing cryo-electron tomography (cryo-ET) and integrating it with cryo-fluorescence microscopy, they achieved micrometer resolution in identifying the glutamatergic synapses along with nanometer resolution to locate AMPA receptors GluA2-subunits using Fab-AuNP conjugates. The findings are summarized with detailed examples of successfully prepared substrates for cryo-ET, specific morphological identification and localization, and the detailed structural organization of excitatory synapses, including synaptic vesicle clusters close to the postsynaptic density and in the cleft.

Strengths

The study advances previous work that used cultured neurons or synaptosomes. Combining cryo-electron tomography (cryo-ET) with fluorescence-guided targeting and labeling with Fab-AuNP conjugates enabled the study of synapses and molecular structures in their native environment without chemical fixation or staining. This preserves their near-native state, offering high specificity and resolution. The methods developed are mostly generalizable, allowing adaptation for identifying and localizing other key molecules at glutamatergic synapses and potentially useful for studying a variety of synapses and cellular structures beyond the scope of this research.

Weaknesses

The preparation and imaging techniques are complex and require highly specialized equipment and expertise, potentially limiting their accessibility and widespread adoption.

Additionally, the methods might need further modifications/tweaks to study other types of synapses or molecular structures effectively.

The reliance on genetically engineered mouse lines and monoclonal, high-affinity antibodies/Fab fragments to specifically label receptors/proteins would limit the wider employment of these methods.

Reviewer #1 (Public review):

Summary:

This paper by Yang et al. established an in vitro triple co-culture BBB model and demonstrated its advantages compared with the mono or double co-culture BBB model. Further, the authors used their established in vitro BBB model and combined it with other methodologies to investigate the specific signaling mechanisms that co-culture with astrocytes but also neurons enhancing the integrity of endothelial cells.

Strengths:

The results persuasively demonstrated that the established triple co-culture BBB model well mimicked several important characteristics of BBB compared with the mono-culture BBB model, including better barrier function and in vivo/in vitro correlation. The use of human-derived immortalized cells made the model construction process faster and more efficient and had a better in vivo correlation without the complications of species differences. This model is expected to be a useful high-throughput evaluation tool for CNS drug development.

Moreover, the authors used a variety of experiments to prove that the triple co-culture model also reflected the interactions between NVU cells, including promoting endothelial cell proliferation and the formation of intercellular junctions. Interestingly, the authors found that neurons also released GDNF to promote barrier properties of brain endothelial cells, as most current research has focused on the promoting effect of astrocytes-derived GDNF on BBB. Meanwhile, the author also validated the functions of GDNF for BBB integrity in vivo by silencing GDNF in mouse brains. Overall, the experiments and data presented support the claim that neurons, alongside astrocytes, contribute to the promoting effects of the barrier function of endothelial cells through GDNF secretion.

Weaknesses:

While the authors explained that the use of human-derived immortalized cells has been justified as more reproducible and efficient in constructing the model, the TEER value of the triple co-culture model remains lower than that of the physiological statement. Future research may need to explore additional methods to further enhance the barrier function of the model.

Reviewer #2 (Public review):

Summary:

Yang and colleagues developed a new in vitro blood-brain barrier model that is relatively simple yet outperforms previous models. By incorporating a neuroblastoma cell line, they demonstrated increased electrical resistance and decreased permeability to small molecules

Strengths:

The authors initially elucidated the soluble mediator responsible for enhancing endothelial functionality, namely GDNF. Subsequently, they elucidated the mechanisms by which GDNF upregulates the expression of VE-cadherin and Claudin-5. They further validated these findings in vivo, and demonstrated predictive value for molecular permeability as well. The study is meticulously conducted and easily comprehensible. The conclusions are firmly supported by the data, and the objectives are successfully achieved. This research is poised to advance future investigations in BBB permeability, leakage, dysfunction, disease modeling, and drug delivery, particularly in high-throughput experiments. I anticipate an enthusiastic reception from the community interested in this area. While other studies have produced similar results with tri-cultures (PMID: 25630899), this study notably enhances electrical resistance compared to previous attempts.

Weaknesses:

The power of this system lies in its simplicity, which is enough to study BBB permeability. However, it also lacks some other important cell-cell interactions such as those involving pericytes. Nonetheless, this is still a valuable tool for high throughput screening.

As with many other similar systems, it has lower TEER values compared to the in vivo counterpart, this is an issue that researchers in the field will have to address in future studies

Reviewer #1 (Public review):

Herzog and colleagues investigated the interactions between working memory (WM) task condition (updating, maintenance) and BMI (body-mass-index), while considering selected dopaminergic genes (COMT, Taq1A, C957T, DARPP-32). Emerging evidence suggest that there might be a specific negative association with BMI in the updating but not maintenance condition, with potential bearings to reversal reward learning in obesity. The inclusion of multiple dopaminergic genes is a strength in the present study, considering the complexity of the interactions between tonic and phasic dopamine across the brain that may distinctly associate with the component processes of WM. Here, the finding was that BMI was negatively associated with WM performance regardless of the condition (updating, maintenance), but in models including moderation by either Taq1A or DARPP-32 (but not by COMT and C957T) an interaction by task condition was observed. Furthermore, a two-way interaction effect between BMI and genotype was observed exclusively in the updating condition. These findings are in line with the accounts by which striatal dopamine as reflected by Taq1A and DARPP-32 play an important role in working memory updating, while cortical dopamine as reflected by COMT is mainly associated with maintenance. The authors conclude that the genetic moderation reflects a compound effect of having high BMI and an advantageous allele in Taq1A or DARPP-32 to working memory updating specifically.

These data increment the accumulating evidence that the dopamine system plays an important role in obesity. The result that Taq1A and DARPP-32 moderated the interaction between WM condition and BMI required intricate post hoc analysis to understand the bearings to updating. The authors found that Taq1A or DARPP-32 genotype moderated the negative association between BMI and WM exclusively in update condition (significant two-way interaction effect), suggesting that the BMI-WM associations in other conditions were similar across genotypes. Importantly, visual inspection of the relationship between WM and BMI (Fig 4 & 5) suggests more prevalent positive effects of the putatively advantageous Taq1A-A1 and DARPP-32-AA genotypes to the overall negative relationship between WM and BMI in updating, but not in the other conditions. Given that an overall negative relationship was statistically supported across all conditions (model 1), a plausible interpretation would be that updating condition stands out in terms of a positive moderation by putative advantageous genotypes, rather than compound negative consequences of BMI and genotype in updating. Statistical testing stratified by Taq1A genotype confirmed that the interaction with task condition was driven by the carriers of the advantageous genotype, whereas stratification by DARPP-32 genotype revealed a significant task-condition interaction in both A/A- and G-carriers. Taken together, the present results highlight inter-subject variability in the associations between obesity, dopamine, and working memory, which can sometimes be captured using blood-based dopamine markers. This finding indicates that not all individuals with obesity show the same patterns of dopamine-related alterations and underscores the necessity to address inter-individual variability in future research and treatment efforts.

Reviewer #2 (Public review):

Summary:

The authors investigated if obesity is associated with elevated working memory deficits. Prior theorizing would suggest that individuals with a higher BMI would be worse at working memory updating, potentially due to impaired dopaminergic signaling in the striatum. However, the authors find that higher BMI was associated with worse working memory performance, irrespective of having to ignore or update new information. To further explore the putative dopaminergic mechanisms, participants are stratified according to genetic polymorphisms in COMT, Taq1A, DARPP and C957T and the ratio of the amino acids phenylalanine and tyrosine, all implicated in dopamine-signaling. They find that carrying specific alleles of Taq1A and DARPP, but not of COMT and C957T, mitigated the otherwise negative relationship between BMI and working memory for updating, but not for maintenance.

The authors put forward several possible mechanistic explanations of these observations, including imbalances in the striatal go/no-go dopamine pathways. However, only future, more direct measures of dopamine signaling can provide a confirmation of these hypotheses.

Strengths:

Differentiating between working memory maintenance (ignoring) and updating is a powerful way to get a deeper insight into specific working memory deficits in individuals with obesity. This way of assessing working memory could potentially be applied to various populations at risk for cognitive or working memory deficits.

By pooling data from three studies, the authors reached a relatively large sample of 320 participants, which enables the assessment of more subtle effects on working memory, including the differentiation between updating and ignoring.

Working memory gating has long implicated striatal dopamine signaling. This paper shows that a specific combination of a high BMI and specific dopamine-related genotypes can selectively moderate working memory updating. More insight into how these risk factors interact can ultimately lead to more tailor-made treatments.

Weaknesses:

The introduction mentions that specific alleles can alter dopamine signaling in various ways. However, the authors are less clear on how they expect these alterations to subsequently affect working memory updating and maintenance in the current study. While I understand that the complexity of these mechanisms might make it challenging to form specific predictions, it would be helpful if the authors acknowledged this uncertainty and clarified that their analyses are exploratory in nature, and they will therefore refrain from any directional hypotheses regarding the genotypes.

The majority of participants seems to fall within the normal BMI-range, whereas the interaction between BMI and genetic variations or amino acid ratio particularly surfaces at higher BMI. As genetic variations are usually associated with small effect sizes, the effective sample size, although large for a behavioral analysis only, might have been too small to detect meaningful effects of particular alleles of COMT and C957T.

The relationships between genetic variations, BMI and specific disturbances in dopamine signaling are complex, as compensating mechanisms might be at play to mitigate any detrimental effects. Future studies that apply more direct measures or manipulations of dopaminergic processes could therefore aid in mechanistically explaining the results.

Reviewer #1 (Public Review):

In the current study, Papandreou et al. developed an iPSC-based midbrain dopaminergic neuronal cell model of Beta-Propeller Protein-Associated Neurodegeneration (BPAN), which is caused by mutations in the WDR45 gene and is known to impair autophagy. They also noted defective autophagy and abnormal BPAN-related gene expression signatures. Further, they performed a drug screening and identified five cardiac glycosides. Treatment with these drugs effectively in improved autophagy defects and restored gene expression. Seeing the autophagy defects and impaired expression of BPAN-related genes adds strength to this study. Importantly, this work shows the value of iPSC-based modeling in studying disease and finding therapeutic strategies for genetic disorders, including BPAN.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors aim to demonstrate that cardiac glycosides restore autophagy flux in an iPSC-derived mDA neuronal model of WDR45 deficiency. They established a patient-derived induced pluripotent stem cell (iPSC)-based midbrain dopaminergic (mDA) neuronal model and performed a medium-throughput drug screen using high-content imaging-based IF analysis. Several compounds were identified that ameliorate disease-specific phenotypes in vitro.

Strengths:

This manuscript engaged in an important topic and yielded some interesting data.

Reviewer #2 (Public review):

Prior work by the Sehgal group has shown that a small group of neurons in the fly brain (anterior posterior (ap) α'β' mushroom body neurons (MBNs)) promote sleep and sleep-dependent appetitive memory specifically under fed conditions (Chouhan et al., (2021) Nature). Here, Li, Chouhan et al. combine cell-specific transcriptomics with measurements of sleep and memory to identify molecular processes underlying this phenomenon. They define transcriptional changes in ap α'β' MBNs and suggest a role for two genes downregulated following memory induction (Polr1F and Regnase-1) in regulating sleep and memory.

The transcriptional analyses in this manuscript are impressive. The authors have now included additional experiments that define acute and developmental roles for Polr1F and Regnase-1 respectively in regulating sleep. They have also provided additional data to strengthen their conclusion that Polr1F knockdown in α'β' mushroom body neurons enhances sleep.

The resubmitted work represents a convincing investigation of two novel sleep-regulatory proteins that may also play important roles in memory formation.

The authors have comprehensively addressed my comments, which I very much appreciate. I congratulate them on this excellent work.

