Reviewer #3 (Public review):

Summary:

Fahrenfort et al. investigate how liberal or conservative criterion placement in a detection task affects the construct validity of neural measures of unconscious cognition and conscious processing. Participants identified instances of "seen" or "unseen" in a detection task, a method known as post hoc sorting. Simulation data convincingly demonstrate that, counterintuitively, a conservative criterion inflates effect sizes of neural measures compared to a liberal criterion. While the impact of criterion shifts on effect size is suggested by signal detection theory, this study is the first to address this explicitly within the consciousness literature. Decoding analysis of data from two EEG experiments further shows that different criteria lead to differential effects on classifier performance in post hoc sorting. The findings underscore the pervasive influence of experimental design and participants report on neural measures of consciousness, revealing that criterion placement poses a critical challenge for researchers.

Strengths and Weaknesses:

One of the strengths of this study is the inclusion of the Perceptual Awareness Scale (PAS), which allows participants to provide more nuanced responses regarding their perceptual experiences. This approach ensures that responses at the lowest awareness level (selection 0) are made only when trials are genuinely unseen. This methodological choice is important as it helps prevent the overestimation of unconscious processing, enhancing the validity of the findings.

A potential area for improvement in this study is the use of single time-points from peak decoding accuracy to generate current source density topography maps. While we recognize that the decoding analysis employed here differs from traditional ERP approaches, the robustness of the findings could be enhanced by exploring current source density over relevant time windows. Event-related peaks, both in terms of timing and amplitude, can sometimes be influenced by noise or variability in trial-averaged EEG data, and a time-window analysis might provide a more comprehensive and stable representation of the underlying neural dynamics.

It is helpful that the authors show the standard error of the mean for the classifier performance over time. A similar indication of a measure of variance in other figures could improve clarity and transparency.<br /> That said, the paper appears solid regarding technical issues overall. The authors also do a commendable job in the discussion by addressing alternative paradigms, such as wagering paradigms, as a possible remedy to the criterion problem (Peters & Lau, 2015; Dienes & Seth, 2010). Their consideration of these alternatives provides a balanced view and strengthens the overall discussion.

Impact of the Work:

This study effectively demonstrates a phenomenon that has been largely unexplored within the consciousness literature. Subjective measures may not reliably capture the construct they aim to measure due to criterion confounds. Future research on neural measures of consciousness should account for this issue, and no-report measures may be necessary until the criterion problem is resolved.

Reviewer #1 (Public review):

Summary:

Urination requires precise coordination between the bladder and external urethral sphincter (EUS), while the neural substrates controlling this coordination remain poorly understood. In this study, Li et al. identify estrogen receptor 1-expressing neurons (ESR1+) in Barrington's nucleus as key regulators that faithfully initiate or suspend urination. Results from peripheral nerve lesions suggest that BarEsr1 neurons play independent roles in controlling bladder contraction and relaxation of the EUS. Finally, the authors performed region-specific retrograde tracing, claiming that distinct populations of BarEsr1 neurons target specific spinal nuclei involved in regulating the bladder and EUS, respectively.

Strength:

Overall, the work is of high quality. The authors integrate several cutting-edge technologies and sophisticated, thorough analyses, including opto-tagged single unit recordings, combined optogenetics, and urodynamics, particularly those following distinct peripheral nerve lesions.

Weakness:

(1) My major concern is the novelty of this study. Keller et al. 2018 have shown that BarEsr1 neurons are active during urination and play an essential role in relaxing the external urethral sphincter (EUS). Minimally, substantial content that merely confirms previous findings (e.g. Figures 1A-E; Figures 3A-E) should be move to the supplementary datasets.

(2) I also have concerns regarding the results showing that the inactivation of BarEsr1 neurons led to the cessation of EUS muscle firing (Figures 2G and S5C). As shown in the cartoon illustration of Figure 8, spinal projections of BarEsr1 neurons contact interneurons (presumably inhibitory) that innervate motor neurons, which in turn excite the EUS. I would therefore expect that the inactivation of BarEsr1 should shift the EUS firing pattern from phasic (as relaxation) to tonic (removal of relaxation), rather than stopping their firing entirely. Could the authors comment on this and provide potential reasons or mechanisms for this finding?

(3) Current evidence is insufficient to support the claim that the majority of BarEsr1 neurons innervate the SPN but not DGC. The current spinal images are uninformative, as the fluorescence reflects the distribution of Esr1- or Crh-expressing neurons in the spinal cord, along with descending BarEsr1 or BarCrh axons. Given the close anatomical proximity of these two nuclei, a more thorough histological analysis is required to demonstrate that the spinal injections were accurately confined to either the SPN or the DGC.

Reviewer #2 (Public review):

Summary:

The authors have performed a rigorous study to assess the role of ESR1+ neurons in the PMC to control the coordination of bladder and sphincter muscles during urination. This is an important extension of previous work defining the role of these brainstem neurons, and convincingly adds to the understanding of their role as master regulators of urination. This is a thorough, well-done study that clarifies how the Pontine micturition center coordinates different muscle groups for efficient urination, but there are some questions and considerations that remain.

Strengths:

These data are thorough and convincing in showing that ESR1+ PMC neurons exert coordinated control over both the bladder and sphincter activity, which is essential for efficient urination. The anatomical distinctions in pelvic versus pudendal control are clear, and it's an advance to understand how this coordination occurs. This work offers a clearer picture of how micturition is driven.

Weaknesses:

The dynamics of how this population of ESR1+ neurons is engaged in natural urination events remains unclear. Not all ESR1+neurons are always engaged, and it is not measured whether this is simply variation in population activity, or if more neurons are engaged during more intense starting bladder pressures, for instance. In particular, the response dynamics of single and doubly-projecting neurons are not defined. Additionally, the model for how these neurons coordinate with CRH+ neuron activity in the PMC is not addressed, although these cell types seem to be engaged at the same time. Lastly, it would be interesting to know how sensory input can likely modulate the activity of these neurons, but this is perhaps a future direction.

Reviewer #3 (Public review):

Summary:

The paper by Li et al explored the role of Estrogen receptor 1 (Esr1) expressing neurons in the pontine micturition center (PMC), a brainstem region also known as Barrington's nucleus (Hou et al 2016, Keller et al 2018). First, the author conducted bulk Ca2+ imaging/unit recording from PMCESR1 to investigate the correlations of PMCESR1 neural activity to voiding behavior in conscious mice and bladder pressure/external urethral muscle activity in urethane anesthetized mice. Next, the authors conducted optogenetics inactivation/activation of PMCESR1 to confirm the contribution to the voiding behavior also conducted peripheral nerve transection together with optogenetics activation to confirm the independent control of bladder pressure and urethral sphincter muscle.

Weaknesses:

(1) The study demonstrates that pelvic nerve transection reduces urinary volume triggered by PMCESR1+ cell photoactivation in freely moving mice. Could the role of pudendal nerve transection also be examined in awake mice to provide a more comprehensive understanding of neural involvement?

(2) While the paper primarily focuses on PMCESR1+ cells in bladder-sphincter coordination, the analysis of PMCESR1+-DGC/SPN neural circuits - given their distinct anatomical projections in the sacral spinal cord - feels underexplored. How do these circuits influence bladder and sphincter function when activated or inhibited? Also, do you have any tracing data to confirm whether bladder-sphincter innervation comes from distinct spinal nuclei?

(3) Although the paper successfully identifies the physiological role of PMCESR1+ cells in bladder-sphincter coordination, the study falls short in examining the electrophysiological properties of PMCESR1+-DGC/SPN cells. A deeper investigation here would strengthen the findings.

(4) The parameters for photoactivation (blue light pulses delivered at 25 Hz for 15 ms, every 30 s) and photoinhibition (pulses at 50 Hz for 20 ms) vary. What drove the selection of these specific parameters? Moreover, for photoactivation experiments, the change in pressure (ΔP = P5 sec - P0 sec) is calculated differently from photoinhibition (Δpressure = Ppeak - Pmin). Can you clarify the reasoning behind these differing approaches?

(5) The discussion could further emphasize how PMCESR1+ cells coordinate bladder contraction and sphincter relaxation to control urination, highlighting their central role in the initiation and suspension of this process.

(6) In Figure 8, The authors analyze the temporal sequence of bladder pressure and EUS bursting during natural voiding and PMC activation-induced voiding. It would be acceptable to consider the existence of a lower spinal reflex circuit, however, the interpretation of the data contains speculation. Bladder pressure measurement is hard to say reflecting efferent pelvic nerve activity in real time. (As a biological system, bladder contraction is mediated by smooth muscle, and does not reflect real-time efferent pelvic nerve activity. As an experimental set-up, bladder pressure measurement has some delays to reflect bladder pressure because of tubing, but EUS bursting has no delay.) Especially for the inactivation experiment, these factors would contribute to the interpretation of data. This reviewer recommends a rewrite of the section considering these limitations. Most of the section is suitable for the results.

Reviewer #1 (Public Review):

Summary:

The study aimed to better understand the role of the H3 protein of the Monkeypox virus (MPXV) in host cell adhesion, identifying a crucial α-helical domain for interaction with heparan sulfate (HS). Using a combination of advanced computational simulations and experimental validations, the authors discovered that this domain is essential for viral adhesion and potentially a new target for developing antiviral therapies.

Strengths:

The study's main strengths include the use of cutting-edge computational tools such as AlphaFold2 and molecular dynamics simulations, combined with robust experimental techniques like single-molecule force spectroscopy and flow cytometry. These methods provided a detailed and reliable view of the interactions between the H3 protein and HS. The study also highlighted the importance of the α-helical domain's electric charge and the influence of the Mg(II) ion in stabilizing this interaction. The work's impact on the field is significant, offering new perspectives for developing antiviral treatments for MPXV and potentially other viruses with similar adhesion mechanisms. The provided methods and data are highly useful for researchers working with viral proteins and protein-polysaccharide interactions, offering a solid foundation for future investigations and therapeutic innovations.

Comments on revised version:

The authors have successfully addressed the questions raised in my review.

Reviewer #2 (Public Review):

Summary:

The manuscript presenting the discovery of a heparan-sulfate (HS) binding domain in monkeypox virus (MPXV) H3 protein as a new anti-poxviral drug target, presented by Bin Zhen and co-workers, is of interest, given that it offers a potentially broad antiviral substance to be used against poxviruses. Using new computational biology techniques, the authors identified a new alpha-helical domain in the H3 protein, which interacts with cell surface HS, and this domain seems to be crucial for H3-HS interaction. Given that this domain is conserved across orthopoxviruses, authors designed protein inhibitors. One of these inhibitors, AI-PoxBlock723, effectively disrupted the H3-HS interaction and inhibited infection with Monkeypox virus and Vaccinia virus. The presented data should be of interest to a diverse audience, given the possibility of an effective anti-poxviral drug.

Strengths:

In my opinion, the experiments done in this work were well-planned and executed. The authors put together several computational methods, to design poxvirus inhibitor molecules, and then they test these molecules for infection inhibition.

Comments on revised version:

The authors have addressed the comments I made in my review.

Reviewer #3 (Public Review):

Summary:

The article is an interesting approach to determining the MPOX receptor using "in silico" tools. The results show the presence of two regions of the H3 protein with a high probability of being involved in the interaction with the HS cell receptor. However, the α-helical region seems to be the most probable, since modifications in this region affect the virus binding to the HS receptor.

Strengths:

In my opinion, it is an informative article with interesting results, generated by a combination of "in silico" and wet science to test the theoretical results. This is a strong point of the article.

Comments on revised version:

After a review of the changes to the manuscript and the author's responses, no further changes are needed.

Reviewer #1 (Public review):

This study of mixed glutamate/GABA transmission from axons of the supramammillary nucleus to dentate gyrus seeks to sort out whether the two transmitters are released from the same or different synaptic vesicles. This conundrum has been examined in other dual-transmission cases and even in this particular pathway there are different views. The authors use a variety of electrophysiological and immunohistochemical methods to reach the surprising (to me) conclusion that glutamate and GABA filled vesicles are distinct yet released from the same nerve terminals. While the strength of the conclusion rests on the abundance of data (approaches) rather than the decisiveness of any one approach, I came away believing that the boutons may indeed produce and release distinct types of vesicles. Accepting the conclusion, one is now left with another conundrum: how can a single bouton sort out VGLUTs and VIAATs to different vesicles, position them in distinct locations with nm precision and recycle them without mixing? And why do it this way instead of with single vesicles having mixed chemical content? For example, could a quantitative argument be made that separate vesicles allow for higher transmitter concentrations? Hopefully, future studies will probe these issues.

Reviewer #2 (Public review):

Summary:

In this study, the authors investigated the release properties of glutamate/GABA co-transmission at the supramammillary nucleus (SuM)-dentate granule cell (DGC) synapses using state -of-the-arts in vitro electrophysiology and anatomical approaches at the light and electron microscopy level. They found that SuM to dentate granule cell synapses, which co-release glutamate and GABA, exhibit distinct differences in paired-pulse ratio, Ca2+ sensitivity, presynaptic receptor modulation, and Ca2+ channel-vesicle coupling configuration for each neurotransmitter. The study shows that glutamate/GABA co-release produces independent glutamatergic and GABAergic synaptic responses, with postsynaptic targets segregated. They show that most SuM boutons form distinct glutamatergic and GABAergic synapses at proximity, characterized by GluN1 and GABAAα1 receptor labeling respectively. Furthermore, they demonstrate that glutamate/GABA co-transmission exhibits distinct short-term plasticity, with glutamate showing frequency-dependent depression and GABA showing frequency-independent stable depression. The authors provide compelling evidence at the anatomical and physiological levels that glutamate and GABA are co-release by different synaptic vesicles within the same synaptic terminal at the SuM-DGC synapses and that the distinct transmission modes of the glutamate and GABA release serve as a frequency-dependent filters of SuM inputs on GC outputs.<br /> This is a fundamental work, that significantly advances our understanding of the mechanism by which the two fast-acting and functionally opposing neurotransmitters glutamate and GABA are co-transmitted at the SuM-DGC synapses and the functional role of this type of Glutamate/GABA co-transmission.

Strengths:

The conclusions of this paper are provided by a large number of compelling data

Reviewer #3 (Public review):

Summary:

In this manuscript, Hirai et al investigated the release properties of glutamate/GABA co-transmission at SuM-GC synapses and reported that glutamate/GABA co-transmission exhibits distinct short-term plasticity with segregated postsynaptic targets. Using optogenetics, whole-cell patch-clamp recordings, and immunohistochemistry, the authors reveal distinct transmission modes of glutamate/GABA co-release as frequency-dependent filters of incoming SuM inputs.

Strengths:

Overall, this study is well-designed and executed; conclusions are supported by the results. This study addressed a long-standing question of whether GABA and glutamate are packaged in the same vesicles and co-released in response to the same stimuli in the SuM-GC synapses (Pedersen et al., 2017; Hashimotodani et al., 2018; Billwiller et al., 2020; Chen et al., 2020; Li et al., 2020; Ajibola et al., 2021). Knowledge gained from this study advances our understanding of neurotransmitter co-release mechanisms and their functional roles in the hippocampal circuits.

Comments on revisions:

The authors have addressed my comments, and now the manuscript is in a good form as it currently stands.

Reviewer #1 (Public review):

This paper reports fossil soft-tissue structures (tail vanes) of pterosaurs, and attempts to relate this to flight performance and other proposed functions for the tail

The paper presents new evidence for soft-tissue strengthening of vanes using exciting new methods.

There is now some discussion of bias in the sample selection method as well as some theory to show how the lattice could have functioned, other than a narrative description.

Reviewer #1 (Public review):

Summary:

Lodhiya et al. demonstrate that antibiotics with distinct mechanisms of action, norfloxacin and streptomycin, cause similar metabolic dysfunction in the model organism Mycobacterium smegmatis. This includes enhanced flux through the TCA cycle and respiration as well as a build-up of reactive oxygen species (ROS) and ATP. Genetic and/or pharmacologic depression of ROS or ATP levels protect M. smegmatis from norfloxacin and streptomycin killing. Because ATP depression is protective, but in some cases does not depress ROS, the authors surmise that excessive ATP is the primary mechanism by which norfloxacin and streptomycin kill M. smegmatis. In general, the experiments are carefully executed; alternative hypotheses are discussed and considered; the data are contextualized within the existing literature.

Strengths:

The authors tackle a problem that is both biologically interesting and medically impactful, namely, the mechanism of antibiotic-induced cell death.

Experiments are carefully executed, for example, numerous dose- and time-dependency studies; multiple, orthogonal readouts for ROS; and several methods for pharmacological and genetic depletion of ATP.

There has been a lot of excitement and controversy in the field, and the authors do a nice job of situating their work in this larger context.

Inherent limitations to some of their approaches are acknowledged and discussed e.g., normalizing ATP levels to viable counts of bacteria.

Weaknesses:

All of the experiments performed here were in the model organism M. smegmatis. As the authors point out, the extent to which these findings apply to other organisms (most notably, slow-growing pathogens like M. tuberculosis) is to be determined. To avoid the perception of overreach, I would recommend substituting "M. smegmatis" for Mycobacteria (especially in the title and abstract).

At first glance, a few of the results in the manuscript seem to conflict with what has been previously reported in the (referenced) literature. In their response to reviewers, the authors addressed my concerns. It would also be ideal to include a few lines in the manuscript briefly addressing these points. (Other readers may have similar concerns)

In the first round of review, I suggested that the authors consider removing Figs. 9 and 10A-B as I believe they distract from the main point of the paper and appear to be the beginning of a new story rather than the end of the current one. I still hold this opinion. However, one of the strengths of the eLife model is that we can agree to disagree.

Reviewer #2 (Public review):

Summary:

The authors are trying to test the hypothesis that ATP bursts are the predominant driver of antibiotic lethality of Mycobacteria

Strengths:

No significant strengths in the current state as it is written.

Weaknesses:

A major weakness is that M. smegmatis has a doubling time of three hours and the authors are trying to conclude that their data would reflect the physiology of M. tuberculossi that has a doubling time of 24 hours. Moreover, the authors try to compare OD measurements with CFU counts and thus observe great variabilities.

Comments on revisions:

I am surprised that the authors simply did not repeat the study in figure one with CFU counts and repeated in triplicate. Since this is M. smegmatis, it would take no longer than two weeks to repeat this experiment and replace the figure. I understand that obtaining CFU counts is much more laborious than OD measurements but it is necessary. Your graph still says that there is 0 bacteria at time 0, yet in your legend it says you started with 600,000 CFU/ml. I don't understand why this experiment was not repeated with CFU counts measured throughout. This is not a big ask since this is M. smegmatis but it appears that the authors do not want to repeat this experiment. Minimally, fix the graph to represent the CFU.

Reviewer #1 (Public review):

Summary:

This is a very nice paper in which the authors addressed the potential for NK cell cellular therapy to treat and potentially eliminate previously established metastases after surgical resections, which are a major cause of death in human cancer patients. To do so they developed a model using the EO771 breast cancer cell line, in which they establish and then resect tumors and the draining lymph node, after which the majority of mice eventually succumb to metastatic disease. They found that when the initiating tumors were resected when still relatively small, adoptive transfers of IL-15/12-conditioned NK cells substantially enhanced the survival of tumor-bearing animals. They then delved into the cellular mechanisms involved. Interestingly and somewhat unexpectedly, the therapeutic effect of the transferred NK cells was dependent on the host's CD8+ T cells. Accordingly, the NK cell therapy contributed to the formation of tumor-specific CD8+ T cells, which protected the recipient animals against tumor re-challenge and were effective in protecting mice from tumor formation when transferred to naive mice. Mechanistically, they used Ifng knockout NK cells to provide evidence that IFNgamma produced by the transferred NK cells was crucial for the accumulation and activation of DCs in the metastatic lung, including expression of CD86, CD40 and MHC genes. In turn, IFNgamma production by NK cells was essential for the induced accumulation of activated CD8 effector T cells and stem cell-like CD8 T cells in the metastatic lung. The authors then expanded their findings from the mouse model to a small clinical trial. They found that inoculations of IL-15/12-conditioned autologous NK cells in patients with various malignancies after resection was safe and showed signs of efficacy.

Strengths:

- Monitoring of long-term metastatic disease and survival after resection used in this paper is a physiological model that closely resembles clinical scenarios more than the animal models usually used, a great strength of the approach.<br /> - Previous literature focused on the notion that NK cells clear metastatic lesions directly, within a short period. The authors' use of a more relevant model and time frame revealed the previously unexplored T cell-dependent mechanism of action of infused NK cells for long-term control of metastatic diseases.<br /> - Also important, the paper provides solid evidence for the contribution of IFNgamma produced by NK cells for activation of dendritic cells and T cells. This is an interesting finding that provokes additional questions concerning the action of the interferon gamma in this context.<br /> - The results from the clinical trial in cancer patients based on the same type of IL-15/12-conditioned NK cell infusions, was encouraging with respect to safety and showed signals of efficacy, which support the translatability of the author's findings.

Future studies in this model could shed even more light on the mechanisms. The authors do not address whether the IL-12 in their cocktail is essential for the effects they see. Relatedly, it was of interest that despite the effectiveness of the transferred IL-15/IL-12 cultured NK cells, the cells failed to persist very long after transfer. Published studies have reported that so-called memory-like NK cells, which are pre-activated with a cocktail of IL-12, IL-18 and IL-15, persist much longer in lympho-depleted mice and patients than IL-2 cultured NK cells. It would be illuminating to compare these two types of NK cell products in the author's model system, and with, or without, lymphodepletion, to identify the critical parameters. If greater persistence occurred with the memory-like NK cell product, it is possible that the NK cells might provide greater benefit, including by directly targeting the tumor.

Reviewer #2 (Public review):

Summary:

The authors show convincing data that increasing NK cell function/frequency can reduce development and progression of metastatic disease after primary tumor resection.

Strengths:

The inclusion of a first-in-human trial highlighting some partial responses of metastatic patients treated with in vitro expanded NK cells is tantalising. It is difficult to perform trials in preventing further metastasis since the timelines are very protracted but more data like these highlighting a role for NK cells in improving local cDC1/T cells anti-tumor immunity will encourage deeper thinking around therapeutic approaches to target endogenous NK cells to achieve the same.

Weaknesses:

As always, more patient data would help increase confidence around the human relevance of the approach.

Comments on revisions:

The authors have addressed all my queries

Reviewer #1 (Public review):

Summary:

Previous work has shown that the evolutionarily-conserved division-orienting protein LGN/ Pins/ GPR1/2 (vertebrates/flies/nematodes) participates in division orientation across a variety of cell types, perhaps most importantly those that undergo asymmetric divisions (ACDs). Micromere formation in echinoids relies on asymmetric cell division at the 16-cell stage, and these authors previously demonstrated a role for the LGN/Pins homolog AGS (Activator of G-protein signaling) in that ACD process. Here they extend that work by investigating and exploiting the question of why echinoids but not other echinoderms form micromeres. Using an impressive combination of phylogenetics and molecular experiments, they determine that much of the difference in ACD and micromere formation in echinoids can be attributed to differences in the AGS C-terminus, in particular a GoLoco domain (GL1) that is missing in most other echinoderms. This work helps explain how AGS works and thereby enhances our understanding of a conserved player in division orientation.

Reviewer #2 (Public review):

This study from Dr. Emura and colleagues addresses the relevance of AGS3 mutations in the execution of asymmetric cell divisions promoting the formation of the micromere during sea-searching development. To this aim, the authors use quantitative imaging approaches to evaluate the localisation of AGS3 mutants truncated at the N-terminal region or at the C-terminal region, and correlate these distributions with the formation of micromere and correct development of embryos to the pluteus stage. The authors also analyse the capacity of these mutated proteins to rescue developmental defects observed upon AGS3 depletion by morpholino antisense nucleotides (MO). Collectively these experiments revealed that the C-terminus of AGS3, coding for four GoLoco motifs binding to cortical Gaphai proteins, is the molecular determinant for cortical localisation of AGS3 at the micromeres and correct pluteus development. Further genetic dissections and expression of chimeric AGS3 mutants carrying shuffled copies of the GoLoco motifs or four copies of the same motifs revealed that the position of GoLoco1 is essential for AGS3 functioning. To understand whether the AGS3-GoLoco1 evolved specifically to promote asymmetric cell divisions, the author analyse chimeric AGS3 variants in which they replaced the sea urchin GoLoco region with orthologs from other echinoids that do not form micromeres, or from Drosophila Pins or human LGN. These analyses corroborate the notion that the GoLoco1 position is crucial for asymmetric AGS3 functions. In the last part of the manuscript, the authors explore whether SpAGS3 interacts with the molecular machinery described to promote asymmetric cell division in eukaryotes, including Insc, NuMA, Par3 and Galphai, and show that all these proteins colocalize at the nascent micromere, together with the fate determinant Vasa. Collectively this evidence highlighted how evolutionarily selected AGS3 modifications are essential to sustain asymmetric divisions and specific developmental programs associated with them.

The manuscript addresses an interesting question and uses elegant genetic approaches associated with imaging analyses to elucidate the molecular mechanisms whereby AGS3 and spindle orientation proteins promote asymmetric divisions and specific developmental programs. This considered, it might be worth clarifying a few aspects of the reported findings.

(1) In some experimental settings, the presence of AGS3 mutants exacerbates the AGS3 deletion by MO (Fig. 4F). Can the author speculate on what can be the molecular explanation?

(2) Imaging analyses of Figure 4B-C suggest that the mutant AGS1111 does not localise at the vegetal cortex while AGS2222 does (Fig. 4C). However these mutants induce similar developmental defects (Fig. 4F) . What could be the reason?

(3) Figure 7 shows the crosstalk between AGS3 and other asymmetry players including NuMA. Vertebrate and Drosophila NuMA are ubiquitously present in tissues and localises to the spindle poles in mitosi. However in Figure 7A and 7E NuMA seems expressed only in a subset of sea urchin embryonic cells. Is this the case?

Reviewer #1 (Public review):

Summary:

The authors aimed to classify hepatocellular carcinoma (HCC) patients into distinct subtypes using a comprehensive multi-omics approach. They employed an innovative consensus clustering method that integrates multiple omics data types, including mRNA, lncRNA, miRNA, DNA methylation, and somatic mutations. The study further sought to validate these subtypes by developing prognostic models using machine learning algorithms and extending the findings through single-cell RNA sequencing (scRNA-seq) to explore the cellular mechanisms driving subtype-specific prognostic differences.

Strengths:

(1) Comprehensive Data Integration: The study's integration of various omics data provides a well-rounded view of the molecular characteristics underlying HCC. This multi-omics approach is a significant strength, as it allows for more accurate and detailed classification of cancer subtypes.

(2) Innovative Methodology: The use of a consensus clustering approach that combines results from 10 different clustering algorithms is a notable methodological advancement. This approach reduces the bias that can result from relying on a single clustering method, enhancing the robustness of the findings.

(3) Machine Learning-Based Prognostic Modeling: The authors rigorously apply a wide array of machine learning algorithms to develop and validate prognostic models, testing 101 different algorithm combinations. This comprehensive approach underscores the study's commitment to identifying the most predictive models, which is a considerable strength.

(4) Validation Across Multiple Cohorts: The external validation of findings in independent cohorts is a critical strength, as it increases the generalizability and reliability of the results. This step is essential for demonstrating the clinical relevance of the proposed subtypes and prognostic models.

Weaknesses:

(1) Inconsistent Storyline:<br /> Despite the extensive data mining and rigorous methodologies, the manuscript suffers from a lack of a coherent and consistent narrative. The transition between different sections, particularly from multi-omics data integration to single-cell validation, feels disjointed. A clearer articulation of how each analysis ties into the overall research question would improve the manuscript.

(2) Questionable Relevance of Immune Cell Activity Analysis:<br /> The evaluation of immune cell activities within the cancer cell model raises concerns about its meaningfulness. The methods used to assess immune function in the tumor microenvironment may not be fully appropriate, potentially limiting the insights gained from this part of the study.

(3) Incomplete Single-Cell RNA-Seq Validation:<br /> The validation of the findings using single-cell RNA-seq data appears insufficient to fully support the study's claims. While the authors make an effort to extend their findings to the single-cell level, the analysis lacks depth. A more comprehensive validation is necessary to substantiate the robustness of the identified subtypes.

(4) Figures and Visualizations:<br /> Several figures in the manuscript are missing necessary information, which affects the clarity of the results. For instance, the pathways in Figure 3A could be clustered to enhance interpretability, the blue bar in Figure 4A is unexplained, and Figure 4B is not discussed in the text. Additionally, the figure legend in Figure 7C lacks detail, and many figure descriptions merely repeat the captions without providing deeper insights.

(5) Appraisal of the Study's Aims and Results:<br /> The authors have set out to achieve an ambitious goal of classifying HCC patients into distinct prognostic subtypes and validating these findings through both bulk and single-cell analyses. While the methodologies employed are innovative and the data integration comprehensive, the study falls short of fully achieving its aims due to inconsistencies in the narrative and incomplete validation. The results partially support the conclusions, but the lack of coherence and depth in certain areas limits the overall impact of the study.

(6) Impact on the Field:<br /> If the identified weaknesses are addressed, this study has the potential to significantly impact the field of HCC research. The multi-omics approach combined with machine learning is a powerful framework that could set a new standard for cancer subtype classification. However, the current state of the manuscript leaves some uncertainty regarding the practical applicability of the findings, particularly in clinical settings.

(6) Additional Context<br /> For readers and researchers, this study offers a valuable look into the potential of integrating multi-omics data with machine learning to improve cancer classification and prognostication. However, readers should be aware of the noted weaknesses, particularly the need for more consistent narrative development and comprehensive validation of the methods. Addressing these issues could greatly enhance the study's utility and relevance to the community.

Reviewer #2 (Public review):

Summary:

Overall, this is a well-executed and insightful study. With some refinement to the presentation and a deeper exploration of the implications, the manuscript will make a significant contribution to the field of cancer genomics and personalized medicine.

Strengths:

The manuscript integrates multi-omics data with machine learning to address the significant heterogeneity of hepatocellular carcinoma (HCC). The use of multiple clustering algorithms and a consensus method strengthens the robustness of the findings. The study successfully develops a prognostic model with excellent predictive accuracy, validated across independent datasets. This adds considerable value to the field, particularly in providing individualized treatment strategies. The identification of two distinct liver cancer subtypes with different biological and metabolic characteristics is well-supported by the data, offering a promising direction for personalized medicine.

Weaknesses:

(1) Consider streamlining the presentation of methods, especially regarding the clustering algorithms and machine learning models. Readers may find it difficult to follow the exact process unless more clearly outlined.

(2) Some figures, such as the signaling pathways and heatmaps, are critical to understanding the study's findings. Ensure that all figures are high quality, easy to interpret, and adequately labeled. You may also want to highlight the key findings within the figure captions more explicitly.

(3) While the manuscript does compare its prognostic model to those previously published, the novelty of the findings could be emphasized more clearly. Discussing the potential limitations of the study (e.g., the reliance on computational models and small sample sizes for scRNA-seq) could strengthen the manuscript.

(4) The manuscript mentions that the data was split into training and validation datasets in a 1:1 ratio. How was the performance verified? Is there an independent test set?

(5) The role of the MIF signaling pathway in subtype differentiation is intriguing, but further mechanistic insights into how this pathway drives the differences between CS1 and CS2 could be discussed in more detail. If experimental evidence for this pathway exists in the literature, it should be mentioned.

(6) Some sentences are quite long and complex, which can affect readability. Breaking them down into shorter, clearer sentences would improve the flow.

Reviewer #1 (Public review):

Summary:

Debeuf et al. introduce a new, fast method for the selection of suitable T cell clones to generate TCR transgenic mice, a method claimed to outperform traditional hybridoma-based approaches. Clone selection is based on the assessment of the expansion and phenotype of cells specific for a known epitope following immune stimulation. The analysis is facilitated by a new software tool for TCR repertoire and function analysis termed DALI. This work also introduces a potentially invaluable TCR transgenic mouse line specific for SARS-CoV-2.

Strengths:

The newly introduced method proved successful in the quick generation of a TCR transgenic mouse line. Clone selection is based on more comprehensive phenotypical information than traditional methods, providing the opportunity for a more rational T-cell clone selection.

The study provides a software tool for TCR repertoire analysis and its linkage with function.

The findings entail general practical implications in the preclinical study of a potentially very broad range of infectious diseases or vaccination.

A novel SARS-CoV-2 spike-specific TCR transgenic mouse line was generated.

Weaknesses:

The authors present a novel method to develop TCR transgenic mice and overcome the limitations of the more traditional method based on hybridomas.

The authors indicate that they did not intend to directly compare their new method with the traditional hybridoma-based approach. However, such comparison becomes inevitable when the classical method is presented as suboptimal and an alternative approach is introduced to address its limitations. Nevertheless, the explanations provided in their rebuttal have helped clarify their position. The intention behind supplementary figure 1 is to illustrate that a clone that appears suitable using traditional assays may fail to produce a successful TCR transgenic line. This is a valid point that I think should be emphasized more clearly in the manuscript, as it highlights the limitations of the traditional method.

However, the main question that remains is whether the proposed new method will reliably resolve this issue. As previously noted, only one mouse line was generated (successfully) from a single candidate, and the method presented to generate their new TCR transgenic line starts from a more advanced point (a well characterized epitope is already known, and tetramers are available to preselect specific clones). Although this approach likely increases the chances of success, it also limits applicability.

The authors suggest that tetramers are not absolutely necessary to select a clone of interest. Testing this hypothesis would have added value to this manuscript, demonstrating the ability to rapidly generate new TCR transgenic lines in response to emerging pathogens, as outlined in the introduction. They propose that, in such cases, mice could be immunised and expanded clones retested for reactivity. However, it is unclear how this strategy differs from the classic method in increasing the chances of selecting an optimal clone.

Regarding the practical value and cost-effectiveness of extensive expression profiling for T cell clone selection, it remains unclear how well a clone chosen for specific traits will retain these features when developed into a TCR transgenic line, or what traits are ideal for different applications. T cell fate is plastic, and various parameters could influence marker expression.

Issues remain concerning the statistical analysis. Data are said to have been analyzed using both parametric and non-parametric tests. The described approach of performing a normality test followed by either parametric or non-parametric tests is not a correct method for statistical data analysis.

Reviewer #2 (Public review):

Summary:

The authors seek to use single-cell sequencing approaches to identify TCRs specific for the SARS CoV2 spike protein, select a candidate TCR for cloning and use it to construct a TCR transgenic mouse. The argument is that this process is less cumbersome than the classical approach, which involves the identification of antigen-reactive T cells in vitro and the construction of T cell hybridomas prior to TCR cloning. TCRs identified by single-cell sequencing that is already paired to transcriptomic data would more rapidly identify TCRs that are likely to contribute to a functional response. The authors successfully identify TCRs that have expanded in response to SARS CoV2 spike protein immunization, bind to MHC tetramers and express genes associated with functional response. They then select a TCR for cloning and construction of a transgenic mouse in order to test the response of resulting T cells in vivo following immunization with spike protein of coronavirus infection.

Strengths:

(1) The study provides proof of principle for the identification and characterization of TCRs based on single-cell sequencing data.

(2) The authors employ a recently developed software tool (DALI) that assists in linking transcriptomic data to individual clones.

(3) The authors successfully generate a TCR transgenic animal derived from the most promising T cell clone (CORSET8) using the TCR sequencing approach.

(4) The authors provide initial evidence that CORSET8 T cells undergo activation and proliferation in vivo in response to immunization or infection.

(5) Procedures are well-described and readily reproducible.

Weaknesses:

(1) The purpose of presenting a failed attempt to generate TCR transgenic mice using a traditional TCR hybridoma method is unclear. The reasons for the failure are uncertain, and the inclusion of this data does not really provide information on the likely success rate of the hybridoma vs single cell approach for TCR identification, as only a single example is provided for either.

(2) There is little information provided regarding the functional differentiation of the CORSET8 T cells following challenge in vivo, including expression of molecules associated with effector function, cytokine production, killing activity and formation of memory. The study would be strengthened by some evidence that CORSET8 T cells are successfully recapitulating the functional features of the endogenous immune response (beyond simply proliferating and expressing CD44). This information is important to evaluate whether the presented sequencing-based identification and selection of TCRs is likely to result in T-cell responses that replicate the criteria for selecting the TCR in the first place.

(3) While I find the argument reasonable that the approach presented here has a lot of likely advantages over traditional approaches for generating TCR transgenic animals, the use of TCR sequencing data to identify TCRs for study in variety of areas, including cancer immunotherapy and autoimmunity, is in broad use. While much of this work opts for alternative methods of TCR expression in primary T cells (i.e. CRISPR or retroviral approaches), the process of generating a TCR transgenic mouse from a cloned TCR is not in itself novel. It would be helpful if the authors could provide a more extensive discussion explaining the novelty of their approach for TCR identification in comparison to other more modern approaches, rather than only hybridoma generation.

Comments on revisions:

The authors have provided additional clarification on the comparisons between the presented method for TCR transgenic generation and the hybridoma method that is more commonly used and added additional verification of the functional response in vivo of T cells expressing the selected TCR. Overall, these additions enhance the evidence that the proposed methods are likely to identify TCRs with a strong immune activation profile and are a reasonable response to the first round of review.

Reviewer #1 (Public review):

Summary:

Jin et al. investigated how the bacterial DNA damage (SOS) response and its regulator protein RecA affects the development of drug resistance under short-term exposure to beta-lactam antibiotics. Canonically, the SOS response is triggered by DNA damage, which results in the induction of error-prone DNA repair mechanisms. These error-prone repair pathways can increase mutagenesis in the cell, leading to the evolution of drug resistance. Thus, inhibiting the SOS regulator RecA has been proposed as means to delay the rise of resistance.

In this paper, the authors deleted the RecA protein from E. coli and exposed this ∆recA strain to selective levels of the beta-lactam antibiotic, ampicillin. After an 8h treatment, they washed the antibiotic away and allowed the surviving cells to recover in regular media. They then measured the minimum inhibitory concentration (MIC) of ampicillin against these treated strains. They note that after just 8 h treatment with ampicillin, the ∆recA had developed higher MICs towards ampicillin, while by contrast, wild-type cells exhibited unchanged MICs. This MIC increase was also observed subsequent generations of bacteria, suggesting that the phenotype is driven by a genetic change.

The authors then used whole genome sequencing (WGS) to identify mutations that accounted for the resistance phenotype. Within resistant populations, they discovered key mutations in the promoter region of the beta-lactamase gene, ampC; in the penicillin-binding protein PBP3 which is the target of ampicillin; and in the AcrB subunit of the AcrAB-TolC efflux machinery. Importantly, mutations in the efflux machinery can impact the resistances towards other antibiotics, not just beta-lactams. To test this, they repeated the MIC experiments with other classes of antibiotics, including kanamycin, chloramphenicol, and rifampicin. Interestingly, they observed that the ∆recA strains pre-treated with ampicillin showed higher MICs towards all other antibiotic tested. This suggests that the mutations conferring resistance to ampicillin are also increasing resistance to other antibiotics.

The authors then performed an impressive series of genetic, microscopy, and transcriptomic experiments to show that this increase in resistance is not driven by the SOS response, but by independent DNA repair and stress response pathways. Specifically, they show that deletion of the recA reduces the bacterium's ability to process reactive oxygen species (ROS) and repair its DNA. These factors drive accumulation of mutations that can confer resistance towards different classes of antibiotics. The conclusions are reasonably well-supported by the data, but some aspects of the data and the model need to be clarified and extended.

Strengths:

A major strength of the paper is the detailed bacterial genetics and transcriptomics that the authors performed to elucidate the molecular pathways responsible for this increased resistance. They systemically deleted or inactivated genes involved in the SOS response in E. coli. They then subjected these mutants the same MIC assays as described previously. Surprisingly, none of the other SOS gene deletions resulted an increase in drug resistance, suggesting that the SOS response is not involved in this phenotype. This led the authors to focus on the localization of DNA PolI, which also participates in DNA damage repair. Using microscopy, they discovered that in the RecA deletion background, PolI co-localizes with the bacterial chromosome at much lower rates than wild-type. This led the authors to conclude that deletion of RecA hinders PolI and DNA repair. Although the authors do not provide a mechanism, this observation is nonetheless valuable for the field and can stimulate further investigations in the future.

