Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors attempt to reconstitute some active zone properties by introducing synaptic ribbon proteins into HEK cells. This "ground-up" approach can be valuable for assessing the necessity of specific proteins in synaptic function. Here, the authors co-transfect a membrane-targeted bassoon, RBP2, calcium channel subunits and Ribeye to generate what they call "synthetic ribbons". The resultant structures show an ability to cluster calcium channels (Figure 4B) and a modest ability to concentrate calcium entry locations (figure 7J). At the light level, the ribeye aggregates look spherical and localize to the membrane through its interaction with the membrane-targeted bassoon. It is a nice proof-of-principle in establishing a useful experimental system for studying calcium channel localization. However, the impact of the study is modest. No new biology is discovered and to call these structures "synthetic ribbons" is an overstatement in the absence of an ultrastructural analysis.

Strengths:

(1) The authors establish a new experimental system for the study of calcium channel localization to active zones.<br /> (2) The clustering of calcium channels to bassoon via RBP2 is a nice confirmation of a previously described interaction between bassoon and calcium channels in a cell-based system<br /> (3) The "ground-up" approach is an attractive one and theoretically allows one to learn a lot about the essential interactions for building a ribbon structure.

Weaknesses:

(1) Are these truly "synthetic ribbons". The ribbon synapse is traditionally defined by its morphology at the EM level. To what extent these structures recapitulate ribbons is not shown. It has been previously shown that Ribeye forms aggregates on its own. Do these structures look any more ribbon-like than ribeye aggregates in the absence of its binding partners?<br /> (2) No new biology is discovered here. The clustering of channels is accomplished by taking advantage of previously described interactions between RBP2, Ca channels and bassoon. The localization of Ribeye to bassoon takes advantage of a previously described interaction between the two. Even the membrane localization of the complexes required the introduction of a membrane-anchoring motif.<br /> (3) The only thing ribbon-specific about these "syn-ribbons" is the expression of ribeye and ribeye does not seem to participate in the localization of other proteins in these complexes. Bsn, Cav1.3 and RBP2 can be found in other neurons.<br /> (4) As the authors point out, RBP2 is not necessary for some Ca channel clustering in hair cells, yet seems to be essential for clustering to bassoon here.<br /> (5) The difference in Ca imaging between SyRibbons and other locations is extremely subtle.<br /> (6) The effect of the expression of palm-Bsn, RBP2 and the combination of the two on Ca-current is ambiguous. It appears that while the combination is larger than the control, it probably isn't significantly different from either of the other two alone (Fig 5). Moreover, expression of Ribeye + the other two showed no effect on Ca current (Figure 7). Also, why is the IV curve right shifted in Figure 7 vs Figure 5?<br /> (7) While some of the IHC is quantified, some of it is simply shown as single images. EV2, EV3 and Figure 4a in particular (4b looks convincing enough on its own, but could also benefit from a larger sample size and quantification)

Reviewer #2 (Public Review):

Summary:

The authors show that co-expression of bassoon, RIBEYE, Cav1.3-alpha1, Cav-beta3, Cav-alpha2delta1, and RBP2 in a heterologus system (HEK293 cells) is sufficient to generate a protein complex resembling a presyanptic ribbon-type active zone both in morphology and in function (in clustering voltage-gated Ca channels and creating sites for localized Ca2+ entry). If the 3 separate Cav gene products are taken as a single protein (i.e. a Ca channel), the conclusion is that the core of a ribbon synapse comprises 4 proteins: bassoon holds the RIBEYE-containing ribbon to the plasma membrane, and RPB2 binds to bassoon and Ca channels, tethering the Ca channels to the presynaptic active zone.

Strengths:

Good use of a heterologous system with generally appropriate controls provides convincing evidence that a presynaptic ribbon-type active zone (without the ability to support exocytosis), with the ability to support localized Ca2+ entry (a key feature of ribbon-type pre-synapses) can be assembled from a few proteins.

Weaknesses:

(1) Relies on over-expression, which almost certainly diminishes the experimentally-measured parameters (e.g. pre-synapse clustering, localization of Ca2+ entry).<br /> (2) Are HEK cells the best model? HEK cells secrete substances and have a studied-endocytitic pathway, but they do not create neurosecretory vesicles. Why didn't the authors try to reconstitute a ribbon synapse in a cell that makes neurosecretory vesicles like a PC12 cell?<br /> (3) Related to 1 and 2: the Ca channel localization observed is significant but not so striking given the presence of Cav protein and measurements of Ca2+ influx distributed across the membrane. Presumably, this is the result of overexpression and an absence of pathways for pre-synaptic targeting of Ca channels. But, still, it was surprising that Ca channel localization was so diffuse. I suppose that the authors tried to reduce the effect of over-expression by using an inducible Cav1.3? Even so, the accessory subunits were constitutively over-expressed.

Reviewer #3 (Public Review):

Summary:

Ribbon synapses are complex molecular assemblies responsible for synaptic vesicle trafficking in sensory cells of the eye and the inner ear. The Ca2+-dependent exocytosis occurs at the active zone (AZ), however, the molecular mechanisms orchestrating the structure and function of the AZs of ribbon synapses are not well understood. To advance in the understanding of those mechanisms, the authors present a novel and interesting experimental strategy pursuing the reconstitution of a minimal active zone of a ribbon synapse within a synapse-naïve cell line: HEK293 cells. The authors have used stably transfected HEK293 cells that express voltage-gated Ca2+ channels subunits (constitutive -CaV beta3 and CaV alpha2 beta1- and inducible CaV1.3 alpha1). They have expressed in those cells several proteins of the ribbon synapse active zone: (1) RIBEYE, (2) a modified version of Bassoon that binds to the plasma membrane through artificial palmitoylation (Palm-Bassoon) and (3) RIM-binding protein 2 (RBP2) to induce the formation of a minimal active zone that they called SyRibbons. The formation of such structures is convincing, however, the evidence of such structures having an impact enhancing Ca2+-currents, as the authors claim, is rather weak in the present version of the study.

Strengths of the study:

(1) The study is carefully carried out using a remarkable combination of (1) superresolution microscopy, to analyze the formation and subcellular distribution of molecular assemblies and (2) functional assessment of voltage-gated Ca2+ channels using patch-clamp recording of Ca2+-currents and fluorometry to correlate Ca2+ influx with the molecular assemblies formed by AZ proteins. The results are of high quality and are in general accompanied of required control experiments.<br /> (2) The method opens new opportunities to further investigate the minimal and basic properties of AZ proteins that are difficult to study using in vivo systems. The cells that operate through ribbon synapses (e.g. photoreceptors and hair cells) are particularly difficult to manipulate, so setting up and validating the use of a heterologous system more suitable for molecular manipulations is highly valuable.<br /> (3) The structures formed by RIBEYE and Palm-Bassoon in HEK293 cells identified by STED nanoscopy are strikingly similar to the AZs of ribbon synapses found in rat inner hair cells (Figure 2).

Weaknesses of the study:

(1) The results obtained in a heterologous system (HEK293 cells) need to be interpreted with caution. They will importantly speed the generation of models and hypothesis that will, however, require in vivo validation.<br /> (2) The authors analyzed the distribution of RIBEYE clusters in different membrane compartments and correctly conclude that RIBEYE clusters are not trapped in any of those compartments, but it is soluble instead. The authors, however, did not carry out a similar analysis for Palm-Bassoon. It is therefore unknown if Palm-Bassoon binds to other membrane compartments besides the plasma membrane. That could occur because in non-neuronal cells GAP43 has been described to be in internal membrane compartments. This should be investigated to document the existence of ectopic internal Synribbons beyond the plasma membrane because it might have implications for interpreting functional data in case Ca2+-channels become part of those internal Synribbons.<br /> (3) The co-expression of RBP2 and Palm-Bassoon induces a rather minor but significant increase in Ca2+-currents (Figure 5). Such an increase does not occur upon expression of (1) Palm-Bassoon alone, (2) RBP2 alone or (3) RIBEYE alone (Figure 5). Intriguingly, the concomitant expression of Palm-Bassoon, RBP2 and RIBEYE does not translate into an increase of Ca2+-currents either (Figure 7).<br /> (4) The authors claim that Ca2+-imaging reveals increased CA2+-signal intensity at synthetic ribbon-type AZs. That claim is a subject of concern because the increase is rather small and it does not correlate with an increase in Ca2+-currents.

Reviewer #1 (Public Review):

Summary:

Protein conformational changes are often critical to protein function, but obtaining structural information about conformational ensembles is a challenge. Over a number of years, the authors of the current manuscript have developed and improved an algorithm, qFit protein, that models multiple conformations into high resolution electron density maps in an automated way. The current manuscript describes the latest improvements to the program, and analyzes the performance of qFit protein in a number of test cases, including classical statistical metrics of data fit like Rfree and the gap between Rwork and Rfree, model geometry, and global and case-by-case assessment of qFit performance at different data resolution cutoffs. The authors have also updated qFit to handle cryo-EM datasets, although the analysis of its performance is more limited due to a limited number of high-resolution test cases and less standardization of deposited/processed data.

Strengths:

The strengths of the manuscript are the careful and extensive analysis of qFit's performance over a variety of metrics and a diversity of test cases, as well as careful discussion of the limitations of qFit. This manuscript also serves as a very useful guide for users in evaluating if and when qFit should be applied during structural refinement.

Reviewer #2 (Public Review):

Summary

The manuscript "Uncovering Protein Ensembles: Automated Multiconformer Model building for X-ray Crystallography and Cryo-EM" by Wankowicz et al. describes updates to qFit, an algorithm for the characterization of conformational heterogeneity of protein molecules based on X-ray diffraction of Cryo-EM data. The work provides a clear description of the algorithm used by qFit. The authors then proceed to validate the performance of qFit by comparing to deposited X-ray entries in the PDB in the 1.2-1.5 Å resolution range as quantified by Rfree, Rwork-Rfree, detailed examination of the conformations introduced by qFit, and performance on stereochemical measures (MolProbity scores). To examine the effect of experimental resolution of X-ray diffraction data, they start from an ultra high-resolution structure (SARS-CoV2 Nsp3 macrodomain) to determine how the loss of resolution (introduced artificially) degrades the ability of qFit to correctly infer the nature and presence of alternate conformations. The authors observe a gradual loss of ability to correctly infer alternate conformations as resolution degrades past 2 Å. The authors repeat this analysis for a larger set of entries in a more automated fashion and again observe that qFit works well for structures with resolutions better than 2 Å, with a rapid loss of accuracy at lower resolution. Finally, the authors examine the performance of qFit on cryo-EM data. Despite a few prominent examples, the authors find only a handful (8) of datasets for which they can confirm a resolution better than 2.0 Å. The performance of qFit on these maps is encouraging and will be of much interest because cryo-EM maps will, presumably, continue to improve and because of the rapid increase in the availability of such data for many supramolecular biological assemblies. As the authors note, practices in cryo-EM analysis are far from uniform, hampering the development and assessment of tools like qFit.

Strengths

qFit improves the quality of refined structures at resolutions better than 2.0 A, in terms of reflecting true conformational heterogeneity and geometry. The algorithm is well-designed and does not introduce spurious or unnecessary conformational heterogeneity. I was able to install and run the program without a problem within a computing cluster environment. The paper is well-written and the validation thorough.<br /> I found the section on cryo-EM particularly enlightening, both because it demonstrates the potential for discovery of conformational heterogeneity from such data by qFit, and because it clearly explains the hurdles towards this becoming common practice, including lack of uniformity in reporting resolution, and differences in map and solvent treatment.

Weaknesses

Due to limitations of past software engineering, the paper lacks a careful comparison to past versions of qFit. In light of the extensive assessment of the current version of qFit, this is a minor concern.

Although qFit can handle supramolecular assemblies and bound organic molecules, analysis in the manuscript is limited to single-chain X-ray structures. I look forward to demonstration of its utility in such cases in future work.

Appraisal & Discussion

Overall, the authors convincingly demonstrate that qFit provides a reliable means to detect and model conformational heterogeneity within high-resolution X-ray diffraction datasets and (based on a smaller sample) in cryo-EM density maps. This represents the state of the art in the field and will be of interest to any structural biologist or biochemist seeking to attain an understanding of the structural basis of the function of their system of interest, including potential allosteric mechanisms-an area where there are still few good solutions. That is, I expect qFit to find widespread use.

Reviewer #3 (Public Review):

Summary:

The authors address a very important issue of going beyond a single-copy model obtained by the two principal experimental methods of structural biology, macromolecular crystallography and cryo electron microscopy (cryo-EM). Such multiconformer model is based on the fact that experimental data from both these methods represent a space- and time-average of a huge number of the molecules in a sample, or even in several samples, and that the respective distributions can be multimodal. Differently from structure prediction methods, this approach is strongly based on accurate high-resolution experimental information and requires validated single-copy high-quality models as input. In overall, the results support the authors' conclusions.

In fact, the method addresses two problems which could be considered separately:

- an automation of construction of multiple conformations when they can be identified visually;<br /> - a determination of multiple conformations when their visual identification is difficult or impossible.

The former is a known problem, when missing alternative conformations may cost a few percent in R-factors. While these conformations are relatively easy to detect and build manually, the current procedure may save significant time being quite efficient, as the test results show. It is an indisputably useful tool for such a goal. The second problem is important from the physical point of view and has been considered first thirty years ago by Burling & Brünger. The manuscript does not specify clearly how much the current tool addresses the second case. To model such maps, the authors introduced errors in structure factors, however, being independent, as in this work, such errors, even quite high, may leave the maps reasonably well interpretable. Obviously, it is impossible to model all kinds of errors and this modeling of noise is appreciated but it would helpful for understanding if the manuscript shows, for example, the worst map when the procedure was successful.

The new procedure deals with a second-order variation in the R-factors, of about 1% or less, like placing riding hydrogen atoms, modeling density deformation or variation of the bulk solvent. In such situations, it is hard to justify model improvement. Keeping Rfree values or their marginal decreasing can be considered as a sign that the model does not overfit data but hardly as a strong argument in favor of the model.

In general, global targets are less appropriate for this kind of problems and local characteristics may be better indicators. Improvement of the model geometry is a good choice. Indeed, yet Cruickshank (1956) showed that averaged density images may lead to a shortening of covalent bonds when interpreting such maps by a single model. However, a total absence of geometric outliers is not necessarily required for the structures solved at a high resolution where diffraction data should have a more freedom to place the atoms where the experiments "see" them.

The key local characteristic for multicomformer models is a closeness of the model map to the experimental one. Actually, the procedure uses a kind of such measure, the Bayesian information criteria (BIC). Unfortunately, the manuscript does not describe how sharply it identifies the best model and how much it changes between the initial and final models; in general, there is no feeling about its values. The Q-score (page 17) can be an appropriate tool for the first problem where the multiple conformations and individual atomic images are clearly separated and not for the second problem where the contributions from neighboring conformations and atoms are merged. In addition to BIC or to even more conventional global target functions such as LS or map correlation, the extreme values of the local difference maps may help to validate, or not, the model.

This described method with the results presented is a strong argument for a need in experimental data and information they contain, differently from a pure structure prediction. This tool is important to produce user-unbiased multiconformer models rapidly and automatically. At the same time, absence of strong density-based validation components may limit its impact.

Strengths:<br /> Addressing an important problem and automatisation of model construction for alternative conformations using high-resolution experimental data.

Weaknesses:<br /> An insufficient validation of the models when no discrete alternative conformations visible and insufficiency of local real-space validation indicators.

Reviewer #1 (Public Review):

Summary:

The authors try to use a gene therapy approach to cure urofacial symptoms in an HSPE2 mutant mouse model.

Strengths:

The authors have convincingly shown the expression of AAV9/HSPE2 in pelvic ganglion and liver tissues. They have also shown the defects in urethra relaxation and bladder muscle contraction in response to EFS in mutant mice, which were reversed in treated mice.

Weaknesses:

It is easy to understand that high expression levels of HPSE2 in the bladder tissue lead to bladder dysfunction in human patients, however, the undetectable level of HPSE2 in AAV9 transfected mice bladders is a big question for the functional correction in those HPSE2 mutated mice.

Reviewer #2 (Public Review):

In this study, Lopes and colleagues provide evidence to support the potential for gene therapy to restore expression of heparanase-2 (Hpse2) in mice mutant for this gene, as occurs in urofacial syndrome. Building on prior studies describing the nature of urinary tract dysfunction in Hpse2 mutant mice, the authors applied a gene therapy approach to determine whether gene replacement could be achieved, and if so, whether restoration of HPSE2 expression could mitigate the urinary tract dysfunction. Using a viral vector-based strategy, shown to be successful for gene replacement in humans, the authors demonstrated dose-dependent viral transduction of pelvic ganglia and liver in wild type mice. No impact on body weight or liver health was noted suggesting the approach was safe. Administration of AAV9/HPSE2 to Hpse2 mutant mice was associated with similar transduction of pelvic ganglia and a corresponding increase in heparanase-2 protein expression in this site. Analysis of bladder outflow tract and bladder body physiology using organ bath studies showed that re-expression of heparanase-2 in Hpse2 mutant mice was associated with restored neurogenic relaxation of the outflow tract and nerve-evoked contraction of the bladder body, albeit with notable variability in the response at lower frequencies across replicates. Differences were noted in the evoked response to carbachol with bladders from Hpse2 mutant male mice showing increased sensitivity upon HPSE2 replacement compared to wild type, but bladders from female mice showing no difference. Based on these findings the authors concluded that AAV9-based HPSE2 replacement is feasible and safe, mitigates some physiological deficits in outflow tract and bladder tissue from Hpse2 mutant mice and provides proof-of-principle for gene replacement approaches for other genes implicated in lower urinary tract disorders. Strengths include a solid experimental design and data in support of some of the conclusions, and discussion of limitations of the approach. Weaknesses include the variability, albeit acknowledged, in some of the functional assessments, and the limited investigation of bladder tissue morphology in Hpse2 mutant mice.

Reviewer #1 (Public Review):

Summary:

This paper suggests to apply intrinsically-motivated exploration for the discovery of robust goal states in gene regulatory networks.

Strengths:

The paper is well written. The biological motivation and the need for such methods are formulated extraordinarily well. The battery of experimental models is impressive.

Weaknesses:

(1) The proposed method is compared to the random search. That says little about the performance with regard to the true steady-state goal sets. The latter could be calculated at least for a few simple ODE (e.g., BIOMD0000000454, `Metabolic Control Analysis: Rereading Reder'). The experiment with 'oscillator circuits' may not be directly interpolated to the other models.

The lack of comparison to the ground truth goal set (attractors of ODE) from arbitrary initial conditions makes it hard to evaluate the true performance/contribution of the method. A part of the used models can be analyzed numerically using JAX, while there are models that can be analyzed analytically.

"...The true versatility of the GRN is unknown and can only be inferred through empirical exploration and proxy metrics....": one could perform a sensitivity analysis of the ODEs, identifying stable equilibria. That could provide a proxy for the ground truth 'versatility'.

(2) The proposed method is based on `Intrinsically Motivated Goal Exploration Processes with Automatic Curriculum Learning', which assumes state action trajectories [s_{t_0:t}, a_{t_0:t}], (2.1 Notations and Assumptions' in the IMGEP paper). However, the models used in the current work do not include external control actions, but rather only the initial conditions can be set. It is not clear from the methods whether IMGEP was adapted to this setting, and how the exploration policy was designed w/o actual time-dependent actions. What does "...generates candidate intervention parameters to achieve the current goal...."<br /> mean considering that interventions 'Sets the initial state...' as explained in Table 2?

(3) Fig 2 shows the phase space for (ERK, RKIPP_RP) without mentioning the typical full scale of ERK, RKIPP_RP. It is unclear whether the path from (0, 0) to (~0.575, ~3.75) at t=1000 is significant on the typical scale of this phase space. is it significant on the typical scale of this phase space?

(4) Table 2:<br /> (a) Where is 'effective intervention' used in the method?<br /> (b) In my opinion 'controllability', 'trainability', and 'versatility' are different terms. If there correspondence is important I would suggest to extend/enhance the column "Proposed Isomorphism". otherwise, it may be confusing. I don't see how this table generalizes generalizes "concepts from dynamical complex systems and behavioral sciences under a common navigation task perspective".

Reviewer #2 (Public Review):

Summary:

Etcheverry et al. present two computational frameworks for exploring the functional capabilities of gene regulatory networks (GRNs). The first is a framework based on intrinsically motivated exploration, here used to reveal the set of steady states achievable by a given gene regulatory network as a function of initial conditions. The second is a behaviorist framework, here used to assess the robustness of steady states to dynamical perturbations experienced along typical trajectories to those steady states. In Figs. 1-5, the authors convincingly show how these frameworks can explore and quantify the diversity of behaviors that can be displayed by GRNs. In Figs. 6-9, the authors present applications of their framework to the analysis and control of GRNs, but the support presented for their case studies is often incomplete.

Following revision, my overall perspective of the paper remains unchanged. The first half of the paper provides solid evidence to support an important conceptual framework. The evidence presented for the use cases in the latter half is incomplete; as the authors note, they are preliminary and meant to be built on in future work. I have included my first round comments below.

Strengths:

Overall, the paper presents an important development for exploring and understanding GRNs/dynamical systems broadly, with solid evidence supporting the first half of their paper in a narratively clear way.

The behaviorist point of view for robustness is potentially of interest to a broad community, and to my knowledge introduces novel considerations for defining robustness in the GRN context.

Some specific weaknesses, mostly concerning incomplete analyses in the second half of the paper:

(1) The analysis presented in Fig. 6 is exciting but preliminary. Are there other appropriate methods for constructing energy landscapes from dynamical trajectories in gene regulatory networks? How do the results in this particular case study compare to other GRNs studied in the paper?

Additionally, it is unclear whether the analysis presented in Fig. 6C is appropriate. In particular, if the pseudopotential landscapes are constructed from statistics of visited states along trajectories to the steady state, then the trajectories derived from dynamical perturbations do not only reflect the underlying pseudo-landscape of the GRN. Instead, they also include contributions from the perturbations themselves.

(2) In Fig. 7, I'm not sure how much is possible to take away from the results as given here, as they depend sensitively on the cohort of 432 (GRN, Z) pairs used. The comparison against random networks is well-motivated. However, as the authors note, comparison between organismal categories is more difficult due to low sample size; for instance, the "plant" and "slime mold" categories each only has 1 associated GRN. Additionally, the "n/a" category is difficult to interpret.

(3) In Fig. 8, it is unclear whether the behavioral catalog generated is important to the intervention design problem of moving a system in one attractor basin to another. The authors note that evolutionary searches or SGD could also be used to solve the problem. Is the analysis somehow enabled by the behavioral catalog in a way that is complementary to those methods? If not, comparison against those methods (or others e.g. optimal control) would strengthen the paper.

(4) The analysis presented in Fig. 9 also is preliminary. The authors note that there exist many algorithms for choosing/identifying the parameter values of a dynamical system that give rise to a desired time series. It would be a stronger result to compare their approach to more sophisticated methods, as opposed to random search and SGD. Other options from the recent literature include Bayesian techniques, sparse nonlinear regression techniques (e.g. SINDy), and evolutionary searches. The authors note that some methods require fine-tuning in order to be successful, but even so, it would be good to know the degree of fine-tuning which is necessary compared to their method. [second round: the authors have included a comparison against CMA-ES, an evolutionary algorithm]

Reviewer #1 (Public Review):

The mechanisms underlying the generation and maintenance of LLPCs have been one of the unresolved issues. In the last few years, several groups have independently generated new genetic tools or models and addressed how LLPCs are generated or maintained in homeostatic conditions or upon immunization or infection. Here, Jing et al. have also established a new PC time stamping system and tried to address the issues above. The authors have found that LLPCs accumulated in the BM PC pool, along with aging, and that LLPCs had unique sufacetome, transcriptome, and BCR clonality. These observations have already been made by other groups (Xu et al. 2020, Robinson et al. 2022, Liu et al. 2022, Koike et al. 2023, Robinson et al. 2023, plus Tellier et al., 2024), therefore it is hard to find significant conceptual advances there. In my opinion, however, genetic analysis of the role of CXCR4 on PC localization or survival in BM (Figure 4 and 5) provided new aspects which have not been addressed in previous studies. Importantly, CXCR4 was required for the maintenance of plasma cells in bone marrow survival niches, conditional loss of which led to rapid mobilization from the bone marrow, reduced plasma cell survival, and reduced antibody titer. Thus, these data suggest that CXCR4-CXCL12 axis is not only important for plasma cell recruitment to the bone marrow but also essential for their lodging on the niches. I think the study is of high quality and the findings should be widely shared in the field.

Reviewer #2 (Public Review):

In this study by Jing, Fooksman, and colleagues, a Blimp1-CreERT2-based genetic tracing study is employed to label plasma cells. Over the course of several months post-tamoxifen treatment, the only remaining labeled cells are long-lived plasma cells. This system provides a way to sort live long-lived plasma cells and compare them to unlabeled plasma cells, which contain a range of short-to-long-lived cells. From this analysis, several observations are made: 1) the turnover rate of plasma cells is greater in the spleen than in the bone marrow; 2) the turnover rate is highest early in life; 3) subtle transcriptional and cell surface marker differences distinguish long- from shorter-lived plasma cells; 4) long-lived plasma cells in the bone marrow are sessile and localize in clusters with each other; 5) CXCR4 is required for plasma cell retention in these clusters and in the bone marrow; 6) Repertoire analysis hints that the selection of long-lived plasma cells is not random for any cell that lands in the bone marrow.

Strengths:

(1) The genetic timestamping approach is a clever and functional way to separate plasma cells of differing longevities.

(2) This approach led to the identification of several markers that could help prospective separation of long-lived plasma cells from others.

(3) Functional labeling of long-lived plasma cells allowed for a higher resolution analysis of transcriptomes and motility than was previously possible.

(4) The genetic system allowed for a revisitation of the importance of CXCR4 in plasma cell retention and survival.

Weaknesses:

(1) Most of the labeling studies, likely for practical reasons, were done on polyclonal rather than antigen-specific plasma cells. The triggers of these responses could vary based on age at the time of exposure, anatomical sites, etc. How these differences might influence markers and transcriptomes, independently of longevity, is not completely known.

(2) The fraction of long-lived plasma cells in the unlabeled fraction varies with age, potentially diluting differences between long- and short-lived plasma cells.

(3) The authors suggest their data favors a model by which plasma cells compete for niche space. Yet there is no evidence presented here that these niches are limiting. While a finite number of plasma cells may occupy a single niche (Figure 2), it may be that these niches overall are abundant in the bone marrow and do not restrict LLPC numbers. Robinson...Tarlinton and colleagues (Immunity, 2023) in fact provide experimental evidence against an extrinsic limit.

(4) The functional importance of the observed transcriptome differences between long- and shorter-lived plasma cells is unknown. An assessment as to whether these differences are conserved in human long- and short-lived bone marrow plasma cells might provide circumstantial supporting evidence that these changes are important for longevity.

Reviewer #3 (Public Review):

Summary:

Long-lived PCs are maintained in a CXCR4-dependent manner.

Strengths:

The reporter mice for fate-mapping can clearly distinguish long-lived PCs from total PCs and greatly contribute to the identification of long-lived PCs.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

How crowding represses the miR-3036-5p is still unclear.

Reviewer #2 (Public Review):

Summary:

Environmental influences on development are ubiquitous, affecting many phenotypes in organisms. However molecular genetic and cellular mechanisms transducing environmental signals are still only barely understood. This study examines part of one such intracellular mechanism in a polyphenic (or dimorphic) aphid.

Strengths:

While other published reports have linked phenotypic plasticity to RNA editing before, this study reports such an interaction in insects. The study uses a wide array of molecular tools to identify connections upstream and downstream of the RNA editing to elucidate the regulatory mechanism, which is illuminating.

Weaknesses:

While this system is intriguing, this report does not foster confidence in its conclusions. Many of the analyses seem based on very small sample sizes. It is itself problematic that sample sizes are not obvious in most figures, although based on Methods section covering RNAseq, they seem to be either 3, 6 or 9, depending on whether stages were pooled, but that point is not made clear. With such small sample sizes, statistical tests of any kind are unreliable. Besides the ambiguity on sample sizes, it's unclear what error bars or whiskers show in plots throughout this study. When sample sizes are small estimates of variance are not reliable. Student's t-test is not appropriate for comparisons with such small sample sizes. Presently, it is not possible to replicate the tests shown in Figures 3, 4 and 6. (Besides the HT-seq reads, other data should also be made publicly available, following the journal's recommendations.) Regardless, effect sizes in some comparisons (Fig 3J, 4A-C, 6E,H) are clearly not large, making confidence in conclusions low. The authors should be cautious about over-interpreting these data.

Reviewer #2 (Public Review):

Summary:

The dominant paradigm in the past decade for modeling the ventral visual stream's response to images has been to train deep neural networks on object classification tasks and regress neural responses from units of these networks. While object classification performance is correlated to variance explained in the neural data, this approach has recently hit a plateau of variance explained, beyond which increases in classification performance do not yield improvements in neural predictivity. This suggests that classification performance may not be a sufficient objective for building better models of the ventral stream. Lindsey & Issa study the role of factorization in predicting neural responses to images, where factorization is the degree to which variables such as object pose and lighting are represented independently in orthogonal subspaces. They propose factorization as a candidate objective for breaking through the plateau suffered by models trained only on object classification. They show the degree of factorization in a model captures aspects of neural variance that classification accuracy alone does not capture, hence factorization may be an objective that could lead to better models of ventral stream. I think the most important figure for a reader to see is Fig. 6.

Strengths:

This paper challenges the dominant approach to modeling neural responses in the ventral stream, which itself is valuable for diversifying the space of ideas.

This paper uses a wide variety of datasets, spanning multiple brain areas and species. The results are consistent across the datasets, which is a great sign of robustness.

The paper uses a large set of models from many prior works. This is impressively thorough and rigorous.

The authors are very transparent, particularly in the supplementary material, showing results on all datasets. This is excellent practice.

Weaknesses:

The authors have addressed many of the weaknesses in the original review. The weaknesses that remain are limitations of the work that cannot be easily addressed. In addition to the limitations stated at the end of the discussion, I'll add two:

(1) This work shows that factorization is correlated with neural similarity, and notably explains some variance in neural similarity that classification accuracy does not explain. This suggests that factorization could be used as an objective (along with classification accuracy) to build better models of the brain. However, this paper does not do that - using factorization to build better models of the brain is left to future work.

Reviewer #3 (Public Review):

Summary:

Object classification serves as a vital normative principle in both the study of the primate ventral visual stream and deep learning. Different models exhibit varying classification performances and organize information differently. Consequently, a thriving research area in computational neuroscience involves identifying meaningful properties of neural representations that act as bridges connecting performance and neural implementation. In the work of Lindsey and Issa, the concept of factorization is explored, which has strong connections with emerging concepts like disentanglement [1,2,3] and abstraction [4,5]. Their primary contributions encompass two facets: (1) The proposition of a straightforward method for quantifying the degree of factorization in visual representations. (2) A comprehensive examination of this quantification through correlation analysis across deep learning models.

To elaborate, their methodology, inspired by prior studies [6], employs visual inputs featuring a foreground object superimposed onto natural backgrounds. Four types of scene variables, such as object pose, are manipulated to induce variations. To assess the level of factorization within a model, they systematically alter one of the scene variables of interest and estimate the proportion of encoding variances attributable to the parameter under consideration.

The central assertion of this research is that factorization represents a normative principle governing biological visual representation. The authors substantiate this claim by demonstrating an increase in factorization from macaque V4 to IT, supported by evidence from correlated analyses revealing a positive correlation between factorization and decoding performance. Furthermore, they advocate for the inclusion of factorization as part of the objective function for training artificial neural networks. To validate this proposal, the authors systematically conduct correlation analyses across a wide spectrum of deep neural networks and datasets sourced from human and monkey subjects. Specifically, their findings indicate that the degree of factorization in a deep model positively correlates with its predictability concerning neural data (i.e., goodness of fit).

Strengths:

The primary strength of this paper is the authors' efforts in systematically conducting analysis across different organisms and recording methods. Also, the definition of factorization is simple and intuitive to understand.

Weaknesses:

Comments on revised version:

I thank the authors for addressing the weaknesses I brought up regarding the manuscript.

Reviewer #1 (Public Review):

Summary:

The authors of the study are trying to show that RNAseq can be used for neoantigen prediction and that the machine learning approach to the prediction can reveal very useful information for the selection of neoantigens for personalized antitumor vaccination.

Strengths:

The authors demonstrated that RNA expression of a neoantigen is a very important factor in the selection of peptides for the creation of personalized vaccines. They proved in vivo that in silico-predicted neoantigens can trigger an antitumor response in mice.

Weaknesses:

The selection of the peptides for vaccination is not clear. Some peptides were selected before and some after processing. What processing is also not clear. The authors didn't provide the full list of peptides before and after processing, please add those. And it wasn't clear that these peptides were previously published. Looking at the previously published table with peptide from B16 F10 (https://www.nature.com/articles/s41598-021-89927-5/tables/3), there are other genes with high expression, e.g. Tab2, Tm9sf3 that have higher expression than Herc6, please clarify the choice.

It's not clear how many mice were used for each group in each experiment, please add this information to the text and figures. It would be good to add this, to aid the understanding of a broader audience.

Please provide information about what software was used for statistical analysis.

Reviewer #2 (Public Review):

Summary:

The authors develop a new neoantigen prediction tool (NAP-CNB) which primarily predicts neoantigens based on expression (RNAseq) and ranks mutations using binding affinity. The validated predicted neoantigens in mice demonstrate that neoantigens with higher expression (but not necessarily the highest immunogenicity) lead to the greatest tumor control.

Strengths:

There is in vivo validation of the neoantigens.<br /> Demonstrates comparability to other prediction algorithms that are commonly used.<br /> Demonstrates that expression holds a higher value than T-cell responses in actual tumor control.

Weaknesses:

Binding affinity does not always predict immune responses or tumor control in vivo which is used as part of the selection criteria.

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.<br /> (1) In Figure 1, a sperm flagellum protein, which is far away 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.

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 provide a genome annotation resource of 33 insects using a motif-blind prediction method for tissue-specific cis-regulatory modules. This is a welcome addition that may facilitate further research in new laboratory systems, and the approach seems to be relatively accurate, although it should be combined with other sources of evidence to be practical.

Strengths:

The paper clearly presents the resource, including the testing of candidate enhancers identified from various insects in Drosophila. This cross-species analysis, and the inherent suggestion that training datasets generated in flies can predict a cis-regulatory activity in distant insects, is interesting. While I can not be sure this approach will prevail in the future, for example with approaches that leverage the prediction of TF binding motifs, the SCRMShaw tool is certainly useful and worth consideration for the large community of genome scientists working on insects.