Reviewer #3 (Public review):

Previous work (Chouhan et al., 2022) from the Sehgal group investigated the relationship between sleep and long-term memory formation by dissecting the role of mushroom body intrinsic neurons, extrinsic neurons, and output neurons during sleep-dependent and sleep-independent memory consolidation. In this manuscript, Li et al., profiled transcriptome in the anterior-posterior (ap) α'/β' neurons and identified genes that are differentially expressed after training in fed condition, which supports sleep-dependent memory formation. By knocking down candidate genes systematically, the authors identified Polr1F and Regnase-1 as two important hits that play potential roles in sleep and memory formation. What is the function of sleep and how to create a memory are two long-standing questions in science. The present study used a new approach to identify novel components that may link sleep and memory consolidation in a specific type of neuron. Importantly, these components implicated that RNA processing may play a role in these processes.

While I am enthusiastic about the innovative approach employed to identify RNA processing genes involved in sleep regulation and memory consolidation, I feel that the data presented in the manuscript is insufficient to support the claim that these two genes establish a definitive link between sleep and memory consolidation. First, the developmental role of Regnase-1 in reducing sleep remains unclear because knocking down Regnase-1 using the GeneSwitch system produced neither acute nor chronic sleep loss phenotype. In the revised manuscript, the author used the Gal80ts to restrict the knockdown of Regnase-1 in adult animals and concluded that Regnase-1 RNAi appears to affect sleep through development. Conducting overexpression experiments of Regnase-1 would lend some credibility to the phenotypes, however, this is not pursued in the revised manuscript. Second, while constitutive Regnase-1 knockdown produced robust phenotypes for both sleep-dependent and sleep-independent memory, it also led to a severe short-term memory phenotype. This raises the possibility that flies with constitutive Regnase-1 knockdown are poor learners, thereby having little memory to consolidate. The defect in learning could be simply caused by chronic sleep loss before training. Thus, this set of results does not substantiate a strong link between sleep and long-term memory consolidation. Lastly, the discussion on the sequential function of training, sleep, and RNA processing on memory consolidation appears speculative based on the present data.

Reviewer #4 (Public review):

Summary:

Li and Chouhan et al. follow up on a previous publication describing the role of anterior-posterior (ap) and medial (m) ɑ′/β′ Kenyon cells in mediating sleep-dependent and sleep-independent memory consolidation, respectively, based on feeding state in Drosophila melanogaster. The authors sequenced bulk RNA of ap ɑ′/β′ Kenyon cells 1h after flies were either trained-fed, trained-starved or untrained-fed and find a small number of genes (59) differentially expressed (3 upregulated, 56 downregulated) between trained-fed and trained-starved conditions. Many of these genes encode proteins involved in the regulation of gene expression. The authors then screened these differentially expressed genes for sleep phenotypes by expressing RNAi hairpins constitutively in ap ɑ′/β′ Kenyon cells and measuring sleep patterns. Two hits were selected for further analysis: Polr1F, which promoted sleep, and Regnase-1, which reduced sleep. The pan-neuronal expression of Polr1F and Regnase-1 RNAi constructs was then temporally restricted to adult flies using the GeneSwitch system. Polr1F sleep phenotypes were still observed, while Regnase-1 sleep phenotypes were not, indicating developmental defects. Appetitive memory was then assessed in flies with constitutive knockdown of Polr1F and Regnase-1 in ap ɑ′/β′ Kenyon cells. Polr1F knockdown did not affect sleep-dependent or sleep-independent memory, while Regnase-1 knockdown disrupted sleep-dependent memory, sleep-independent memory, as well as learning. Polr1F knockdown increased pre-ribosomal RNA transcripts in the brain, as measured by qPCR, in line with its predicted role as part of the RNA polymerase I complex. A puromycin incorporation assay to fluorescently label newly synthesized proteins also indicated higher levels of bulk translation upon Polr1F knockdown. Regnase-1 knockdown did not lead to observable changes in measurements of bulk translation.

Strengths:

The proposed involvement of RNA processing genes in regulating sleep and memory processes is interesting, and relatively unexplored. The methods are satisfactory.

Weaknesses:

The main weakness of the paper is in the overinterpretation of their results, particularly relating to the proposed link between sleep and memory consolidation, as stated in the title. Constitutive Polr1F knockdown in ap ɑ′/β′ Kenyon cells had no effect on appetitive long-term memory, while constitutive Regnase-1 knockdown affected both learning and memory. Since the effects of constitutive Regnase-1 knockdown on sleep could be attributed to developmental defects, it is quite plausible that these same developmental defects are what drive the observed learning and memory phenotypes. In this case, an alternative explanation of the authors' findings is that constitutive Regnase-1 knockdown disrupts the entire functioning of ap ɑ′/β′ Kenyon cells, and as a consequence behaviors involving these neurons (i.e. learning, memory and sleep) are disrupted. It will be important to provide further evidence of the function of RNA processing genes in memory in order to substantiate the memory link proposed by the authors.

Reviewer #1 (Public review):

This is a very nice study of Belidae weevils using anchored phylogenomics that presents a new backbone for the family and explores, despite a limited taxon sampling, several evolutionary aspects of the group. I find that the methodology is appropriate, and all analytical steps are well presented. The paper is well written and presents interesting aspects of Belidae systematics and evolution. The major weakness of the study being the very limited taxon sampling that has deep implications for the discussion of ancestral estimations.

Reviewer #2 (Public review):

Summary:

The authors used a combination of anchored hybrid enrichment and Sanger sequencing to construct a phylogenomic data set for the weevil family Belidae. Using evidence from fossils and previous studies they are able to estimate a phylogenetic tree with a range of dates for each node - a timetree. They use this to reconstruct the history of the belids' geographic distributions and associations with their hostplants. They infer that the belids' association with conifers pre-dates the rise of the angiosperms. They offer an interpretation of belid history in terms of the breakup of Gondwanaland, but acknowledge that they cannot rule out alternative interpretations that invoke dispersal.

Strengths:

The strength of any molecular-phylogenetic study hinges on four things: the extent of the sampling of taxa; the extent of the sampling of loci (DNA sequences) per genome; the quality of the analysis; and - most subjectively - the importance and interest of the evolutionary questions the study allows the authors to address. The first two of these, sampling of taxa and loci, impose a tradeoff: with finite resources, do you add more taxa or more loci? The authors follow a reasonable compromise here, obtaining a solid anchored-enrichment phylogenomic data set (423 genes, >97 kpb) for 33 taxa, but also doing additional analyses that included 13 additional taxa from which only Sanger sequencing data from 4 genes was available. The taxon sampling was pretty solid, including all 7 tribes and a majority of genera in the group. The analyses also seemed to be solid - exemplary, even, given the data available.

This leaves the subjective question of how interesting the results are. The very scale of the task that faces systematists in general, and beetle systematists in particular, presents a daunting challenge to the reader's attention: there are so many taxa, and even a sophisticated reader may never have heard of any of them. Thus it's often the case that such studies are ignored by virtually everyone outside a tiny cadre of fellow specialists. The authors of the present study make an unusually strong case for the broader interest and importance of their investigation and of its focal taxon, the belid weevils.

The belids are of special interest because - in a world churning with change and upheaval, geologically and evolutionarily - relatively little seems to have been going on with them, at least with some of them, for the last hundred million years or so. The authors make a good case that the Araucaria-feeding belid lineages found in present-day Australasia and South America have been feeding on Araucaria continuously since the days when it was a dominant tree taxon nearly worldwide, before it was largely replaced by angiosperms. Thus these lineages plausibly offer a modern glimpse of an ancient ecological community.

Comments on current version:

The MS was already in pretty good shape last time around, and the authors have made most of the minor revisions and copy-edits suggested by the reviewers. There may be a few remaining points of disagreement with the reviewers, but these seem to be minor matters of opinion and nothing that ought to delay publication.

Reviewer #1 (Public Review):

Summary:

The study investigated how root cap cell corpse removal affects the ability of microbes to colonize Arabidopsis thaliana plants. The findings demonstrate how programmed cell death and its control in root cap cells affect the establishment of symbiotic relationships between plants and fungi. Key details on molecular mechanisms and transcription factors involved are also given. The study suggests reevaluating microbiome assembly from the root tip, thus challenging traditional ideas about this process. While the work presents a key foundation, more research along the root axis is recommended to gain a better understanding of the spatial and temporal aspects of microbiome recruitment.

Comments on revised version:

The authors have positively addressed all the critical points I raised in the previous review.

Reviewer #2 (Public Review):

Summary:

The authors identify the root cap as an important key region for establishing microbial symbioses with roots. By highlighting for the first time the crucial importance of tight regulation of a specific form of programmed cell death of root cap cells and the clearance of their cell corpses, they start unraveling the molecular mechanisms and its regulation at the root cap (e.g. by identifying an important transcription factor) for the establishment of symbioses with fungi (and potentially also bacterial microbiomes).

Strengths:

It is often believed that the recruitment of plant microbiomes occurs from bulk soil to rhizosphere to endosphere. These authors demonstrate that we have to re-think microbiome assembly as a process starting and regulated at the root tip and proceeding along the root axis.

Comments on revised version:

The authors have addressed all critical points in their revision.

Reviewer #1 (Public review):

In this manuscript, the role of orexin receptors in dopamine transmission is studied. It extends previous findings suggesting an interplay of these two systems in regulating behaviour by first characterising the expression of orexin receptors in the midbrain and then disrupting orexin transmission in dopaminergic neurons by deleting its predominant receptor, OX1R (Ox1R fl/fl, Dat-Cre tg/wt mice). Electrophysiological and calcium imaging data suggest that orexin A acutely and directly stimulates SN and VTA dopaminergic neurons, but does not seem to induce c-Fos expression. Behavioural effects of depleting OX1R from dopaminergic neurons includes enhanced novelty-induced locomotion and exploration, relative to littermate controls (Ox1R fl/fl, Dat-Cre wt/wt). However, no difference between groups is observed in tests that measure reward processing, anxiety, and energy homeostasis. To test whether depletion of OX1R alters overall orexin-triggered activation across the brain, PET imaging is used in OX1R∆DAT knockout and control mice. This analysis reveals that several regions show a higher neuronal activation after orexin injection in OX1R∆DAT mice, but the authors focus their follow up study on the dorsal bed nucleus of the stria terminalis (BNST) and lateral paragigantocellular nucleus (LPGi). Dopaminergic inputs and expression of dopamine receptors type-1 and -2 (DRD1 & DRD2) is assessed and compared to control demonstrating moderate decrease of DRD1 and DRD2 expression in BNST of OX1R∆DAT mice and unaltered expression of DRD2, with absence of DRD1 expression in LPGi of both groups. Overall, this study is valuable for the information it provides on orexin receptor expression and function on behaviour and for the new tools it generated for the specific study of this receptor in dopaminergic circuits.

Strengths:

The use of a transgenic line that lacks OX1R in dopamine-transporter expressing neurons is a strong approach to dissect the direct role of orexin in modulating dopamine signalling in the brain. The battery of behavioural assays to study this line provides a valuable source of information for researchers interested in the role of orexin in animal physiology.

Weaknesses:

This study falls short in providing evidence for an anatomical substrate of the altered behaviour observed in mice lacking orexin receptor subtype 1 in dopaminergic neurons. How orexin transmission in dopaminergic neurons regulates the expression of postsynaptic dopamine receptors (as observed in BNST of OX1R∆DAT mice) is an intriguing question poorly discussed. Whether disruption of orexin activity alters dopamine release in target areas is an important point not addressed.

Reviewer #2 (Public review):

Summary:

This manuscript examines expression of orexin receptors in midbrain - with a focus on dopamine neurons - and uses several fairly sophisticated manipulation techniques to explore the role of this peptide neurotransmitter in reward-related behaviors. Specifically, in situ hybridization is used to show that dopamine neurons predominantly express orexin receptor 1 subtype and then go on to delete this receptor in dopamine transporter-expressing using a transgenic strategy. Ex vivo calcium imaging of midbrain neurons is used to show that, in the absence of this receptor, orexin is no longer able to excite dopamine neurons of the substantia nigra.