In order to understand how RecA deletion affects cellular physiology, the authors performed RNA-seq on ampicillin-treated strains. Crucially, they discovered that in the RecA deletion strain, genes associated with antioxidative activity (cysJ, cysI, cysH, soda, sufD) and Base Excision Repair repair (mutH, mutY, mutM), which repairs oxidized forms of guanine, were all downregulated. The authors conclude that down-regulation of these genes might result in elevated levels of reactive oxygen species in the cells, which in turn, might drive the rise of resistance. Experimentally, they further demonstrated that treating the ∆recA strain with an antioxidant GSH prevents the rise of MICs. These observations will be useful for more detailed mechanistic follow-ups in the future.

Weaknesses:

Throughout the paper, the authors use language suggesting that ampicillin treatment of the ∆recA strain induces higher levels of mutagenesis inside the cells, leading to the rapid rise of resistance mutations. However, as the authors note, the mutants enriched by ampicillin selection can play a role in efflux and can thus change a bacterium's sensitivity to a wide range of antibiotics, in what is known as cross-resistance. The current data is not clear on whether the elevated "mutagenesis" is driven ampicillin selection or by a bona fide increase in mutation rate.

Furthermore, on a technical level, the authors employed WGS to identify resistance mutations in the treated ampicillin-treated wild-type and ∆recA strains. However, the WGS methodology described in the paper is inconsistent. Notably, wild-type WGS samples were picked from non-selective plates, while ΔrecA WGS isolates were picked from selective plates with 50 μg/mL ampicillin. Such an approach biases the frequency and identity of the mutations seen in the WGS and cannot be used to support the idea that ampicillin treatment induces higher levels of mutagenesis.

Finally, it is important to establish what the basal mutation rates of both the WT and ∆recA strains are. Currently, only the ampicillin-treated populations were reported. It is possible that the ∆recA strain has inherently higher mutagenesis than WT, with a larger subpopulation of resistant clones. Thus, ampicillin treatment might not in fact induce higher mutagenesis in ∆recA.

Comments on revisions:

Thank you for responding to the concerns raised previously. The manuscript overall has improved.

Reviewer #2 (Public review):

Summary:

This study aims to demonstrate that E. coli can acquire rapid antibiotic resistance mutations in the absence of a DNA damage response. The authors employed a modified Adaptive Laboratory Evolution (ALE) workflow to investigate this, initiating the process by diluting an overnight culture 50-fold into an ampicillin selection medium. They present evidence that a recA- strain develops ampicillin resistance mutations more rapidly than the wild-type, as indicated by the Minimum Inhibitory Concentration (MIC) and mutation frequency. Whole-genome sequencing of recA- colonies resistant to ampicillin showed predominant inactivation of genes involved in the multi-drug efflux pump system, contrasting with wild-type mutations that seem to activate the chromosomal ampC cryptic promoter. Further analysis of mutants, including a lexA3 mutant incapable of inducing the SOS response, led the authors to conclude that the rapid evolution of antibiotic resistance occurs via an SOS-independent mechanism in the absence of recA. RNA sequencing suggests that antioxidative response genes drive the rapid evolution of antibiotic resistance in the recA- strain. They assert that rapid evolution is facilitated by compromised DNA repair, transcriptional repression of antioxidative stress genes, and excessive ROS accumulation.

Strengths:

The experiments are well-executed and the data appear reliable. It is evident that the inactivation of recA promotes faster evolutionary responses, although the exact mechanisms driving this acceleration remain elusive and deserve further investigation.

Weaknesses:

Some conclusions are overstated. For instance, the conclusion regarding the LexA3 allele, indicating that rapid evolution occurs in an SOS-independent manner (line 217), contradicts the introductory statement that attributes evolution to compromised DNA repair. The claim made in the discussion of Figure 3 that the hindrance of DNA repair in recA- is crucial for rapid evolution is at best suggestive, not demonstrative. Additionally, the interpretation of the PolI data implies its role, yet it remains speculative. In Figure 2A table, mutations in amp promoters are leading to amino acid changes! The authors' assertion that ampicillin significantly influences persistence pathways in the wild-type strain, affecting quorum sensing, flagellar assembly, biofilm formation, and bacterial chemotaxis, lacks empirical validation. Figure 1G suggests that recA cells treated with ampicillin exhibit a strong mutator phenotype; however, it remains unclear if this can be linked to the mutations identified in Figure 2's sequencing analysis.

Reviewer #3 (Public review):

Summary:

In the present work, Zhang et al investigate involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance evolution in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term (transient) drug resistance evolution can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by accumulation of reactive oxygen species and compromised DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance).

Strengths:

The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to comprised DNA repair and oxidative stress. Antibiotic resistance evolution under transient stress is poorly studied, so the authors' work is a nice mechanistic contribution to this field.

Weaknesses:

The authors do not show any direct evidence of altered mutation rate or accumulated DNA damage in their model.

Reviewer #1 (Public review):

Summary:

In this manuscript Spott et al. combine SARS-CoV-2 genomic data alongside granular mobility data to retrospectively evaluate the spread of SARS-CoV-2 alpha lineages throughout Germany and specifically Thuringia. They further prospectively identified districts with strong mobility links to the first district in which BQ.1.1 was observed to direct additional surveillance efforts to these districts. The additional surveillance effort resulted in the earlier identification of BQ.1.1 in districts with strong links to the district in which BQ.1.1 was first observed.

Strengths:

There are two important strengths of this work. The first, is the scale and detail in the data that has been generated an analyzed as part of this study. Specifically, the authors use 6,500 SARS-CoV-2 sequences and district level mobility data within Thuringia. I applaud the authors for making a subset of their analyses public e.g. on the associated micro react page.

Further, the main focus of the article is on the potential utility of mobility-directed surveillance sequence. While I may certainly be mistaken, I have not seen this proposed elsewhere, at least in the context of SARS-CoV-2. The authors were further able to test this concept in a real world setting during the emergence of BQ.1.1 and compare it to the "gold standard" of random sampling. This is a unique real-world evaluation of a novel surveillance sequencing strategy and there is considerable value in publishing this analysis. Given the increased focus on optimizing sampling strategies for genomic surveillance, this work provides a novel strategy and will hopefully motivate additional modeling and real-world implementations.

Weaknesses:

The article is quite strong and I find the analyses to generally be rigorous. Limitations of the analysis, particularly due to the fact that BQ.1.1 remained a low-prevalence variant, are adequately addressed. The results do not provide quantitative, definitive proof that mobility-guided sampling is an optimal strategy, but they also do not claim to nor do I think they need to to make an important contribution to the field.

Reviewer #2 (Public review):

In the manuscript, the authors combine SARS-CoV-2 sequence data from a state in Germany and mobility data to help in understanding the movement of virus and the potential to help decide where to focus sequencing. The global expansion in sequencing capability is a key outcome of the public health response. However, there remains uncertainty how to maximise the insights the sequence data can give. Improved ability to predict the movement of emergent variants would be a useful public health outcome.

However, I remain unconvinced that changing surveillance strategies is necessarily sensible as it remains unclear what the ultimate benefit of variant hunting is. Decisions to adapt surveillance strategies should not be taken lightly as there are substantial benefits of maintaining a stable and as representative as possible, system over time. It's unclear what public health action would result of detecting a few more sequences of a variant. Once a variant has been identified (arguably anywhere in the world/region), we already have the necessary information to motivate the development of updated vaccines/monoclonals.

Reviewer #1 (Public review):

Summary:

This paper describes a new in vitro model for DRG neurons that recapitulates several key differences between the peripheral and central branches of DRG axons in vivo. These differences include morphology (with one branch being thinner than the other), and regenerative capacity (with the peripheral branch displaying higher regenerative capacity). The authors analyze the abundance of various microtubule-associated protein (MAPs) in each branch, as well as the microtubule dynamics in each branch, and find significant differences between branches. Importantly, they found that a well-known conditioning paradigm (prior lesion of the peripheral branch improves the regenerative capacity of the central branch) is not only reproduced in this system but also leads to loss of the asymmetry of MAPs between branches. Zooming in on one MAP that shows differential abundance between the axons, they find that the severing enzyme Spastin is required for the asymmetry in microtubule dynamics and in regenerative capacity following a conditioning lesion.

Strengths:

The establishment of an experimental system that recapitulates DRG axon asymmetry in vitro is an important step that is likely to be useful for other studies. In addition, identifying key molecular signatures that differ between central and peripheral branches, and determining how they are lost following a conditioning lesion adds to our understanding of why peripheral axons have a better regenerative capacity. Last, the author's use of an in vivo model system to support some of their in vitro findings is a strength of this work.

Weaknesses:

The main weakness of the manuscript is that to a large degree, one of its main conclusions (MAP symmetry underlies differences in regenerative capacity) relies mainly on a correlation, without firmly establishing a causal link. However, this weakness is relatively minor because (1) it is partially addressed with the Spastin KO and (2) there isn't a trivial way to show a causal relationship in this case.

Reviewer #2 (Public review):

Summary:

The authors set out to develop a tissue culture method in which to study the different regenerative abilities of the central and peripheral branch of sensory axons. Neurons developed a small and large branch, which have different regenerative abilities, different transport rates, and different microtubule properties. The study provides convincing evidence that the two axonal branches differ in a way to correspond to in vivo. The different regenerative abilities of the two branches are an important observation because until now it has not been clear whether this difference is intrinsic to the neuron and axons or due to differences in the environment surrounding the axons. The authors have then looked for molecular explanations of the differences between the branches. They find different transport rates and different microtubule dynamics. The different microtubule dynamics are explained by differing levels of spastin, an enzyme that severs microtubules encouraging dynamics.

Strengths:

The differences between the two branches are clearly shown, together with differences in transport, microtubule dynamics, and regeneration. The in vitro model is novel and could be widely used. The methods used are robust and generally accepted.

Weaknesses:

In order for the method to be used it needs to be better described. For instance what proportion of neurons develop just two axonal branches, one of which is different? How selective are the researchers in finding appropriate neurons?

Reviewer #3 (Public review):

Summary:

In this manuscript, Costa and colleagues investigate how asymmetry in dorsal root ganglion (DRG) neurons is established. The authors developed an in vitro system that mimics the pseudo-unipolar morphology and asymmetry of DRG neurons during the regeneration of the peripheral and central branch axons. They suggest that central-like DRG axons exhibit a higher density of growing microtubules. By reducing the polymerization of microtubules in these central-like axons, they were able to eliminate the asymmetry in DRG neurons.

Strengths:

The authors point out a distinct microtubule-associated protein signature that differentiates between DRG neurons' central and peripheral axonal branches. Experimental results demonstrate that genetic deletion of spastin eliminated the differences in microtubule dynamics and axon regeneration between the central and peripheral branches.

Weaknesses:

While some of the data are compelling, experimental evidence only partially supports the main claims.

In its current form, the study is primarily descriptive and lacks convincing mechanistic insights. It misses important controls and further validation using 3D in vitro models.

Given the heterogeneity of dorsal root ganglion (DRG) neurons, it is unclear whether the in vitro model described in this study can be applied to all major classes of DRG neurons. Also unclear is the inconsistency with embryonic DRG cultures with embryonic (E)16 from rats and E13 from mice (spastin knockout and wild-type controls). Furthermore, the authors stated (line 393) that only a small subset of cultured DRG neurons exhibited a pseudo-unipolar morphology. The authors should include the percentage of the neurons that exhibit a pseudo-unipolar morphology.

The significance of studying microtubule polymerization to DRG asymmetry in vitro is questionable, especially considering the model's validity. The authors might consider eliminating the in vitro data and instead focus on characterizing DRG asymmetry in vivo both before and after a conditioning lesion. If the authors choose to retain the in vitro data, classifying the central and peripheral-like branches in cultured DRG neurons will require further in-depth characterization. Additional validation should be performed in adult DRG neuron cultures not aged in vitro.

The comparison of asymmetry associated with a regenerative response between in vitro and in vivo paradigms has significant limitations due to the nature of the in vitro culture system. When cultured in isolation, DRG neurons fail to form functional connections with appropriate postsynaptic target neurons (the central branch) or to differentiate the peripheral domains associated with the innervation of target organs. Rather than growing neurons on a flat, hard surface like glass, more physiologically relevant substrates and/or culturing conditions should be considered. This approach could help eliminate potential artifacts caused by plating adult DRG neurons on a flat surface. Additionally, the authors should consider replicating their findings in a 3D culture model or using dorsal root ganglia explants, where both centrally and peripherally projecting axons are present.

Panels 5H-J require additional processing with astrocyte markers to accurately define the lesion borders. Furthermore, including a lower magnification would facilitate a direct comparison of the lesion site. The use of cholera toxin subunit B (CTB) to trace dorsal column sensory axons is prone to misinterpretation, as the tracer accumulates at the axon's tip. This limitation makes it extremely challenging to distinguish between regenerating and degenerating axons.

Reviewer #1 (Public review):

Summary of Key Findings:

The authors identified 20 ancient molluscan linkage groups (MLGs) that are largely conserved in other molluscan groups but highly dynamic and rearranged in chitons. This contrasts with the stability seen in other animal groups.

Significant chromosome rearrangements, fusions, and duplications were observed in chitons, particularly in the most basal clades like Lepidopleurida, indicating that chitons undergo more extensive genomic changes than expected.

Chitons exhibit extremely high levels of genomic heterozygosity, exceeding that of other molluscan species and even Lepidoptera. This presents challenges for assembling high-quality genomes but also points to genetic diversity as a driver of evolutionary processes.

Partial genome duplications, particularly in Liolophura japonica, extend the knowledge of gene duplication events within the broader Mollusca clade.

The paper speculates that these genomic rearrangements may contribute to maintaining species boundaries in sympatric and parapatric radiations, as observed in certain Acanthochitona species.

Strengths:

The use of high-quality genomic data, including four de novo genome assemblies, provides robust evidence for the conclusions.

The research challenges the common assumption that chitons are evolutionarily conservative, showing that their genomes are highly dynamic despite their morphological stasis.

The study adds to the understanding of how chromosomal rearrangements might contribute to speciation, a concept that can be applied to other taxa.

Limitations:

The paper acknowledges that the limited availability of high-quality genomes across molluscs may restrict the scope of comparative analyses. More genomic data from other molluscan groups could strengthen the conclusions.

The role of high heterozygosity in chitons is highlighted, but more information is needed to clarify how this affects genome assembly and evolutionary outcomes.

Implications for Future Research:

The research raises important questions about the relationship between genomic instability and phenotypic stasis, which can inform studies in other animal groups.

The findings call for a re-evaluation of how we define and measure biodiversity, particularly in "neglected" clades like chitons. Further studies could focus on linking the observed genomic changes to specific adaptive traits or ecological niches.

Reviewer #2 (Public review):

Summary:

The authors provide four new annotated genomes for an important taxon within Mollusca known as Polyplacophora (chitons). They provide an impressive analysis showing syntenic relationships between the chromosomes of these four genomes but also other available chiton genome sequences and analysis of 20 molluscan linkage groups to expand this analysis across Mollusca.

Strengths:

The authors have selected particular chiton species for genome sequencing and annotation that expand what is known about genomes across portions of chiton phylogenetic diversity lacking genome sequences. The manuscript is well-written and illustrated in a concise manner. The figures are mostly clear, allowing a reader to visually compare the syntenic relationships of chromosomes, especially within chitons. Their phylogenetic analysis provides a simple manner to map important events in molluscan genome evolution. This study greatly expands what is known about molluscan and chiton comparative genomics.

Weaknesses:

I am not especially convinced that chitons have experienced more substantial genomic rearrangements or other genomic events than other molluscan classes, and for this reason, I did not personally find the title compelling: "Still waters run deep: Large scale genome rearrangements in the evolution of morphologically conservative Polyplacophora." Are the documented events "large scale genomic rearrangements"? It seems that mostly they found two cases of chromosome fusion, plus one apparent case of whole genome duplication. What do they mean by "Still waters run deep"? I have no idea. I guess they consider chitons to be morphologically conservative in their appearance and lifestyle so they are calling attention to this apparent paradox. However, most chiton genomes seem to be relatively conserved, but there are unexpected chromosome fusion events within a particular genus, Acanthochitona. Likewise, they found a large-scale gene duplication event in Acanthopleurinae, a different subfamily of chitons, which is quite interesting but these seem to be geologically recent events that do not especially represent the general pattern of genome evolution across this ancient molluscan taxon.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

The study by Liu et al provides a functional analysis of lnc-FANCI-2 in cervical carcinogenesis, building on their previous discovery of FANCI-2 being upregulated in cervical cancer by HPV E7.

The authors conducted a comprehensive investigation by knocking out (KO) FANCI-2 in CaSki cells and assessing viral gene expression, cellular morphology, altered protein expression and secretion, altered RNA expression through RNA sequencing (verification of which by RT-PCR is well appreciated), protein binding, etc. Verification experiments by RT-PCR, Western blot, etc are notable strengths of the study.

The KO and KD were related to increased Ras signaling and EMT and reduced IFN-y/a responses.

Although the large amount of data is well acknowledged, it is a limitation that most data come from CaSki cells, in which FANCI-2 localization is different from SiHa cells and cancer tissues (Figure 1). The cytoplasmic versus nuclear localization is somewhat puzzling.

Reviewer #3 (Public review):

Summary:

A long noncoding RNA, lnc-FANCI-2, was reported to be regulated by HPV E7 oncoprotein and a cell transcription factor, YY1 by this group. The current study focuses on the function of lnc-FANCI-2 in HPV-16 positive cervical cancer is to intrinsically regulate RAS signaling, thereby facilitating our further understanding of additional cellular alterations during HPV oncogenesis. The authors used advanced technical approaches such as KO, transcriptome and (IRPCRP) and LC- MS/MS analyses in the current study and concluded that KO Inc-FANCI-2 significantly increases RAS signaling, especially phosphorylation of Akt and Erk1/2.

Strengths:

(1) HPV E6E7 are required for full immortalization and maintenance of the malignant phenotype of cervical cancer, but they are NOT sufficient for full transformation and tumorigenesis. This study helps further understanding of other cellular alterations in HPV oncogenesis.

(2) lnc-FANCI-2 is upregulated in cervical lesion progression from CIN1, CIN2-3 to cervical cancer, cancer cell lines, and HPV transduced cell lines.

(3) Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression.

(4) Proteomic profiling of cytosolic and secreted proteins showed inhibition of MCAM, PODXL2, and ECM1 and increased levels of ADAM8 and TIMP2 in KO cells.

(5) RNA-seq analyses revealed that KO cells exhibited significantly increased RAS signaling but decreased IFN pathways.

(6) Increased phosphorylated Akt and Erk1/2, IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decreased RAC3 were observed in KO cells.

Weaknesses:

(1) The authors observed the increased Inc-FANCI-2 in HPV 16 and 18 transduced cells, and other cervical cancer tissues as well, HPV-18 positive HeLa cells exhibited different expressions of Inc-FANCI-2.

(2) Previous studies and data in the current showed a steadily increased Inc-FANCI-2 during cancer progression, however, the authors did not observe significant changes in cell behaviors (both morphology and proliferation) in KO Inc-FANCI-2.

(3) The authors observed the significant changes of RAS signaling (downstream) in KO cells, but they provided limited interpretations of how these results contributed to full transformation or tumorigenesis in HPV-positive cancer.

Reviewer #1 (Public review):

Summary:

The manuscript by Rühling et al analyzes the mode of entry of S. aureus into mammalian cells in culture. The authors propose a novel mechanism of rapid entry that involves the release of calcium from lysosomes via NAADP-stimulated activation of TPC1, which in turn causes lysosomal exocytosis; exocytic release of lysosomal acid sphingomyelinase (ASM) is then envisaged to convert exofacial sphingomyelin to ceramide. These events not only induce the rapid entry of the bacteria into the host cells but are also described to alter the fate of the intracellular S. aureus, facilitating escape from the endocytic vacuole to the cytosol.

Strengths:

The proposed mechanism is novel and could have important biological consequences.

Weaknesses:

Unfortunately, the evidence provided is unconvincing and insufficient to document the multiple, complex steps suggested. In fact, there appear to be numerous internal inconsistencies that detract from the validity of the conclusions, which were reached mostly based on the use of pharmacological agents of imperfect specificity.

Firstly, the release of calcium from lysosomes is not demonstrated. Localized changes in the immediate vicinity of lysosomes need to be measured to ascertain that these organelles are the source of cytosolic calcium changes. In fact, 9-phenantrol, which the authors find to be the most potent inhibitor of invasion and hence of the putative calcium changes, is not a blocker of lysosomal calcium release but instead blocks plasmalemmal TRPM4 channels. On the other hand, invasion is seemingly independent of external calcium. These findings are inconsistent with each other and point to non-specific effects of 9-phenantrol. The fact that ionomycin decreases invasion efficiency is taken as additional evidence of the importance of lysosomal calcium release. It is not clear how these observations support involvement of lysosomal calcium release and exocytosis; in fact treatment with the ionophore should itself have induced lysosomal exocytosis and stimulated, rather than inhibited invasion. Yet, manipulations that increase and others that decrease cytosolic calcium both inhibited invasion.

The proposed role of NAADP is based on the effects of "knocking out" TPC1 and on the pharmacological effects of Ned-19. It is noteworthy that TPC2, rather than TPC1, is generally believed to be the primary TPC isoform of lysosomes. Moreover, the gene ablation accomplished in the TPC1 "knockouts" is only partial and rather unsatisfactory. Definitive conclusions about the role of TPC1 can only be reached with proper, full knockouts. Even the pharmacological approach is unconvincing because the high doses of Ned-19 used should have blocked both TPC isoforms and presumably precluded invasion. Instead, invasion is reduced by only ≈50%. A much greater inhibition was reported using 9-phenantrol, the blocker of plasmalemmal calcium channels. How is the selective involvement of lysosomal TPC1 channels justified?

Invoking an elevation of NAADP as the mediator of calcium release requires measurements of the changes in NAADP concentration in response to the bacteria. This was not performed. Instead, the authors analyzed the possible contribution of putative NAADP-generating systems and reported that the most active of these, CD38, was without effect, while the elimination of SARM1, another potential source of NAADP, had a very modest (≈20%) inhibitory effect that may have been due to clonal variation, which was not ruled out. In view of these data, the conclusion that NAADP is involved in the invasion process seems unwarranted.

The involvement of lysosomal secretion is, again, predicated largely on the basis of pharmacological evidence. No direct evidence is provided for the insertion of lysosomal components into the plasma membrane, or for the release of lysosomal contents to the medium. Instead, inhibition of lysosomal exocytosis by vacuolin-1 is the sole source of evidence. However, vacuolin-1 is by no means a specific inhibitor of lysosomal secretion: it is now known to act primarily as a PIKfyve inhibitor and to cause massive distortion of the endocytic compartment, including gross swelling of endolysosomes. The modest (20-25%) inhibition observed when using synaptotagmin 7 knockout cells is similarly not convincing proof of the requirement for lysosomal secretion.

ASM is proposed to play a central role in the rapid invasion process. As above, most of the evidence offered in this regard is pharmacological and often inconsistent between inhibitors or among cell types. Some drugs affect some of the cells, but not others. It is difficult to reach general conclusions regarding the role of ASM. The argument is made even more complex by the authors' use of exogenous sphingomyelinase (beta-toxin). Pretreatment with the toxin decreased invasion efficiency, a seemingly paradoxical result. Incidentally, the effectiveness of the added toxin is never quantified/validated by directly measuring the generation of ceramide or the disappearance of SM.

The use of fluorescent analogs of sphingomyelin and ceramide is not well justified and it is unclear what conclusions can be derived from these observations. Despite the low resolution of the images provided, it appears as if the labeled lipids are largely in endomembrane compartments, where they would presumably be inaccessible to the secreted ASM. Moreover, considering the location of the BODIPY probe, the authors would be unable to distinguish intact sphingomyelin from its breakdown product, ceramide. What can be concluded from these experiments? Incidentally, the authors report only 10% of BODIPY-positive events after 10 min. What are the implications of this finding? That 90% of the invasion events are unrelated to sphingomyelin, ASM, and ceramide?

It is also unclear how the authors can distinguish lysenin entry into ruptured vacuoles from the entry of RFP-CWT, used as a criterion of bacterial escape. Surely the molecular weights of the probes are not sufficiently different to prevent the latter one from traversing the permeabilized membrane until such time that the bacteria escape from the vacuole.

Both SMase inhibitors (Figure 4C) and SMase pretreatment increased bacterial escape from the vacuole. The former should prevent SM hydrolysis and formation of ceramide, while the latter treatment should have the exact opposite effects, yet the end result is the same. What can one conclude regarding the need and role of the SMase products in the escape process?

Reviewer #2 (Public review):

Summary:

In this manuscript, Ruhling et al propose a rapid uptake pathway that is dependent on lysosomal exocytosis, lysosomal Ca2+ and acid sphingomyelinase, and further suggest that the intracellular trafficking and fate of the pathogen is dictated by the mode of entry.

The evidence provided is solid, methods used are appropriate and results largely support their conclusions, but can be substantiated further as detailed below. The weakness is a reliance on chemical inhibitors that can be non-specific to delineate critical steps.

Specific comments:

A large number of experiments rely on treatment with chemical inhibitors. While this approach is reasonable, many of the inhibitors employed such as amitriptyline and vacuolin1 have other or non-defined cellular targets and pleiotropic effects cannot be ruled out. Given the centrality of ASM for the manuscript, it will be important to replicate some key results with ASM KO cells.

Most experiments are done in HeLa cells. Given the pathway is projected as generic, it will be important to further characterize cell type specificity for the process. Some evidence for a similar mechanism in other cell types S. aureus infects, perhaps phagocytic cell type, might be good.

I'm a little confused about the role of ASM on the surface. Presumably, it converts SM to ceramide, as the final model suggests. Overexpression of b-toxin results in the near complete absence of SM on phagosomes (having representative images will help appreciate this), but why is phagosomal SM detected at high levels in untreated conditions? If bacteria are engulfed by SM-containing membrane compartments, what role does ASM play on the surface? If surface SM is necessary for phagosomal escape within the cell, do the authors imply that ASM is tuning the surface SM levels to a certain optimal range? Alternatively, can there be additional roles for ASM on the cell surface? Can surface SM levels be visualized (for example, in Figure 4 E, F)?

Related to that, why is ASM activity on the cell surface important? Its role in non-infectious or other contexts can be discussed.

If SM removal is so crucial for uptake, can exocytosis of lysosomes alone provide sufficient ASM for SM removal? How much or to what extent is lysosomal exocytosis enhanced by initial signaling events? Do the authors envisage the early events in their model happening in localized confines of the PM, this can be discussed.

How are inhibitor doses determined? How efficient is the removal of extracellular bacteria at 10 min? It will be good to substantiate the cfu experiments for infectivity with imaging-based methods. Are the roles of TPC1 and TPC2 redundant? If so, why does silencing TPC1 alone result in a decrease in infectivity? For these and other assays, it would be better to show raw values for infectivity. Please show alterations in lysosomal Ca2+ at the doses of inhibitors indicated. Is lysosomal Ca2+ released upon S. aureus binding to the cell surface? Will be good to directly visualize this.

The precise identification of cytosolic vs phagosomal bacteria is not very easy to appreciate. The methods section indicates how this distinction is made, but how do the authors deal with partial overlaps and ambiguities generally associated with such analyses? Please show respective images. The number of events (individual bacteria) for the live cell imaging data should be clearly mentioned.

In the phagosome maturation experiments, what is the proportion of bacteria in Rab5 or Rab7 compartments at each time point? Will the decreased Rab7 association be accompanied by increased Rab5? Showing raw values and images will help appreciate such differences. Given the expertise and tools available in live cell imaging, can the authors trace Rab5 and Rab7 positive compartment times for the same bacteria?

The results with longer-term infection are interesting. Live cell imaging suggests that ASM-inhibited cells show accelerated phagosomal escape that reduces by 6 hpi. Where are the bacteria at this time point ? Presumably, they should have reached lysosomes. The relationship between cytosolic escape, replication, and host cell death is interesting, but the evidence, as presented is correlative for the populations. Given the use of live cell imaging, can the authors show these events in the same cell?

Given the inherent heterogeneity in uptake processes and the use of inhibitors in most experiments, the distinction between ASM-dependent and independent pathways might not be as clear-cut as the authors suggest. Some caution here will be good. Can the authors estimate what fraction of intracellular bacteria are taken up ASM-dependent?

Reviewer #1 (Public review):

Summary:

Liang and Guan have studied the transport mechanism of Melbiose transporter MelB using the string method in collective variables and replica-exchange umbrella sampling simulations. The authors study the mechanism of substrate binding to the outward-facing state, conformational change of the transporter from outward-facing to inward-facing, and substrate unbinding from inward-facing state. In their analysis, they also highlight the effects of mutant D59C and the effect of sodium binding on the substrate transport process.

Strengths:

The authors employ a combination of string method and replica-exchange umbrella sampling simulation techniques to provide a complete map of the free energy landscape for sodium-coupled melibiose transport in MelB.

Weaknesses:

(1) Free energy barriers appear to be very high for a substrate transport process. In Figure 3, the transitions from IF (Inward facing) to OF (Outward facing) state appear to have a barrier of 12 kcal/mol. Other systems with mutant or sodium unbound have even higher barriers. This does not seem consistent with previous studies where transport mechanisms of transporters have been explored using molecular dynamics.

(2) Figure 2b: The PMF between images 20-30 shows the conformation change from OF to IF, where the occluded (OC) state is the highest barrier for transition. However, OC state is usually a stable conformation and should be in a local minimum. There should be free energy barriers between OF and OC and in between OC and IF.

(3) String method pathway is usually not the only transport pathway and alternate lower energy pathways should be explored. The free energy surface looks like it has not deviated from the string pathway. Longer simulations can help in the exploration of lower free energy pathways.

(4) The conformational change in transporters from OF to IF state is a complicated multi-step process. First, only 10 images in the string pathway are used to capture the transition from OF to IF state. I am not sure is this number is enough to capture the process. Second, the authors have used geodesic interpolation algorithm to generate the intermediate images. However, looking at Figure 3B, it looks like the transition pathway has not captured the occluded (OC) conformation, where the transport tunnel is closed at both the ends. Transporters typically follow a stepwise conformational change mechanism where OF state transitions to OC and then to IF state. It appears that the interpolation algorithm has created a hourglass-like state, where IF gates are opening and OF gates are closing simultaneously thereby creating a state where the transport tunnel is open on both sides of the membrane. These states are usually associated with high energy. References 30-42 cited in the manuscript reveal a distinct OC state for different transporters.

Reviewer #2 (Public review):

Summary:

The manuscript by Liang and Guan provides an impressive attempt to characterize the conformational free energy landscape of a melibiose permease (MelB), a symporter member of major facilitator superfamily (MFS) of transporters. Although similar studies have been conducted previously for other members of MFS, each member or subfamily has its own unique features that make the employment of such methods quite challenging. While the methodology is indeed impressive, characterizing the coupling between large-scale conformational changes and substrate binding in membrane transporters is quite challenging and requires a sophisticated methodology. The conclusions obtained from the three sets of path-optimization and free energy calculations done by the authors are generally supported by the provided data and certainly add to our understanding of how sodium binding facilitates the transport of melibiose in MelB. However, the data is not generated reliably which questions the relevance of the conclusions as well. I particularly have some concerns regarding the implementation of the methodology that I will discuss below.

(1) In enhanced sampling techniques, often much attention is given to the sampling algorithm. Although the sampling algorithm is quite important and this manuscript has chosen an excellent pair: string method with swarms of trajectories (SMwST) and replica-exchange umbrella sampling (REUS) for this task, there are other important factors that must be taken into account. More specifically, the collective variables used and the preparation of initial conformations for sampling. I have objectives for both of these (particularly the latter) that I detail below. Overall, I am not confident that the free energy profiles generated (summarized in Figure 5) are reliable, and unfortunately, much of the data presented in this manuscript heavily relies on these free energy profiles.

(2) The authors state that they have had an advantage over other similar studies in that they had two endpoints of the string to work from experimental data. I agree that this is an advantage. However, this could lead to some dangerous flaws in the methodology if not appropriately taken into account. Proteins such as membrane transporters have many slow degrees of freedom that can be fully captured within tens of nanoseconds (90 ns was the simulation time used here for the REUS). Biased sampling allows us to overcome this challenge to some extent, but it is virtually impossible to take into account all slow degrees of freedom in the enhanced sampling protocol (e.g., the collective variables used here do not represent anything related to sidechain dynamics). Therefore, if one mixes initial conformations that form different initial structures (e.g., an OF state and an IF state from two different PDB files), it is very likely that despite all equilibration and relaxation during SMwST and REUS simulations, the conformations that come from different sources never truly mix. This is dangerous in that it is quite difficult to detect such inconsistencies and from a theoretical point of view it makes the free energy calculations impossible. Methods such as WHAM and its various offshoots all rely on overlap between neighboring windows to calculate the free energy difference between two windows and the overlap should be in all dimensions and not just the ones that we use for biasing. This is related to well-known issues such as hidden barriers and metastability. If one uses two different structures to generate the initial conformations, then the authors need to show their sampling has been long enough to allow the two sets of conformations to mix and overlap in all dimensions, which is a difficult task to do.

(3) I also have concerns regarding the choice of collective variables. The authors have split the residues in each transmembrane helix into the cyto- and periplasmic sides. Then they have calculated the mass center distance between the cytoplasmic sides of certain pairs of helices and have also done the same for the periplasmic side. Given the shape of a helix, this does not seem to be an ideal choice since rather than the rotational motion of the helix, this captures more the translational motion of the helix. However, the transmembrane helices are more likely to undergo rotational motion than the translational one.

(4) Convergence: String method convergence data does not show strong evidence for convergence (Figure S2) in my opinion. REUS convergence is also not discussed. No information is provided on the exchange rate or overlap between the windows.

Reviewer #3 (Public review):

The paper from Liang and Guan details the calculation of the potential mean force for the transition between two key states of the melibiose (Mel) transporter MelB. The authors used the string method along with replica-exchange umbrella sampling to model the transition between the outward and inward-facing Mel-free states, including the binding and subsequent release of Mel. They find a barrier of ~6.8 kcal/mol and an overall free-energy difference of ~6.4 kcal/mol. They also investigate the same process without the co-transported Na+, finding a higher barrier, while in the D59C mutant, the barrier is nearly eliminated.

I found this to be an interesting and technically competent paper. I was disappointed actually to see that the authors didn't try to complete the cycle. I realize this is beyond the scope of the study as presented.

The results are in qualitative agreement with expectations from experiments. Could the authors try to make this comparison more quantitative? For example, by determining the diffusivity along the path, the authors could estimate transition rates.

Relatedly, could the authors comment on how typical concentration gradients of Mel and Na+ would affect these numbers?

Reviewer #1 (Public review):

In their manuscript "PDGFRRa signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking" Forman and colleagues use iMEPM cells to characterize the effects of PDGF signaling on alternative splicing. They first perform RNA-seq using a one-hour stimulation with Pdgf-AA in control and Srsf3 knockdown cells. While Srsf3 manipulation results in a sizeable number of DE genes, PDGF does not. They then turn to examine alternative splicing, due to findings from this lab. They find that both PDGF and Srsf3 contribute much more to splicing than transcription. They find that the vast majority of PDGF-mediated alternative splicing depends upon Srsf3 activity and that skipped exons are the most common events with PDGF stimulation typically promoting exon skipping in the presence of Srsf3. They used eCLIP to identify RNA regions bound to Srsf3. Under both PDGF conditions, the majority of peaks were in exons with +PDGF having a substantially greater number of these peaks. Interestingly, they find differential enrichment of sequence motifs and GC content in stimulated versus unstimulated cells. They examine 2 transcripts encoding PI3K pathway (enriched in their GO analysis) members: Becn1 and Wdr81. They then go on to examine PDGFRRa and Rab5, an endosomal marker, colocalization. They propose a model in which Srsf3 functions downstream of PDGFRRa signaling to, in part, regulate PDGFRa trafficking to the endosome. The findings are novel and shed light on the mechanisms of PDGF signaling and will be broadly of interest. This lab previously identified the importance of PDGF naling on alternative splicing. The combination of RNA-seq and eCLIP is an exceptional way to comprehensively analyze this effect. The results will be of great utility to those studying PDGF signaling or neural crest biology.

Comments on the revised version:

The authors have fully addressed my previous comments and I have no further concerns.

Reviewer #2 (Public review):

Summary:

This manuscript builds upon the work of a previous study published by the group (Dennison, 2021) to further elucidate the coregulatory axis of Srsf3 and PDGFRa on craniofacial development. The authors in this study investigated the molecular mechanisms by which PDGFRa signaling activates the RNA-binding protein Srsf3 to regulate alternative splicing (AS) and gene expression (GE) necessary for craniofacial development. PDGFRa signaling-mediated Srsf3 phosphorylation drives its translocation into the nucleus and affect binding affinity to different proteins and RNA, but the exact molecular mechanisms were not known. The authors performed RNA sequencing on immortalized mouse embryonic mesenchyme (MEPM) cells treated with shRNA targeting 3' UTR of Srsf3 or scramble shRNA (to probe AS and DE events that are Srsf3 dependent) and with and without PDGF-AA ligand treatment (to probe AS and DE events that are PDGFRa signaling dependent). They found that PDGFRa signaling has more effect on AS than on DE. A matching eCLIP-seq experiment was performed to investigate how Srsf3 binding sites change with and without PDGFRa signaling.

Strengths:

(1) The work builds well upon the previous data and the authors employ a variety of appropriate techniques to answer their research questions.

(2) The authors show that Srsf3 binding pattern within the transcript as well as binding motifs change significantly upon PDGFRa signaling, providing a mechanistic explanation for the significant changes in AS.

(3) By combining RNA-seq and eCLIP datasets together, the authors identified a list of genes that are directly bound by Srsf3 and undergo changes in GE and/or AS. Two examples are Becn1 and Wdr81, which are involved in early endosomal trafficking.

Weaknesses:

(1) The authors identify two genes whose AS are directly regulated by Srsf3 and involved in endosomal trafficking; however, they do not validate the differential AS results and whether changes in these genes can affect endosomal trafficking. In Figure 6, they show that PDGFRa signaling is involved in endosome size and Rab5 colocalization, but do not show how Srsf3 and the two genes are involved.

(2) The proposed model does not account for other proteins mediating the activation of Srsf3 after Akt phosphorylation. How do we know this is a direct effect (and not secondary or tertiary effect)?

This is a thoroughly revised manuscript. I would like to congratulate the authors to have invested a lot of time, resources, new data, and a more refined discussion to make this a compelling piece of work. I have no further concerns.

Reviewer #1 (Public review):

Summary:

The manuscript titled "Household clustering and seasonal genetic variation of Plasmodium falciparum at the community-level in The Gambia" presents a valuable genetic spatio-temporal analysis of malaria-infected individuals from four villages in The Gambia, covering the period between December 2014 and May 2017. The majority of samples were analyzed using a SNP barcode with the Spotmalaria panel, with a subset validated through WGS. Identity-by-descent (IBD) was calculated as a measure of genetic relatedness and spatio-temporal patterns of the proportion of highly related infections were investigated. Related clusters were detected at the household level, but only within a short time period.

Strengths:

This study offers a valuable dataset, particularly due to its longitudinal design and the inclusion of asymptomatic cases. The laboratory analysis using the Spotmalaria platform combined and supplemented with WGS is solid, and the authors show a linear correlation between the IBD values determined with both methods, although other studies have reported that at least 200 SNPs are required for IBD analysis. Data-analysis pipelines were created for (1) variant filtering for WGS and subsequent IBD analysis, and (2) creating a consensus barcode from the spot malaria panel and WGS data and subsequent SNP filtering and IBD analysis.

Weaknesses:

Further refining the data could enhance its impact on both the scientific community and malaria control efforts in The Gambia.

(1) The manuscript would benefit from improved clarity and better explanation of results to help readers follow more easily. Despite familiarity with genotyping, WGS, and IBD analysis, I found myself needing to reread sections. While the figures are generally clear and well-presented, the text could be more digestible. The aims and objectives need clearer articulation, especially regarding the rationale for using both SNP barcode and WGS (is it to validate the approach with the barcode, or is it to have less missing data?). In several analyses, the purpose is not immediately obvious and could be clarified.

(2) Some key results are only mentioned briefly in the text without corresponding figures or tables in the main manuscript, referring only to supplementary figures, which are usually meant for additional detail, but not main results. For example, data on drug resistance markers should be included in a table or figure in the main manuscript.