Weaknesses:

While the authors made the effort to provide access to the SCRMShaw annotations via the RedFly database, the usefulness of this resource is somewhat limited at the moment. First, it is possible to generate tables of annotated elements with coordinates, but it would be more useful to allow downloads of the 33 genome annotations in GFF (or equivalent) format, with SCRMshaw predictions appearing as a new feature. Also, I should note that unlike most species some annotations seem to have issues in the current RedFly implementation. For example, Vcar and Jcoen turn empty.

Reviewer #2 (Public Review):

Summary:

The ability of researchers to identify and compare enhancers across different species is an important facet of understanding gene regulation across development and evolution. Many traditional methods of enhancer identification involve sequence alignments and manual annotations, limiting the ability to expand the scope of regulatory investigations into many species. In order to overcome this obstacle, the authors apply a previously published machine learning method called SCRMshaw to predict enhancers across 33 insect species, using D. melanogaster as a reference. SCRMshaw operates through the selection of a few dozen training loci in a reference genome, marking genomic loci in other species that are significantly enriched with similar k-mer distributions relative to randomly selected genomic backgrounds. Upon identification of predicted enhancer regions, the authors perform post-processing step filtering and identify the most likely predicted enhancer candidates based on the proximity of an orthologous target gene. They then perform reporter gene analysis to validate selected predicted enhancers from other species in D. melanogaster. The analysis of the expression patterns returned variable results across the selected predicted regions.

Strengths:

The authors provide annotations of predicted regions across dozens of insect species, with the intention of expanding and refining the annotations for use by the scientific field. This is useful, as researchers will be able to use the identified annotations for their own work or as a benchmark for future methods. This work also showcases the flexible and versatile nature of SCRMshaw, which can readily obtain predictions using training sets of genomic loci requiring only a few dozen annotations as input. SCRMshaw does not require sequence alignments of the enhancers and can operate without prior knowledge of the cis-regulatory sequence rules such as transcription factor binding motifs, making it a useful tool to explore the evolution of enhancers in further distant and less well-studied species.

Weaknesses:

This work provides predicted enhancer annotations across many insect species, with reporter gene analysis being conducted on selected regions to test the predictions. However, the code for the SCRMshaw analysis pipeline used in this work is not made available, making reproducibility of this work difficult. Additionally, while the authors claim the predicted enhancers are available within the REDfly database, the predicted enhancer coordinates are currently not downloadable as Supplementary Material or from a linked resource.

The authors do not validate or benchmark the application of SCRMshaw against other published methods, nor do they seek to apply SCRMshaw under a variety of conditions to confirm the robustness of the returned predicted enhancers across species. Since SCRMshaw relies on an established k-mer enrichment of the training loci, its performance is presumably highly sensitive to the selection of training regions as well as the statistical power of the given k-mer counts. The authors do not justify their selection of training regions by which they perform predictions.

While there is an attempt made to report and validate the annotated predicted enhancers using previously published data and tools, the validation lacks the depth to conclude with confidence that the predicted set of regions across each species is of high quality. In vivo, reporter assays were conducted to anecdotally confirm the validity of a few selected regions experimentally, but even these results are difficult to interpret. There is no large-scale attempt to assess the conservation of enhancer function across all annotated species.

Lastly, it is suggested that predicted regions are derived from the shared presence of sequence features such as transcription factor binding motifs, detected through k-mer enrichment via SCRMshaw. This assumption has not been examined, although there are public motif discovery tools that would be appropriate to discover whether SCRMshaw is assigning predicted regions based on previously understood motif grammar, or due to other sequence patterns captured by k-mer count distributions. Understanding the sequence-derived nature of what drives predictions is within the scope of this work and would boost confidence in the predicted enhancers, even if it is limited to a few training examples for the sake of clarity of interpretation.

Reviewer #3 (Public Review):

Summary:

In this ambitious paper, the authors develop an unparalleled community resource of insect genome regulatory annotations spanning five insect orders. They employ their previously-developed SCRMshaw method for computational cross-species enhancer prediction, drawing on available training datasets of validated enhancer sequence and expression from Drosophila melanogaster, which had been previously shown to perform well across select holometabolous insects (representing 160-345MY divergence). In this work, they expand regulatory sequence annotation to 33 insect genomes spanning Holometabola and Hemiptera, which is even more distantly related to the fly model. They perform multiple downstream analyses of sets of predicted enhancers to assess the true-positive rate of predictions; the independent comparisons of real predictions with simulated predictions and with chromatin accessibility data, as well as the functional validation through reporter gene analysis, strengthen their conclusions that their annotation pipeline achieves a high true-positive rate and can be used across long divergence times to computationally annotate regulatory genome regions, an ability that has been previously inaccessible for non-model insects and now is possible across the many newly-sequenced insect scaffold-level genomes.

Strengths:

This work fills a large gap in current methods and resources for predicting regulatory regions of the genome, a task that has long lagged behind that of coding region prediction and analysis.

Despite technical constraints in working outside of well-developed model insect systems, the authors creatively draw on existing resources to scaffold a pipeline and independently assess the likelihood of prediction validity.

The established database will be a welcome community resource in its current state, and even more so as the authors continue to expand their annotations to more insect genomes as they indicate. Their available analysis pipeline itself will be useful to the community as well for research groups that may want to undertake their own regulatory genome annotation.

Weaknesses:

The rates of predicted true positive enhancer identification vary widely across the genomes included here based on the simulations and comparison to datasets of accessible chromatin in a manner that doesn't map neatly onto phylogenetic distance. At this point, it is unclear why these patterns may arise, although this may become more clear as regulatory annotation is undertaken for more genomes.

Functional assessment of predicted enhancers was performed through reporter gene assays primarily in Drosophila melanogaster imaginal discs, a system amenable to transgenics. Unfortunately, this mode of canonical imaginal disc development is only representative of a subset of all holometabolous insects; therefore, it is difficult to interpret reporter gene expression in a fly imaginal disc as evidence of a true positive enhancer that would be active in its native species whose adult appendages develop differently through the larval stage (for example, Coleopteran and Lepidopteran legs). However, the reporter gene assays from other tissues do offer strong evidence of true positive enhancer detection, and constraints on transgenic experiments in other systems mean that this approach is the best available.

Reviewer #3 (Public Review):

Overall:

ExoIII has been described and commercialized as a dsDNA specific nuclease. Several lines of evidence, albeit incomplete, have indicated this may not be entirely true. Therefore, Wang et al comprehensively characterize the endonuclease and exonuclease enzymatic activities of ExoIII on ssDNA. A strength of the manuscript is the testing of popular kits that utilize ExoIII and coming up with and testing practical solutions (e.g., addition of SSB proteins ExoIII variants such as K121A and varied assay conditions).

Comments:

(1) The footprint of ExoIII on DNA is expected to be quite a bit larger than 5-nt, see structure in manuscript reference #5. Therefore, the substrate design in Figure 1A seems inappropriate for studying the enzymatic activity and it seems likely that ExoIII would be interacting with the FAM and/or BHQ1 ends as well as the DNA. Could this cause quenching? Would this represent real ssDNA activity? Is this figure/data necessary for the manuscript?<br /> (2) Based on the descriptions in the text, it seems there is activity with some of the other nucleases in 1C, 1F, and 1I other than ExoIII and Cas12a. Can this be plotted on a scale that allows the reader to see these relative to one other?<br /> (3) The sequence alignment in Figure 2N and corresponding text indicate a region of ExoIII lacking in APE1 that may be responsible for their differences in substrate specificity in regards to ssDNA. Does the mutational analysis support this hypothesis?

Reviewer #2 (Public Review):

Summary:

This paper describes some experiments addressing 3' exonuclease and 3' trimming activity of bacterial exonuclease III. The quantitative activity is in fact very low, despite claims to the contrary. The work is of low interest with regard to biology, but possibly of use for methods development. Thus the paper seems better suited to a methods forum.

Strengths:

Technical approaches.

Comments on revised version:

All concerns have been addressed.

Reviewer #1 (Public Review):

Summary:

Ciliary rootlet is a structure associated with the ciliary basal body (centriole) with beautiful striation observed by electron microscopy. It has been known for more than a century, but its function and protein arrangement is still unknown. This work reconstructed near-atomic resolution 3D structure of the rootlet using cryo-electron tomography, discovered a number of interesting filamentous structures inside and built molecular model of the rootlet.

Strengths:

The authors exploited the current possible ability of cryo-ET and used it appropriately to describe 3D structure of the rootlet. They carefully conducted subtomogram averaging and classification, which enabled an unprecedented detailed view of this structure. The dual use of (nearly) intact rootlet from cilia and extracted (demembraned) rootlet enabled them to describe with confidence how D1/D2/A bands form periodic structures and cross with longitudinal filaments, which are likely coiled-coil.

Weaknesses:

Some more clarifications in the method and indications in figures were needed in the original version. The authors addressed them in the revision.

Reviewer #3 (Public Review):

Summary:

The study offers a compelling molecular model for the organization of rootlets, a critical organelle that links cilia to the basal body. Striations have been observed in rootlets, but their assembly, composition, and function remain unknown. While previous research has explored rootlet structure and organization, this study delivers an unprecedented level of resolution, valuable to the centrosome and cilia field. The authors isolated rootlets from mice's eyes. They apply EM to partially purified rootlets (first negative stain, then cryoET). From these micrographs, they observed striations along the membranes along the rootlet but no regular spacing was observed.

The thickness of the sample and membranes prevented good contrast in the tomograms. Thus they further purified the rootlets using detergent, which allowed them to obtain cryoET micrographs of the rootlets with greater details. The tomograms were segmented and further processed to improve the features of the rootlet structures. From their analysis, they described 3 regular cross-striations and amorphous densities, which are connected perpendicularly to filaments along the length of the rootlets. They propose that various proteins provide the striations and rootletin (mouse homolog of human c-nap1) forms parallel coiled coils that run along the rootlet. Overall their data provide a detailed model for the molecular organization of the rootlet.

The major strength is that this high-quality study uses state-of-the-art cryo-electron tomography, sub-tomogram averaging, and image analysis to provide a model of the molecular organization of rootlets. The micrographs are exceptional, with excellent contrast and details, which also implies the sample preparation was well optimized to provide excellent samples for cryo-ET. The manuscript is also clear and accessible.

This research marks a significant step forward in our understanding of rootlets' molecular organization.

Reviewer #1 (Public Review):

Summary:

This finding shows a connection between cancer associated beta-catenin mutations extracellular vesicle secretion. A link between the beta-catenin mutation and expression of trafficking and exocytosis machinery. They used a multidisciplinary approach to explore expression levels of relevant proteins and single particle imaging to directly explore the release of extracellular vesicles. These results suggest a role of extracellular vesicles in immune evasion in liver cancer with the role needing to be further explored in other forms of cancer. I find this work to be compelling and of strong significance.

Strengths:

This paper uses multidisciplinary methods to demonstrate a compelling role of beta-catenin mutations in suppressing EV secretion in tumors. The results and imaging are extremely convincing and compelling.

Reviewer #2 (Public Review):

Summary:

Dantzer and colleagues are investigating the pivotal role of ß-catenin, a gene that undergoes mutation in various cancer cells, and its influence on promoting the evasion of immune cells. In their initial experiments, the authors developed a HepG2 mutated ß-catenin KD model, conducting transcriptional and proteomic analyses. The results revealed that the silencing of mutated ß-catenin in HepG2 cells led to an up-regulation in the expression of exosome biogenesis genes.

Furthermore, the researchers verified that these KD cells exhibited an increased production of exosomes, with the mutant form of ß-catenin concurrently decreasing the expression of SDC4 and Rab27a. Intriguingly, applying a GSK inhibitor to the cells resulted in reduced expression of SDC4 and Rab27a. Subsequent findings indicated that mutated ß-catenin actively facilitates immune escape through exosomes, and silencing exosome biogenesis correlates with a decrease in immune cell infiltration.<br /> In a crucial clinical correlation, the study demonstrated that patients with ß-catenin mutations exhibited low levels of exosome biogenesis.

Strengths:

Overall, the data robustly supports the outlined conclusions, and the study is commendably designed and executed. However, there are a few suggestions for manuscript improvement.

Weaknesses: No weakness

Reviewer #3 (Public Review):

Summary:

In this very important study by Dantzer et al., 'Emerging role of oncogenic b-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma' the authors define a role for oncogenic b-catenin on exosome biology and explore the link between reduce exosome secretion and tumor immune cell evasion. Using transcriptional and proteomic analysis of hepatocellular carcinoma cells with either oncogenic or wildtype b-catenin the authors find that oncogenic b-catenin negatively regulates exosome biogenesis.

The authors can provide compelling evidence that oncogenic b-catenin in different hepatocellular carcinoma cells negatively regulates exosome biogenesis and secretion, by downregulation of, amongst others, SDC4 and RAB27A, two proteins involved in exosome biogenesis. The authors corroborate these results by inducing b-catenin activation using CHIR99021 in a hepatocarcinoma cell line with non-oncogenic bCatenin (Huh7 cells). The authors can further demonstrate convincingly that reduction in exosome release by hepatocarcinoma spheroids leads to a reduction in immune cell infiltration into the tumor spheroid.

Strengths:

This is a very important and well-conceived study, that appeals to a readership beyond the field of hepatocarcinoma. The authors demonstrate a compelling link between oncogenic bCatenin and exosome biogenesis. Their results are convincing and with well-designed control experiments. The authors included various complementary lines of investigation to verify their findings.

Weaknesses:

One limitation of this study is that the mechanistic relationship of exosome release and how they affect immune cells remains to be elucidated. In this context, the authors conclusions rest on the assumption that hepatocarcinoma immune evasion is based exclusively on the reduced number of exosomes. However, the authors do not analyze exosome composition between exosomes of wildtype and oncogenic background, which could be different.

Reviewer #1 (Public Review):

Summary:

The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the s1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.

Strengths:

The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.

Comments on revised version:

The authors have satisfactorily addressed my comments and this has significantly improved the clarity of the manuscript.

The only point that I would like to inquire about is the role of EL4 in modulating Na+ entry. In the simulations do the authors see no role of EL4 in controlling Na+ entry. It is particularly intriguing as some studies in the recent past displayed charged mutations in EL4 of dDAT, SERT and GAT1 as being detrimental for substrate entry/uptake. It would therefore be nice to add a small discussion if there is any role for EL4 in Na+ entry.

Reviewer #2 (Public Review):

Summary

Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be co-transported with the substrate, GABA (and a co-transported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281A-E283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.

Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.

Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.

Strengths

- MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.

- A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.

- The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.

Weaknesses

- Assessing the effects of Na+ binding on the large scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to a AF2 model that's not well packed or really a feature of the open outward conformation.

- Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.

Reviewer #1 (Public Review):

Summary:

This study investigated the phosphoryl transfer mechanism of the enzyme adenylate kinase, using SCC-DFTB quantum mechanical/molecular mechanical (QM/MM) simulations, along with kinetic studies exploring the temperature and pH dependence of the enzyme's activity, as well as the effects of various active site mutants. Based on a broad free energy landscape near the transition state, the authors proposed the existence of wide transition states (TS), characterized by the transferring phosphoryl group adopting a meta-phosphate-like geometry with asymmetric bond distances to the nucleophilic and leaving oxygens. In support of this finding, kinetic experiments were conducted with Ca2+ ions at different temperatures and pH, which revealed a reduced entropy of activation and unique pH-dependence of the catalyzed reaction.

Strengths:

A combined application of simulation and experiments is a strength.

Weaknesses:

The conclusion that the enzyme-catalyzed reaction involves a wide transition state is not sufficiently clarified with some concerns about the determined free energy profiles compared to the experimental estimate. (See Recommendations for the authors.)

Reviewer #2 (Public Review):

Summary:

The authors report results of QM/MM simulations and kinetic measurements for the phosphoryl-transfer step in adenylate kinase. The main assertion of the paper is that a wide transition state ensemble is a key concept in enzyme catalysis as a strategy to circumvent entropic barriers. This assertion is based on observation of a "structurally wide" set of energetically equivalent configurations that lie along the reaction coordinate in QM/MM simulations, together with kinetic measurements that suggest a decrease of the entropy of activation.

Strengths:

The study combines theoretical calculations and supporting experiments.

Weaknesses:

The current paper hypothesizes a "wide" transition state ensemble as a catalytic strategy and key concept in enzyme catalysis. Overall, it is not clear the degree to which this hypothesis is fully supported by the data. The reasons are as follows:

(1) Enzyme catalysis reflects a rate enhancement with respect to a baseline reaction in solution. In order to assert that something is part of a catalytic strategy of an enzyme, it would be necessary to demonstrate from simulations that the activation entropy for the baseline reaction is indeed greater and the transition state ensemble less "wide". Alternatively stated, when indicating there is a "wide transition state ensemble" for the enzyme system - one needs to indicate that is with respect to the non-enzymatic reaction. However, these simulations were not performed and the comparisons not demonstrated. The authors state "This chemical step would take about 7000 years without the enzyme" making it impossible to measure; nonetheless, the simulations of the nonenzymatic reaction would be fairly straight forward to perform in order to demonstrate this key concept that is central to the paper. Rather, the authors examine the reaction in the absence of a catalytically important Mg ion.

(2) The observation of a "wide conformational ensemble" is not a quantitative measure of entropy. In order to make a meaningful computational prediction of the entropic contribution to the activation free energy, one would need to perform free energy simulations over a range of temperatures (for the enzymatic and non-enzymatic systems). Such simulations were not performed, and the entropy of activation was thus not quantified by the computational predictions. The authors instead use a wider TS ensemble as a proxy for larger entropy, and miss an opportunity to compare directly to the experimental measurements.

Reviewer #3 (Public Review):

Summary:

By conducting QM/MM free energy simulations, the authors aimed to characterize the mechanism and transition state for the phosphoryl transfer in adenylate kinase. The qualitative reliability of the QM/MM results has been supported by several interesting experimental kinetic studies. However, the interpretation of the QM/MM results is not well supported by the current calculations.

Strengths:

The QM/MM free energy simulations have been carefully conducted. The accuracy of the semi-empirical QM/MM results was further supported by DFT/MM calculations, as well as qualitatively by several experimental studies.

Weaknesses:

(1) One key issue is the definition of the transition state ensemble. The authors appear to define this by simply considering structures that lie within a given free energy range from the barrier. However, this is not the rigorous definition of transition state ensemble, which should be defined in terms of committor distribution. This is not simply an issue of semantics, since only a rigorous definition allows a fair comparison between different cases - such as the transition state in an enzyme vs in solution, or with and without the metal ion. For a chemical reaction in a complex environment, it is also possible that many other variables (in addition to the breaking and forming P-O bonds) should be considered when one measures the diversity in the conformational ensemble.

In the revised ms, the authors included committor analysis. However, the discussion of the result is very brief. In particular, if we use the common definition of the transition state ensemble (TSE) as those featuring the committor around 0.5, the reaction coordinate of the TSE would span a much narrower range than those listed in Table 1. This point should be carefully addressed.

(2) While the experimental observation that the activation entropy differs significantly with and without the Ca2+ ion is interesting, it is difficult to connect this result with the "wide" transition state ensemble observed in the QM/MM simulations so far. Even without considering the definition of the transition state ensemble mentioned above, it is unlikely that a broader range of P-O distances would explain the substantial difference in the activation entropy measured in the experiment. Since the difference is sufficiently large, it should be possible to compute the value by repeating the free energy simulations at different temperatures, which would lead to a much more direct evaluation of the QM/MM model/result and the interpretation.

Reviewer #1 (Public Review):

Continuous attractor networks endowed with some sort of adaptation in the dynamics, whether that be through synaptic depression or firing rate adaptation, are fast becoming the leading candidate models to explain many aspects of hippocampal place cell dynamics, from hippocampal replay during immobility to theta sequences during run. Here, the authors show that a continuous attractor network endowed with spike frequency adaptation and subject to feedforward external inputs is able to account for several previously unaccounted aspects of theta sequences, including (1) sequences that move both forwards and backwards, (2) sequences that alternate between two arms of a T-maze, (3) speed modulation of place cell firing frequency, and (4) the persistence of phase information across hippocampal inactivations.

I think the main result of the paper (findings (1) and (2)) are likely to be of interest to the hippocampal community, as well as to the wider community interested in mechanisms of neural sequences. In addition, the manuscript is generally well written and the analytics are impressive. However, several issues should be addressed, which I outline below.

Major comments:

In real data, population firing rate is strongly modulated by theta (i.e., cells collectively prefer a certain phase of theta - see review paper Buzsaki, 2002) and largely oscillates at theta frequency during run. With respect to this cyclical firing rate, theta sweeps resemble "Nike" check marks, with the sweep backwards preceding the sweep forwards within each cycle before the activity is quenched at the end of the cycle. I am concerned that (1) the summed population firing rate of the model does not oscillate at theta frequency, and (2) as the authors state, the oscillatory tracking state must begin with a forward sweep. With regards to (1), can the authors show theta phase spike preference plots for the population to see if they match data? With regards to (2), can the authors show what happens if the bump is made to sweep backwards first, as it appears to do within each cycle?

I could not find the width of the external input mentioned anywhere in the text or in the table of parameters. The implication is that it is unclear to me whether, during the oscillatory tracking state, the external input is large compared to the size of the bump, so that the bump lives within a window circumscribed by the external input and so bounces off the interior walls of the input during the oscillatory tracking phase, or whether the bump is continuously pulled back and forth by the external input, in which case it could be comparable to the size of the bump. My guess based on Fig 2c is that it is the latter. Please clarify and comment.

I would argue that the "constant cycling" of theta sweeps down the arms of a T-maze was roughly predicted by Romani & Tsodyks, 2015, Figure 7. While their cycling spans several theta cycles, it nonetheless alternates by a similar mechanism, in that adaptation (in this case synaptic depression) prevents the subsequent sweep of activity from taking the same arm as the previous sweep. I believe the authors should cite this model in this context and consider the fact that both synaptic depression and spike frequency adaptation are both possible mechanisms for this phenomenon. But I certainly give the authors credit for showing how this constant cycling can occur across individual theta cycles.

The authors make an unsubstantiated claim in the paragraph beginning with line 413 that the Tsodyks and Romani (2015) model could not account for forwards and backwards sweeps. Both the firing rate adaptation and synaptic depression are symmetry breaking models that should in theory be able to push sweeps of activity in both directions, so it is far from obvious to me that both forward and backward sweeps are not possible in the Tsodyks and Romani model. The authors should either prove that this is the case (with theory or simulation) or excise this statement from the manuscript.

The section on the speed dependence of theta (starting with line 327) was very hard to understand. Can the authors show a more graphical explanation of the phenomenon? Perhaps a version of Fig 2f for slow and fast speeds, and point out that cells in the latter case fire with higher frequency than in the former?

I had a hard time understanding how the Zugaro et al., (2005) hippocampal inactivation experiment was accounted for by the model. My intuition is that while the bump position is determined partially by the location of the external input, it is also determined by the immediate history of the bump dynamics as computed via the local dynamics within the hippocampus (recurrent dynamics and spike rate adaptation). So that if the hippocampus is inactivated for an arbitrary length of time, there is nothing to keep track of where the bump should be when the activity comes back on line. Can the authors please explain more how the model accounts for this?

Can the authors comment on why the sweep lengths oscillate in the bottom panel of Fig 5b during starting at time 0.5 seconds before crossing the choice point of the T-maze? Is this oscillation in sweep length another prediction of the model? If so, it should definitely be remarked upon and included in the discussion section.

Perhaps I missed this, but I'm curious whether the authors have considered what factors might modulate the adaptation strength. In particular, might rat speed modulate adaptation strength? If so, would have interesting predictions for theta sequences at low vs high speeds.

I think the paper has a number of predictions that would be especially interesting to experimentalists but are sort of scattered throughout the manuscript. It would be beneficial to have them listed more prominently in a separate section in the discussion. This should include (1) a prediction that the bump height in the forward direction should be higher than in the backward direction, (2) predictions about bimodal and unimodal cells starting with line 366, (3) prediction of another possible kind of theta cycling, this time in the form of sweep length (see comment above), etc.

Reviewer #2 (Public Review):

In this work, the authors elaborate on an analytically tractable, continuous-attractor model to study an idealized neural network with realistic spiking phase precession/procession. The key ingredient of this analysis is the inclusion of a mechanism for slow firing-rate adaptation in addition to the otherwise fast continuous-attractor dynamics. The latter continuous-attractor dynamics classically arises from a combination of translation invariance and nonlinear rate normalization.

For strong adaptation/weak external input, the network naturally exhibits an internally generated, travelling-wave dynamics along the attractor with some characteristic speed. For small adaptation/strong external stimulus, the network recovers the classical externally driven continuous-attractor dynamics. Crucially, when both adaptation and external input are moderate, there is a competition with the internally generated and externally generated mechanisms leading to an oscillatory tracking regime. In this tracking regime, the population firing profile oscillates around the neural field tracking the position of the stimulus. The authors demonstrate by a combination of analytical and computational arguments that oscillatory tracking corresponds to realistic phase precession/procession. In particular the authors can account for the emergence of unimodal and bimodal cells, as well as some other experimental observations with respect the dependence of phase precession/procession on the animal's locomotion.

The strengths of this work are at least three-fold: 1) Given its simplicity, the proposed model has a surprisingly large explanatory power of the various experimental observations. 2) The mechanism responsible for the emergence of precession/procession can be understood as a simple yet rather illuminating competition between internally driven and externally driven dynamical trends. 3) Amazingly, and under some adequate simplifying assumptions, a great deal of analysis can be treated exactly, which allows for a detailed understanding of all parametric dependencies. This exact treatment culminates with a full characterization of the phase space of the network dynamics, as well as the computation of various quantities of interest, including characteristic speeds and oscillating frequencies.

As mentioned by the authors themselves, the main limitation of this work is that it deals with a very idealized model and it remains to see how the proposed dynamical behaviors would persists in more realistic models. For example, the model is based on a continuous attractor model that assumes perfect translation-invariance of the network connectivity pattern. Would the oscillating tracking behavior persist in the presence of connection heterogeneities? Another limitation is that the system needs to be tuned to exhibit oscillation within the theta range and that this tuning involves a priori variable parameters such as the external input strength. Is the oscillating-tracking behavior overtly sensitive to input strength variations? The author mentioned that an external pacemaker can serve to drive oscillation within the desired theta band but there is no evidence presented supporting this. A final and perhaps secondary limitation has to do with the choice of parameter, namely the time constant of neural firing which is chosen around 3ms. This seems rather short given that the fast time scale of rate models (excluding synaptic processes) is usually given by the membrane time constant, which is typically about 15ms. I suspect this latter point can easily be addressed.

Reviewer #1 (Public Review):

This study exploits novel agent (IMT) that inhibits mitochondrial activity in combination with venetoclax. While the concept is not novel, the agent is novel (inhibitor of the mitochondrial RNA polymerase, described in Nature in other tumor models), and quest for safe mitochondrial inhibitors is highly warranted. The strength is in vivo activity data shown in CLDX and in one of the two AML PDX models tested, and the apparent safety of the combination. However, the impact on survival is impressive in CLDX but not in PDX, and unclear why Ven-sensitive PDX is resistant to combination (opposite what cell line data show). The paper is lacking mechanistic data beyond Seahorse and standard apoptosis assays, and even transcriptome analysis from PDX cells is poorly analyzed. There is no real evidence that this agent overcome Ven resistance, which could be done for example in primary AML cells. Finally, no on-target pharmacodynamic endpoints are measured in vivo to support the activity of the compound on mitochondrial activity at the doses used (which are safe). These multiple weaknesses significantly reduce my enthusiasm for this manuscript.

The cell line data show additive/synergistic effects of IMT and Ven on cell viability in p53-WT cells. However, no mechanisms of synergy beyond OCR are shown, which is a missed opportunity.

No data are shown in primary AML cells in vitro. This could address venetoclax-resistant AML cells with distinct genomic profiles.

The in vivo CLDX model (MV4;11) data is quite impressive, showing reduction of tumor burden and meaningful extension of survival in combination cohort. It is unclear why venetoclax used at highest dose normally sued in vivo (100mg/kg) did not show any impact on survival in this Ven-sensitive model. It is disappointing that no biomarkers of mitochondrial activity (for example, simple pAMPK, or levels of mitochondrial subunits) are shown to support on-target pharmacodynamic activity. However, efficacy in human PDX is less impressive, for example in Fig 6C the combination has extended survival from 96 to 112 days, possibly due to early stopping of treatment (around day 30); and no extension of survival is seen in another PDX in Fig 7. Still, this is indicative of combinatorial activity in TP53-mutant PDX. There is however discrepancy with in vitro studies that show no impact of combination in TP53 mutant cells and synergy in TP53-wt cells, and the opposite findings in vivo, which is not explained. Overall, the activity of the combination is modest. The safety is encouraging, but again, no pharmacodynamic measurements are shown to support that IMT at least partially inhibited mitochondrial activity in AML cells.

In Discussion the statement that inhibition of POLRMT can overcome venetoclax resistance is not supported by the data, as no additive effects are seen in vitro in TP53 mutant cells, and no other resistant models (such as primary AML cells) are tested. In vivo as stated above there is some activity in TP53 mutant PDX but this alone cannot be sued to justify this strong statement. Also, the sentence that "...we were able to reduce the tumor burden in all (cell- and patient-derived) xenografted mice treated with a combination of IMT and venetoclax" is not supported by data in Fig 7.

Reviewer #2 (Public Review):

Summary:

The manuscript by Arabanian and colleagues presents studies showing how inhibition of mitochondrial transcription and replication with a novel inhibitor of the mitochondrial polymerase, IMT, can promote AML cell death in combination with the Bcl2 inhibitor venetoclax. They further show that this combinatorial efficacy is evident in vivo in both the AML cell line MV411 and in a PDX model. Given the multiple studies showing the importance of Oxphos in maintaining AML cell survival, the current studies provide an additional strategy to inhibit Oxphos and thus improve the therapeutic management of AML.

Strengths:

A novel aspect of this work is that IMT is a new class of mitochondrial inhibitor that acts by inhibiting the mitochondrial polymerase. In addition, the demonstration of therapeutic efficacy both in vitro and in vivo (including with PDX), together with some data showing minimal toxicity, adds to the impact of this work. Their overall conclusion that IMT increases the potency of Vex in treating AMLs is supported.

Weaknesses:

There are several deficiencies that should be addressed to substantiate the rigor and impact of this study. Of most importance, they need to show that IMT actually inhibits the mitochondrial polymerase in AML cells, and there are additional concerns with their models that if addressed would improve the ability of IMT to be developed clinically.

Reviewer #1 (Public Review):

Rebecca R.G. et al. set to determine the function of grid cells. They present an interesting case claiming that the spatial periodicity seen in the grid pattern provides a parsimonious solution to the task of coding 2D trajectories using sequential cell activation. Thus, this work defines a probable function grid cells may serve (here, the function is coding 2D trajectories), and proves that the grid pattern is a solution to that function. This approach is somewhat reminiscent in concept to previous works that defined a probable function of grid cells (e.g., path integration) and constructed normative models for that function that yield a grid pattern. However, the model presented here gives clear geometric reasoning to its case.

Stemming from 4 axioms, the authors present a concise demonstration of the mathematical reasoning underlying their case. The argument is interesting and the reasoning is valid, and this work is a valuable addition to the ongoing body of work discussing the function of grid cells.

However, the case uses several assumptions that need to be clearly stated as assumptions, clarified, and elaborated on: Most importantly, the choice of grid function is grounded in two assumptions:<br /> (1) that the grid function relies on the activation of cell sequences, and<br /> (2) that the grid function is related to the coding of trajectories. While these are interesting and valid suggestions, since they are used as the basis of the argument, the current justification could be strengthened (references 28-30 deal with the hippocampus, reference 31 is interesting but cannot hold the whole case).

The work further leans on the assumption that sequences in the same direction should be similar regardless of their position in space, it is not clear why that should necessarily be the case, and how the position is extracted for similar sequences in different positions. The authors also strengthen their model with the requirement that grid cells should code for infinite space. However, the grid pattern anchors to borders and might be used to code navigated areas locally. Finally, referencing ref. 14, the authors claim that no existing theory for the emergence of grid cell firing that unifies the experimental observations on periodic firing patterns and their distortions under a single framework. However, that same reference presents exactly that - a mathematical model of pairwise interactions that unifies experimental observations. The authors should clarify this point.

Reviewer #2 (Public Review):

Summary:

In this work, the authors consider why grid cells might exhibit hexagonal symmetry - i.e., for what behavioral function might this hexagonal pattern be uniquely suited? The authors propose that this function is the encoding of spatial trajectories in 2D space. To support their argument, the authors first introduce a set of definitions and axioms, which then lead to their conclusion that a hexagonal pattern is the most efficient or parsimonious pattern one could use to uniquely label different 2D trajectories using sequences of cells. The authors then go through a set of classic experimental results in the grid cell literature - e.g. that the grid modules exhibit a multiplicative scaling, that the grid pattern expands with novelty or is warped by reward, etc. - and describe how these results are either consistent with or predicted by their theory. Overall, this paper asks a very interesting question and provides an intriguing answer. However, the theory appears to be extremely flexible and very similar to ideas that have been previously proposed regarding grid cell function.

Major strengths:

The general idea behind the paper is very interesting - why *does* the grid pattern take the form of a hexagonal grid? This is a question that has been raised many times; finding a truly satisfying answer is difficult but of great interest to many in the field. The authors' main assertion that the answer to this question has to do with the ability of a hexagonal arrangement of neurons to uniquely encode 2D trajectories is an intriguing suggestion. It is also impressive that the authors considered such a wide range of experimental results in relation to their theory.

Major weaknesses:

One major weakness I perceive is that the paper overstates what it delivers, to an extent that I think it can be a bit confusing to determine what the contributions of the paper are. In the introduction, the authors claim to provide "mathematical proof that ... the nature of the problem being solved by grid cells is coding of trajectories in 2-D space using cell sequences. By doing so, we offer a specific answer to the question of why grid cell firing patterns are observed in the mammalian brain." This paper does not provide proof of what grid cells are doing to support behavior or provide the true answer as to why grid patterns are found in the brain. The authors offer some intriguing suggestions or proposals as to why this might be based on what hexagonal patterns could be good for, but I believe that the language should be clarified to be more in line with what the authors present and what the strength of their evidence is.

Relatedly, the authors claim that they find a teleological reason for the existence of grid cells - that is, discover the function that they are used for. However, in the paper, they seem to instead assume a function based on what is known and generally predicted for grid cells (encode position), and then show that for this specific function, grid cells have several attractive properties.

There is also some other work that seems very relevant, as it discusses specific computational advantages of a grid cell code but was not cited here: https://www.nature.com/articles/nn.2901.

A second major weakness was that some of the claims in the section in which they compared their theory to data seemed either confusing or a bit weak. I am not a mathematician, so I was not able to follow all of the logic of the various axioms, remarks, or definitions to understand how the authors got to their final conclusion, so perhaps that is part of the problem. But below I list some specific examples where I could not follow why their theory predicted the experimental result, or how their theory ultimately operated any differently from the conventional understanding of grid cell coding. In some cases, it also seemed that the general idea was so flexible that it perhaps didn't hold much predictive power, as extra details seemed to be added as necessary to make the theory fit with the data.