The authors proceed to use this same model to study the effect of orexin receptor 1 deletion on a series of behavioral tests, namely, novelty-induced locomotion and exploration, anxiety-related behavior, preference for sweet solutions, cocaine-induced conditioned place preference, and energy metabolism. Of these, the most consistent effects are seen in the tests of novelty-induced locomotion and exploration in which the mice with orexin 1 receptor deletion are observed to show greater levels of exploration, relative to wild-type, when placed in a novel environment, an effect that is augmented after icv administration of orexin.

In the final part of the paper, the authors use PET imaging to compare brain-wide activity patterns in the mutant mice compared to wildtype. They find differences in several areas both under control conditions (i.e., after injection of saline) as well as after injection of orexin. They focus in on changes in dorsal bed nucleus of stria terminalis (dBNST) and the lateral paragigantocellular nucleus (LPGi) and perform analysis of the dopaminergic projections to these areas. They provide anatomical evidence that these regions are innervated by dopamine fibers from midbrain, are activated by orexin in control, but not mutant mice, and that dopamine receptors are present. Thus, they argue these anatomical data support the hypothesis that behavioral effects of orexin receptor 1 deletion in dopamine neurons are due to changes in dopamine signaling in these areas.

Strengths:

Understanding how orexin interacts with the dopamine system is an important question and this paper contains several novel findings along these lines. Specifically:

(1) Distribution of orexin receptor subtypes in VTA and SN is explored thoroughly.<br /> (2) Use of the genetic model that knocks out a specific orexin receptor subtype from dopamine-transporter-expressing neurons is a useful model and helps to narrow down the behavioral significance of this interaction.<br /> (3) PET studies showing how central administration of orexin evokes dopamine release across the brain is intriguing, especially that two key areas are pursued - BNST and LPGi - where the dopamine projection is not as well described/understood.

Weaknesses:

The role of the orexin-dopamine interaction is not explored in enough detail. The manuscript presents several related findings, but the combination of anatomy and manipulation studies do not quite tell a cogent story. Ideally, one would like to see the authors focus on a specific behavioral parameter and show that one of their final target areas (dBNST or LPGi) was responsible or at least correlated with this behavioral readout.

In many places in the Results, insufficient explanation and statistical reporting is provided. Throughout the Results - especially in the section on behavior although not restricted to this part - statements are made without statistical tests presented to back up the claims, e.g., "Compared to controls, Ox1RΔDAT 143 mice did not show significant changes in spontaneous locomotor activity in home cages" (L143) and "In a hole-board test, female Ox1RΔDAT mice showed increased nose pokes into the holes in early (1st and 2nd) sessions compared to control mice" (L151). In other places, ANOVAs are mentioned but full results including main effects and interactions are not described in detail, e.g., in F3-S3, only a single p-value is presented and it is difficult to know if this is the interaction term or a post hoc test (L205). These and all other statements need statistics included in the text as support. Addition of these statistical details was also requested by the editor.

In the presentation of reward processing this is particularly important as no statistical tests are shown to demonstrate that controls show a cocaine-induced preference or a sucrose preference. Here, one option would be to perform one-sample t-tests showing that the data were different to zero (no preference). As it is, the claim that "Both of the control and Ox1RΔDAT groups showed a preference for cocaine injection" is not yet statistically supported.

Reviewer #1 (Public review):

Summary:

The authors investigated the anatomical features of the synaptic boutons in layer 1 of the human temporal neocortex. They examined the size of each synapse, the macular or perforated appearance, the size of the synaptic active zone, the number and volume of the mitochondria, and the number of synaptic and dense core vesicles, also differentiating between the readily releasable, the recycling, and the resting pool of synaptic vesicles. The coverage of the synapse by astrocytic processes was also assessed, and all the above parameters were compared to other layers of the human temporal neocortex. The authors conclude that the subcellular morphology of the layer 1 synapses are suitable for the functions of the neocortical layer, i.e. the synaptic integration within the cortical column. The low glial coverage of the synapses might allow increased glutamate spillover from the synapses, enhancing synpatic crosstalk within this cortical layer.

Strengths:

The strengths of this paper are the abundant and very precious data about the fine structure of the human neocortical layer 1. Quantitative electron microscopy data (especially that derived from the human brain) are very valuable since this is a highly time- and energy-consuming work. The techniques used to obtain the data, as well as the analyses and the statistics performed by the authors are all solid, strengthen this manuscript, and mainly support the conclusions drawn in the discussion.

Weaknesses:

There are several weaknesses in this work. First, the authors should check and review extensively for improvements to the use of English. Second, several additional analyses performed on the existing data could substantially elevate the value of the data presented. Much more information could be gained from the existing data about the functions of the investigated layer, of the cortical column, and about the information processing of the human neocortex. Third, several methodological concerns weaken the conclusions drawn from the results.

Reviewer #2 (Public review):

Summary:

The study of Rollenhagen et al. examines the ultrastructural features of Layer 1 of the human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as far as possible from the epilepsy focus, and as such considered to be non-epileptic. The analyses included 4 patients with different ages, sex, medication, and onset of epilepsy. The manuscript is a follow-on study with 3 previous publications from the same authors on different layers of the temporal cortex:

Layer 4 - Yakoubi et al 2019 eLife<br /> Layer 5 - Yakoubi et al 2019 Cerebral Cortex<br /> Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex.

They find, that the L1 synaptic boutons mainly have a single active zone, a very large pool of synaptic vesicles, and are mostly devoid of astrocytic coverage.

Strengths:

The manuscript is well-written and easy to read. The Results section gives a detailed set of figures showing many morphological parameters of synaptic boutons and glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in the human brain are still very limited, the current manuscript has substantial relevance.

The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analysis is clear and precise.

Weaknesses:

One of the main findings of this paper is that "low degree of astrocytic coverage of L1 SBs suggests that glutamate spillover and as a consequence synaptic cross-talk may occur at the majority of synaptic complexes in L1". However, the authors only quantified the volume ratio of astrocytes in all 6 layers, which is not necessarily the same as the glial coverage of synapses. In order to strengthen this statement, the authors could provide 3D data (that they have from the aligned serial sections) detailing the percentage of synapses that have glial processes in close proximity to the synaptic cleft, that would prevent spillover.

A specific statement is missing on whether only glutamatergic boutons were analysed in this MS, or GABAergic boutons were also included. There is a statement, that they can be distinguished from glutamatergic ones, but it would be useful to state it clearly in the Abstract, Results, and Methods section what sort of boutons were analysed. Also, what is the percentage of those boutons from the total bouton population in L1?

Synaptic vesicle diameter (that has been established to be ~40nm independent of species) can properly be measured with EM tomography only, as it provides the possibility to find the largest diameter of every given vesicle. Measuring it in 50 nm thick sections results in underestimation (just like here the values are ~25 nm) as the measured diameter will be smaller than the true diameter if the vesicle is not cut in the middle, (which is the least probable scenario). The authors have the EM tomography data set for measuring the vesicle diameter properly.

It is a bit misleading to call vesicle populations at certain arbitrary distances from the presynaptic active zone as readily releasable pool, recycling pool, and resting pool, as these are functional categories, and cannot directly be translated to vesicles at certain distances. Indeed, it is debated whether the morphologically docked vesicles are the ones, that are readily releasable, as further molecular steps, such as proper priming are also a prerequisite for release.

Tissue shrinkage due to aldehyde fixation is a well-documented phenomenon that needs compensation when dealing with density values. The authors cite Korogod et al 2015 - which actually draws attention to the problem comparing aldehyde fixed and non-fixed tissue, still the data is non-compensated in the manuscript. Since all the previous publications from this lab are based on aldehyde fixed non-compensated data, and for this sake, this dataset should be kept as it is for comparative purposes, it would be important to provide a scaling factor applicable to be able to compare these data to other publications.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The study provides only morphological details, these can be useful in the future when combined with functional assessments or computational approaches. The authors emphasize the importance of their findings on astrocytic coverage and suggest important implications for glutamate spillover. However, the percentage of synapses that form tripartite synapses has not been quantified, the authors' functional claims are based solely on volumetric fraction measurements.

The distinction between excitatory and inhibitory synapses is not clear, they should be analyzed separately.

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

Reviewer #1 (Public review):

Assessment:

This important work advances our understanding of navigation and path integration in mammals by using a clever behavioral paradigm. The paper provides compelling evidence that mice are able to create and use a cognitive map to find "short cuts" in an environment, using only the location of rewards relative to the point of entry to the environment and path integration, and need not rely on visual landmarks.

Summary:

The authors have designed a novel experimental apparatus called the 'Hidden Food Maze (HFM)' and a beautiful suite of behavioral experiments using this apparatus to investigate the interplay between allothetic and idiothetic cues in navigation. The results presented provide a clear demonstration of the central claim of the paper, namely that mice only need a fixed start location and path integration to develop a cognitive map. The experiments and analyses conducted to test the main claim of the paper -- that the animals have formed a cognitive map -- are conclusive. While I think the results are quite interesting and sound, one issue that needs to be addressed is the framing how landmarks are used (or not), as discussed below, although I believe this will be a straight forward issue for the authors to address.

Strengths:

The 90 degree rotationally symmetric design and use of 4 distal landmarks and 4 quadrants with their corresponding rotationally equivalent locations (REL) lends itself to teasing apart the influence of path integration and landmark-based navigation in a clever way. The authors use a really complete set of experiments and associated controls to show that mice can use a start location and path integration to develop a cognitive map and generate shortcut routes to new locations.

Weaknesses:

There were no major weaknesses identified that were not addressed during revisions.

Reviewer #3 (Public review):

Summary:

How is it that animals find learned food locations in their daily life? Do they use landmarks to home in on these learned locations or do they learn a path based on self-motion (turn left, take ten steps forward, turn right, etc.). This study carefully examines this question in a well designed behavioral apparatus. A key finding is that to support the observed behavior in the hidden food arena, mice appear to not use the distal cues that are present in the environment for performing this task. Removal of such cues did not change the learning rate, for example. In a clever analysis of whether the resulting cognitive map based on self-motion cues could allow a mouse to take a shortcut, it was found that indeed they are. The work nicely shows the evolution of the rodent's learning of the task, and the role of active sensing in the targeted reduction of uncertainty of food location proximal to its expected location.

Strengths:

A convincing demonstration that mice can synthesize a cognitive map for the finding of a static reward using body frame-based cues. Showing that uncertainty of final target location is resolved by an active sensing process of probing holes proximal to the expected location. Showing that changing the position of entry into the arena rotates the anticipated location of the reward in a manner consistent with failure to use distal cues.

Weaknesses:

The task is low stakes, and thus the failure to use distal cues at most costs the animal a delay in finding the food; this delay is likely unimportant to the animal, and the pre-training procedure is likely to make it clear to the animal's that distal cues are unreliable even if desirable to use. Thus, it is unclear whether this result would generalize to a situation where the animal may be under some time pressure, urgency due to food (or water) restriction, or due to predatory threat, or situations where distal cues are reliable. In such cases, the use of distal cues to make locating the reward robust to changing start locations may be more likely to be observed.