(3) The study uses samples from 2 different studies. While these are conducted in the same villages, their study design is not the same, which should be addressed in the interpretation and discussion of the results. Between Dec 2014 and Sept 2016, sampling was conducted only in 2 villages and at less frequent intervals than between Oct 2016 to May 2017. The authors should assess how this might have impacted their temporal analysis and conclusions drawn. In addition, it should be clarified why and for exactly in which analysis the samples from Dec 2016 - May 2017 were excluded as this is a large proportion of your samples.

(4) Based on which criteria were samples selected for WGS? Did the spatiotemporal spread of the WGS samples match the rest of the genotyped samples? I.e. were random samples selected from all times and places, or was it samples from specific times/places selected for WGS?

(5) The manuscript would benefit from additional detail in the methods section.

(6) Since the authors only do the genotype replacement and build consensus barcode for 199 samples, there is a bias between the samples with consensus barcode and those with only the genotyping barcode. How did this impact the analysis?

(7) The linear correlation between IBD-values of barcode vs genome is clear. However, since you do not use absolute values of IBD, but a classification of related (>=0.5 IBD) vs. unrelated (<0.5), it would be good to assess the agreement of this classification between the 2 barcodes. In Figure S6 there seem to be quite some samples that would be classified as unrelated by the consensus barcode, while they have IBD>0.5 in the Genome-IBD; in other words, the barcode seems to be underestimating relatedness.<br /> a. How sensitive is this correlation to the nr of SNPs in the barcode?

(8) With the sole focus on IBD, a measure of genetic relatedness, some of the conclusions from the results are speculative.<br /> a. Why not include other measures such as genetic diversity, which relates to allele frequency analysis at the population level (using, for example, nucleotide diversity)? IBD and the proportion of highly related pairs are not a measure of genetic diversity. Please revise the manuscript and figures accordingly.<br /> b. Additionally, define what you mean by "recombinatorial genetic diversity" and explain how it relates to IBD and individual-level relatedness.<br /> c. Recombination is one potential factor contributing to the loss of relatedness over time. There are several other factors that could contribute, such as mobility/gene flow, or study-specific limitations such as low numbers of samples in the low transmission season and many months apart from the high transmission samples.<br /> d. By including other measures such as linkage disequilibrium you could further support the statements related to recombination driving the loss of relatedness.

(9) While the authors conclude there is no seasonal pattern in the drug-resistant markers, one can observe a big fluctuation in the dhps haplotypes, which go down from 75% to 20% and then up and down again later. The authors should investigate this in more detail, as dhps is related to SP resistance, which could be important for seasonal malaria chemoprofylaxis, especially since the mutations in dhfr seem near-fixed in the population, indicating high levels of SP resistance at some of the time points.

(10) I recommend that raw data from genotyping and WGS should be deposited in a public repository.

Reviewer #2 (Public review):

Summary:

Malaria transmission in the Gambia is highly seasonal, whereby periods of intense transmission at the beginning of the rainy season are interspersed by long periods of low to no transmission. This raises several questions about how this transmission pattern impacts the spatiotemporal distribution of circulating parasite strains. Knowledge of these dynamics may allow the identification of key units for targeted control strategies, the evaluation of the effect of selection/drift on parasite phenotypes (e.g., the emergence or loss of drug resistance genotypes), and analyze, through the parasites' genetic nature, the duration of chronic infections persisting during the dry season. Using a combination of barcodes and whole genome analysis, the authors try to answer these questions by making clever use of the different recombination rates, as measured through the proportion of genomes with identity-by-descent (IBD), to investigate the spatiotemporal relatedness of parasite strains at different spatial (i.e., individual, household, village, and region) and temporal (i.e., high, low, and the corresponding the transitions) levels. The authors show that a large fraction of infections are polygenomic and stable over time, resulting in high recombinational diversity (Figure 2). Since the number of recombination events is expected to increase with time or with the number of mosquito bites, IBD allows them to investigate the connectivity between spatial levels and to measure the fraction of effective recombinational events over time. The authors demonstrate the epidemiological connectivity between villages by showing the presence of related genotypes, a higher probability of finding similar genotypes within the same household, and how parasite-relatedness gradually disappears over time (Figure 3). Moreover, they show that transmission intensity increases during the transition from dry to wet seasons (Figure 4). If there is no drug selection during the dry season and if resistance incurs a fitness cost it is possible that alleles associated with drug resistance may change in frequency. The authors looked at the frequencies of six drug-resistance haplotypes (aat1, crt, dhfr, dhps, kelch13, and mdr1), and found no evidence of changes in allele frequencies associated with seasonality. They also find chronic infections lasting from one month to one and a half years with no dependence on age or gender.

The use of genomic information and IBD analytic tools provides the Control Program with important metrics for malaria control policies, for example, identifying target populations for malaria control and evaluation of malaria control programs.

Strength:

The authors use a combination of high-quality barcodes (425 barcodes representing 101 bi-allelic SNPs) and 199 high-quality genome sequences to infer the fraction of the genome with shared Identity by Descent (IBD) (i.e. a metric of recombination rate) over several time points covering two years. The barcode and whole genome sequence combination allows full use of a large dataset, and to confidently infer the relatedness of parasite isolates at various spatiotemporal scales.

Reviewer #3 (Public review):

This study aimed to investigate the impact of seasonality on the malaria parasite population genetic. To achieve this, the researchers conducted a longitudinal study in a region characterized by seasonal malaria transmission. Over a 2.5-year period, blood samples were collected from 1,516 participants residing in four villages in the Upper River Region of The Gambia and tested the samples for malaria parasite positivity. The parasites from the positive samples were genotyped using a genetic barcode and/or whole genome sequencing, followed by a genetic relatedness analysis.

The study identified three key findings:

(1) The parasite population continuously recombines, with no single genotype dominating, in contrast to viral populations;

(2) The relatedness of parasites is influenced by both spatial and temporal distances; and

(3) The lowest genetic relatedness among parasites occurs during the transition from low to high transmission seasons. The authors suggest that this latter finding reflects the increased recombination associated with sexual reproduction in mosquitoes.

The results section is well-structured, and the figures are clear and self-explanatory. The methods are adequately described, providing a solid foundation for the findings. While there are no unexpected results, it is reassuring to see the anticipated outcomes supported by actual data. The conclusions are generally well-supported; however, the discussion on the burden of asymptomatic infections falls outside the scope of the data, as no specific analysis was conducted on this aspect and was not stated as part of the aims of the study. Nonetheless, the recommendation to target asymptomatic infections is logical and relevant.

Reviewer #1 (Public review):

Summary:

This manuscript describes a novel magnetic steering technique to target human adipose derived mesenchymal stem cells (hAMSC) or induce pluripotent stem cells to the TM (iPSC-TM). The authors show that delivery of the stem cells lowered IOP, increased outflow facility, and increased TM cellularity.

Strengths:

The technique is novel and shows promise as a novel therapeutic to lower IOP in glaucoma. hAMSC are able to lower IOP below the baseline as well as increase outflow facility above baseline with no tumorigenicity. These data will have a positive impact on the field and will guide further research using hAMSC in glaucoma models.

Weaknesses:

The transgenic mouse model of glaucoma the authors used did not show ocular hypertensive phenotypes at 6-7 months of age as previously reported. Therefore, if there is no pathology in these animals the authors did not show a restoration of function, but rather a decrease in pressure below normal IOP.

Reviewer #2 (Public review):

Summary:

This observational study investigates the efficacy of intracameral injected human stem cells as a means to re-functionalize the trabecular meshwork for the restoration of intraocular pressure homeostasis. Using a murine model of glaucoma, human adipose-derived mesenchymal stem cells are shown to be biologically safer and functionally superior at eliciting a sustained reduction in intraocular pressure (IOP). The authors conclude that the use of human adipose-derived mesenchymal stem cells has the potential for long-term treatment of ocular hypertension in glaucoma.

Strengths:

A noted strength is the use of a magnetic steering technique to direct injected stem cells to the iridocorneal angle. An additional strength is the comparison of efficacy between two distinct sources of stem cells: human adipose-derived mesenchymal vs. induced pluripotent cell derivatives. Utilizing both in vivo and ex vivo methodology coupled with histological evidence of introduced stem cell localization provides a consistent and compelling argument for a sustainable impact exogenous stem cells may have on the re-functionalization of a pathologically compromised TM.

Weaknesses:

A noted weakness of the study, as pointed out by the authors, includes the unanticipated failure of the genetic model to develop glaucoma-related pathology (elevated IOP, TM cell changes). While this is most unfortunate, it does temper the conclusion that exogenous human adipose derived mesenchymal stem cells may restore TM cell function. Given that TM cell function was not altered in their genetic model, it is difficult to say with any certainty that the introduced stem cells would be capable of restoring pathologically altered TM function. A restoration effect remains to be seen. Another noted complication to these findings is the observation that sham intracameral-injected saline control animals all showed elevated IOP and reduced outflow facility, compared to WT or Tg untreated animals, which allowed for more robust statistically significant outcomes. Additional comments/concerns that the authors may wish to address are elaborated in the Private Review section.

Reviewer #3 (Public review):

Summary:

The purpose of the current manuscript was to investigate a magnetic cell steering technique for efficiency and tissue-specific targeting, using two types of stem cells, in a mouse model of glaucoma. As the authors point out, trabecular meshwork (TM) cell therapy is an active area of research for treating elevated intraocular pressure as observed in glaucoma. Thus, further studies determining the ideal cell choice for TM cell therapy is warranted. The experimental protocol of the manuscript involved the injection of either human adipose derived mesenchymal stem cells (hAMSCs) or induced pluripotent cell derivatives (iPSC-TM cells) into a previously reported mouse glaucoma model, the transgenic MYOCY437H mice and wild-type littermates followed by the magnetic cell steering. Numerous outcome measures were assessed and quantified including IOP, outflow facility, TM cellularity, retention of stem cells, and the inner wall BM of Schlemm's canal.

Strengths:

All of these analyses were carefully carried out and appropriate statistical methods were employed. The study has clearly shown that the hAMSCs are the cells of choice over the iPSC-TM cells, the latter of which caused tumors in the anterior chamber. The hAMSCs were shown to be retained in the anterior segment over time and this resulted in increased cellular density in the TM region and a reduction in IOP and outflow facility. These are all interesting findings and there is substantial data to support it.

Weaknesses:

However, where the study falls short is in the MYOCY437H mouse model of glaucoma that was employed. The authors clearly state that a major limitation of the study is that this model, in their hands, did not exhibit glaucomatous features as previously reported, such as a significant increase in IOP, which was part of the overall purpose of the study. The authors state that it is possible that "the transgene was silenced in the original breeders". The authors did not show PCR, western blot, or immuno of angle tissue of the tg to determine transgenic expression (increased expression of MYOC was shown in the angle tissue of the transgenics in the original paper by Zode et al, 2011). This should be investigated given that these mice were rederived. Thus, it is clearly possible that these are not transgenic mice. If indeed they are transgenics, the authors may want to consider the fact that in the Zode paper, the most significant IOP elevation in the mutant mice was observed at night and thus this could be examined by the authors. Other glaucomatous features of these mice could also have been investigated such as loss of RGCs, to further determine their transgenic phenotype. Finally, while increased cellular density in the TM region was observed, proliferative markers could be employed to determine if the transplanted cells are proliferating.

Reviewer #1 (Public review):

Summary:

The manuscript "Interplay of condensate material properties and chromatin heterogeneity governs nuclear condensate ripening" presents experiments and theory to explain the dynamic behavior of nuclear condensates. The authors present experimental data that shows the size of multiple artificially induced condensates as a function of time for various conditions. They identify different dynamic regimes, which all differ from traditional Ostwald ripening. By careful analysis and comparison with a quantitative model, the authors conclude that the elastic effects of the chromatin are relevant and the interplay between (heterogeneous) elasticity and surface tension governs the droplets' behavior. However, since they apply a simple model to a complex system, I think that the work is sometimes prone to over-interpretation, which I detail below. In summary, since droplet growth in a heterogeneous, elastic environment is unavoidable for condensates, this work achieves an important step toward understanding this complex setting. The work will likely stimulate more experiments (using different methods or alternative settings) as well as theory (accounting for additional effects, like spatial correlations).

Strengths:

A particularly strong point of the work is the tight integration between experiment and theory. Both parts are explained well at an appropriate level with more details in the methods section and the supplementary information. I cannot comment much on the experiments, but they seem convincing to me and the authors quantify the relevant parameters. Concerning the theory, they derive a model at the appropriate level of description. The analysis of the model is performed and explained well. Even though spatial correlations are not taken into account, the model will serve as a useful basis for developing more complicated models in the future. It is also worth mentioning that the clear classification into different growth regimes is helpful since such results, with qualitative predictions for parameter dependencies, likely also hold in more complex scenarios.

Weaknesses:

I think that the manuscript would profit from more precise definitions and explanations in multiple points, as detailed below. Clearly, not all these points can be fully incorporated in a model at this point, but I think it would be helpful to mention weaknesses in the manuscript and to discuss the results a bit more carefully.

(1) The viscosity analysis likely over-interprets the data. First, the FRAP curves do not show clear exponential behavior. For Figure 1C, there are at least two time scales and it is not clear to me why the shorter time scale right after bleaching is not analyzed. If the measured time scale were based on the early recovery, the differences between the two cases would likely be very small. For Figure 1D, the recovery is marginal, so it is not clear how reliable the measurements are. More generally, the analysis was performed on condensates of very different sizes, which can surely affect the measurements; see https://doi.org/10.7554/eLife.68620 for many details on using FRAP to analyze condensate dynamics. Second, the relaxation dynamics are likely not purely diffusive in a viscous environment since many condensates show elastic properties (https://doi.org/10.1126/science.aaw4951). I could very well imagine that the measured recovery time is related to the viscoelastic time scale. Third, the assumption of the Stokes-Einstein-Sutherland equation to relate diffusivity and viscosity is questionable because of viscoelasticity and the fact that the material is clearly interacting, so free diffusion is probably not expected.

(2) A large part of the paper is spent on the difference between different dynamic regimes, which are called "fusion", "ripening", and "diffusion-based" (with slightly different wording in different parts). First, I would welcome consistent language, e.g., using either fusion or coalescence. Second, I would welcome an early, unambiguous definition of the regimes. A definition is given at the end of page 2, but this definition is not clear to me: Does the definition pertain to entire experiments (e.g., is something called "fusion" if any condensates fuse at any time in the experiment?), or are these labels used for different parts of the experiment (e.g., would the data in Figure 1H first be classified as "ripening" and then "diffusion-based")? More generally, the categorization seems to depend on the observed system size (or condensate count) and time scale. Third, I find the definition of the ripening time a bit strange since it is clearly correlated with droplet size. Is this dependency carefully analyzed in the subsequent parts?

(3) The effect of the elastic properties of the chromatin is described by a Neo-Hookean model, but the strains R/\xi used in the theory are of the order of 100, which is huge. At such high strains, the Neo-Hookean model essentially has a constant pressure 5E/6, so the mesh size \xi does not matter. It is not clear to me whether chromatin actually exhibits such behavior, and I find it curious that the authors varied the stiffness E but not the mesh size \xi when explaining the experiments in the last section although likely both parameters are affected by the experimental perturbations. In any case, https://doi.org/10.1073/pnas.2102014118 shows that non-linear elastic effects related to breakage and cavitation could set in, which might also be relevant to the problem described here. In particular, the nucleation barrier discussed in the later part of the present manuscript might actually be a cavitation barrier due to elastic confinement. In any case, I would welcome a more thorough discussion of these aspects (in particular the large strains).

(4) The description of nucleation on page 7 is sloppy and might be misleading. First, at first reading I understood the text as if droplets of any radius could nucleate with probability p_nuc related to Eq. 7. This must be wrong since large droplets have ΔG<0 implying p_nuc > 1. Most likely, the nucleation rate only pertains to the critical radius (which is what might be meant by R_0, but it is unclear from the description). In this case, the critical radius and its dependence on parameters should probably be discussed. It might also help to give the value of the supersaturation S in terms of the involved concentrations, and it should be clarified whether P_E depends on R_0 or not (this might also relate to the cavitation barrier raised in point 3 above). Secondly, it is a bit problematic that E is sampled from a normal distribution, which allows for negative stiffnesses! More importantly, the exact sampling protocol is important since sampling more frequently (in the simulations) leads to a larger chance of hitting a soft surrounding, which facilitates nucleation. I could not find any details on the sampling in the numerical simulations, but I am convinced that it is a crucial aspect. I did find a graphical representation of the situation in Figure S4A, but I think it is misleading since there is no explicit space in the model and stiffnesses are not correlated.

Reviewer #2 (Public review):

Summary:

The authors used a chemical linker to induce phase separation in U2OS cell nuclei with two different proteins, a coiled-coil protein (Mad1) and a disordered domain (from LAF-1), whose condensates were purported to have different material properties. First, they performed Fluorescence Recovery After Photobleaching (FRAP) and estimated the viscosity via the Stokes-Einstein equation. Combined with droplet fusion assays, this yielded an estimate of the surface tension, wherein the disordered condensates were found to have 130 times higher surface tension than the coiled-coil condensates. Confocal fluorescence microscopy was used to follow condensates over time, enabling classification of growth events as either fusion-, ripening-, or diffusion-based, and subsequent comparison of the relative abundances of these growth events between the two condensate types. Coiled-coil condensates grew primarily by diffusive processes, whereas disordered condensates grew primarily by ripening processes. The coarsening rates were described by growth exponents extracted from power-law fits of average normalized condensate radius over time. In both cases, these growth exponents were smaller than those predicted by theory, leading the authors to propose that nuclear condensate growth is generally suppressed by chromatin mechanics, as found in previous studies albeit with different exponents. The authors developed a theory to understand how the extent of this effect may depend on condensate material properties like surface tension. Treating chromatin as a neo-Hookean elastic solid, the authors assume a form of mechanical pressure that plateaus with increasing condensate size, and the resulting theory is used to analyze the observed condensate growth dynamics. A linearized extension of the theory is used to distinguish between suppressed, elastic, and Ostwald ripening. Finally, the authors consider the impact of different chromatin environments on condensate growth patterns and dynamics, which is achieved experimentally with another cell type (HeLa) and with a drug that decondenses chromatin (TSA). They find that condensate growth patterns are not significantly changed in either condensate type, but that the number of condensates nucleated and their related growth exponent are more sensitive to variations in chromatin stiffness in the coiled-coil system due to its low surface tension.

Strengths:

This work provides evidence that nuclear condensates can coarsen not only by fusion but also by continuous diffusive growth processes, predominant in coiled-coil condensates, and ripening, predominant in disordered condensates. Across these different condensate types and coarsening mechanisms, the authors find growth exponents lower than theoretical expectations, reinforcing the notion that elastic media can suppress condensate growth in the nucleus. Combined with theory, these observed differences in growth patterns and rates are argued to originate from differences in material properties, namely, surface tension relative to local chromatin stiffness. The authors further suggest that the few ripening events that are seen in coiled-coil condensates may be elastic in nature due to gradients in chromatin stiffness as opposed to Ostwald ripening. If this assertion proves to be robust, it would mark an early observation of elastic ripening in living cells.

Weaknesses:

(1) The assertion that nuclear condensates experience an external pressure from the chromatin network implies that chromatin should be excluded from the condensates (Nott et al., Molecular Cell (2015); Shin et al., Cell (2018)). This has not been shown or discussed here. While Movie 1 suggests the coiled-coil condensates may exclude chromatin, Movie 2 suggests the disordered condensates do not. LAF-1, as an RNA helicase, interacts with RNA, and RNA can be associated with chromatin in the nucleus. RNA can also modulate droplet viscosity. The authors' analysis of the disordered condensate data only makes sense if these condensates exclude chromatin, which they have not demonstrated, and which appears not to be the case.

(2) Critical physical parameters like viscosity and surface tension have not been directly measured but rather are estimated indirectly using FRAP and the Stokes-Einstein equation. While not uncommon in the field, this approach is flawed as droplet viscosity is not simply determined by the size of the composing particles. Rather, in polymeric systems, viscosity strongly depends on the local protein concentration and intermolecular interactions (Rubinstein & Semenov Macromolecules (2001)). This unjustified approach propagates to the surface tension estimate since only the ratio of viscosity to surface tension is explicitly measured. Since the paper's conclusions strongly hinge on the magnitude of the surface tension, a more accurate estimate or direct measurement of this salient material property is called for.

(3) The phase diagram of growth modes very much depends on the assumption of neo-Hookean elasticity of the chromatin network. This assumption is poorly justified and calls into question the general conclusions about possible growth phases. The authors need to either provide evidence for neo-Hookean elasticity, or, alternatively, consider a model in which strain stiffening or thinning continues as droplets grow, which would likely lead to very different conclusions, and acknowledge this uncertainty.

(4) There is limited data for the elastic ripening claim. In Figure 3E, only one data point resides in the elastic ripening (δ < 0) range, with a few data points very close to zero.

(5) The authors claim that "our work shows that the elastic chromatin network can stabilize condensates against Ostwald ripening but only when condensate surface tension is low." This claim also depends on the details of the chosen neo-Hookean model of chromatic elasticity, and it is not studied here whether these results are robust to other models.

(6) It is also not clear how the total number of Mad1 proteins and LAF-1 disordered regions change while the condensates evolve with time. As the experiments span longer than 6 hours, continued protein production could lead to altered condensate coarsening dynamics. For example, continued production of Mad1 can lead to the growth of all Mad1 condensates, mimicking the diffusive growth process.

Reviewer #1 (Public review):

Summary:

Enterobacteriaceae produce microcins to target their competitors. Using informatics approaches, the authors identified 12 new microcins. They expressed them in E. coli, demonstrating that the microcins have antimicrobial activity against other microbes, including plant pathogens and the ESKAPE pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.

Strengths:

Overall, this study has the merit of identifying new potential antimicrobial molecules that could be used to target important pathogens. The bioinformatics analysis, the expression system used, and the antimicrobial assays performed are solid, and the data presented are convincing. This work will set the basis for new studies to investigate the potential role of these microcins in vivo.

Weaknesses:

The work has been performed in vitro, which is a valid approach for identifying the antimicrobial peptides and assessing their antimicrobial activity. Future studies will need to address whether these new microcins exhibit antimicrobial activity in vivo (e.g., in the context of infection models), and to identify the targets (receptor and mechanisms of action) for the new microcins.

Reviewer #2 (Public review):

Mortzfeld et al. describe their study of class IIb microcins. Furthering our awareness of the presence and action of microcins is an important line of research. However, several issues related to the premise, sequence analysis, and validation require attention to support the claims.

(1) Previous studies have been published on the broader distribution of microcins across bacteria. The software has been published for their identification. Comparison to this software and/or discussion of previous work should be included to place this work in the context of the field.

(2) It is not clear how immunity proteins were identified and there does not appear to be functional confirmation to show these predicted immunity proteins are real. Thus, it is premature to state that immunity genes have been found. This may also confound some of the validation studies below if proper immunity proteins have not been included.

(3) Please show the nt alignment used to generate the tree. Without seeing it, one would guess that the sequences are either quite similar (making the results from this study less novel) or there would be concerns that the phylogenetic relationship derived from the nt alignment is spurious.

(4) Figure 1 B-C: There are numerous branches that do not have phylogenetic support (values <50%). These are not statistically valid phylogenetic relationships and should be collapsed. The resulting tree should be used in the description of clades.

(5) The discovered microcins are not being directly tested since they are expressed heterologous and reliant on non-native modification systems. The results present the statement that novel microcins have been validated. This should be described accordingly.

(6) The key finding of this paper is the claim that 12 novel class IIb microcins have been validated. To substantiate this claim, original images showing evidence of antibacterial activity must be made available rather than a presence/absence chart. The negative controls for this table are unclear and should be included with the original images.

(7) Further data for the purified microcin is needed. The purification method described is standard practice and should allow for product quantification, which should be included. Standard practice includes an SDS page showing the purity of the microcin, or at least the TEV digest to show microcin has been produced, and importantly a control sample (scrambled sequence, empty vector purification, etc) to show that observed activity (Figure 2B) is not from a purification carry over. This data should be included to support that microcin has been purified and is active.

Reviewer #3 (Public review):

Summary:

In this study, several novel class IIb microcin biosynthetic gene clusters have been discovered by specific homology searches and manual curation. Using a specific E. coli expression system, the microcins were expressed and conjugated to monoglycosylated enterobactin as siderophore moiety. While this synthetic biology approach cannot account for other siderophores being coupled to the microcin core peptide in the original producing strains, it nonetheless allows for a general screening for the activity of the heterologously produced compounds. Through this approach, the activity of several predicted microcins has been confirmed and three novel class IIb microcin clades were identified.

Strengths:

The experimental design is sound, the results are corroborated by suitable controls, and the findings have a high level of novelty and significance. Furthermore, the comments of the initial round of peer review have been answered satisfactorily by the authors.

Reviewer #1 (Public review):

Summary:

This study investigates the role of CD131, a receptor subunit for GM-CSF and IL-3, in ulcerative colitis pathogenesis using a DSS-induced murine colitis model. By comparing wild-type and CD131-deficient mice, the authors demonstrate that CD131 contributes to DSS-induced colitis, working in concert with tissue-infiltrating macrophages.

Strengths:

The research shows that CD131's influence on macrophage and T cell chemotaxis is mediated by CCL4. The authors conclude by proposing a pro-inflammatory role for CD131 in murine colitis and suggest potential clinical relevance in human inflammatory bowel disease.

Weaknesses:

The statistical association between increased CD131 expression and clinical IBD was not observed in Table 1, indicating that the main results from animal experiments were not reproduced in human subjects. Additionally, due to the absence of experimental results regarding the downstream signaling pathways through CD131, it is difficult to infer the precise differentiated outcomes of this study. Furthermore, the effects of CD131 on immune cells other than macrophages were not presented, and the results specific to macrophage-selective CD131 were not shown. Therefore, I conclude that it is challenging to provide a detailed review as there is a lack of supporting evidence for the core arguments made in this paper.

Reviewer #2 (Public review):

Summary:

This study investigates the potential role of CD131, a cytokine receptor subunit shared by GM-CSF and IL-3, in intestinal inflammation. Using heterozygous mice with an inactivating mutation on this gene, the study demonstrates ameliorated inflammation, associated with less infiltration of macrophages. Moreover, the depletion of macrophages prevented many of the inflammatory effects of DSS and made both WT and mutant mice equivalent in terms of inflammation severity. Correlative data showing increased CD131+ cells in tissues of patients with ulcerative colitis is also demonstrating, evidence for plausibility for these pathways in human disease.

Strengths:

The phenotype of mutant mice seems quite robust and the pathways proposed, GM-CSF signaling in macrophages with CCL4 as a downstream pathway, are all plausible and concordant with existing models. Many of the experiments included meaningful endpoints and were overall well performed.

Weaknesses:

(1) Experimental rigor was lacking in this manuscript, which provided limited or no details on the number of independent iterations that each experiment was done, the number of animals per group, the number of technical or biological replicates in each graph, etc.

(2) Details of animal model validation showing that this particular mutant allele results in a lack of CD131 protein expression were not shown. Moreover, since the paper uses heterozygous mice, it is critical to show that at the protein level, there is indeed reduced expression of CD131 in het mice compared to controls (many heterozygous states do not lead to appreciable protein depletion).

(3) Another major weakness is that the paper asserts a causal relationship between CD131 signaling and CCL4 production: the data shown indicates that the phenotypes of CCL4 deficiency (through Ab blockade) and CD131 partial deficiency (in het mice) are similar. However, this does not establish that CD131 signaling acts through CCL4.

(4) Lastly, while the paper claims that CD131 acts through macrophage recruitment, the evidence is circumstantial and not direct. DSS-induced acute colitis is largely mediated by macrophages, so any manipulation associated with less severe inflammation is accompanied by lesser macrophage infiltration in this model: this does not directly establish that CD131 acts directly on macrophages, which would require cell-specific knockout or complex cell reconstitution experiments.

Reviewer #1 (Public review):

Summary:

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

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

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

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

Strengths:

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

Weaknesses:

Suggestions for refinement:

The initial experiment, inducing global hypo-methylation by eliminating DNMT1 in HAP1 cells, is intriguing and warrants more detailed description. How many genes experience mis-regulation or aberrant expression? What phenotypic changes occur in these cells? Why did the authors focus on L1TD1? Providing some of this data would be helpful to understand the rationale behind the thorough analysis of L1TD1.

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

Reviewer #2 (Public review):

In this study, Kavaklıoğlu et al. investigated and presented evidence for a role for domesticated transposon protein L1TD1 in enabling its ancestral relative, L1 ORF1p, to retrotranspose in HAP1 human tumor cells. The authors provided insight into the molecular function of L1TD1 and shed some clarifying light on previous studies that showed somewhat contradictory outcomes surrounding L1TD1 expression. Here, L1TD1 expression was correlated with L1 activation in a hypomethylation dependent manner, due to DNMT1 deletion in HAP1 cell line. The authors then identified L1TD1 associated RNAs using RIP-Seq, which display a disconnect between transcript and protein abundance (via Tandem Mass Tag multiplex mass spectrometry analysis). The one exception was for L1TD1 itself, is consistent with a model in which the RNA transcripts associated with L1TD1 are not directly regulated at the translation level. Instead, the authors found L1TD1 protein associated with L1-RNPs and this interaction is associated with increased L1 retrotransposition, at least in the contexts of HAP1 cells. Overall, these results support a model in which L1TD1 is restrained by DNA methylation, but in the absence of this repressive mark, L1TD1 is expression, and collaborates with L1 ORF1p (either directly or through interaction with L1 RNA, which remains unclear based on current results), leads to enhances L1 retrotransposition. These results establish feasibility of this relationship existing in vivo in either development or disease, or both.

Comments on revised version:

In general, the authors did an acceptable job addressing the major concerns throughout the manuscript. This revision is much clearer and has improved in terms of logical progression.

Reviewer #1 (Public review):

The authors used a novel multi-dimensional experience sampling (mDES) approach to identify data-driven patterns of experience samples that they use to interrogate fMRI data collected during naturalistic movie-watching data. They identify a set of multi-sensory features of a set of movies that delineate low-dimensional gradients of BOLD fMRI signal patterns that have previously been linked to fundamental axes of cortical organization.

Reviewer #2 (Public review):

The present study explores how thoughts map onto brain activity, a notoriously challenging question because of the dynamic, subjective, and abstract nature of thoughts. To tackle this question, the authors collected continuous thought ratings from participants watching a movie, and additionally made use of an open-source fMRI dataset recorded during movie watching as well as five established gradients of brain variation as identified in resting state data. Using a voxel-space approach, the results show that episodic knowledge, verbal detail, and sensory engagement of thoughts commonly modulate visual and auditory cortex, while intrusive distraction modulates the frontoparietal network. Additionally, sensory engagement mapped onto a gradient from primary to association cortex, while episodic knowledge mapped onto a gradient from the dorsal attention network to visual cortex. Building on the association between behavioral performance and neural activation, the authors conclude that sensory coupling to external input and frontoparietal executive control are key to comprehension in naturalistic settings.

The manuscript stands out for its methodological advancements in quantifying thoughts over time and its aim to study the implementation of thoughts in the brain during naturalistic movie watching.

Strengths:

(1) The study raises a question that has been difficult to study in naturalistic settings so far but is key to understanding human cognition, namely how thoughts map onto brain activation.

(2) The thought ratings introduce a novel method for continuously tracking thoughts, promising utility beyond this study.

(3) The authors used diverse data types, metrics, and analyses to substantiate the effects of thinking from multiple perspectives.

Reviewer #3 (Public review):

This study attempted to investigate the relations between processing in the human brain during movie watching and corresponding thought processes. This is a highly interesting question, as movie watching presents a semi-constrained task, combining naturally occurring thoughts and common processing of sensory inputs across participants. This task is inherently difficult because in order to know what participants are thinking at any given moment, one has to interrupt the same thought process which is the object of study.

This study attempts to deal with this issue by aggregating staggered experience sampling data across participants in one behavioral study and using the population level thought patterns to model brain activity in different participants in an open access fMRI dataset.

The behavioral data consist of 120 participants who watched 3 11-minute movie clips. Participants responded to the mDES questionnaire: 16 visual scales characterizing ongoing thought 5 times, two minutes apart, in each clip. The 16 items are first reduced to 4 factors using PCA, and their levels are compared across the different movies. The factors are "episodic knowledge", "intrusive distraction", "verbal detail", and "sensory engagement". The factors differ between the clips, and distraction is negatively correlated with movie comprehension and sensory engagement is positively correlated with comprehension.

The components are aggregated across participants (transforming single subject mDES answers into PCA space and concatenating responses of different participants) and are used as regressors in a GLM analysis. This analysis identifies brain regions corresponding to the components. The resulting brain maps reveal activations that are consistent with the proposed mental processes (e.g. negative loading for intrusion in frontoparietal network, positive loadings for visual and auditory cortices for sensory engagement).

Then, the coordinates for brain regions which were significant for more than one component are entered into a paper search in neurosynth. It is not clear what this analysis demonstrates beyond the fact that sensory engagement contained both visual and auditory components.

The next analysis projected group-averaged brain activation onto gradients (based on previous work) and used gradient timecourses to predict the behavioral report timecourses. This revealed that high activations in gradient 1 (sensory→association) predicted high sensory engagement, and that "episodic knowledge" thought patterns were predicted by increased visual cortex activations. Then, permutation tests were performed to see whether these thought pattern related activations corresponded to well defined regions on a given cluster.

In conclusion, this study tackles a highly interesting subject and does it creatively and expertly.

Reviewer #1 (Public review):

Summary:

This paper represents a huge amount of work on a condition whose patients' health and well-being have not always been prioritized, and only relatively recently has the immune dysregulation seen in patients with Down Syndrome (DS) been garnering major research interest.

This paper provides an unparalleled examination of immune disorder in patients with DS. The authors also report the results from a clinical trial with the JAK inhibitor tofacitinib in DS patients.

Strengths:

This manuscript report an herculean effort and provides an unparalleled examination of immune disorder in a large number of patients with DS.

Weaknesses:

Not a major weakness but, apart from finding an elevation of CD4 T central memory cells and more differentiated plasmablast, several of the alteration reported in this manuscript had already been suggested by a few case reports and very small series. On the other hand, the number of patients (and controls) utilized for this study is remarkable and allows to draw much firmer conclusions.

Comments on revised version:

I don't have any further comments.

Reviewer #2 (Public review):

In this manuscript, Rachubinski and colleagues provide a comprehensive clinical, immunological, and autoantibody assessment of autoimmune/inflammatory manifestations of patients with Down syndrome (DS) in a large number of patients with this disorder. These analyses confirm prior results of excess interferon and cytokine signals in DS patients and extend these observations to highlight early-onset immunological aberrancies, far before symptoms occur, as well as characterizing novel autoantibody reactivities in this patient population. Then, the authors report the interim analysis of an open label, Phase II, clinical trial of the JAK1/3 inhibitor, tofacitinib, that aims to define the safety, clinical efficacy, and immunological outcomes of DS patients who suffer from inflammatory conditions of the skin. The clinical trial analysis indicates that the treatment is tolerated without serious adverse effects and that the majority of patients have experienced clinical improvement or remission in their corresponding clinical cutaneous manifestations as well as improvement or normalization of aberrant immunological signals such as cytokines.

The major strength of the study is the recruitment and uniform, systematic evaluation of an impressive number of DS patients. Moreover, the promising early results from the tofacitinib clinical trial pave the way for analysis of a larger number of patients within the Phase II trial and otherwise, which may lead to improved clinical outcomes of affected patients. An inherent weakness of such studies is the descriptive nature of several parameters and the relatively small size of tofacitinib-treated DS patients. However, the descriptive nature of some of the correlative research analyses are of scientific interest and are useful to generate hypotheses for future additional (including mechanistic) work and treatment of 10 DS patients in a formal clinical trial at interim analysis is not a trivial task for a disease like this. The manuscript achieves the aims of the authors and the results support their conclusions. The authors appropriately acknowledge areas that require more research and areas that are not well understood. The results are represented in a useful manner and statistical methods and analyses appear sound.

Comments on revised version:

The authors have satisfactorily addressed my comments in the revised manuscript.

Reviewer #3 (Public review):

Summary:

Individuals with Down syndrome (DS) have high rates of autoimmunity and can have exaggerated immune responses to infection that can unfortunately cause significant medical complications. Prior studies from these authors and others have convincingly demonstrated that individuals with DS have immune dysregulation including increased Type I IFN activity, elevated production of inflammatory cytokines (hypercytokinemia), increased autoantibodies, and populations of dysregulated adaptive immune cells that pre-dispose to autoimmunity. Prior studies have demonstrated that using JAK inhibitors to treat patient samples in vitro, in small case series of patients, and in mouse models of DS leads to improvement of immune phenotype and/or clinical disease. This manuscript provides two major advances in our understanding of the immune dysregulation and therapy for patients. First, they perform deep immune phenotyping on several hundred individuals with DS and demonstrate that immune dysregulation is present from infancy. Second, they report promising interim analysis of a Phase II clinical trial of a JAK inhibitor in 10 people with DS and moderate to severe skin autoimmunity.

Strengths and weaknesses:

The relatively large cohort and careful clinical annotation here provides new insights into the immune phenotype of patients with DS. For example, it is interesting that regardless of autoimmune disease or autoantibody status, individuals with DS have elevated cytokines and CRP. Analysis of the cohorts by age demonstrated that some cytokines are significant elevated in people with DS starting in infancy (e.g., IL-9 and IL-17C). Nearly all adults with DS in this study had autoantibodies (98%) and most had six or more autoantibodies (63%), which differed significantly from euploid study participants. This implies that all patients with DS might benefit from early intervention with therapy to reduce inflammation. However, it is also worth considering that an alternative interpretation that since hypercytokinemia does not vary based on disease state in individuals with DS, that this may not be a key factor driving autoimmunity (although it may be relevant for other clinical symptoms such as neuroinflammation).

Small case series have suggested the benefit of JAK inhibitors to treat autoimmunity in DS. This is the first report of a prospective clinical trial to test a JAK inhibitor in this setting. The clinical trial entry criteria included moderate to severe autoimmune skin disease in patients aged 12-50 years with DS, and treatment was with the JAK1/3 inhibitor tofacitinib. This clinical trial is a critically important step for the field. The early results support that treatment is well tolerated with improvement of interferon scores in patients and reduction of autoantibodies. Most patients experienced clinical improvement, with alopecia areata having the greatest response. Treatment may not affect all skin disease equally, for example of the 5 patients with hidradenitis suppurativa, only 1 showed clinical improvement based on skin score. While very promising, the clinical trial results reported here are preliminary and based on interim analysis of 10 patients at 16 weeks. Individuals with DS have a lifelong risk of immune dysregulation and thus it is unclear how long therapy, if of benefit, would need to be continued. Results of longer-term therapy will be informative when considering the risks/benefits of this therapy.

Comments on revised version:

The authors have made appropriate revisions to this important contribution to the literature.

Reviewer #1 (Public review):

This study by Popli et al. evaluated the function of Atg14, an autophagy protein, in reproductive function using a conditional knockout mouse model. The authors showed that female mice lacking Atg14 were infertile partly due to defective embryo transport function of the oviduct and faulty uterine receptivity and decidualization using PgrCre/+;Atg14f/f mice. The findings from this work are exciting and novel. The authors demonstrated that a loss of Atg14 led to an excessive pyroptosis in the oviductal epithelial cells that compromises cellular integrity and structure, impeding the transport function of the oviduct. In addition, the authors use both genetic and pharmacological approaches to test the hypothesis. Therefore, the findings from this study are high-impact and likely reproducible. However, there are multiple major concerns that need to be addressed to improve the quality of the work.

Reviewer #2 (Public review):

In this manuscript, Popli et al investigated the roles of autophagy related gene, Atg14, in the female reproductive tract (FRT) using conditional knockout mouse models. By ablation of Atg14 in both oviduct and uterus with PR-Cre (Atg14 cKO), authors discovered that such females are completely infertile. They went on to show that Atg14 cKO females have impaired embryo implantation as well as embryo transport from oviduct to uterus. Further analysis showed that Atg14 cKO leads to increased pyroptosis in oviduct, which disrupts oviduct epithelial integrity and leads to obstructive oviduct lumen and impaired embryo transport. The authors concluded that Atg14 is critical for maintaining the oviduct homeostasis and keeping the inflammation under check to enable proper embryo transport.