I don't quite follow how, for at least some of their model predictions, the 'sequence code of trajectories' theory differs from the general attractor network theory. It seems from the introduction that these theories are meant to serve different purposes, but the section of the paper in which the authors claim that various experimental results are predicted by their theory makes this comparison difficult for me to understand. For example, in the section describing the effect of environmental manipulations in a familiar environment, the authors state that the experimental results make sense if one assumes that sequences are anchored to landmarks. But this sounds just like the classic attractor-network interpretation of grid cell activity - that it's a spatial metric that becomes anchored to landmarks.

It was not clear to me why their theory predicted the field size/spacing ratio or the orientation of the grid pattern to the wall.

I don't understand how repeated advancement of one unit to the next, as shown in Figure 4E, would cause the change in grid spacing near a reward.

I don't follow how this theory predicts the finding that the grid pattern expands with novelty. The authors propose that this occurs because the animals are not paying attention to fine spatial details, and thus only need a low-resolution spatial map that eventually turns into a higher-resolution one. But it's not clear to me why one needs to invoke the sequence coding hypothesis to make this point.

The last section, which describes that the grid spacing of different modules is scaled by the square root of 2, says that this is predicted if the resolution is doubled or halved. I am not sure if this is specifically a prediction of the sequence coding theory the authors put forth though since it's unclear why the resolution should be doubled or halved across modules (as opposed to changed by another factor).

Reviewer #3 (Public Review):

The manuscript presents an intriguing explanation for why grid cell firing fields do {\em not} lie on a lattice whose axes aligned to the walls of a square arena. This observation, by itself, merits the manuscript's dissemination to the journals audience.

The presentation is quirky (but keep the quirkiness!).

But let me recast the problem presented by the authors as one of combinatorics. Given repeating, spatially separated firing fields across cells, one obtains temporal sequences of grid cells firing. Label these cells by integers from $[n]$. Any two cells firing in succession should uniquely identify one of six directions (from the hexagonal lattice) in which the agent is currently moving.

Now, take the symmetric group $\Sigma$ of cyclic permutations on $n$ elements.<br /> We ask whether there are cyclic permutations of $[n]$ such that

So, for instance, $(4,2,3,1)$ would not be counted as a valid permutation of $(1,2,3,4)$, as $(2,3)$ and $(1,4)$ are adjacent.

Furthermore, given $[n]$, are there two distinct cyclic permutations such that {\em no} adjacencies are preserved when considering any pair of permutations (among the triple of the original ordered sequence and the two permutations)? In other words, if we consider the permutation required to take the first permutation into the second, that permutation should not preserve any adjacencies.

{\bf Key question}: is there any difference between the solution to the combinatorics problem sketched above and the result in the manuscript? Specifically, the text argues that for $n=7$ there is only {\em one} solution.

Ideally, one would strive to obtain a closed-form solution for the number of such permutations as a function of $n$.

Joint Public Review:

The overall goal of this manuscript is to understand how Notch signaling is activated in specific regions of the endocardium, including the OFT and AVC, that undergo EMT to form the endocardial cushions. Using dofetilide to transiently block circulation in E9.5 mice, the authors show that Notch receptor cleavage still occurs in the valve-forming regions due to mechanical sheer stress as Notch ligand expression and oxygen levels are unaffected. The authors go on to show that changes in lipid membrane structure activate mTOR signaling, which causes phosphorylation of PKC and Notch receptor cleavage.

The strengths of the manuscript include the dual pharmacological and genetic approaches to block blood flow in the mouse, the inclusion of many controls including those for hypoxia, the quality of the imaging, and the clarity of the text. However, several weaknesses were noted surrounding the main claims where the supporting data are incomplete.

PKC - Notch1 activation:

(1) Does deletion of Prkce and Prkch affect blood flow, and if so, might that be suppressing Notch1 activation indirectly?

(2) It would be helpful to visualize the expression of prkce and prkch by in situ hybridization in E9.5 embryos.

(3) PMA experiments: Line 223-224: A major concern is related to the conclusion that "blood flow activates Notch in the cushion endocardium via the mTORC2-PKC signaling pathway". To make that claim, the authors show that a pharmacological activation with a potent PKC activator, PMA, rescues NICD levels in the AVC in dofetilide-treated embryos. This claim would also need proof that a lack of blood flow alters the activity of mTORC2 to phosphorylate the targets of PKC phosphorylation. Also, this observation does not explain the link between PKC activity and Notch activation.

(4) In addition, the authors hypothesise that shear stress lies upstream of PKC and Notch activation, and that because shear stress is highest at the valve-forming regions, PKC and Notch activity is localised to the valve-forming regions. Since PMA treatment affects the entire endocardium which expresses Notch1, NICD should be seen in areas outside of the AVC in the PMA+dofetilide condition. Please clarify.

Lipid Membrane:

(1) It is not clear how the authors think that the addition of cholesterol changes the lipid membrane structure or alters Cav-1 distribution. Can this be addressed? Does adding cholesterol make the membrane more stiff? Does increased stiffness result from higher shear stress?

(2) The loss of blood flow apparently affects Cav1 membrane localization and causes a redistribution from the luminal compartment to lateral cell adhesion sites. Cholesterol treatment of dofetilide-treated hearts (lacking blood flow) rescued Cav1 localization to luminal membrane microdomains and rescued NICD expression. It remains unclear how the general addition of cholesterol would result in a rescue of regionalized membrane distribution within the AVC and in high-shear stress areas.

(3) The authors do not show the entire heart in that rescue treatment condition (cholesterol in dofetilide-treated hearts). Also, there is no quantification of that rescue in Figure 4B. Currently, only overview images of the heart are shown but high-resolution images on a subcellular scale (such as electron microscopy) are needed to resolve and show membrane microdomains of caveolae with Cav1 distribution. This is important because Cav-1could have functions independent of caveolae (eg. Lolo et al., https://doi.org/10.1038/s41556-022-01034-3).

Figure Legends, missing data, and clarity:

(1) The number of embryos used in each experiment is not clear in the text or figure legends. In general, figure legends are incomplete (for instance in Figure 1).

(2) Line 204: The authors refer to unpublished endocardial RNAseq data from E9.5 embryos. These data must be provided with this manuscript if it is referred to in any way in the text.

(3) Figure 1 shows Dll4 transcript levels, which do not necessarily correlate with protein levels. It would be important to show quantifications of these patterns as Notch/Dll4 levels are cycling and may vary with time and between different hearts.

(4) Line 212-214: The authors describe cardiac cushion defects due to the loss of blood flow and refer to some quantifications that are not completely shown in Figure 3. For instance, quantifications for cushion cellularity and cardiac defects at three hours (after the start of treatment?) are missing.

(5) Related to Figure 5. The work would be strengthened by quantification of the effects of dofetilide and verapamil on heartbeat at the doses applied. Is the verapamil dosage used here similar to the dose used in the clinic?

Overstated Claims:

(1) The authors claim that the lipid microstructure/mTORC2/PKC/Notch pathway is responsive to shear stress, rather than other mechanical forces or myocardial function. Their conclusions seem to be extrapolated from various in vitro studies using non-endocardial cells. To solidify this claim, the authors would need additional biomechanical data, which could be obtained via theoretical modelling or using mouse heart valve explants. This issue could also be addressed by the authors simply softening their conclusions.

(2) Line 263-264: In the discussion, the authors conclude that "Strong fluid shear stress in the AVC and OFT promotes the formation of caveolae on the luminal surface of the endocardial cells, which enhances PKCε phosphorylation by mTORC2." This link was shown rather indirectly, rather than by direct evidence, and therefore the conclusion should be softened. For example, the authors could state that their data are consistent with this model.

(3) In the Discussion, it says: "Mammalian embryonic endocardium undergoes extensive EMT to form valve primordia while zebrafish valves are primarily the product of endocardial infolding (Duchemin et al., 2019)." In the paper cited, Duchemin and colleagues described the formation of the zebrafish outflow tract valve. The zebrafish atrioventricular valve primordia is formed via partial EMT through Dll-Notch signaling (Paolini et al. Cell Reports 2021) and the collective cell migration of endocardial cells into the cardiac jelly. Then, a small subset of cells that have migrated into the cardiac jelly give rise to the valve interstitial cells, while the remainder undergo mesenchymal-to-endothelial transition and become endothelial cells that line the sinus of the atrioventricular valve (Chow et al., doi: 10.1371/journal.pbio.3001505). The authors should modify this part of the Discussion and cite the relevant zebrafish literature.

Reviewer #2 (Public Review):

Summary:

The authors demonstrated that maternal choline supplementation (MCS) improved spatial memory, reduced a marker of hyperexcitability/epilepsy (FosB expression), and reduced oxidative stress (as measured by restored NeuN expression) in an Alzheimer's disease mouse model. This multidisciplinary study spanned behavior, EEG, and histological measures and constituted a large amount of work. Overall, the results supported that MCS does have important effects on hippocampal function, which may substantially impact human AD.

Strengths:

The strength of the group was the ability to monitor the incidence of interictal spikes (IIS) over the course of 1.2-6 months in the Tg2576 Alzheimer's disease model, combined with meaningful behavioral and histological measures. The authors were able to demonstrate MCS had protective effects in Tg2576 mice, which was particularly convincing in the hippocampal novel object location task.

Weaknesses:

Although choline deficiency was associated with impaired learning and elevated FosB expression, consistent with increased hyperexcitability, IIS was reduced with both low and high choline diets. Although not necessarily a weakness, it complicates the interpretation and requires further evaluation.

Joint Public Review:

Chartampila et al. describe the effect of early-life choline supplementation on cognitive functions and epileptic activity in a mouse model of Alzheimer's disease. The cognitive abilities were assessed by the novel object recognition test and the novel object location test, performed in the same cohort of mice at 3 months and 6 months of age. Neuronal loss was tested using NeuN immunoreactivity, and neuronal hyperexcitability was examined using deltaFosB and video-EEG recordings, providing multi-level correlations between these different parameters.

The study was designed as a 6-month follow-up, with repeated behavioral and EEG measurements through disease development and multilevel correlations providing valuable and interesting findings on AD progression and the effect of early-life choline supplementation. Moreover, the behavioral data that suggest an adverse effect of low choline in WT mice are interesting and important also beyond the context of AD, highlighting the dramatic effect of diet on the phenotypes of animal.

Reviewer #1 (Public Review):

Summary:

The authors propose a new method to quantitatively assess morphogenetic processes during organismal development. They apply their method to ascidian morphogenesis and thus find that gastrulation is a two-step process.

The method applies to morphogenetic changes of surfaces. It consists of the following steps: first, surface deformations are quantified based on microscopy images without requiring cellular segmentation and tracking. This is achieved by mapping, at each time point, a polygonal mesh initially defined on a sphere to the surface of the embryo. The mapped vertices of this polygonal mesh then serve as (Lagrangian) markers for the embryonic surface. From these, one can infer the deformation of the surface, which can be expressed in terms of the strain tensor at each point of the surface. Changes in the strain tensor give the strain rate, which captures the morphogenetic processes. Second, at each time point, the strain rate field is decomposed in terms of spherical harmonics. Finally, the evolution of the weights of the various spherical harmonics in the decomposition is analysed via wavelet analysis. The authors apply their workflow to ascidian development between 4 and 8.7 hpf. From their analysis, they find clear indications for gastrulation and neurulation and identify two sub-phases of gastrulation, namely, endoderm invagination and 'blastophore closure'.

Strengths:

The combination of various tools allows the authors to obtain a quantitative description of the developing embryo without the necessity of identifying fiducial markers. Visual inspection shows that their method works well. Furthermore, this quantification then allows for an unbiased identification of different morphogenetic phases.

Weaknesses:

At times, the explanation of the method is hard to follow, unless the reader is already familiar with concepts like level-set methods or wavelet transforms. Furthermore, the software for performing the determination of Lagrangian markers or the subsequent spectral analysis does not seem to be available to the readers.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors proposed a method to quantitatively analyze 3D live imaging data of early developing embryos, using ascidian development as an example. For this purpose, the previously proposed level set method was used to computationally track the temporal evolution of reference points introduced on the embryo surface. Then, from the obtained three-dimensional trajectories, the velocity field was obtained, from which the strain rate field was computed according to the idea of continuum mechanics. The information in the strain rate field was reduced to a scalar field, determined by taking the square root of the sum of the squares of the eigenvalues. The scalar field is then further decomposed into a spectrum using spherical harmonics. In this paper, the authors focused on the modes with lower order with real coefficients. The time evolution of these modes was analyzed using wavelet transforms. The authors claimed that the results reflected the developmental stages of ascidian embryos.

Strengths:

In this way, this manuscript proposes a pipeline of analyses combining various methods. The strength of this method lies in its ability to quantitatively analyze the deformation of the entire embryo without the requirement for cellular segmentation and tracking.

Weaknesses:

The limitations of the proposed analysis pipeline are not clearly indicated. Claims such as the identification of developmental stages need more quantitative validation. In addition, it is not clearly shown how the proposed method can distinguish between the superposition of individual cell behavior and the collective behavior of cells.

Reviewer #1 (Public Review):

Summary:

In this human neuroimaging and electrophysiology study, the authors aimed to characterize the effects of a period of visual deprivation in the sensitive period on excitatory and inhibitory balance in the visual cortex. They attempted to do so by comparing neurochemistry conditions ('eyes open', 'eyes closed') and resting state, and visually evoked EEG activity between ten congenital cataract patients with recovered sight (CC), and ten age-matched control participants (SC) with normal sight.

First, they used magnetic resonance spectroscopy to measure in vivo neurochemistry from two locations, the primary location of interest in the visual cortex, and a control location in the frontal cortex. Such voxels are used to provide a control for the spatial specificity of any effects because the single-voxel MRS method provides a single sampling location. Using MR-visible proxies of excitatory and inhibitory neurotransmission, Glx and GABA+ respectively, the authors report no group effects in GABA+ or Glx, no difference in the functional conditions 'eyes closed' and 'eyes open'. They found an effect of the group in the ratio of Glx/GABA+ and no similar effect in the control voxel location. They then performed multiple exploratory correlations between MRS measures and visual acuity, and reported a weak positive correlation between the 'eyes open' condition and visual acuity in CC participants.

The same participants then took part in an EEG experiment. The authors selected only two electrodes placed in the visual cortex for analysis and reported a group difference in an EEG index of neural activity, the aperiodic intercept, as well as the aperiodic slope, considered a proxy for cortical inhibition. They report an exploratory correlation between the aperiodic intercept and Glx in one out of three EEG conditions.

The authors report the difference in E/I ratio, and interpret the lower E/I ratio as representing an adaptation to visual deprivation, which would have initially caused a higher E/I ratio. Although intriguing, the strength of evidence in support of this view is not strong. Amongst the limitations are the low sample size, a critical control cohort that could provide evidence for a higher E/I ratio in CC patients without recovered sight for example, and lower data quality in the control voxel.

Strengths of study:

How sensitive period experience shapes the developing brain is an enduring and important question in neuroscience. This question has been particularly difficult to investigate in humans. The authors recruited a small number of sight-recovered participants with bilateral congenital cataracts to investigate the effect of sensitive period deprivation on the balance of excitation and inhibition in the visual brain using measures of brain chemistry and brain electrophysiology. The research is novel, and the paper was interesting and well-written.

Limitations:

- Low sample size. Ten for CC and ten for SC, and a further two SC participants were rejected due to a lack of frontal control voxel data. The sample size limits the statistical power of the dataset and increases the likelihood of effect inflation.

- Lack of specific control cohort. The control cohort has normal vision. The control cohort is not specific enough to distinguish between people with sight loss due to different causes and patients with congenital cataracts with co-morbidities. Further data from more specific populations, such as patients whose cataracts have not been removed, with developmental cataracts, or congenitally blind participants, would greatly improve the interpretability of the main finding. The lack of a more specific control cohort is a major caveat that limits a conclusive interpretation of the results.

- MRS data quality differences. Data quality in the control voxel appears worse than in the visual cortex voxel. The frontal cortex MRS spectrum shows far broader linewidth than the visual cortex (Supplementary Figures). Compared to the visual voxel, the frontal cortex voxel has less defined Glx and GABA+ peaks; lower GABA+ and Glx concentrations, lower NAA SNR values; lower NAA concentrations. If the data quality is a lot worse in the FC, then small effects may not be detectable.

- Because of the direction of the difference in E/I, the authors interpret their findings as representing signatures of sight improvement after surgery without further evidence, either within the study or from the literature. However, the literature suggests that plasticity and visual deprivation drive the E/I index up rather than down. Decreasing GABA+ is thought to facilitate experience-dependent remodelling. What evidence is there that cortical inhibition increases in response to a visual cortex that is over-sensitised due to congenital cataracts? Without further experimental or literature support this interpretation remains very speculative.

- Heterogeneity in the patient group. Congenital cataract (CC) patients experienced a variety of duration of visual impairment and were of different ages. They presented with co-morbidities (absorbed lens, strabismus, nystagmus). Strabismus has been associated with abnormalities in GABAergic inhibition in the visual cortex. The possible interactions with residual vision and confounds of co-morbidities are not experimentally controlled for in the correlations, and not discussed.

- Multiple exploratory correlations were performed to relate MRS measures to visual acuity (shown in Supplementary Materials), and only specific ones were shown in the main document. The authors describe the analysis as exploratory in the 'Methods' section. Furthermore, the correlation between visual acuity and E/I metric is weak, and not corrected for multiple comparisons. The results should be presented as preliminary, as no strong conclusions can be made from them. They can provide a hypothesis to test in a future study.

- P.16 Given the correlation of the aperiodic intercept with age ("Age negatively correlated with the aperiodic intercept across CC and SC individuals, that is, a flattening of the intercept was observed with age"), age needs to be controlled for in the correlation between neurochemistry and the aperiodic intercept. Glx has also been shown to negatively correlate with age.

- Multiple exploratory correlations were performed to relate MRS to EEG measures (shown in Supplementary Materials), and only specific ones were shown in the main document. Given the multiple measures from the MRS, the correlations with the EEG measures were exploratory, as stated in the text, p.16, and in Figure 4. Yet the introduction said that there was a prior hypothesis "We further hypothesized that neurotransmitter changes would relate to changes in the slope and intercept of the EEG aperiodic activity in the same subjects." It would be great if the text could be revised for consistency and the analysis described as exploratory.

- The analysis for the EEG needs to take more advantage of the available data. As far as I understand, only two electrodes were used, yet far more were available as seen in their previous study (Ossandon et al., 2023). The spatial specificity is not established. The authors could use the frontal cortex electrode (FP1, FP2) signals as a control for spatial specificity in the group effects, or even better, all available electrodes and correct for multiple comparisons. Furthermore, they could use the aperiodic intercept vs Glx in SC to evaluate the specificity of the correlation to CC.

Reviewer #2 (Public Review):

Summary:

The manuscript reports non-invasive measures of activity and neurochemical profiles of the visual cortex in congenitally blind patients who recovered vision through the surgical removal of bilateral dense cataracts. The declared aim of the study is to find out how restoring visual function after several months or years of complete blindness impacts the balance between excitation and inhibition in the visual cortex.

Strengths:

The findings are undoubtedly useful for the community, as they contribute towards characterising the many ways this special population differs from normally sighted individuals. The combination of MRS and EEG measures is a promising strategy to estimate a fundamental physiological parameter - the balance between excitation and inhibition in the visual cortex, which animal studies show to be heavily dependent upon early visual experience. Thus, the reported results pave the way for further studies, which may use a similar approach to evaluate more patients and control groups.

Weaknesses:

The main issue is the lack of an appropriate comparison group or condition to delineate the effect of sight recovery (as opposed to the effect of congenital blindness). Few previous studies suggested an increased excitation/Inhibition ratio in the visual cortex of congenitally blind patients; the present study reports a decreased E/I ratio instead. The authors claim that this implies a change of E/I ratio following sight recovery. However, supporting this claim would require showing a shift of E/I after vs. before the sight-recovery surgery, or at least it would require comparing patients who did and did not undergo the sight-recovery surgery (as common in the field).

MR Spectroscopy shows a reduced GLX/GABA ratio in patients vs. sighted controls; however, this finding remains rather isolated, not corroborated by other observations. The difference between patients and controls only emerges for the GLX/GABA ratio, but there is no accompanying difference in either the GLX or the GABA concentrations. There is an attempt to relate the MRS data with acuity measurements and electrophysiological indices, but the explorative correlational analyses do not help to build a coherent picture. A bland correlation between GLX/GABA and visual impairment is reported, but this is specific to the patients' group (N=10) and would not hold across groups (the correlation is positive, predicting the lowest GLX/GABA ratio values for the sighted controls - the opposite of what is found). There is also a strong correlation between GLX concentrations and the EEG power at the lowest temporal frequencies. Although this relation is intriguing, it only holds for a very specific combination of parameters (of the many tested): only with eyes open, only in the patient group.

For these reasons, the reported findings do not allow us to draw firm conclusions on the relation between EEG parameters and E/I ratio or on the impact of early (vs. late) visual experience on the excitation/inhibition ratio of the human visual cortex.

Reviewer #3 (Public Review):

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

My expertise is in EEG (particularly in the decomposition of periodic and aperiodic activity) and statistical methods. I have several major concerns in terms of methodological and statistical approaches along with the (over)interpretation of the results. These major concerns are detailed below.

(1) Variability in visual deprivation:

- The document states a large variability in the duration of visual deprivation (probably also the age at restoration), with significant implications for the sensitivity period's impact on visual circuit development. The variability and its potential effects on the outcomes need thorough exploration and discussion.

(2) Sample size:

- The small sample size is a major concern as it may not provide sufficient power to detect subtle effects and/or overestimate significant effects, which then tend not to generalize to new data. One of the biggest drivers of the replication crisis in neuroscience.

- The main problem with the correlation analyses between MRS and EEG measures is that the sample size is simply too small to conduct such an analysis. Moreover, it is unclear from the methods section that this analysis was only conducted in the patient group (which the reviewer assumed from the plots), and not explained why this was done only in the patient group. I would highly recommend removing these correlation analyses.

(3) Statistical concerns:

- The statistical analyses, particularly the correlations drawn from a small sample, may not provide reliable estimates (see https://www.sciencedirect.com/science/article/pii/S0092656613000858, which clearly describes this problem).

- Statistical analyses for the MRS: The authors should consider some additional permutation statistics, which are more suitable for small sample sizes. The current statistical model (2x2) design ANOVA is not ideal for such small sample sizes. Moreover, it is unclear why the condition (EO & EC) was chosen as a predictor and not the brain region (visual & frontal) or neurochemicals. Finally, the authors did not provide any information on the alpha level nor any information on correction for multiple comparisons (in the methods section). Finally, even if the groups are matched w.r.t. age, the time between surgery and measurement, the duration of visual deprivation, (and sex?), these should be included as covariates as it has been shown that these are highly related to the measurements of interest (especially for the EEG measurements) and the age range of the current study is large.

- EEG statistical analyses: The same critique as for the MRS statistical analyses applies to the EEG analysis. In addition: was the 2x3 ANOVA conducted for EO and EC independently? This seems to be inconsistent with the approach in the MRS analyses, in which the authors chose EO & EC as predictors in their 2x2 ANOVA.

- Figure 4: The authors report a p-value of >0.999 with a correlation coefficient of -0.42 with a sample size of 10 subjects. This can't be correct (it should be around: p = 0.22). All statistical analyses should be checked.

- Figure 2c. Eyes closed condition: The highest score of the *Glx/GABA ratio seems to be ~3.6. In subplot 2a, there seem to be 3 subjects that show a Glx/GABA ratio score > 3.6. How can this be explained? There is also a discrepancy for the eyes-closed condition.

(4) Interpretation of aperiodic signal:

- Several recent papers demonstrated that the aperiodic signal measured in EEG or ECoG is related to various important aspects such as age, skull thickness, electrode impedance, as well as cognition. Thus, currently, very little is known about the underlying effects which influence the aperiodic intercept and slope. The entire interpretation of the aperiodic slope as a proxy for E/I is based on a computational model and simulation (as described in the Gao et al. paper).

- Especially the aperiodic intercept is a very sensitive measure to many influences (e.g. skull thickness, electrode impedance...). As crucial results (correlation aperiodic intercept and MRS measures) are facing this problem, this needs to be reevaluated. It is safer to make statements on the aperiodic slope than intercept. In theory, some of the potentially confounding measures are available to the authors (e.g. skull thickness can be computed from T1w images; electrode impedances are usually acquired alongside the EEG data) and could be therefore controlled.

- The authors wrote: "Higher frequencies (such as 20-40 Hz) have been predominantly associated with local circuit activity and feedforward signaling (Bastos et al., 2018; Van Kerkoerle et al., 2014); the increased 20-40 Hz slope may therefore signal increased spontaneous spiking activity in local networks. We speculate that the steeper slope of the aperiodic activity for the lower frequency range (1-20 Hz) in CC individuals reflects the concomitant increase in inhibition." The authors confuse the interpretation of periodic and aperiodic signals. This section refers to the interpretation of the periodic signal (higher frequencies). This interpretation can not simply be translated to the aperiodic signal (slope).

- The authors further wrote: We used the slope of the aperiodic (1/f) component of the EEG spectrum as an estimate of E/I ratio (Gao et al., 2017; Medel et al., 2020; Muthukumaraswamy & Liley, 2018). This is a highly speculative interpretation with very little empirical evidence. These papers were conducted with ECoG data (mostly in animals) and mostly under anesthesia. Thus, these studies only allow an indirect interpretation by what the 1/f slope in EEG measurements is actually influenced.

(5) Problems with EEG preprocessing and analysis:

- It seems that the authors did not identify bad channels nor address the line noise issue (even a problem if a low pass filter of below-the-line noise was applied).

- What was the percentage of segments that needed to be rejected due to the 120μV criteria? This should be reported specifically for EO & EC and controls and patients.

- The authors downsampled the data to 60Hz to "to match the stimulation rate". What is the intention of this? Because the subsequent spectral analyses are conflated by this choice (see Nyquist theorem).

- "Subsequently, baseline removal was conducted by subtracting the mean activity across the length of an epoch from every data point." The actual baseline time segment should be specified.

- "We excluded the alpha range (8-14 Hz) for this fit to avoid biasing the results due to documented differences in alpha activity between CC and SC individuals (Bottari et al., 2016; Ossandón et al., 2023; Pant et al., 2023)." This does not really make sense, as the FOOOF algorithm first fits the 1/f slope, for which the alpha activity is not relevant.

- The model fits of the 1/f fitting for EO, EC, and both participant groups should be reported.

(6) Validity of GABA measurements and results:

- According the a newer study by the authors of the Gannet toolbox (https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/nbm.5076), the reliability and reproducibility of the gamma-aminobutyric acid (GABA) measurement can vary significantly depending on acquisition and modeling parameter. Thus, did the author address these challenges? Furthermore, the authors wrote: "We confirmed the within-subject stability of metabolite quantification by testing a subset of the sighted controls (n=6) 2-4 weeks apart. Looking at the supplementary Figure 5 (which would be rather plotted as ICC or Blant-Altman plots), the within-subject stability compared to between-subject variability seems not to be great. Furthermore, I don't think such a small sample size qualifies for a rigorous assessment of stability.

- "Why might an enhanced inhibitory drive, as indicated by the lower Glx/GABA ratio" Is this interpretation really warranted, as the results of the group differences in the Glx/GABA ratio seem to be rather driven by a decreased Glx concentration in CC rather than an increased GABA (see Figure 2).

- Glx concentration predicted the aperiodic intercept in CC individuals' visual cortices during ambient and flickering visual stimulation. Why specifically investigate the Glx concentration, when the paper is about E/I ratio?

(7) Interpretation of the correlation between MRS measurements and EEG aperiodic signal:

- The authors wrote: "The intercept of the aperiodic activity was highly correlated with the Glx concentration during rest with eyes open and during flickering stimulation (also see Supplementary Material S11). Based on the assumption that the aperiodic intercept reflects broadband firing (Manning et al., 2009; Winawer et al., 2013), this suggests that the Glx concentration might be related to broadband firing in CC individuals during active and passive visual stimulation." These results should not be interpreted (or with very caution) for several reasons (see also problem with influences on aperiodic intercept and small sample size). This is a result of the exploratory analyses of correlating every EEG parameter with every MRS parameter. This requires well-powered replication before any interpretation can be provided. Furthermore and importantly: why should this be specifically only in CC patients, but not in the SC control group?

(8) Language and presentation:

- The manuscript requires language improvements and correction of numerous typos. Over-simplifications and unclear statements are present, which could mislead or confuse readers (see also interpretation of aperiodic signal).

- The authors state that "Together, the present results provide strong evidence for experience-dependent development of the E/I ratio in the human visual cortex, with consequences for behavior." The results of the study do not provide any strong evidence, because of the small sample size and exploratory analyses approach and not accounting for possible confounding factors.

- "Our results imply a change in neurotransmitter concentrations as a consequence of *restoring* vision following congenital blindness." This is a speculative statement to infer a causal relationship on cross-sectional data.

- In the limitation section, the authors wrote: "The sample size of the present study is relatively high for the rare population , but undoubtedly, overall, rather small." This sentence should be rewritten, as the study is plein underpowered. The further justification "We nevertheless think that our results are valid. Our findings neurochemically (Glx andGABA+ concentration), and anatomically (visual cortex) specific. The MRS parameters varied with parameters of the aperiodic EEG activity and visual acuity. The group differences for the EEG assessments corresponded to those of a larger sample of CC individuals (n=38) (Ossandón et al., 2023), and effects of chronological age were as expected from the literature." These statements do not provide any validation or justification of small samples. Furthermore, the current data set is a subset of an earlier published paper by the same authors "The EEG data sets reported here were part of data published earlier (Ossandón et al., 2023; Pant et al., 2023)." Thus, the statement "The group differences for the EEG assessments corresponded to those of a larger sample of CC individuals (n=38) " is a circular argument and should be avoided.

Reviewer #1 (Public Review):

This study identifies two behavioral processes that underlie learned pathogen avoidance behavior in C. elegans: exiting and re-entry of pathogenic bacterial lawns. Long-term behavioral tracking indicates that animals increase the prevalence of both behaviors over long-term exposure to the pathogen Pseudomonas aeruginosa. Using an optogenetic silencing screen, the authors identify groups of neurons, whose activity regulates lawn occupancy. Surprisingly, they find that optogenetic inhibition of neurons during only the first two hours of pathogen exposure can establish subsequent long-term changes in pathogen aversion. By leveraging a compressed sensing approach, the authors define a set of neurons involved in either lawn exit or lawn re-entry behavior using a constrained set of transgenic lines that drive Arch-3 expression in overlapping groups of neurons. They then measure the calcium activity of the candidate neurons involved in lawn re-entry in freely moving animals using GCaMP, and observe a reduction in their neural activity after exposure to a pathogen. Optogenetic inhibition of AIY and SIA neurons during acute pathogen exposure in naïve animals delays lawn entry whereas activating these neurons in animals previously exposed to pathogen enhances lawn entry, albeit transiently.

This work is missing several controls that are necessary to substantiate their claims. My most important concern is that the optogenetic screen for neurons that alter pathogenic lawn occupancy does not have an accompanying control on non-pathogenic OP50 bacteria. Hence, it remains unclear whether these neuronal inhibition experiments lead to pathogen-specific or generalized lawn-leaving alterations. For strains that show statistical differences between - and + ATR conditions, the authors should perform follow-up validation experiments on non-pathogenic OP50 lawns to ensure that the observed effect is PA14-specific. Similarly, neuronal inhibition experiments in Figures 5E and H are only performed with naïve animals on PA14 - we need to see the latency to re-entry on OP50 as well, to make general conclusions about these neurons' role in pathogen-specific avoidance.

My second major concern is regarding the calcium imaging experiments of candidate neurons involved in lawn re-entry behavior. Although the data shows that AIY, AVK, and SIA/SIB neurons all show reduced activity following pathogen exposure, the authors do not relate these activity changes to changes in behavior. Given the well-established links between these cells and forward locomotion, it is essential to not only report differences in activity but also in the relationship between this activity and locomotory behavior. If animals are paused outside of the pathogen lawn, these neurons may show low activity simply because the animals are not moving forward. Other forward-modulated neurons may also show this pattern of reduced activity if the animals remain paused. Given that the authors have recorded neural activity before and after contact with pathogenic bacteria in freely moving animals, they should also provide an analysis of the relationship between proximity to the lawn and the activity of these neurons.

This work is missing methodological descriptions that are necessary for the correct interpretation of the results shown here. Figure 2 suggests that the determination of statistical significance across the optogenetic inhibition screen will be found in the Methods, but this information is not to be found there. At various points in the text, authors refer to "exit rate", "rate constant", and "entry rate". These metrics seem derived from an averaged measurement across many individual animals in one lawn evacuation assay plate. However "latency to re-entry" is only defined on a per-animal basis in the lawn re-exposure assay. These differences should be clearly stated in the methods section to avoid confusion and to ensure that statistics are computed correctly.

This work also contains mislabeled graphs and incorrect correspondence with the text, which make it difficult to follow the authors 'claims. The text suggests that Pdop-2::Arch3 and Pmpz-1::Arch3 show increased exit rates, whereas Figure 2 shows that Pflp-4::Arch3 but not Pmpz-1::Arch3 has increased exit rate. The authors should also make a greater effort to correctly and clearly label which type of behavioral experiment is used to generate each figure and describe the differences in experimental design in the main text, figure legends, and methods. Figure 2E depicts trajectories of animals leaving a lawn over a 2.5-minute interval but it is unclear when this time window occurs within the 18-hour lawn leaving assay. Likewise, Figure 2H depicts a 30-minute time window which has an unclear relationship to the overall time course of lawn leaving. This figure legend is also mislabeled as "Infected/Healthy", whereas it should be labeled "-/+ ATR".

This work raises the interesting possibility that different sets of neurons control lawn exit and lawn re-entry behaviors following pathogen exposure. However, the authors never directly test this claim. To rigorously show this, the authors would need to show that lawn-exit-promoting neurons (CEPs, HSNs, RIAs, RIDs, SIAs) are dispensable for lawn re-entry behavior and that lawn re-entry promoting neurons (AVK, SIA, AIY, MI) are dispensable for lawn exit behavior in pathogen-exposed animals. The authors identify AVK neurons as important for modulating lawn re-entry behavior by brief inhibition at the start of pathogen exposure but fail to find that these neurons are required for increased latency to re-entry in naïve animals (Figure 5D). Recent work from Marquina-Solis et al (2024) shows that chronic silencing of these neurons delays pathogen lawn leaving, due to impaired release of flp-1 neuropeptide. Authors may wish to connect their work more closely with the existing literature by investigating the behavioral process by which AVK contributes to lawn evacuation.