Reviewer #1 (Public review):

Summary:

This study focuses on characterizing a previously identified gene, encoding the secreted protein Ppe1, that may play a role in rice infection by the blast fungus Magnaporthe oryzae. Magnaporthe oryzae is a hemibiotrophic fungus that infects living host cells before causing disease. Infection begins with the development of a specialized infection cell, the appressorium, on the host leaf surface. The appressorium generates enormous internal turgor that acts on a thin penetration peg at the appressorial base, forcing it through the leaf cuticle. Once through this barrier, the peg elaborates into bulbous invasive hyphae that colonizes the first infected cell before moving to neighboring cells via plasmodesmata. During this initial biotrophic growth stage, invasive hyphae invaginate the host plasma membrane, which surrounds growing hyphae as the extra-invasive hyphae membrane (EIHM). To avoid detection, the fungus secretes apoplastic effectors into the EIHM matrix via the conventional ER-Golgi secretion pathway. The fungus also forms a plant-derived structure called the biotrophic interfacial complex (BIC) that receives cytoplasmic effectors through an unconventional secretion route before they are delivered into the host cell. Together, these secreted effector proteins act to evade or suppress host innate immune responses. Here the authors contribute to our understanding of M. oryzae infection biology by showing how Ppe1, which localizes to both the appressorial penetration peg and to the appressorial-like transpressoria associated with invasive hyphal movements into adjacent cells, maximizes host cell penetration and disease development and is thus a novel contributor to rice blast disease.

Strengths:

A major goal of M. oryzae research is to understand how the fungus causes disease, either by determining the physiological underpinnings of the fungal infection cycle or by identifying effectors and their host targets. Such new knowledge may point the way to novel mitigation strategies. Here, the authors make an interesting discovery that bridges both fungal physiology and effector biology research by showing how a secreted protein Ppe1, initially considered an effector with potential host targets, associates with its own penetration peg (and transpressoria) to facilitate host invasion. In a previous study, the authors had identified a small family of small secreted proteins that may function as effectors. Here they suggest Ppe1 (and, later in the manuscript, Ppe2/3/5) localizes outside the penetration peg when appressoria develops on surfaces that permit penetration, but not on artificial hard surfaces that prevent peg penetration. Deleting the PPE1 gene reduced (although did not abolish) penetration, and a fraction of those that penetrated developed invasive hyphae that were reduced in growth compared to WT. Using fluorescent markers, the authors show that Ppe1 forms a ring underneath appressoria, likely where the peg emerges, which remained after invasive hyphae had developed. The ring structure is smaller than the width of the appressorium and also lies within the septin ring known to form during peg development. This so-called penetration ring also formed at the transpressorial penetration point as invasive hyphae moved to adjacent cells. This structure is novel, and required for optimum penetration during infection. Furthermore, Ppe1, which carries a functional signal peptide, may form on the periphery of the peg, together suggesting it is secreted and associated with the peg to facilitate penetration. Staining with aniline blue also suggests Ppe1 is outside the peg. Together, the strength of the work lies in identifying a novel appressorial penetration ring structure required for full virulence.

Weaknesses:

The main weakness of the paper is that, although Ppe1 is associated with the peg and optimizes penetration, the function of Ppe1 is not known. The work starts off considering Ppe1 a secreted effector, then a facilitator of penetration by associating with the peg, but what role it plays here is only often speculated about. For example, the authors consider at various times that it may have a structural role, a signaling role orchestrating invasive hyphae development, or a tethering role between the peg and the invaginated host plasma membrane (called throughout the host cytoplasmic membrane, a novel term that is not explained). However, more effort should be expended to determine which of these alternative roles is the most likely. Otherwise, as it stands, the paper describes an interesting phenomenon (the appressorial ring) but provides no understanding of its function.

The inability to nail down the function of Ppe1 likely stems from two underlying assumptions with weak support. Firstly, the authors assume that Ppe1 is secreted and associated with the peg to form a penetration ring between the plant cell wall and cytoplasm membrane. However, the authors do not demonstrate it is secreted (for instance by blocking Ppe1 secretion and its association with the peg using brefeldin A). Also, they do not sufficiently show that Ppe1 localizes on the periphery of the peg. This is because confocal microscopy is not powerful enough to see the peg. The association they are seeing (for example in Figure 4) shows localization to the bottom of the appressorium and around the primary hyphae, but the peg cannot be seen. Here, the authors will need to use SEM, perhaps in conjunction with gold labeling of Ppe1, to show it is associating with the peg and, indeed, is external to the peg (rather than internal, as a structural role in peg rigidity might predict). It would also be interesting to repeat the microscopy in Figure 4C but at much earlier time points, just as the peg is penetrating but before invasive hyphae have developed - Where is Ppe1 then? Finally, the authors speculate, but do not show, that Ppe1 anchors penetration pegs on the plant cytoplasm membrane. Doing so may require FM4-64 staining, as used in Figure 2 of Kankanala et al, 2007 (DOI: 10.1105/tpc.106.046300), to show connections between Ppe1 and host membranes. Note that the authors also do not show that the penetration ring is a platform for effector delivery, as speculated in the Discussion.

Secondly, the authors assume Ppe1 is required for host infection due to its association with the peg. However, its role in infection is minor. The majority of appressoria produced by the mutant strain penetrate host cells and elaborate invasive hyphae, and lesion sizes are only marginally reduced compared to WT (in fact, the lesion density of the 70-15 WT strain itself seems reduced compared to what would be expected from this strain). The authors did not analyze the lesions for spores to confirm that the mutant strains were non-pathogenic (non-pathogenic mutants sometimes form small pinprick-like lesions that do not sporulate). Thus, the pathogenicity phenotype of the knockout mutant is weak, which could contribute to the inability to accurately define the molecular and cellular function of Ppe1.

In summary, it is important that the role of Ppe1 in infection be determined.

Reviewer #2 (Public review):

The article focuses on the study of Magnaporthe oryzae, the fungal pathogen responsible for rice blast disease, which poses a significant threat to global food security. The research delves into the infection mechanisms of the pathogen, particularly the role of penetration pegs and the formation of a penetration ring in the invasion process. The study highlights the persistent localization of Ppe1 and its homologs to the penetration ring, suggesting its function as a structural feature that facilitates the transition of penetration pegs into invasive hyphae. The article provides a thorough examination of the infection process of M. oryzae, from the attachment of conidia to the development of appressoria and the formation of invasive hyphae. The discovery of the penetration ring as a structural element that aids in the invasion process is a significant contribution to the understanding of plant-pathogen interactions. The experimental methods are well-documented, allowing for reproducibility and validation of the results.

Reviewer #1 (Public Review):

The paper itself has a reasonable aim, to compare the inputs to the hippocampus from cortical regions across mammals. But for some reason, the conclusions that are reached are very limited. We know for example that the main laboratory rodents investigated, rats and mice, are nocturnal, live in underground tunnels, and have a very wide field of view with no fovea. In contrast, primates have a highly developed cortical system for vision and a fovea, and so have very different capabilities to rodents, as they have an ability to identify people or objects at a distance, and to remember where they have been seen. Despite this major difference in the visual cortical processing in these different mammals, somehow important points are missed in this paper about how the cortical processing is organised in these different mammals, and how this is reflected in the anatomy.

Reviewer #2 (Public Review):

Summary:

The manuscript emphasizes a phylogenetic conservation of the hippocampal region and primary sensory cortical regions in mammalian species. The authors then propose that the evident species-specific differences in behavior and memory-related functions may be due to differences in type and amount of cortico-hippocampal connectivity.

Strengths:

The authors are well-established researchers with a long history of excellent results and publications. The question (co-influence of cortical and hippocampal connections) is potentially interesting.

Weaknesses:

The treatment is very broad and macro scale, ignoring the likelihood that hippocampal-cortical connectivity and behavioral outcomes result from multiple differences at a more micro-scale. The designated "mammalian" sample is also broad. Thus, it can appear incomplete as a sample, and incompletely discussed.

Reviewer #1 (Public review):

Summary:

The "number sense" refers to an imprecise and noisy representation of number. Many researchers propose that the number sense confers a fixed (exogenous) subjective representation of number that adheres to scalar variability, whereby the variance of the representation of number is linear in the number.

This manuscript investigates whether the representation of number is fixed, as usually assumed in the literature, or whether it is endogenous. The two dimensions on which the authors investigate this endogeneity are the subject's prior beliefs about stimuli values and the task objective. Using two experimental tasks, the authors collect data that are shown to violate scalar variability and are instead consistent with a model of optimal encoding and decoding, where the encoding phase depends endogenously on prior and task objectives. I believe the paper asks a critically important question. The literature in cognitive science, psychology, and increasingly in economics, has provided growing empirical evidence of decision-making consistent with efficient coding. However, the precise model mechanics can differ substantially across studies. This point was made forcefully in a paper by Ma and Woodford (2020, Behavioral & Brain Sciences), who argue that different researchers make different assumptions about the objective function and resource constraints across efficient coding models, leading to a proliferation of different models with ad-hoc assumptions. Thus, the possibility that optimal coding depends endogenously on the prior and the objective of the task, opens the door to a more parsimonious framework in which assumptions of the model can be constrained by environmental features. Along these lines, one of the authors' conclusions is that the degree of variability in subjective responses increases sublinearly in the width of the prior. And importantly, the degree of this sublinearity differs across the two tasks, in a manner that is consistent with a unified efficient coding model.

Comments:

(1) Modeling and implementation of estimation task

The biggest concern I have with the paper is about the experimental implementation and theoretical account of the estimation task. The salient features of the experimental data (Figure 1C) are that the standard deviations of subjects' estimated quantities are hump-shaped in the true stimulus x and that the standard deviation, conditional on the true stimulus x, is increasing in prior width. The authors attribute these features to a Bayesian encoding and decoding model in which the internal representation of the quantity is noisy, and the degree of noise depends on the prior - as in models of efficient coding (Wei and Stocker 2015 Nature Neuro; Bhui and Gershman 2018 Psych Review; Hahn and Wei 2024 Nature Neuro).

The concern I have is about the final "step" in the model, where the authors assume there is an additional layer of motor noise in selecting the response. The authors posit that the subject's selection of the response is drawn from a Gaussian with a mean set to the optimally decoded estimate x*(r), and variance set to a free parameter sigma_0^2. However, the authors also assume that the Gaussian distribution is "truncated to the prior range." This truncation is a nontrivial assumption, and I believe that on its own, it can explain many features of the data.

To see this, assume that there is no noise in the internal representation of x, there is only motor noise. This corresponds to a special case of the authors' model in which υ is set to 0. The model then reduces to a simple account in which responses are drawn from a Gaussian distribution centered at the true value of x, but with asymmetric noise due to the truncation. I simulated such a model with sigma_0=7. The resulting standard deviations of responses for each value of x (based on 1000 draws for each value of x), across the three different priors, reproduce the salient patterns of the standard deviation in Figure 1C: i) within each condition, the standard deviation is hump-shaped and peaks at x=60 and ii) conditional on x, standard deviation increases in prior width. The takeaway is that this simple model with only truncated motor noise - and without any noisy or efficient coding of internal representations - provides an alternative channel through which the prior affects behavior.

Of course, this does not imply that subjects' coding is not described by the efficient encoding and decoding model posited by the authors. However, it does suggest an important alternative mechanism for the authors' theoretical results in the estimation task. Moreover, some of the quantitative conclusions about the differences in behavior with the discrimination task would be greatly affected by the assumption of truncated motor noise.

Turning to the experiment, a basic question is whether such a truncation was actually implemented in the design. That is, was the range of the slider bar set to the range of the prior? (The methods section states that the size on the screen of the slider was proportional to the prior width, but it was unclear whether the bounds of the slider bar changed with the prior). If the slider bar range did depend on the prior, then it becomes difficult to interpret the data. If not, then perhaps one can perform analyses to understand how much the motor noise is responsible for the dependence of the standard deviation on both x and the prior width. Indeed, the authors emphasize that their model is best fit at α=0.48, which would seem to imply that the best fitting value of υ is strictly positive. However, it would be important to clarify whether the estimation procedure allowed for υ=0, or whether this noise parameter was constrained to be positive (i.e., clarify whether the estimation assumed noisy and efficient coding of internal representations).