The authors have barely addressed most of my concerns in this revised version with a few minor issues remaining to be addressed:<br /> (1) The authors tried to address my first concern regarding the statement that "autophagy is critical for maintaining the oviduct homeostasis". The revised statement in Line 53-54 "we report that Atg14-dependent autophagy plays a crucial role in maintaining..." is still not correct. It should be corrected as " we report that autophagy-related protein Atg14 plays a crucial role in maintaining...".<br /> (2) Line 349-351 described 80-90% of blastocysts retrieved from oviducts of cKO mice, which is in consistent with Figure 3B (showing more than 98%).<br /> (3) Line 447, "Fig. 5E" should be Fig. 6A. In addition, grammar error in the next sentence.<br /> (4) In Figure 6D, why the composition of blastocysts in chemical treated group do not add up to 100%.

Reviewer #3 (Public review):

Summary:

The manuscript by Pooja Popli and co-authors tested the importance of Atg14 in the female reproductive tract by conditionally deleting Atg14 use PrCre and also Foxj1cre. The authors showed that loss of Atg14 leads to infertility due to the retention of embryos within the oviduct. The authors further concluded that the retention of embryos within the oviduct is due to pyroptosis in oviduct cells leading to defective cellular integrity. The revised manuscript has included new experimental data (Figs. S2B, 5B, 5C, and S3) that satisfied the concerns of this reviewer. The manuscript should provide important advancement to the field.

Reviewer #1 (Public review):

Summary:

The anatomical connectivity of the claustrum and the role of its output projections has, thus far, not been studied in detail. The aim of this study was to map the outputs of the endopiriform (EN) region of the claustrum complex, and understand their functional role. Here the authors have combined sophisticated intersectional viral tracing techniques, and ex vivo electrophysiology to map the neural circuitry of EN outputs to vCA1, and shown that optogenetic inhibition of the EN→vCA1 projection impairs both social and object recognition memory. Interestingly the authors find that the EN neurons target inhibitory interneurons providing a mechanism for feedforward inhibition of vCA1.

Strengths:

The strength of this study was the application of a multilevel analysis approach combining a number of state-of-the-art techniques to dissect the contribution of the EN→vCA1 to memory function.

In addition the authors conducted behavioural analysis of locomotor activity, anxiety and fear memory, and complemented the analysis of discrimination with more detailed description of the patterns of exploratory behaviour.

Reviewer #2 (Public review):

Summary:

Yamawaki et al., conducted a series of neuroanatomical tracing and whole cell recording experiments to elucidate and characterise a relatively unknown pathway between the endopiriform (EN) and CA1 of the ventral hippocampus (vCA1) and to assess its functional role in social and object recognition using fibre photometry and dual vector chemogenetics. The main findings were that the EN sends robust projections to the vCA1 that collateralise to the prefrontal cortex, lateral entorhinal cortex and piriform cortex, and these EN projection neurons terminate in the stratum lacunosum-moleculare (SLM) layer of distal vCA1, synapsing onto GABAergic neurons that span across the Pyramidal-Stratum Radiatum (SR) and SR-SML borders. It was also demonstrated that EN input disynaptically inhibits vCA1 pyramidal neurons. vCA1 projecting EN neurons receive afferent input from piriform cortex, and from within EN. Finally, fibre photometry experiments revealed that vCA1 projecting EN neurons are most active when mice explore novel objects or conspecifics, and pathway-specific chemogenetic inhibition led to an impairment in the ability to discriminate between novel vs. familiar objects and conspecifics.

Revision 1:<br /> The authors have addressed most of my concerns, but a few weaknesses remain :

(1) I expected to see the addition of raw interaction times with objects and conspecifics for each phase of social testing (pre-test, sociability test, social discrimination), as per my comment on including raw data. However, the authors only provided total distance traveled and velocity, and total interaction time in Figure S9, which is less informative.

(2) The authors observed increased activity in vCA1-projecting EN neurons tracking with the preferred object during the pre-test (object-object exploration) phase of the social tests, and the summary schematic (Figure 9A) depicts animals as showing a preference for one object over the other (although they are identical) in both the social and object recognition tests. However, in the chemogenetic experiment, the data (Fig S9B) indicate that animals did not show this preference for one object over another, making the expected baseline for this task unclear. This also raises an important question of whether the lack of effect from chemogenetic inhibition of vCA1-projecting EN neurons could be attributed to the absence of this baseline preference.<br /> Additionally, the finding that vCA1-projecting EN activity is associated with the preferred object exploration appears to counter the authors' argument that novelty engages this circuit (since both objects are novel in this instance). This discrepancy warrants further discussion.

Reviewer #2 (Public review):

Summary:

This study aimed to test experimentally a theoretical framework that aims to explain the perception of tinnitus, i.e., the perception of a phantom sound in the absence of external stimuli, through differences in auditory predictive coding patterns. To this aim, the researchers compared the neural activity preceding and following the perception of a sound using MEG in two different studies. The sounds could be highly predictable or random, depending on the experimental condition. They revealed that individuals with tinnitus and controls had different anticipatory predictions. This finding is a major step in characterizing the top-down mechanisms underlying sound perception in individuals with tinnitus.

Strengths:

This article uses an elegant, well-constructed paradigm to assess the neural dynamics underlying auditory prediction. The findings presented in the first experiment were partially replicated in the second experiment, which included 80 participants. This large number of participants for an MEG study ensures very good statistical power and a strong level of evidence. The authors used advanced analysis techniques - Multivariate Pattern Analysis (MVPA) and classifier weights projection - to determine the neural patterns underlying the anticipation and perception of a sound for individuals with or without tinnitus. The authors evidenced different auditory prediction patterns associated with tinnitus. Overall, the conclusions of this paper are well supported, and the limitations of the study are clearly addressed and discussed.

Weaknesses:

Even though the authors took care of matching the participants in age and sex, the control could be more precise. Tinnitus is associated with various comorbidities, such as hearing loss, anxiety, depression, or sleep disorders. The authors assessed individuals' hearing thresholds with a pure tone audiogram, but they did not take into account the high frequencies (6 kHz to 16 kHz) in the patient/control matching. Moreover, other hearing dysfunctions, such as speech-in-noise deficits or hyperacusis, could have been taken into account to reinforce their claim that the observed predictive pattern was not linked to hearing deficits. Mental health and sleep disorders could also have been considered more precisely, as they were accounted for only indirectly with the score of the 10-item mini-TQ questionnaire evaluating tinnitus distress. Lastly, testing the links between the individuals' scores in auditory prediction and tinnitus characteristics, such as pitch, loudness, duration, and occurrence (how often it is perceived during the day), would have been highly informative.

Comments on revisions:

Thank you for your responses. There are a few remaining points that, if addressed, could further enhance the manuscript:

- While the manuscript acknowledges the limitation of not matching groups on hearing thresholds in Study 1, a deeper analysis of participants' hearing abilities and their impact on MEG results, similar to that conducted in Study 2, would be valuable. Specifically, including a linear model that considers all frequencies, group membership, and their interactions could highlight differences across groups. Additionally, examining the effect of high-frequency hearing loss on prediction scores, as performed in Study 2, would strengthen the analysis, particularly given the trend noted (line 719). Such an addition could make a significant contribution to the literature by exploring how hearing abilities may influence prediction patterns.

- The connection with the hippocampal regions (line 864) remains somewhat unclear. While the inclusion of the Paquette reference appropriately links temporal region activity with tinnitus, it does not fully support the statement: "An increased focus on hippocampal regions, e.g., in fMRI, patient, or animal studies, could be a worthwhile complement to our MEG work, given the outstanding relevance of medial temporal areas in the formation of associations in statistical learning paradigms"

- Authors should add a comparison of participants mini-TQ scores on both studies<br /> - Authors should add significant level on Fig 6.B as in Fig 3.C, and a n.s on Fig 6.D

Reviewer #1 (Public review):

Summary:

Kimura et al performed a saturation mutagenesis study of CDKN2A to assess functionality of all possible missense variants and compare them to previously identified pathogenic variants. They also compared their assay result with those from in silico predictors.

Strengths:

CDKN2A is an important gene that modulate cell cycle and apoptosis, therefore it is critical to accurately assess functionality of missense variants. Overall, the paper reads well and touches upon major discoveries in a logical manner.

Weaknesses:

The paper lacks proper details for experiments and basic data, leaving the results less convincing. Analyses are superficial and does not provide variant-level resolution. Many of which were addressed during the revision process.

Comments on revisions

The manuscript was improved during the revision process.

Reviewer #2 (Public review):

Summary:

This study describes a deep mutational scan across CDKN2A using suppression of cell proliferation in pancreatic adenocarcinoma cells as a readout for CDKN2A function. The results are also compared to in silico variant predictors currently utilized by the current diagnostic frameworks to gauge these predictors' performance. The authors also functionally classify CDKN2A somatic mutations in cancers across different tissues

Review:

The goal of this paper was to perform functional classification of missense mutations in CDKN2A in order to generate a resource to aid in clinical interpretation of CDKN2A genetic variants identified in clinical sequencing. In our initial review, we concluded that this paper was difficult to review because there was a lack of primary data and experimental detail. The authors have significantly improved the clarity, methodological detail and data exposition in this revision, facilitating a fuller scientific review. Based on the data provided we do not think the functional characterization of CDKN2A variants is robust or complete enough to meet the stated goal of aiding clinical variant interpretation. We think the underlying assay could be used for this purpose but different experimental design choices and more replication would be required for these data to be useful. Alternatively, the authors could also focus on novel CDKN2A variants as there seems to be potential gain of function mutations that are simply lumped into "neutral" that may have important biological implications.

Major concerns:

Low experimental concordance. The p-value scatter plot (Figure 2 Figure Supplement 3A) across 560 variants shows low collinearity indicating poor replicability. These data should be shown in log2fold changes, but even after model fitting with the gamma GLM still show low concordance which casts strong doubt on the function scores.<br /> The more detailed methods provided indicate that the growth suppression experiment is done in 156 pools with each pool consisting of the 20 variants corresponding to one of the 156 aa positions in CKDN2A. There are several serious problems with this design.

Batch effects in each of the pools preventing comparison across different residues. We think this is a serious design flaw and not standard for how these deep mutational scans are done. The standard would be to combine all 156 pools in a single experiment. Given the sequencing strategy of dividing up CDKN2A into 3 segments, the 156 pools could easily have been collapsed into 3 (1 to 53, 54 to 110, 111 to 156). This would significantly minimize variation in handling between variants at each residue and would be more manageable for performance of further replicates of the screen for reproducibility purposes. The huge variation in confluency time 16-40 days for each pool suggest that this batch effect is a strong source of variation in the experiment

Lack of experimental/biological replication: The functional assay was only performed once on all 156 CDKN2A residues and was repeated for only 28 out of 156 residues, with only ~80% concordance in functional classification between the first and second screens. This is not sufficiently robust for variant interpretation. Why was the experiment not performed more than once for most aa sites?

For the screen, the methods section states that PANC-1 cells were infected at MOI=1 while the standard is an MOI of 0.3-0.5 to minimize multiple variants integrating into a single cell. At an MOI =1 under a Poisson process which captures viral integration, ~25% of cells would have more than 1 lentiviral integrant. So in 25% of the cells the effect of a variant would be confounded by one or more other variants adding noise to the assay.

While the authors provide more explanation of the gamma GLM, we strongly advise that the heatmap and replicate correlations be shown with the log2 fold changes rather than the fit output of the p-values.

In this study, the authors only classify variants into the categories "neutral", "indeterminate", or "deleterious" but they do not address CDKN2A gain-of-function variants that may lead to decreased proliferation. For example, there is no discussion on variants at residue 104, whose proliferation values mostly consist of higher magnitude negative log2fold change values. These variants are defined as neutral but from the one replicate of the experiment performed, they appear to be potential gain-of-function variants.

Joint Public Review:

Automatically identifying single cell types in heterogeneous mixed cell populations holds great promise to characterize mixed cell populations and to discover new rules of spatial organization and cell-cell communication. Although the current manuscript focuses on the application of quality control of iPSC cultures, the same approach can be extended to a wealth of other applications including in depth study of the spatial context. The simple and high-content assay democratizes use and enables adoption by other labs.

The authors also propose a new nucleocentric phenotyping pipeline, where a convolutional neural network is trained on the nucleus and some margins around it. This nucleocentric approach improves classification performance at high densities because nuclear segmentation is less prone to errors in dense cultures.

The manuscript is supported by comprehensive experimental and computational validations that raises the bar beyond the current state of the art in the field of high-content phenotyping and makes this manuscript especially compelling. These include (i) Explicitly assessing replication biases (batch effects); (ii) Direct comparison of feature-based (a la cell profiling) versus deep-learning-based classification (which is not trivial/obvious for the application of cell profiling); (iii) Systematic assessment of the contribution of each fluorescent channel; (iv) Evaluation of cell-density dependency; (v) explicit examination of mistakes in classification; (vi) Evaluating the performance of different spatial contexts around the cell/nucleus; (vii) generalization of models trained on cultures containing a single cell type (mono-cultures) to mixed co-cultures; (viii) application to multiple classification tasks.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

However, the authors should conduct a more thorough computational analysis to complement their manuscript. While the identification of improved multi-point mutants is commendable, the manuscript lacks a detailed investigation into the mechanisms by which these mutations enhance protein properties. The authors briefly mention that some physicochemical characteristics of the mutants are unusual, but they do not delve into why these mutations result in improved performance. Could computational techniques, such as molecular dynamics simulations, be employed to explore the effects of these mutations? Additionally, the authors claim that their method is efficient. However, the selected VHH is relatively short (<150 AA), resulting in lower computational costs. It remains unclear whether the computational cost of this approach would still be acceptable when designing larger proteins (>1000 AA). Besides, the design process involves a large number of prediction tasks, including the properties of both single-site saturation and multi-point mutants. The computational load is closely tied to the protein length and the number of mutation sites. Could the authors analyze the model's capability boundaries in this regard and discuss how scalable their approach is when dealing with larger proteins or more complex mutation tasks?

Reviewer #2 (Public review):

In this paper, the authors aim to explore whether an AI model trained on natural protein data can aid in designing proteins that are resistant to extreme environments. While this is an interesting attempt, the study's computational contributions are weak, and the design of the computational experiments appears arbitrary.

(1) The writing throughout the paper is poor. This leaves the reader confused.

(2) The main technical issue the authors address is whether AI can identify protein mutations that adapt to extreme environments based solely on natural protein data. However, the introduction could be more concise and focused on the key points to better clarify the significance of this question.

(3) The authors did not develop a new model but instead used their previously developed Pro-PRIME model. This significantly weakens the novelty and contribution of this work.

(4) The computational experiments are not well-justified. For instance, the authors used a zero-shot setting for single-point mutation experiments but opted for fine-tuning in multiple-point mutation experiments. There is no clear explanation for this discrepancy. How does the model perform in zero-shot settings for multiple-point mutations? How would fine-tuning affect single-point mutation results? The choice of these strategies seems arbitrary and lacks sufficient discussion.

Reviewer #1 (Public review):

Summary:

In this manuscript, Arimura et al describe MagIC-Cryo-EM, an innovative method for immune-selective concentrating of native molecules and macromolecular complexes for Cryo-EM imaging and single-particle analysis. Typically, Cryo-EM imaging requires much larger concentrations of biomolecules than that are feasible to achieve by conventional biochemical fractionation. Overall, this manuscript is meticulously and clearly written and may become a great asset to other electron microscopists and chromatin researchers.

Strengths:

Previously, Arimura et al. (Mol. Cell 2021) isolated from Xenopus extract and resolved by Cryo-EM a sub-class of native nucleosomes conjugated containing histone H1.8 at the on-dyad position, similar to that previously observed by other researchers with reconstituted nucleosomes. Here they sought to analyze immuno-selected nucleosomes aiming to observe specific modes of H1.8 positioning (e.g. on-dyad and off-dyad) and potentially reveal structural motifs responsible for the decreased affinity of H1.8 for the interphase chromatin compared to metaphase chromosomes. The main strength of this work is a clever and novel methodological design, in particular the engineered protein spacers to separate captured nucleosomes from streptavidin beads for a clear imaging. The authors provide a detailed step-by-step description of MagIC-Cryo-EM procedure including nucleosome isolation, preparation of GFP nanobody attached magnetic beads, optimization of the spacer length, concentration of the nucleosomes on graphene grids, data collection and analysis, including their new DUSTER method to filter-out low signal particles. This tour de force methodology should facilitate considering of MagIC-Cryo-EM by other electron microscopists especially for analysis of native nucleosome complexes.<br /> In pursue of biologically important new structures, the immune-selected H1.8-containing nucleosomes were solved at about 4A resolution; their structure appears to be very similar to the previously determined structure of H1.8-reconstituted nucleosomes. There were no apparent differences between the metaphase and interphase complexes suggesting that the on-dyad and off-dyad positioning does not explain the differences in H1.8 - nucleosome binding. However, they were able to identify and solve complexes of H1.8-GFP with histone chaperone NPM2 in a closed and open conformation providing mechanistic insights for H1-NPM2 binding and the reduced affinity of H1.8 to interphase chromatin as compared to metaphase chromosomes.

Weaknesses:

Still, I feel that there are certain limitations and potential artifacts resulting from formaldehyde fixation, use of bacterial-expressed recombinant H1.8-GFP, and potential effects of magnetic beads and/or spacer on protein structure, that should be more explicitly discussed. Also, the GFP-pulled down H1.8 nucleosomes should be better characterized biochemically to determine the actual linker DNA lengths (which are known to have a strong effect of linker histone affinity) and presence or absence of other factors such as HMG proteins that may compete with linker histones and cause the multiplicity of nucleosome structural classes (such as shown on Fig. 3F) for which the association with H1.8 is uncertain.

Reviewer #2 (Public review):

Summary:

The authors present a straightforward and convincing demonstration of a reagent and workflow that they collectively term "MagIC-cryo-EM", in which magnetic nanobeads combined with affinity linkers are used to specifically immobilize and locally concentrate complexes that contain a protein-of-interest. As a proof of concept, they localize, image, and reconstruct H1.8-bound nucleosomes reconstructed from frog egg extracts. The authors additionally devised an image-processing workflow termed "DuSTER", which increases the true positive detections of the partially ordered NPM2 complex. The analysis of the NPM2 complex {plus minus} H1.8 was challenging because only ~60 kDa of protein mass was ordered. Overall, single-particle cryo-EM practitioners should find this study useful.

Strengths:

The rationale is very logical and the data are convincing.

Weaknesses: I have seen an earlier version of this study at a conference. The conference presentation was much easier to follow than the current manuscript. It is as if this manuscript had undergone review at another journal and includes additional experiments to satisfy previous reviewers. Specifically, the NPM2 results don't seem to add much to the main story (MagIC-cryo-EM), and read more like an addendum. The authors could probably publish the NPM2 results separately, which would make the core MagIC results (sans DusTER) easier to read.

Reviewer #3 (Public review):

Summary:

In this paper, Arimura et al report a new method, termed MagIC-Cryo-EM, which refers to the method of using magnetic beads to capture specific proteins out of a lysate via, followed immunoprecipitation and deposition on EM grids. The so-enriched proteins can be analzyed structurally. Importantly, the nanoparticles are further functionalized with protein-based spacers, to avoid a distorted halo around the particles. This is a very elegant approach and allows the resolution of the stucture of small amounts of native proteins at atomistic resolution.<br /> Here, the authors apply this method to study the chromatosome formation from nucleosomes and the oocyte-specific linker histone H1.8. This allows them to resolve H1.8-containing chromatomosomes from oocyte extract in both interphase and metaphase conditions at 4.3 A resolution, which reveal a common structure with H1 placed right at the dyad and contacting both entry-and exit linker DNA.<br /> They then investigate the origin of H1.8 loss during interphase. They identify a non-nucleosomal H1.8-containing complex from interphase preparations. To resolve its structure, the authors develop a protocol (DuSTER) to exclude particles with ambiguous center, revealing particles with five-fold symmetry, that matches the chaperone NPM2. MS and WB confirms that the protein is present in interphase samples but not metaphase. The authors further separate two isoforms, an open and closed form that coexist. Additional densities in the open form suggest that this might be bound H1.8.

Strengths:

Together this is an important addition to the suite of cryoEM methods, with broad applications. The authors demonstrate the method using interesting applications, showing that the methods work and they can get high resolution structures from nucleosomes in complex with H1 from native environments.

Weaknesses:

The structures of the NPM2 chaperone is less well resolved, and some of the interpretation in this part seems only weakly justified.

Reviewer #1 (Public review):

The manuscript by Sayeed et al. uses a comprehensive series of multi-omics approaches to demonstrate that late-stage human cytomegalovirus (HCMV) infection leads to a marked disruption of TEAD1 activity, a concomitant loss of TEAD1-DNA interactions, and extensive chromatin remodeling. The data are thoroughly presented and provide evidence for the role of TEAD1 in the cellular response to HCMV infection. However, a key question remains unresolved: is the observed disruption of TEAD1 activity a direct consequence of HCMV infection, or could it be secondary to the broader innate antiviral response? In this respect, the study would benefit from experiments that assess the effect of TEAD1 overexpression or knockdown/deletion on HCMV replication dynamics. Such functional assays could help delineate whether TEAD1 perturbation directly influences viral replication or is part of a downstream/indirect cellular response, providing deeper mechanistic insights.

Reviewer #2 (Public review):

Summary:

This work uses genomic and biochemical approaches for HCMV infection in human fibroblasts and retinal epithelial cell lines, followed by comparisons and some validations using strategies such as immunoblots. Based on these analyses, they propose several mechanisms that could contribute to the HCMV-induced diseases, including closing of TEAD1-occupying domains and reduced TEAD1 transcript and protein levels, decreased YAP1 and phospho-YAP1 levels, and exclusion of TEAD1 exon 6.

Strengths:

The genomics experiments were done in duplicates and data analyses show good technical reproducibility. Data analyses are performed to show changes at the transcript and chromatin level changes, followed by some Western blot validations.

Weaknesses:

This work, at the current stage, is quite correlative since no functional studies are done to show any causal links. For readers who are outside the field, some clarifications of the system and design need to be stated.

Reviewer #1 (Public review):

Summary<br /> Roseman et al. use a new inhibitor of the maintenance DNA methyltransferase DNMT1 to probe the role of methylation on binding of the CTCF protein, which is known to be involved chromatin loop formation. As previous reported, and as expected based on our knowledge that CTCF binding is methylation-sensitive, the authors find that loss of methylation leads to additional CTCF binding sites and increased loop formation. By comparing novel loops with the binding of the pre-mRNA splicing factor SON, which localizes to the nuclear speckle compartment, they propose that these reactivated loops localize to near speckles. This behavior is dependent on CTCF whereas degradation of two speckle proteins does not affect CTCF binding or loop formation. The authors propose a model in which DNA methylation controls the association of genome regions with speckles via CTCF-mediated insulation.

Strengths<br /> The strengths of the study are 1) the use of a new, specific DNMT1 inhibitor and 2) the observation that genes whose expression is sensitive to DNMT1 inhibition and dependent on CTCF (cluster 2) show higher association with SON than genes which are sensitive to DNMT1 inhibition but are CTCF insensitive, is in line with the authors' general model.

Weaknesses<br /> There are a number of significant weaknesses that as a whole undermine many of the key conclusions, including the overall mechanistic model of a direct regulatory role of DNA methylation on CTCF-mediated speckle association of chromatin loops.

(1) The authors frequently make quasi-quantitative statements but do not actually provide the quantitative data, which they actually all have in hand. To give a few examples: "reactivated CTCF sites were largely methylated (p. 4/5), "many CTCF binding motifs enriched..." (p.5), "a large subset of reactivated peaks..."(p.5), "increase in strength upon DNMT1 inhibition" (p.5); "a greater total number....." (p.7). These statements are all made based on actual numbers and the authors should mention the numbers in the text to give an impression of the extent of these changes (see below) and to clarify what the qualitative terms like "largely", "many", "large", and "increase" mean. This is an issue throughout the manuscript and not limited to the above examples.<br /> Related to this issue, many of the comparisons which the authors interpret to show differences in behavior seem quite minor. For example, visual inspection suggests that the difference in loop strength shown in figure 1E is something like from 0 to 0.1 for K562 cells and a little less for KCT116 cells. What is a positive control here to give a sense of whether these minor changes are relevant. Another example is on p. 7, where the authors claim that CTCF partners of reactivated peaks tend to engage in a "greater number" of looping partners, but inspection of Figure 2A shows a very minor difference from maybe 7 to 7.5 partners. While a Mann-Whitney test may call this difference significant and give a significant P value, likely due to high sample number, it is questionable that this is a biologically relevant difference.

(2) The data to support the central claim of localization of reactivated loops to speckles is not overly convincing. The overlap with SON Cut&Tag (figure 2F) is partial at best and although it is better with the publicly available TSA-seq data, the latter is less sensitive than Cut&Tag and more difficult to interpret. It would be helpful to validate these data with FISH experiments to directly demonstrate and measure the association of loops with speckles (see below).

(3) It is not clear that the authors have indeed disrupted speckles from cells by degrading SON and SRRM2. Speckles contain a large number of proteins and considering their phase separated nature stronger evidence for their complete removal is needed. Note that the data published in ref 58 suffers from the same caveat.

(4) The authors ascribe a direct regulatory role to DNA methylation in controlling the association of some CTCF-mediated loops to speckles (p. 20). However, an active regulatory role of speckle association has not been demonstrated and the observed data are equally explainable by a more parsimonious model in which DNA methylation regulates gene expression via looping and that the association with speckles is merely an indirect bystander effect of the activated genes because we know that active genes are generally associated with speckles. The proposed mechanism of a regulatory role of DNA methylation in controlling speckle association is not convincingly demonstrated by the data. As a consequence, the title of the paper is also misleading.

(5) As a minor point, the authors imply on p. 15 that ablation of speckles leads to misregulation of genes by altering transcription. This is not shown as the authors only measure RNA abundance, which may be affected by depletion of constitutive splicing factors, but not transcription. The authors would need to show direct effects on transcription.

Reviewer #2 (Public review):

Summary:<br /> CTCF is one of the most well-characterized regulators of chromatin architecture in mammals. Given that CTCF is an essential protein, understanding how its binding is regulated is a very active area of research. It has been known for decades that CTCF is sensitive to 5-cystosine DNA methylation (5meC) in certain contexts. Moreover, at genomic imprints and in certain oncogenes, 5meC-mediated CTCF antagonism has very important gene regulatory implications. A number of labs (eg, Schubeler and Stamatoyannopoulos) have assessed the impact of DNA methylation on CTCF binding, but it is important to also interrogate the effect on chromatin organization (ie, looping). Here, Roseman and colleagues used a DNMT1 inhibitor in two established human cancer lines (HCT116 [colon] and K562 [leukemia]), and performed CTCF ChIPseq and HiChIP. They showed that "reactivated" CTCF sites-that is, bound in the absence of 5meC-are enriched in gene bodies, participate in many looping events, and intriguingly, appear associated with nuclear speckles. This last aspect suggests that these reactivated loops might play an important role in increased gene transcription. They showed a number of genes that are upregulated in the DNA hypomethylated state actually require CTCF binding, which is an important result.

Strengths:<br /> Overall, I found the paper to be succinctly written and the data presented clearly. The relationship between CTCF binding in gene bodies and association with nuclear speckles is an interesting result. Another strong point of the paper was combining DNMT1 inhibition with CTCF degradation.

Weaknesses:<br /> The most problematic aspect of this paper in my view is the insufficient evidence for the association of "reactivated" CTCF binding sites with nuclear speckles needs to be more diligently demonstrated (see Major Comment). One unfortunate aspect was that this paper neglected to discuss findings from our recent paper, wherein we also performed CTCF HiChIP in a DNA methylation mutant (Monteagudo-Sanchez et al., 2024 PMID: 39180406). It is true, this is a relatively recent publication, although the BioRxiv version has been available since fall 2023. I do not wish to accuse the authors of actively disregarding our study, but I do insist that they refer to it in a revised version. Moreover, there are a number of differences between the studies such that I find them more complementary rather than overlapping. To wit, the species (mouse vs human), the cell type (pluripotent vs human cancer), the use of a CTCF degron, and the conclusions of the paper (we did not make a link with nuclear speckles). Furthermore, we used a constitutive DNMT knockout which is not viable in most cell types (HCT116 cells being an exception), and in the discussion mentioned the advantage of using degron technology:

"With high-resolution techniques, such as HiChIP or Micro-C (119-121), a degron system can be coupled with an assessment of the cis-regulatory interactome (118). Such techniques could be adapted for DNA methylation degrons (eg, DNMT1) in differentiated cell types in order to gauge the impact of 5meC on the 3D genome."

The authors here used a DNMT1 inhibitor, which for intents and purposes, is akin to a DNMT1 degron, thus I was happy to see a study employ such a technique. A comparison between the findings from the two studies would strengthen the current manuscript, in addition to being more ethically responsible.

Reviewer #1 (Public review):

Summary:

In organisms with open mitosis, nuclear envelope breakdown at mitotic entry and re-assembly of the nuclear envelope at the end of mitosis are important, highly regulated processes. One key regulator of nuclear envelope re-assembly is the BAF (Barrier-to-Autointegration) protein, which contributes to cross-linking of chromosomes to the nuclear envelope. Crucially, BAF has to be in a dephosphorylated form to carry out this function, and PP2A has been shown to be the phosphatase that dephosphorylates BAF. The Ankle2/LEM4 protein has previously been identified as an important regulator of PP2A in the dephosphorylation of BAF but its precise function is not fully understood, and Li and colleagues set out to investigate the function of Ankle2/LEM4 in both Drosophila flies and Drosophila cell lines.

Strengths:

The authors use a combination of biochemical and imaging techniques to understand the biology of Ankle2/LEM4. On the whole, the experiments are well conducted and the results look convincing. A particular strength of this manuscript is that the authors are able to study both cellular phenotypes and organismal effects of their mutants by studying both Drosophila D-mel cells and whole flies.

The work presented in this manuscript significantly enhances our understanding of how Ankle2/LEM4 supports BAF dephosphorylation at the end of mitosis. Particularly interesting is the finding that Ankle2/LEM4 appears to be a bona fide PP2A regulatory protein in Drosophila, as well as the localisation of Ankle2/LEM4 and how this is influenced by the interaction between Ankle2 and the ER protein Vap33. It would be interesting to see, though, whether these insights are conserved in mammalian cells, e.g. does mammalian Vap33 also interact with LEM4? Is LEM4 also a part of the PP2A holoenzyme complex in mammalian cells?

Weaknesses:

This work is certainly impactful but more discussion and comparison of the Drosophila versus mammalian cell system would be helpful. Also, to attract the largest possible readership, the Ankle2 protein should be referred to as Ankle2/LEM4 throughout the paper to make it clear that this is the same molecule.

A schematic model at the end of the final figure would be very useful to summarise the findings.

Reviewer #2 (Public review):

The authors first identify Ankle2 as a regulatory subunit and direct interactor of PP2A, showing they interact both in vitro and in vivo to promote BAF dephosphorylation. The Ankyrin domain of Ankle2 is important for the interaction with PP2A. They then show Ankle2 also interacts with the ER protein Vap33 through FFAT motifs and they particularly co-localize during mitosis. The recruitment of Ankle2 to Vap33 is essential to ER and nuclear envelop membrane in telophase while earlier in mitosis, it relies on the C terminus but not the FFAT motifs for recruitments to the nuclear membrane and spindle envelop in early mitosis. The molecular determinants and receptors are currently not known. The authors check the function of the PP2A recruitment to Ankle2/Vap33 in the context of embryos and show this recruitment pathway is functionally important. While the Ankle2/Vap33 interaction is dispensable in adult flies -looking at wing development, the PP2A/Ankle2 interaction is essential for correct wing and fly development. Overall, this is a very complete paper that reveals the molecular mechanism of PP2A recruitment to Ankle2 and studies both the cellular and the physiological effect of this interaction in the context of fly development.

Strengths:

The paper is well written and the narrative is well-developed. The figures are of high quality, well-controlled, clearly labelled, and easy to understand. They support the claims made by the authors.

Weaknesses:

The study would benefit from being discussed in the context of what is already known on Ankle2 biology in C.elegans and human cells. It is important to highlight the structures shown in the paper are alphafold models, rather than validated structures.

Reviewer #3 (Public review):

Summary:

The authors were interested in how Ankle2 regulates nuclear envelope reformation after cell division. Other published manuscripts, including those from the authors, show without a doubt that Ankle2 plays a role in this critical process. However, the mechanism by which Ankle2 functions was unclear. Previous work using worms and humans (Asencio et al., 2012) established that human ANKLE2 could bind endogenous PP2A subunits. The binding was direct and was mediated through a region before and including the first ankyrin repeat in human ANKLE2. In addition to its interaction with PP2A, Asencio et al., 2012 also show that ANKLE2 regulates VRK1 kinase activity. Together PP2A and VRK1 regulate BAF phosphorylation for proper nuclear envelope reformation. Here, the authors provide more evidence for interaction with PP2A by also mapping the domain of interaction to the ankyrin repeat in Drosophila. In addition, the ankyrin repeat is essential for nuclear envelope reformation after division. They show that Ankle2 can bind in a PP2A complex without other known regulatory subunits of PP2A. The authors also identify a novel interaction with ER protein Vap33, but functional relevance for this interaction in nuclear envelope reformation is not provided in the manuscript, which the authors explicitly state. This manuscript does not comment on the activity of Ballchen/VRK1 in relation to Ankle2 loss and BAF phosphorylation or nuclear envelope reformation, even though links were previously shown by multiple studies (Asencio et al., Link et al., Apridita Sebastian et al.,). Nuclear envelope defects were rescued by the reduction of VRK1 in two of these manuscripts. It is possible that BAF phosphorylation phenotypes can be contributed by both PP2A inactivity and VRK1 overactivity due to the loss of Ankle2.

Strengths:

This manuscript is a useful finding linking Ankle2 function during nuclear envelope reformation to the PP2A complex. The authors present solid data showing that Ankle2 can form a complex with PP2A-29B and Mts and generate a phosphoproteomic resource that is fundamentally important to understanding Ankle2 biology.

Weaknesses:

However, the main findings/conclusions about subcellular localization might be incomplete since they are drawn from overexpression experiments. In addition, throughout the text, some conclusions are overstated or are not supported by data.

Reviewer #1 (Public review):

Summary:

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

Strengths:

To my knowledge, this is the first characterization of the role of CG14545 genes. Each experiment seems to be well-designed and adequately controlled.

Weaknesses:

However, the conclusions from each experiment are somewhat separate, and the functional relationships between Sakura's functions are not well established. In other words, although the loss of Sakura in the germline causes pleiotropic effects, the cause-and-effect relationships between the individual defects remain unclear.

Reviewer #2 (Public review):

In this study, the authors identified CG14545 (and named it Sakura), as a key gene essential for Drosophila oogenesis. Genetic analyses revealed that Sakura is vital for both oogenesis progression and ultimate female fertility, playing a central role in the renewal and differentiation of germ stem cells (GSC).

The absence of Sakura disrupts the Dpp/BMP signaling pathway, resulting in abnormal bam gene expression, which impairs GSC differentiation and leads to GSC loss. Additionally, Sakura is critical for maintaining normal levels of piRNAs. Also, the authors convincingly demonstrate that Sakura physically interacts with Otu, identifying the specific domains necessary for this interaction, suggesting a cooperative role in germline regulation. Importantly, the loss of otu produces similar defects to those observed in Sakura mutants, highlighting their functional collaboration.

The authors provide compelling evidence that Sakura is a critical regulator of germ cell fate, maintenance, and differentiation in Drosophila. This regulatory role is mediated through the modulation of pMad and Bam expression. However, the phenotypes observed in the germarium appear to stem from reduced pMad levels, which subsequently trigger premature and ectopic expression of Bam. This aberrant Bam expression could lead to increased CycA levels and altered transcriptional regulation, impacting piRNA expression. Given Sakura's role in pMad expression, it would be insightful to investigate whether overexpression of Mad or pMad could mitigate these phenotypic defects (UAS-Mad line is available at Bloomington Drosophila Stock Center).

A major concern is the overstated role of Sakura in regulating Orb. The data does not reveal mislocalized Orb; rather, a mislocalized oocyte and cytoskeletal breakdown, which may be secondary consequences of defects in oocyte polarity and structure rather than direct misregulation of Orb. The conclusion that Sakura is necessary for Orb localization is not supported by the data. Orb still localizes to the oocyte until about stage 6. In the later stage, it looks like the cytoskeleton is broken down and the oocyte is not positioned properly, however, there is still Orb localization in the ~8-stage egg chamber in the oocyte. This phenotype points towards a defect in the transport of Orb and possibly all other factors that need to localize to the oocyte due to cytoskeletal breakdown, not Orb regulation directly. While this result is very interesting it needs further evaluation on the underlying mechanism. For example, the decrease in E-cadherin levels leads to a similar phenotype and Bam is known to regulate E-cadherin expression. Is Bam expressed in these later knockdowns?

The manuscript would benefit from a more balanced interpretation of the data concerning Sakura's role in Orb regulation. Furthermore, a more expanded discussion on Sakura's potential role in pMad regulation is needed. For example, since Otu and Bam are involved in translational regulation, do the authors think that Mad is not translated and therefore it is the reason for less pMad? Currently the discussion presents just a summary of the results and not an extension of possible interpretation discussed in context of present literature.

Reviewer #3 (Public review):

In this very thorough study, the authors characterize the function of a novel Drosophila gene, which they name Sakura. They start with the observation that sakura expression is predicted to be highly enriched in the ovary and they generate an anti-sakura antibody, a line with a GFP-tagged sakura transgene, and a sakura null allele to investigate sakura localization and function directly. They confirm the prediction that it is primarily expressed in the ovary and, specifically, that it is expressed in germ cells, and find that about 2/3 of the mutants lack germ cells completely and the remaining have tumorous ovaries. Further investigation reveals that Sakura is required for piRNA-mediated repression of transposons in germ cells. They also find evidence that sakura is important for germ cell specification during development and germline stem cell maintenance during adulthood. However, despite the role of sakura in maintaining germline stem cells, they find that sakura mutant germ cells also fail to differentiate properly such that mutant germline stem cell clones have an increased number of "GSC-like" cells. They attribute this phenotype to a failure in the repression of Bam by dpp signaling. Lastly, they demonstrate that sakura physically interacts with otu and that sakura and otu mutants have similar germ cell phenotypes. Overall, this study helps to advance the field by providing a characterization of a novel gene that is required for oogenesis. The data are generally high-quality and the new lines and reagents they generated will be useful for the field. However, there are some weaknesses and I would recommend that they address the comments in the Recommendations for the authors section below.

Reviewer #1 (Public review):

Summary:

This study aimed to determine whether bacterial translation inhibitors affect mitochondria through the same mechanisms. Using mitoribosome profiling, the authors found that most antibiotics, except telithromycin, act similarly in both systems. These insights could help in the development of antibiotics with reduced mitochondrial toxicity.<br /> They also identified potential novel mitochondrial translation events, proposing new initiation sites for MT-ND1 and MT-ND5. These insights not only challenge existing annotations but also open new avenues for research on mitochondrial function.

Strengths:

Ribosome profiling is a state-of-the-art method for monitoring the translatome at very high resolution. Using mitoribosome profiling, the authors convincingly demonstrate that most of the analyzed antibiotics act in the same way on both bacterial and mitochondrial ribosomes, except for telithromycin. Additionally, the authors report possible alternative translation events, raising new questions about the mechanisms behind mitochondrial initiation and start codon recognition in mammals.

Weaknesses:

The main weaknesses of this study are:<br /> - While the authors highlight an interesting difference in the inhibitory mechanism of telithromycin on bacterial and mitochondrial ribosomes, mechanistic explanations or hypotheses are lacking.<br /> - The assignment of alternative start codons in MT-ND1 and MT-ND5 is very interesting but does not seem to fully align with structural data.<br /> - The newly proposed translation events in the ncRNAs are preliminary and should be further substantiated with additional evidence or interpreted with more caution.

Reviewer #2 (Public review):

In this study, the authors set out to explore how antibiotics known to inhibit bacterial protein synthesis also affect mitoribosomes in HEK cells. They achieved this through mitoribosome profiling, where RNase I and Mnase were used to generate mitoribosome-protected fragments, followed by sequencing to map the regions where translation arrest occurs. This profiling identified the codon-specific impact of antibiotics on mitochondrial translation.