If the authors work through these criticisms, this work can become an important contribution to the field of pathogen learning in C. elegans. However, in its current form, this work remains incomplete.

Reviewer #2 (Public Review):

In this manuscript, Hallacy et al. used a compressed sensing-based optogenetic screening method to investigate the crucial neurons that regulate pathogenic avoidance behavior in C. elegans. They further substantiate their findings using complementary optogenetic activation and imaging techniques to confirm the roles of the key neurons identified through extensive screening efforts. Notably, they identified AIY and SIA as pivotal neurons in the dynamic process of pathogenic avoidance. Their significant discovery is the delayed or stalled reentry process, which drives avoidance behavior; to my knowledge, this dynamic has not been previously documented. Additionally, the successful integration of quantitative optogenetic tools and compressed sensing algorithms is noteworthy, demonstrating the potential for obtaining highly quantitative data from the C. elegans nervous system. This approach is quite rare in this field, yet it represents a promising direction for studying this simple nervous system.

However, the paper's main weakness lies in its lack of a detailed mechanism explaining how the delayed reentry process directly influences the actual locomotor output that results in avoidance. The term 'delayed reentry' is used as a dynamic metric for quantifying the screening, yet the causal link between this metric and the mechanistic output remains unclear. Despite this, the study is well-structured, with comprehensive control experiments, and is very well constructed.

Reviewer #3 (Public Review):

Summary:

Using a compressed sensing-based approach applied previously by the author's group, the authors conducted an initial screen for neurons that when optogenetically down-regulated, influenced learned pathogen avoidance consisting of two component behaviors, exit from the bacterial lawn and lawn re-entry. Authors found that 4 classes of neurons AVK, SIA, AIY, and MI were inferred over a wide range of sparsity parameters, thereby indicating the importance of lawn re-entry. They found six classes of neurons required for lawn exit. The authors then went on to further analyze the neurons for the re-entry behavior, and conducted calcium imaging of those neurons in the freely behaving animals. They found that the activities of AIY and SIA neurons decreased after the animals that had been exposed to the pathogenic bacteria tried to re-enter the bacterial lawn. They also found that when those neurons of the animals that had not been exposed to pathogenic bacteria were downregulated by optogenetics, those operated animals increased the latency of the re-entry, which is a similar behavioral modification to that of the animals that had been exposed to the pathogen. Conversely, those neurons of the animals that were exposed to pathogenic bacteria were up-regulated by optogenetics, those animals showed a shortened latency of the re-entry, which is similar to the behavior observed in the animals not exposed to pathogen.

Strengths:

This is overall a very nice piece of work. Most importantly, an initial screening of neurons was conducted by a compressed sensing-based approach previously applied by the same group. It is also worth emphasizing that this compressed analysis is applicable when the behavior of interest involves a small number of neurons, as the authors pointed out in the Introduction Session. Therefore, the readers should keep in mind that the validation and significance of this work heavily depend on the justification of scarcity parameters that the authors chose. Nevertheless, this work is well justified because neurons identified by the initial screening were thoroughly analyzed by various methods including calcium imaging and optogenetic manipulation of neuronal activities and behavioral analyses using an animal-tracking system.

Weaknesses:

My only concern is that the authors should be more careful about describing their "compressed sensing-based approach". Authors often cite their previous Nature Methods paper, but should explain more because this method is critical for this manuscript. Also, this analysis is based on the hypothesis that only a small number of neurons are responsible for a given behavior. Authors should explain more about how to determine scarcity parameters, for example.

Reviewer #1 (Public Review):

Summary:

The study characterized the cellular and molecular mechanisms of spike timing-dependent long-term depression (t-LTD) at the synapses between excitatory afferents from lateral (LPP) and medial (MPP) perforant pathways to granule cells (GC) of the dentate gyrus (DG) in mice.

Strengths:

The electrophysiological experiments are thorough. The experiments are systematically reported and support the conclusions drawn.<br /> This study extends current knowledge by elucidating additional plasticity mechanisms at PP-GC synapses, complementing existing literature.

Weaknesses:

To more conclusively define the pivotal role of astrocytes in modulating t-LTD at MPP and LPP GC synapses through SNARE protein-dependent glutamate release, as posited in this study, the authors could adopt additional methods, such as alternative mouse models designed to regulate SNARE-dependent exocytosis, as well as optogenetic or chemogenetic strategies for precise astrocyte manipulation during t-LTD induction. This would provide more direct evidence of the influence of astrocytic activity on synaptic plasticity.

Reviewer #2 (Public Review):

Summary:

This work reports the existence of spike timing-dependent long-term depression (t-LTD) of excitatory synaptic strength at two synapses of the dentate gyrus granule cell, which are differently connected to the entorhinal cortex via either the lateral or medial perforant pathways (LPP or MPP, respectively). Using patch-clamp electrophysiological recording of tLTD in combination with either pharmacology or a genetically modified mouse model, they provide information on the differences in the molecular mechanism underlying this t-LTD at the two synapses.

Strengths:

The two synapses analyzed in this study have been understudied. This new data thus provides interesting new information on a plasticity process at these synapses, and the authors demonstrate subtle differences in the underlying molecular mechanisms at play. Experiments are in general well controlled and provide robust data that are properly interpreted.

Weaknesses:

- Caution should be taken in the interpretation of the results to extrapolate to adult brain as the data were obtained in P13-21 days old mice, a period during which synapses are still maturing and highly plastic.<br /> - In experiments where the drug FK506 or thapsigargin are loaded intracellularly, the concentrations used are as high as for extracellular application. Could there be an error of interpretation when stating that the targeted actors are necessarily in the post-synaptic neuron? Is it not possible for the drug to diffuse out of the cell as it is evident that it can enter the cell when applied extracellularly?<br /> - The experiments implicating glutamate release from astrocytes in t-LTD would require additional controls to better support the conclusions made by the authors. As the data stand, it is not clear how the authors identified astrocytes to load BAPTA and if dnSNARE expression in astrocytes does not indirectly perturb glutamate release in neurons.

Significance:

While this is the first report of t-LTD at these synapses, this plasticity process has been mechanistically well investigated at other synapses in the hippocampus and in the cortex. Nevertheless, this new data suggests that mechanistic differences in the induction of t-LTD at these two DG synapses could contribute to the differences in the physiological influence of the LPP and MPP pathways.

Reviewer #3 (Public Review):

Coatl et al. investigated the mechanisms of synaptic plasticity of two important hippocampal synapses, the excitatory afferents from lateral and medial perforant pathways (LPP and MPP, respectively) of the entorhinal cortex (EC) connecting to granule cells of the hippocampal dentate gyrus (DG). They find that these two different EC-DG synaptic connections in mice show a presynaptically expressed form of long-term depression (LTD) requiring postsynaptic calcium, eCB synthesis, CB1R activation, astrocyte activity, and metabotropic glutamate receptor activation. Interestingly, LTD at MPP-GC synapses requires ionotropic NMDAR activation whereas LTD at LPP-GC synapse is NMDAR independent. Thus, they discovered two novel forms of t-LTD that require astrocytes at EC-GC synapses. Although plasticity of EC-DG granule cell (GC) synapses has been studied using classical protocols, These are the first analysis of the synaptic plasticity induced by spike timing dependent protocols at these synapses. Interestingly, the data also indicate that t-LTD at each type of synapse require different group I mGluRs, with LPP-GC synapses dependent on mGluR5 and MPP-GC t-LTD requiring mGluR1.

The authors performed a detailed analysis of the coefficient of variation of the EPSP slopes, miniature responses and different approaches (failure rate, PPRs, CV, and mEPSP frequency and amplitude analysis) they demonstrate a decrease in the probability of neurotransmitter release and a presynaptic locus for these two forms of LTD at both types of synapses. By using elegant electrophysiological experiments and taking advantage of the conditional dominant-negative (dn) SNARE mice in which doxycycline administration blocks exocytosis and impairs vesicle release by astrocytes, they demonstrate that both LTD forms require the release of gliotransmitters from astrocytes. These data add in an interesting way to the ongoing discussion on whether LTD induced by STDP participates in refining synapses potentially weakening excitatory synapses under the control of different astrocytic networks. The conclusions of this paper are mostly well supported by data, but some aspects the results must be clarified and extended.

(1) It should be clarified whether present results are obtained with or without the functional inhibitory synapse activation. It is not clear if GABAergic synapses are blocked or not. If GABAergic synapses are not blocked authors must discuss whether the LTD of the EPSPs is due to a decrease in glutamatergic receptor activation or an increase in GABAergic receptor activation. Moreover, it should be recommended to analyze not only the EPSPs but also the EPSCs to address whether the decrease in synaptic transmission is caused by a decrease in the input resistance or by a decrease in the space constant (lambda).<br /> (2) Authors show that Thapsigargin loaded in the postsynaptic neuron prevents the induction of LTD at both synapses. Analyzing the effects of blocking postsynaptic IP3Rs (Heparin in the patch pipette) and Ryanodine receptors (Ruthenium red in the patch pipette) is recommended for a deeper analysis of the mechanism implicated in the induction of this novel forms of LTD in the hippocampus.<br /> (3) Authors nicely demonstrate that CB1R activation is required in these forms of LTD by blocking CB1Rs with AM251, however an interesting unanswered question is whether CB1R activation is sufficient to induce this synaptic plasticity. This reviewer suggests studying whether applying puffs of the CB1R agonist, WIN 55,212-2, could induce these forms of LTD.<br /> (4) Finally, adding a last figure with a cartoon summarizing the proposed model of action in these novel forms of LTD would add a positive value and would help the reading of the manuscript, especially in those aspects related with the discussion of the results.

The extension of these results would improve the manuscript which provides interesting results showing two novel forms of presynaptic t-LTD in the brain synapses with different action mechanisms probably implicated in the different aspects of information processing.

Reviewer #1 (Public Review):

Summary:

The authors provide solid evidence with a mouse model as well as supporting in vitro and analysis of clinical samples that loss of Fak increases the development of BRAF V600E-induced dysplastic lesions and carcinomas in the cecum via downregulation of Egfr-mediated Erk phosphorylation. This fine-tuning of Erk phosphorylation increases the expression of Lrg4 mRNA expression and promotes Lrg4 stability through downregulation of the E3 ubiquitin ligase Nedd4. The high Lrg4 expression correlates with an increased intestinal stem cell transcriptional signature that the authors suggest drives higher rates of transformation. This provides important insight that factors such as FAK may be able to modulate MAPK-driven tumorigenesis in specific circumstances. The data presented here are largely specific to the cecum. While these specific findings may ultimately have practical implications for human CRC outside the cecum and even therapeutic implications, these remain unexplored and will be a point for future investigations.

Strengths:

The authors use a mouse model (intestinal specific BRAF V600E +/- Fak knockout) as well as supporting in vitro analyses and clinical sample characterization to support their model. For both in vitro and in vivo studies, the authors use a combination of genetic and pharmacologic (including EGFR, FAK, and MEK inhibitors) tools to modulate the MAPK pathway. They also use a combination of transcriptional (RNA-Seq) and protein (IHC and Western blotting) readouts to support their proposed model. Importantly, they use a distinct mouse model (mutant Kras) to demonstrate their findings with Fak loss are specific to instances where EGFR can modulate ERK activation, providing strong evidence for their model. Finally, they also correlate their findings in the murine model with patient samples and with trends in the TCGA database. Collectively, these create a solid and convincing basis for their proposed model.

Weaknesses:

(1) The murine data is largely confined to the cecum. While the analysis of the cecum is appropriate based on the cecum specificity of their phenotype, they often use these findings to make broader generalizations about the nature of tumorigenesis in the intestinal epithelia and in CRC more generally. In my opinion, there was insufficient evidence presented supporting the extension of the proposed model beyond the cecum. While this is a weakness, it could be part of a growing effort to characterize left and right-sided malignancies as related but separate disease processes.

(2) The authors generally do a good job of focusing their analysis on the cecum and supporting their model. For example, Figure 5A examines different colon compartments, including the cecum. However, the authors fail to demonstrate that Fak loss only promotes Lrg4 upregulation in the cecum, where they observe an increase in BRAF V600E dysplasia and carcinoma. This is again seen in Figure 6A, where they only characterize Nedd4 expression in the cecum and not other compartments of the colon.

(3) The authors evaluate a broad range of tissues, including normal colonic mucosa, polyps, pre-cancerous dysplastic lesions, adenocarcinomas, and adenocarcinoma cell lines. While this breadth is a strength of the paper, the authors, at times, equate experimental observations in each of these conditions, despite the difference in the biology of these tissues/cells. For example, in their mouse model, they equate the development of dysplastic lesions and carcinoma lesions. This makes it difficult to accurately interpret their data and conclusions.

(4) In Figure 5i, this experiment was only completed in one cell line (HT29), despite the conclusion that Lrg4 expression is increased by decreased ERK phosphorylation due to protein stabilization. HT29 cells are a transformed human CRC cell line, quite different than a pre-malignant cecum intestinal epithelial cell. While convincing, the authors could have performed this key experiment in non-transformed murine cecal organoids (as they did for other experiments in Figure 5E), which would better recapitulate the mouse and pre-malignant setting to explain their mouse phenotype.

(5) While a large portion of the discussion focusses on the therapeutic implications of these findings, the authors only really investigate tumorigenesis. They likely have additional investigations planned for future manuscripts.

Reviewer #2 (Public Review):

Summary:

The manuscript by Gao et al. described a study identifying the role of FAK in fine-tuning the activation levels of ERK signaling in BRAF-V600E-driven colorectal cancer. The authors generated new mouse models combining Vill-Cre mediated BRAF-V600E expression with FAK deletion. Analyses of intestinal tumor phenotypes revealed that FAK-loss promotes BRAF-V600E-induced tumor formation, specifically in the cecum. Interestingly, these tumors closely resemble human sessile serrated adenoma/polyps. Using bioinformatics analysis, the authors found that FAK deletion upregulates the intestinal stem cell and fetal-type transcriptomic signatures compared to mice expressing BRAF-V600E alone. In addition, FAK-loss decreases the phosphorylation of ERK whereas it increases the expression of Lgr4 at both mRNA and protein levels. To mechanistically connect FAK-mediated downregulation of ERK and upregulation of Lgr4 in the context of BRAF-V600E mutation, results from biochemical experiments showed that MEK inhibitor treatment decreases the expression of NEDD4, a previously identified ubiquitin E3 ligase of Lgr4, which coincides with increased Lgr4 protein expression both in cells and in vivo. Moreover, the FAK-dependent modulation of ERK signaling is specific to BRAF-V600E-driven tumorigenesis only as knockout of FAK has no effect in Vill-Cre/KRAS-G12D mice. Collectively, the authors proposed a "just right" model in that a tunable FAK expression controls the optimal level of ERK pathway output needed for BRAF-V600E-induced cecal tumor formation.

Strengths:

This study provides new insights into the mechanisms underlying the serrated pathway-driven tumorigenesis in colorectal cancer. The newly established mouse model with compound mutations of BRAF and FAK offers a useful resource for future studies of the serrated pathway. The conclusions of this paper are mostly supported by data.

Weaknesses:

However, some aspects of the paper can be strengthened with additional mechanistically focused experiments.

(1) Some of the conclusions of the paper mainly rely on bioinformatic analyses of RNA-seq data. For example, it has been noted in several places in the paper that the knockout of FAK in Vill-Cre/BRAF-V600E mice does not affect the transcriptional outcome downstream of ERK while ERK phosphorylation levels are decreased. This statement is based on the lack of significant difference in the MAPK signature according to GSEA. However, whereas a significant enrichment of certain pathways can be used as support evidence, the lack of enrichment does not necessarily indicate those pathways are not involved. Other experiments are needed to examine the expression of ERK target genes to confirm. Similarly, the upregulation of fetal stem cell signature in FAK knockout mice needs to be verified using other methods besides GSEA.

(2) According to Figure 5i, the half-life of Lgr4 is around 48 hours in HT29 cells. However, it has been reported by at least two other publications cited in this paper (Ref. 44 and 45) that the half-life of Lgr4 is much shorter. This discrepancy is not explained.

(3) The effect of decreased ERK signaling on NEDD4 expression has only been briefly explored in Figure 6. The mechanisms by which FAK-loss and/or inhibition of MEK/ERK activity regulate NEDD4 expression are currently unclear. Moreover, the levels of NEDD4 expression are only analyzed in one mouse per group in Figure 6a. Quantitative analysis of NEDD4 as well as Lgr4 expression in additional numbers of mice will provide more solid support for the inverse correlation between NEDD4 and Lgr4 proteins. Since MEK inhibitor treatment also increases Lgr4 mRNA expression as shown in Figure 5f-g, the relative contribution of this altered mRNA expression vs. NEDD4L-mediated ubiquitination has not been investigated.

(4) It is an interesting finding that knockout FAK has no effect on KRAS-G12D-driven hyperplasia as shown in Figure 7. However, additional studies are needed to further explore the potential mechanisms by which FAK-loss specifically decreases EGFR/ERK signaling in the context of BRAF-V600E mutation.

Reviewer #3 (Public Review):

Summary:

Right-sided colorectal Cancer (CRC) is very different from left-sided CRC. Therefore it is important to model this cancer in mice and find new molecular targets. A broad set of data exists on FAK (Focal Adhesion Kinase) being important in colorectal cancer. However, this has focussed on APC mutant CRC which tends to be left-sided. BRAF mutation is common in right-sided CRC (and is rarely mutated with APC). Therefore the authors have tested whether FAK is important in this context. The authors show that FAK deletion surprisingly accelerates BRAF mutant CRC. Tumours arise in the proximal colon (which recapitulates BRAF mutant right-sided CRC). There are low for Lgr5 and high for foetal programmes. Mechanistically they suggest a pathway from FAK to NEDD4 to Lgr4 may underpin this phenotype.

Strengths:

Strong genetic data from FAK revealed that there is an acceleration of tumourigenesis and mice now develop proximal colon tumours and can be viewed as a good model of right-sided CRC.<br /> The expression data between humans and mice is strong.

Weaknesses:

The functional mechanism of how FAK loss promotes tumourigenesis is still quite correlative. An alternative hypothesis is that it drives inflammation in the proximal colon that drives tumourigenesis.

We still did not know the functional role for LGR4 (loss leads to a loss of paneth cells in homeostasis) so I'm not sure you can hypothesise a stem cell role.

Reviewer #2 (Public Review):

Summary:

This interesting study challenges the dogma regarding the link between bacterial metabolism decrease and tolerance to aminoglycosides (AG). The authors demonstrate that mutants well-known for being tolerant to AG, such as those of complexes I and II, are not so due to a decrease in the proton motive force (PMF) and thus antibiotic uptake, as previously reported in the literature.

Strengths:

This is a complete study that employs several read-outs.

In this revised version, the authors have carefully addressed all the reviewers' comments. I appreciate the effort made in this new version to clarify that this study does not refute the PMF-dependent mechanism of aminoglycoside uptake (in the discussion_ lines 731-734_).

The addition of the requested experiments using lower concentrations of aminoglycosides is a considerable improvement as it allows for comparison with previously published results.

Reviewer #1 (Public Review):

Summary:

Wang and colleagues presented an investigation of pig-origin bacteria Bacillus velezensis HBXN2020, for its released genome sequence, in vivo safety issue, probiotic effects in vitro, and protection against Salmonella infection in a murine model. Various techniques and assays are performed; the main results are all descriptive, without new insight advancing the field or a mechanistic understanding of the observed protection.

Strengths:

An extensive study on probiotic property of the Bacillus velezensis strain HBXN2020

Weaknesses:

The main results are descriptive without mechanistic insight. Additionally, most of the results and analysis parts are separated without a link or a story-telling way to deliver a concise message.

Reviewer #2 (Public Review):

Summary:

In this study, Wang and colleagues study the potential probiotic effects of Bacillus velezensis. Bacillus species have potential benefit to serve as probiotics due to their ability to form endospores and synthesize secondary metabolites. B. velezensis has been shown to have probiotic effects in plants and animals but data for human use are scarce, particularly with respect to salmonella-induced colitis. In this work, the authors identify a strain of B. velezensis and test it for its ability to control colitis in mice.

Key findings:

(1) The authors sequence an isolate for B. velezensis - HBXN2020 and describe its genome (roughly 4 mb, 46% GC-content etc).<br /> (2) The authors next describe the growth of this strain in broth culture and survival under acid and temperature stress. The susceptibility of HBXN2020 was tested against various antibiotics and against various pathogenic bacteria. In the case of the latter, the authors set out to determine if HBXN2020 could directly inhibit the growth of pathogenic bacteria. Convincing data, indicating that this is indeed the case, are presented.<br /> (3) To determine the safety profile of BHXN2020 (for possible use as a probiotic), the authors infected the strain in mice and monitored weight, together with cytokine profiles. Infected mice displayed no significant weight loss and expression of inflammatory cytokines remained unchanged. Blood cell profiles of infected mice were consistent with that of uninfected mice. No significant differences in tissues, including the colon were observed.<br /> (4) Next, the authors tested the ability to HBXN2020 to inhibit growth of Salmonella typhimurium (STm) and demonstrate that HBXN2020 inhibits STm in a dose dependent manner. Following this, the authors infect mice with STm to induce colitis and measure the ability of HBXN2020 to control colitis. The first outcome measure was a reduction in STm in faeces. Consistent with this, HBXN2020 reduced STm loads in the ileum, cecum, and colon. Colon length was also affected by HBXN2020 treatment. In addition, treatment with HBXN2020 reduced the appearance colon pathological features associated with colitis, together with a reduction in inflammatory cytokines.<br /> (5) After noting the beneficial (and anti-inflammatory effects) of HBXN2020, the authors set out to investigate effects on microbiota during treatment. Using a variety of algorithms, the authors demonstrate that upon HXBN2020 treatment, microbiota composition is restored to levels akin to that seen in healthy mice.<br /> (6) Finally, the authors assessed the effect of using HBXN2020 as prophylactic treatment for colitis by first treating mice with the spores and then infecting with STm. Their data indicate that treatment with HBXN2020 reduced colitis. A similar beneficial impact was seen with the gut microbiota.

Strengths:

(1) Good use of in vitro and animal models to demonstrate a beneficial probiotic effect.<br /> (2) Most observations are supported using multiple approaches.<br /> (3) Mouse experiments are very convincing.

Weaknesses:

(1) Whilst a beneficial effect is observed, there no investigation of the mechanism that underpins this.<br /> (2) Mouse experiments would have benefited from the use of standard anti-inflammatory therapies to control colitis. That way the authors could compare their approach of using bacillus spores that current gold standard for treatment.

Reviewer #3 (Public Review):

Summary:

The manuscript by Wang et al. investigates the effects of B. velezensis HBXN2020 in alleviating S. Typhimurium-induced mouse colitis. The results showed that B. velezensis HBXN2020 could alleviate bacterial colitis by enhancing intestinal homeostasis (decreasing harmful bacteria and enhancing the abundance of Lactobacillus and Akkermansia) and gut barrier integrity and reducing inflammation.

Strengths:

B. velezensis HBXN2020 is a novel species of Bacillus that can produce a great variety of secondary metabolites and exhibit high antibacterial activity against several pathogens. B. velezensis HBXN2020 is able to form endospores and has strong anti-stress capabilities. B. velezensis HBXN2020 has a synergistic effect with other beneficial microorganisms, which can improve intestinal homeostasis.

Weaknesses:

Few studies about the clinical application of Bacillus velezensis. Thus, more studies are still needed to explore the effectiveness of Bacillus velezensis before clinical application.

Reviewer #1 (Public Review):

Summary:

Duan et al analyzed brain imaging data in UKBK and found a pattern in brain structure changes by aging. They identified two patterns and found links that can be differentiated by the categorization.

Strengths:

This discovery harbors substantial impacts in aging and brain structure and function.

Weaknesses:

Therefore, the study requires more validation efforts. Most importantly, data underlying the stratification of two groups are not obvious and lack further details. Can they also stratified by different method? i.e. PCA?

Any external data can be used for validation?

Other previous discoveries or claims supporting the results of the study should be explored to support the conclusion.

Sex was merely used as a covariate. Were there sex-differences during brain aging? Sex ratio difference in group 1 and 2?

Although statistically significant, Fig 3 shows minimal differences. LTL and phenoAge is displayed in adjusted values but what is the actual values that differ between pattern 1 and 2?

It is not intuitive to link gene expression result shown in Fig 8 and brain structure and functional differences between pattern 1 and 2. Any overlap of genes identified from analyses shown in Fig 6 (GWAS) and 8 (gene expression)?

Reviewer #2 (Public Review):

Summary:

The authors aimed to understand the heterogeneity of brain aging by analyzing brain imaging data. Based on the concept of structural brain aging, they divided participants into two groups based on the volume and rate of decrease of gray matter volume (GMV). The group with rapid brain aging showed accelerated biological aging and cognitive decline and was found to be vulnerable to certain neuropsychiatric disorders. Furthermore, the authors claimed the existence of a "last in, first out" mirroring pattern between brain aging and brain development, which they argued is more pronounced in the group with rapid brain aging. Lastly, the authors identified genetic differences between the two groups and speculated that the cause of rapid brain aging may lie in genetic differences.

Strengths:

The authors supported their claims by analyzing a large amount of data using various statistical techniques. There seems to be no doubt about the quality and quantity of the data. Additionally, they demonstrated their strength in integrating diverse data through various analysis techniques to conclude.

Weaknesses:

The authors provided appropriate answers to the reviewers' questions and revised the manuscript accordingly, and as a result, the paper has been edited to be more easily understood.

Reviewer #1 (Public Review):

Summary:

In previous work the Elias group has shown that leptin sensing PMv neurons make connections with the neuroendocrine reproductive axis and are involved in reproductive function/s. Sáenz de Miera et al. build on this body of work to investigate the sufficiency of leptin sensing PMv neurons to evoke the release of luteinizing hormone. The team further investigates how glutamate signaling from leptin-sensing neurons can influence pubertal timing in females, along with mature estrous cycles. Genetic ablation of Slc17a6 (Vglut2) from LepRb-expressing cells resulted in a delay of the first estrus cycle post pubertal transition, along with a significantly lengthened estrous cycle in mature females. However, this deficit did not lengthen the latency to birth of the first litter in experimental dams. Restoration of leptin signaling in LepRb PMv neurons that was previously shown to induce puberty and instate reproductive function in LepRb knock-out female mice (Mahany et al., 2018). Here, Sáenz de Miera et al. use a combined genetic and viral strategy to demonstrate that glutamate signaling in LepRb PMv neurons is required for sexual maturation in LepRb knock-out female mice.

Strengths:

Most of the experiments performed in this manuscript are well justified and rigorously tested. The genetic method to simultaneously remove glutamate signaling and restore the leptin receptor in LepRb PMv neurons was well executed and showed that glutamate signaling in LepRb PMv neurons is necessary for leptin-dependent fertility.

Weaknesses:

Analysis of experimentally induced luteinizing hormone release could be confounded by spontaneous pulses of luteinizing hormone that are independent of LepRb PMv neurons.

Reviewer #2 (Public Review):

Summary:

This is a very well-written manuscript by Saenz de Meira and colleagues on a careful study reporting on the key role of glutamate transporter vGlut2 expression in the neurons of the ventral perimammillary nucleus (PMv) of the hypothalamus expressing the leptin receptor LepRb in energy homeostasis, puberty, and estrous cyclicity. The authors first show using cre-dependent chemogenetic viral tools that the selective activation of the PMv LepRb induces luteinizing hormone (LH) release. Then the authors demonstrate that the selective invalidation of vGlut2 in LepRb-expressing cells in the all body induces obesity and mild alteration of sexual maturation in both sexes and blunted estrous cyclicity in females. Finally, the authors knock out vGlut2 in PMv neurons in which they reintroduce LepRb expression in an otherwise LepRb-null background using an AAV Cre approach. This latter very elegant experiment shows that while the sole re-expression of LepRb in PMv neurons in LepRb-null mice was shown before to restore puberty onset, deleting vGlut2 in LepRb-expressing PMv neurons blunts this effect.

Strengths:

The authors employ state-of-the-art methods and their conclusions are robustly supported by the results.

Weaknesses:

None identified. Only minor comments have been formulated.

Reviewer #3 (Public Review):

Summary:

The authors examined the effects of glutamate release from PMv LepR neurons in the regulation of puberty and reproduction in female mice.

Strengths:

Multiple genetic mouse models were utilized to either manipulate PMv LepR neuron activities or to delete glutamate vesicle transporters from LepR neurons. The authors have been quite rigorous in validating these models and exploring potential contaminations. Most of the data presented are solid and convincing and support the conclusion.

Comments on revised version:

The authors have addressed most of my comments.

Reviewer #2 (Public Review):

In this study, Sekulovski and colleagues report refinements to an in vitro model of human amnion formation. Working with 3D cultures and BMP4 to induce differentiation, the authors chart the time course of amnion induction in human pluripotent stem cells in their system using immunofluorescence and RNA-seq. They carry out validation through comparison of their data to existing embryo datasets, and through immunostaining of post-implantation marmoset embryos. Functional experiments show that the transcription factor TFAP2C drives the amnion differentiation program once it has been initiated.

There is currently great interest in the development of in vitro models of human embryonic development. While it is known that the amnion plays an important structural supporting role for the embryo, its other functions, such as morphogen production and differentiation potential, are not fully understood. Since a number of aspects of amnion development are specific to primates, models of amniogenesis will be valuable for the study of human development. Advantages of this model include its efficiency and the purity of the cell populations produced, a significant degree of synchrony in the differentiation process, benchmarking with single-cell data and immunocytochemistry from primate embryos, and identification of key markers of specific phases of differentiation. Weaknesses are the absence of other embryonic tissues in the model, and overinterpretation of certain findings, in particular relating bulk RNA-seq results to scRNA-seq data from published analyses of primate embryos and results from limited (though high quality) embryo immunostainings.

Reviewer #3 (Public Review):

In this work, the authors tried to profile time-dependent changes in gene and protein expression during BMP-induced amnion differentiation from hPSCs. The authors depicted a GATA3 - TFAP2A - ISL1/HAND1 order of amniotic gene activation, which provides a more detailed temporary trajectory of amnion differentiation compared to previous works. As a primary goal of this study, the above temporal gene/protein activation order is amply supported by experimental data. However, the mechanistic insights on amniotic fate decision, as well as the transcriptomic analysis comparing amnion-like cells from this work and other works remain limited. While this work allows us to see more details of amnion differentiation and understand how different transcription factors were turned on in a sequence and might be useful for benchmarking the identity of amnion in ex utero cultured human embryos/embryoids, it provides limited insights on how amnion cells might diverge from primitive streak / mesoderm-like cells, despite some transcriptional similarity they shared, during early development.

[Editors' note: In the revised manuscript, the authors have added new results and made textual revisions that address the reviewers' concerns. These changes have significantly enhanced the clarity, quality, and impact of the study. ]

Reviewer #1 (Public Review):

Summary:

The work by Zeng et al. comprehensively explored the differences in the effects of leaf and soil microbes on the seed germination, seedling survival and seedling growth of an invasive forb, Ageratina Adenophora, and found evidence of stronger adverse effects of leaf microbes on Ageratina compared with soil microbes. By further DNA sequencing and fungal strain cultivation, the authors were able to identify some of the key microbial guilds that may facilitate such negative and positive feedbacks.

Strengths:

(1) The theoretic framework is well-established;<br /> (2) Relating the direction of plant-microbe feedback to certain microbial guild is always hard, but the authors had done a great job in identifying and interpreting such relationships.

Weaknesses:

(1) Allelopathic effects can't be directly accounted for;<br /> (2) The fungal strains accumulated in dead seedlings may also accumulate in live seedlings, thus more evidence is needed to validate the claim by the authors that Allophoma and Alternaria can increase seedling mortality.

Reviewer #1 (Public Review):

Summary:

The authors addressed the influence of DKK2 on colorectal cancer (CRC) metastasis to the liver using an orthotopic model transferring AKP-mutant organoids into the spleens of wild-type animals. They found that DKK2 expression in tumor cells led to enhanced liver metastasis and poor survival in mice. Mechanistically, they associate Dkk2-deficiency in donor AKP tumor organoids with reduced Paneth-like cell properties, particularly Lz1 and Lyz2, and defects in glycolysis. Quantitative gene expression analysis showed no significant changes in Hnf4a1 expression upon Dkk2 deletion. Ingenuity Pathway Analysis of RNA-Seq data and ATAC-seq data point to a Hnf4a1 motif as a potential target. They also show that HNF4a binds to the promoter region of Sox9, which leads to LYZ expression and upregulation of Paneth-like properties. By analyzing available scRNA data from human CRC data, the authors found higher expression of LYZ in metastatic and primary tumor samples compared to normal colonic tissue; reinforcing their proposed link, HNF4a was highly expressed in LYZ+ cancer cells compared to LYZ- cancer cells.

Strengths:

Overall, this study contributes a novel mechanistic pathway that may be related to metastatic progression in CRC.

Weaknesses:

The main concerns are related to incremental gains, missing in vivo support for several of their conclusions in murine models, and missing human data analyses. Additionally, methods and statistical analyses require further clarification.

Main comments:

(1) Novelty<br /> The authors previously described the role of DKK2 in primary CRC, correlating increased DKK2 levels to higher Src phosphorylation and HNF4a1 degradation, which in turn enhances LGR5 expression and "stemness" of cancer cells, resulting in tumor progression (PMID: 33997693). A role for DKK2 in metastasis has also been previously described (sarcoma, PMID: 23204234).

(2) Mouse data<br /> a) The authors analyzed liver mets, but the main differences between AKT and AKP/Dkk2 KO organoids could arise during the initial tumor cell egress from the intestinal tissue (which cannot be addressed in their splenic injection model), or during pre-liver stages, such as endothelial attachment. While the analysis of liver mets is interesting, given that Paneths cells play a role in the intestinal stem cell niche, it is questionable whether a study that does not involve the intestine can appropriately address this pathway in CRC metastasis.<br /> b) The overall number of Paneth cells found in the scRNA-seq analysis of liver mets was strikingly low (17 cells, Figure 3), and assuming that these cells are driving the differences seems somewhat far-fetched. Adding to this concern is inappropriate gating in the flow plot shown in Figure 6. This should be addressed experimentally and in the interpretation of data.<br /> c) Figures 3, 5, and 6 show the individual gene analyses with unclear statistical data. It seems that the p-values were not adjusted, and it is unclear how they reached significance in several graphs. Additionally, it was not stated how many animals per group and cells per animal/group were included in the analyses.<br /> d) Figure 6 suggests a signaling cascade in which the absence of DKK2 leads to enhanced HNF4A expression, which in turn results in reduced Sox9 expression and hence reduced expression of Paneth cell properties. It is therefore crucial that the authors perform in vivo (splenic organoid injection) loss-of-function experiments, knockdown of Sox9 expression in AKP organoids, and Sox9 overexpression experiments in AKP/Dkk2 KO organoids to demonstrate Sox9 as the central downstream transcription factor regulating liver CRC metastasis.<br /> e) Given the previous description of the role of DKK2 in primary CRC, it is important to define the step of liver metastasis affected by Dkk2 deficiency in the metastasis model. Does it affect extravasation, liver survival, etc.?