(2) Differences across tasks

A main takeaway from the paper is that optimal coding depends on the expected reward function in each task. This is the explanation for why the degree of sublinearity between standard deviation and prior width changes across the estimation and discrimination task. But besides the two different reward functions, there are also other differences across the two tasks. For example, the estimation task involves a single array of dots, whereas the discrimination task involves a pair of sequences of Arabic numerals. Related to the discussion above, in the estimation task the response scale is continuous whereas in the discrimination task, responses are binary. Is it possible that these other differences in the task could contribute to the observed different degrees of sublinearity? It is likely beyond the scope of the paper to incorporate these differences into the model, but such differences across the two tasks should be discussed as potential drivers of differences in observed behavior.

If it becomes too difficult to interpret the data from the estimation task due to the slider bar varying with the prior range, then which of the paper's conclusions would still follow when restricting the analysis to the discrimination task?

(3) Placement literature

One closely related experiment to the discrimination task in the current paper can be found in Frydman and Jin (2022 Quarterly Journal of Economics). Those authors also experimentally vary the width of a uniform prior in a discrimination task using Arabic numerals, in order to test principles of efficient coding. Consistent with the current findings, Frydman and Jin find that subjects exhibit greater precision when making judgments about numbers drawn from a narrower distribution. However, what the current manuscript does is it goes beyond Frydman and Jin by modeling and experimentally varying task objectives to understand and test the effects on optimal coding. This contribution should be highlighted and contrasted against the earlier experimental work of Frydman and Jin to better articulate the novelty of the current manuscript.

Reviewer #2 (Public review):

Summary:

This paper provides an ingenious experimental test of an efficient coding objective based on optimization as a task success. The key idea is that different tasks (estimation vs discrimination) will, under the proposed model, lead to a different scaling between the encoding precision and the width of the prior distribution. Empirical evidence in two tasks involving number perception supports this idea.

Strengths:

- The paper provides an elegant test of a prediction made by a certain class of efficient coding models previously investigated theoretically by the authors.

The results in experiments and modeling suggest that competing efficient coding models, optimizing mutual information alone, may be incomplete by missing the role of the task.

Weaknesses:

- The claims would be more strongly validated if data were present at more than two widths in the discrimination experiment.

- A very strong prediction of the model -- which determines encoding entirely from prior and task -- is that Fisher Information is uniform throughout the range, strongly at odds with the traditional assumption of imprecision increasing with the numerosity (Weber/Fechner law). This prediction should be checked against the data collected. It may not be trivial to determine this in the Estimation experiment, but should be feasible in the Discrimination experiment in the Wide condition: Is there really no difference in discriminability at numbers close to 10 vs numbers close to 90? Figure 2 collapses over those, so it's not evident whether such a difference holds or not. I'd have loved to look into this in reviewing, but the authors have not yet made their data publicly available - I strongly encourage them to do so.

Importantly, the inverse u-shaped pattern in Figure 1 is itself compatible with a Weber's-law-based encoding, as shown by simulation in Figure 5d in Hahn&Wei [1]. This suggests a potential competing variant account, in apparent qualitative agreement with the findings reported: the encoding is compatible with Fisher's law, and only a single scalar, the magnitude of sensory noise, is optimized for the task for the loss function (3). As this account would be substantially more in line with traditional accounts of numerosity perception - while still exhibiting task-dependence of encoding as proposed by the authors - it would be worth investigating if it can be ruled out based on the data gathered for this paper.

References:

[1] Hahn & Wei, A unifying theory explains seemingly contradictory biases in perceptual estimation, Nature Neuroscience 2024

Reviewer #3 (Public review):

Summary:

This work demonstrates that people's imprecision in numeric perception varies with the stimulus context and task goal. By measuring imprecision across different widths of uniform prior distributions in estimation and discrimination tasks, the authors find that imprecision changes sublinearly with prior width, challenging previous range normalization models. They further show that these changes align with the efficient encoding model, where decision-makers balance expected rewards and encoding costs optimally.

Strengths:

The experimental design is straightforward, controlling the mean of the number distribution while varying the prior width. By assessing estimation errors and discrimination accuracy, the authors effectively highlight how imprecision adjusts across conditions.

The model's predictions align well with the data, with the exponential terms (1/2 and 3/4) of imprecision changes matching the empirical results impressively.

Weaknesses:

Some details in the model section are unclear. Specifically, I'm puzzled by the Wiener process assumption where r∣x∼N(m(x)T,s^2T). Does this imply that both the representation of number x and the noise are nearly zero at the beginning, increasing as observation time progresses? This seems counterintuitive, and a clearer explanation would be helpful.

The authors explore range normalization models with Gaussian representation, but another common approach is the logarithmic representation (Barretto-García et al., 2023; Khaw et al., 2021). Could the logarithmic representation similarly lead to sublinearity in noise and distribution width?

Additionally, Heng et al. (2020) found that subjects did not alter their encoding strategy across different task goals, which seems inconsistent with the fully adaptive representation proposed here. I didn't find the analysis of participants' temporal dynamics of adaptation. The behavioral results in the manuscript seem to imply that the subjects adopted different coding schemes in a very short period of time. Yet in previous studies of adaptation, experimental results seem to be more supportive of a partial adaptive behavior (Bujold et al., 2021; Heng et al., 2020), which might balance experimental and real-world prior distributions. Analyzing temporal dynamics might provide more insight. Noting that the authors informed subjects about the shape of the prior distribution before the experiment, do the results in this manuscript suggest a top-down rapid modulation of number representation?

Barretto-García, M., De Hollander, G., Grueschow, M., Polanía, R., Woodford, M., & Ruff, C. C. (2023). Individual risk attitudes arise from noise in neurocognitive magnitude representations. Nature Human Behaviour, 7(9), 1551-1567. https://doi.org/10.1038/s41562-023-01643-4

Bujold, P. M., Ferrari-Toniolo, S., & Schultz, W. (2021). Adaptation of utility functions to reward distribution in rhesus monkeys. Cognition, 214, 104764. https://doi.org/10.1016/j.cognition.2021.104764

Heng, J. A., Woodford, M., & Polania, R. (2020). Efficient sampling and noisy decisions. eLife, 9, e54962. https://doi.org/10.7554/eLife.54962

Khaw, M. W., Li, Z., & Woodford, M. (2021). Cognitive Imprecision and Small-Stakes Risk Aversion. The Review of Economic Studies, 88(4), 1979-2013. https://doi.org/10.1093/restud/rdaa044

Reviewer #1 (Public review):

Summary:

In this work, the authors recorded the dynamics of the 5-HT with fiber photometry from CA1 in one hemisphere and LFP from CA1 in the other hemisphere. They observed an ultra-slow oscillation in the 5-HT signal during both wakefulness and NREM sleep. The authors have studied different phases of the ultra-slow oscillation to examine the potential difference in the occurrence of some behavioral state-related physiological phenomena (hippocampal ripples, EMG, and inter-area coherence).

Strengths:

The relation between the falling/rising phase of the ultra-slow oscillation and the ripples is sufficiently shown. There are some minor concerns about the observed relations that should be addressed with some further analysis.

Systematic observations have started to establish a strong relation between the dynamics of neural activity across the brain and measures of behavioral arousal. Such relations span a wide range of temporal scales that are heavily inter-related. Ultra-slow time scales are specifically understudied due to technical limitations and neuromodulatory systems are the strongest mechanistic candidates for controlling/modulating the neural dynamics at these time scales. The hypothesis of the relation between a specific time scale and one certain neuromodulator (5-HT in this manuscript) could have a significant impact on the understanding of the hierarchy in the temporal scales of neural activity.

Weaknesses:

One major caveat of the study is that different neuromodulators are strongly correlated across all time scales and related to this, the authors need to discuss this point further and provide more evidence from the literature (if any) that suggests similar ultra-slow oscillations are weaker or lack from similar signals recorded for other neuromodulators such as Ach and NA.

A major question that has been left out from the study and discussion is how the same level of serotonin before and after the peak could be differentially related to the opposite observed phenomenon. What are the possible parallel mechanisms for distinguishing between the rising and falling phases? Any neurophysiological evidence for sensing the direction of change in serotonin concentration (or any other neuromodulator), and is there any physiological functionality for such mechanisms?

Reviewer #2 (Public review):

Summary:

In their study, Cooper et al. investigated the spontaneous fluctuations in extracellular 5-HT release in the CA1 region of the hippocampus using GRAB5-HT3.0. Their findings revealed the presence of ultra-low frequency (less than 0.05 Hz) oscillations in 5-HT levels during both NREM sleep and wakefulness. The phase of these 5-HT oscillations was found to be related to the timing of hippocampal ripples, microarousals, electromyogram (EMG) activity, and hippocampal-cortical coherence. In particular, ripples were observed to occur with greater frequency during the descending phase of 5-HT oscillations, and stronger ripples were noted to occur in proximity to the 5-HT peak during NREM. Microarousal and EMG peaks occurred with greater frequency during the ascending phase of 5-HT oscillations. Additionally, the strongest coherence between the hippocampus and cortex was observed during the ascending phase of 5-HT oscillations. These patterns were observed in both NREM sleep and the awake state, with a greater prevalence in NREM. The authors posit that 5-HT oscillations may temporally segregate internal processing (e.g., memory consolidation) and responsiveness to external stimuli in the brain.

Strengths:

The findings of this research are novel and intriguing. Slow brain oscillations lasting tens of seconds have been suggested to exist, but to my knowledge they have never been analyzed in such a clear way. Furthermore, although it is likely that ultra-slow neuromodulator oscillations exist, this is the first report of such oscillations, and the greatest strength of this study is that it has clarified this phenomenon both statistically and phenomenologically.

Weaknesses:

As with any paper, this one has some limitations. While there is no particular need to pursue them, I will describe ten of them below, including future directions:

(1) Contralateral recordings: 5-HT levels and electrophysiological recordings were obtained from opposite hemispheres due to technical limitations. Ipsilateral simultaneous recordings may show more direct relationships.

(2) Sample size: The number of mice used in the experiments is relatively small (n=6). Validation with a larger sample size would be desirable.

(3) Lack of causality: The observed associations show correlations, not direct causal relationships, between 5-HT oscillations and neural activity patterns.

(4) Limited behavioral states: The study focuses primarily on sleep and quiet wakefulness. Investigation of 5-HT oscillations during a wider range of behavioral states (e.g., exploratory behavior, learning tasks) may provide a more complete understanding.

(5) Generalizability to other brain regions: The study focuses on the CA1 region of the hippocampus. It's unclear whether similar 5-HT oscillation patterns exist in other brain regions.

(6) Long-term effects not assessed: Long-term effects of ultra-low 5-HT oscillations (e.g., on memory consolidation or learning) were not assessed.

(7) Possible species differences: It's uncertain whether the findings in mice apply to other mammals, including humans.

(8) Technical limitations: The temporal resolution and sensitivity of the GRAB5-HT3.0 sensor may not capture faster 5-HT dynamics.

(9) Interactions with other neuromodulators: The study does not explore interactions with other neuromodulators (e.g., norepinephrine, acetylcholine) or their potential ultraslow oscillations.

(10) Limited exploration of functional significance: While the study suggests a potential role for 5-HT oscillations in memory consolidation and arousal, direct tests of these functional implications are not included.

Reviewer #3 (Public review):

Summary:

The activity of serotonin (5-HT) releasing neurons as well as 5-HT levels in brain structures targeted by serotonergic axons are known to fluctuate substantially across the animal's sleep/wake cycle, with high 5-HT levels during wakefulness (WAKE), intermediate levels during non-REM sleep (NREM) and very low levels during REM sleep. Recent studies have shown that during NREM, the activity of 5-HT neurons in raphe nuclei oscillates at very low frequencies (0.01 - 0.05 Hz) and this ultraslow oscillation is negatively coupled to broadband EEG power. However, how exactly this 5-HT oscillation affects neural activity in downstream structures is unclear.