The study finds that most antibiotics tested inhibit mitochondrial translation similarly to their bacterial counterparts, except telithromycin, which exhibited distinct stalling patterns. Specifically, chloramphenicol and linezolid selectively inhibited translation when certain amino acids were in the penultimate position of the nascent peptide, which aligns with their known bacterial mechanism. Telithromycin stalls translation at an R/K-X-R/K motif in bacteria, and the study demonstrated a preference for arresting at an R/K/A-X-K motif in mitochondria. Additionally, alternative translation initiation sites were identified in MT-ND1 and MT-ND5, with non-canonical start codons. Overall, the paper presents a comprehensive analysis of antibiotics in the context of mitochondrial translation toxicity, and the identification of alternative translation initiation sites will provide valuable insights for researchers in the mitochondrial translation field.

From my perspective as a structural biologist working on the human mitoribosome, I appreciate the use of mitoribosome profiling to explore off-target antibiotic effects and the discovery of alternative mitochondrial translation initiation sites. However, the description is somewhat limited by a focus on this single methodology. The authors could strengthen their discussion by incorporating structural approaches, which have contributed significantly to the field. For example, antibiotics such as paromomycin and linezolid have been modeled in the human mitoribosome (PMID: 25838379), while streptomycin has been resolved (10.7554/eLife.77460), and erythromycin was previously discussed (PMID: 24675956). The reason we can now describe off-target effects more meaningfully is due to the availability of fully modified human mitoribosome structures, including mitochondria-specific modifications and their roles in stabilizing the decoding center and binding ligands, mRNA, and tRNAs (10.1038/s41467-024-48163-x).<br /> These and other relevant studies should be acknowledged throughout the paper to provide additional context.

Reviewer #3 (Public review):

Summary:

Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provides another application of the technique to further study on mitochondrial translation.

Strengths:

This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells.<br /> The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and written well.

Weaknesses:

This is a novel and interesting study, however, most of the conclusion comes from mitoribosome profiling analysis, as a result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

Reviewer #1 (Public review):

Summary:

Qin and colleagues analysed data from the Human Connectome Project on four right-handed subgroups with different gyrification patterns in Heschl's gyrus. Based on these groups, the authors highlight the structure-function relationship of planum temporale asymmetry in lateralised language processing at the group level and next at the individual level. In particular, the authors propose that especially microstructural asymmetries are related to functional auditory language asymmetries in the planum temporale.

Strengths:

The study is interesting because of an ongoing and long-standing debate about the relationship between structural and functional brain asymmetries, and in particular whether structural brain asymmetries can be seen as markers of functional language brain lateralisation.

In this debate, the relationship between Heschl's gyrus asymmetry and planum temporale asymmetry is rare and therefore valuable here. A large sample size and inter-rater reliability support the findings.

Weaknesses:

The authors highlight the microstructural results, but could also emphasise on their interesting macrostructural results.

Reviewer #1 (Public review):

This work from Cui, Pan, Fan et al explores memory impairment in chronic pain mouse models, a topic of great interest for the neurobiology field. In particular, the work starts from a very interesting observation, that WT mice can be divided in susceptible and unsusceptible to memory impairment upon modelling chronic pain with CCI. This observation represents the basis of the work where the authors identify the sphingosine receptor S1PR1 as down-regulated in the dentate gyrus of susceptible animals and demonstrate through an elegant range of experiments involving AAV mediated knockdown or overexpression of S1PR1 that this receptor is involved in the memory impairment observed with chronic pain. Importantly for translational purposes, they also show that activation of S1PR1 through a pharmacological paradigm is able to rescue the memory impairment phenotype.

The authors also link these defects to reduced dendritic branching and reduced number of mature excitatory synapses in the DG to the memory phenotype.

They then proceed to explore possible mechanisms downstream of S1PR1 that could explain this reduction in dendritic spines. They identify integrin α2 as an interactor of S1PR1 and show a reduction in several proteins involved in actin dynamic, which is crucial for dendritic spine formation and plasticity.

They thus hypothesize that the interaction between S1PR1 and Integrin α2 is fundamental for the activation of Rac1 and Cdc42 and consequently for the polymerisation of actin; a reduction in this pathway upon chronic pain would thus lead to impaired actin polymerisation, synapse formation and thus impaired memory.

The work is of great interest and the experiments are of very good quality with results of great importance.

Comments on revisions:

The authors have replied satisfactorily to my previous concerns.

Reviewer #2 (Public review):

Summary:

The study investigates the molecular mechanisms underlying chronic pain-related memory impairment by focusing on S1P/S1PR1 signaling in the dentate gyrus (DG) of the hippocampus. Through behavioural tests (Y-maze and Morris water maze) and RNA-seq analysis, the researchers segregated chronic pain mice into memory impairment-susceptible and -unsusceptible subpopulations. They discovered that S1P/S1PR1 signaling is crucial for determining susceptibility to memory impairment, with decreased S1PR1 expression linked to structural plasticity changes and memory deficits.

Knockdown of S1PR1 in the DG induced a susceptible phenotype, while overexpression or pharmacological activation of S1PR1 promoted resistance to memory impairment and restored normal synaptic structure. The study identifies actin cytoskeleton-related pathways, including ITGA2 and its downstream Rac1/Cdc42 signaling, as key mediators of S1PR1's effects, offering new insights and potential therapeutic targets for chronic pain-related cognitive dysfunction.

This manuscript consists of a comprehensive investigation and significant findings. The study provides novel insights into the molecular mechanisms of chronic pain-related memory impairment, highlighting the critical role of S1P/S1PR1 signaling in the hippocampal dentate gyrus. The clear identification of S1P/S1PR1 as a potential therapeutic target offers promising avenues for future research and treatment strategies. The manuscript is well-structured, methodologically sound, and presents valuable contributions to the field.

Strengths:

(1) The manuscript is well-structured and written in clear, concise language. The flow of information is logical and easy to follow.

(2) The segregation of mice into memory impairment-susceptible and -unsusceptible subpopulations is innovative and well-justified. The statistical analyses are robust and appropriate for the data.

(3) The detailed examination of S1PR1 expression and its impact on synaptic plasticity and actin cytoskeleton reorganization is impressive. The findings are significant and contribute to the understanding of chronic pain-related memory impairment.

Comments on revisions:

The authors have satisfactorily addressed all the issues raised.

Reviewer #1 (Public review):

Summary:

The paper addresses the knowledge gap between the representation of goal direction in the central complex and how motor systems stabilize movement toward that goal. The authors focused on two descending neurons, DNa01 and 02, and showed that they play different roles in steering the fly toward a goal. They also explored the connectome data to propose a model to explain how these DNs could mediate response to lateralized sensory inputs. They finally used lateralized optogenetic activation/inactivation experiments to test the roles of these neurons in mediating turnings in freely walking flies.

Strengths:

The experiments are well-designed and controlled. The experiment in Figure 4 is elegant, and the authors put a lot of effort into ensuring that ATP puffs do not accidentally activate the DNs. They also have explained complex experiments well. I only have minor comments for the authors.

Weaknesses:

(1) I do not fully understand how the authors extracted the correlation functions from the population data in Figure 1. Since the ipsilateral DNs are anti-correlated with the contralateral ones, I expected that the average will drop to zero when they are pooled together (e.g., 1E-G). Of course, this will not be the case if all the data in Figure 1 are collected from the same brain hemisphere. It would be helpful if the authors could explain this.

(2) What constitutes the goal directions in Figures 1-3 and 8, as the authors could not use EPG activity as a proxy for goal directions? If these experiments were done in the dark, without landmarks, one would expect the fly's heading to drift randomly at times, and they would not engage the DNa01/02 for turning. Do the walking trajectories in these experiments qualify as menotactic bouts?

(3) In Figure 2B, the authors mentioned that DNa02 overpredicts and 01 underpredicts rapid turning and provided single examples. It would be nice to see more population-level quantification to support this claim.

Reviewer #2 (Public review):

The data is largely electrophysiological recordings coupled with behavioral measurements (technically impressive) and some gain-of-function experiments in freely walking flies. Loss-of-function was tested but had minimal effect, which is not surprising in a system with partially redundant control mechanisms. The data is also consistent with/complementary to subsequent manuscripts (Yang 2023, Feng 2024, and Ros 2024) showing additional descending neurons with contributions to steering in walking and flying.

The experiments are well executed, the results interesting, and the description clear. Some hypotheses based on connectome anatomy are tested: the insights on the pre-synaptic side - how sensory and central complex heading circuits converge onto these DNs are stronger than the suggestions about biomechanical mechanisms for how turning happens on the motor side.

Of particular interest is the idea that different sensory cues can converge on a common motor program. The turn-toward or turn-away mechanism is initiated by valence rather than whether the stimulus was odor or temperature or memory of heading. The idea that animals choose a direction based on external sensory information and then maintain that direction as a heading through a more internal, goal-based memory mechanism, is interesting but it is hard to separate conclusively.

The "see-saw", where left-right symmetry is broken to allow a turn, presumably by excitation on one side and inhibition of the other leg motor modules, is interesting but not well explained here. How hyperpolarization affects motor outputs is not clear.

The statement near Figure 5B that "DNa02 activity was higher on the side ipsilateral to the attractive stimulus, but contralateral to the aversive stimulus" is really important - and only possible to see because of the dual recordings.

Reviewer #3 (Public review):

Summary:

Rayshubskiy et al. performed whole-cell recordings from descending neurons (DNs) of fruit flies to characterize their role in steering. Two DNs implicated in "walking control" and "steering control" by previous studies (Namiki et al., 2018, Cande et al., 2018, Chen et al., 2018) were chosen by the authors for further characterization. In-vivo whole-cell recordings from DNa01 and DNa02 showed that their activity predicts spontaneous ipsilateral turning events. The recordings also showed that while DNa02 predicts transient turns DNa01 predicts slow sustained turns. However, optogenetic activation or inactivation showed relatively subtle phenotypes for both neurons (consistent with data in other recent preprints, Yang et al 2023 and Feng et al 2024). The authors also further characterized DNa02 with respect to its inputs and showed a functional connection with olfactory and thermosensory inputs as well as with the head-direction system. DNa01 is not characterized to this extent.

Strengths:

(1) In-vivo recordings and especially dual recordings are extremely challenging in Drosophila and provide a much higher resolution DN characterization than other recent studies that have relied on behavior or calcium imaging. Especially impressive are the simultaneous recordings from bilateral DNs (Figure 3). These bilateral recordings show clearly that DNa02 cells not only fire more during ipsilateral turning events but that they get inhibited during contralateral turns. In line with this observation, the difference between left and right DNa02 neuronal activity is a much better predictor of turning events compared to individual DNa02 activity.

(2) Another technical feat in this work is driving local excitation in the head-direction neuronal ensemble (PEN-1 neurons), while simultaneously imaging its activity and performing whole-cell recordings from DNa02 (Figure 4). This impressive approach provided a way to causally relate changes in the head-direction system to DNa02 activity. Indeed, DNa02 activity could predict the rate at which an artificially triggered bump in the PEN-1 ring attractor returns to its previous stable point.

(3) The authors also support the above observations with connectomics analysis and provide circuit motifs that can explain how the head direction system (as well as external olfactory/thermal stimuli) communicated with DNa02. All these results unequivocally put DNa02 as an essential DN in steering control, both during exploratory navigation as well as stimulus-directed turns.

Weaknesses:

(1) I understand that the first version of this preprint was already on biorxiv in 2020, and some of the "weaknesses" I list are likely a reflection of the fact that I'm tasked to review this manuscript in late 2024 (more than 4 years later). But given this is a 2024 updated version it suffers from laying out the results in contemporary terms. For instance, the manuscript lacks any reference to the DNp09 circuit implicated in object-directed turning and upstream to DNa02 even though the authors cite one of the papers where this was analyzed (Braun et al, 2024). More importantly, these studies (both Braun et al 2024 and Sapkal et al 2024) along with recent work from the authors' lab (Yang et al 2023) and other labs (Feng et al 2024) provide a view that the entire suite of leg kinematics changes required for turning are orchestrated by populations of heterogeneous interconnected DNs. Moreover, these studies also show that this DN-DN network has some degree of hierarchy with some DNs being upstream to other DNs. In this contemporary view of steering control, DNa02 (like DNg13 from Yang et al 2023) is a downstream DN that is recruited by hierarchically upstream DNs like DNa03, DNp09, etc. In this view, DNa02 is likely to be involved in most turning events, but by itself unable to drive all the motor outputs required for the said events. This reasoning could be used while discussing the lack of major phenotypes with DNa02 activation or inactivation observed in the current study, which is in stark contrast to strong phenotypes observed in the case of hierarchically upstream DNs like DNp09 or DNa03. In the section, "Contributions of single descending neuron types to steering behavior": the authors start off by asking if individual DNs can make measurable contributions to steering behavior. Once more, any citations to DNp09 or DNa03 - two DNs that are clearly shown to drive strong turning-on activation (Bidaye et al, 2020, Feng et al 2024) - are lacking. Besides misleading the reader, such statements also digress the results away from contemporary knowledge in the field. I appreciate that the brief discussion in the section titled "Ensemble codes for steering" tries to cover these recent updates. However, I think this would serve a better purpose in the introduction and help guide the results.

(2) The second major weakness is the lack of any immunohistochemistry (IHC) images quantifying the expression of the genetic tools used in these studies. Even though the main split-Gal4 tools for DNa01 and DNa02 were previously reported by Namiki et al, 2018, it is important to document the expression with the effectors used in this work and explicitly mention the expression in any ectopic neurons. Similarly, for any experiments where drivers were combined together (double recordings, functional connectivity) or modified for stochastic expression (Figure 8), IHC images are absolutely necessary. Without this evidence, it is difficult to trust many of the results (especially in the case of behavioral experiments in Figure 8). For example, the DNa01 genetic driver used by the authors is also expressed in some neurons in the nerve cord (as shown on the Flylight webpage of Janelia Research Campus). One wonders if all or part of the results described in Figure 8 are due to DNa01 manipulation or manipulation of the nerve cord neurons. The same applies for optic lobe neurons in the DNa02 driver.

(3) The paper starts off with a comparative analysis of the roles of DNa01 and DNa02 during steering. Unfortunately, after this initial analysis, DNa01 is largely ignored for further characterization (e.g. with respect to inputs, connectomics, etc.), only to return in the final figure for behavioral characterization where DNa01 seems to have a stronger silencing phenotype compared to DNa02. I couldn't find an explanation for this imbalance in the characterization of DNa01 versus DNa02. Is this due to technical reasons? Or was it an informed decision due to some results? In addition to being a biased characterization, this also results in the manuscript lacking a coherent thread, which in turn makes it a bit inaccessible to the non-specialist.

(4) There seems to be a discrepancy with regard to what is emphasized in the main text and what is shown in Figures S3/S4 in relation to the role of these DNs in backward walking. There are only two sentences in the main text where these figures are cited.<br /> a) "DNa01 and DNa02 firing rate increases were not consistently followed by large changes in forward velocity (Figs. 1G and S3)."<br /> b) "We found that rotational velocity was consistently related to the difference in right-left firing rates (Fig. 3B). This relationship was essentially linear through its entire dynamic range, and was consistent across paired recordings (Fig. 3C). It was also consistent during backward walking, as well as forward walking (Fig. S4)."<br /> These main text sentences imply the role of the difference between left and right DNa02 in turning. However, the actual plots in the Figures S3 and S4 and their respective legends seem to imply a role in "backward walking". For instance, see this sentence from the legend of Figure S3 "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward. When (firing rateDNa02>>firing rateDNa01), the fly is also often moving backward, but forward movement is still more common overall, and so the net effect is that forward velocity is small but still positive when (firing rateDNa02>>firing rateDNa01). Note that when we condition our analysis on behavior rather than neural activity, we do see that backward walking is associated with a large firing rate differential (Fig. S4)." This sort of discrepancy in what is emphasized in the text, versus what is emphasized in the figures, ends up confusing the reader. More importantly, I do not agree with any of these conclusions regarding the implication of backward walking. Both Figures S3 and S4 are riddled with caveats, misinterpretations, and small sample sizes. As a result, I actually support the authors' decision to not infer too much from these figures in the "main text". In fact, I would recommend going one step further and removing/modifying these figures to focus on the role of "rotational velocity". Please find my concerns about these two figures below:<br /> a) In Figures S3 and S4, every heat map has a different scale for the same parameter: forward velocity. S3A is -10 to +10mm/s. S3B is -6 to +6 S4B (left) is -12 to +12 and S4B (right) is -4 to +4. Since the authors are trying to depict results based on the color-coding this is highly problematic.<br /> b) Figure S3A legend "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward." There are also several instances when ΔvoltageDNa02= ΔvoltageDNa01 and both are low (lower left quadrant) when the fly is typically moving backwards. So in my opinion, this figure in fact suggests DNa02 has no role in backward velocity control.<br /> c) Based on the example traces in S4A, every time the fly walks backwards it is also turning. Based on this it is important to show absolute rotational velocity in Figure S4C. It could be that the fly is turning around the backward peak which would change the interpretation from Figure S4C. Also, it is important to note that the backward velocities in S4A are unprecedentedly high. No previous reports show flies walking backwards at such high velocities (for example see Chen et al 2018, Nat Comm. for backward walking velocities on a similar setup).<br /> d) In my opinion, Figure S4D showing that right-left DNa02 correlates with rotational velocity, regardless of whether the fly is in a forward or backward walking state, is the only important and conclusive result in Figures S3/S4. These figures should be rearranged to only emphasize this panel.

(5) Figure 3 shows a really nice analysis of the bilateral DNa02 recordings data. While Figure S5 shows that authors have a similar dataset for DNa01, a similar level analysis (Figures 3D, E) is not done for DNa01 data. Is there a reason why this is not done?

(6) In Figure 4 since the authors have trials where bump-jump led to turning in the opposite direction to the DNa02 being recorded, I wonder if the authors could quantify hyperpolarization in DNa02 as is predicted from connectomics data in Figure 7.

(7) Figure 6 suggests that DNa02 contains information about latent steering drives. This is really interesting. However, in order to unequivocally claim this, a higher-resolution postural analysis might be needed. Especially given that DNa02 activation does not reliably evoke ipsilateral turning, these "latent" steering events could actually contain significant postural changes driven by DNa02 (making them "not latent"). Without this information, at least the authors need to explicitly mention this caveat.

(8) Figure 7 would really benefit from connectome data with synapse numbers (or weighted arrows) and a corresponding analysis of DNa01.

(9) In Figure 8E, the most obvious neuronal silencing phenotype is decreased sideways velocity in the case of DNa01 optogenetic silencing. In Figure S2, the inverse filter for sideways velocity for DNa01 had a higher amplitude than the rotational velocity filter. Taken together, does this point at some role for DNa01 in sideways velocity specifically?

(10) In Figure 8G, the effect on inner hind leg stance prolongation is very weak, and given the huge sample size, hard to interpret. Also, it is not clear how this fits with the role of DNa01 in slow sustained turning based on recordings.

Reviewer #1 (Public review):

In this paper, Wu et al. investigated the physiological roles of CCDC113 in sperm flagellum and HTCA stabilization by using CRISPR/Cas knockouts mouse models, co-IP and single sperm imaging. They find that CCDC113 localizes in the linker region among radial spokes, the nexin-dynein regulatory complex (N-DRC), and doublet microtubules (DMTs) RS, N-DRC and DMTs and interacts with axoneme-associated proteins CFAP57 and CFAP91, acting as an adaptor protein that facilitates the linkage between RS, N-DRC and DMTs within the sperm axoneme. They show the disruption of CCDC113 produced spermatozoa with disorganized sperm flagella and CFAP91, DRC2 could not colocalize with DMTs in Ccdc113-/- spermatozoa. Interestingly, the data also indicate that CCDC113 could localize on the HTCA region, and interact with HTCA-associated proteins. The knockout of Ccdc113 could also produce acephalic spermatozoa. By using Sun5 and Centlein knockout mouse models, the authors further find SUN5 and CENTLEIN are indispensable for the docking of CCDC113 to the implantation site on the sperm head. Overall, the experiments were designed properly and performed well to support the authors' observation in each part. Furthermore, the study's findings offer valuable insights into the physiological and developmental roles of CCDC113 in the male germ line, which can provide insight into impaired sperm development and male infertility. The conclusions of this paper are mostly well supported by data, but some points need to be clarified and discussed.

(1) In Fig. 1, a sperm flagellum protein, which is far way from CCDC113, should be selected as a negative control to exclude artificial effects in co-IP experiments.<br /> (2) Whether the detachment of sperm head and tail in Ccdc113-/- mice is a secondary effect of the sperm flagellum defects? The author should discuss this point.<br /> (3) Given that some cytoplasm materials could be observed in Ccdc113-/- spermatozoa (Fig. 5A), whether CCDC113 is also essential for cytoplasmic removal?<br /> (4) Although CCDC113 could not bind to PMFBP1, the localization of CCDC113 in Pmfbp1-/- spermatozoa should be also detected to clarify the relationship between CCDC113 and SUN5-CENTLEIN-PMFBP1.

Comments on revisions:

The authors addressed all my concerns. The manuscript was greatly improved.

Reviewer #2 (Public review):

Summary:

In the present study, the authors select the coiled-coil protein CCDC113 and revealed its expression in the stages of spermatogenesis in the testis as well as in the different steps of spermiogenesis with expression also mapped in the different parts of the epididymis. Gene deletion led to male infertility in CRISPR-Cas9 KO mice and PAS staining showed defects mapped in the different stages of the seminiferous cycle and through the different steps of spermiogenesis. EM and IF with several markers of testis germ cells and spermatozoa in the epididymis indicated defects in flagella and head-to-tail coupling for flagella as well as acephaly. The authors' co-IP experiments of expressed CCDC113 in HEK293T cells indicated an association with CFAP91 and DRC2 as well as SUN5 and CENTLEIN.

The authors propose that CCDC113 connects CFAP91 and DRC2 to doublet microtubules of the axoneme and CCDC113's association with SUN5 and CENTLEIN to stabilize the sperm flagellum head-to-tail coupling apparatus. Extensive experiments mapping CCDC13 during postnatal development are reported as well as negative co-IP experiments and studies with SUN5 KO mice as well as CENTLEIN KO mice.

Strengths:

The authors provide compelling observations to indicate the relevance of CCDC113 to flagellum formation with potential protein partners. The data are relevant to sperm flagella formation and its coupling to the sperm head.

Weaknesses:

The authors' observations are consistent with the model proposed but the authors' conclusions for the mechanism may require direct demonstration in sperm flagella. The Walton et al paper shows human CCDC96/113 in cilia of human respiratory epithelia. An application of such methodology to the proteins indicated by Wu et al for the sperm axoneme and head-tail coupling apparatus is eagerly awaited as a follow-up study.

Reviewer #1 (Public review):

Summary:

The authors aimed to investigate the oscillatory activity of GnRH neurones in freely behaving mice. By utilising GCaMP fiber photometry, they sought to record real-time neuronal activity to understand the patterns and dynamics of GnRH neuron firing and their implications for reproductive physiology.

Strengths:

- The use of GCaMP fiber photometry allows for high temporal resolution recordings of neuronal activity, providing real-time data on the dynamics of GnRH neurones.<br /> - Recording in freely behaving animals ensures that the findings are physiologically relevant and not artifacts of a controlled laboratory environment.<br /> - The authors used statistical methods to characterise the oscillatory patterns, ensuring the reliability of their findings.

Weaknesses:

- While the study identifies distinct oscillatory patterns in GnRH neurones' calcium dynamics, it falls short in exploring the functional implications of these patterns for GnRH pulsatility and overall reproductive physiology.<br /> - The study lacks broader discussion to include comparisons with existing studies on GnRH neurone activity and pulsatility and highlight how the findings of this study align with or differ from previous research and what novel contributions are made.<br /> - The authors aimed to characterise the oscillatory activity of GnRH neurons and successfully identified distinct oscillatory patterns. The results support the conclusion that GnRH neurons exhibit complex oscillatory behaviours, which are critical for understanding their role in reproductive physiology. However, it has not been made clear what exactly do the authors mean by "multi-dimensional oscillatory patterns" and how has this been shown.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors report GCaMP fiber-photometry recordings from the GnRH neuron distal projections in the ventral arcuate nucleus. The recording are taken from intact, male and female, freely behaving mice. The report three patterns of neuronal activity:

1) abrupt increases in the Ca2+ signals that are perfectly correlated with LH pulses.

2) a gradual, yet fluctuating (with a slow ultradian frequency), increase in activity, which is associated with the onset of the LH surge in female animals.

3) clustered (high frequency) baseline activity in both female and male animals.

Strengths:

The GCaMP fiber-photometry recordings reported here are the first direct recordings from GnRH neurones in free behaving mice. These recordings suggest a rich repertoire of activity, including the integration of distinct "surge" and "pulse" generation signals, and an ultradian rhythm during the onset of the surge.

Weaknesses:

The data analysis methods used for the characterisation of the oscillatory behaviour could be complemented with more advanced wavelet methods to quantify and analyse how the frequency content of the observed Ca2+ signal changes over the cycle.

Joint Public Reviews:

Summary:

This paper studies the genetic factors contributing to childhood obesity. Through a comprehensive analysis integrating genome-wide association study (GWAS) data with 3D genomic datasets across 57 human cell types, consisting of Capture-C/Hi-C, ATAC-seq, and RNA-seq, the study identifies significant genetic contributions to obesity using stratified LD score regression, emphasizing the enrichment of genetic signals in pancreatic alpha cells and identification of significant effector genes at obesity-associated loci such as BDNF, ADCY3, TMEM18, and FTO. Additionally, the study implicated ALKAL2, a gene responsive to inflammation in nerve nociceptors, as a novel effector gene at the TMEM18 locus, suggesting a role for inflammatory and neurological pathways in obesity's pathogenesis which was supported through colocalization analysis using eQTL derived from the GTEx dataset. This comprehensive genomic analysis sheds light on the complex genetic architecture of childhood obesity, highlighting the importance of cellular context for future research and the development of more effective strategies.

Strengths:

Overall, the paper has several strengths, including leveraging large-scale, multi-modal datasets, using appropriate computational tools, and in-depth discussion of their significant results.

Reviewer #1 (Public review):

In this manuscript, Zhang et al. report a genetic screen to identify novel transcriptional regulators that coordinate mitochondrial biogenesis. They performed an RNAi-based modifier screen wherein they systematically knocked down all known transcription factors in the developing Drosophila eye, which was sensitized and had decreased mitochondrial DNA content. Through this screen, they identify CG1603 as a potential regulator of mitochondrial volume. They show that protein levels of mitochondrial proteins like TFAM, SDHA, and other mitochondrial proteins and mtDNA content are downregulated in CG1603 mutants. RNA-Seq and ChIP-Seq further show that CG1603 binds to the promoter regions of several known nuclear-encoded mitochondrial genes and regulates their expression. Finally, they also identified YL-1 as an upstream regulator of CG1603. Most studies have focused on PGC-1α as a master regulator of mitochondrial biogenesis. which seems to be a context-dependent regulator. Also, PGC-1α mediated regulation does not explain the regulation of 1100 genes that are required for mitochondrial biogenesis. Therefore, identifying new regulators in this work is crucial for the advancement of our understanding of mitochondrial biogenesis.

Reviewer #2 (Public review):

Summary:

In this study, the authors identified nuclear genome-encoded transcription factors that regulate mtDNA maintenance and mitochondrial biogenesis. They started with an RNAi screening in developing Drosophila eyes with reduced mtDNA content and identified several putative candidate genes. Subsequently, using ChIP-seq data, they built a potential regulatory network that seems to govern mitochondrial biogenesis. Next, they focused on a candidate gene, CG1603 /clifford, for further characterization. Based on the expression of different markers, such as TFAM and SDHA, in RNAi and overexpression clones in the midgut, they argued that CG1603 promotes mitochondrial biogenesis and the expression of ETC complex genes. They used a CG1603 mutant to show reduced mtDNA and mitochondrial protein levels. Clonal analyses showed a reduction in mitochondrial biogenesis and membrane potential upon loss of CG1603. They further showed that the protein is localized to the mitochondria, and binds to polytene chromosomes in the salivary gland. Based on the RNA-seq results from the mutants and the ChIP data, the authors argued that the nucleus-encoded mitochondrial genes are downregulated >2 folds in the CG1603 mutants and that the regulatory elements bound by CG1603 are related to ETC biogenesis. Finally, they showed that YL-1, another candidate in the network, is an upstream regulator of CG1603. The screening strategy was well-designed, and the follow-up experiments were nicely executed.

Comments on revisions:

The authors have addressed my previous comments satisfactorily.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

However, given that S1P is upstream N NF-κB signaling, it is unclear if it offers conceptual innovations as compared to previous studies from the same team (Palazzo et al. 2020; 2022, 2023)

Reviewer #2 (Public review):

Summary:

Sphingosine-1-phosphate (S1P) metabolic and signaling genes are expressed highly in retinal Müller glia (MG) cells. This study tested how S1P signaling regulates glial phenotype, dedifferentiation of, reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs using in vivo chick retina. Major techniques used are Sc-RNASeq and immunohistochemistry to determine the gene expression and proliferation of MG cells that co-label with signaling antibodies or mRNA FISH following treating the in vivo eyes with various S1P signaling antagonists, agonists, and signal modulators. The major conclusions drawn are supported by the results presented. However, the methodology they have used to modulate the S1P pathway using various chemical drugs raises questions about the outcomes and whether those are the real effects of S1P receptor modulation or S1P synthesis inhibition.

Strengths:

- Use of elaborated single-cell RNAseq expression data.<br /> - Use of FISH for S1P receptors and kinase as a good quality antibody is not available.<br /> - Use of EdU assay in combination with IHC<br /> - Comparison with human and Zebrafish Sc-RNA data

Weaknesses:

The methodology is not very clean. A number of drugs (inhibitors/ antagonists/agonists signal modulators) are used to modulate S1P expression or signaling in the retina without evidence that these drugs are reaching the target cells. No alternative evaluation if the drugs, in fact, are effective. The drug solubility in the vehicle and in the vitreous is not provided, and how did they decide on using a single dose of each drug to have the optimal expected effect on the S1P pathway?

Reviewer #1 (Public review):

Summary:

Shrestha et al report an investigation of mechanisms underlying gustatory preference for carboxylic acids in Drosophila. They begin with a screen of selected IR mutants, identifying 5 candidates - 2 IR co-receptors and 3 other IRs - whose loss of function causes defects in feeding preference for one or more of the three tested carboxylic acids. The requirement for IR51b, IR94a, and IR94h in carboxylic acid responses is evaluated in more detail using behavior, electrophysiology (labellar sensilla), and calcium imaging (pharyngeal neurons). The behavioral valence of IR94a and IR94h neurons is assessed using optogenetics. Overall the study uses a variety of approaches to test and validate the requirement of IRs in pharyngeal carboxylic acid taste.

Strengths:

The involvement of the identified IRs in gustatory responses to carboxylic acids is very clear from this study. The authors use mutants and transgenic rescue experiments and evaluate outcomes using electrophysiology, behavior, and imaging. Complementary approaches of loss-of-function and artificial activation support the main conclusion that the identified pharyngeal neurons sense carboxylic acids and convey a positive behavioral valence.

Weaknesses:

Some aspects of expression analysis and calcium imaging need to be clarified to better support the conclusions.

(1) The conclusion of two parallel IR-mediated pathways rests on expression analysis of Ir94a-GAL4 and Ir94h-GAL4 lines and the observation that Ir51b expression driven by either can rescue the Ir51b mutant phenotype. However, the expression analysis is not as rigorous as it needs to be for such a conclusion. Prior work found co-expression of Ir94a and Ir94h in the LSO. Here, the co-expression of the two drivers has not been examined, and Ir94a-GAL4 does not appear to be expressed in the LSO. Given the challenges in validating expression patterns in pharyngeal organs, the possibility that the drivers do not entirely capture endogenous expression cannot be ruled out. Rescue experiments using feeding preference or single-cell imaging don't suffice as validation. Plus, the expression of Ir51b could not be defined.

(2) The description of methods and results for the ex vivo calcium imaging is not satisfactory. Details about which cells are being analyzed, and in which organs are not included. No solvent stimulus is tested. The temporal dynamics of the responses are not presented. Movies of the imaging are not included as supplementary information - it would be important to visualize those with what was considered modest movement.

(3) The observed differences in phenotypes of Ir25a and Ir76b mutants are intriguing, as are those between the co-receptor mutants and Ir51b, Ir94a, and Ir94h, but have not been sufficiently considered. Prior studies have also found roles for other response modes (OFF response), other IRs and GRs, and other organs (labellum, tarsi) in behavioral responses to carboxylic acids. Overall, the authors' model may be overly simplistic, and the discussion does not do justice to how their model reconciles with the body of work that already exists.

Reviewer #2 (Public review):

Shrestha et al investigated the role of IR receptors in the detection of 3 carboxylic acids in adult Drosophila. A low concentration of either of these carboxylic acids added to 2 mM sucrose (1% lactic acid (LA), citric acid (CA), or glycolic acid (GA)) stimulates the consumption of adult flies in choice conditions. The authors use this behavioral test to screen the impact of mutations within 33 receptors belonging to the IR family, a large family of receptors derived from glutamate receptors and expressed both in the olfactory and gustatory sensilla of insects. Within the panel of mutants tested, they observed that 3 receptors (IR25a, IR51b, and IR76b) impaired the detection of LA, CA, and GA, and that 2 others impacted the detection of CA and GA (IR94a and IR94h). Interestingly, impairing IR51b, IR94a, and IR94h did not affect the electrophysiological responses of external gustatory sensilla to LA, CA, and GA. Thanks to the use of GAL4 strains associated with these receptors and thanks to the use of poxn mutants (which do not develop external gustatory sensilla but still have functional internal receptors), they show evidence that IR94a and IR94h are only expressed in two clusters of gustatory neurons of the pharynx, respectively in the VCSO (ventral cibarial sense organ) and in the VCSO + LSO (labral sense organ). As for IR51b, the GAL4 approach was not successful but RT-PCR made on different parts of the insect showed an expression both in the pharyngeal organs and in peripheral receptors. These main findings are then complemented by a host of additional experiments meant to better understand the respective roles of IR94a and IR94h, by using optogenetics and brain calcium imaging using GCamp6. They also report a failed attempt to co-express IR51b, IR94a, and IR94h into external receptors, a co-expression which did not confer the capability of bitter-sensitive cells (expressing GR33a-GAL4) to detect either of the carboxylic acids. These data complete and expand previous observations made on this group and others, and dot to 2 new IR receptors which show an unsuspected specific expression, into organs that still remain difficult to study.

The conclusions of this paper are supported by the data presented, but it remains difficult to make general conclusions as concerns the mechanisms by which carboxylic acids are detected.

(1) All experiments were done with 1% of carboxylic acids. What is the dose dependency of the behavioral responses to these acids, and is it conceivable that other receptors are involved at other concentrations?

(2) One result needs to be better discussed and hypotheses proposed - which is why the mutations of most receptors lead to a loss of detection (mutant flies become incapable of detecting the acid) while mutations in IR94a and IR94h make CA and GA potent deterrents. Does it mean that CA and GA are detected by another set of receptors that, when activated, make flies actively avoid CA and GA? In that case, do the authors think that testing receptors one by one is enough to uncover all the receptors participating in the detection of these substances?

(3) The paper needs to be updated with a recent paper published by Guillemin et al (2024), indicating that LA is detected externally by a combination of IR94e, IR76b and IR25a. IR25a might help to form a fully functional receptor in GR33a neurons (a former study from Chen et al (2017) indicate that IR25a is expressed in all gustatory neurons of the pharynx).

(4) Although it was not the main focus of the paper, it would have been most interesting if the cells expressing IR94a and IR94h were identified, and placed on the functional map proposed by the group of Dahanukar (Chen et al 2017 Cell Reports, Chen et al 2019 Cell Reports).

Reviewer #3 (Public review):

Summary:

In this work, the authors investigated the molecular and cellular basis of sour taste perception in Drosophila melanogaster, focusing on identifying receptors that mediate attractive responses to certain carboxylic acids. It builds on previous work from the same group that had identified the IR co-receptors IR25a and IR76b for this sensory process, screening a set of mutants in IRs to identify three, IR51b, IR94a, and IR94h, required for feeding preference responses to some or all of the tested acids.

Strengths:

The work is of interest because it assigns sensory roles to IRs of previously unknown function, in particular IR94a and IR94h, and points to pharyngeal neurons in which these receptors are expressed as the relevant sensory neurons (potentially with different roles for IR94a- and IR94h-expressing neurons). The work combines elegant genetics, simple but effective feeding and taste assays, chemo-/opto-genetic activation, and some calcium imaging. Overall the presented data look solid and well-controlled.

Weaknesses:

The in situ expression analysis relies entirely on transgenic driver lines for IR94a and IR94h (which had been previously described, though not fully cited in this work). Importantly, given that many of the behavioral experiments (genetic rescue, physiology, artificial activation) use the IR94a and IR94h GAL4 driver lines, it would be helpful to validate that these faithfully reflect IR94a and IR94h expression (as far as I can tell, such validation wasn't done in the original papers describing these lines as part of a large collection of IR drivers). For IR51b, pharyngeal expression is concluded indirectly from non-quantitative RT-PCR analysis (genetic reporters did not work). The lack of direct detection of gene/protein expression (for example, through RNA FISH, immunofluorescence, or protein tagging) would have made for a more complete characterization of these receptors (for example, there is no direct evidence that they also express IR25a and IR76b, as one might expect). Finally, the relationship of IR94a and IR94h neurons to other types of pharyngeal neurons remains unclear, as are their projection patterns in the SEZ.

Conceptually, the work is of interest mostly to those in the immediate field; there have been a very large number of studies in the past decade (several from this lab) characterizing the contributions of different IRs to various chemosensory processes. The current work doesn't lend much insight into the nature of the minimal functional unit of gustatory IRs (reconstitution of a functional IR in a heterologous neuron/cell has not been achieved here, but this is a limitation of many other previous studies), nor to how different pharyngeal sensory pathways might collaborate to control behavior. Nevertheless, the findings provide a useful contribution to the literature.

Reviewer #2 (Public review):

Summary:

Salt stress is a significant and growing concern for agriculture in some parts of the world. While the effects of sodium excess have been studied in Arabidopsis and (many) crop species, most studies have focused on Na uptake, toxicity and overall effects on yield, rather than on developmental responses to excess Na, per se. The work by Ishka and colleagues aims to fill this gap.

Working from an existing dataset that exposed a diverse panel of A. thaliana accessions to control, moderate, and severe salt stress, the authors identify candidate loci associated with altering the root:shoot ratio under salt stress. Following a series of molecular assays, they characterize a DUF247 protein which they dub SR3G, which appears to be a negative regulator of root growth under salt stress.

Overall, this is a well-executed study which demonstrates the functional role played by a single gene in plant response to salt stress in Arabidopsis.

Review of revised manuscript:

The authors have addressed my point-by-point comments to my satisfaction. In the cases where they have changed their manuscript language, clarified figures, or added analyses I have no further comment. In some cases, there is a fruitful back-and-forth discussion of methodology which I think will be of interest to readers.

I have nothing to add during this round of review. I think that the paper and associated discussion will make a nice contribution to the field

Reviewer #1 (Public review):

Summary:

The authors aim to assess the effect of salt stress on root:shoot ratio, identify the underlying genetic mechanisms, and evaluate their contribution to salt tolerance. To this end, the authors systematically quantified natural variations in salt-induced changes in root:shoot ratio. This innovative approach considers the coordination of root and shoot growth rather than exploring biomass and the development of each organ separately. Using this approach, the authors identified a gene cluster encoding eight paralog genes with a domain-of-unknown-function 247 (DUF247), with the majority of SNPs clustering into SR3G (At3g50160). In the manuscript, the authors utilized an integrative approach that includes genomic, genetic, evolutionary, histological, and physiological assays to functionally assess the contribution of their genes of interest to salt tolerance and root development.

Comments on revisions:

As the authors correctly noted, variations across samples, genotypes, or experiments make achieving statistical significance challenging. Should the authors choose to emphasize trends across experiments to draw biological conclusions, careful revisions of the text, including titles and figure legends, will be necessary to address some of the inconsistencies between figures (see examples below). However, I would caution that this approach may dilute the overall impact of the work on SR3G function and regulation. Therefore, I strongly recommend pursuing additional experimental evidence wherever possible to strengthen the conclusions.