(3) Human data<br /> Can the authors address whether the expression of Dkk2 changes in human CRC and whether mutations in Dkk2 as correlated with metastatic disease or CRC stage?

(4) Bioinformatic analysis<br /> The authors did not provide sufficient information on bioinformatic analyses. The authors did not include information about the software, cutoffs, or scripts used to make their analyses or output those figures in the manuscript, which challenges the interpretation and assessment of the results. Terms like "Quantitative gene expression analyses" (line 136) "visualized in a Uniform Approximation and Projection" (line 178) do not explain what was inputted and the analyses that were executed. There are multiple forms to align, preprocess, and visualize bulk, single cell, ATAC, and ChIP-seq data, and depending on which was used, the results vary greatly. For example, in the single-cell data, the authors did not inform how many cells were sequenced, nor how many cells had after alignment and quality filtering (RNA count, mt count, etc.), so the result on Paneth+ to Goblet+ percent in lines 184 and 185 cannot be reached because it depends on this information. The absence of a clustering cutoff for the single-cell data is concerning since this greatly affects the resulting cluster number (https://www.nature.com/articles/s41592-023-01933-9). The authors should provide a comprehensive explanation of all the data analyses and the steps used to obtain those results.

(5) Clarity of methods and experimental approaches<br /> The methods were incomplete and they require clarification.

Reviewer #2 (Public Review):

Summary:

The authors propose that DKK2 is necessary for the metastasis of colon cancer organoids. They then claim that DKK2 mediates this effect by permitting the generation of lysozyme-positive Paneth-like cells within the tumor microenvironmental niche. They argue that these lysozyme-positive cells have Paneth-like properties in both mouse and human contexts. They then implicate HNF4A as the causal factor responsive to DKK2 to generate lysozyme-positive cells through Sox9.

Strengths:

The use of a genetically defined organoid line is state-of-the-art. The data in Figure 1 and the dependence of DKK2 for splenic injection and liver engraftment, as well as the long-term effect on animal survival, are interesting and convincing. The rescue using DKK2 administration for some of their phenotype in vitro is good. The inclusion and analysis of human data sets help explore the role of DKK2 in human cancer and help ground the overall work in a clinical context.

Weaknesses:

In this work by Shin et al., the authors expand upon prior work regarding the role of Dickkopf-2 in colorectal cancer (CRC) progression and the necessity of a Paneth-like population in driving CRC metastasis. The general topic of metastatic requirements for colon cancer is of general interest. However, much of the work focuses on characterizing cell populations in a mouse model of hepatic outgrowth via splenic transplantation. In particular, the concept of Paneth-like cells is primarily based on transcriptional programs seen in single-cell RNA sequencing data and needs more validation. Although including human samples is important for potential generality, the strength could be improved by doing immunohistochemistry in primary and metastatic lesions for Lyz+ cancer cells. Experiments that further bolster the causal role of Paneth-like CRC cells in metastasis are needed.

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):

Summary:

In this manuscript, Popli et al investigated the roles of the 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), the authors discovered that such females are completely infertile. They went on to show that Atg14 cKO females have impaired embryo implantation and uterus receptivity due to impaired response to P4 stimulation and stromal decidualization. In addition to the uterus defect, the authors also discovered that early embryos are trapped inside the oviduct and cannot be efficiently transported to the uterus in these females. They went on to show that oviduct epithelium in Atg14 cKO females showed increased pyroptosis, which disrupts oviduct epithelial integrity and leads to obstructive oviduct lumen and impaired embryo transport. Therefore, the authors concluded that autophagy is critical for maintaining the oviduct homeostasis and keeping the inflammation under check to enable proper embryo transport.

Strengths:

This study revealed an important and unexpected role of the autophagy-related gene Atg14 in preventing pyroptosis and maintaining oviduct epithelial integrity, which is poorly studied in the field of reproductive biology. The study is well designed to test the roles of ATG14 in mouse oviduct and uterus. The experimental data in general support the conclusion and the interpretations are mostly accurate. This work should be of interest to reproductive biologists and scientists in the field of autophagy and pyroptosis.

Weaknesses:

Despite the strengths, there are several major weaknesses raising concerns. In addition, the mismatched figure panels, the undefined acronyms, and the poor description/presentation of some of the data significantly hinder the readability of the manuscript.

(1) In the abstract, the authors stated that "autophagy is critical for maintaining the oviduct homeostasis and keeping the inflammation under check to enable embryo transport". This statement is not substantiated. Although Atg14 is an autophagy-related gene and plays a critical role in oviduct homeostasis, the authors did not show a direct link between autophagy and pyroptosis/oviduct integrity. In addition, the authors pointed out in the last paragraph of the introduction that none of the other autophagy-related genes (ATG16L, FIP200, BECN1) exhibited any discernable impact on oviduct function. Therefore, the oviduct defect is caused by Atg14 specifically, not necessarily by autophagy.

(2) In lines 412-414, the authors stated that "Atg14 ablation in the oviduct causes activation of pyroptosis", which is also not supported by the experimental data. The authors did not show that Atg14 is expressed in oviduct cells. PR-Cre is also not specific in oviduct cells. It is possible that Atg14 knockout in other PR-expressing tissues (such as the uterus) indirectly activates pyroptosis in the oviduct. More experiments will be required to support this claim. In line with the no defect when Atg14 is knocked out in oviduct ciliary cells, it will be good to use the secretory cells Cre, such as Pax8-Cre, to demonstrate that Atg14 functions in the secretory cells of the oviduct thus supporting this conclusion.

(3) With FOXJ1-Cre, the authors attempted to specifically knockout Atg14 in ciliary cells, but there are no clear fertility and embryo implantation defects in Foxj1/Atg14 cKO mice. The author should provide the verification data to show that Atg14 had been effectively depleted in ciliary cells if Atg14 is normally expressed.

(4) In lines 307-313, the author tested whether ATG14 is required for the decidualization of HESCs. The author stated that "Control siRNA transfected cells when treated with EPC seemed to change their morphological transformation from fibroblastic to epithelioid (Fig. 2E) and had increased expression of the decidualization markers IGFBP1 and PRL by day three only (Fig. 2F)". First, the labels in Figure 2 are not corresponding to the description in the text. Second, the morphology of the HESCs in control and Atg14 siRNA group showed no obvious difference even at day 3 and day 6. The author should point out the difference in each panel and explain in the text or figure legend.

(5) In lines 332-336, the authors pointed out that the cKO mice oviduct lining shows marked eosinophilic cytoplasmic change, but there's no data to support the claim. In addition, the authors further described that "some of the cells showed degenerative changes with cytoplasmic vacuolization and nuclear pyknosis, loss of nuclear polarity, and loss of distinct cell borders giving an appearance of fusion of cells (Fig. 3D)". First, Figure 3D did not show all these phenotypes and it is likely a mismatch to Figure 3E. Even in Figure 3E, it is not obvious to notice all the phenotypes described here. The figure legend is overly simple, and there's no explanation of the arrowheads in the panel. More data/images are required to support the claim here and provide a clear indication and explanation in the figure legend.

(6) In lines 317-325, it is rather confusing about the description of the portion of embryos from the oviduct and uterus. In addition, the total number of embryos was not provided. I would recommend presenting the numerical data to show the average embryos from the oviduct and uterus instead of using the percentage data in Figures 3A and 5G.

(7) In lines 389-391, authors tested whether Polyphyllin VI treatment led to activated pyroptosis and blocked embryo transport. Although Figures 5F-G showed the expected embryo transport defect, the authors did not show the pyroptosis and oviduct morphology. It will be important to show that the Polyphyllin VI treatment indeed led to oviduct pyroptosis and lumen disruption.

(8) In line 378, it would be better to include a description of pyroptosis and its molecular mechanisms to help readers to better understand your experiments. Alternatively, you can add it in the introduction.

(9) Please make sure to provide definitions for the acronyms such as FRT, HESCs, GSDMD, etc.

(10) It is rather confusing to use oviducal cell plasticity in this manuscript. The work illustrated the oviducal epithelial integrity, not the plasticity.

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 using 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 manuscript has some interesting findings, however there are also areas that could be improved.

Strengths:

The importance of Atg14 and autophagy in the female reproductive tract is incompletely understood. The manuscript also provides partial evidence about a new mechanism linking Atg14 to pyropotosis.

Weaknesses:

(1) It is not clear why the loss of Atg14 selectively induces Pyroptosis within oviduct cells but not in other cellular compartments. The authors should demonstrate that these events are not happening in uterine cells.

(2) The manuscript never showed any effect on the autophagy upon loss of Atg14. Is there any effect on autophagy upon Atg14 loss? If so does that contribute to the observation?

(3) It is not clear what the authors meant by cellular plasticity and integrity. There is no evidence provided in that aspect that the plasticity of oviduct cells is lost. Similarly, more experimental evidence is necessary for the conclusion about cellular integrity.

(4) The mitochondrial phenotype shown in Figure 3 didn't appear as severe as it is described in the results section. The analyses should be more thorough. They should include multiple frames (in supplemental information) showing mitochondrial morphology in multiple cells. The authors should also test that aspect in uterine cells. The authors should measure Feret's diagram. Difference in membrane potential etc. for a definitive conclusion.

(5) The comment that the loss of Atg14 and pyroptosis leads to the narrowing of the lumen in the oviduct should be experimentally shown.

(6) The manuscript never showed the proper mechanism through which Atg14 loss induces pyroptosis. The authors should link the mechanism.

Reviewer #1 (Public Review):

This paper discusses the identification of viral genes in publicly available DNA and RNA sequencing datasets. In many cases, these datasets have been assembled into contigs. Many viral genes were identified and contigs containing genes from more than one type of virus were more common than expected. The analysis appears to be sound and the results presented will be of great interest to the community.

The strengths of the paper are in the analysis itself, which is detailed, complex, and on a very large scale. To my knowledge, the identification of DNA viral proteins in sequencing datasets not deliberately infected with viruses has not previously been performed on this scale. Many proteins were identified which are at the limit of our current capacity to detect divergent proteins. I think the use of multiple methodologies strengthens the study, as it increases the depth of the results. The authors are also clear about the limitations of their study and give many caveats about their results, which is excellent.

I have two major concerns about the study. The first is the presentation, which in places makes it difficult to tell exactly how and why the analysis has been performed. I do not think it would be possible to reproduce this analysis based only on the information presented in the Materials and Methods section. This makes it difficult to assess the exact details of the method and whether they are appropriate. I would appreciate something like a flow chart to show, for each SRA dataset and each assembled contig, the exact steps taken for classification and the hierarchy of tools, plus the threshold values, applied to the results. An overview of the results at the beginning of the results section would also be helpful - how many proteins were identified, what were their host species, how many contigs were assembled and how many of these were chimeric, etc.

My second concern is that it is not clear how each protein was determined to be either viral or non-viral or how contigs were assigned as chimeric or non-chimeric. Positive and negative controls are not mentioned and false positive or negative rates are not calculated. Given that many of the identified proteins are highly divergent from known viral proteins, it would be good to see how likely it is that a random protein would be assigned as viral, or a viral protein as non-viral. Chimeric contigs could occur due to misassembly or endogenous viral elements, it seems like viruses in these categories may have been filtered using Cenote Taker but no checks are described to confirm that the filtering was successful.

Overall, I think that the study is useful and of interest, but I think more clarity in the presentation of the results would increase the value of the paper for many readers.

Reviewer #2 (Public Review):

Summary:

A large-scale computational analysis of published sequences of various animal species provides evidence for extensive gene transfer amongst DNA viruses.

Strengths:

The study provides evidence for a large number of previously uncharacterized DNA viruses and supports a model whereby DNA viruses have evolved by combining distinct shared replication modules and some of these evolutionary oddities likely remain in the biosphere. The work provides a useful repository and potential framework for additional virus discovery efforts.

Weaknesses:

This is an entirely computational story, with very limited experimental validation. A large number of often confusing new acronyms are introduced that may be "cute" (such as the reference to the delicious half-smoke sausage) but are not particularly useful. This is not helped by the somewhat "telegraphic" presentation of the data that is sometimes difficult to digest. Not all paragraphs deliver what they promise. For example under the title "Polyomaviruses and papillomaviruses" there is no discussion of papillomaviruses. Overall, however, these weaknesses do not diminish my enthusiasm for this paper, which will be an important resource for computational and non-computational virus hunters.

Reviewer #3 (Public Review):

Summary:

Buck et al., set out to characterize small DNA tumor viruses through the generation and analysis of ~100,000 public sequencing datasets from the SRA and other databases. Using a variety of powerful bioinformatic methods including alignment-based searches, statistical modelling, and structure-aware detection, the authors successfully classify novel protein sequences which support the occurrence of evolutionary gene transfer between DNA virus families. The authors propose a naming scheme to better capture viral diversity and uncover novel chimeric viruses, those containing genes from multiple established virus families. Additional analysis using the generated dataset was performed to search for DNA and RNA viruses of interest, demonstrating the utility of generated datasets for exploratory screens. The assembled sequencing datasets are publicly available, providing invaluable resources for current and future investigations within this subfield.

Strengths:

The scope of data analysis (100,000+ SRA records and additional libraries) is substantial, and the authors have contributed to further insight into the modularity of previously uncharacterized viral genomes, through computationally demanding advanced bioinformatics analyses in addition to extensive manual inspection.

The publicly available resources generated as a result of these analyses provide useful data for further experiments to inspect viral diversity and modularity. Other scanning experiments and further investigation of biologically relevant viruses using these contigs may uncover, for example, animal reservoirs or novel recombinant viruses of significance.

Novel instances of genomic modularity provide excellent starting points for understanding virus evolutionary pathways and gene transfer events.

Weaknesses:

Overall, the methods section of this paper requires more detail.

The inclusion criteria for which "SRA" datasets were or were not utilized within this study are poorly defined. This means the comprehensiveness of the study for a given search space of the SRA is not defined, and the results are ultimately not reproducible, or expandable. For example, are all vertebrate RNA-seq samples processed? Or just aquatic vertebrate RNA-seq? Were samples randomly sampled from a more comprehensive data set? What is the make-up of the search space and how much was DNA-seq or RNA-seq? This section should be expanded and explicit accounting provided for how dataset selection was performed. This would provide additional confidence in the results and conclusions, as well as allow for future analysis to be conducted.

Hallmark virus genes require further clarification, as it is unclear what genes are utilized as bait, or in the initial search process. The reported "Hallmark gene sets" are not described in a systematic way. What is the sensitivity and specificity of these gene sets? Was there a validation of the performance characteristics (ROC) for this gene set with different tools? How is this expected to be utilized? Which kinds of viruses are excluded/missed? Are viroids included?

For the Tailtomavirus, additional information is needed for sufficient confidence. Was this "chimeric" genomic arrangement detected in a single library? This raises a greater issue of how technical artifacts, which may appear as chimeric assemblies, are ruled out in the workflow. If two viral genomes share a k-mer of length greater than the assembly k, the graph may become merged. Are there read pairs that span all regions of the genome? Is there evidence for multiple homologous viruses with synteny between them that supports the combination of these genes as an evolving genome, or is this an anomalous observation? Read alignments should be included and Bandage graph visualization for all cases of chimeric assemblies and active steps to disprove the baseline hypotheses that these are technical artifacts of genome assembly.

Justification for exclusion of endogenized sequences is not included and must be described, as small DNA tumor viruses may endogenize into the host genome as part of their life cycle. How is such an integration resolved from an evolutionary "endogenization"? What's the biological justification for this step?

Additional supporting information, clear presentation, and context are needed to strengthen results and conclusions.

Basic reporting of global statistics, such as the total number of viruses found per family, should be included in the main text to better support the scope of the results. How many viruses (per family) were previously known, and therefore what is the magnitude of the expansion performed here?

Additional parameters and information should be included in bioinformatic tool outputs to provide greater clarity and interpretation of results. For example, reporting the "BLASTp E-val", as for the PolB homology (BLASTp 6E-12) is not informative, and does not tell the reader this is (we assume) an expectancy value. For each such case please report, the top database hit accession, percent identity, query coverage, and E-value. Otherwise, a judgment cannot be adequately made regarding the quality of evidence for homology. Similarly, for HHpred what does the number represent - confidence, identity, or coverage?

Some findings described in the Results section may require revision. Several of the Nidoviruses (Nidovirus takifugu, Nidovirus hypomesus, Nidovirus ambystoma, etc...) have been previously described by three groups, first by Edgar et al., (https://www.nature.com/articles/s41586-021-04332-2), then Miller et al., (https://academic.oup.com/ve/article/7/2/veab050/6290018) and then Lauber et al., (https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012163). This is now the 4th description of the same set of viruses. These sequences are in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/OV442424.1), although it is unclear why they're not returned as BLAST hits. Miller also described the Togavirus co-segment previously.

It is also uncertain what is being described with HelPol/maldviruses which was not previously described in distantly similar relatives. How many were described in the previous literature and how many are described by this work?

Co-phylogenies should be used to convey gene transfer and flow clearly to support the conclusions made in the text.

Statements such as, "The group encompasses a surprising degree of genomic diversity...", should be supported by additional information to strengthen conclusions (e.g., what the expected diversity is). What is the measurement for genomic diversity here, and why is this surprising? There is overall a lack of quantification to support the conclusions made throughout the paper.

Reviewer #1 (Public Review):

Summary of the work: In this work, Fruchard et. al. study the enzyme Tgt and how it modifies guanine in tRNAs to queuosine (Q), essential for Vibrio cholerae's growth under aminoglycoside stress. Q's role in codon decoding efficiency and its proteomic effects during antibiotic exposure is examined, revealing Q modification impacts tyrosine codon decoding and influences RsxA translation, affecting the SoxR oxidative stress response. The research proposes Q modification's regulation under environmental cues reprograms the translation of genes with tyrosine codon bias, including DNA repair factors, crucial for bacterial antibiotic response.

The experiments are well-designed and conducted and the conclusions, for the most part, are well supported by the data. However, a few clarifications will significantly strengthen the manuscript.

Major:<br /> Figure S4 A-D. These growth curves are important data and should be presented in the main figures. Moreover, given that it is not possible to make a rsxA mutant, I wonder if it would be possible to connect rsx and tgt using the following experiment: expression of tgt results in resistance to TOB (in B), while expression of only rsx lower resistance to TOB (in D). Then simultaneous overexpression of both tgt/rsx in the WT strain should have either no effect on TOB resistance or increased resistance, relative to the WT. Perhaps the authors have done this, and if so, the data should be included as it will significantly strengthen their model.

Figure S4 - Is there a rationale for why it is possible to make rsx mutants in E. coli, but not in V. cholerae? For example, does E. coli have a second gene/protein that is redundant in function to rsxA, while V. cholerae does not? I think your data hint at this, since in the right panel growth data, your double mutant does not fully rescue back to rsx single mutant levels, suggesting another factor in tgt mutant also acts to lower resistance to TOB. If so, perhaps a line or two in text will be helpful for readers.

-For growth curves in Figure 2 and relative comparisons like in Figure 5D and Figure S4 (and others in the paper), statistics and error bars, along with replicate information should be provided.

-Figure 6A - Is the transcript fold change in linear or log? If linear, then tgt expression should not be classified as being upregulated in TOB. It is barely up by ~2-fold with TOB- 0.6....which is a mild phenotype, at best.

-Line 779- 780: "This indicates that sub-MIC TOB possibly induces tgt expression through the stringent response activation." To me, the data presented in this figure, do not support this statement. The experiment is indirect.

-Figure 3B and D. - These samples only have tobramycin, correct? The legend says both carbenicillin and tobramycin.

-Figure 5. The color schemes in bars do not match up with the color scheme in cartoons below panels B and C. That makes it confusing to read. Please fix.

-A lot of abbreviations have been used. This makes reading a bit cumbersome. Ideally, less abbreviations will be used.

Reviewer #2 (Public Review):

Fruchard et al. investigate the role of the queuosine (Q) modification of the tRNA (Q-tRNA) in the human pathogen Vibrio cholerae. First, the authors state that the absence of Q-modified tRNAs (tgt mutant) increases the translation of TAT codons and proteins with a high TAT codon bias. Second, the absence of Q increases rsxA translation, because rsxA gene has a high TAT codon bias. Third, increased RsxA in the absence of Q inhibits SoxR response, reducing resistance towards the antibiotic tobramycin (TOB). Authors also predict in silico which genes harbor a higher TAT bias and found that among them are some involved in DNA repair, experimentally observing that a tgt mutant is more resistant to UV than the wt strain. It is worth noting that authors employ a wide variety of techniques, both experimental and bioinformatic. However, some aspects of the work need to be clarified or reevaluated.

(1) The statement that the absence of Q increases the translation of TAT codons and proteins encoded by TAT-enriched genes presents the following problems that should be addressed:

(1.1) The increase in TAT codon translation in the absence of Q is not supported by proteomics, since there was no detected statistical difference for TAT codon usage in proteins differentially expressed. Furthermore, there are some problems regarding the statistics of proteomics. Some proteins shown in Table S1 have adjusted p-values higher than their p-values, which makes no sense. Maybe there is a mistake in the adjusted p-value calculation. In addition, it is not common to assume that proteins that are quantitatively present in one condition and absent in another are differentially abundant proteins. Proteomics data software typically addresses this issue and applies some corrections. It would be advisable to review that.

(1.2) Problems with the interpretation of Ribo-seq data (Figure 4D). On the one hand, the Ribo-seq data should be corrected (normalized) with the RNA-seq data in each of the conditions to obtain ribosome profiling data, since some genes could have more transcription in some of the conditions studied. In other articles in which this technique is used (such as in Tuorto et al., EMBO J. 2018; doi: 10.15252/embj.201899777), it is interpreted that those positions in which the ribosome moves most slowly and therefore less efficiently translated), are the most abundant. Assuming this interpretation, according to the hypothesis proposed in this work, the fragments enriched in TAT codons should have been less abundant in the absence of Q-tRNA (tgt mutant) in the Rib-seq experiment. However, what is observed is that TAT-enriched fragments are more abundant in the tgt mutant, and yet the Ribo-seq results are interpreted as RNA-seq, stating that this is because the genes corresponding to those sequences have greater expression in the absence of Q. On the other hand, it would be interesting to calculate the mean of the protein levels encoded by the transcripts with high and low ribosome profiling data.

(1.3) This statement is contrary to most previously reported studies on this topic in eukaryotes and bacteria, in which ribosome profiling experiments, among others, indicate that translation of TAT codons is slower (or unaffected) than translation of the TAC codons, and the same phenomenon is observed for the rest of the NAC/T codons. This is completely opposed to the results showed in Figure 4. However, the results of these studies are either not mentioned or not discussed in this work. Some examples of articles that should be discussed in this work:<br /> - "Queuosine-modified tRNAs confer nutritional control of protein translation" (Tuorto et al., 2018; 10.15252/embj.201899777)<br /> - "Preferential import of queuosine-modified tRNAs into Trypanosoma brucei mitochondrion is critical for organellar protein synthesis" (Kulkarni et al., 2021; doi:10.1093/nar/gkab567.<br /> - "Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed" (Cirzi et al., 2023; 10.15252/embj.2022112507)<br /> - "Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth" (Zhao et al., 2023; 10.1016/j.cell.2023.10.026)<br /> - "tRNA queuosine modification is involved in biofilm formation and virulence in bacteria" (Diaz-Rullo and Gonzalez-Pastor, 2023; doi: 10.1093/nar/gkad667). In this work, the authors indicate that Q-tRNA increases NAT codon translation in most bacterial species. Could the regulation of TAT codon-enriched proteins by Q-tRNAs in V. cholerae an exception? In addition, authors use a bioinformatic method to identify genes enriched in NAT codons similar to the one used in this work, and to find in which biological process are involved the genes whose expression is affected by Q-tRNAs (as discussed for the phenotype of UV resistance). It will be worth discussing all of this.

(1.4) It is proposed that the stress produced by the TOB antibiotic causes greater translation of genes enriched in TAT codons. On the one hand, it is shown that the GFP-TAT version (gene enriched in TAT codons) and the RsxA-TAT-GFP protein (native gene naturally enriched in TAT) are expressed more, compared to their versions enriched in TAC in a tgt mutant than in a wt, in the presence of TBO (Fig. 5C). However, in the absence of TOB, and in a wt context, although the two versions of GFP have a similar expression level (Fig. 3SD), the same does not occur with RsxA, whose RsxA-TAT form (the native one) is expressed significantly more than the RsxA-TAC version (Fig. 3SA). How can it be explained that in a wt context, in which there are also tRNA Q-modification, a gene naturally enriched in TAT is translated better than the same gene enriched in TAC? It would be expected that in the presence of Q-tRNAs the two versions would be translated equally (as happens with GFP) or even the TAT version would be less translated. On the other hand, in the presence of TOB the fluorescence of WT GFP(TAT) is higher than the fluorescence of WT GFP(TAC) (Figure S3E) (mean fluorescence data for RsxA-GFP version in the presence of TOB is not shown). These results may indicate that the apparent better translation of TAT versions could be due to indirect effects rather from TAT codon translation.

(2) Another problem is related to the already known role of Q in prevention of stop codon readthrough, which is not discuss at all in the work. In the absence of Q, stop codon readthrough is increased. In addition, it is known that aminoglycosides (such as tobramycin) also increase stop codon readthrough ("Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides"; Wanger and Green, 2023; 10.7554/eLife.52611). Absence of Q and presence of aminoglycosides can be synergic, producing devastating increases in stop codon readthrough and a large alteration of global gene expression. All of these needs to be discussed in the work. Moreover, it is known that stop codon readthrough can alter gene expression and mRNA sequence context all influence the likelihood of stop codon readthrough. Thus, this process could also affect to the expression of recoded GFP and RsxA versions.

(3) The statement about that the TOB resistance depends on RsxA translation, which is related to the presence of Q, also presents some problems:

(3.1) It is observed that the absence of tgt produces a growth defect in V. cholerae when exposed to TOB (Figure 1A), and it is stated that this is mediated by an increase in the translation of RsxA, because its gene is TAT enriched. However, in Figure S4F, it is shown that the same phenotype is observed in E. coli, but its rsxA gene is not enriched in TAT codons. Therefore, the growth defect observed in the tgt mutant in the presence of TOB may not be due to the increase in the translation of TAT codons of the rsxA gene in the absence of Q. This phenotype is very interesting, but it may be related to another molecular process regulated by Q. Maybe the role of Q in preventing stop codon readthrough is important in this process, reducing cellular stress in the presence of TOB and growing better.

(3.2) All experiments related to the effect of Q on the translation of TAT codons have been performed with the tgt mutant strain. Considering that the authors have a pSEVA-tgt plasmid to overexpress this gene, they would have to show whether tgt overexpression in a wt strain produces a decrease in the translation of proteins encoded by TAT-enriched genes such as RsxA. This experiment would allow them to conclude that Q reduces RsxA levels, increasing resistance to TOB.

(3.3) On the other hand, Fig. 1B shows that when the wt and tgt strains compete, both overexpressing tgt, the tgt mutant strain grows better in the presence of TOB. This result is not very well understood, since according to the hypothesis proposed, the absence of modification by Q of the tRNA would increase the translation of genes enriched in TAT, therefore, a strain with a higher proportion of Q-modified tRNAs as in the case of the wt strain overexpressing tgt would express the rsxA gene less than the tgt strain overexpressing tgt and would therefore grow better in the presence of TOB. For all these reasons, it would be necessary to evaluate the effect of tgt overexpression on the translation of RsxA.

(3.4) According to Figure 1I, the overexpression of tRNA-Tyr(GUA) caused a better growth of tgt mutant in comparison to WT. If the growth defect observed in tgt mutant in the presence of TOB is due to a better translation of the TAT codons of rsxA gene, the overexpression of tRNA-Tyr(GUA) in the tgt mutant should have resulted in even better RsxA translation a worse growth, but not the opposite result.

(4) It cannot be stated that DNA repair is more efficient in the tgt mutant of V. cholerae, as indicated in the text of the article and in Fig 7. The authors only observe that the tgt mutant is more resistant to UV radiation and it is suggested that the reason may be TAT bias of DNA repair genes. To validate the hypothesis that UV resistance is increased because DNA repair genes are TAT biased, it would be necessary to check if DNA repair is affected by Q. UV not only produces DNA damage, but also oxidative stress. Therefore, maybe this phenotype is due to the increase in proteins related to oxidative stress controlled by RsxA, such as the superoxide dismutase encoded by sodA. It is also stated that these repair genes were found up for the tgt mutant in the Ribo-seq data, with unchanged transcription levels. Again, it is necessary to clarify this interpretation of the Ribo-seq data, since the fact that they are more represented in a tgt mutant perhaps means that translation is slower in those transcripts. Has it been observed in proteomics (wt vs tgt in the absence of TOB) whether these proteins involved in repair are more expressed in a tgt mutant?

(5) The authors demonstrate that in E. coli the tgt mutant does not show greater resistance to UV radiation (Fig. 7D), unlike what happens in V. cholerae. It should be discussed that in previous works it has been observed that overexpression in E. coli of the tgt gene or the queF gene (Q biosynthesis) is involved in greater resistance to UV radiation (Morgante et al., Environ Microbiol, 2015 doi: 10.1111/1462-2920.12505; and Díaz-Rullo et al., Front Microbiol. 2021 doi: 10.3389/fmicb.2021.723874). As an explanation, it was proposed (Diaz-Rullo and Gonzalez-Pastor, NAR 2023 doi: 10.1093/nar/gkad667) that the observed increase in the capacity to form biofilms in strains that overexpress genes related to Q modification of tRNA would be related to this greater resistance to UV radiation.

Reviewer #3 (Public Review):

Summary:

In this manuscript the authors begin with the interesting phenotype of sub-inhibitory concentrations of the aminoglycoside tobramycin proving toxic to a knockout of the tRNA-guanine transglycosylase (Tgt) of the important human pathogen, Vibrio cholerae. Tgt is important for incorporating queuosine (Q) in place of guanosine at the wobble position of GUN codons. The authors go on to define a mechanism of action where environmental stressors control expression of tgt to control translational decoding of particularly tyrosine codons, skewing the balance from TAC towards TAT decoding in the absence of the enzyme. The authors use advanced proteomics and ribosome profiling to reveal that the loss of tgt results in increased translation of proteins like RsxA and a cohort of DNA repair factors, whose genes harbor an excess of TAT codons in many cases. These findings are bolstered by a series of molecular reporters, mass spectrometry, and tRNA overexpression strains to provide support for a model where Tgt serves as a molecular pivot point to reprogram translational output in response to stress.

Strengths:

The manuscript has many strengths. The authors use a variety of strains, assays, and advanced techniques to discover a mechanism of action for Tgt in mediating tolerance to sub-inhibitory concentrations of tobramycin. They observe a clear phenotype for a tRNA modification in facilitating reprogramming of the translational response, and the manuscript certainly has value in defining how microbes tolerate antibiotics.

Weaknesses:

The conclusions of the manuscript are mostly very well-supported by the data, but in some places control experiments or peripheral findings cloud precise conclusions. Some additional clarification, discussion, or even experimental extension could be useful in strengthening these areas.

(1) The authors have created and used a variety of relevant molecular tools. In some cases, using these tools in additional assays as controls would be helpful. For example, testing for compensation of the observed phenotypes by overexpression of the Tyrosine tRNA(GUA) in Figure 2A with the 6xTAT strain, Figure 5C with the rxsA-GFP fusion, and/or Figure 7B with UV stress would provide additional information of the ability of tRNA overexpression to compensate for the defect in these situations.<br /> (2) The authors present a clear story with a reprogramming towards TAT codons in the knockout strain, particularly regarding tobramycin treatment. The control experiments often hint at other codons also contributing to the observed phenotypes (e.g., His or Asp), yet these effects are mostly ignored in the discussion. It would be helpful to discuss these findings at a minimum in the discussion section, or possibly experimentally address the role of His or Asp by overexpression of these tRNAs together with Tyrosine tRNA(GUA) in an experiment like that of Figure 1I to see if a more "wild type" phenotype would present. In fact, the synergy of Tyr, His, and/or Asp codons likely helps to explain the effects observed with the DNA repair genes in later experiments.<br /> (3) Regarding Figure 6D, the APB northern blot feels like an afterthought. It was loaded with different amounts of RNA as input and some samples are repeated three times, but Δcrp only once. Collectively, it makes this experiment very difficult to assess.

Minor Points:<br /> (4) Fig S2B, do the authors have a hypothesis why the Asp and Phe tRNAs lead to a growth decrease in the untreated samples? It appears like Phe(GAA) partially compensates for the defect.<br /> (5) Lines 655 to 660 seem more appropriate as speculation in the discussion rather than as a conclusion in the results, where no direct experiments are performed. The authors might take advantage of the "Ideas and Speculation" section that eLife allows.

Reviewer #1 (Public Review):

Summary:

In this paper, the researchers aimed to address whether bees causally understand string-pulling through a series of experiments. I first briefly summarize what they did:

- In experiment 1, the researchers trained bees without string and then presented them with flowers in the test phase that either had connected or disconnected strings, to determine what their preference was without any training. Bees did not show any preference.

- In experiment 2, bees were trained to have experience with string and then tested on their choice between connected vs. disconnected string.

- experiment 3 was similar except that instead of having one option which was an attached string broken in the middle, the string was completely disconnected from the flower.

- In experiment 4, bees were trained on green strings and tested on white strings to determine if they generalize across color.

- In experiment 5, bees were trained on blue strings and tested on white strings.

- In experiment 6, bees were trained where black tape covered the area between the string and the flower (i.e. so they would not be able to see/ learn whether it was connected or disconnected).

- In experiments 2-6, bees chose the connected string in the test phase.

- In experiment 7, bees were trained as in experiment 3 and then tested where the string was either disconnected or coiled i.e. still being 'functional' but appearing different.

- In experiment 8, bees were trained as before and then tested on a string that was in a different coiled orientation, either connected or disconnected.

- In experiments 7 and 8 the bees showed no preference.

Strengths:

I appreciate the amount of work that has gone into this study and think it contains a nice, thorough set of experiments. I enjoyed reading the paper and felt that overall it was well-written and clear. I think experiment 1 shows that bees do not have an untrained understanding of the function of the string in this context. The rest of the experiments indicate that with training, bees have a preference for unbroken over broken string and likely use visual cues learned during training to make this choice. They also show that as in other contexts, bees readily generalize across different colors.

Weaknesses:

(1) I think there are 2 key pieces of information that can be taken from the test phase - the bees' first choice and then their behavior across the whole test. I think the first choice is critical in terms of what the bee has learned from the training phase - then their behavior from this point is informed by the feedback they obtain during the test phase. I think both pieces of information are worth considering, but their behavior across the entire test phase is giving different information than their first choice, and this distinction could be made more explicit.