The present study addresses this gap by replicating the observation of the ultraslow oscillation in the 5-HT system, and further observing that hippocampal sharp wave-ripples (SWRs), biomarkers of offline memory processing, occur preferentially in barrages on the falling phase of the 5-HT oscillation during both wakefulness and NREM sleep. In contrast, the raising phase of the 5-HT oscillation is associated with microarousals during NREM and increased muscular activity during WAKE. Finally, the raising 5-HT phase was also found to be associated with increased synchrony between the hippocampus and neocortex. Overall, the study constitutes a valuable contribution to the field by reporting a close association between raising 5-HT and arousal, as well as between falling 5-HT and offline memory processes.

Strengths:

The study makes compelling use of the state-of-the-art methodology to address its aims: the genetically encoded 5-HT sensor used in the study is ideal for capturing the ultraslow 5-HT dynamics and the novel detection method for SWRs outperforms current state-of-the-art algorithms and will be useful to many scientists in the field. Explicit validation of both of these methods is a particular strength of this study.

The analytical methods used in the article are appropriate and are convincingly applied, the use of a general linear mixed model for statistical analysis is a particularly welcome choice as it guards against pseudoreplication while preserving statistical power.

Overall, the manuscript makes a strong case for distinct sub-states across WAKE and NREM, associated with different phases of the 5-HT oscillation.

Weaknesses:

All of the evidence presented in the study is correlational. While the study mostly avoids claims of causality, it would still benefit from establishing whether the 5-HT oscillation has a direct role in the modulation of SWR rate via e.g. optogenetic activation/inactivation of 5-HT axons. As it stands, the possibility that 5-HT levels and SWRs are modulated by the same upstream mechanism cannot be excluded.

Reviewer #1 (Public review):

Summary:

In the manuscript by Hoisington et al., the authors utilized a novel conditional neuronal prosap2-interacting protein 1 (Prosapip1) knockout mouse to delineate the effects of both neuronal and dorsal hippocampal (dHP)-specific knockout of Prosapip1 impacts biochemical and electrophysiological neuroadaptations within the dHP that may mediate behaviors associated with this brain region.

Strengths:

(1) Methodological Strengths

a. The generation and use of a conditional neuronal knockout of Prosapip1 is a strength. These mice will be useful for anyone interested in studying or comparing and contrasting the effects of loss of Prosapip1 in different brain regions or in non-neuronal tissues.

b. The use of biochemical, electrophysiological, and behavioral approaches are a strength. By providing data across multiple domains, a picture begins to emerge about the mechanistic role for Prosapip1. While questions still remain, the use of the 3 domains is a strength.

c. The use of both global, constitutive neuronal loss of Prosapip1 and postnatal dHP-specific knockout of Prosapip1 help support and validate the behavioral conclusions.

(2) Strengths of the results

a. It is interesting that loss of Prosapip1 leads to specific alterations in the expression of GluN2B and PSD95 but not GluA1 or GluN2A in a post-homogenization fraction that the author's term a "synaptic" fraction. Therefore, these results suggest protein-specific modulation of glutamatergic receptors within a "synaptic" fraction.

b. The electrophysiological data demonstrate an NMDAR-dependent alteration in measures of hippocampal synaptic plasticity, including long-term potentiation (LTP) and NMDAR input/output. These data correspond with the biochemical data demonstrating a biochemical effect on GluN2B localization. Therefore, the conclusion that loss of Prosapip1 influences NMDAR function is well supported.

c. The behavioral data suggest deficits in memory in particular novel object recognition and spatial memory, in the Prosapip1 knockout mice. These data are strongly bolstered by both the pan-neuronal knockout and the dHP Cre transduction.

Weaknesses:

(1) Methodological Weaknesses

a. The synapsin-Cre mice may more broadly express Cre-recombinase than just in neuronal tissues. Specifically, according to Jackson Laboratories, there is a concern with these mice expressing Cre-recombinase germline. As the human protein atlas suggests that Prosapip1 protein is expressed extraneuronally, validation of neuron or at least brain-specific knockout would be helpful in interpreting the data. Having said that, the data demonstrating that the brain region-specific knockout has similar behavioral impacts helps alleviate this concern somewhat; however, there are no biochemical or electrophysiological readouts from these animals, and therefore an alternative mechanism in this adult knockout cannot be excluded.

b. The use of the word synaptic and the crude fractionation make some of the data difficult to interpret/contextualize. It is unclear how a single centrifugation that eliminates the staining of a nuclear protein can be considered a "synaptic" fraction. This is highlighted by the presence of GAPDH in this fraction which is a cytosolically-enriched protein. While GAPDH may be associated with some membranes it is not a synaptic protein. There is no quantification of GAPDH against total protein to validate that it is not enriched in this fraction over control. Moreover, it should not be used as a loading control in the synaptic fraction. There are multiple different ways to enrich membranes, extrasynaptic fractions, and PSDs and a better discussion on the caveats of the biochemical fractionation is a minimum to help contextualize the changes in PSD95 and GluN2B.

c. Also, the word synaptosomal on page 7 is not correct. One issue is this is more than synaptosomes and another issue is synaptosomes are exclusively presynaptic terminals. The correct term to use is synaptoneurosome, which includes both pre and postsynaptic components. Moreover, as stated above, this may contain these components but is most likely not a pure or even enriched fraction.

d. The age at which the mice underwent injection of the Cre virus was not mentioned.

(2) Weaknesses of results

a. There were no measures of GluN1 or GluA2 in the biochemical assays. As GluN1 is the obligate subunit, how it is impacted by the loss of Prosapip1 may help contextualize the fact that GluN2B, but not GluN2A, is altered. Moreover, as GluA2 has different calcium permeance, alterations in it may be informative.

b. While there was no difference in GluA1 expression in the "synaptic" fraction, it does not mean that AMPAR function is not impacted by the loss of Prosapip1. This is particularly important as Prosapip1 may interact with kinases or phosphatases or their targeting proteins. Therefore, measuring AMPAR function electrophysiologically or synaptic protein phosphorylation would be informative.

c. There is a lack of mechanistic data on what specifically and how GluN2B and PSD95 expression is altered. This is due to some of the challenges with interpreting the biochemical fractionation and a lack of results regarding changes in protein posttranslational modifications.

d. The loss of social novelty measures in both the global and dHP-specific Prosapip1 knockout mice were not very robust. As they were consistently lost in both approaches and as there were other consistent memory deficits, this does not impact the conclusions, but may be important to temper discussion to match these smaller deficits within this domain.

e. Alterations in presynaptic paired-pulse ratio measures are intriguing and may point to a role for Prosapip1 in synapse development, as discussed in the manuscript. It would be interesting to delineate if these PPR changes also occur in the adult knockout to help detail the specific Prosapip1-induced neuroadaptations that link to the alterations in novelty-induced behaviors.

Reviewer #2 (Public review):

Summary:

The authors provide valuable findings characterizing a Prosapip1 conditional knockout mouse and the effects of knockout on hippocampal excitatory transmission, NMDAR transmission, and several learning behaviors. Furthermore, the authors selectively and conditionally knockout Prosapip1 in the dorsal hippocampus and show that it is required for the same spatial learning and memory assessed in the conditional knockout mice. The study uncovers how Prosapip1 is involved PSD organization and is a functional and critical player in dorsal Hippocampal LTP via its interaction with GluN2B subunits.

Strengths:

The study is well-controlled and detailed, and the data in the paper match the conclusions.

Weaknesses:

Some statistical information is lacking.

Reviewer #1 (Public review):

The authors investigate the function and neural circuitry of reentrant signals in the visual cortex. Recurrent signaling is thought to be necessary to common types of perceptual experience that are defined by long-range relationships or prior expectations. Contour illusions - where perceptual objects are implied by stimuli characteristics - are a good example of this. The perception of these illusions is thought to emerge as recurrent signals from higher cortical areas feedback onto the early visual cortex, to tell the early visual cortex that it should be seeing object contours where none are actually present.

The authors test the involvement of reentrant cortical activity in this kind of perception using a drug challenge. Reentrance in the visual cortex is thought to rely on NMDAR-mediated glutamate signalling. The authors accordingly employ an NMDA antagonist to stop this mechanism, looking for the effect of this manipulation on visually evoked activity recorded in EEG.

The motivating hypothesis for the paper is that NMDA antagonism should stop recurrent activity and that this should degrade perceptual activity supporting the perception of a contour illusion, but not other types of visual experience. Results in fact show the opposite. Rather than degrading cortical activity evoked by the illusion, memantine makes it more likely that machine learning classification of EEG will correctly infer the presence of the illusion.

On the face of it, this is confusing, and the paper currently does not entirely resolve this confusion. But there are relatively easy ways to improve this. The authors would be well served by entertaining more possible outcomes in the introduction - there's good reason to expect a positive effect of memantine on perceptual brain activity, and I provide details on this below. The authors also need to further emphasize that the directional expectations that motivated E1 were, of course, adapted after the results from this experiment emerged. The authors presumably at least entertained the notion that E2 would reproduce E1 - meaning that E2 was motivated by a priori expectations that were ultimately met by the data.

I broadly find the paper interesting, graceful, and creative. The hypotheses are clear and compelling, the techniques for both manipulation of brain state and observation of that impact are cutting edge and well suited, and the paper draws clear and convincing conclusions that are made necessary by the results. The work sits at the very interesting crux of systems neuroscience, neuroimaging, and pharmacology. I believe the paper can be improved in revision, but my suggestions are largely concerning presentation and nuance of interpretation.

(1) I miss some treatment of the lack of behavioural correlate. What does it mean that metamine benefits EEG classification accuracy without improving performance? One possibility here is that there is an improvement in response latency, rather than perceptual sensitivity. Is there any hint of that in the RT results? In some sort of combined measure of RT and accuracy?

(2) An explanation is missing, about why memantine impacts the decoding of illusion but not collinearity. At a systems level, how would this work? How would NMDAR antagonism selectively impact long-range connectivity, but not lateral connectivity? Is this supported by our understanding of laminar connectivity and neurochemistry in the visual cortex?

(3) The motivating idea for the paper is that the NMDAR antagonist might disrupt the modulation of the AMPA-mediated glu signal. This is in line with the motivating logic for Self et al., 2012, where NMDAR and AMPAR efficacy in macacque V1 was manipulated via microinfusion. But this logic seems to conflict with a broader understanding of NMDA antagonism. NMDA antagonism appears to generally have the net effect of increasing glu (and ACh) in the cortex through a selective effect on inhibitory GABA-ergic cells (eg. Olney, Newcomer, & Farber, 1999). Memantine, in particular, has a specific impact on extrasynaptic NMDARs (that is in contrast to ketamine; Milnerwood et al, 2010, Neuron), and this type of receptor is prominent in GABA cells (eg. Yao et al., 2022, JoN). The effect of NMDA antagonists on GABAergic cells generally appears to be much stronger than the effect on glutamergic cells (at least in the hippocampus; eg. Grunze et al., 1996).

This all means that it's reasonable to expect that memantine might have a benefit to visually evoked activity. This idea is raised in the GD of the paper, based on a separate literature from that I mentioned above. But all of this could be better spelled out earlier in the paper, so that the result observed in the paper can be interpreted by the reader in this broader context.

To my mind, the challenging task is for the authors to explain why memantine causes an increase in EEG decoding, where microinfusion of an NMDA antagonist into V1 reduced the neural signal Self et al., 2012. This might be as simple as the change in drug... memantine's specific efficacy on extrasynaptic NMDA receptors might not be shared with whatever NMDA antagonist was used in Self et al. 2012. Ketamine and memantine are already known to differ in this way.