(1) Given the phenotypic differences shown in Figures S17A-B, 10A-C, and 6A, the statement that "SR3G does not play a role in plant development under non-stress conditions" (lines 680-681) requires revision to better reflect the observed data.<br /> (2) I agree with the authors that detecting expression differences in lowly expressed genes can be challenging. However, as demonstrated in the reference provided (Lu et al., 2023), a significant reduction in WRKY75 expression is observed in T-DNA insertion mutant alleles of WRKY75. In contrast, Fig. 9B in the current manuscript shows no reduction in WRKY75 expression in the two mutant alleles selected by the authors, which suggests that these alleles cannot be classified as loss-of-function mutants (line 745). Additionally, the authors note that the wrky75 mutant exhibits reduced main root length under salt stress, consistent with the phenotype reported by Lu et al. (2023). However, other phenotypic discrepancies exist between the two studies. For example, 1) Lu et al. (2023) report that w¬rky75 root length is comparable to WT under control conditions, whereas the current manuscript shows that wrky75 root growth is significantly lower than WT; 2) under salt stress, Lu et al. (2023) show that wrky75 accumulates higher levels of Na+, whereas the current study finds Na+ levels in wrky75 indistinguishable from WT. To confirm the loss of WRKY75 function in these T-DNA insertion alleles the authors should provide additional evidence (e.g., Western blot analysis).

Reviewer #1 (Public review):

Summary:

In this study, the authors used a chronic murine dietary restriction model to study the effects of chronic malnutrition on controls of bacterial infection and overall immunity, including cellularity and functions of different immune cell types. They further attempted to determine whether refeeding can revert the infection susceptibility and immunodeficiency. Although refeeding here improves anthropometric deficits, the authors of this study show that this is insufficient to recover the impairments across the immune cell compartments.

Strengths:

The manuscript is well-written and conceived around a valid scientific question. The data supports the idea that malnutrition contributes to infection susceptibility and causes some immunological changes. The malnourished mouse model also displayed growth and development delays. The work's significance is well justified. Immunological studies in the malnourished cohort (human and mice) are scarce, so this could add valuable information.

Weaknesses:

The assays on myeloid cells are limited, and the study is descriptive and overstated. The authors claim that "this work identifies a novel cellular link between prior nutritional state and immunocompetency, highlighting dysregulated myelopoiesis as a major." However, after reviewing the entire manuscript, I found no cellular mechanism defining the link between nutritional state and immunocompetency.

Reviewer #2 (Public review):

Summary:

Sukhina et al. use a chronic murine dietary restriction model to investigate the cellular mechanisms underlying nutritionally acquired immunodeficiency as well as the consequences of a refeeding intervention. The authors report a substantial impact of undernutrition on the myeloid compartment, which is not rescued by refeeding despite rescue of other phenotypes including lymphocyte levels, and which is associated with maintained partial susceptibility to bacterial infection.

Strengths:

Overall, this is a nicely executed study with appropriate numbers of mice, robust phenotypes, and interesting conclusions, and the text is very well-written. The authors' conclusions are generally well-supported by their data.

Weaknesses:

There is little evaluation of known critical drivers of myelopoiesis (e.g. PMID 20535209, 26072330, 29218601) over the course of the 40% diet, which would be of interest with regard to comparing this chronic model to other more short-term models of undernutrition.

Further, the microbiota, which is well-established to be regulated by undernutrition (e.g. PMID 22674549, 27339978, etc.), and also well-established to be a critical regulator of hematopoiesis/myelopoiesis (e.g. PMID 27879260, 27799160, etc.), is completely ignored here.

Reviewer #3 (Public review):

Summary:

Sukhina et al are trying to understand the impacts of malnutrition on immunity. They model malnutrition with a diet switch from ad libitum to 40% caloric restriction (CR) in post-weaned mice. They test impacts on immune function with listeriosis. They then test whether re-feeding corrects these defects and find aspects of emergency myelopoiesis that remain defective after a precedent period of 40% CR. Overall, this is a very interesting observational study on the impacts of sudden prolonged exposure to less caloric intake.

Strengths:

The study is rigorously done. The observation of lasting defects after a bout of 40% CR is quite interesting. Overall, I think the topic and findings are of interest.

Weaknesses:

While the observations are interesting, in this reviewer's opinion, there is both a lack of mechanistic understanding of the phenomena and also some lack of resolution/detail about the phenomena itself. Addressing the following major issues would be helpful towards aspects of both:

(1) Is it calories, per se, or macro/micronutrients that drive these phenotypes observed with 40% CR. At the least, I would want to see isocaloric diets (primarily protein, fat, or carbs) and then some of the same readouts after 40% CR. Ie does low energy with relatively more eg protein prevent immunosuppression (as is commonly suggested)? Micronutrients would be harder to test experimentally and may be out of the scope of this study. However, it is worth noting that many of the malnutrition-associated diseases are micronutrient deficiencies.

(2) Is immunosuppression a function of a certain weight loss threshold? Or something else? Some idea of either the tempo of immunosuppression (happens at 1, in which weight loss is detected; vs 2-3, when body length and condition appear to diverge; or 5 weeks), or grade of CR (40% vs 60% vs 80%) would be helpful since the mechanism of immunosuppression overall is unclear (but nailing it may be beyond the scope of this communication).

(3) Does an obese mouse that gets 40% CR also become immunodeficient? As it stands, this ad libitum --> 40% CR model perhaps best models problems in the industrial world (as opposed to always being 40% CR from weaning, as might be more common in the developing world), and so modeling an obese person losing a lot of weight from CR (like would be achieved with GLP-1 drugs now) would be valuable to understanding generalizability.

(4) Generalizing this phenomenon as "bacterial" with listeriosis, which is more like a virus in many ways (intracellular phase, requires type I IFN, etc.) and cannot be given by the natural route of infection in mice, may not be most accurate. I would want to see an experiment with E.Coli, or some other bacteria, to test the statement of generalizability (ie is it bacteria, or type I IFN-pathway dominant infections, like viruses). If this is unique listeriosis, it doesn't undermine the story as it is at all, but it would just require some word-smithing.

(5) Previous reports (which the authors cite) implicate Leptin, the levels of which scale with fat mass, as "permissive" of a larger immune compartment (immune compartment as "luxury function" idea). Is their phenotype also leptin-mediated (ie leptin AAV)?

(6) The inability of re-feeding to "rescue" the myeloid compartment is really interesting. Can the authors do a bone marrow transplantation (CR-->ad libitum) to test if this effect is intrinsic to the CR-experienced bone marrow?

(7) Is the defect in emergency myelopoiesis a defect in G-CSF? Ie if the authors injected G-CSF in CR animals, do they equivalently mobilize neutrophils? Does G-CSF supplementation (as one does in humans) rescue host defense against Listeria in the CR or re-feeding paradigms?

Reviewer #1 (Public review):

Syngnathid fishes (seahorses, pipefishes, and seadragons) present very particular and elaborated features among teleosts and a major challenge is to understand the cellular and molecular mechanisms that permitted such innovations and adaptations. The study provides a valuable new resource to investigate the morphogenetic basis of four main traits characterizing syngnathids, including the elongated snout, toothlessness, dermal armor and male pregnancy. More particularly, the authors have focused on a late stage of pipefish organogenesis to perform single-cell RNA-sequencing (scRNA-seq) completed by in situ hybridization analyses to identify molecular pathways implicated in the formation of the different specific traits.<br /> The first set of data explores the scRNA-seq atlas composed of 35,785 cells from two samples of gulf pipefish embryos that authors have been able to classify into major cell types characterizing vertebrate organogenesis, including epithelial, connective, neural and muscle progenitors. To affirm identities and discover potential properties of clusters, authors primarily use KEGG analysis that reveals enriched genetic pathways in each cell types. After revisions, the authors have provided extended supplementary files to well interpret the dataset and some statements have been clarified. I thank the authors for the revisions/completions of ISH results compared to initial submission.

To conclude, the scRNA-seq dataset in this unconventional model organism will be useful for the community and will provide clues for future research to understand the extraordinary evolution of the Syngnathidae family.

Reviewer #2 (Public review):

Summary:

The authors present the first single-cell atlas for syngathid fishes, providing a resource for future evolution & development studies in this group.

Strengths:

The concept here is simple and I find the manuscript to be well written. I like the in situ hybridization of marker genes >> this is really nice. I also appreciate the gene co-expression analysis to identify modules of expression. There are no explicit hypotheses tested in the manuscript, but the discovery of these cell types should have value in this organism and in the determination of morphological novelties in seahorses and their relatives.

Weaknesses:

I think there are a few computational analyses that might improve the generality of the results.

(1) The cell types: The authors use marker gene analysis and KEGG pathways to identify cell types. I'd suggest a tool like SAMap (https://elifesciences.org/articles/66747) which compares single cell data sets from distinct organisms to identify 'homologous' cell types -- I imagine the zebrafish developmental atlases could serve as a reasonable comparative reference.

(2) Trajectory analyses: Authors suggest that their analyses might identify progenitor cell states and perhaps related differentiated states. They might explore cytoTRACE and/or pseudotime-based trajectory analyses to more fully delineate these ideas.

(3) Cell-cell communication: I think it's very difficult to identify 'tooth primordium' cell types, because cell types won't be defined by organ in this way. for instance dental glia will cluster with other glia, dental mesenchyme will likely cluster with other mesenchymal cell types. so the histology and ISH in most convincing in this regard. having said this, given the known signaling interactions in the developing tooth (and in development generally) the authors might explore cell-cell communication analysis (e.g., CellChat) to identify cell types that may be interacting.

Comments on revisions:

I feel essentially the same about this manuscript. it's a useful resource for future experimental forays into this unique system. The team made improvements to deal with comments from other reviewers related to quality of confirmatory in situ hybridization. This is good.

Regarding their response that one can't use CellChat if you're not working in mice or human, this is inaccurate. the assumption one makes is that ligand-receptor pairs and signaling pathways have conserved functions across animals (vertebrates). It's the same assumption the authors make when using the KEGG pathway to score enrichment of pathways in clusters. CellChat used in fishes in Johnson et al 2023 Nature Communications | ( 2023) 14:4891.

Reviewer #3 (Public review):

Summary:

This study established a single-cell RNA sequencing atlas of pipefish embryos. The results obtained identified unique gene expression patterns for pipefish-specific characteristics, such as fgf22 in the tip of the palatoquadrate and Meckel's cartilage, broadly informing the genetic mechanisms underlying morphological novelty in teleost fishes. The data obtained are unique and novel, potentially important in understanding fish diversity. Thus, I would enthusiastically support this manuscript if the authors improve it to generate stronger and more convincing conclusions than the current forms.

Weakness:

Regarding the expression of sfrp1a and bmp4 dorsal to the elongating ethmoid plate and surrounding the ceratohyal: Are their expression patterns spatially extended or broader compared to the pipefish ancestor? Is there a much closer species available to compare gene expression patterns with pipefish? Did the authors consider using other species closely related to pipefish for ISH? Sfrp1a and bmp4 may be expressed in the same regions of much more closely related species without face elongation. I understand that embryos of such species are not always accessible, but it is also hard to argue responsible genes for a specific phenotype by only comparing gene expression patterns between distantly related species (e.g., pipefish vs. zebrafish). Due to the same reason, I would not directly compare/argue gene expression patterns between pipefish and mice, although I should admit that mice gene expression patterns are sometimes helpful to make a hypothesis of fish evolution. Alternatively, can the authors conduct ISH in other species of pipefish? If the expression patterns of sfrp1a and bmp4 are common among fishes with face elongation, the conclusion would become more solid. If these embryos are not available, is it possible to reduce the amount of Wnt and BMP signal using Crispr/Cas, MO, or chemical inhibitor? I do think that there are several ways to test the Wnt and/or BMP hypothesis in face elongation.

Reviewer #1 (Public review):

Summary:

This study investigates an intriguing question in cognitive control from a temporal dynamics perspective: why does concurrent verbal working memory load eliminate the color-word Stroop effect? Through a series of thorough data analyses, the authors propose that verbal working memory load occupies the stimulus-response mapping resources represented by theta-band activity, thereby disrupting the mapping process for task-irrelevant distractors. This reduces the response tendency to the distractors, ultimately leading to the elimination of the Stroop effect.

Strengths:

The behavioral and neural evidence presented in the manuscript is solid, and the findings have valuable theoretical implications for research on Stroop conflict processing.

Weaknesses:

There are several areas where the manuscript could be improved.

Major Comments:

(1) In the Results section, the rationale behind selecting the beta band for the central (C3, CP3, Cz, CP4, C4) regions and the theta band for the fronto-central (Fz, FCz, Cz) regions is not clearly explained in the main text. This information is only mentioned in the figure captions. Additionally, why was the beta band chosen for the S-ROI fronto-central region and the theta band for the S-ROI central region? Was this choice influenced by the MVPA results?

(2) In the Data Analysis section, line 424 states: "Only trials that were correct in both the memory task and the Stroop task were included in all subsequent analyses. In addition, trials in which response times (RTs) deviated by more than three standard deviations from the condition mean were excluded from behavioral analyses." The percentage of excluded trials should be reported. Also, for the EEG-related analyses, were the same trials excluded, or were different criteria applied?

(3) In the Methods section, line 493 mentions: "A 400-200 ms pre-stimulus time window was selected as the baseline time window." What is the justification in the literature for choosing the 400-200 ms pre-stimulus window as the baseline? Why was the 200-0 ms pre-stimulus period not considered?

(4) Is the primary innovation of this study limited to the methodology, such as employing MVPA and RSA to establish the relationship between late theta activity and behavior?

(5) On page 14, lines 280-287, the authors discuss a specific pattern observed in the alpha band. However, the manuscript does not provide the corresponding results to substantiate this discussion. It is recommended to include these results as supplementary material.

(6) On page 16, lines 323-328, the authors provide a generalized explanation of the findings. According to load theory, stimuli compete for resources only when represented in the same form. Since the pre-memorized Chinese characters are represented semantically in working memory, this explanation lacks a critical premise: that semantic-response mapping is also represented semantically during processing.

(7) The classic Stroop task includes both a manual and a vocal version. Since stimulus-response mapping in the vocal version is more automatic than in the manual version, it is unclear whether the findings of this study would generalize to the impact of working memory load on the Stroop effect in the vocal version.

(8) While the discussion section provides a comprehensive analysis of the study's results, the authors could further elaborate on the theoretical and practical contributions of this work.

Reviewer #2 (Public review):

Summary:

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strengths:

(1) The experiment is well-designed.

(2) The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Weaknesses:

(1) As the researchers mentioned, a previous study reported a diminished Stroop effect with concurrent working memory tasks to memorize meaningless visual shapes rather than memorize Chinese characters as in the study. My main concern is that lower-level graphic processing when memorizing visual shapes also influences the Stroop effect. The stage of Stroop conflict processing affected by the working memory load may depend on the specific content of the concurrent working memory task. If that's the case, I sense that the generalization of this finding may be limited.

(2) The P1 and N450 components are sensitive to congruency in previous studies as mentioned by the researchers, but the results in the present study did not replicate them. This raised concerns about data quality and needs to be explained.

Reviewer #1 (Public review):

Summary:

Assessment of cardiac LEC transcriptomes post-MI may yield new targets to improve lymphatic function. scRNAseq is a valid approach as cardiac LECs are rare compared to blood vessel endothelial cells.

Strengths:

Extensive bioinformatics approaches employed by the group

Weaknesses:

Too few cells included in scRNAseq data set and the spatial transcriptomics data that was exploited has little relevance, or rather specificity, for cardiac lymphatics. This study seems more a collection of preliminary transcriptomic data than a true scientific report to help advance the field.

Comments on revisions:

Thank you for the revision that helps clarify some outstanding questions.

(1) I still have questions relating to the relevance of the spatial maps generated and shown in fig 3C. They are supposedly generated using a 'molecular finger print' specific to each sub-cluster of LECs. However, given that at early stages postMI most populations are exceedingly rare in your analyses, could you please explain or comment on the relevance of the spatial maps?

(2) Fig 3 s1 would indicate that the population CaII is the majoritarian one in healthy hearts, while quantifications in Fig 3A show that rather the LEC Co subpopulation is majoritarian. Further, in mouse hearts histological analyses have demonstrated that cardiac lymphatics are restricted to the outer layers of the heart. This is not seen in your spatial maps. This seems to be the case only for the LEc Co population in healthy hearts, but not for other subpopulation signatures. Please explain.

(3) Further, the population of CaI, with 1 cell analysed in d3, but appears very prevalent in the spatial maps at d3. Please explain.

(4) In your list of 12 genes used as matrix anchors to identify LEC subpopulations in your screens, it is not apparent how LEC CaI, II and III differ so much as to allow selective detection of subpopulations. This similitude of profiles is supported by Fig 2F, and further explanations are needed to explain how the spatial maps of LEC ca subpopulations appear as distinct as shown in fig 3 S1 and Fig 3C.

Reviewer #2 (Public review):

Summary:

This study integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points post-MI. They identified four transcriptionally distinct subtypes of lymphatic endothelial cells and localized them in space. They observed that LECs subgroups are localized in different zones of infarcted heart with functions. Specifically, they demonstrated that LEC ca III may be involved in directly regulating myocardial injuries in the infarcted zone concerning metabolic stress, while LEC ca II may be related to the rapid immune inflammatory responses of the border zone in the early stage of MI. LEC ca I and LEC collection mainly participate in regulating myocardial tissue edema resolution in the middle and late stages post-MI. Finally, cell trajectory and Cell-Chat analyses further identified that LECs may regulate myocardial edema through Aqp1, and likely affect macrophage infiltration through the galectin9-CD44 pathway. The authors concluded that their study revealed the dynamic transcriptional heterogeneity distribution of LECs in different regions of the infarcted heart and that LECs formed different functional subgroups that may exert different bioeffects in myocardial tissue post-MI.

Strengths:

The study addresses a significant clinical challenge, and the results are of great translational value. All experiments were carefully performed, and their data support the conclusion.

Editors' comments (Public review):

Weaknesses:

(1) Figure 7C, 7E, 7I, and "Figure7-figure supplement 1 ": All data in these data panels are based on only n=3, which is insufficient. Sample sizes of n=3 are too low to correctly assess normality of distribution and, as a consequence, do not allow to select the appropriate parametric/non-parametric tests. Accordingly, no statistical comparison can be performed and all p values and symbols currently indicating statistically significant differences between groups must be removed.

(2) Figure 3A, 3B, or 3C: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(3) Figure 4 E and 4F: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(4) Figure 5: No information about n numbers per group is provided. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

Reviewer #1 (Public review):

Summary:

In the work Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly-developed in vitro KO model, with live imaging experiments, electrophysiological recordings and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with higher number of docked vesicles and shorter AZ. The evaluation of presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted, and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that results in chronic deletion of SNX4 in neurons. A convincing sequence of experimental techniques are applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at presynaptic site is relevant and of high interest in the field of synaptic physiology and on the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow to address the physiological roles of SNX4 at synaptic site and speculate on the underlying molecular mechanisms. The novel experiments on autophagy progression as well as spontaneous neurotransmission are well conducted, although do not assist for the explanation of the molecular mechanism underneath.

Comments on revisions:

Other implementations in the revised version are quite limited and would benefit from a more detailed presentation and description. i.e.: Sholl analysis in the new figure 1h, is presented with no definition of number of cells employed and standard deviations of the replication. The "simil" Sholl analysis performed on VAMP2 is still puzzling and some explanations on the reason for the constant value of VAMP2 fluorescent signal from less than 0 to 160 µm from the cell body is to be added. How is the increased number of active synapses explained? How is this related to shorter AZ and higher number of docked vesicles?

Reviewer #2 (Public review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

Comments on revisions:

I am fine with revision in principle. the authors have addressed my concerns.

Reviewer #3 (Public review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4.

Comments on revisions:

The authors have addressed a couple of the more major concerns with the manuscript, however many of the original weaknesses remain. The primary weakness being the lack of mechanism. It is disappointing that real-time VAMP2 trafficking was not investigated, and the authors justification as to why the experiment was not performed was not convincing (especially since this is the approach that all other groups employ to examine SV cargo trafficking). In a number of instances "contractual constraints" are referred to as an explanation for not performing additional experiments. It was unclear whether this refers to licencing issues with the mouse line or the lack of personnel to perform the work. Regardless it still leaves this work as somewhat incomplete.

Reviewer #1 (Public review):

Summary:

Arafi et al. present results of studies designed to better understand the effects of mutations in the presenilin-1 (PSEN1) gene on proteolytic processing of the amyloid precursor protein (APP). This is important because APP processing can result in the production of the amyloid β-protein (Aβ), a key pathologic protein in Alzheimer's disease (AD). Aβ exists in various forms that differ in amino acid sequence and assembly state. The predominant forms of Aβ are Aβ40 and Aβ42, which are 40 and 42 amino acids in length, respectively. Shorter and longer forms derive from processive proteolysis of the Aβ region of APP by the heterotetramer β-secretase, within which presenilin 1 possesses the active site of the enzyme. Each form may become toxic if it assembles into non-natively folded, oligomeric, or fibrillar structures. A deep mechanistic understanding of enzyme-substrate interactions is a first step toward the design and successful use of small-molecule therapeutics for AD.

The key finding of Arafi et al. is that three PSEN mutations display unusual profiles of effects on Aβ production that have novel implications for the stalled E-S complex hypothesis. PSEN1 F386S is unique in that initial ε cleavage is not reduced compared with WT PSEN1; only certain trimming steps are deficient, results consistent with FLIM experiments that reveal stabilized E-S complexes only in Aβ-rich regions in the cell. In contrast, PSEN1 A431E and A434T display very little ε cleavage and therefore very little overall Aβ production, suggesting a limited role of Aβ in the pathogenesis of these two mutants and pointing to stalled E-S complexes as the common factor. For the biochemist, this may not be surprising, but in the context of understanding and treating AD, it is immense because it shifts the paradigm from targeting the results of γ-secretase action, viz., Aβ oligomers and fibrils, to targeting initial Aβ production at the molecular level. It is the equivalent of taking cancer treatment from simple removal of tumorous tissue to prevention of tumor formation and growth. Arafi et al. have provided us with a blueprint for the design of small-molecule inhibitors of γ-secretase. The significance of this achievement cannot be overstated.

Strengths and weaknesses:

The comprehensiveness and rigor of the study are notable. Rarely have I reviewed a manuscript reporting the results of so many orthogonal experiments, all of which support the authors' hypotheses, and of so many excellent controls. In addition, as found in clinical trial reports, the limitations of the study were discussed explicitly. None of these significantly affected the conclusions of the study.

Reviewer #2 (Public review):

Summary:

The work by Arafi et al. shows the effect of Familial Alzheimer's Disease presenilin-1 mutants on endoproteinase and carboxylase activity. They have elegantly demonstrated how some mutants alter each step of processing. Together with FLIM experiments, this study provides additional evidence to support their 'stalled complex hypotheses'.

Strengths:

This is a beautiful biochemical work. The approach is comprehensive.

Weaknesses:

(1) It appears that the purified g-secretase complex generates the same amount of Ab40 and Ab42, which is quite different in cellular and biochemical studies. Is there any explanation for this?

(2) It has been reported the Ab production lines from Ab49 and Ab48 can be crossed with various combinations (PMID: 23291095 and PMID: 38843321). How does the production line crossing impact the interpretation of this work?

(3) In Figure 5, did the authors look at the protein levels of PS1 mutations and C99-720, as well as secreted Ab species? Do the different amounts of PS1 full-length and PS1-NTF/CTF influence FILM results?

(4) It is interesting that both Ab40 and Ab42 Elisa kits detect Ab43. Have the authors tested other kits in the market? It might change the interpretation of some published work.

Reviewer #1 (Public review):

In this study, Rosenblum et al introduce a novel and automatic way of calculating sleep cycles from human EEG. Previous results have shown that the slope of the non-oscillatory component of the power spectrum (called the aperiodic or fractal component) changes with sleep stage. Building on this, the authors present an algorithm that extracts the continuous-time fluctuations in the fractal slope and propose that peaks in this variable can be used to identify sleep cycle limits. Cycles defined in this way are termed "fractal cycles". The main focus of the article is a comparison of "fractal" and "classical" (ie defined manually based on the hypnogram) sleep cycles in numerous datasets.

The manuscript amply illustrates through examples the strong overlap between fractal and classical cycle identification. Accordingly, a high percentage (81%) can be matched one-to-one between methods and sleep cycle duration is well correlated (around R = 0.5). Moreover, the methods track certain global changes in sleep structure in different populations: shorter cycles in children and longer cycles in patients medicated with REM-suppressing anti-depressants. Finally, a major strength of the results is that they show similar agreement between fractal and classical sleep cycle length in 5 different data sets, showing that it is robust to changes in recording settings and methods.

The match between fractal and classical cycles is not one-to-one. For example, the fractal method identifies a correlation between age and cycle duration in adults that is not apparent with the classical method.<br /> The difference between the fractal and classical methods appear to be linked to the uncertain definition of sleep cycles since they are tied to when exactly the cycle begins/ends and whether or not to count cycles during fractured sleep architecture at sleep onset. Moreover, the discrepancies between the two are on the order of that found between classical cycles defined manually or via an automatic algorithm.

Overall the fractal cycle is an attractive method to study sleep architecture since it dispenses with time-consuming and potentially subjective manual identification of sleep cycles. However, given its difference with the classical method, it is unlikely that fractal scoring will be able to replace classical scoring directly. By providing a complementary quantification, it will likely contribute to refining the definition of sleep cycles that is currently ambiguous in certain cases. Moreover, it has the potential to be applied on animal studies which rarely deal with sleep cycle structure.

Reviewer #2 (Public review):

Summary:

This study focused on using strictly the slope of the power spectral density (PSD) to perform automated sleep scoring and evaluation of the durations of sleep cycles. The method appears to work well because the slope of the PSD is highest during slow-wave sleep, and lowest during waking and REM sleep. Therefore, when smoothed and analyzed across time, there are cyclical variations in the slope of the PSD, fit using an IRASA (Irregularly resampled auto-spectral analysis) algorithm proposed by Wen & Liu (2016).

Strengths:

The main novelty of the study is that the non-fractal (oscillatory) components of the PSD that are more typically used during sleep scoring can be essentially ignored because the key information is already contained within the fractal (slope) component. The authors show that for the most part, results are fairly consistent between this and conventional sleep scoring, but in some cases show disagreements that may be scientifically interesting.

Weaknesses:

The previous weaknesses were well-addressed by the authors in the revised manuscript. I will note that from the fractal cycle perspective, waking and REM sleep are not very dissimilar. Combining these states underlies some of the key results of this study.

Reviewer #1 (Public review):

Summary:

This study investigates an intriguing question in cognitive control from a temporal dynamics perspective: why does concurrent verbal working memory load eliminate the color-word Stroop effect? Through a series of thorough data analyses, the authors propose that verbal working memory load occupies the stimulus-response mapping resources represented by theta-band activity, thereby disrupting the mapping process for task-irrelevant distractors. This reduces the response tendency to the distractors, ultimately leading to the elimination of the Stroop effect.

Strengths:

The behavioral and neural evidence presented in the manuscript is solid, and the findings have valuable theoretical implications for research on Stroop conflict processing.

Comments on revisions:

The authors have addressed all concerns

Reviewer #2 (Public review):

Summary

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strength

The experiment is well designed.<br /> The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Comments on revisions:

All my concerns have been properly addressed, no further comments.

Reviewer #1 (Public review):

Summary:

The authors define a new metric for visual displays, derived from psychophysical response times, called visual homogeneity (VH). They attempt to show that VH is explanatory of response times across multiple visual tasks. They use fMRI to find visual cortex regions with VH-correlated activity. On this basis, they declare a new visual region in human brain, area VH, whose purpose is to represent VH for the purpose of visual search and symmetry tasks.

Link to original review: https://elifesciences.org/reviewed-preprints/93033v2/reviews#peer-review-0

Comments on latest version:

Authors rebuttal: We agree that visual homogeneity is similar to existing concepts such as target saliency, memorability etc. We have proposed it as a separate concept because visual homogeneity has an independent empirical measure (the reciprocal of target-absent search time in oddball search, or the reciprocal of same response time in a same-different task, etc) that may or may not be the same as other empirical measures such as saliency and memorability. Investigating these possibilities is beyond the scope of our study but would be interesting for future work. We have now clarified this in the revised manuscript (Discussion, p. 42).

Reviewer response to rebuttal: Neither the original ms nor the comments on that ms pretended that "visual homogeneity" was entirely separate from target saliency etc. So this is a response to a criticism that was never made. What the authors do claim, and what the comments question, is that they have successfully subsumed long-recognized psychophysical concepts like target saliency etc. under a new, uber-concept, "visual homogeneity" that explains psychophysical experimental results in a more unified and satisfying way. This subsumption of several well-established psychophysical concepts under a new, unified category is what reviewers objected to.

Authors rebuttal: However, we'd like to emphasize that the question of whether visual homogeneity is novel or related to existing concepts misses entirely the key contribution of our study.

Reviewer response to rebuttal: Sorry, but the claim of a new uber-concept in psychophysics, "visual homogeneity", is a major claim of the paper. The fact that it is not the only claim made does not absolve the authors from having to prove it satisfactorily.

"Authors rebuttal: "In addition, the large regions of VH correlations identified in Experiments 1 and 2 vs. Experiments 3 and 4 are barely overlapping. This undermines the claim that VH is a universal quantity, represented in a newly discovered area of visual cortex, that underlies a wide variety of visual tasks and functions."<br /> • We respectfully disagree with your assertion. First of all, there is partial overlap between the VH regions, for which there are several other obvious explanations that must be considered first before dismissing VH outright as a flawed construct. We acknowledge these alternatives in the Results (p. 27), and the relevant text is reproduced below.

"We note that it is not straightforward to interpret the overlap between the VH regions identified in Experiments 2 & 4. The lack of overlap could be due to stimulus differences (natural images in Experiment 2 vs silhouettes in Experiment 4), visual field differences (items in the periphery in Experiment 2 vs items at the fovea in Experiment 4) and even due to different participants in the two experiments. There is evidence supporting all these possibilities: stimulus differences (Yue et al., 2014), visual field differences (Kravitz et al., 2013) as well as individual differences can all change the locus of neural activations in object-selective cortex (Weiner and Grill-Spector, 2012a; Glezer and Riesenhuber, 2013). We speculate that testing the same participants on search and symmetry tasks using similar stimuli and display properties would reveal even larger overlap in the VH regions that drive behavior."

Reviewer response to rebuttal: The authors are saying that their results merely look unconvincing (weak overlap between VH regions defined in different experiments) because there were confounding differences between their experiments, in subject population, stimuli, etc. That is possible, but in that case it is up to the authors to show that their definition of a new "area VH" is convincing when the confounding differences are resolved, e.g. by using the same stimuli in the different experiments they attempt to agglomerate here. That would require new experiments, and none are offered in this revision.

Authors rebuttal: • Thank you for carefully thinking through our logic. We agree that a distance-to-centre calculation is entirely unnecessary as an explanation for target-present visual search. The similarity between target and distractor, so there is nothing new to explain here. However, this is a narrow and selective interpretation of our findings because you are focusing only on our results on target-present searches, which are only half of all our data. The other half is the target-absent responses which previously have had no clear explanation. You are also missing the fact that we are explaining same-different and symmetry tasks as well using the same visual homogeneity computation. We urge you to think more deeply about the problem of how to decide whether an oddball is present or not in the first place. How do we actually solve this task?

Reviewer response to rebuttal: It is the role of the authors to think deeply about their paper and on that basis present a clear and compelling case that readers can understand quickly and agree with. That is not done here.

Authors rebuttal: There must be some underlying representation and decision process. Our study shows that a distance-to-centre computation can actually serve as a decision variable to solve disparate property-based visual tasks. These tasks pose a major challenge to standard models of decision-making because the underlying representation and decision variable have been unclear. Our study resolves this challenge by proposing a novel computation that can be used by the brain to solve all these disparate tasks, and bring these tasks into the ambit of standard theories of decision-making.

Reviewer response to rebuttal: There is only a "challenge" if you accept the authors' a priori assumption that all of these tasks must have a common explanation and rely on a single neural mechanism. I do not accept that assumption, and I don't think the authors provide evidence to support the assumption. There is nothing "unclear" about how search, oddball, etc. have been thoroughly explained, separately, in the psychophysical literature that spans more than a century.

Authors rebuttal: • You are indeed correct in noting that both Experiment 1 & 2 involve oddball search, and so at the superficial level, it looks circular that the oddball search data of Experiment 1 is being used to explain the oddball search data of Experiment 2.<br /> However a deeper scrutiny reveals more fundamental differences: Experiment 1 consisted of only oddball search with the target appearing on the left or right, whereas Experiment 2 consisted of oddball search with the target either present or completely absent. In fact, we were merely using the search dissimilarities from Experiment 1 to reconstruct the underlying object representation, because it is well-known that neural dissimilarities are predicted well by search dissimilarities (Sripati & Olson, 2009; Zhivago et al, 2014).

Reviewer response to rebuttal: Here again the authors cite differences between their multiple experiments as a virtue that supports their conclusions. Instead, the experiments should have been designed for maximum similarity if the authors intended to explain them with the same theory.

Authors rebuttal: To thoroughly refute any lingering concern about circularity, we reasoned that the model predictions for Experiment 2 could have been obtained by a distance-to-center computation on any brain like object representation. To this end, we used object representations from deep neural networks pretrained on object categorization, whose representations are known to match well with the brain, and asked if a distance-to-centre computation on these representations could predict the search data in Experiment 2. This was indeed the case, and these results are now included an additional section in Supplementary Material (Section S1).

Reviewer response to rebuttal: The authors' claims are about human performance and how it is based on the human brain. Their claims are not well supported by the human experiments that they performed. It serves no purpose to redo the same experiments in silico, which cannot provide stronger evidence that compensates for what was lacking in the human data.

Authors rebuttal: "Confirming the generality of visual homogeneity<br /> We performed several additional analyses to confirm the generality of our results, and to reject alternate explanations.

First, it could be argued that our results are circular because they involve taking oddball search times from Experiment 1 and using them to explain search response times in Experiment 2. This is a superficial concern since we are using the search dissimilarities from Experiment 1 only as a proxy for the underlying neural representation, based on previous reports that neural dissimilarities closely match oddball search dissimilarities (Sripati and Olson, 2010; Zhivago and Arun, 2014). Nonetheless, to thoroughly refute this possibility, we reasoned that we would get similar predictions of the target present/absent responses in Experiment using any other brain-like object representation. To confirm this, we replaced the object representations derived from Experiment 1 with object representations derived from deep neural networks pretrained for object categorization, and asked if distance-to-center computations could predict the target present/absent responses in Experiment 2. This was indeed the case (Section S1).

Second, we wondered whether the nonlinear optimization process of finding the best-fitting center could be yielding disparate optimal centres each time. To investigate this, we repeated the optimization procedure with many randomly initialized starting points, and obtained the same best-fitting center each time (see Methods).

Third, to confirm that the above model fits are not due to overfitting, we performed a leave-one-out cross validation analysis. We left out all target-present and target-absent searches involving a particular image, and then predicted these searches by calculating visual homogeneity estimated from all other images. This too yielded similar positive and negative correlations (r = 0.63, p < 0.0001 for target-present, r = -0.63, p < 0.001 for target-absent).

Fourth, if heterogeneous displays indeed elicit similar neural responses due to mixing, then their average distance to other objects must be related to their visual homogeneity. We confirmed that this was indeed the case, suggesting that the average distance of an object from all other objects in visual search can predict visual homogeneity (Section S1).

Fifth, the above results are based on taking the neural response to oddball arrays to be the average of the target and distractor responses. To confirm that averaging was indeed the optimal choice, we repeated the above analysis by assuming a range of relative weights between the target and distractor. The best correlation was obtained for almost equal weights in the lateral occipital (LO) region, consistent with averaging and its role in the underlying perceptual representation (Section S1).

Finally, we performed several additional experiments on a larger set of natural objects as well as on silhouette shapes. In all cases, present/absent responses were explained using visual homogeneity (Section S2)."

Reviewer response to rebuttal: The authors can experiment on side questions for as long as they please, but none of the results described above answer the concern about how center-fitting undercuts the evidentiary value of their main results.

Authors rebuttal: • While it is true that the optimal center needs to be found by fitting to the data, there no particular mystery to the algorithm: we are simply performing a standard gradient-descent to maximize the fit to the data. We have described the algorithm clearly and are making our codes public. We find the algorithm to yield stable optimal centers despite many randomly initialized starting points. We find the optimal center to be able to predict responses to entirely novel images that were excluded during model training. We are making no assumption about the location of centre with respect to individual points. Therefore, we see no cause for concern regarding the center-finding algorithm.

Reviewer response to rebuttal: The point of the original comment was that center-fitting should not be done in the first place because it introduces unknowable effects.

•Authors rebuttal: Most visual tasks, such as finding an animal, are thought to involve building a decision boundary on some underlying neural representation. Even visual search has been portrayed as a signal-detection problem where a particular target is to be discriminated from a distractor. However none of these formulations work in the case of property-based visual tasks, where there is no unique feature to look for.<br /> We are proposing that, when we view a search array, the neural response to the search array can be deduced from the neural responses to the individual elements using well-known rules, and that decisions about an oddball target being present or absent can be made by computing the distance of this neural response from some canonical mean firing rate of a population of neurons. This distance to center computation is what we denote as visual homogeneity. We have revised our manuscript throughout to make this clearer and we hope that this helps you understand the logic better.<br /> • You are absolutely correct that the stimulus complexity should matter, but there are no good empirically derived measures for stimulus complexity, other than subjective ratings which are complex on their own and could be based on any number of other cognitive and semantic factors. But considering what factors are correlated with target-absent response times is entirely different from asking what decision variable or template is being used by participants to solve the task.

Reviewer response to rebuttal: If stimulus complexity is what matters, as the authors agree here, then it is incumbent on them to measure stimulus complexity. The difficulty of measuring stimulus complexity does not justify avoiding the problem with an analysis that ignores complexity.

Authors rebuttal: • We have provided empirical proof for our claims, by showing that target-present response times in a visual search task are correlated with "different" responses in the same-different task, and that target-absent response times in the visual search task are correlated with "same" responses in the same-different task (Section S4).

Reviewer response to rebuttal: Sorry, but there is still no reason to think that same-different judgments are based on a mythical boundary halfway between the two. If there is a boundary, it will be close to the same end of the continuum, where subjects might conceivably miss some tiny difference between two stimuli. The vast majority of "different" stimuli will be entirely different from the same stimulus, producing no confusability, and certainly not a decision boundary halfway between two extremes.

Authors rebuttal: • Again, the opposite correlations between target present/absent search times with VH are the crucial empirical validation of our claims that a distance-to-center calculation explain how we perform these property-based tasks. The VH predictions do not fully explain the data. We have explicitly acknowledged this shortcoming, so we are hardly dismissing it as a problem.

Reviewer response to rebuttal: The authors' acknowledgement of flaws in the ms does not argue in favor of publication, but rather just the opposite.

Authors rebuttal: • Finding an oddball, deciding if two items are same or different and symmetry tasks are disparate visual tasks that do not fit neatly into standard models of decision-making. The key conceptual advance of our study is that we propose a plausible neural representation and decision variable that allows all three property-based visual tasks to be reconciled with standard models of decision-making.

Reviewer response to rebuttal: The original comment stands as written. Same/different will have a boundary very close to the "same" end of the continuum. The boundary is only halfway between two choices if the stimulus design forces the boundary to be there, as in the motion and cat/dog experiments.

Authors rebuttal: "There is no inherent middle point boundary between target present and target absent. Instead, in both types of trial, maximum information is present when target and distractors are most dissimilar, and minimum information is present when target and distractors are most similar. The point of greatest similarity occurs at then limit of any metric for similarity. Correspondingly, there is no middle point dip in information that would produce greater difficulty and higher response times. Instead, task difficulty and response times increase monotonically with similarity between targets and distractors, for both target present and target absent decisions. Thus, in Figs. 2F and 2G, response times appear to be highest for animals, which share the largest numbers of closely similar distractors."<br /> • Your alternative explanation rests on vague factors like "maximum information" which cannot be quantified. By contrast we are proposing a concrete, falsifiable model for three property-based tasks - same/different, oddball present/absent and object symmetry. Any argument based solely on item similarity to explain visual search or symmetry responses cannot explain systematic variations observed for target-absent arrays and for symmetric objects, for the reasons explained earlier.