In addition, while the bees' first choice is reported, no statistics are presented for their preferences.

(2) It seemed to me that the bees might not only be using visual feedback but also motor feedback. This would not explain their behavior in the first test choice, but could explain some of their subsequent behavior. For example, bees might learn during training that there is some friction/weight associated with pulling the string, but in cases where the string is separated from the flower, this would presumably feel different to the bee in terms of the physical feedback it is receiving. I'd be interested to see some of these test videos (perhaps these could be shared as supplementary material, in addition to the training videos already uploaded), to see what the bees' behavior looks like after they attempt to pull a disconnected string.

(3) I think the statistics section needs to be made clearer (more in private comments).

(4) I think the paper would be made stronger by considering the natural context in which the bee performs this behavior. Bees manipulate flowers in all kinds of contexts and scrabble with their legs to achieve nectar rewards. Rather than thinking that it is pulling a string, my guess would be that the bee learns that a particular motor pattern within their usual foraging repertoire (scrabbling with legs), leads to a reward. I don't think this makes the behavior any less interesting - in fact, I think considering the behavior through an ecological lens can help make better sense of it.

Reviewer #2 (Public Review):

Summary:

The authors wanted to see if bumblebees could succeed in the string-pulling paradigm with broken strings. They found that bumblebees can learn to pull strings and that they have a preference to pull on intact strings vs broken ones. The authors conclude that bumblebees use image matching to complete the string-pulling task.

Strengths:

The study has an excellent experimental design and contributes to our understanding of what information bumblebees use to solve a string-pulling task.

Weaknesses:

Overall, I think the manuscript is good, but it is missing some context. Why do bumblebees rely on image matching rather than causal reasoning? Could it have something to do with their ecology? And how is the task relevant for bumblebees in the wild? Does the test translate to any real-life situations? Is pulling a natural behaviour that bees do? Does image matching have adaptive significance?

Reviewer #3 (Public Review):

Summary:

This paper presents bees with varying levels of experience with a choice task where bees have to choose to pull either a connected or unconnected string, each attached to a yellow flower containing sugar water. Bees without experience of string pulling did not choose the connected string above chance (experiment 1), but with experience of horizontal string pulling (as in the right-hand panel of Figure 4) bees did choose the connected string above chance (experiments 2-3), even when the string colour changed between training and test (experiments 4-5). Bees that were not provided with perceptual-motor feedback (i.e they could not observe that each pull of the string moved the flower) during training still learned to string pull and then chose the connected string option above chance (experiment 6). Bees with normal experience of string pulling then failed to discriminate between connected and unconnected strings when the strings were coiled or looped, rather than presented straight (experiments 7-8).

Weaknesses:

The authors have only provided video of some of the conditions where the bees succeeded. In general, I think a video explaining each condition and then showing a clip of a typical performance would make it much easier to follow the study designs for scholars. Videos of the conditions bees failed at would be highly useful in order to compare different hypotheses for how the bees are solving this problem. I also think it is highly important to code the videos for switching behaviours. When solving the connected vs unconnected string tasks, when bees were observed pulling the unconnected string, did they quickly switch to the other string? Or did they continue to pull the wrong string? This would help discriminate the use of perceptual-motor feedback from other hypotheses.

The experiments are also not described well, for my below comments I have assumed that different groups of bees were tested for experiments 1-8, and that experiment 6 was run as described in line 331, where bees were given string-pulling training without perceptual feedback rather than how it is described in Figure 4B, which describes bees as receiving string pulling training with feedback.

The authors suggest the bees' performance is best explained by what they term 'image matching'. However, experiment 6 does not seem to support this without assuming retroactive image matching after the problem is solved. The logic of experiment 6 is described as "This was to ensure that the bees could not see the familiar "lollipop shape" while pulling strings....If the bees prefer to pull the connected strings, this would indicate that bees memorize the arrangement of strings-connected flowers in this task." I disagree with this second sentence, removing perceptual feedback during training would prevent bees memorising the lollipop shape, because, while solving the task, they don't actually see a string connected to a yellow flower, due to the black barrier. At the end of the task, the string is now behind the bee, so unless the bee is turning around and encoding this object retrospectively as the image to match, it seems hard to imagine how the bee learns the lollipop shape.

Despite this, the authors go on to describe image matching as one of their main findings. For this claim, I would suggest the authors run another experiment, identical to experiment 6 but with a black panel behind the bee, such that the string the bee pulls behind itself disappears from view. There is now no image to match at any point from the bee's perspective so it should now fail the connectivity task.

Strengths:

Despite these issues, this is a fascinating dataset. Experiments 1 and 2 show that the bees are not learning to discriminate between connected and unconnected stimuli rapidly in the first trials of the test. Instead, it is clear that experience in string pulling is needed to discriminate between connected and unconnected strings. What aspect of this experience is important? Experiment 6 suggests it is not image matching (when no image is provided during problem-solving, but only afterward, bees still attend to string connectivity) and casts doubt on perceptual-motor feedback (unless from the bee's perspective, they do actually get feedback that pulling the string moves the flower, video is needed here). Experiments 7 and 8 rule out means-end understanding because if the bees are capable of imagining the effect of their actions on the string and then planning out their actions (as hypotheses such as insight, means-end understanding and string connectivity suggest), they should solve these tasks.

If the authors can compare the bees' performance in a more detailed way to other species, and run the experiment suggested, this will be a highly exciting paper

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.

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. While the analysis is informative and could be useful for the community, some interpretations appear superficial and data must be completed to confirm identities and properties. Notably, supplementary information should be provided to show quality control data corresponding to the final cell atlas including the UMAP showing the sample source of the cells, violin plots of gene count, UMI count, and mitochondrial fraction for the overall dataset and by cluster, and expression profiles on UMAP of selected markers characterizing cluster identities.

The second set of data aims to correlate the scRNA-seq analysis with in situ hybridizations (ISH) in two different pipefish (gulf and bay) species to identify and characterize markers spatially, and validate cell types and signaling pathways active in them. While the approach is rational, the authors must complete the data and optimize labeling protocols to support their statements. One major concern is the quality of ISH stainings and images; embryos show a high degree of pigmentation that could hide part of the expression profile, and only subparts and hardly detectable tissues/stainings are presented. The authors should provide clear and good-quality images of ISH labeling on whole-mount specimens, highlighting the magnification regions and all other organs/structures (positive controls) expressing the marker of interest along the axis. Moreover, ISH probes have been designed and produced on gulf pipefish genome and cDNA respectively, while ISH labeling has been performed indifferently on bay or gulf pipefish embryos and larvae. The authors should specify stages and species on figure panels and should ensure sequence alignment of the probe-targeted sequences in the two species to validate ISH stainings in the bay pipefish. Moreover, spatiotemporal gene expression being a very dynamic process during embryogenesis, interpretations based on undefined embryonic and larval stages of pipefish development and compared to 3dpf zebrafish are insufficient to hypothesize on developmental specificities of pipefish features, such as on the absence of tooth primordia that could represent a very discrete and transient cell population. The ISH analyses would require a clean and precise spatiotemporal expression comparison of markers at the level of the entire pipefish and zebrafish specimens at well-defined stages, otherwise, the arguments proposed on teleost innovations and adaptations turn out to be very speculative.

To conclude, whereas the scRNA-seq dataset in this unconventional model organism will be useful for the community, the spatiotemporal and comparative expression analyses have to be thoroughly pushed forward to support the claims. Addressing these points is absolutely necessary to validate the data and to give new insights 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: The 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 an organ in this way. For instance, dental glia will cluster with other glia, and dental mesenchyme will likely cluster with other mesenchymal cell types. So the histology and ISH is 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.

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.

Weaknesses:

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:

Given the cost of producing action potentials and transmitting them along axons, it has always seemed a bit strange that there are synaptic failures: when a spike arrives at a synapse, about half the time nothing happens. This paper proposes a perfectly reasonable explanation: reducing failures (or, more generally, reducing noise) is costly. Four possible mechanisms are proposed, each associated with a different cost, with costs of the form 1/sigma_i^rho where sigma_i is the failure-induced variability at synapse i and rho is an exponent. The four different mechanisms produce four different values of rho.

What is interesting about the study is that the model makes experimental predictions about the relationship between learning rate, variability and presynaptic firing rate. Those predictions are consistent with experimental data, making it a strong candidate model. The fact that the predictions come from reasonable biological mechanisms make it a very strong candidate model and suggest several experiments to test it further.

Interestingly, the predictions made by this model are nearly indistinguishable from the predictions made by a normative model (Synaptic plasticity as Bayesian inference. Aitchison it al., Nature Neurosci. 24:565-571 (2021). As pointed out by the authors, working out whether the brain is using Bayesian inference to tune learning rules, or it just looks like it's Bayesian inference but the root cause is cost minimization, will be an interesting avenue for future research.

Finally, the authors relate their cost of reliability to the cost used in variational Bayesian inference. Intriguingly, the biophysical cost provides an upper bound on the variational cost. This is intellectually satisfying, as it answers a "why" question: why would evolution evolve to produce the kind of costs seen in the brain?

Strengths:

This paper provides a strong mix of theoretical analysis, simulations and comparison to experiments. And the extended appendices, which are very easy to read, provide additional mathematical insight.

Weaknesses:

None.

Reviewer #2 (Public Review):

Summary

This manuscript argues about the similarity between two frameworks describing synaptic plasticity. In the Bayesian inference perspective, due to the noise and the limited available pre- and postsynaptic information, synapses can only have an estimate of what should be their weight. The belief about those weights is described by their mean and variance. In the energy efficient perspective, synaptic parameters (individual means and variances) are adapted such that the neural network achieves some task while penalizing large mean weights as well as small weight variances. Interestingly, the authors show both numerically and analytically the strong link between those two frameworks. In particular, both frameworks predict that (a) synaptic variances should decrease when the input firing rate increases and (b) that the learning rate should increase when the weight variances increase. Both predictions have some experimental support.

Strengths

(1) Overall, the paper is very well written and the arguments are clearly presented.

(2) The tight link between the Bayesian inference perspective and the energy efficiency perspective is elegant and well supported, both with numerical simulations as well as with analytical arguments.

(3) I also particularly appreciate the derivation of the reliability cost terms as a function of the different biophysical mechanisms (calcium efflux, vesicle membrane, actin and trafficking). Independently of the proposed mapping between the Bayesian inference perspective and the energy efficiency perspective, those reliability costs (expressed as power-law relationships) will be important for further studies on synaptic energetics.

Weaknesses

(1) As recognised by the authors, the correspondence between the entropy term in the variational inference description and the reliability cost in the energetic description is strong, but not perfect. Indeed, the entropy term scales as -log(sigma) while reliability cost scales as sigma^(-rho).

(2) Even though this is not the main point of the paper, I appreciate the effort made by the authors to look for experimental data that could in principle validate the Bayesian/energetic frameworks. A stronger validation will be an interesting avenue for future research.

Reviewer #1 (Public Review):

This study conducted a series of experiments to comprehensively support the allocentric rather than egocentric visual spatial reference updating for the path-integration mechanism in the control of target-oriented locomotion. Authors firstly manipulated the waiting time before walking to tease apart the influence from spatial working memory in guiding locomotion. They demonstrated that the intrinsic bias in perceiving distance remained constant during walking and that the establishment of a new spatial layout in the brain took a relatively longer time beyond the visual-spatial working memory. In the following experiments, the authors then uncovered that the strength of the intrinsic bias in distance perception along the horizontal direction is reduced when participants' attention is distracted, implying that world-centered path integration requires attentional effort. This study also revealed horizontal-vertical asymmetry in a spatial coding scheme that bears a resemblance to the locomotion control in other animal species such as desert ants.

The revised version of the study effectively situates the research within the broader context of terrestrial navigation, focusing on the movement of land-based creatures and offers a clearer explanation for the potential neurological basis of the human brain's allocentric odometer. Previous feedback has been thoroughly considered, and additional details have been incorporated into the presentation of the results.

Reviewer #3 (Public Review):

This study investigated what kind of reference (allocentric or egocentric) frame we used for perception in darkness. This question is essential and was not addressed much before. The authors compared the perception in the walking condition with that in the stationary condition, which successfully separated the contribution of self-movement to the spatial representation. In addition, the authors also carefully manipulated the contribution of the waiting period, attentional load, vestibular input, testing task, and walking direction (forward or backward) to examine the nature of the reference frame in darkness systematically.

I am a bit confused by Figure 2b. Allocentric coordinate refers to the representation of the distance and direction of an object relative to other objects but not relative to the observer. In Figure 2, however, the authors assumed that the perceived target was located on the interception between the intrinsic bias curve and the viewing line from the NEW eye position to the target. This suggests that the perceived object depends on the observer's new location, which seems odd with the allocentric coordinate hypothesis.

According to Fig 2b, the perceived size should be left-shifted and lifted up in the walking condition compared to that in the stationary condition. However, in Figure 3C and Fig 4, the perceived size was the same height as that in the baseline condition.

Is the left-shifted perceived distance possibly reflecting a kind of compensation mechanism? Participants could not see the target's location but knew they had moved forward. Therefore, their brain automatically compensates for this self-movement when judging the location of a target. This would perfectly predict the left-shifted but not upward-shifted data in Fig 3C. A similar compensation mechanism exists for size constancy in which we tend to compensate for distance in computing object size.

According to Fig 2a, the target, perceived target, and eye should be aligned in one straight line. This means that connecting the physical targets and the corresponding perceived target results in straight lines that converge at the eye position. This seems, however, unlikely in Figure 3c.

Reviewer #2 (Public Review):

Summary:

The goal of this study is to clarify how the brain simultaneously represents item-specific temporal information and item-independent boundary information. The authors report spectral EEG data from intracranial patients performing a delayed free recall task. They perform cosine similarity analyses on principal components derived from gamma band power across stimulus duration. The authors find that similarity between items in serial position 1 (SP1) and all other within-list items decreases as a function of serial position, consistent with temporal context models. The authors find that across-list item similarity to SP1 is greatest for SP1 items relative to items from other serial positions, an effect that is greater in medial parietal lobe compared to lateral temporal cortex and hippocampus. The authors conclude that their findings suggest that perceptual boundary information is represented in medial parietal lobe. Despite a robust dataset, the methodological limitations of the study design prevent strong interpretations from being made from these data. The same-serial position across-list similarity may be driven by attentional mechanisms that are distinct from boundary information.

Strengths:

(1) The motivation of the study is strong as how both temporal contextual drift and event boundaries contribute to memory mechanisms is an important open question.

(2) The dataset of spectral EEG data from 99 intracranial patients provides the opportunity for precise spatiotemporal investigation of neural memory mechanisms.

Weaknesses:

The goal of reconciling temporal context and event boundary mechanisms is timely and would be of interest; however, an attentional account can still be used to explain the findings. This alternative account is not considered in the manuscript.

(1) The issue related to interpreting the SP1 similarity effects as reflecting boundary specific representations remains in the revised manuscript. The authors suggest that because cross-list SP1 similarity is found in recalled items that this supports the boundary interpretation. However, the effects could still be explained by variability in attention that is not specific to an event-boundary per se. As both subsequently recalled items and primacy items tend to recruit more gamma power than non-recalled and non-primacy items, recalled items will tend to have greater similarity with one another. It does not necessarily follow though that that this similarity is due to a "boundary representation."

(2) The authors partly addressed my concern regarding the comparison of recalled pairs. How did the authors account for the fact that the same participants do not contribute equally to all ROIs? If only participants who have electrodes in all ROIs are included, are the effects consistent?

Reviewer #1 (Public Review):

Summary:

This study applied pattern similarity analyses to intracranial EEG recordings to determine how neural drift is related to memory performance in a free recall task. The authors compared neural similarity within and across lists, in order to contrast signals related to contextual drift vs. the onset of event boundaries. They find that within-list neural differentiation in the lateral temporal cortex correlates with probability of word recall; in contrast, across-list pattern similarity in the medial parietal lobe correlates with recall for items near event boundaries (early-list serial positions). This primacy effect persists for the first three items of a list. Medial parietal similarity is also enhanced across lists for end-of-list items, however this effect then predicts forgetting. The authors do not find that within- or across-list pattern similarity in the hippocampus is related to recall probability.

Strengths:

The authors use a large dataset of human intracranial electrophysiological recordings, which gives them high statistical power to compare neural activity and memory across three important memory encoding regions. In so doing, the authors seek to address a timely and important question about the neural mechanisms that underlie the formation of memories for events.

The use of both within and across event pattern similarity analyses, combined with linear mixed effects modeling, is a marriage of techniques that is novel and translatable in principle to other types of data.

Weaknesses:

In several instances the paper does not address apparent inconsistencies between the prior literature and the findings. For example, the first main finding is that recalled items have more differentiated lateral temporal cortex representations within lists than not recalled items. This seems to be the opposite of the prediction from temporal context models that are used to motivate the paper-context models would predict that greater contextual similarity within a list should lead to greater memory through enhanced temporal clustering in recall. This is what El-Kalliny et al (2019) found, using a highly similar design (free recall, intracranial recordings from the lateral temporal lobe). The authors never address this contradiction in any depth in order to reconcile it with the previous literature and with the motivating theoretical model.

The way that the authors conduct the analysis of medial parietal neural similarity at boundaries leads to results that cannot be conclusively interpreted. The authors report enhanced similarity across lists for the first item in each list, which they interpret as reflecting a qualitatively distinct boundary signal. However, this finding can readily be explained by contextual drift if one assumes that whatever happens at the start of each list is similar or identical across lists (for example, a get ready prompt or reminder of instructions). In other words, this is analogous to presenting the same item at the start of every single list, in which case it is not surprising that the parietal (or any neural) representation would be similar to itself at the start of every list. So, a qualitatively unique boundary representation would not be necessary to explain this result. The authors do not include analyses to rule this out, which makes it difficult to interpret a key finding.

There is a similar absence of interpretation with respect to the previous literature for the data showing enhanced boundary-related similarity in the medial parietal cortex. The authors' interpretation seems to be that they have identified a boundary-specific signal that reflects a large and abrupt change in context, however another plausible interpretation is that enhanced similarity in the medial parietal cortex is related to a representation of a schema for the task structure that has been acquired across repeated instances.

The authors do not directly compare their model to other models that could explain how variability in neural activity predicts memory. One example is the neural fatigue hypothesis, which the authors mention, however there are no analyses or data to suggest that their data is better fit by a boundary/contextual drift mechanism as opposed to neural fatigue.

Reviewer #3 (Public Review):

Summary:

In this study, the authors analyzed data from 99 individuals with implanted electrodes who were performing a word-list recall task. Because the task involves successively encoding and then recalling 25 lists in a row, they were able to measure the similarity in neural responses for items within the same list as well as items across different lists, allowing them to test hypotheses about the impact of between-list boundaries on neural responses. They find that, in addition to slow drift in responses across items within a list and changes across lists, there is boundary-related structure in the medial parietal lobe such that early items in each list show similarity (for recalled items) and late items in each list show similarity (for not recalled items).

Strengths:

The dataset used in this paper is substantially larger than most iEEG datasets, allowing for the detection of nuanced differences between item positions and for analyses of individual differences in boundary-related responses. There are excellent visualizations of the similarity structure between items for each region, and this work connects to a growing literature on the role of event boundaries in structuring neural responses.

Weaknesses:

(1) The visualization in Fig 1B claims that the prediction of the temporal context model is that nearby items in the presented sequence should have similar representations; that is, nearby items within a list should be similar, and the end of a list should look similar to the beginning of the next list. First, it's unclear to me if this is exactly what TCM would predict for this dataset, since lists are separated by ~60 seconds of distractor and retrieval tasks, rather than simply by a brief event boundary. Second, the authors do not actually test this model of continuous similarity across lists. After examining smooth drift in the within-list analysis (Fig 2), the across-list analyses (Figs 3-5) use a model with a "list distance" regressor that predicts discrete changes between lists. The authors state that it is not possible to replace this list distance regressor with an item distance regressor (which would be a straight line in Fig 3D rather than stair-steps) because this would be too collinear with the boundary proximity regressor, but I do not understand why these regressors would be collinear at all (since the boundary proximity regressor does not systematically increase or decrease across items).

(2) There is no theoretical or quantitative justification for the specific forms of the boundary proximity models, For initial items, a model of e^(1-d) is used (with d being serial position), but it is not stated how the falloff scale of this model was selected (as opposed to e.g. e^((1-d)/2)). For final items, a different linear model of d/#items is used, which seems to have a somewhat different interpretation, since it changes at a constant rate across all items rather than only modeling items near the final boundary. Confusingly, the schematic in Fig 1B shows symmetric effects at initial and final boundaries, despite two different models being used and the authors' assertion in their response that they do not believe these processes are symmetric.

(3) It is unclear to me whether the authors believe that the observed similarity after boundaries is due to an active process in which "the medial parietal lobe uses drift-resets" to reinstate a boundary-related context, or that this similarity is simply because "the context for the first item may be the boundary itself", and therefore this effect would emerge naturally from a temporal context model that incorporates the full task structure as the "items."

Reviewer #3 (Public Review):

Summary:

The authors food-deprived male and female mice and observed a much stronger reduction of leptin levels, energy consumption in the visual cortex, and visual coding performance in males than females. This indicates a sex-specific strategy for the regulation of the energy budget in the face of low food availability.

Strengths:

This study extends a previous study demonstrating the effect of food deprivation on visual processing in males, by providing a set of clear experimental results, demonstrating the sex-specific difference. It also provides hypotheses about the strategy used by females to reduce energy budget based on the literature.

Weaknesses:

The authors do not provide evidence that females are not impacted by visually guided behaviors contrary to what was shown in males in the previous study.

Reviewer #1 (Public Review):

Padamsey et al. followed up on their previous study in which they found that male mice sacrifice visual cortex computation precision to save energy in periods of food restriction (Padamsey et al. 2021, Neuron). In the present study, the authors find that female mice show much lower levels of adaptation in response to food restriction on the level of metabolic signaling and visual cortex computation. This is an important finding for understanding sex differences in adaptation to food scarcity and also impacts the interpretation of studies employing food restriction in behavioral analyses and learning paradigms.

Strengths:

The manuscript is, in general, very clear and the conclusions are straightforward. The experiments are performed in the same conditions for males and females and the authors did not find differences in the behavioral states of male and female mice that could explain differences in energy consumption. Moreover, they show that visual cortex in both males and females does not change its baseline energy consumption in the dark, therefore the adjustment of energy budget in males only targets visual processing.

Weaknesses:

The number of experiments is insufficient to compare the effects of food restriction in males and females directly, which is discussed by the authors: to address this point they use Bayes factor analysis to provide an estimate of the likelihood that females and males indeed differ in terms of energy metabolism and sensory processing adaptions during food restriction.

Reviewer #2 (Public Review):

Summary:

Padamsey et al build up on previous significant work from the same group which demonstrated robust changes in the visual cortex in male mice from long-term (2-3 weeks) food restriction. Here, the authors extend this finding and reveal striking sex-specific differences in the way the brain responds to food restriction. The measures included the whole-body measure of serum leptin levels, and V1-specific measures of activity of key molecular players (AMPK and PPARα), gene expression patterns, ATP usage in V1, and the sharpness of visual stimulus encoding (orientation tuning). All measures supported the conclusion that the female mouse brain (unlike in males) does not change its energy usage and cortical functional properties on comparable food restriction.

While the effect of food restriction on more peripheral tissue such as muscle and bones has been well studied, this result contributes to our understanding of how the brain responds to food restriction. This result is particularly significant given that the brain consumes a large fraction of the body's energy consumption (20%), with the cortex accounting for half of that amount. The sex-specific differences found here are also relevant for studies using food restriction to investigate cortical function.

Strengths:

The study uses a wide range of approaches mentioned above which converge on the same conclusion, strengthening the core claim of the study.

Weaknesses:

Since the absence of a significant effect does not prove the absence of any changes, the study cannot claim that the female mouse brain does not change in response to food restriction. However, the authors do not make this claim. Instead, they make the well-supported claim that there is a sex-specific difference in the response of V1 to food restriction.

Reviewer #2 (Public Review):

Summary:

The study investigates whether speech and music processing involve specific or shared brain networks. Using intracranial EEG recordings from 18 epilepsy patients, it examines neural responses to speech and music. The authors found that most neural activity is shared between speech and music processing, without specific regional brain selectivity. Furthermore, domain-selective responses to speech or music are limited to frequency-specific coherent oscillations. The findings challenge the notion of anatomically distinct regions for different cognitive functions in the auditory process.

Strengths:

(1) This study uses a relatively large corpus of intracranial EEG data, which provides high spatiotemporal resolution neural recordings, allowing for more precise and dynamic analysis of brain responses. The use of continuous speech and music enhances ecological validity compared to artificial or segmented stimuli.

(2) This study uses multiple frequency bands in addition to just high-frequency activity (HFA), which has been the focus of many existing studies in the literature. This allows for a more comprehensive analysis of neural processing across the entire spectrum. The heterogeneity across different frequency bands also indicates that different frequency components of the neural activity may reflect different underlying neural computations.

(3) This study also adds empirical evidence towards distributed representation versus domain-specificity. It challenges the traditional view of highly specialized, anatomically distinct regions for different cognitive functions. Instead, the study suggests a more integrated and overlapping neural network for processing complex stimuli like speech and music.

Weaknesses:

While this study is overall convincing, there are still some weaknesses in the methods and analyses that limit the implication of the work.

The study's main approach, focusing primarily on the grand comparison of response amplitudes between speech and music, may overlook intricate details in neural coding. Speech and music are not entirely orthogonal with each other at different levels of analysis: at the high-level abstraction, these are two different categories of cognitive processes; at the low-level acoustics, they overlap a lot; at intermediate levels, they may also share similar features. For example, the study doesn't adequately address whether purely melodic elements in music correlate with intonations in speech at the neural level. A more granular analysis, dissecting stimuli into distinct features like pitch, phonetics, timbre, and linguistic elements, could unveil more nuanced shared, and unique neural processes between speech and music. Prior research indicates potential overlap in neural coding for certain intermediate features in speech and music (Sankaran et al. 2023), suggesting that a simple averaged response comparison might not fully capture the complexity of neural encoding. Further delineation of phonetic, melodic, linguistic, and other coding, along with an analysis of how different informational aspects (phonetic, linguistic, melodic, etc) are represented in shared neural activities, could enhance our understanding of these processes and strengthen the study's conclusions.

While classifying electrodes into 3 categories provides valuable insights, it may not fully capture the complexity of the neural response distribution to speech and music. A more nuanced and continuous approach could reveal subtler gradations in neural response, rather than imposing categorical boundaries. This could be done by computing continuous metrics, like unique variances explained by each category or by each acoustic feature, etc. Incorporating such a continuum could enhance our understanding of the neural representation of speech and music, providing a more detailed and comprehensive picture of cortical processing. This goes back to my first comment that the selected set of stimuli may not fully exploit the entire space of speech and music, and there are possible exemplars that violate the preference map here. For example, this study only considered a specific set of multi-instrumental music, it is not clear to me if other types of music would result in different response profiles in individual channels. It is also not clear if a foreign language that the listeners cannot comprehend would evoke similar response profiles. On the contrary, breaking down into the neural coding of more fundamental feature representations that constitute speech and music, and analyzing the unique contribution of each feature would give a more comprehensive understanding.

The paper's emphasis on shared and overlapping neural activity, as observed through sEEG electrodes, provides valuable insights. It is probably true that domain-specificity for speech and music does not exist at such a macro scale. However, it's important to consider that each electrode records from a large neuronal population, encompassing thousands of neurons. This broad recording scope might mask more granular, non-overlapping feature representations at the single neuron level. Thus, while the study suggests shared neural underpinnings for speech and music perception at a macroscopic level, it cannot definitively rule out the possibility of distinct, non-overlapping neural representations at the microscale of local neuronal circuits for features that are distinctly associated with speech and music. This distinction is crucial for fully understanding the neural mechanisms underlying speech and music perception that merit future endeavors with more advanced large-scale neuronal recordings.

Reviewer #1 (Public Review):

Summary:

In this study, the authors examined the extent to which processing of speech and music depends on neural networks that are either specific to a domain or general in nature. They conducted comprehensive intracranial EEG recordings on 18 epilepsy patients as they listened to natural, continuous forms of speech and music. This enabled an exploration of brain activity at both the frequency-specific and network levels across a broad spectrum. Utilizing statistical methods, the researchers classified neural responses to auditory stimuli into categories of shared, preferred, and domain-selective types. It was observed that a significant portion of both focal and network-level brain activity is commonly shared between the processing of speech and music. However, neural responses that are selectively responsive to speech or music are confined to distributed, frequency-specific areas. The authors highlight the crucial role of using natural auditory stimuli in research and the need to explore the extensive spectral characteristics inherent in the processing of speech and music.

Strengths:

The study's strengths include its high-quality sEEG data from a substantial number of patients, covering a majority of brain regions. This extensive cortical coverage grants the authors the ability to address their research questions with high spatial resolution, marking an advantage over previous studies. They performed thorough analyses across the entire cortical coverage and a wide frequency range of neural signals. The primary analyses, including spectral analysis, temporal response function calculation, and connectivity analysis, are presented straightforwardly. These analyses, as well as figures, innovatively display how neural responses, in each frequency band and region/electrode, are 'selective' (according to the authors' definition) to speech or music stimuli. The findings are summarized in a manner that efficiently communicates information to readers. This research offers valuable insights into the cortical selectivity of speech and music processing, making it a noteworthy reference for those interested in this field. Overall, this research offers a valuable dataset and carries out extensive yet clear analyses, amounting to an impressive empirical investigation into the cortical selectivity of speech and music. It is recommended for readers who are keen on understanding the nuances of selectivity and generality in the processing of speech and music to refer to this study's data and its summarized findings.

Weaknesses:

(1) The study employed longer speech and music stimuli, thereby promising improved ecological validity as compared to prior research, a point emphasized by the authors. However, it failed to differentiate between neural responses to the diverse content or local structures within speech and music. The authors considered the potential limitation of treating these extensive speech and music stimuli as stationary signals, neglecting their complex musical or linguistic structural details and temporal variations across local structures such as sentences and phrases. This balanced perspective offered by the authors aids readers in better understanding the context of the study and highlights potential areas for expansion and further considerations.

(2) In contrast to previous studies that employed short stimulus segments along with various control stimuli to ensure that observed selectivity for speech or music was not merely due to low-level acoustic properties, this study used longer, ecological stimuli. However, the control stimuli used in this study, such as tone or syllable sequences, do not align with the low-level acoustic properties of the speech and music stimuli. This mismatch raises concerns that the differences or selectivity between speech and music observed in this study might be attributable to these basic acoustic characteristics rather than to more complex processing factors specific to speech or music. However, this should not deter readers from recognizing the study's strengths, namely, the use of iEEG recordings that offer high spatial resolution and extensive cortical coverage.

(3) The concept of selectivity - shared, preferred, and domain-selective - may not present sufficient theoretical accuracy. It is appreciated that the authors put effort into clearly defining their operational measurement on 'selectivity'. Later, the authors further mentioned the specific indication of their analyses. However, the authors' categorization of neural sites/regions as shared, preferred, or domain-selective regarding speech and music processing essentially resembles a traditional ANOVA test with posthoc analysis. While this categorization gives meaningful context to the results, the mere presence of significant differences among control stimuli, a segment of speech, and a piece of music does not present a strong case that a region is specifically selective to a type of stimulus like speech. The narrative of the manuscript could potentially lead to an overgeneralized interpretation of their findings as being broadly applicable to speech or music, if a reader does not delve into the details.

(4) The authors' approach, akin to mapping a 'receptive field' by correlating stimulus properties with neural responses to ascertain functional selectivity for speech and music, presents potential issues. If cortical regions exhibit heightened responses to one type of stimulus over another, it doesn't automatically imply selectivity or preference for that stimulus. The explanation could lie in functional aspects, such as a region's sensitivity to temporal units of a specific duration, be it music, speech, or even movie segments, and its role in chunking such units (e.g., around 500 ms), which might be more prevalent in music than in speech, or vice versa in the current study. This study does not delve into the functional mechanisms of how speech and music are processed across different musical or linguistic hierarchical levels but merely demonstrates differences in neural responses to various stimuli over a 10-minute span.

Reviewer #3 (Public Review):

Summary:

Te Rietmolen et al., investigated the selectivity of cortical responses to speech and music stimuli using neurosurgical stereo EEG in humans. The authors address two basic questions: 1. Are speech and music responses localized in the brain or distributed; 2. Are these responses selective and domain specific or rather domain general and shared. To investigate this, the study proposes a nomenclature of shared responses (speech and music responses are not significantly different), domain selective (one domain is significant from baseline and the other is not), domain preferred (both are significant from baseline but one is larger than the other and significantly different from each other). The authors employ this framework using neural responses across the spectrum (rather than focusing on high gamma), providing evidence for a low level of selectivity across spectral signatures. To investigate the nature of the underlying representations they use encoding models to predict neural responses (low and high frequency) given a feature space of the stimulus envelope or peak rate (by time delay) and find stronger encoding for both in the low frequency neural responses. The top encoding electrodes are used as seeds for a pair-wise connectivity (coherence) in order to repeat the shared/selective/preferred analysis across the spectra, suggesting low selectivity. Spectral power and connectivity are also analyzed on the level of regional patient population to rule out (and depict) any effects driven by a select few patients. Across analyses the authors consistently show a paucity of domain selective responses and when evident these selective responses were not represented across the entire cortical region. The authors argue that speech and music mostly rely on shared neural resources.

Strengths:

I found this manuscript to be rigorous providing compelling and clear evidence towards shared neural signatures for speech and music. The use of intracranial recordings provides an important spatial and temporal resolution that lends itself to the power, connectivity and encoding analyses. The statistics and methods employed are rigorous and reliable, estimated based on permutation approaches and cross-validation/regularization was employed and reported properly. The analysis of measures across the entire spectra in both power, coherence and encoding models provides a comprehensive view of responses that no doubt will benefit the community as an invaluable resource. Analysis on the level of patient population (feasible with their high N) per region also supports the generalizability of the conclusions across a relatively large cohort of patients. Last but not least, I believe the framework of selective, preferred, and shared is a welcome lens through which to investigate cortical function.

Weaknesses:

I did not find methodological weaknesses in the current version of the manuscript. I do believe that it is important to highlight that the data is limited to passively listening to naturalistic speech and music. The speech and music stimuli are not completely controlled with varying key acoustic features (inherent to the different domains). Overall, I found the differences in stimulus and lack of attentional controls (passive listening) to be minor weaknesses that would not dramatically change the results or conclusions.