(4) The paper's proposal is that the effect of memantine is mediated by an impact on the efficacy of reentrant signaling in visual cortex. But perhaps the best-known impact of NMDAR manipulation is on LTP, in the hippocampus particularly but also broadly. Perception and identification of the kanisza illusion may be sensitive to learning (eg. Maertens & Pollmann, 2005; Gellatly, 1982; Rubin, Nakayama, Shapley, 1997); what argues against an account of the results from an effect on perceptual learning? Generally, the paper proposes a very specific mechanism through which the drug influences perception. This is motivated by results from Self et al 2012 where an NMDA antagonist was infused into V1. But oral memantine will, of course, have a whole-brain effect, and some of these effects are well characterized and - on the surface - appear as potential sources of change in illusion perception. The paper needs some treatment of the known ancillary effects of diffuse NMDAR antagonism to convince the reader that the account provided is better than the other possibilities.

(5) The cross-decoding approach to data analysis concerns me a little. The approach adopted here is to train models on a localizer task, in this case, a task where participants matched a kanisza figure to a target template (E1) or discriminated one of the three relevant stimuli features (E2). The resulting model was subsequently employed to classify the stimuli seen during separate tasks - an AB task in E1, and a feature discrimination task in E2. This scheme makes the localizer task very important. If models built from this task have any bias, this will taint classifier accuracy in the analysis of experimental data. My concern is that the emergence of the kanisza illusion in the localizer task was probably quite salient, respective to changes in stimuli rotation or collinearity. If the model was better at detecting the illusion to begin with, the data pattern - where drug manipulation impacts classification in this condition but not other conditions - may simply reflect model insensitivity to non-illusion features.

I am also vaguely worried by manipulations implemented in the main task that do not emerge in the localizer - the use of RSVP in E1 and manipulation of the base rate and staircasing in E2. This all starts to introduce the possibility that localizer and experimental data just don't correspond, that this generates low classification accuracy in the experimental results and ineffective classification in some conditions (ie. when stimuli are masked; would collinearity decoding in the unmasked condition potentially differ if classification accuracy were not at a floor? See Figure 3c upper, Figure 5c lower).

What is the motivation for the use of localizer validation at all? The same hypotheses can be tested using within-experiment cross-validation, rather than validation from a model built on localizer data. The argument may be that this kind of modelling will necessarily employ a smaller dataset, but, while true, this effect can be minimized at the expense of computational cost - many-fold cross-validation will mean that the vast majority of data contributes to model building in each instance.

It would be compelling if results were to reproduce when classification was validated in this kind of way. This kind of analysis would fit very well into the supplementary material.

Reviewer #2 (Public review):

Summary:

In this paper, the authors investigate the role of NMDA-receptors in recurrent processing. In doing so, the authors present data from two studies, where they attempt to decode different stimulus features, namely contrast, collinearity, and illusory contours. The latter of which the authors claim relies uniquely on recurrent processing. Therefore, to test whether NMDA receptors are particularly involved in recurrent processing they administer a NMDA-antagonist to see whether the decoding of illusory contours is specifically perturbed, and leaves the decoding of other features intact. They further aim to disentangle the role of NMDA-receptors by manipulating visibility and task relevance of the decoded features

In the first experiment, the authors decode two targets, the first was always presented clearly, the second's visibility was manipulated by presenting it after a short lag rather than a long lag (inducing attentional blink), as well as masking the target on half the trials. First, they find for target 1 clear evidence for the NMDA-receptor increasing (rather than decreasing) decoding performance of illusory contours. They move on to analyse target 2 to explore the manipulations of lag and masking. Here they find that masking reduced decoding of all three stimulus features, but only the lag reduced decoding of illusory contours. Importantly, the NMDA-antagonist improved decoding only in the unmasked, long lag condition, in the cluster analyses. However, the interaction with the lag condition was not significant, and the effect on decoding was primarily present in the later decoding time window, and not significant when exploring the peak of the decoding time window.

The second experiment was highly similar, but got rid of the lag manipulation, and replaced it with a manipulation of task relevance. Notably, masking did not abolish the decoding of illusory contours completely, in contrast to the first experiment. More importantly, they find that the NMDA-receptor now clearly increases decoding of illusory contours, particularly when the illusory contours are not masked. No effect of task relevance is found.

Taken together the authors state that evidence is found for NMDA-receptors role in recurrent processing.

Strengths:

This is an interesting study using state-of-the-art methods in combination with drug manipulation to study recurrent processing. Their analysis methods are state-of-the-art, and the question that they are trying to address is topical and interesting to a wide research audience, encompassing both researchers interested in visual perception and consciousness, as well as those interested in perturbed vision as found in psychiatric disorders.

Weaknesses:

The experimental design is somewhat complicated, which can make it difficult to match the authors' claims to the actual evidence that is provided. I have some reservations about the paper which are born out of a few issues.<br /> (1) The title, abstract, and introduction hide their counterintuitive finding of increased decoding, presumably as it was unexpected.<br /> (2) Their analysis choices are sometimes unclear, making it difficult to assess whether the analyses are sensible.<br /> (3) The appropriate tests for the interactions that the authors claim they found are often lacking.

To start off, I think the reader is being a bit tricked when reading the paper. Perhaps my priors are too strong, but I assumed, just like the authors, that NMDA-receptors would disrupt recurrent processing, in line with previous work. However, due to the continuous use of the ambiguous word 'affected' rather than the more clear increased or perturbed recurrent processing, the reader is left guessing what is actually found. That's until they read the results and discussion finding that decoding is actually improved. This seems like a really big deal, and I strongly urge the authors to reword their title, abstract, and introduction to make clear they hypothesized a disruption in decoding in the illusion condition, but found the opposite, namely an increase in decoding. I want to encourage the authors that this is still a fascinating finding.

Apologies if I have missed it, but it is not clear to me whether participants were given the drug or placebo during the localiser task. If they are given the drug this makes me question the logic of their analysis approach. How can one study the presence of a process, if their very means of detecting that process (the localiser) was disrupted in the first place? If participants were not given a drug during the localiser task, please make that clear. I'll proceed with the rest of my comments assuming the latter is the case. But if the former, please note that I am not sure how to interpret their findings in this paper.

The main purpose of the paper is to study recurrent processing. The extent to which this study achieves this aim is completely dependent to what extent we can interpret decoding of illusory contours as uniquely capturing recurrent processing. While I am sure illusory contours rely on recurrent processing, it does not follow that decoding of illusory contours capture recurrent processing alone. Indeed, if the drug selectively manipulates recurrent processing, it's not obvious to me why the authors find the interaction with masking in experiment 2. Recurrent processing seems to still be happening in the masked condition, but is not affected by the NMDA-receptor here, so where does that leave us in interpreting the role of NMDA-receptors in recurrent processing? If the authors can not strengthen the claim that the effects are completely driven by affecting recurrent processing, I suggest that the paper will shift its focus to making claims about the encoding of illusory contours, rather than making primary claims about recurrent processing.

An additional claim is being made with regards to the effects of the drug manipulation. The authors state that this effect is only present when the stimulus is 1) consciously accessed, and 2) attended. The evidence for claim 1 is not supported by experiment 1, as the masking manipulation did not interact in the cluster-analyses, and the analyses focussing on the peak of the timing window do not show a significant effect either. There is evidence for this claim coming from experiment 2 as masking interacts with the drug condition. Evidence for the second claim (about task relevance) is not presented, as there is no interaction with the task condition. A classical error seems to be made here, where interactions are not properly tested. Instead, the presence of a significant effect in one condition but not the other is taken as sufficient evidence for an interaction, which is not appropriate. I therefore urge the authors to dampen the claim about the importance of attending to the decoded features. Alternatively, I suggest the authors run their interactions of interest on the time-courses and conduct the appropriate cluster-based analyses.

How were the length of the peak-timing windows established in Figure 1E? My understanding is that this forms the training-time window for the further decoding analyses, so it is important to justify why they have different lengths, and how they are determined. The same goes for the peak AUC time windows for the interaction analyses. A number of claims in the paper rely on the interactions found in these post-hoc analyses, so the 223- to 323 time window needs justification.

Reviewer #3 (Public review):

Summary:

In this study, Stein and colleagues use a clever masking/attentional blink paradigm using Kanisza stimuli, coupled with EEG decoding and the NMDA antagonist memantine, to isolate putative neural markers of feedforward, lateral, and feedback processing.

In two elegant experiments, they show that memantine selective influences EEG decoding of only illusory Kanisza surfaces (but not contour continuation or raw contrast), only when unmasked, only when attention is available (not when "blinked"), and only when task-relevant.

This neatly implicates NMDA receptors in the feedback mechanisms that are believed to be involved in inferring illusory Kanisza surfaces, and builds a difficult bridge between the large body of human perceptual experiments and pharmacological and neurophysiological work in animals.

Strengths:

Three key strengths of the paper are<br /> (1) The elegant and thorough experimental design, which includes internal replication of some key findings.<br /> (2) The clear pattern of results across the full set of experiments.<br /> (3) The clear writing and presentation of results.

The paper effectively reports a 4-way interaction, with memantine only influencing decoding of surfaces (1) that are unmasked (2), with attention available (3) and task-relevant (4). Nevertheless, the results are very clear, with a clear separation between null effects on other conditions and quite a strong (and thus highly selective) effect on this one intersection of conditions. This makes the pattern of findings very convincing.

Weaknesses:

Overall this is an impressive and important paper. However, to my mind, there are two minor weaknesses.

First, despite its clear pattern of neural effects, there is no corresponding perceptual effect. Although the manipulation fits neatly within the conceptual framework, and there are many reasons for not finding such an effect (floor and ceiling effects, narrow perceptual tasks, etc), this does leave open the possibility that the observation is entirely epiphenomenal, and that the mechanisms being recorded here are not actually causally involved in perception per se.

Second, although it is clear that there is an effect on decoding in this particular condition, what that means is not entirely clear - particularly since performance improves, rather than decreases. It should be noted here that improvements in decoding performance do not necessarily need to map onto functional improvements, and we should all be careful to remain agnostic about what is driving classifier performance. Here too, the effect of memantine on decoding might be epiphenomenal - unrelated to the information carried in the neural population, but somehow changing the balance of how that is electrically aggregated on the surface of the skull. *Something* is changing, but that might be a neurochemical or electrical side-effect unrelated to actual processing (particularly since no corresponding behavioural impact is observed.)

Reviewer #1 (Public review):

Summary:

This study investigated the role of transcriptional and translational controls of gene expression in dorsal root ganglia and lumbar spinal cord in neuropathic pain in mice. Using ribosome profiling (Ribo-seq) and translating ribosome affinity purification (TRAP), they show changes in transcriptomic and translational gene expression at the peripheral and central levels rapidly after nerve injury. While translational changes in gene expression remained elevated for more than two months in both DRGs and the spinal cord, transcriptomic regulation was absent in the spinal cord long after the onset of neuropathy. Disrupting mRNA translation in dorsal horn neurons using antisense oligonucleotides reduced mechanical withdrawal threshold and facial expression of pain. Using fluorescent noncanonical amino acid tagging (FUNCAT), the authors further show that de novo protein expression primarily occurs in inhibitory neurons in the superficial dorsal horn after nerve injury. Accordingly, a selective increase in translational control of gene expression in spinal inhibitory neurons, or a subset of mainly inhibitory neurons expressing parvalbumin (PV), using transgenic mice, led to a decrease in the excitability of PV neurons and mechanical allodynia. In contrast, decreasing the translational control of spinal PV neurons prevented the alteration of the electrophysiological properties of the PV cells induced by nerve injury.

Strengths:

This is a well-written article that uncovers a previously unappreciated role of gene expression control in PV neurons, which seems to play an important part in the loss of inhibitory control of spinal circuits typically seen after peripheral nerve injury. The conclusions are generally well supported by the data.

Weaknesses:

The study would benefit from further clarifications in the methods section and a deeper analysis of gene expression changes in mRNA expression and ribosomal footprint observed after nerve injury.