Reviewer response to rebuttal: There is nothing vague about this comment. The authors use an analysis that assumes a decision boundary at the centerpoint of their arbitrarily defined stimulus space. This assumption is not supported, and it is unlikely, considering that subjects are likely to notice all but the smallest variations between same and different stimuli, putting the boundary nearly at the same end of the continuum, not the very middle.

Authors rebuttal: "(1) The area VH boundaries from different experiments are nearly completely non-overlapping.

In line with their theory that VH is a single continuum with a decision boundary somewhere in the middle, the authors use fMRI searchlight to find an area whose responses positively correlate with homogeneity, as calculated across all of their target present and target absent arrays. They report VH-correlated activity in regions anterior to LO. However, the VH defined by symmetry Experiments 3 and 4 (VHsymmetry) is substantially anterior to LO, while the VH defined by target detection Experiments 1 and 2 (VHdetection) is almost immediately adjacent to LO. Fig. S13 shows that VHsymmetry and VHdetection are nearly non-overlapping. This is a fundamental problem with the claim of discovering a new area that represents a new quantity that explains response times across multiple visual tasks. In addition, it is hard to understand why VHsymmetry does not show up in a straightforward subtraction between symmetric and asymmetric objects, which should show a clear difference in homogeneity."

• We respectfully disagree. The partial overlap between the VH regions identified in Experiments 1 & 2 can hardly be taken as evidence against the quantity VH itself, because there are several other obvious alternate explanations for this partial overlap, as summarized earlier as well. The VH region does show up in a straightforward subtraction between symmetric and asymmetric objects (Section S7), so we are not sure what the Reviewer is referring to here.

Reviewer response to rebuttal: In disagreeing with the comment quoted above, the authors are maintaining that a new functional area of cerebral cortex can be declared even if that area changes location on the cortical map from one experiment to another. That position is patently absurd.

Authors rebuttal: "(3) Definition of the boundaries and purpose of a new visual area in the brain requires circumspection, abundant and convergent evidence, and careful controls.

Even if the VH metric, as defined and calculated by the authors here, is a meaningful quantity, it is a bold claim that a large cortical area just anterior to LO is devoted to calculating this metric as its major task. Vision involves much more than target detection and symmetry detection. Cortex anterior to LO is bound to perform a much wider range of visual functionalities. If the reported correlations can be clarified and supported, it would be more circumspect to treat them as one byproduct of unknown visual processing in cortex anterior to LO, rather than treating them as the defining purpose for a large area of visual cortex."

• We totally agree with you that reporting a new brain region would require careful interpretation and abundant and converging evidence. However, this requires many studies worth of work, and historically category-selective regions like the FFA have achieved consensus only after they were replicated and confirmed across many studies. We believe our proposal for the computation of a quantity like visual homogeneity is conceptually novel, and our study represents a first step that provides some converging evidence (through replicable results across different experiments) for such a region. We have reworked our manuscript to make this point clearer (Discussion, p 32).

Reviewer response to rebuttal: Indeed, declaring a new brain area depends on much more work than is done here. Thus, the appropriate course here is to wait before claiming to have identified a new cortical area.

Reviewer #2 (Public review):

Summary:

This study proposes visual homogeneity as a novel visual property that enables observers perform to several seemingly disparate visual tasks, such as finding an odd item, deciding if two items are same, or judging if an object is symmetric. In Exp 1, the reaction times on several objects were measured in human subjects. In Exp 2, visual homogeneity of each object was calculated based on the reaction time data. The visual homogeneity scores predicted reaction times. This value was also correlated with the BOLD signals in a specific region anterior to LO. Similar methods were used to analyze reaction time and fMRI data in a symmetry detection task. It is concluded that visual homogeneity is an important feature that enables observers to solve these two tasks.

Strengths:

(1) The writing is very clear. The presentation of the study is informative.

(2) This study includes several behavioral and fMRI experiments. I appreciate the scientific rigor of the authors.

Weaknesses:

Before addressing the manuscript itself, I would like to comment the review process first. Having read the lasted revised manuscript, I shared many of the concerns raised by the two reviewers in the last two rounds of review. It appears that the authors have disagreed with the majority of comments made by the two reviewers. If so, I strongly recommend that the authors proceed to make this revision as a Version of Record and conclude this review process. According to eLife's policy that the authors have the right to make a Version of Record at any time during the review process, and I fully respect that right. However, I also ask that the authors respect the reviewer's right to retain the comments regarding this paper.

Beside that, I still have several further questions about this study.

(1) My main concern with this paper is the way visual homogeneity is computed. On page 10, lines 188-192, it says: "we then asked if there is any point in this multidimensional representation such that distances from this point to the target-present and target-absent response vectors can accurately predict the target-present and target-absent response times with a positive and negative correlation respectively (see Methods)". This is also true for the symmetry detection task. If I understand correctly, the reference point in this perceptual space was found by deliberating satisfying the negative and positive correlations in response times. And then on page 10, lines 200-205, it shows that the positive and negative correlations actually exist. This logic is confusing. The positive and negative correlations emerge only because this method is optimized to do so. It seems more reasonable to identify the reference point of this perceptual space independently, without using the reaction time data. Otherwise, the inference process sounds circular. A simple way is to just use the mean point of all objects in Exp 1, without any optimization towards reaction time data.<br /> I raised this question in my initial review. However, the authors did not address whether the positive and negative correlations still hold if the mean point is defined as the reference point without any optimization. The authors also argue that it is similar to a case of fitting a straight line. It is fine that the authors insist on the straight line (e.g., correlation). However, I would not call "straight line correlations" a "quantitative model" as a high-profile journals like eLife. Please remove all related arguments of a novel quantitative model.

(2) Visual homogeneity (at least given the current form) is an unnecessary term. It is similar to distractor heterogeneity/distractor variability/distractor saliency in literature. However, the authors attempt to claim it as a novel concept. Both R1 and me raised this question in the very first review. However, the authors refused to revise the manuscript. In the last review, I mentioned this and provided some example sentences claiming novelty. The authors only revised the last sentence of the abstract, and even did not bother to revise the last sentence of significance: "we show that these tasks can be solved using a simple property WE DEFINE as visual homogeneity". Also, lines 851 still shows "we have defined a NOVEL image property, visual homogeneity...". I am confused about whether the authors agree or disagree that "visual homogeneity is an unnecessary term". If the authors agree, they should completely remove the related phrase throughout the paper. If not, they should keep all these and state the reasons. I don't think this is a correct approach to revising a manuscript.

(3) If the authors agree that visual homogeneity is not new, I suggest a complete rewrite of the title, abstract, significance, and introduction. Let me ask a simple question, can we remove "visual homogeneity" and use some more well-established term like "image feature similarity"? If yes, visual homogeneity is unnecessary.

(4) If I understand it correctly, one of the key findings of this paper is "the response times for target-present searches were positively correlated with visual homogeneity. By contrast, the response times for target-absent searches were negatively correlated with visual homogeneity" (lines 204-207). I think the authors have already acknowledged that this positive correlation is not surprising at all because it reflects the classic target-distractor similarity effect. If this is the case, please completely remove the positive correlation as a novel prediction and finding.

(5) In my last review, I mentioned the seminal paper by Duncan and Humphreys (1989) has clearly stated that "difficulty increases with increased similarity of targets to nontargets and decreased similarity between nontargets" (the sentence in their abstract). Here, "similarity between nontargets" is the same as the visual homogeneity defined here. Similar effects have been shown in Duncan (1989) and Nagy, Neriani, and Young (2005). See also the inconsistent results in Nagy& Thomas, 2003, Vicent, Baddeley, Troscianko&Gilchrist, 2009. More recently, Wei Ji Ma has systematically investigated the effects of heterogeneous distractors in visual search. I think the introduction part of Wei Ji Ma's paper (2020) provides a nice summary of this line of research.

Thanks to the authors' revision, I now better understand the negative correlation. The between-distrator similarity mentioned above describes the heterogeneity of distractors WITHIN an image. However, if I understand it correctly, this study aims to address the negative correlation of reaction time and target-absent stimuli ACROSS images. In other words, why do humans show a shorter reaction time to an image of four pigeons than to an image of four dogs (as shown in Figure 2C), simply because the later image is closer to the reference point of the image space. In this sense, this negative correlation is indeed not the same as distractor heterogeneity. However, this is known as the saliency effect or oddball effects. For example, it seems quite natural to me that humans respond faster to a fish image if the image set contains many images of four-leg dogs that look very different from fish. If this is indeed a saliency effect, why should we define a new term "visual homogeneity"?

(6) The section "key predictions" is quite straightforward. I understand the logic of positive and negative correlations. However, what is the physical meaning of "decision boundary" (Fig. 1G) here? How does the "decision boundary" map on the image space?

(7) In my opinion, one of the advantages of this study is the fMRI dataset, which is valuable because previous studies did not collect fMRI data. The key contribution may be the novel brain region associated with display heterogeneity. If this is the case, I would suggest using a more parametric way to measure this region. For example, one can use Gabor stimuli and systematically manipulate the variations of multiple Gabor stimuli, the same logic also applies to motion direction. If this study uses static Gabor, random dot motion, object images that span from low-level to high-level visual stimuli, and consistently shows that the stimulus heterogeneity is encoded in one brain region, I would say this finding is valuable. But this sounds another experiment. In other words, it is insufficient to claim a new brain region given the current form of the manuscript.

References:

* Duncan, J., & Humphreys, G. W. (1989). Visual search and stimulus similarity. Psychological Review, 96(3), 433-458. doi: 10.1037/0033-295x.96.3.433<br /> * Duncan, J. (1989). Boundary conditions on parallel processing in human vision. Perception, 18(4), 457-469. doi: 10.1068/p180457<br /> * Nagy, A. L., Neriani, K. E., & Young, T. L. (2005). Effects of target and distractor heterogeneity on search for a color target. Vision Research, 45(14), 1885-1899. doi: 10.1016/j.visres.2005.01.007<br /> * Nagy, A. L., & Thomas, G. (2003). Distractor heterogeneity, attention, and color in visual search. Vision Research, 43(14), 1541-1552. doi: 10.1016/s0042-6989(03)00234-7<br /> * Vincent, B., Baddeley, R., Troscianko, T., & Gilchrist, I. (2009). Optimal feature integration in visual search. Journal of Vision, 9(5), 15-15. doi: 10.1167/9.5.15<br /> * Singh, A., Mihali, A., Chou, W. C., & Ma, W. J. (2023). A Computational Approach to Search in Visual Working Memory.<br /> * Mihali, A., & Ma, W. J. (2020). The psychophysics of visual search with heterogeneous distractors. BioRxiv, 2020-08.<br /> * Calder-Travis, J., & Ma, W. J. (2020). Explaining the effects of distractor statistics in visual search. Journal of Vision, 20(13), 11-11.

Reviewer #3 (Public review):

Summary of the review process from the Reviewing Editor:

The authors and the reviewers did not agree on several important points made in this paper. The reviewers were critical of the operationalisation of the concept of visual homogeneity (VH), and questioned its validity. For instance, they found it unsatisfying that VH was not calculated on the basis of images themselves, but on the basis of reaction times instead. The authors responded by providing further explanation and argumentation for the importance of this novel concept, but the reviewers were not persuaded. The reviewers also pointed out some data features that did not fit the theory (e.g., overlapping VH between present and absent stimuli), which the authors acknowledge as a point that needs further refining. Finally, the reviewers pointed out that the new so-called visual homogeneity brain region does not overlap very much in the two studies, to which the authors have responded that it is remarkable that there is even partial overlap, given the many confounding differences between the two studies. Altogether, the authors have greatly elaborated their case for VH as an important concept, but the reviewers were not persuaded, and we conclude that the current evidence does not yet meet the high bar for declaring that a novel image property, visual homogeneity, is computed in a localised brain region.

Reviewer #1 (Public review):

The authors explain that an action potential that reach an axon terminal emits a small electrical field as it "annihilates". This happens even though there is no gap junction, at chemical synapses. The generated electrical field is simulated to show that it can affect a nearby, disconnected target membrane by tens of microvolts for tenths of a microsecond. Longer effects are simulated for target locations a few microns away.

To simulate action potentials (APs), the paper does not use the standard Hodgkin-Huxley formalism because it fails to explain AP collision. Instead it uses the Tasaki and Matsumoto (TM) model which is simplified to only models APs with three parameters and as a membrane transition between two states of resting versus excited. The authors expand the strictly binary, discrete TM method to a Relaxing Tasaki Model (RTM) that models the relaxation of the membrane potential after an AP. They find that the membrane leak can be neglected in determining AP propagation and that the capacitive currents dominate the process.

The strength of the work is that authors identified an important interaction between neurons that is neglected by the standard models. A weakness of the proposed approach is the assumptions that it makes. For instance, the external medium is modeled as a homogeneous conductive medium, which may be further explored to properly account for biological processes. To the authors' credit, the external medium can be largely varying and could be left out from the general model, only to be modeled specific instances.

The authors provide convincing evidence by performing experiments to record action potential propagation and collision properties and then developing a theoretical framework to simulate effect of their annihilation on nearby membranes. They provide both experimental evidence and rigorous mathematical and computer simulation findings to support their claims. The work has a potential of explaining significant electrical interaction between nerve centers that are connected via a large number of parallel fibers.

Comments on revisions:

The authors responded to all of my previous concerns and significantly improved the manuscript.

Reviewer #2 (Public review):

In this study, the authors measured extracellular electrical features of colliding APs travelling in different directions down an isolated earthworm axon. They then used these features to build a model of the potential ephaptic effects of AP annihilation, i.e. the electrical signals produced by colliding/annihilating APs that may influence neighbouring tissue. The model was then applied to some different hypothetical scenarios involving synaptic connections. In a revised version of the manuscript, it was also applied, with success, to published experimental data on the cerebellar basket cell-to-Purkinje cell pinceau connection. The conclusion is that an annihilating AP at a presynaptic terminal can emphatically influence the voltage of a postsynaptic cell (this is, presumably, the 'electrical coupling between neurons' of the title), and that the nature of this influence depends on the physical configuration of the synapse.

As an experimental neuroscientist who has never used computational approaches, I am unable to comment on the rigour of the analytical approaches that form the bulk of this paper. The experimental approaches appear very well carried out, and the data showing equal conduction velocity of anti- and orthodromically propagating APs in every preparation is now convincing.

The conclusions drawn from the synaptic modelling have been considerably strengthened by the new Figure 5. Here, the authors' model - including AP annihilation at a synaptic terminal - is used to predict the amplitude and direction of experimentally observed effects at the cerebellar basket cell-to-Purkinje cell synapse (Blot & Barbour 2014). One particular form of the model (RTM with tau=0.5ms and realistic non-excitability of the terminal) matches the experimental data extremely well. This is a much more convincing demonstration that the authors' model of ephaptic effects can quantitatively explain key features of experimental data pertaining to synaptic function. As such, the implications for the relevance of ephaptic coupling at different synaptic contacts may be widespread and important.

However, it appears that all of the models in the new Fig5 involve annihilating APs, yet only one fits the data closely. A key question, which should be addressed if at all possible, is what happens to the predictive power of the best-fitting model in Fig5 if the annihilation, and only the annihilation, is removed? In other words, can the authors show that it is specifically the ephaptic effects of AP annihilation, rather than other ephaptic effects of, say AP waveform/amplitude/propagation, that explain the synaptic effects measured in Blot & Barbour (2014)? This would appear to be a necessary demonstration to fully support the claims of the title.

Reviewer #1 (Public review):

Summary:

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

Strengths:

The data show the effects of ornithine on taste: in two-bottle and briefer intake tests, adding ornithine results in a higher intake of most, but not all, stimuli tests. Bilateral nerve cuts or the addition of GPRC6A antagonists decrease this effect. Small effects of ornithine are shown in whole-nerve recordings.

Weaknesses:

The conclusion seems to be that the authors have found evidence for ornithine acting as a taste modifier through the GPRC6A receptor expressed on the anterior tongue. It is hard to separate their conclusions from the possibility that any effects are additive rather than modulatory. Animals did prefer ornithine to water when presented by itself. Additionally, the authors refer to evidence that ornithine is activating the T1R1-T1R3 amino acid taste receptor, possibly at higher concentrations than they use for most of the study, although this seems speculative. It is striking that the largest effects on taste are found with the other amino acid (umami) stimuli, leading to the possibility that these are largely synergistic effects taking place at the tas1r receptor heterodimer.

Reviewer #2 (Public review):

Summary:

The authors used rats to determine the receptor for a food-related perception (kokumi) that has been characterized in humans. They employ a combination of behavioral, electrophysiological, and immunohistochemical results to support their conclusion that ornithine-mediated kokumi effects are mediated by the GPRC6A receptor. They complemented the rat data with some human psychophysical data. I find the results intriguing, but believe that the authors overinterpret their data.

Strengths:

The authors examined a new and exciting taste enhancer (ornithine). They used a variety of experimental approaches in rats to document the impact of ornithine on taste preference and peripheral taste nerve recordings. Further, they provided evidence pointing to a potential receptor for ornithine.

Weaknesses:

The authors have not established that the rat is an appropriate model system for studying kokumi. Their measurements do not provide insight into any of the established effects of kokumi on human flavor perception. The small study on humans is difficult to compare to the rat study because the authors made completely different types of measurements. Thus, I think that the authors need to substantially scale back the scope of their interpretations. These weaknesses diminish the likely impact of the work on the field of flavor perception.

Reviewer #3 (Public review):

Summary:

In this study, the authors set out to investigate whether GPRC6A mediates kokumi taste initiated by the amino acid L-ornithine. They used Wistar rats, a standard laboratory strain, as the primary model and also performed an informative taste test in humans, in which miso soup was supplemented with various concentrations of L-ornithine. The findings are valuable and overall the evidence is solid. L-Ornithine should be considered to be a useful test substance in future studies of kokumi taste and the class C G protein-coupled receptor known as GPRC6A (C6A) along with its homolog, the calcium-sensing receptor (CaSR) should be considered candidate mediators of kokumi taste.

Strengths:

The overall experimental design is solid based on two bottle preference tests in rats. After determining the optimal concentration for L-Ornithine (1 mM) in the presence of MSG, it was added to various tastants, including inosine 5'-monophosphate; monosodium glutamate (MSG); mono-potassium glutamate (MPG); intralipos (a soybean oil emulsion); sucrose; sodium chloride (NaCl); citric acid and quinine hydrochloride. Robust effects of ornithine were observed in the cases of IMP, MSG, MPG, and sucrose, and little or no effects were observed in the cases of sodium chloride, citric acid, and quinine HCl. The researchers then focused on the preference for Ornithine-containing MSG solutions. The inclusion of the C6A inhibitors Calindol (0.3 mM but not 0.06 mM) or the gallate derivative EGCG (0.1 mM but not 0.03 mM) eliminated the preference for solutions that contained Ornithine in addition to MSG. The researchers next performed transections of the chord tympani nerves (with sham operation controls) in anesthetized rats to identify the role of the chorda tympani branches of the facial nerves (cranial nerve VII) in the preference for Ornithine-containing MSG solutions. This finding implicates the anterior half-two thirds of the tongue in ornithine-induced kokumi taste. They then used electrical recordings from intact chorda tympani nerves in anesthetized rats to demonstrate that ornithine enhanced MSG-induced responses following the application of tastants to the anterior surface of the tongue. They went on to show that this enhanced response was insensitive to amiloride, selected to inhibit 'salt tastant' responses mediated by the epithelial Na+ channel, but eliminated by Calindol. Finally, they performed immunohistochemistry on sections of rat tongue demonstrating C6A positive spindle-shaped cells in fungiform papillae that partially overlapped in its distribution with the IP3 type-3 receptor, used as a marker of Type-II cells, but not with (i) gustducin, the G protein partner of Tas1 receptors (T1Rs), used as a marker of a subset of type-II cells; or (ii) 5-HT (serotonin) and Synaptosome-associated protein 25 kDa (SNAP-25) used as markers of Type-III cells.

Weaknesses:

The researchers undertook what turned out to be largely confirmatory studies in rats with respect to their previously published work on Ornithine and C6A in mice (Mizuta et al Nutrients 2021).

The authors point out that animal models pose some difficulties of interpretation in studies of taste and raise the possibility in the Discussion that umami substances may enhance the taste response to ornithine (Line 271, Page 9). They miss an opportunity to outline the experimental results from the study that favor their preferred interpretation that ornithine is a taste enhancer rather than a tastant.

At least two other receptors in addition to C6A might mediate taste responses to ornithine: (i) the CaSR, which binds and responds to multiple L-amino acids (Conigrave et al, PNAS 2000), and which has been previously reported to mediate kokumi taste (Ohsu et al., JBC 2010) as well as responses to Ornithine (Shin et al., Cell Signaling 2020); and (ii) T1R1/T1R3 heterodimers which also respond to L-amino acids and exhibit enhanced responses to IMP (Nelson et al., Nature 2001). While the experimental results as a whole favor the authors' interpretation that C6A mediates the Ornithine responses, they do not make clear either the nature of the 'receptor identification problem' in the Introduction or the way in which they approached that problem in the Results and Discussion sections. It would be helpful to show that a specific inhibitor of the CaSR failed to block the ornithine response. In addition, while they showed that C6A-positive cells were clearly distinct from gustducin-positive, and thus T1R-positive cells, they missed an opportunity to clearly differentiate C6A-expressing taste cells and CaSR-expressing taste cells in the rat tongue sections.

It would have been helpful to include a positive control kokumi substance in the two-bottle preference experiment (e.g., one of the known gamma-glutamyl peptides such as gamma-glu-Val-Gly or glutathione), to compare the relative potencies of the control kokumi compound and Ornithine, and to compare the sensitivities of the two responses to C6A and CaSR inhibitors.

The results demonstrate that enhancement of the chorda tympani nerve response to MSG occurs at substantially greater Ornithine concentrations (10 and 30 mM) than were required to observe differences in the two bottle preference experiments (1.0 mM; Figure 2). The discrepancy requires careful discussion and if necessary further experiments using the two-bottle preference format.

Reviewer #1 (Public review):

Summary:

This work proposes a new approach to analyse cell-count data from multiple brain regions. Collecting such data can be expensive and time-intensive, so, more often than not, the dimensionality of the data is larger than the number of samples. The authors argue that Bayesian methods are much better suited to correctly analyse such data compared to classical (frequentist) statistical methods. They define a hierarchical structure, partial pooling, in which each observation contributes to the population estimate to more accurately explain the variance in the data. They present two case studies in which their method proves more sensitive in identifying regions where there are significant differences between conditions, which otherwise would be hidden.

Strengths:

The model is presented clearly, and the advantages of the hierarchical structure are strongly justified. Two alternative ways are presented to account for the presence of zero counts. The first involves the use of a horseshoe prior, which is the more flexible option, while the second involves a modified Poisson likelihood, which is better suited to datasets with a large number of zero counts, perhaps due to experimental artifacts. The results show a clear advantage of the Bayesian method for both case studies.

The code is freely available, and it does not require a high-performance cluster to execute for smaller datasets. As Bayesian statistical methods become more accessible in various scientific fields, the whole scientific community will benefit from the transition away from p-values. Hierarchical Bayesian models are an especially useful tool that can be applied to many different experimental designs. However, while conceptually intuitive, their implementation can be difficult. The authors provide a good framework with room for improvement.

Weaknesses:

Alternative possibilities are discussed regarding the prior and likelihood of the model. Given that the second case study inspired the introduction of the zero-inflation likelihood, it is not clear how applicable the general methodology is to various datasets. If every unique dataset requires a tailored prior or likelihood to produce the best results, the methodology will not easily replace more traditional statistical analyses that can be applied in a straightforward manner. Furthermore, the differences between the results produced by the two Bayesian models in case study 2 are not discussed. In specific regions, the models provide conflicting results (e.g., regions MH, VPMpc, RCH, SCH, etc.), which are not addressed by the authors. A third case study would have provided further evidence for the generalizability of the methodology.

Reviewer #2 (Public review):

Summary:

This is a well-written methodology paper applying a Bayesian framework to the statistics of cell counts in brain slices. A sharpening of the bounds on measured quantities is demonstrated over existing frequentist methods and therefore the work is a contribution to the field.

Strengths:

As well as a mathematical description of the approach, the code used is provided in a linked repository.

Weaknesses:

A clearer link between the experimental data and model-structure terminology would be a benefit to the non-expert reader.

Reviewer #1 (Public review):

Summary:

Loh and colleagues investigate valence encoding in the mesolimbic dopamine system. Using an elegant approach, they show that sucrose, which normally evokes strong dopamine neuron activity and release in the nucleus accumbens, is made aversive via conditioned taste aversion, the same sucrose stimulus later evokes much less dopamine neuron activity and release. Thus, dopamine activity can dynamically track the changing valence of an unconditioned stimulus. These results are important for helping clarify valence and value related questions that are the matter of ongoing debate regarding dopamine functions in the field.

Strengths:

This is an elegant way to ask this question, the within subject's design and the continuity of the stimulus is a strong way to remove a lot of the common confounds that make it difficult to interpret valence-related questions. I think these are valuable studies that help tie up questions in the field while also setting up a number of interesting future directions. There are number of control experiments and tweaks to the design that help eliminate a number of competing hypotheses regarding the results. The data are clearly presented and contextualized.

Weaknesses for consideration:

The focus on one relatively understudied region of the rat striatum for dopamine recordings could potentially limit generalization of the findings. While this can be determined in future studies, the implications should be further discussed in the current manuscript.

Reviewer #2 (Public review):

Summary:

Koh et al. report an interesting manuscript studying dopamine binding in the lateral accumbens shell of rats across the course of conditioned taste aversion. The question being asked here is how does the dopamine system respond to aversion? The authors take advantage of unique properties of taste aversion learning (notably, within-subjects remapping of valence to the same physical stimulus) to address this.

They combine a well controlled behavioural design (including key, unpaired controls) with fibre photometry of dopamine binding via GrabDA and of dopamine neuron activity by gCaMP, careful analyses of behaviour (e.g., head movements; home cage ingestion), the authors show that, 1) conditioned taste aversion of sucrose suppresses the activity of VTA dopamine neurons and lateral shell dopamine binding to subsequent presentations of the sucrose tastant; 2) this pattern of activity was similar to the innately aversive tastant quinine; 3) dopamine responses were negatively correlated with behavioural (inferred taste reactivity) reactivity; and 4) dopamine responses tracked the contingency of between sucrose and illness because these responses recovered across extinction of the conditioned taste aversion.

Strengths:

There are important strengths here. The use of a well-controlled design, the measurement of both dopamine binding and VTA dopamine neuron activity, the inclusion of an extinction manipulation; and the thorough reporting of the data. I was not especially surprised by these results, but these data are a potentially important piece of the dopamine puzzle (e.g., as the authors note, salience-based argument struggles to explain these data).

Weaknesses for consideration:

(1) The focus here is on the lateral shell. This is a poorly investigated region in the context of the questions being asked here. Indeed, I suspect many readers might expect a focus on the medial shell. So, I think this focus is important. But, I think it does warrant greater attention in both the introduction and discussion. We do know from past work that there can be extensive compartmentalisation of dopamine responses to appetitive and aversive events and many of the inconsistent findings in the literature can be reconciled by careful examination of where dopamine is assessed. I do think readers would benefit from acknowledgement this - for example it is entirely reasonable to suppose that the findings here may be specific to the lateral shell.

(2) Relatedly, I think readers would benefit from an explicit rationale for studying the lateral shell as well as consideration of this in the discussion. We know that there are anatomical (PMID: 17574681), functional (PMID: 10357457), and cellular (PMID: 7906426) differences between the lateral shell and the rest of the ventral striatum. Critically, we know that profiles of dopamine binding during ingestive behaviours there can be highly dissimilar to the rest of ventral striatum (PMID: 32669355). I do think these points are worth considering.

(3) I found the data to be very thoughtfully analysed. But in places I was somewhat unsure:<br /> (a) Please indicate clearly in the text when photometry data show averages across trials versus when they show averages across animals.<br /> (b) I did struggle with the correlation analyses, for two reasons.<br /> (i) First, the key finding here is that the dopamine response to intraoral sucrose is suppressed by taste aversion. So, this will significantly restrict the range of dopamine transients, making interpretation of the correlations difficult.

(ii) Second, the authors report correlations by combining data across groups/conditions. I understand why the authors have done this, but it does risk obscuring differences between the groups. So, my question is: what happens to this trend when the correlations are computed separately for each group? I suspect other readers will share the same question. I think reporting these separate correlations would be very helpful for the field - regardless of the outcome.

(4) Figure 1A is not as helpful as it might be. I do think readers would expect a more precise reporting of GCaMP expression in TH+ and TH- neurons. I also note that many of the nuances in terms of compartmentalisation of dopamine signalling discussed above apply to ventral tegmental area dopamine neurons (e.g. medial v lateral) and this is worth acknowledging when interpreting.

Reviewer #3 (Public review):

Summary:

This study helps to clarify the mixed literature on dopamine responses to aversive stimuli. While it is well accepted that dopamine in the ventral striatum increases in response to various rewarding and appetitive stimuli, aversive stimuli have been shown to evoke phasic increases or decreasing depending on the exact aversive stimuli, behavioral paradigm, and/or dopamine recording method and location examined. Here the authors use a well-designed set of experiments to show differential responses to an appetitive primary reward (sucrose) that later becomes a conditioned aversive stimulus (sucrose previously paired with lithium chloride in a conditioned taste aversion paradigm). The results are interesting and add valuable data to the question of how the mesolimbic dopamine system encodes aversive stimuli, however, the conclusions are strongly stated given that the current data do not necessarily align with prior conflicting data in terms of recording location, and it is not clear exactly how to interpret the generally biphasic dopamine response to the CTA-sucrose which also evolves over exposures within a single session.

Strengths:

• The authors nicely demonstrate that their two aversive stimuli examined, quinine and sucrose following CTA, evoked aversive facial expressions and paw movements that differed from those following rewarding sucrose to support that the stimuli experienced by the rats differ in valence.

• Examined dopamine responses to the exact same sensory stimuli conditioned to have opposing valences, avoiding standard confounds of appetitive and aversive stimuli being sensed by different sensory modalities (i.e., sweet taste vs. electric shock).

• The authors examined multiple measurements of dopamine activity - cell body calcium (GCaMP6f) in midbrain and release in NAc (Grab-DA2h), which is useful as the prior mixed literature on aversive dopamine responses comes from a variety of recording methods.

• Correlations between sucrose preference and dopamine signals demonstrate behavioral relevance of the differential dopamine signals.

• The delayed testing experiment in Figure 7 nicely controls for the effect of time to demonstrate that the "rewarding" dopamine response to sucrose only recovers after multiple extinction sucrose exposures to extinguish the CTA.

Weaknesses for consideration:

• Regional differences in dopamine signaling to aversive stimuli are mentioned in the introduction and discussion. For instance, the idea that dopamine encodes salience is strongly argued against in the discussion, but the paper cited as arguing for that (Kutlu et al. 2021) is recording from the medial core in mice. Given other papers cited in the text about the regional differences in dopamine signaling in the NAc and from different populations of dopamine neurons in midbrain, it's important to mention this distinction wrt to salience signaling. Relatedly, the text says that the lateral NAc shell was targeted for accumbens recordings, but the histology figure looks like the majority of fibers were in the anterior lateral core of NAc. For the current paper to be a convincing last word on the issue, it would be extremely helpful to have similar recordings done in other parts of the NAc to do a more thorough comparison against other studies.

• Dopamine release in the NAc never dips below baseline for the conditioned sucrose. Is it possible to really consider this as a signal for valence per se, as opposed to it being a weaker response relative to the original sucrose response?

• Related to this, the main measure of the dopamine signal here, "mean z-score," obscures the temporal dynamics of the aversive dopamine response across a trial. This measure is used to claim that sucrose after CTA is "suppressing" dopamine neuron activity and release, which is true relative to the positive valence sucrose response. However, both GRAB-DA and cell-body GCaMP measurements show clear increases after onset of sucrose infusion before dipping back to baseline or slightly below in the average of all example experiments displayed. One could point to these data to argue either that aversive stimuli cause phasic increases in dopamine (due to the initial increase) or decreases (due to the delayed dip below baseline) depending on the measurement window. Some discussion of the dynamics of the response and how it relates to the prior literature would be useful.<br /> - Would this delayed below-baseline dip be visible with a shorter infusion time?<br /> - Does the max of the increase or the dip of the decrease better correlate with the behavioral measures of aversion (orofacial, paw movements) or sucrose preference than "mean z-score" measure used here?<br /> - The authors argue strongly in the discussion against the idea that dopamine is encoding "salience." Could this initial peak (also seen in the first few trials of quinine delivery, fig 1c color plot) be a "salience" response?

• Related to this, the color plots showing individual trials show a reduction in the increases to positive valence sucrose across conditioning day trials and a flip from infusion-onset increase to delayed increases across test day trials. This evolution across days makes it appear that the last few conditioning day trials would be impossible to discriminate from the first few test day trials in the CTA-paired. Presumably, from strength of CTA as a paradigm, the sucrose is already aversive to the animals at the first trial of test day. Why do the authors think the response evolves across this session?

• Given that most of the work is using a conditioned aversive stimulus, the comparison to a primary aversive tastant quinine is useful. However, the authors saw basically no dopamine response to a primary aversive tastant quinine (measured only with GRAB-DA) and saw less noticeable decreases following CTA for NAc recordings with GRAB-DA2h than with cell body GCaMP. Given that they are using the high-affinity version of the GRAB sensor, this calls into question whether this is a true difference in release vs. soma activity or issue of high affinity release sensor making decreases in dopamine levels more difficult to observe.

Reviewer #1 (Public review):

Summary:

The authors have used full-length single-cell sequencing on a sorted population of human fetal retina to delineate expression patterns associated with the progression of progenitors to rod and cone photoreceptors. They find that rod and cone precursors contain a mix of rod/cone determinants, with a bias in both amounts and isoform balance likely deciding the ultimate cell fate. Markers of early rod/cone hybrids are clarified, and a gradient of lncRNAs is uncovered in maturing cones. Comparison of early rods and cones exposes an enriched MYCN regulon, as well as expression of SYK, which may contribute to tumor initiation in RB1 deficient cone precursors.

Strengths:

(1) The insight into how cone and rod transcripts are mixed together at first is important and clarifies a long-standing notion in the field.

(2) The discovery of distinct active vs inactive mRNA isoforms for rod and cone determinants is crucial to understanding how cells make the decision to form one or the other cell type. This is only really possible with full-length scRNAseq analysis.

(3) New markers of subpopulations are also uncovered, such as CHRNA1 in rod/cone hybrids that seem to give rise to either rods or cones.

(4) Regulon analyses provide insight into key transcription factor programs linked to rod or cone fates.

(5) The gradient of lncRNAs in maturing cones is novel, and while the functional significance is unclear, it opens up a new line of questioning around photoreceptor maturation.

(6) The finding that SYK mRNA is naturally expressed in cone precursors is novel, as previously it was assumed that SYK expression required epigenetic rewiring in tumors.

Weaknesses:

(1) The writing is very difficult to follow. The nomenclature is confusing and there are contradictory statements that need to be clarified.

(2) The drug data is not enough to conclude that SYK inhibition is sufficient to prevent the division of RB1 null cone precursors. Drugs are never completely specific so validation is critical to make the conclusion drawn in the paper.

Reviewer #2 (Public review):

Summary:

The authors used deep full-length single-cell sequencing to study human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.

Strengths:

This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging full-length sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing photoreceptors across the fovea and peripheral retina, particularly characterizing SYK's role in the proliferative response of cones in the RB loss background. The study provides an in-depth analysis of developing human photoreceptors, with the authors conducting thorough analyses using full-length single-cell RNA sequencing. The strength of the study lies in its design, which integrates single-cell full-length RNA-seq, long-read RNA-seq, and follow-up histological and functional experiments to provide compelling evidence supporting their conclusions. The model of cell type-dependent splicing for NRL and THRB is particularly intriguing. Moreover, the potential involvement of the SYK and MYC pathways with RB in cone progenitor cells aligns with previous literature, offering additional insights into RB development.

Weaknesses:

The manuscript feels somewhat unfocused, with a lack of a strong connection between the analysis of developing photoreceptors, which constitutes the bulk of the manuscript, and the discussion on retinoblastoma. Additionally, given the recent publication of several single-cell studies on the developing human retina, it is important for the authors to cross-validate their findings and adjust their statements where appropriate.

Reviewer #3 (Public review):

Summary:

The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.

Strengths:

The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.

Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq studies of human retinal development.

Weaknesses:

The weaknesses identified are relatively minor. This is a technically strong and thorough study, that is broadly useful to investigators studying retinal development and retinoblastoma.

Reviewer #1 (Public review):

Summary:

This manuscript reports the investigation of PriC activity during DNA replication initiation in Escherichia coli. It is reported that PriC is necessary for the growth and control of DNA replication initiation under diverse conditions where helicase loading is perturbed at the chromosome origin oriC. A model is proposed where PriC loads helicase onto ssDNA at the open complex formed by DnaA at oriC. Reconstituted helicase loading assays in vitro support the model. The manuscript is well-written and has a logical narrative.

Major Questions/Comments:

An important observation here is that a ΔpriC mutant alone displays under-replication, suggesting that this helicase loading pathway is physiologically relevant. Has this PriC phenotype been reported previously? If not, would it be possible to confirm this result using an independent experimental approach (e.g. marker frequency analysis or fluorescent reporter-operator systems)?

Is PriA necessary for the observed PriC activity at oriC? Is there evidence that PriC functions independently of PriA in vivo?

Is PriC helicase loading activity in vivo at the origin direct (the genetic analysis leaves other possibilities tenable)? Could PriC enrichment at oriC be detected using chromatin immunoprecipitation?

Reviewer #2 (Public review):

This is a great paper. Yoshida et al. convincingly show that DnaA does not exclusively do loading of the replicative helicase at the E. coli oriC, but that PriC can also perform this function. Importantly, PriC seems to contribute to helicase loading even in wt cells albeit to a much lesser degree than DnaA. On the other hand, PriC takes a larger role in helicase loading during aberrant initiation, i.e. when the origin sequence is truncated or when the properties of initiation proteins are suboptimal. Here highlighted by mutations in dnaA or dnaC.

This is a major finding because it clearly demonstrates that the two roles of DnaA in the initiation process can be separated into initially forming an open complex at the DUE region by binding/nucleation onto DnaA-boxes and second by loading of the helicase. Whereas these two functions are normally assumed to be coupled, the present data clearly show that they can be separated and that PriC can perform at least part of the helicase loading provided that an area of duplex opening is formed by DnaA.

This puts into question the interpretation of a large body of previous work on mutagenesis of oriC and dnaA to find a minimal oriC/DnaA complex in many bacteria. In other words, mutants in which oriC is truncated/mutated may support the initiation of replication and cell viability only in the presence of PriC. Such mutants are capable of generating single-strand openings but may fail to load the helicase in the absence of PriC. Similarly, dnaA mutants may generate an aberrant complex on oriC that trigger strand opening but are incapable of loading DnaB unless PriC is present.

In the present work, the sequence of experiments presented is logical and the manuscript is clearly written and easy to follow. The very last part regarding PriC in cSDR replication does not add much to the story and may be omitted.

Reviewer #3 (Public review):

Summary:

At the abandoned replication fork, loading of DnaB helicase requires assistance from PriABC, repA, and other protein partners, but it does not require replication initiator protein, DnaA. In contrast, nucleotide-dependent DnaA binding at the specific functional elements is fundamental for helicase loading, leading to the DUE region's opening. However, the authors questioned in this study that in case of impeding replication at the bacterial chromosomal origins, oriC, a strategy similar to an abandoned replication fork for loading DnaB via bypassing the DnaA interaction step could be functional. The study by Yoshida et al. suggests that PriC could promote DnaB helicase loading on the chromosomal oriC ssDNA without interacting with the DnaA protein. However, the conclusions drawn from the primarily qualitative data presented in the study could be slightly overwhelming and need supportive evidence.

Strengths:

Understanding the mechanism of how DNA replication restarts via reloading the replisomes onto abandoned DNA replication forks is crucial. Notably, this knowledge becomes crucial to understanding how bacterial cells maintain DNA replication from a stalled replication fork when challenging or non-permissive conditions prevail. This critical study combines experiments to address a fundamental question of how DnaB helicase loading could occur when replication initiation impedes at the chromosomal origin, leading to replication restart.