Reviewer #1 (Public Review):

Summary:

In this article, the authors investigate whether the connectivity of the hippocampus is altered in individuals with aphantasia ¬- people who have reduced mental imagery abilities and where some describe having no imagery, and others describe having vague and dim imagery. The study investigated this question using a fMRI paradigm, where 14 people with aphantasia and 14 controls were tested, and the researchers were particularly interested in the key regions of the hippocampus and the visual-perceptual cortices. Participants were interviewed using the Autobiographical Interview regarding their autobiographical memories (AMs), and internal and external details were scored. In addition, participants were queried on their perceived difficulty in recalling memories, imagining, and spatial navigation, and their confidence regarding autobiographical memories was also measured. Results showed that participants with aphantasia reported significantly fewer internal details (but not external details) compared to controls; that they had lower confidence in their AMs; and that they reported finding remembering and imagining in general more difficult than controls. Results from the fMRI section showed that people with aphantasia displayed decreased hippocampal and increased visual-perceptual cortex activation during AM retrieval compared to controls. In contrast, controls showed strong negative functional connectivity between hippocampus and the visual cortex. Moreover, resting state connectivity between the hippocampus and visual cortex predicted better visualisation skills. The authors conclude that their study provides evidence for the important role of visual imagery in detail-rich vivid AM, and that this function is supported by the connectivity between the hippocampus and visual cortex. This study extends previous findings of reduced episodic memory details in people with aphantasia, and enables us to start theorising about the neural underpinnings of this finding.

The data provided good support for the conclusion that the authors draw, namely that there is a 'tight link between visual imagery and our ability to retrieve vivid and detail-rich personal past events'. However, as the authors also point out, the exact nature of this relationship is difficult to infer from this study alone, as the slow temporal resolution of fMRI cannot establish the directionality between the hippocampus and the visual-perceptual cortex. This is an exciting future avenue to explore.

Strengths:

A great strength of this study is that it introduces a fMRI paradigm in addition to the autobiographical interview, paralleling work done on episodic memory in cognitive science (e.g. Addis and Schacter, 2007, https://doi.org/10.1016%2Fj.neuropsychologia.2006.10.016 ), which has examined episodic and semantic memory in relation to imagination (future simulation) in non-aphantasic participants as well as clinical populations. Future work could build on this study, and for example use the recombination paradigm (Addis et al. 2009, 10.1016/j.neuropsychologia.2008.10.026 ), which would shed further light on the ability of people with aphantasia to both remember and imagine events. Future work could also build on the interesting findings regarding spatial navigation, which together with previous findings in aphantasia (e.g. Bainbridge et al., 2021, https://doi.org/10.1016/j.cortex.2020.11.014 ) strongly suggests that spatial abilities in people with aphantasia are unaffected. This can shed further light on the different neural pathways of spatial and object memory in general. In general, this study opens up a multitude of new avenues to explore and is likely to have a great impact on the field of aphantasia research.

Weaknesses:

A weakness of the study is that some of the questions used are a bit vague, and no objective measure is used, which could have been more informative. For example, the spatial navigation question (reported as 'How difficult is it typically for you to orient you spatially?' could have been more nuanced to tap into whether participants relied mostly on cognitive maps (likely supported by the hippocampus) or landmarks. It would also have been interesting to conduct a spatial navigation task, as participants do not necessarily have insight to their spatial navigation abilities (they could have been overconfident or underconfident in their abilities). Secondly, the question 'how difficult is it typically for you to use your imagination?' could also be more nuanced, as imagination is used in a variety of ways, and we only have reason to hypothesise that people with aphantasia might have difficulties in some cases (i.e. sensory imagination involving perceptual details). It is unlikely that people with aphantasia would have more difficulty than controls to use their imagination to imagine counterfactual situations and engage in counterfactual thought (de Brigard et al., 2013, https://doi.org/10.1016%2Fj.neuropsychologia.2013.01.015) due to its non-sensory nature, but the question used does not distinguish between these types of imagination. Again, this is a ripe area for future research. The general phrasing of 'how difficult is [x]' could also potentially bias participants towards more negative answers, something which ought to be controlled for in future research.

Reviewer #2 (Public Review):

Summary:

This study investigates to what extent neural processing of autobiographical memory retrieval is altered in people who are unable to generate mental images ('aphantasia'). Self-report as well as objective measures were used to establish that the aphantasia group indeed had lower imagery vividness than the control group. The aphantasia group also reported fewer sensory and emotional details of autobiographical memories. In terms of brain activity, compared to controls, aphantasics had a reduction in activity in the hippocampus and an increase in the activity in visual cortex during autobiographical memory retrieval. For controls, these two regions were also functionally connected during autobiographical memory retrieval, which did not seem to be the case for aphantasics. Finally, resting-state connectivity between visual cortex and hippocampus was positively related to autobiographical vividness in the control group but negatively in the aphantasia group. The results are in line with the idea that aphantasia is caused by an increase in noise within the visual system combined with a decrease in top-down communication from the hippocampus.

Recent years have seen a lot of interest in the influence of aphantasia on other cognitive functions and one of the most consistent findings is deficits in autobiographical memory. This is one of the first studies to investigate the neural correlates underlying this difference, thereby substantially increasing our understanding of aphantasia and the relationship between mental imagery and autobiographical memory.

Strengths:

One of the major strengths of this study is the use of both self-report as well as objective measures to quantify imagery ability. Furthermore, the fMRI analyses are hypothesis-driven and reveal unambiguous results, with alterations in hippocampal and visual cortex processing seeming to underlie the deficits in autobiographical memory.

Weaknesses:

In terms of weaknesses, the control task, doing mathematical sums, also differs from the autobiographical memory task in aspects that are unrelated to imagery or memory, such as self-relevance and emotional salience, which makes it hard to conclude that the differences in activity are reflecting only the cognitive processes under investigation. However, given that the most important comparisons are between groups of participants, this does not diminish the main conclusions about aphantasia.

Overall, I believe that this is a timely and important contribution to the field and will inspire novel avenues for further investigation.

Reviewer #1 (Public Review):

Summary:

This is an experimentally soundly designed work and a very well-written manuscript. There is a very clear logic that drives the reader from one experiment to the next, the experimental design is clearly explained throughout and the relevance of the acquired data is well analyzed and supports the claims made by the authors. The authors made an evident effort to combine imaging, genetic, and molecular data to describe previously unknown early embryonic movement patterns and to identify regulatory mechanisms that control several aspects of it.

Strengths:

The authors develop a new method to analyze, quantitatively, the onset of movement during the latter embryonic stages of Drosophila development. This setup allows for a high throughput analysis of general movement dynamics based on the capture of variations of light intensity reflected by the embryo. This setup is capable of imaging several embryos simultaneously and provides a detailed measure of movement over time, which proves to be very useful for further discoveries in the manuscript. This setup already provides a thorough and quantifiable description of a process that is little known and identifies two different phases during late embryonic movements: a myogenic phase and a neurogenic phase, which they elegantly prove is dependent on neuronal activity by knocking down action potentials across the nervous system.

However, in this system, movement is detected as a whole, and no further description of the type of movement is provided beyond frequency and amplitude; it would be interesting to know from the authors if a more precise description of the movements that take place at this stage can be achieved with this method (e.g. motion patterns across the A-P body axis).

Importantly, this highly quantitative experimental setup is an excellent system for performing screenings of motion regulators during late embryonic development, and its use could be extended to search for different modulators of the process, beyond miRNAs (genetic mutants, drugs, etc.).

Using their newly established motion detection pipeline, the authors identify miR-2b-1 as required for proper larval and embryonic motion, and identify an overall reduction in the quantity of both myogenic and neurogenic movements, as well as an increased frequency in neurogenic movement "pulses".

Focusing on the neurogenic movement phenotype the authors use in situ probes and perform RT-PCR on FACS-sorted CNS cells to unambiguously detect miR-2b-1 expression in the embryonic nervous system. The neurogenic motion defects observed in miR-2b-1 mutant embryos and early larvae can be completely rescued by the expression of ectopic miR-2b-1 specifically in the nervous system, providing solid evidence of the requirement and sufficiency of miR-2b-1 expressed in the nervous system to regulate these phases of movement.

To explore the mechanism through which miR-2b-1 impacts embryonic movement, the authors use a state-of-the-art bioinformatic approach to identify potential targets of miR-2b-1, and find that the expression levels of an uncharacterized gene, CG3638, are indeed regulated by miR-2b-1. Furthermore, they prove that by knocking down the expression of CG3638 in a miR-2b-1 mutant background, the neurogenic embryonic movement defects are rescued, pointing that the repression of CG3638 by miR-2b-1 is necessary for correct motion patterns in wild-type embryos. Therefore, this paper provides the first functional characterization of CG3638, and names this gene Motor.

Finally, the authors aim to discriminate which elements of the embryonic motor system miR-2b-1/Motor are required. Using directed overexpression of miR-2b-1 and Motor knockdown in the motor neurons and the chordotonal (sensory) organs, they prove that the miR-2b-1/Motor regulatory axis is specifically required in the sensory organs to promote normal embryonic and larval movement.

Weaknesses:

The initial screening to identify miRNAs involved in motion behaviors is performed in early larval movement. The logic presented by the authors is clear - it is assumed that early larval movement cannot proceed normally in the absence of previous embryonic motion - and ultimately helped them identify a miRNA required for modulation of embryonic movement. However, it is possible that certain miRNAs play a role in the modulation of embryonic movement while being dispensable for early L1 behaviors. Such regulators might have been missed with the current screening setup.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript, "A microRNA that controls the emergence of embryonic movement" by Menzies, Chagas, and Alonso provides evidence that Drosophila miR-2b-1 is expressed in neurons and controls the expression of the predicted chloride channel CG3638, here named "Motor". Loss of the miRNA leads to movement phenotypes that can be rescued by downregulation of Motor; using specific drivers, the authors show that a larval movement phenotype (slower movement) can be rescued by knockdown of Motor in the chordotonal organs, suggesting that the increase in Motor found in the chordotonal organs is likely the root of the movement defects. Overall, I found the data presented in the manuscript of reasonable quality and are well enough supported by the presented data.

The genetic and phenotypic analysis seems to be correct. The nicest part of the manuscript is the connection between the loss of a miRNA and finding its likely target in generating a phenotype. The authors also develop some protocols for the analysis of the movement phenotypes which may be useful for others.

Reviewer #1 (Public Review):

Summary:

The authors in this manuscript performed scRNA-seq on a cohort of 15 early-stage cervical cancer patients with a mixture of adeno- and squamous cell carcinoma, HPV status, and several samples that were upstaged at the time of surgery. From their analyses they identified differential cell populations in both immune and tumour subsets related to stage, HPV status, and whether a sample was adenocarcinoma or squamous cell. Putative microenvironmental signaling was explored as a potential explanation for their differential cell populations. Through these analyses the authors also identified SLC26A3 as a potential biomarker for later stage/lymph node metastasis which was verified by IHC and IF. The dataset is likely useful for the community, however, the strong claims made are not adequately supported by the data and would require additional functional validation.

Strengths:

The dataset could be useful for the community.<br /> SLC26A3 could potentially be a useful marker to predict lymph node metastasis with further study.

Weaknesses:

The link between the background in the introduction and the actual study and findings is often tenuous or not clearly explained. A re-working of the intro to better set up and link to the study questions would be beneficial.

For the sequencing, which kit was used on the Novaseq6000?

Additional details are needed for the analysis pipeline. How were batch effects identified/dealt with, what were the precise functions and settings for each step of the analysis, how was clustering performed and how were clusters validated etc. Currently, all that is given is software and sometimes function names which are entirely inadequate to be able to assess the validity of the analysis pipeline. This could alternatively be answered by providing annotated copies of the scripts used for analysis as a supplement.

For Cell type annotation, please provide the complete list of "selected gene markers" that were used for annotation.

No statistics are given for the claims on cell proportion differences throughout the paper (for cell types early, epithelial sub-clusters later, and immune cell subsets further on). This should be a multivariate analysis to account for ADC/SCC, HPV+/- and Early/Late stage.

The Y-axis label is missing from the proportion histograms in Figure 2D. In these same panels, the bars change widths on the right side. If these are exclusively in ADC, show it with a 0 bar for SCC, not doubling the width which visually makes them appear more important by taking up more area on the plot.

Throughout the manuscript, informatic predictions (differentiation potential, malignancy score, stemness, and trajectory) are presented as though they're concrete facts rather than the predictions they are. Strong conclusions are drawn on the basis of these predictions which do not have adequate data to support. These conclusions which touch on essentially all of the major claims made in the manuscript would need functional data to validate, or the claims need to be very substantially softened as they lack concrete support. Indeed, the fact that most of the genes examined that were characteristic of a given cluster did not show the expected expression patterns in IHC highlights the fact that such predictions require validation to be able to draw proper inferences.

The cluster Epi_10_CYSTM1 which is the basis for much of the paper is present in a single individual (with a single cell coming from another person), and heavily unconnected from the rest of the epithelial populations. If so much emphasis is placed on it, the existence of this cluster as a true subset of cells requires validation.

Claims based on survival analysis of TCGA for Epi_10_CYSTM1 are based on a non-significant p-value, though there is a slight trend in that direction.

The claim "The identification of Epi_10_CYSTM1 as the only cell cluster found in patients with stage IIICp raises the possibility that this cluster may be a potential marker to diagnose patients with lymph node metastasis." This is incorrect according to the sample distributions which clearly show cells from the patient who has EPI_10_CYSTM1 in multiple other clusters. This is then used as justification for SLC26A3 which appears to be associated with associated with late stage, however, in the images SLC26A3 appears to be broadly expressed in later tumours rather than restricted to a minor subset as it should be if it were actually related to the EPI_10_CYSTM1 cluster.

The authors claim that cytotoxic T cells express KRT17, and KRT19. This likely represents a mis-clustering of epithelial cells.

Multiple claims are made for specific activities based on GO term biological process analysis which while not contradictory to the data, certainly are by no means the only explanation for it, nor directly supported.

Reviewer #2 (Public Review):

Summary:

Peng et al. present a study using scRNA-seq to examine phenotypic properties of cervical cancer, contrasting features of both adenocarcinomas (ADC) and squamous cell carcinoma (SCC), and HPV-positive and negative tumours. They propose several key findings: unique malignant phenotypes in ADC with elevated stemness and aggressive features, interactions of these populations with immune cells to promote an immunosuppressive TME, and SLC26A3 as a biomarker for metastatic (>=Stage III ) tumours.

Strengths:

This study provides a valuable resource of scRNA-seq data from a well-curated collection of patient samples. The analysis provides a high-level view of the cellular composition of cervical cancers. The authors introduce some mechanistic explanations of immunosuppression and the involvement of regulatory T cells that are intriguing.

Weaknesses:

I believe that many of the proposed conclusions are over-interpretations or unwarranted generalizations of the single-cell analysis. These conclusions are often based on populations in the scRNA-seq data that are described as enriched or specific to a given group of samples (eg. ADC). This conclusion is based on the percentage of cells in that population belonging to the given group; for example, a cluster of cells that dominantly come from ADC. The data includes multiple samples for each group, but statistical approaches are never used to demonstrate the reproducibility of these claims.

This leads to problematic conclusions. For example, the "ADC-specific" Epi_10_CYSTM1 cluster, which is a central focus of the paper, only contains cells from one of the 11 ADC samples and represents only a small fraction of the malignant cells from that sample (Sample 7, Figure 2A). Yet, this population is used to derive SLC26A3 as a potential biomarker. SLC26A3 transcripts were only detected in this small population of cells (none of the other ADC samples), which makes me question the specificity of the IHC staining on the validation cohort.

This is compounded by technical aspects of the analysis that hinder interpretation. For example, it is clear that the clustering does not perfectly segregate cell types. In Figures 2B and D, it is evident that C4 and C5 contain mixtures of cell type (eg. half of C4 is EPCAM+/CD3-, the other half EPCAM-/CD3+). These contaminations are carried forward into subclustering and are not addressed. Rather, it is claimed that there is a T cell population that is CD3- and EPCAM+, which does not seem likely.

Reviewer #1 (Public Review):

Summary:

This manuscript from Clayton and co-authors, entitled "Mechanism of dimer selectivity and binding cooperativity of BRAF inhibitors", aims at clarifying the molecular mechanism of BRAF dimer selectivity. Indeed, first generation BRAF inhibitors, targeting monomeric BRAFV600E, are ineffective in treating resistant dimeric BRAF isoforms. Here, the authors employed molecular dynamics simulations to study the conformational dynamics of monomeric and dimeric BRAF, in the presence and absence of inhibitors. Multi-microseconds MD simulations showed an inward shift of the αC helix in the BRAFV600E mutant dimer. This helped identify a hydrogen bond between the inhibitors and the BRAF residue Glu501 as critical for dimer compatibility. The stability of the aforementioned interaction seems to be important to distinguish between dimer-selective and equipotent inhibitors.

Strengths:

The study is overall valuable and robust. The authors used the recently developed particle mesh Ewald constant pH molecular dynamics, a state-of-the-art method, to investigate the correct histidines protonation considering the dynamics of the protein. Then, multi-microsecond simulations showed differences in the flexibility of the αC helix and DFG motif. The dimerization restricts the αC position in the inward conformation, in agreement with the result that dimer-compatible inhibitors are able to stabilize the αC-in state. Noteworthy, the MD simulations were used to study the interactions between the inhibitors and the protein, suggesting a critical role for a hydrogen bond with Glu501. Finally, simulations of a mixed state of BRAF (one protomer bound to the inhibitor and the other apo) indicate that the ability to stabilize the inward αC state of the apo protomer could be at the basis of the positive cooperativity of PHI1.

Weaknesses:

Regarding the analyses of the mixed state simulations, the DFG dihedral probability densities for the apo protomer (Fig. 5a right) are highly overlapping. It is not convincing that a slight shift can support the conclusion that the binding in one protomer is enough to shift the DFG motif outward allosterically. Moreover, the DFG dihedral time-series for the apo protomer (Supplementary Figure 9) clearly shows that the measured quantities are affected by significant fluctuations and poor consistency between the three replicates. The apo protomer of the mixed state simulations could be affected by the same problem that the authors pointed out in the case of the apo dimer simulations, where the amount of sampling is insufficient to model the DFG-out/-in transition properly. There is similar concern with the Lys483-Glu501 salt bridge measured for the apo protomers of the mixed simulations. As it can be observed from the probabilities bar plot (Fig. 5a middle), the standard deviation is too high to support a significant role for this interaction in the allosteric modulation of the apo protomer.

Reviewer #2 (Public Review):

Summary:

The authors employ molecular dynamics simulations to understand the selectivity of FDA approved inhibitors within dimeric and monomeric BRAF species. Through these comprehensive simulations, they shed light on the selectivity of BRAF inhibitors by delineating the main structural changes occurring during dimerization and inhibitor action. Notably, they identify the two pivotal elements in this process: the movement and conformational changes involving the alpha-C helix and the formation of a hydrogen bond involving the Glu-501 residue. These findings find support in the analyses of various structures crystallized from dimers and co-crystallized monomers in the presence of inhibitors. The elucidation of this mechanism holds significant potential for advancing our understanding of kinase signalling and the development of future BRAF inhibitor drugs.

Strengths:

The authors employ a diverse array of computational techniques to characterize the binding sites and interactions between inhibitors and the active site of BRAF in both dimeric and monomeric forms. They combine traditional and advanced molecular dynamics simulation techniques such as CpHMD (All-atom continuous constant pH molecular dynamics) to provide mechanistic explanations. Additionally, the paper introduces methods for identifying and characterizing the formation of the hydrogen bond involving the Glu501 residue without the need for extensive molecular dynamics simulations. This approach facilitates the rapid identification of future BRAF inhibitor candidates.

Weaknesses:

Despite the use of molecular dynamics yields crucial structural insights and outlines a mechanism to elucidate dimer selectivity and cooperativity in these systems, the authors could consider adoption of free energy methods to estimate the values of hydrogen bond energies and hydrophobic interactions, thereby enhancing the depth of their analysis.

Reviewer #1 (Public Review):

Summary:

In the manuscript by Tie et.al., the authors couple the methodology which they have developed to measure LQ (localization quotient) of proteins within the Golgi apparatus along with RUSH based cargo release to quantify the speed of different cargos traveling through Golgi stacks in nocodazole induced Golgi ministacks to differentiate between cisternal progression vs stable compartment model of the Golgi apparatus. The debate between cisternal progression model and stable compartment model has been intense and going on for decades and important to understand the basic way of function/organization of the Golgi apparatus. As per the stable compartment model, cisterna are stable structures and cargo moves along the Golgi apparatus in vesicular carriers. While as per cisternal progression model, Golgi cisterna themselves mature acquiring new identity from the cis face to the trans face and act as transport carriers themselves. In this work, authors provide a missing part regarding intra-Golgi speed for transport of different cargoes as well as the speed of TGN exit and based on the differences in the transport velocities for different cargoes tested favor a stable compartment model. The argument which authors make is that if there is cisternal progression, all the cargoes should have a similar intra-Golgi transport speed which is essentially the rate at which the Golgi cisterna mature. Furthermore, using a combination of BFA and Nocodazole treatments authors show that the compartments remain stable in cells for at least 30-60 minutes after BFA treatment.

Strengths:

The method to accurately measure localization of a protein within the Golgi stack is rigorously tested in the previous publications from the same authors and in combination with pulse chase approaches has been used to quantify transport velocities of cargoes through the Golgi. This is a novel aspect in this paper and differences in intra-Golgi velocities for different cargoes tested makes a case for a stable compartment model.

Weaknesses:

Experiments are only tested in one cell line (HeLa cells) and predominantly derived from experimental paradigm using RUSH assays where a secretory cargo is released in a wave (not the most physiological condition) and therefore additional approaches would make a more compelling case for the model.

Reviewer #2 (Public Review):

Summary:

This manuscript describes the use of quantitative imaging approaches, which have been a key element of the labs work over the past years, to address one of the major unresolved discussions in trafficking: intra-Golgi transport. The approach used has been clearly described in the labs previous papers, and is thus clearly described. The authors clearly address the weaknesses in this manuscript and do not overstate the conclusions drawn from the data. The only weakness not addressed is the concept of blocking COPI transport with BFA, which is a strong inhibitor and causes general disruption of the system. This is an interesting element of the paper, which I think could be improved upon by using more specific COPI inhibitors instead, although I understand that this is not necessarily straightforward.

I commend the authors on their clear and precise presentation of this body of work, incorporating mathematical modelling with a fundamental question in cell biology. In all, I think that this is a very robust body of work, that provides a sound conclusion in support of the stable compartment model for the Golgi.

General points:

The manuscript contains a lot of background in its results sections, and the authors may wish to consider rebalancing the text: The section beginning at Line 175 is about 90% background and 10% data. Could some data currently in supplementary be included here to redress this balance, or this part combined with another?

Reviewer #3 (Public Review):

The manuscript by Tie et al. provides a quantitative assessment of intra-Golgi transport of diverse cargos. Quantitative approaches using fluorescence microscopy of RUSH synchronized cargos, namely GLIM and measurement of Golgi residence time, previously developed by the author's team (publications from 20216 to 2022), are being used here.

Most of the results have been already published by the same team in 2016, 2017, 2020 and 2021. In this manuscript, very few new data have been added. The authors have put together measurements of intra-Golgi transport kinetics and Golgi residence time of many cargos. The quantitative results are supported by a large number of Golgi mini-stacks/cells analyzed. They are discussed with regard to the intra-Golgi transport models being debated in the field, namely the cisternal maturation/progression model and the stable compartments model. However, over the past decades, the cisternal progression model has been mostly accepted thanks to many experimental data.

The authors show that different cargos have distinct intra-Golgi transport kinetics and that the Golgi residence time of glycosyltransferases is high. From this and the experiment using brefeldinA, the authors suggest that the rim progression model, adapted from the stable compartments model, fits with their experimental data.

Strengths:

The major strength of this manuscript is to put together many quantitative results that the authors previously obtained and to discuss them to give food for thought about the intra-Golgi transport mechanism.<br /> The analysis by fluorescence microscopy of intra-Golgi transport is tough and is a tour de force of the authors even if their approach show limitations, which are clearly stated. Their work is remarkable in regards to the numbers of Golgi markers and secretory cargos which have been analyzed.

Weaknesses:

As previously mentioned, most of the data provided here were already published and thus accessible for the community. Is there is a need to publish them again?<br /> The authors' discussion about the intra-Golgi transport model is rather simplistic. In the introduction, there is no mention of the most recent models, namely the rapid partitioning and the rim progression models. To my opinion, the tubular connections between cisternae and the diffusion/biochemical properties of cargos are not enough taken into account to interpret the results. Indeed, tubular connections and biochemical properties of the cargos may affect their transit through the Golgi and the kinetics with which they reach the TGN for Golgi exit.<br /> Nocodazole is being used to form Golgi mini-stacks, which are necessary to allow intra-Golgi measurement. The use of nocodazole might affect cellular homeostasis but this is clearly stated by the authors and is acceptable as we need to perturb the system to conduct this analysis. However, the manual selection of the Golgi mini-stack being analyzed raises a major concern. As far as I understood, the authors select the mini-stacks where the cargo and the Golgi reference markers are clearly detectable and separated, which might introduce a bias in the analysis.<br /> The terms 'Golgi residence time ' is being used but it corresponds to the residence time in the trans-cisterna only as the cargo has been accumulated in the trans-Golgi thanks to a 20{degree sign}C block. The kinetics of disappearance of the protein of interest is then monitored after 20{degree sign}C to 37{degree sign}C switch.<br /> Another concern also lies in the differences that would be introduced by different expression levels of the cargo on the kinetics of their intra-Golgi transport and of their packaging into post-Golgi carriers.

Reviewer #1 (Public Review):

Summary:

This work presents an in-depth characterization of the factors that influence the structural dynamics of the Clostridium botulinum guanidine-IV riboswitch (riboG). Using a single-molecule FRET, the authors demonstrate that riboG undergoes ligand and Mg2+ dependent conformational changes consistent with dynamic formation of a kissing loop (KL) in the aptamer domain. Formation of the KL is attenuated by Mg2+ and Gua+ ligand at physiological concentrations as well as the length of the RNA. Interestingly, the KL is most stable in the context of just the aptamer domain compared to longer RNAs capable of forming the terminator stem. To attenuate transcription, binding of Gua+ and formation of the KL must occur rapidly after transcription of the aptamer domain but before transcription of the rest of the terminator stem.

Strengths:

(1) Single molecule FRET microscopy is well suited to unveil the conformational dynamics of KL formation and the authors provide a wealth of data to examine the effect of the ligand and ions on riboswitch dynamics. The addition of complementary transcriptional readthrough assays provides further support the author's proposed model of how the riboswitch dynamics contribute to function.<br /> (2) The single-molecule data strongly support that the effect of Gua+ ligand and Mg2+ influence the RNA structure differently for varying lengths of the RNA. The authors also demonstrate that this is specific for Mg2+ as Na+ and K+ ions have little effect.<br /> (3) The PLOR method utilized is clever and well adapted for both dual labeling of RNAs and examining RNA at various lengths to mimic co-transcriptional folding. Using PLOR, they demonstrate that a change in the structural dynamics and ligand binding can occur after extension of the RNA transcript by a single nucleotide. Such a tight window of regulation has intriguing implications for kinetically controlled riboswitches.<br /> (4) In the revised version, the authors utilized multiple destabilizing and compensatory mutations to strengthen their structural interpretation of the KL structure and dynamics and cementing their conclusions.

Reviewer #2 (Public Review):

Summary:

Gao et al., used single-molecule FRET and step-wise transcription methods to study the conformations of the recently reported guanidine-IV class of bacterial riboswitches that upregulate transcription in the presence of elevated guanidine. Using three riboswitch lengths, the authors analyzed the distributions and transitions between different conformers in response to different Mg2+ and guanidine concentrations. These data led to a three-state kinetic model for the structural switching of this novel class of riboswitches whose structures remain unavailable. Using the PLOR method that the authors previously invented, they further examined the conformations, ligand responses, and gene-regulatory outcomes at discrete transcript lengths along the path of vectorial transcription. These analyses uncover that the riboswitch exhibits differential sensitivity to ligand-induced conformational switching at different steps of transcription, and identify a short window where the regulatory outcome is most sensitive to ligand binding.

Strengths:

Dual internal labeling of long RNA transcripts remains technically very challenging, but essential for smFRET analyses of RNA conformations. The authors should be commended for achieving very highly quality and purity in their labelled RNA samples. The data are extensive, robust, thorough, and meticulously controlled. The interpretations are logical and conservative. The writing is reasonably clear and illustrations are of high quality. The findings are significant because the paradigm uncovered here for this relatively simple riboswitch class is likely also employed in numerous other kinetically regulated riboswitches. The ability to quantitatively assess RNA conformations and ligand responses at multiple discrete points along the path towards the full transcript provides a rare and powerful glimpse into co-transcriptional RNA folding, ligand-binding, and conformational switching.

Weaknesses:

The use of T7 RNA polymerase instead of a near cognate bacterial RNA polymerase in the termination/antitermination assays is a significant caveat. It is understandable as T7 RNA polymerase is much more robust than its bacterial counterparts, which probably will not survive the extensive washes required by the PLOR method. The major conclusions should still hold, as the RNA conformations are probed by smFRET at static, halted complexes instead of on the fly. However, potential effects of the cognate RNA polymerase cannot be discerned here, including transcriptional rates, pausing, and interactions between the nascent transcript and the RNA exit channel, if any. The authors should refrain from discussing potential effects from the DNA template or the T7 RNA polymerase, as these elements are not cognate with the riboswitch under study.

Reviewer #3 (Public Review):

Summary:

In this article, Gao et. al. uses single-molecule FRET (smFRET) and position-specific labelling of RNA (PLOR) to dissect the folding and behavioral ligand sensing of the Guanidine-IV riboswitch in the presence and absence of the ligand guanidine and the cation Mg2+. Results provided valuable information on the mechanistic aspects of the riboswitch, including the confirmation on the kissing loop present in the structure as essential for folding and riboswitch activity. Co-transcriptional investigations of the system provided key information on the ligand-sensing behavior and ligand-binding window of the riboswitch. A plausible folding model of the Guanidine-IV riboswitch was proposed as a final result. The evidence presented here sheds additional light into the mode of action of transcriptional riboswitches.

Strengths:

The investigations were very thorough, providing data that supports the conclusions. The use of smFRET and PLOR to investigate RNA folding has been shown to be a valuable tool to the understand of folding and behavior properties of these structured RNA molecules. The co-transcriptional analysis brought important information on how the riboswitch works, including the ligand-sensing and the binding window that promotes the structural switch. The fact that investigations were done with the aptamer domain, aptamer domain + terminator/anti-terminator region, and the full length riboswitch were essential to inform how each domain contributes to the final structural state if in the presence of the ligand and Mg2+.

Weaknesses:

The system has its own flaws when comparing to physiological conditions. The RNA polymerase used (the study uses T7 RNA polymerase) is different from the bacterial RNA polymerase, not only on complexity, but also in transcriptional speed, that can direct interfere with folding and ligand-sensing. Additionally, rNTPs concentrations were much lower than physiological concentrations during transcription, likely causing a change in the polymerase transcriptional speed. These important aspects and how they could interfere with results are important to be addressed to the broad audience. Another point of consideration to be aware is that the bulky fluorophores attached to the nucleotides can interfere with folding to some extent.

Reviewer #1 (Public Review):

This study explored the relationship between sustained attention and substance use from ages 14 to 23 in a large longitudinal dataset. They found behaviour and brain connectivity associated with poorer sustained attention at age 14 predicted subsequent increase in cannabis and cigarette smoking from ages 14-23. They concluded that the brain network of sustained attention is a robust biomarker for vulnerability to substance use. The big strength of the study is a substantial sample size and validation of the generalization to an external dataset. In addition, various methods/models were used to prove the relationship between sustained attention and substance use over time.

Reviewer #2 (Public Review):

Weng and colleagues investigated the relationship between sustained attention and substance use in a large cohort across three longitudinal visits (ages 14, 19, and 23). They employed a stop signal task to assess sustained attention and utilized the Timeline Followback self-report questionnaire to measure substance use. They assessed the linear relationship between sustained attention-associated functional connections and substance use at an earlier visit (age 14 or 19). Subsequently, they utilized this relationship along with the functional connection profile at a later age (age 19 or 23) to predict substance use at those respective ages. The authors found that connections in association with reduced sustained attention predicted subsequent increases in substance use, a conclusion validated in an external dataset. Altogether, the authors suggest that sustained attention could serve as a robust biomarker for predicting future substance use.

This study by Weng and colleagues focused on an important topic of substance use prediction in adolescence/early adulthood. While the study largely achieves its aims, several points merit further clarification:

(1) Regarding connectome-based predictive modeling, an assumption is that connections associated with sustained attention remain consistent across age groups. However, this assumption might be challenged by observed differences in the sustained attention network profile (i.e., connections and related connection strength) across age groups (Figures 2 G-I, Fig. 3 G_I). It's unclear how such differences might impact the prediction results.

(2) Another assumption of the connectome-based predictive modeling is that the relationship between sustained attention network and substance use is linear, and remains linear over development. Such linear evidence from either the literature or their data would be of help.

(3) Heterogeneity in results suggests individual variability that is not fully captured by group-level analyses. For instance, Figure 1A shows decreasing ICV (better-sustained attention) with age on the group level, while there are both increasing and decreasing patterns on the individual level via visual inspection. Figure 7 demonstrates another example in which the group with a high level of sustained attention has a lower risk of substance use at a later age compared to that in the group with a low level of sustained attention. However, there are individuals in the high sustained attention group who have substance use scores as high as those in the low sustained attention group. This is important to take into consideration and could be a potential future direction for research.

The above-mentioned points might partly explain the significant but low correlations between the observed and predicted ICV as shown in Figure 4. Addressing these limitations would help enhance the study's conclusions and guide future research efforts.

Reviewer #3 (Public Review):

Summary:

Weng and colleagues investigated the association between attention-related connectivity and substance use. They conducted a study with a sizable sample of over 1,000 participants, collecting longitudinal data at ages 14, 19, and 23. Their findings indicate that behaviors and brain connectivity linked to sustained attention at age 14 forecasted subsequent increases in cigarette and cannabis use from ages 14 to 23. However, early substance use did not predict future attention levels or attention-related connectivity strength.

Strengths:

The study's primary strength lies in its large sample size and longitudinal design spanning three time-points. A robust predictive analysis was employed, demonstrating that diminished sustained attention behavior and connectivity strength predict substance use, while early substance use does not forecast future attention-related behavior or connectivity strength.

Weaknesses:

It's questionable whether the prediction approach (i.e., CPM), even when combined with longitudinal data, can establish causality. I recommend removing the term 'consequence' in the abstract and replacing it with 'predict'. Additionally, the paper could benefit from enhanced rigor through additional analyses, such as testing various thresholds and conducting lagged effect analyses with covariate regression.