Antisense oligonucleotides used to reduce translation by disrupting eIF4E expression were administered i.c.v. It is unknown if the authors controlled for locomotor deficits, which might add confounds in the interpretation of behavioral results. A more local route should have been preferable to avoid targeting brain regions, which could potentially affect behavior.

Only female mice were used for Ribo-Seq, TRAP, FUNCAT, and electrophysiology, but both sexes were used for behavior experiments.

The conditional KO of 4E-BP1 using transgenic animals should be total in the targeted cells. However, only a partial reduction is reported in Figure S2 in GAD2, PV, Vglut2, or Tac1 cells. Again, proper methods for quantification of fluorescence in these experiments are lacking.

The elegant knockdown of eIF4E using AAV-mediated shRNAmir shows a recovery of the electrophysiological intrinsic properties of PV neurons after injury. It is unclear if such manipulation would be sufficient to reverse mechanical allodynia in vivo.

Reviewer #2 (Public review):

Summary:

I reviewed the manuscript titled "Translational Control in the Spinal Cord Regulates Gene Expression and Pain Hypersensitivity in the Chronic Phase of Neuropathic Pain." This manuscript compares transcription and translation in the spinal cord during the acute and chronic phases of neuropathic pain induced by surgical nerve injury. The authors chose to focus their investigation on translation in the chronic phase due to its greater impact on gene expression in the spinal cord compared to transcription.

(1) The study is significant because the molecular mechanisms underlying chronic pain remain elusive. The role of translational regulation in the spinal cord has not been investigated in neuroplasticity and chronic pain mouse models. The manuscript is innovative and technically robust. The authors employed several cutting-edge techniques such as Rio-seq, TRAP-seq, slice electrophysiology, and viral approaches. Despite the technical complexity, the manuscript is well-written. The authors demonstrated that inhibition of eIF4E alleviates pain hypersensitivity, that de novo protein synthesis is more pronounced in inhibitory interneurons, and that manipulating mTOR-eIF4E pathways alters mechanical sensitivity and neuroplasticity.

(2) Strengths: innovation (conceptual and technical levels), data support the conclusions.

Weakness:

Confusion about the sex of the animals. It is unclear whether eIF4E ASO affects translation and which cells. It is not determined that modulating translation in PV+ neurons impacts neuropathic pain behaviors.

Reviewer #3 (Public review):

Summary:

This study provides evidence for translational changes in inhibitory spinal dorsal horn neurons following chronic nerve injury. Gene expression changes have been widely studied in the context of pain induction and provided key insights into the adaptation of the nervous system in the early phases of chronic pain. Whereas this is interesting biologically, most patients will arrive in the clinic beyond the acute phase of their injury, thus limiting the translational relevance of these studies. Recent studies have extended this work to highlight the difference between acute and chronic pain states, potentially explaining the cascading factors leading to chronic pain, and hopefully how to prevent this in vulnerable populations. The present study suggests that translational changes within spinal inhibitory populations could underlie long-term chronic pain, leading to decreased inhibition and heightened pain thresholds.

Strengths:

The approaches used and the broad outcomes of the manuscript are interesting and could be an exciting development in the field. The authors are using approaches more common in molecular biology and extending these into neuroscientific research, getting into the detail of how pathology could impact gene expression differentially across the course of an injury. This could open up new areas of research to selectively target not only defined populations but additionally help alleviate pain symptoms once an injury has already reached the maintenance phase. There is an opportunity to delve into what must be a very large data set and learn more about what genes are differentially translated and how this could affect circuit function.

Weaknesses:

Whereas the authors approach a key question in pain chronicity, the manuscript falls a little short of providing any conclusive data.

The manuscript was in some areas very difficult to follow. Terminology was not always consistent or clear, and the flow of the manuscript could use some attention to highlight key areas. Whereas the overall message is clear in the summary, this would not necessarily be the case when reading the manuscript alone.

The study claims to show that translational control mechanisms in the spinal cord play a role in mediating neuropathic pain hypersensitivity, but the studies presented do not fully support this statement. The authors instead provide some correlation between translation and behavioural reflex excitability (namely vfh and Hargreaves).

It is difficult to fully interpret the work, as there are a number of inconsistencies, namely the range of timings pre- and post-injury, lack of controls for manipulations, the use of shmiRNA versus lineage deletions, and lack of detailed somatosensory testing. It is not completely clear how this work could be translatable as is, without a deeper understanding of how translational control affects circuit function and whether all of this is necessarily bad for the system, or whether this is a positive homeostatic adaptation to the hyperexcitability of the circuit following injury.

A large portion of the work is focussed on showing an inhibitory-selective change in translation following chronic nerve injury. The evidence for this is however lacking. Statistics to show that translational effects are restricted to inhibitory subpopulations are inadequate. The author's choice of transgenic lines is not clear and seems to rely on availability rather than hypothesis.

Reviewer #1 (Public Review):

In the article by Dearlove et al., the authors present evidence in strong support of nucleotide ubiquitylation by DTX3L, suggesting it is a promiscuous E3 ligase with capacity to ubiquitylate ADP ribose and nucleotides. The authors include data to identify the likely site of attachment and the requirements for nucleotide modification.

While this discovery potentially reveals a whole new mechanism by which nucleotide function can be regulated in cells, there are some weaknesses that should be considered. Is there any evidence of nucleotide ubiquitylation occurring cells? It seems possible, but evidence in support of this would strengthen the manuscript. The NMR data could also be strengthened as the binding interface is not reported or mapped onto the structure/model, this seems of considerable interest given that highly related proteins do have the same activity.

The paper is for the most part well well-written and is potentially highly significant

Comments on revised version:

The revised manuscript has addressed many of the concerns raised and clarified a number of points. As a result the manuscript is improved.

The primary concern that remains is the absence of biological function for Ub-ssDNA/RNA and the inability to detect it in cells. Despite this the manuscript will be of interest to those in the ubiquitin field and will likely provoke further studies and the development of tools to better assess the cellular relevance. As a result this manuscript is important.

Minor issue:<br /> Figure 1A - the authors have now included the constructs used but it would be more informative if the authors lined up the various constructs under the relevant domains in the full-length protein.

Reviewer #2 (Public Review):

The manuscript by Dearlove et al. entitled "DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids" reports a novel activity of a DELTEX E3 ligase family member, DTX3L, which can conjugate ubiquitin to the 3' hydroxyl of single-stranded oligonucleotides via an ester linkage. The findings that unmodified oligonucleotides can act as substrates for direct ubiquitylation and the identification of DTX3 as the enzyme capable of performing such oligonucleotide modification are novel, intriguing, and impactful because they represent a significant expansion of our view of the ubiquitin biology. The authors perform a detailed and diligent biochemical characterization of this novel activity, and key claims made in the article are well supported by experimental data. However, the studies leave room for some healthy skepticism about the physiological significance of the unique activity of DTX3 and DTX3L described by the authors because DTX3/DTX3L can also robustly attach ubiquitin to the ADP ribose moiety of NAD or ADP-ribosylated substrates. The study could be strengthened by a more direct and quantitative comparison between ubiquitylation of unmodified oligonucleotides by DTX3/DTX3L with the ubiquitylation of ADP-ribose, the activity that DTX3 and DTX3L share with the other members of the DELTEX family.

Comment on revised version:

In my opinion, reviewers' comments are constructively addressed by the authors in the revised manuscript, which further strengthens the revised submission and makes it an important contribution to the field. Specifically, the authors perform a direct quantitative comparison of two distinct ubiquitylation substrates, unmodified oligonucleotides and fluorescently labeled NADH and report that kcat/Km is 5-fold higher for unmodified oligos compared to NADH. This observation suggests that ubiquitylation of unmodified oligos is not a minor artifactual side reaction in vitro and that unmodified oligonucleotides may very well turn out to be the physiological substrates of the enzyme. However, the true identity of the physiological substrates and the functionally relevant modification site(s) remain to be established in further studies.

Reviewer #1 (Public review):

Summary:

Tian et al. describes how TIPE regulates melanoma progression, stemness, and glycolysis. The authors link high TIPE expression to increased melanoma cell proliferation and tumor growth. TIPE causes dimerization of PKM2, as well as translocation of PKM2 to the nucleus, thereby activating HIF-1alpha. TIPE promotes the phosphorylation of S37 on PKM2 in an ERK-dependent manner. TIPE is shown to increase stem-like phenotype markers. The expression of TIPE is positively correlated with the levels of PKM2 Ser37 phosphorylation in murine and clinical tissue samples. Taken together, the authors demonstrate how TIPE impacts melanoma progression, stemness, and glycolysis through dimeric PKM2 and HIF-1alpha crosstalk.

The authors manipulated TIPE expression using both shRNA and overexpression approaches throughout the manuscript. Using these models, they provide strong evidence of the involvement of TIPE in mediating PKM2 Ser37 phosphorylation and dimerization. The authors also used mutants of PKM2 at S37A to block its interaction with TIPE and HIF-1alpha. In addition, an ERK inhibitor (U0126) was used to block the phosphorylation of Ser37 on PKM2. The authors show how dimerization of PKM2 by TIPE causes nuclear import of PKM2 and activation of HIF-1alpha and target genes. Pyridoxine was used to induce PKM2 dimer formation, while TEPP-46 was used to suppress PKM2 dimer formation. TIPE maintains stem cell phenotypes by increasing expression of stem-like markers. Furthermore, the relationship between TIPE and Ser37 PKM2 was demonstrated in murine and clinical tissue samples.

The evaluation of how TIPE causes metabolic reprogramming can be better assessed using isotope tracing experiments and improved bioenergetic analysis.

Reviewer #2 (Public review):

In this article, Tian et al present a convincing analysis of the molecular mechanisms underpinning TIPE-mediated regulation of glycolysis and tumor growth in melanoma. The authors begin by confirming TIPE expression in melanoma cell lines and identify "high" and "low" expressing models for functional analysis. They show that TIPE depletion slows tumour growth in vivo, and using both knockdown and over expression approaches, show that this is associated with changes in glycolysis in vitro. Compelling data using multiple independent approaches is presented to support an interaction between TIPE and the glycolysis regulator PKM2, and over-expression of TIPE promoted nuclear translocation of PKM2 dimers. Mechanistically, the authors also demonstrate that PKM2 is required for TIPE-mediated activation of HIF1a transcriptional activity, as assessed using an HRE-promoter reporter assay, and that TIPE-mediated PKM2 dimerization is p-ERK dependent. Finally, the dependence of TIPE activity on PKM2 dimerization was demonstrated on tumor growth in vivo and in regulation of glycolysis in vitro, and ectopic expression of HIF1a could rescue inhibition of PKM2 dimerization in TIPE overexpressing cells and reduced induction of general cancer stem cell markers, showing a clear role for HIF1a in this pathway.

The detailed mechanistic analysis of TIPE mediated regulation of PKM2 to control aerobic glycolysis and tumor growth is a major strength of the study and provides new insights into the molecular mechanisms that underpin the Warburg effect in melanoma cells. The main conclusions of this paper are well supported by data, however further investigation of a potential oncogenic effect of TIPE in melanoma patients is warranted to support the tumor promoting role of TIPE identified in the experimental models. Analysis of patient samples showed a significant increase in TIPE protein levels in primary melanoma compared to benign skin tumours, and a further increase upon metastatic progression. Moreover, TIPE levels correlate with proliferation (Ki67) and hypoxia gene sets in the TCGA melanoma patient dataset. However, the authors note in the discussion that high TIPE expression associates with better survival outcomes in the TCGA melanoma patients and these data should be included in this paper. Further investigation of how TIPE-mediated regulation of glycolysis contributes to melanoma progression is warranted to confirm the authors claims of a potential oncogenic function. Regardless, the new insights into the molecular mechanisms underpinning TIPE-mediated aerobic glycolysis in melanoma are convincing and will likely generate interest in the cancer metabolism field.