Weaknesses:

The term colony formation used for a spotting assay could be misleading for apparent reasons. Both assess cell viability and growth; while colony formation is quantitative, spotting is qualitative. Particularly in this study, where differences appear minor but draw significant conclusions, the colony formation assays representing growth versus moderate or severe inhibition are a more precise measure of viability.

Figure 2<br /> The reduced number of two oriC copies per cell in the dnaA46priC-deficient strain was considered moderate inhibition. When combined with the data suggested by the dnaAC2priC-deficient strain containing two origins in cells with or without PriC (indicating no inhibition)-the conclusion was drawn that PriC rescue blocked replication via assisting DnaC-dependent DnaB loading step at oriC ssDNA.

The results provided by Saifi B, Ferat JL. PLoS One. 2012;7(3):e33613 suggests the idea that in an asynchronous DnaA46 ts culture, the rate by which dividing cells start accumulating arrested replication forks might differ (indicated by the two subpopulations, one with single oriC and the other with two oriC). DnaA46 protein has significantly reduced ATP binding at 42C, and growing the strain at 42C for 40-80 minutes before releasing them at 30 C for 5 minutes has the probability that the two subpopulations may have differences in the active ATP-DnaA. The above could be why only 50% of cells contain two oriC. Releasing cells for more time before adding rifampicin and cephalexin could increase the number of cells with two oriCs. In contrast, DnaC2 cells have inactive helicase loader at 42 C but intact DnaA-ATP population (WT-DnaA at 42 or 30 C should not differ in ATP-binding). Once released at 30 C, the reduced but active DnaC population could assist in loading DnaB to DnaA, engaged in normal replication initiation, and thus should appear with two oriC in a PriC-independent manner.

Broadly, the evidence provided by the authors may support the primary hypothesis. Still, it could call for an alternative hypothesis: PriC involvement in stabilizing the DnaA-DnaB complex (this possibility could exist here). To prove that the conclusions made from the set of experiments in Figures 2 and 3, which laid the foundations for supporting the primary hypothesis, require insights using on/off rates of DnaB loading onto DnaA and the stability of the complexes in the presence or absence of PriC, I have a few other reasons to consider the latter arguments.

Figure 3<br /> One should consider the fact that dnA46 is present in these cells. Overexpressing pdnaAFH could produce mixed multimers containing subunits of DnaA46 (reduced ATP binding) and DnaAFH (reduced DnaB binding). Both have intact DnaA-DnaA oligomerization ability. The cooperativity between the two functions by a subpopulation of two DnaA variants may compensate for the individual deficiencies, making a population of an active protein, which in the presence of PriC could lead to the promotion of the stable DnaA: DnaBC complexes, able to initiate replication. In the light of results presented in Hayashi et al. and J Biol Chem. 2020 Aug 7;295(32):11131-11143, where mutant DnaBL160A identified was shown to be impaired in DnaA binding but contained an active helicase function and still inhibited for growth; how one could explain the hypothesis presented in this manuscript. If PriC-assisted helicase loading could bypass DnaA interaction, then how growth inhibition in a strain carrying DnaBL160A should be described. However, seeing the results in light of the alternative possibility that PriC assists in stabilizing the DnaA: DnaBC complex is more compatible with the previously published data.

Figure 4<br /> Overexpression of DiaA could contribute to removing a higher number of DnaA populations. This could be more aggravated in the absence of PriC (DiaA could titrate out more DnaA)- the complex formed between DnaA: DnaBC is not stable, therefore reduced DUE opening and replication initiation leading to growth inhibition (Fig. 4A ∆priC-pNA135). Figure 7C: Again, in the absence of PriC, the reduced stability of DnaA: DnaBC complex leaves more DnaA to titrate out by DiaA, and thus less Form I*. However, adding PriC stabilizes the DnaA: DnaBC hetero-complexes, with reduced DnaA titration by DiaA, producing additional Form I*. Adding a panel with DnaBL160A that does not interact with DnaA but contains helicase activity could be helpful. Would the inclusion of PriC increase the ability of mutant helicase to produce additional Form I*?

Figure 5<br /> The interpretation is that colony formation of the Left-oriC ∆priC double mutant was markedly compromised at 37˚C (Figure 5B), and 256 the growth defects of the Left-oriC mutant at 25{degree sign}C and 30{degree sign}C were aggravated. However, prima facia, the relative differences in the growth of cells containing and lacking PriC are similar. Quantitative colony-forming data is required to claim these results. Otherwise, it is slightly confusing.

A minor suggestion is to include cells expressing PriC using plasmid DNA to show that adding PriC should reverse the growth defect of dnaA46 and dnaC2 strains at non-permissive temperatures. The same should be added at other appropriate places.

Reviewer #1 (Public review):

The authors present an important work where they model some of the complex interactions between immune cells, fibroblasts and cancer cells. The model takes into account the increased ECM production of cancer-associated fibroblasts. These fibres trap the cancer but also protect it from immune system cells. In this way, these fibroblasts' actions both promote and hinder cancer growth. By exploring different scenarios, the authors can model different cancer fates depending on the parameters regulating cancer cells, immune system cells and fibroblasts. In this way, the model explores non-trivial scenarios. An important weakness of this study is that, though it is inspired by NSCLC tumors, it is restricted to modelling circular tumor lesions and does not explore the formation of ramified tumors, as in NSCLC. In this way, is only a general model and it is not clear how it can be adapted to simulate more realistic tumor morphologies.

Reviewer #2 (Public review):

Summary:

The authors develop a computational model (and a simplified version thereof) to treat an extremely important issue regarding tumor growth. Specifically, it has been argued that fibroblasts have the ability to support tumor growth by creating physical conditions in the tumor microenvironment that prevent the relevant immune cells from entering into contact with, and ultimately killing, the cancer cells. This inhibition is referred to as immune exclusion. The computational approach follows standard procedures in the formulation of models for mixtures of different material species, adapted to the problem at hand by making a variety of assumptions as to the activity of different types of fibroblasts, namely "normal" versus "cancer-associated". The model itself is relatively complex, but the authors do a convincing job of analyzing possible behaviors and attempting to relate these to experimental observations.

Strengths:

As mentioned, the authors do an excellent job of analyzing the behavior of their model both in its full form (which includes spatial variation of the concentrations of the different cellular species) and in its simplified mean field form. The model itself is formulated based on established physical principles, although the extent to which some of these principles apply to active biological systems is not clear (see Weaknesses). The results of the model do offer some significant insights into the critical factors which determine how fibroblasts might affect tumor growth; these insights could lead to new experimental ways of unraveling these complex sets of issues and enhancing immunotherapy.

Weaknesses:

Models of the form being studied here rely on a large number of assumptions regarding cellular behavior. Some of these seemed questionable, based on what we have learned about active systems. The problem of T cell infiltration as well as the patterning of the extracellular matrix (ECM) by fibroblasts necessarily involve understanding cell motion and cell interactions due e.g. to cell signaling. Adopting an approach based purely on physical systems driven by free energies alone does not consider the special role that active processes can play, both in motility itself and in the type of self-organization that can occur due to these cell-cell interactions. This to me is the primary weakness of this paper.

A separate weakness concerns the assumption that fibroblasts affect T cell behavior primarily by just making a more dense ECM. There are a number of papers in the cancer literature (see, for some examples, Carstens, J., Correa de Sampaio, P., Yang, D. et al. Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun 8, 15095 (2017); Sun, Xiujie, Bogang Wu, Huai-Chin Chiang, Hui Deng, Xiaowen Zhang, Wei Xiong, Junquan Liu et al. "Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion." Nature 599, no. 7886 (2021): 673-678) that seem to indicate that density alone is not a sufficient indicator of T cell behavior. Instead, the organization of the ECM (for example, its anisotropy) could be playing a much more essential role than is given credit for here. This possibility is hinted at in the Discussion section but deserves much more emphasis.

Finally, the mixed version of the model is, from a general perspective, not very different from many other published models treating the ecology of the tumor microenvironment (for a survey, see Arabameri A, Asemani D, Hadjati J (2018), A structural methodology for modeling immune-tumor interactions including pro-and anti-tumor factors for clinical applications. Math Biosci 304:48-61). There are even papers in this literature that specifically investigate effects due to allowing cancer cells to instigate changes in other cells from being tumor-inhibiting to tumor-promoting. This feature occurs not only for fibroblasts but also for example for macrophages which can change their polarization from M1 to M2. There needed to be some more detailed comparison with this existing literature.

Reviewer #1 (Public review):

Summary:

In this manuscript by Obray et al., the authors show that adolescent ethanol exposure increases mechanical allodynia in adulthood. Additionally, they show that BLA-mediated inhibition of the prelimbic cortex is reduced, resulting in increased excitability in neurons that then project to vlPAG. This effect was mediated by BLA inputs onto PV interneurons. The primary finding of the manuscript is that these AIE-induced changes further impact acute pain processing in the BLA-PrL-vlPAG circuit, albeit behavioral readouts after inducing acute pain were not different between AIE rats and controls. These results provide novel insights into how AIE can have long-lasting effects on pain-related behaviors and neurophysiology. In this manuscript by Obray et al., the authors show that adolescent ethanol exposure increases mechanical allodynia in adulthood. Additionally, they show that BLA-mediated inhibition of the prelimbic cortex is reduced, resulting in increased excitability in neurons that then project to vlPAG. This effect was mediated by BLA inputs onto PV interneurons. The primary finding of the manuscript is that these AIE-induced changes further impact acute pain processing in the BLA-PrL-vlPAG circuit, albeit behavioral readouts after inducing acute pain were not different between AIE rats and controls. These results provide novel insights into how AIE can have long-lasting effects on pain-related behaviors and neurophysiology.

Strengths:

The manuscript was very well written and the experiments were rigorously conducted. The inclusion of both behavioral and neurophysiological circuit recordings was appropriate and compelling. The attention to SABV and appropriate controls was well thought out. The Discussion provided novel ideas for how to think about AIE and chronic pain and proposed several interesting mechanisms. This was a very well-executed set of experiments.

Weaknesses:

There is a mild disconnect between behavioral readout (reflexive pain) and neural circuits of interest (emotional). Considering that this circuit is likely engaged in the aversiveness of pain, it would have been interesting to see how carrageenan and/or AIE impacted non-reflexive pain measures. Perhaps this would reveal a potentiated or dysregulated phenotype that matches the neurophysiological changes reported. However, this critique does not take away from the value of the paper or its conclusions.

Reviewer #2 (Public review):

Summary:

The study by Obray et al. entitled "Adolescent alcohol exposure promotes mechanical allodynia and alters synaptic function at inputs from the basolateral amygdala to the prelimbic cortex" investigated how adolescent intermittent ethanol exposure (AIE) affects the BLA -> PL circuit, with an emphasis on PAG projecting PL neurons, and how AIE changes mechanical and thermal nociception. The authors found that AIE increased mechanical, but not thermal nociception, and an injection of an inflammatory agent did not produce changes in an ethanol-dependent manner. Physiologically, a variety of AIE-specific effects were found in PL neuron firing at BLA synapses, suggestive of AIE-induced alterations in neurotransmission at BLA-PVIN synapses.

Strengths:

This was a comprehensive examination of the effects of AIE on this neural circuit, with an in-depth dissection of the various neuronal connections within the PL.

Sex was included as a biological variable, yet there were little to no sex differences in AIE's effects, suggestive of similar adaptations in males and females.

Reviewer #3 (Public review):

Summary:

Obray et al. investigate the long-lasting effects of adolescent intermittent ethanol (AIE) in rats, a model of alcohol dependence, on a neural circuit within the prefrontal cortex. The studies are focused on inputs from the basolateral amygdala (BLA) onto parvalbumin (PV) interneurons and pyramidal cells that project to the periaqueductal gray (PAG). The authors found that AIE increased BLA excitatory drive onto parvalbumin interneurons and increased BLA feedforward inhibition onto PAG-projecting neurons.

Strengths:

Fully powered cohorts of male and female rodents are used, and the design incorporates both AIE and an acute pain model. The authors used several electrophysiological techniques to assess synaptic strength and excitability from a few complimentary angles. The design and statistical analysis are sound, and the strength of evidence supporting synaptic changes following AIE results is solid.

Weaknesses:

(1) There is incomplete evidence supporting some of the conclusions drawn in this manuscript. The authors claim that the changes in feedforward inhibition onto pyramidal cells are due to the changes in parvalbumin interneurons, but evidence is not provided to support that idea. PV cells do not spontaneously fire action potentials spontaneously in slices (nor do they receive high levels of BLA activity while at rest in slices). It is possible that spontaneous GABA release from PV cells is increased after AIE but the authors did not report sIPSC frequency. Second, the authors did not determine that PV cells mediate the feedforward BLA op-IPSCs and changes following AIE (this would require manipulation to reduce/block PV-IN activity). This limitation in results and interpretation is important because prior work shows BLA-PFC feedforward IPSCs can be driven by somatostatin cells. Cholecystokinin cells are also abundant basket cells in PFC and have been recently shown to mediate feedforward inhibition from the thalamus and ventral hippocampus, so it's also possible that CCK cells are involved in the effects observed here.

(2) The authors conclude that the changes in this circuit likely mediate long-lasting hyperalgesia, but this is not addressed experimentally. In some ways, the focused nature of the study is a benefit in this regard, as there is extensive prior literature linking this circuit with pain behaviors in alternative models (e.g., SNI), but it should be noted that these studies have not assessed hyperalgesia stemming from prior alcohol exposure. While the current studies do not include a causative behavioral manipulation, the strength of the association between BLA-PL-PAG function and hyperalgesia could be bolstered by current data if there were relationships detected between electrophysiological properties and hyperalgesia. Have the authors assessed this? In addition, this study is limited by not addressing the specificity of synaptic adaptations to the BLA-PL-PAG circuit. For instance, PL neurons send reciprocal projections to BLA and send direct projections to the locus coeruleus (which the authors note is an important downstream node of the PAG for regulating pain).

(3) I have some concerns about methodology. First, 5-ms is a long light pulse for optogenetics and might induce action-potential independent release. Does TTX alone block op-EPSCs under these conditions? Second, PV cells express a high degree of calcium-permeable AMPA receptors, which display inward rectification at positive holding potentials due to blockade from intracellular polyamines. Typically, this is controlled/promoted by including spermine in the internal solution, but I do not believe the authors did that. Nonetheless, the relatively low A/N ratios for this cell type suggest that CP-AMPA receptors were not sampled with the +40/+40 design of this experiment, raising concerns that the majority of AMPA receptors in these cells were not sampled during this experiment. Finally, it should be noted that asEPSC frequency can also reflect changes in a number of functional/detectable synapses. This measurement is also fairly susceptible to differences in inter-animal differences in ChR2 expression. There are other techniques for assessing presynaptic release probability (e.g., PPR, MK-801 sensitivity) that would improve the interpretation of these studies if that is intended to be a point of emphasis.

(4) In a few places in the manuscript, results following voluntary drinking experiments (especially Salling et al. and Sicher et al.) are discussed without clear distinction from prior work in vapor models of dependence

(5) Discussion (lines 416-420). The authors describe some differing results with the literature and mention that the maximum current injection might be a factor. To me, this does not seem like the most important factor and potentially undercuts the relevance of the findings. Are the cells undergoing a depolarization block? Did the authors observe any changes in the rheobase or AP threshold? On the other hand, a more likely difference between this and previous work is that the proportion of PAG-projecting cells is relatively low, so previous work in L5 likely sampled many types of pyramidal cells that project to other areas. This is a key example where additional studies by the current group assessing a distinct or parallel set of pyramidal cells would aid in the interpretation of these results and help to place them within the existing literature. Along these lines, PAG-projecting neurons are Type A cells with significant hyperpolarization sag. Previous studies showed that adolescent binge drinking stunts the development of HCN channel function and ensuing hyperpolarization sag. Have the authors observed this in PAG-projecting cells? Another interesting membrane property worth exploring with the existing data set is the afterhyperpolarization / SK channel function.

Reviewer #1 (Public Review):

Otero-Coronel et al. address an important question for neuroscience - how does a premotor neuron capable of directly controlling behavior integrate multiple sources of sensory inputs to inform action selection? For this, they focused on the teleost Mauthner cell, long known to be at the core of a fast escape circuit. What is particularly interesting in this work is the naturalistic approach they took. Classically, the M-cell was characterized, both behaviorally and physiologically, using an unimodal sensory space. Here the authors make the effort (substantial!) to study the physiology of the M-cell taking into account both the visual and auditory inputs. They performed well-informed electrophysiological approaches to decipher how the M-cell integrates the information of two sensory modalities depending on the strength and temporal relation between them.

The empirical results are convincing and well-supported. The manuscript is well-written and organized. The experimental approaches and the selection of stimulus parameters are clear and informed by the bibliography. The major finding is that multisensory integration increases the certainty of environmental information in an inherently noisy environment.

Reviewer #2 (Public Review):

In this manuscript, Otero-Coronel and colleagues use a combination of acoustic stimuli and electrical stimulation of the tectum to study MSI in the M-cells of adult goldfish. They first perform a necessary piece of groundwork in calibrating tectal stimulation for maximal M-cell MSI, and then characterize this MSI with slightly varying tectal and acoustic inputs. Next, they quantify the magnitude and timing of FFI that each type of input has on the M-cell, finding that both the tectum and the auditory system drive FFI, but that FFI decays more slowly for auditory signals. These are novel results that would be of interest to a broader sensory neuroscience community. By then providing pairs of stimuli separated by 50ms, they assess the ability of the first stimulus to suppress responses to the second, finding that acoustic stimuli strongly suppress subsequent acoustic responses in the M-cell, that they weakly suppress subsequent tectal stimulation, and that tectal stimulation does not appreciably inhibit subsequent stimuli of either type. Finally, they show that M-cell physiology mirrors previously reported behavioural data in which stronger stimuli underwent less integration.

The manuscript is generally well-written and clear. The discussion of results is appropriately broad and open-ended. It's a good document. Our major concerns regarding the study's validity are captured in the individual comments below. In terms of impact, the most compelling new observation is the quantification of the FFI from the two sources and the logical extension of these FFI dynamics to M-cell physiology during MSI. It is also nice, but unsurprising, to see that the relationship between stimulus strength that MSI is similar for M-cell physiology to what has previously been shown for behavior. While we find the results interesting, we think that they will be of greatest interest to those specifically interested in M-cell physiology and function.

Reviewer #1 (Public Review):

This work addresses how to quantify functional compensation throughout the aging process and identifies brain regions that engage in compensatory mechanisms during the Cattell task, a measure of fluid cognition. The authors find that regions of the frontal cortex and cuneus showed unique effects of both age and performance. Interestingly, these two regions demonstrated differential activation patterns taking into account both age and performance. Specifically, the researchers found that the relationship between performance and activation in the cuneal ROI was strongest in older adults, however, this was not found in younger adults. These findings suggest that specifically within the cuneus, greater activation is needed by older adults to maintain performance, suggestive of functional compensation.

The conclusions derived from the study are well supported by the data. The authors validated the use of the in-scanner Cattell task by demonstrating high reliability in the same sample with the standard out-of-scanner version. Some strengths of the study include the large sample size and wide age range of participants. The authors use a stringent Bayes factor of 20 to assess the strength of evidence. The authors used a whole-brain approach to define regions of interest (ROIs) based on activation patterns that were jointly related to age and performance. Overall, the methods are technically sound and support the authors' conclusions.

Comment from Reviewing Editor: In the revised manuscript, the authors have addressed the weaknesses previously identified by reviewer 1.

Reviewer #2 (Public Review):

This work by Knights et al., makes use of the Cam-CAN dataset to investigate functional compensation during a fluid processing task in older adults, in a fairly large sample of approximately 200 healthy adults ranging from 19 to 87. Using univariate methods, the authors identify two brain regions in which activity increases as a function of both age and performance and conduct further investigations to assess whether the activity of these regions provides information regarding task difficulty. The authors conclude that the cuneal cortex - a region of the brain previously implicated in visual attention - shows evidence of compensation in older adults.

The conclusions of the paper are well supported by the data, and the authors use appropriate statistical analyses. The use of multivariate methods over the last 20 years has demonstrated many effects that would have been missed using more traditional univariate analysis techniques. The data set is also of an appropriate size, and as the authors note, fluid processing is an extremely important domain in the field of cognition in aging, due to its steep decline over aging.

Comment from Reviewing Editor: It would have been nice to see an analysis of a more crystallised intelligence task included too, as a contrast since this is an area that does not demonstrate such a decline (and perhaps continues to improve over aging). This comment does not take away the important contributions of the manuscript.

Reviewer #3 (Public Review):

This neuroimaging study investigated how brain activity related to visual pattern-based reasoning changes over the adult lifespan, addressing the topic of functional compensation in older age. To this end, the authors employed a version of the Cattell task, which probes visual pattern recognition for identifying commonalities and differences within sets of abstract objects in order to infer the odd object among a given set. Using a state-of-the-art univariate analysis approach on fMRI data from a large lifespan sample, the authors identified brain regions in which the activation contrast between hard and easy Cattell task conditions was modulated by both age and performance. Regions identified comprised prefrontal areas and bilateral cuneus. Applying a multivariate decoding approach to activity in these regions, the authors went on to show that only in older adults, the cuneus, but not the prefrontal regions, carried information about the task condition (hard vs. easy) beyond that already provided by activity patterns of voxels that showed a univariate main effect of task difficulty. This was taken as compelling evidence for task-specific compensatory activity in the cuneus in advanced age.

The study is well-motivated and well-written. The authors used appropriate, rigorous methods that allowed them to control for a range of possible confounds or alternative explanations. Laudable aspects include the large sample with a wide and even age distribution, the validation of the in-scanner task performance against previous results obtained with a more standard version outside the scanner, and the control for vascular age-related differences in hemodynamic activity via a BOLD signal amplitude measure obtained from a separate resting-state fMRI scan. Overall, the conclusions are well-supported by the data.

Comment from Reviewing Editor: The revised manuscript has addressed the points raised during the review of the original submission.

Reviewer #1 (Public review):

Summary:

The authors sought to identify unknown factors involved in the repair of uracil in DNA through a CRISPR knockout screen.

Strengths:

The screen identified both known and unknown proteins involved in DNA repair resulting from uracil or modified uracil base incorporation into DNA. The conclusion is that the protein activity of METTL3, which converts A nucleotides to 6mA nucleotides, plays a role in the DNA damage/repair response. The importance of METTL3 in DNA repair, and its colocalization with a known DNA repair enzyme, UNG2, is well characterized.

Weaknesses:

This reviewer identified no major weaknesses in this study. The manuscript could be improved by tightening the text throughout, and more accurate and consistent word choice around the origin of U and 6mA in DNA. The dUTP nucleotide is misincorporated into DNA, and 6mA is formed by methylation of the A base present in DNA. Using words like 6mA "deposition in DNA" seems to imply it results from incorporation of a methylated dATP nucleotide during DNA synthesis.

Reviewer #2 (Public review):

Summary:

In this work, the authors performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA, and identified unexpected factors, such as the RNA m6A methyltransferase METTL3, as required to overcome floxuridine-driven cytotoxicity in mammalian cells. Interestingly, the observed N6-methyladenosine was embedded in DNA, which has been reported as DNA 6mA in mammalian genomes and is currently confirmed with mass spectrometry in this model. Therefore, this work consolidated the functional role of mammalian genomic DNA 6mA, and supported with solid evidence to uncover the METTL3-6mA-UNG2 axis in response to DNA base damage.

Strengths:

In this work, the authors took an unbiased, genome-wide CRISPR approach to identify novel factors involved in uracil repair with potential clinical interest.

The authors designed elegant experiments to confirm the METTL3 works through genomic DNA, adding the methylation into DNA (6mA) but not the RNA (m6A), in this base damage repair context. The authors employ different enzymes, such as RNase A, RNase H, DNase, and liquid chromatography coupled to tandem mass spectrometry to validate that METTL3 deposits 6mA in DNA in response to agents that increase genomic uracil.

They also have the Mettl3-KO and the METTL3 inhibition results to support their conclusion.

Weaknesses:

Although this study demonstrates that METTL3-dependent 6mA deposition in DNA is functionally relevant to DNA damage repair in mammalian cells, there are still several concerns and issues that need to be improved to strengthen this research.

First, in the whole paper, the authors never claim or mention the mammalian cell lines contamination testing result, which is the fundamental assay that has to be done for the mammalian cell lines DNA 6mA study.

Second, in the whole work, the authors have not supplied any genomic sequencing data to support their conclusions. Although the sequencing of DNA 6mA in mammalian models is challenging, recent breakthroughs in sequencing techniques, such as DR-Seq or NT/NAME-seq, have lowered the bar and improved a lot in the 6mA sequencing assay. Therefore, the authors should consider employing the sequencing methods to further confirm the functional role of 6mA in base repair.

Third, the authors used the METTL3 inhibitor and Mettl3-KO to validate the METTL3-6mA-UNG2 functional roles. However, the catalytic mutant and rescue of Mettl3 may be the further experiments to confirm the conclusion.

Reviewer #3 (Public review):

Summary:

The authors are showing evidence that they claim establishes the controversial epigenetic mark, DNA 6mA, as promoting genome stability.

Strengths:

The identification of a poorly understood protein, METTL3, and its subsequent characterization in DDR is of high quality and interesting.

Weaknesses:

(1) The very presence of 6mA (DNA) in mammalian DNA is still highly controversial and numerous studies have been conclusively shown to have reported the presence of 6mA due to technical artifacts and bacterial contamination. Thus, to my knowledge there is no clear evidence for 6mA as an epigenetic mark in mammals, and consequently, no evidence of writers and readers of 6mA. None of this is mentioned in the introduction. Much of the introduction can be reduced, but a paragraph clearly stating the controversy and lack of evidence for 6mA in mammals needs to be added, otherwise, the reader is given an entirely distorted view of the field.

These concerns must also be clearly in the limitations section and even in the results section which fails to nuance the authors' findings.

(2) What is the motivation for using HT-29 cells? Moreover, the materials and methods do not state how the authors controlled for bacterial contamination, which has been the most common cause of erroneous 6mA signals to date. Did the authors routinely check for mycoplasma?

(3) The single cell imaging of 6mA in various cells is nice. The results are confirmed by mass spec as an orthogonal approach. Another orthogonal and quantitative approach to assessing 6mA levels would be PacBio. Similarly, it is unclear why the authors have not performed dot-blots of 6mA for genomic DNA from the given cell lines.

(4) The results of Figure 3 need further investigation and validation. If the results are correct the authors are suggesting that the majority of 6mA in their cell lines is present in the DNA, and not the RNA, which is completely contrary to every other study of 6mA in mammalian cells that I am aware of. This could suggest that the antibody is not, in fact, binding to 6mA, but to unmodified adenine, which would explain why the signal disappears after DNAse treatment. Indeed, binding of 6mA to unmethylated DNA is a commonly known problem with most 6mA antibodies and is well described elsewhere.

(5) Given the lack of orthologous validation of the observed DNA 6mA and the lack of evidence supporting the presence of 6mA in mammalian DNA and consequently any functional role for 6mA in mammalian biology, the manuscript's conclusions need to be toned down significantly, and the inherent difficulty in assessing 6mA accurately in mammals acknowledged throughout.

Reviewer #1 (Public review):

Summary:

In this study, the authors describe the construction of an extremely large-scale anatomical model of juvenile rat somatosensory cortex (excluding the barrel region), which extends earlier iterations of these models by expanding across multiple interconnected cortical areas. The models are constructed in a way to maintain biological detail from a granular scale - for example, individual cell morphologies are maintained, and synaptic connectivity is founded on anatomical contacts. The authors use this model to investigate a variety of properties, from cell-type specific targeting (where the model results are compared to findings from recent large-scale electron microscopy studies) to network metrics. The model is also intended to serve as a platform and resource for the community by being a foundation for simulations of neuronal circuit activity and for additional anatomical studies that rely on the detailed knowledge of cellular identity and connectivity.

Strengths:

As the authors point out, the combination of scale and granularity of their model are what make this study valuable and unique. The comparisons with recent electron microscopy findings are some of the most compelling results presented in the study, showing that certain connectivity patterns can arise directly from the anatomical configuration, while other discrepancies highlight where more selective targeting rules (perhaps based on molecular cues) are likely employed. They also describe intriguing effects of cortical thickness and curvature on circuit connectivity and characterize the magnitude of those effects on different cortical layers.

The detailed construction of the model is drawn on wide range of data sources (cellular and synaptic density measures, neuronal morphologies, cellular composition measures, brain geometry, etc.) that are integrated together; other data sources are used for comparison and validation. This consolidation and comparison also represents a valuable contribution to the overall understanding of the modeled system.

Weaknesses:

The scale of the model, which is a primary strength, also can carry some drawbacks. In order to integrate all the diverse data sources together, many specific decisions must be made about, for example, translating findings from different species or regions to the modeled system, or deciding which aspects of the system can be assumed to be same and which should vary. All these decisions will have effects on the predicted results from the model, which could limit the types of conclusions that can be made (both by the others and by others in the community who may wish to use the model for their own work). However, the public release of the models and most of the associated tools does provide others a somewhat easier path to modify and evaluate this iteration of the model for their own studies.

Overall, the model presented in this study represents an enormous amount of work and stands as the basis for other work by the same group as well as a unique resource for the community, even while acknowledging that it may be somewhat unwieldy for the community to employ due to the weight of its manifold specific construction decisions, size, and complexity.

Reviewer #2 (Public review):

Summary:

The authors build a colossal anatomical model of juvenile rat non-barrel primary somatosensory cortex, including inputs from the thalamus. This enhances past models by incorporating information on the shape of the cortex and estimated densities of various types of excitatory and inhibitory neuron across layers. This is intended to enable analysis of the micro- and mesoscopic organisation of cortical connectivity and to be a base anatomical model for large-scale simulations of physiology.

Strengths:

• The authors incorporate many diverse data sources on morphology and connectivity.<br /> • This paper takes on the challenging task of linking micro- and meso-scale connectivity<br /> • By building in the shape of the cortex, the authors were able to link cortical geometry to connectivity. In particular they make an unexpected prediction that cortical conicality affects the modularity of local connectivity, which should be testable.<br /> • The author's analysis of the model led to the interesting prediction that layer 5 neurons' connect local modules, which may be testable in the future, and provide a basis to link from detailed anatomy to functional computations.<br /> • The visualisation of the anatomy in various forms is excellent<br /> • The model is openly shared

Weaknesses:

• There is no effort to determine how specific or generalisable the findings here are to other parts of cortex.<br /> • Although there is a link to physiological modelling in another paper, there is no clear pathway to go from this type of model to understanding how the specific function of the modelled areas may emerge here (and not in other cortical areas).<br /> • Some of the decisions seem a little ad-hoc, and the means to assess those decisions is not always easily available to the reader<br /> • The shape of the juvenile cortex - a key novelty of this work - was based on merely a scalar reduction of the adult cortex. This is very surprising, and surely an oversimplification. Huge efforts have gone into modelling the complex nonlinear development of cortex, by teams including the developing Human Connectome Project. For such a fundamental aspect of this work, why isn't it possible to reconstruct the shape of this relatively small part of juvenile rat cortex?<br /> • The same relative laminar depths are used for all subregions. This will have a large impact on the model. However, relative laminar depths can change drastically across the cortex (see e.g. many papers by Palomero-Gallagher, Zilles and colleagues). The authors should incorporate the real laminar depths, or, failing that, show evidence to show that the laminar depth differences across the subregions included in the model are negligible.<br /> • The authors perform an affine mapping between mouse and rat cortex. This is again surprising. In human imaging, affine mappings are insufficient to map between two individual brains of the same species, and nonlinear transformations are instead used. That an affine transformation should be considered sufficient to map between two different species is then very surprising. For some models, this may be fine, but there is a supposed emphasis here on biological precision in terms of anatomical location.<br /> o Live nature of the model. This is such a colossal model, and effort, that I worry that it may be quite difficult to update in light of new data. For example, how much person and compute time would it take to update the model to account for different layer sizes across subregions? Or to more precisely account for the shape of juvenile rat cortex?

Reviewer #3 (Public review):

This manuscript reports a detailed model of the rat non-barrel somatosensory cortex, consisting of 4.2 million morphologically and biophysically detailed neuron models, arranged in space and connected according to highly sophisticated rules informed by diverse experimental data. Due to its breadth and sophistication the model will undoubtedly be of interest to the community, and the reporting of anatomical details of modeling in this paper is important for understanding all the assumptions and procedures involved in constructing the model. While a useful contribution to this field, the model and the manuscript could be improved by employing data more directly and comparing simple features of the model's connectivity - in particular, connection probabilities - with relevant experimental data.

The manuscript is overall well-written, but contains a substantial number of confusing or unclear statements, and some important information is not provided.

Comments on revisions:

The authors mostly addressed all my points and improved the paper substantially. I do not have further extensive comments except one general point below.

Regarding section 2.3 and metrics of connectivity like pairwise connection probabilities, it is great that the authors rewrote that section and added comparisons with experimental data in Figs. 4 and S9. Unfortunately, what one finds when direct comparisons are made is that the modeled pairwise connectivity is quite different from the data. Fig. S9 shows that the model's results do not agree with data in about half of the cases (purple and red arrows). Similarly large discrepancies can be seen for some other metrics, like in Fig. S10B and S10C1,C2. (And similar concerns apply to thalamocortical connections in section 2.5, where it looks like little to no data are available to verify the pairwise connectivity between the thalamic and cortical neurons via a direct comparison.)

This is concerning since this model forms the basis for multiple other studies of cortical dynamics and function by the same group and potentially others in the community, with multiple papers relying on it, whereas basic properties of connectivity are apparently not captured well.

On the other hand, this is also a "glass half full" situation, showing that the sophisticated algorithms for establishing connections, developed by the authors, are working well in at least half of the connection types explored. It is therefore imperative that the authors continue refining these algorithms to capture the remaining half in future iterations and producing improved models that the community can better rely on.

Please also note that Fig. S11 does not have a caption.

Reviewer #1 (Public review):

Summary:

The manuscript "Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation" by Cho and colleagues investigates de novo tight junction formation during the differentiation of immortalized human HaCaT keratinocytes to granular-like cells, as well as during epithelial remodeling that occurs upon the apoptotic of individual cells in confluent monolayers of the representative epithelial cell line EpH4. The authors demonstrate the involvement of Rho-ROCK with well-conducted experiments and convincing images. Moreover, they unravel the underlying molecular mechanism, with Rho-ROCK activity activating the transmembrane serine protease Matriptase, which in turn leads to the cleavage of EpCAM and TROP2, respectively, releasing Claudins from EpCAM/TROP2/Claudin complexes at the cell membrane to become available for polymerization and de novo tight junction formation. These functional studies in the two different cell culture systems are complemented by localization studies of the according proteins in the stratified mouse epidermis in vivo.

In total, these are new and very intriguing and interesting findings that add important new insights into the molecular mechanisms of tight junction formation, identifying Matriptase as the "missing link" in the cascade of formerly described regulators. The involvement of TROP2/EpCAM/Claudin has been reported recently (Szabo et al., Biol. Open 2022; Bugge lab), and Matriptase had been formerly described to be required for in tight junction formation as well, again from the Bugge lab. Yet, the functional correlation/epistasis between them, and their relation to Rho signaling, had not been known thus far.

However, experiments addressing the role of Matriptase require a little more work.

Strengths:

Convincing functional studies in two different cell culture systems, complemented by supporting protein localization studies in vivo. The manuscript is clearly written and most data are convincingly demonstrated, with beautiful images and movies.

Weaknesses:

The central finding that Rho signaling leads to increased Matriptase activity needs to be more rigorously demonstrated (e.g. western blot specifically detecting the activated version or distinguishing between the full-length/inactive and processed/active version).

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate how epithelia maintain intercellular barrier function despite and during cellular rearrangements upon e.g. apoptotic extrusion in simple epithelia or regenerative turnover in stratified epithelia like this epidermis. A fundamental question in epithelial biology. Previous literature has shown that Rho-mediated local regulation of actomyosin is essential not only for cellular rearrangement itself but also for directly controlling tight junction barrier function. The molecular mechanics however remained unclear. Here the authors use extensive fluorescent imaging of fixed and live cells together with genetic and drug-mediated interference to show that Rho activation is required and sufficient to form novo tight junctional strands at intercellular contacts in epidermal keratinocytes (HaCat) and mammary epithelial cells. After having confirmed previous literature they then show that Rho activation activates the transmembrane protease Matriptase which cleaves EpCAM and TROP2, two claudin-binding transmembrane proteins, to release claudins and enable claudin strand formation and therefore tight junction barrier function.

Strengths:

The presented mechanism is shown to be relevant for epithelial barriers being conserved in simple and stratifying epithelial cells and mainly differs due to tissue-specific expression of EpCAM and TROP2. The authors present careful state-of-the-art imaging and logical experiments that convincingly support the statements and conclusion. The manuscript is well-written and easy to follow.

Weaknesses:

Whereas the in vitro evidence of the presented mechanism is strongly supported by the data, the in vivo confirmation is mostly based on the predicted distribution of TROP2. Whereas the causality of Rho-mediated Matriptase activation has been nicely demonstrated it remains unclear how Rho activates Matriptase.

Reviewer #1 (Public review):

Summary:

The ingenious design in this study achieved the observation of 3D cell spheroids from an additional lateral view and gained more comprehensive information than the traditional one angle of imaging, which extensively extended the methods to investigate cell behaviors in the growth or migration of tumor organoids in the present study. I believe that this study opens an avenue and provides an opportunity to characterize the spheroid formation dynamics from different angles, in particular side-view with high resolution, in other organoids study in the future.

Reviewer #2 (Public review):

Summary:

The author developed a new device to overcome current limitations in the imaging process of 3D spheroidal structures. In particular, they created a system to follow in real-time tumour spheroid formation, fusion and cell migration without disrupting their integrity. The system has also been exploited to test the effects of a therapeutic agent (chemotherapy) and immune cells.

Strengths:

The system allows the in situ observation of the 3D structures along the 3 axes (x,y and z) without disrupting the integrity of the spheroids; in a time-lapse manner it is possible to follow the formation of the 3D structure and the spheroids fusion from multiple angles, allowing a better understanding of the cell aggregation/growth and kinetic of the cells.

Interestingly the system allows the analysis of cell migration/ escape from the 3D structure analysing not only the morphological changes in the periphery of the spheroids but also from the inner region demonstrating that the proliferating cells in the periphery of the structure are more involved in the migration and dissemination process. The application of the system in the study of the effects of doxorubicin and NK cells would give new insights in the description of the response of tumor 3D structure to killing agents.

Reviewer #1 (Public review):

Epigenetic regulation complex (PRC2) is essential for neural crest specification, and its misregulation has been shown to cause severe craniofacial defects. This study shows that Eed, a core PRC2 component, is critical for craniofacial osteoblast differentiation and mesenchymal proliferation after neural crest induction. Using mouse genetics and single-cell RNA sequencing, the researcher found that conditional knockout of Eed leads to significant craniofacial hypoplasia, impaired osteogenesis, and reduced proliferation of mesenchymal cells in post-migratory neural crest populations.

Overall, the study is superficial and descriptive. No in-depth mechanism was analyzed and the phenotype analysis is not comprehensive.

Reviewer #2 (Public review):

Summary:

The role of PRC2 in post-neural crest induction was not well understood. This work developed an elegant mouse genetic system to conditionally deplete EED upon SOX10 activation. Substantial developmental defects were identified for craniofacial and bone development. The authors also performed extensive single-cell RNA sequencing to analyze differentiation gene expression changes upon conditional EED disruption.

Strengths:

(1) Elegant genetic system to ablate EED post neural crest induction.

(2) Single-cell RNA-seq analysis is extremely suitable for studying the cell type-specific gene expression changes in developmental systems.

Weaknesses:

(1) Although this study is well designed and contains state-of-the-art single-cell RNA-seq analysis, it lacks the mechanistic depth in the EED/PRC2-mediated epigenetic repression. This is largely because no epigenomic data was shown.

(2) The mouse model of conditional loss of EZH2 in neural crest has been previously reported, as the authors pointed out in the discussion. What is novel in this study to disrupt EED? Perhaps a more detailed comparison of the two mouse models would be beneficial.

(3) The presentation of the single-cell RNA-seq data may need improvement. The complexity of the many cell types blurs the importance of which cell types are affected the most by EED disruption.

(4) While it's easy to identify PRC2/EED target genes using published epigenomic data, it would be nice to tease out the direct versus indirect effects in the gene expression changes (e.g Figure 4e).