Reviewer #1 (Public Review):

In this manuscript, Yoo et al describe the role of a specialized cell type found in muscle, Fibro-adipogenic progenitors (FAPs), in promoting regeneration following sciatic nerve injury. Using single-cell transcriptomics, they characterize the expression profiles of FAPs at various times after nerve crush or denervation. Their results reveal that a population of these muscle-resident mesenchymal progenitors up-regulate the receptors for GDNF, which is secreted by Schwann cells following crush injury, suggesting that FAPs respond to this growth factor. They also find that FAPs increase expression of BDNF, which promotes nerve regeneration. The authors demonstrate FAP production of BDNF in vivo is upregulated in response to injection of GDNF and that conditional deletion of BDNF in FAPs results in delayed nerve regeneration after crush injury, primarily due to lagging remyelination. Finally, they also find reduced BDNF expression following crush injury in aged mice, suggesting a potential mechanism to explain the decrease in peripheral nerve regenerative capability in aged animals. These results are very interesting and novel and provide important insights into the mechanisms regulating peripheral nerve regeneration, which has important clinical implications for understanding and treating nerve injuries. However, there are a few concerns that the authors need to address.

Given that only a fraction of the FAPs express BDNF after injury, the authors need to demonstrate the specificity of the Prrx1-Cre for FAPs. This is particularly important because muscle stem cell also express GDNF receptors (Fig. 3C & D) and myogenic progenitors/satellite cells produce BDNF after nerve injury (Griesbeck et al., 1995 (PMID 8531223); Omura et al., 2005 (PMID 16221288)). Moreover, as the authors point out, there are multipotent mesenchymal precursor cells in the nerve that migrate into the surrounding tissue following nerve injury and contribute to regeneration (Carr et al, PMID 30503141). Therefore, there are multiple possible sources of BDNF, highlighting the need to clearly demonstrate that FAP-derived BDNF is essential.

Similarly, the authors should provide some evidence that BDNF protein is produced by FAPs. All of their data for BDNF expression is based on mRNA expression and that appears to only be increased in a small subset of FAPs. Perhaps an immunostaining could be done to demonstrate up-regulation of BDNF in FAPs after injury.

The suggestion that Schwann cell-derived GDNF is responsible for up-regulation of BDNF in the FAPs is indirect, based largely on the data showing that injection of GDNF into the muscle is sufficient to up-regulate BDNF (Fig. 4F & G). However, to more directly connect the 2 observations in a causal way, the authors should inject a Ret/GDNF antagonist, such as a Ret-Fc construct, then measure the BDNF levels.

In assessing the regeneration after nerve crush, the authors focus on remyelination, for example, assessing CMAP and g-ratios. However, they should also quantify axon regeneration, which can be done distal to the crush injury at earlier time points, before the 6 weeks scored in their study. Evaluating axon regeneration, which occurs prior to remyelination, would be especially useful because BDNF can act on both Schwann cells, to promote myelination, and axons, enhancing survival and growth. They could also evaluate the stability of the neuromuscular junctions, particularly if a denervation was done with the conditional knock outs, although that may be a bit beyond the scope of this study.

Reviewer #2 (Public Review):

Summary:

Yoo and colleagues studied the cellular mechanism allowing fibro-adipogenic progenitors (FAPs), muscle resident mesenchymal progenitors, to contribute to nerve regeneration upon regenerative injury. In addition to their expected role in the maintenance of muscle tissue, FAPs also contribute to the maturation and maintenance of neural tissue. After nerve injury, they prevent dying back loss of motor neurons. Consistently, muscle denervation activates FAPs, suggesting that FAPs can sense the injured distal peripheral nerve.

A transcriptomic database was established using flow cytometry protocols and single-cell RNA-seq. FAPs were isolated from sciatic nerve crush (SNC), considered a regenerative condition, and compared to a non-regenerative condition consisting of denervation-affected muscles (DEN) at different time points after injury: early (3 and 7 days post-injury, dpi) and late (14 and 28 dpi), when the regeneration process has started to resolve. Transcriptome changes of the nine different conditions were compared: non-injured, 3, 7, 14, and 28 days after injury. Bioinformatic analysis and other filters were applied, including UMAP plots, hierarchical clustering analysis using differentially expressed genes (DEGs), volcano plots, and RNA velocity analysis. In addition to most of the supplementary material, the first three and a half central figures consist of the analysis of the transcriptome changes comparing the different conditions. Overall, the data indicate similar DEGs after both types of injury at early stages. Still, just after SNC, the gene expression pattern reaches similar levels compared to non-injured, meaning the injured process is resolved. For example, the Interleukin6/Stat3 pathway is upregulated in both injury models but downregulated at 28 days just in SNC. When focusing on the comparison between 28 dpi between both types of injury, it indicates a role of FAPs in the resolution of inflammation in SNC and participation of FAPs in fibrosis and inflammation in DEN at 28 dpi. Genes related to wound healing were enriched in both.

With the question in mind of how FAPs are sensing injury, the authors identified a subset of FAPs relevant to regeneration in the SNC model. The unsupervised clustering of FAPs cells considering the nine different types of samples resulted in seven clusters of FAPs. Cluster one was exclusive to non-injury animals or regenerated samples. Clusters two and three were exclusive to the early injured or denervated nerve, suggesting that cluster one senses injury and clusters two and three are derived from it. Among the highest DEGs in cluster one were the GDNF receptors Ret and Gfra1. It is known that GDNF is released by Schwann cells after nerve injury in the literature. Also, gene expression analysis in clusters two and three predicts RTK involvement and GDNF signaling. Altogether, transcriptomic data suggest that GDNF is the mechanism by which FAPs sense nerve injury.

On the other hand, they found BDNF expression limited to cluster two of injured FAPs, suggesting that FAPs respond to GDNF by secreting BDNF. Although the specific role of secreted BDNF by FAPs in nerve regeneration is unknown, BDNF is known to have a regenerative influence on injured sciatic nerves by promoting both axonal growth and myelination. Consistent with their hypothesis, the analysis of gene expression in Schwann cells (sorted using the Plp1CreER Rosatd tomato mouse) and FAPs after injury indicates an initial increase in GDNF gene expression in early time points after injury in Schwann cells, followed by increased expression of BDNF in FAPs. Using conditional knock-out of BDNF in low limb FAPs (Prrx1Cre; Bdnffl/fl), they were able to demonstrate that nerve regeneration is impaired in Prrx1Cre; Bdnffl/fl, by delayed myelinization of axons.

Strengths:

I found the article well-written and cleverly maximized the interpretation and analysis of single-cell transcriptome data. Their findings illuminate how growth factors allow communication between cells responding to injury to promote regeneration. I find the data generated by the authors sufficient to support their model and claims,

Weaknesses:

Although, I find the data the authors generated enough for their claims. I do see them as relatively poor, and a complementary analysis of protein expression would strengthen the paper through immunostaining of the different genes mentioned for FAPs and Schwann cells. The model is entirely supported by measuring mRNA levels and negative regulation of gene expression in specific cells. Additionally, what happens to the structure of the neuromuscular junction after regeneration when GDNF or BDNF expression is reduced? The determination of decreasing levels of FAPs BDNF mRNA during aging is interesting; is the gain of BDNF expression in FAPs reverting the phenotype?

Reviewer #3 (Public Review):

Summary:

The manuscript by Kyusang Yoo et al. "Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis" investigates the role and mechanisms of fibro-adipogenic progenitors (FAPs), that are muscle-resident mesenchymal progenitors, in the maturation and maintenance of the neuromuscular system. There is earlier evidence that absence of FAPs or its functional decline with age cause smaller regenerated myofibers. Role of FAPs on peripheral nerve regeneration is very poorly studied. This study has translational importance because traumatic injury to the peripheral nerve can cause lifelong paralysis of the injured limb.

This manuscript provides data indicating that GDNF-BDNF axis plays an important role in peripheral nerve regeneration and function.

Strengths:

Because the role of FAPs on peripheral nerve regeneration is very poorly studied this investigation is a major step towards understanding the mechanism on the role of FAPs. They use scRNA-seq, animal models, and cKO mice that is also important. This study has translational importance because traumatic injury to the peripheral nerve can cause lifelong paralysis of the injured limb.<br /> This is an interesting and original study focusing on the role of FAPs and indicating that GDNF-BDNF axis plays an important role in peripheral nerve regeneration and function.

Weaknesses:

In Fig. 1 and 2 authors provide data on scRNA seq and this is important information reporting the finding of RET and GFRa1 transcripts in the subpopulation of FAP cells. However, authors provide no data on the expression of RET and GFRa1 proteins in FAP cells.<br /> Another problem is the lack of information showing that GDNF secreted by Schwann cells can activate RET and its down-stream signaling in FAP cells.<br /> There is no direct experimental proof that GDNF activating GFRa1-RET signaling triggers BDNF upregulation In FAP cells.<br /> The data that GDNF signaling is inducing the synthesis and secretion of BDNF is also not conclusive.

Reviewer #1 (Public Review):

Summary:

Using a mouse model of head and neck cancer, Barr et al show that tumor-infiltrating nerves connect to brain regions via the ipsilateral trigeminal ganglion, and they demonstrate the effect this has on behavior. The authors show that there are neurites surrounding the tumors using a WGA assay and show that the brain regions that are involved in this tumor-containing circuit have elevated Fos and FosB expression and increased calcium response. Behaviorally, tumor-bearing mice have decreased nest building and wheel running and increased anhedonia. The behavior, Fos expression, and heightened calcium activity were all decreased in tumor-bearing mice following nociceptor neuron elimination.

Strengths:

This paper establishes that sensory neurons innervate head and neck cancers and that these tumors impact select brain areas. This paper also establishes that behavior is altered following these tumors and that drugs to treat pain restore some but not all of the behavior. The results from the experiments (predominantly gene and protein expression assays, cFos expression, and calcium imaging) support their behavioral findings both with and without drug treatment.

Weaknesses:

Study suggests that the effects of their tumor models of mouse behavioral are largely non-specific to the tumor as most behaviors are rescued by analgesic treatment. So, most of the changes were likely due to site-specific pain and not a unique signal from the tumor.

Reviewer #2 (Public Review):

Summary:

Cancer treatments are not just about the tumor - there is an ever-increasing need for treating pain, fatigue, and anhedonia resulting from the disease as patients are undergoing successful but prolonged bouts with cancer. Using an implantable oral tumor model in the mouse, Barr et al describe neural infiltration of tumors, and posit that these nerve fibers are transmitting pain and other sensory signals to the brain that reduce pleasure and motivation. These findings are in part supported by anatomical and transcriptional changes in the tumor that suggest sensory innervation, neural tracing, and neural activity measurements. Further, the authors conduct behavior assays in tumor-bearing animals and inhibit/ablate pain sensory neurons to suggest the involvement of local sensory innervation of tumors in mediating cancer-induced malaise.

Strengths:

• This is an important area of research that may have implications for improving the quality of life of cancer patients.

• The studies use a combination of approaches (tracing and anatomy, transcriptional, neural activity recordings, behavior assays, loss-of-function) to support their claims.

• Tracing experiments suggest that tumor-innervating afferents are connected to brain nuclei involved in oral pain sensing. Consistent with this, the authors observed increased neural activity in those brain areas of tumor-bearing animals. It should be noted that some of these brain nuclei have also been implicated in cancer-induced behavioral alterations in non-head and neck tumor models.

• Experiments are for the most part well-controlled, and approaches are validated.

• The paper is well-written and the layout was easy to follow.

Weaknesses:

• The main claim is that tumor-infiltrating nerves underlie cancer-induced behavioral alterations, but the experimental interventions are not specific enough to support this. For example, all TRPV1 neurons, including those innervating the skin and internal organs, are ablated to examine sensory innervation of the tumor. Within the context of cancer, behavioral changes may be due to systemic inflammation, which may alter TRPV1 afferents outside the local proximity of tumor cells. A direct test of the claims of this paper would be to selectively inhibit/ablate nerve fibers innervating the tumor or mouth region.

• Behavioral results from TRPV1 neuron ablation studies are in part confounded by differing tumor sizes in ablated versus control mice. Are the differences in behavior potentially explained by the ablated animals having significantly smaller tumors? The differences in tumor sizes are not negligible. One way to examine this possibility might be to correlate behavioral outcomes with tumor size.

Reviewer #3 (Public Review):

Summary:

The authors have tested for and demonstrated a physical (i.e., sensory nerves to the brain) connection between tumors and parts of the brain. This can explain why there is an increase in depressive disorders in HNSCC patients. While connections such as this have been suspected, this is a novel demonstration pointing to sensory neurons that is accompanied by a remarkable amount of complementary data.

Strengths:

There is substantial evidence provided for the hypotheses tested. The data are largely quite convincing.

Weaknesses:

The authors mention in their Discussion the need for additional experiments. Could they also include / comment on the potential impact on the anti-tumor immune system in their model?

Minor:

The authors mention the importance of inflammation contributing to pain in cancer but do not clearly highlight how this may play a role in their model. Can this be clarified?

The tumor model apparently requires isoflurane injection prior to tumor growth measurements. This is different from most other transplantable types of tumors used in the literature. Was this treatment also given to control (i.e., non-tumor) mice at the same time points? If not, can the authors comment on the impact of isoflurane (if any) in their model?

The authors emphasize in several places that this is a male mouse model. They mention this as a limitation in the Discussion. Was there an original reason why they only tested male mice?

Reviewer #1 (Public Review):

Summary:

This work by Passlick and colleagues set out to reveal the mechanism by which short bouts of ischemia perturb glutamate signalling. This manuscript builds upon previous work in the field that reported a paradoxical increase in synaptic transmission following acute, transient ischemia termed ischemic or anoxic long-term potentiation. Despite these observations, how this occurs and the involvement of glutamate release and uptake mechanisms remains unanswered.

Here the authors employed two distinct chemical ischemia models, one lasting 2 minutes, the other 5 minutes. Recording evoked field excitatory postsynaptic potentials in acute brain slices, the authors revealed that shorter bouts of ischemia resulted in a transient decrease in postsynaptic responses followed by an overshoot and long-term potentiation. Longer bouts of chemical ischemia (5 minutes), however, resulted in synaptic failure that did not return to baseline levels over 50 minutes of recording (Figure 1).

Two-photon imaging of fluorescent glutamate sensor iGluSnFR expressed in astrocytes matched postsynaptic responses with shorter ischemia resulting in a transient dip before the increase in extracellular glutamate which was not the case with prolonged ischemia (Figure 2).

Mechanistically, the authors show that these increased glutamate levels and postsynaptic responses were not due to changes in glutamate clearance (Figure 3). Next using a competitive antagonist for AMPA postsynaptic AMPA receptors the authors show that synaptic glutamate release was enhanced by 2 minute chemical ischemia.

Taken together, these data reveal the underlying mechanism regarding ischemic long-term potentiation, highlighting presynaptic release as the primary culprit. Additionally, the authors show relative insensitivity of glutamate uptake mechanisms during ischemia, highlighting the resilience of astrocytes to this metabolic challenge.

Strengths:

This manuscript uses robust and modern techniques to address the mechanism by which ischemia influences synaptic transmission in the hippocampus.

The data are of high quality, with adequately powered sample sizes to address their hypotheses.

Weaknesses:

The question of the physiological relevance of short bouts of ischemia remains.

The precise mechanisms underlying the shift between ischemia-induced long-term potentiation and long-term failure of synaptic responses were not addressed. Could this be cell death?

Sex differences are not addressed or considered.

Reviewer #2 (Public Review):

Summary:

To investigate the impact of chemical ischemia induced by blocking mitochondrial function and glycolysis, the authors measured extracellular field potentials, performed whole-cell patch-clamp recordings, and measured glutamate release with optical techniques. They found that shorter two-minute-lasting blockade of energy production initially blocked synaptic transmission but subsequently caused a potentiation of synaptic transmission due to increased glutamate release. In contrast, longer five-minute-lasting blockage of energy production caused a sustained decrease of synaptic transmission. A correlation between the increase of intracellular potassium concentration and the response upon chemical ischemia indicates that the severity of the ischemia determines whether synapses potentiate or depress upon chemical ischemia. A subsequent mechanistic analysis revealed that the speed of uptake of glutamate is unchanged. An increase in the duration of the fiber volley reflecting the extracellular voltage of the action potentials of the axon bundle was interpreted as an action potential broadening, which could provide a mechanistic explanation. In summary, the data convincingly demonstrate that synaptic potentiation induced by chemical ischemia is caused by increased glutamate release.

Strengths:

The manuscript is well-written and the experiments are carefully designed. The results are exciting, novel, and important for the field. The main strength of the manuscript is the combination of electrophysiological recordings and optical glutamate imaging. The main conclusion of increased glutamate release was furthermore supported with an independent approach relying on a low-affinity competitive antagonist of glutamate receptors. The data are of exceptional quality. Several important controls were carefully performed, such as the stability of the recordings and the size of the extracellular space. The number of experiments is sufficient for the conclusions. The careful data analysis justifies the classification of two types of responses, namely synaptic potentiation and depression after chemical ischemia. Except for the duration of the presynaptic action potentials (see below weaknesses) the data are carefully discussed and the conclusions are justified.

Weaknesses:

The weaknesses are minor and only relate to the interpretation of some of the data regarding the presynaptic mechanisms causing the potentiation of release. The authors measured the fiber volley, which reflects the extracellular voltage of the compound action potential of the fiber bundle. The half-duration of the fiber volley was increased, which could be due to the action potential broadening of the individual axons but could also be due to differences in conduction velocity. We are therefore skeptical whether the conclusion of action broadening is justified.

Reviewer #3 (Public Review):

Summary:

This valuable study shows that shorter episodes (2 minutes duration) of energy depletion, as it occurs in ischemia, could lead to long-lasting dysregulation of synaptic transmission with presynaptic alterations of glutamate release at the CA3-CA1 synapses. A longer duration of chemical ischemia (5 minutes) permanently suppresses synaptic transmission. By using electrophysiological approaches, including field and patch clamp recordings, combined with imaging studies, the authors demonstrated that 2 minutes of chemical ischemia leads to a prolonged potentiation of synaptic activity with a long-lasting increase of glutamate release from presynaptic terminals. This was observed as an increase in iGluSnFR fluorescence, a sensor for glutamate expressed selectively on hippocampal astrocytes by viral injection. The increase in iGluSnFR fluorescence upon 2-minute chemical ischemia could not be ascribed to an altered glutamate uptake, which is unaffected by both 2-minute and 5-minute chemical ischemia. The presynaptic increase in glutamate release upon short episodes of chemical ischemia is confirmed by a reduced inhibitory effect of the competitive antagonist gamma-D-glutamylglycine on AMPA receptor-mediated postsynaptic responses. Fiber volley durations in field recording are prolonged in slices exposed to 2 min chemical ischemia. The authors interpret this data as an indication that the increase in glutamate release could be ascribed to a prolongation of the presynaptic action potential possibly due to inactivation of voltage-dependent K+ channels. However, more direct evidence is needed to support this hypothesis fully. This research highlights an important mechanism by which altered ionic homeostasis underlying metabolic failure can impact on neuronal activity. Moreover, it also showed a different vulnerability of mechanisms involved in glutamatergic transmission with a marked resilience of glutamate uptake to chemical ischemia.

Strengths:

(1) The authors use a variety of experimental techniques ranging from electrophysiology to imaging to study the contribution of several mechanisms underlying the effect of chemical ischemia on synaptic transmission.

(2) The experiments are appropriately designed and clearly described in the figures and in the text.

(3) The controls are appropriate.

Weaknesses:

- The data on fiber volley duration should be supported by more direct measurements to prove that chemical ischemia increases presynaptic Ca2+ influx due to a presynaptic broadening of action potentials. Given the influence that positioning of the stimulating and recording electrode can have on the fiber volley properties, I found this data insufficient to support the assumption of a relationship between increased iGluSnFR fluorescence, action potential broadening, and increased presynaptic Ca2+ levels.

- The results are obtained in an ex-vivo preparation, it would be interesting to assess if they could be replicated in vivo models of cerebral ischemia.

Impact:

This study provides a more comprehensive view of the long-term effects of energy depletion during short episodes of experimental ischemia leading to the notion that not only post-synaptic changes, as reported by others, but also presynaptic changes are responsible for long-lasting modification of synaptic transmission. Interestingly, the direction of synaptic changes is bidirectional and dependent on the duration of chemical ischemia, indicating that different mechanisms involved in synaptic transmission are differently affected by energy depletion.

Reviewer #1 (Public Review):

Summary

The work by She et al. investigates the role of SRFS2 in the MyoD+ progenitor cells during development. Deletion of SRFS2 in MyoD+ progenitor cells resulted in a defect in the directional migration of these cells and resulted in the presence of myoD+ progenitor in both nonmuscle and muscle tissues. The authors showed a defect in gene program regulation ECM, cell migration, cytoskeletal organization, and skeletal muscle differentiation by scRNA-seq. The authors further showed that many of these processes are regulated by a downstream target of SRFS2, the serine-threonine kinase Aurka. Finally, the authors showed that SRFS2 acts as a splicing factor and could contribute to differentiation by controlling the splicing of muscle-specific transcripts. This study addresses an important question in skeletal muscle development by focusing on the pathways and factors that regulate the migration of myoD+ progenitors and the impact of this process in skeletal muscle differentiation. This work is interesting but requires experimental evidence to support the findings.

Strengths

The regulators of myod+progenitor migration during skeletal muscle development is not completely understood. This work demonstrates that SRFS2 and aura kinase are key players in the process. Combining knockout and reporter lines in mice, the authors perform a detailed analysis of skeletal muscle cells to demonstrate the specific defects in SRFS2 in skeletal muscle development.

Weaknesses

This work explores an interesting question on regulating myoD+ progenitors and the defects of this process in skeletal muscle differentiation by SRFS2 but spreads out in many directions rather than focusing on the key defects. A number of approaches are used, but they lack the robust mechanistic analysis of the defects that result in muscle differentiation. Specifically, the role of SRFS2 on splicing appears to be a misfit here and does not explain the primary defects in the migration of myoD+ progenitors. There are concerns about the scRNA-seq and many transcripts in muscle biology that are not expressed in muscle cells. Focusing on main defects and additional experimental evidence to clear the fusion vs. precocious differentiation vs. reduced differentiation will strengthen this work.

(1) The analysis of RNA-seq data (Figure 2) is limited, and it is unclear how it relates to the work presented in this MS. The Go enrichment analysis is combined for both up and down-regulated DEG, thus making it difficult to understand the impact differently in both directions. Stac2 is a predominant neuronal isoform (while Stac3 is the muscle), and the Symm gene is not found in the HGNC or other databases. Could the authors provide the approved name for this gene? The premise of this work is based on defects in ECM processes resulting in the mis-targeting of the muscle progenitors to the nonmuscle regions. Which ECM proteins are differentially expressed?

(2) Could authors quantify the muscle progenitors dispersed in nonmuscle regions before their differentiation? Which nonmuscle tissues MyoD+ progenitors are seen? Most of the tDT staining in the enlarged sections appears to be punctate without any nuclear staining seen in these cells (Figure 3 B, D E-F). Could authors provide high-resolution images? Also, in the diaphragm cross-sections in mutants, tdT labeling appears to be missing in some areas within the myofibers defined as cavities by the authors (marked by white arrows, Figure 3H). Could this polarized localization of tDT be contributing to specific defects?

(3) Is there a difference in the levels of tDT in the myoD" muscle progenitors that are mis-targeted vs the others that are present in the muscle tissues?

(4) scRNA is unsuitable for myotubes and myofibers due to their size exclusion from microfluidics. Could authors explain the basis for scRNA-seq vs SnRNA-seq in this work? How are SKM defined in scRNA-data in Figure 4? As the myofibers are small in KO, could the increased level of late differentiation markers be due to the enrichment of these small myotubes/myofibers in scRNA? A different approach, such as ISH/IF with the myogenic markers at E9.5-10.5, may be able to resolve if these markers are prematurely induced.

(5) TNC is a marker for tenocytes and is absent in skeletal muscle cells. The authors mentioned a downregulation of TNC in the KO SKM derived clusters. This suggests a contamination of the tenocytes in the control cells. In spite of the downregulation of multiple ECM genes showed by scRNA-seq data, the ECM staining by laminin in KO in Figure 3 appears to be similar to controls.

(6) The expression of many fusion genes, such as myomaker and myomerger, is reduced in KO, suggesting a primary fusion defect vs a primary differentiation defect. Many mature myofiber proteins exhibit an increased expression in disease states, suggesting them as a compensatory mechanism. Authors need to provide additional experimental evidence supporting precocious differentiation as the primary defect.

(7) The fusion defects in KO are also evident in siRNA knockdown for SRSF2 and Aurka in C2C12, which mostly exhibits mononucleated myocytes in knockdowns. Also, a fusion index needs to be provided.

(8) The last section of the role of SRSF2 on splicing appears to be a misfit in this study. Authors describe the Bin1 isoforms in centronuclear myopathy, but exon17 is not involved in myopathy. Is exon17 exclusion seen in other diseases/ splicing studies?

Reviewer #2 (Public Review):

Summary:

This study was aimed to study the role of SRSF2 in governing MyoD progenitors to specific muscle regions. The Results confirmed the role of SRSF2 in controlling myogenic differentiation through the regulation of targeted genes and alternative splicing during skeletal muscle development.

Strengths:

The study used different methods and techniques to achieve aims and support the conclusions such as RNA sequencing analysis, Gene Ontology analysis, immunostaining analysis.<br /> This study provides novel findings that SRSF2 controls the myogenic differentiation of MyoD+ progenitors, using transgenic mouse model and in vitro studies.

Weaknesses:

Although unbiased sequencing methods were used, their findings about SRSF2 served as a transcriptional regulator and functioned in alternative splicing events are not novel.<br /> The introductions and discussion is not clearly written. The authors did not raise clear scientific questions in the introduction part. The last paragraph is only copy-paste of the abstract. The discussion part is mainly the repeat of their results without clear discussion.

Reviewer #3 (Public Review):

Summary:

This study employs an optogenetics approach aimed at activating oncogene (KRASG12V) expression in a single somatic cell, with a focus on following the progression of activated cell to examine tumourigenesis probabilities under altered tissue environments. The research explores the role of stemness factors (VENTX/NANOG/OCT4) in facilitating oncogenic RAS (KRASG12V)-driven malignant transformations. Although the evidence provided are incomplete, the authors propose an important mechanism whereby reactivation of re-programming factors correlates with the increased likelihood of a mutant cell undergoing malignant transformation.

Strengths:

· Innovative Use of Optogenetics: The application of optogenetics for precise activation of KRAS in a single cell is valuable to the field of cancer biology, offering an opportunity to uncover insight into cellular responses to oncogenic mutations.<br /> · Important Observations: The findings concerning stemness factors' role in promoting oncogenic transformation are important, contributing data to the field of cancer biology.

Weaknesses:

Lack of Methodological Clarity: The manuscript lacks detailed descriptions of methodologies, making it difficult to fully evaluate the experimental design and reproducibility, rendering incomplete evidence to support the conclusion. Improving methodological transparency and data presentation will crucially strengthen the paper's contributions to understanding the complex processes of tumourigenesis.<br /> Sub-optimal Data Presentation and Quality:

The resolution of images throughout the manuscript are too low. Images presented in Figure 2 and Figure 4 are of very low resolution. It is very hard to distinguish individual cells and in which tissue they might reside.<br /> Lack of quantitative data and control condition data obtained from images of higher magnification limits the ability to robustly support the conclusions.

Here are some details:<br /> · Tissue specificity of the cells express KRASG12V oncogene: In this study, the ubiquitin promoter was used to drive oncogenic KRASG12V expression. Despite this, the authors claim to activate KRAS in a single brain cell based on their localized photo-activation strategy. However, upon reviewing the methods section, the description was provided that 'Localized uncaging was performed by illumination for 7 minutes on a Nikon Ti microscope equipped with a light source peaking at 405 nm, Figure 1. The size of the uncaging region was controlled by an iris that defines a circular illumination with a diameter of approximately 80 μm.' It is surprising that an epi-fluorescent microscope with an illumination diameter of around 80μm can induce activation in a single brain cell beneath skin tissue. Additionally, given that the half-life for mTFP maturation is around 60 minutes, it is likely that more cells from a variety of different lineages could be activated, but the fluorescence would not be visible until more than 1-hour post-illumination. Authors might want to provide more evidence to support their claim on the single cell KRAS activation.<br /> · Stability of cCYC: The manuscript does not provide information on the half-life and stability of cCYC. Understanding these properties is crucial for evaluating the system's reliability and the likelihood of leakiness, which could significantly influence the study's outcomes.<br /> · Metastatic Dissemination claim: Typically, metastatic cancer cells migrate to and proliferate within specific niches that are conducive to outgrowth, such as the caudal hematopoietic tissue (CHT) or liver. In figure 3 A, an image showing the presence of mTFP expressing cells in both the head and tail regions of the larva, with additional positive dots located at the fin fold. This is interpreted as "metastasis" by the authors. However, the absence of a supportive cellular compartment within the fin-fold tissue makes the presence of mTFP-positive metastatic cells there particularly puzzling. This distribution raises concerns about the spatial specificity of the optogenetic activation protocol.<br /> The unexpected locations of these signals suggest potential ectopic activation of the KRAS oncogene, which could be occurring alongside or instead of targeted activation. This issue is critical as it could affect the interpretation of whether the observed mTFP signal expansion over time is due to actual cell proliferation and infiltration, or merely a result of ectopic RAS transgene activation.<br /> · Image Resolution Concerns: The cells depicted in Figure 3C β, which appear to be near the surface of the yolk sac and not within the digestive system as suggested in the MS, underscore the necessity for higher-resolution imaging. Without clearer images, it is challenging to ascertain the exact locations and states of these cells, thus complicating the assessment of experimental results.<br /> · The cell transplantation experiment is lacking protocol details: The manuscript does not adequately describe the experimental protocols used for cell transplantation, particularly concerning the origin and selection of cells used for injection into individual larvae. This omission makes it difficult to evaluate the reliability and reproducibility of the results. Such as the source of transplanted cells:<br /> • If the cells are derived from hyperplastic growths in larvae where RAS and VX (presumably VENTX) were locally activated, the manuscript fails to mention any use of fluorescence-activated cell sorting (FACS) to enrich mTFP-positive cells. Such a method would be crucial for ensuring the specificity of the cells being studied and the validity of the results.<br /> • If the cells are obtained from whole larvae with induced RAS + VX expression, it is notable and somewhat surprising that the larvae survived up to six days post-induction (6dpi) before cells were harvested for transplantation. This survival rate and the subsequent ability to obtain single cell suspensions raise questions about the heterogeneity of the RAS + VX expressing cells that transplanted.<br /> · Unclear Experimental Conditions in Figure S3B: The images in Figure S3B lack crucial details about the experimental conditions. It is not specified whether the activation of KRAS was targeted to specific cells or involved whole-body exposure. This information is essential for interpreting the scope and implications of the results accurately.<br /> · Contrasting Data in Figure S3C compared to literature: The graph in Figure S3C indicates that KRAS or KRAS + DEX induction did not result in any form of hyperplastic growth. This observation starkly contrasts with previous literature where oncogenic KRAS expression in zebrafish led to significant hyper-proliferation and abnormal growth, as evidenced by studies such as those published in and Neoplasia (2018), DOI: 10.1016/j.neo.2018.10.002; Molecular Cancer (2015), DOI: 10.1186/s12943-015-0288-2; Disease Models & Mechanisms (2014) DOI: 10.1242/dmm.007831. The lack of expected hyperplasia raises questions about the experimental setup or the specific conditions under which KRAS was expressed. The authors should provide detailed descriptions of the conditions under which the experiments were conducted in Figure S3B and clarifying the reasons for the discrepancies observed in Figure S3C are crucial. The authors should discuss potential reasons for the deviation from previous reports.

Further comments:

Throughout the study, KRAS-activated cell expansion and metastasis are two key phenotypes discussed that Ventx is promoting. However, the authors did not perform any experiments to directly show that KRAS+ cells proliferate only in Ventx-activated conditions. The authors also did not show any morphological features or time-lapse videos demonstrating that KRAS+ cells are motile, even though zebrafish is an excellent model for in vivo live imaging. This seems to be a missed opportunity for providing convincing evidence to support the authors' conclusions.

There were minimal experimental details provided for the qPCR data presented in the supplementary figures S5 and S6, therefore, it is hard to evaluate result obtained.

Reviewer #1 (Public Review):

Scerbo et al. developed an approach based on the oncogene kRasG12V and a reprogramming factor to induce deterministic and reproducible malignant transformation in a single cell. The activation of kRasG12V alone is not sufficient in their hands to initiate carcinogenesis, but when combined with the transient activation of a reprogramming factor (such as Ventx, Nanog, or Oct4), it significantly increases the probability of malignant transformation. This combination of oncogene and reprogramming factor may alter the epigenetic and functional state of the cell, leading to the development of tumors within a short period of time. The use of these two factors allows for the controlled manipulation of a single cell to study the cellular and molecular events involved in the early stages of tumorigenesis. The authors then performed allotransplantations of allegedly single fluorescent TICs in recipient larvae and found a large number of fluorescent cells in distant locations, claiming that these cells have all originated from the single transplanted TIC and migrated away. The number of fluorescent cells showed in the recipient larve just after two days is not compatible with a normal cell cycle length and more likely represents the progeny of more than one transplanted cell. The ability to migrate from the injection site should be documented by time-lapse microscopy. Then, the authors conclude that "By allowing for specific and reproducible single cell malignant transformation in vivo, their optogenetic approach opens the way for a quantitative study of the initial stages of cancer at the single cell level". However, the evidence for these claims are weak and further characterization should be performed to:

(1) show that they are actually activating the oncogene in a single cell (the magnification is too low and it is difficult to distinguish a single nucleus, labelling of the cell membrane may help to demonstrate that they are effectively activating the oncogene in, or transplanting, a single cell)<br /> (2) the expression of the genes used as markers of tumorigenesis is performed in whole larvae, with only a few transformed cells in them. Changes should be confirmed in FACS sorted fluorescent cells<br /> (3) the histology of the so called "tumor masses" is not showing malignant transformation, but at the most just hyperplasia. In the brain, the sections are not perfectly symmetrical and the increase of cellularity on one side of the optic tectum is compatible with this asymmetry.<br /> (4) The number of fluorescent cells found dispersed in the larve transplanted with one single TIC after 48 hours will require a very fast cell cycle to generate over 50 cells. Do we have an idea of the cell cycle features of the transplanted TICs?

Reviewer #2 (Public Review):

Summary:

In the work by Scerbo et al, the authors aim to better understand the open question of what factors constrain cells that are genetically predisposed to form cancer (e.g. those with a potentially cancer-causing mutation like activated Ras) to only infrequently undergo this malignant transformation, with a focus on the influence of embryonic or pluripotency factors (e.g. VENTX/NANOG). Using genetically defined zebrafish models, the authors can inducibly express the KRASG12V oncogene using a combination of Cre/Lox transgenes further controlled by optogenetically inducible Cre-activated (CreER fusion that becomes active with light-induced uncaging of a tamoxifen-analogue in a targeted region of the zebrafish embryo). They further show that transient expression and activation of a pluripotency factor (e.g. Ventx fused to a GR receptor that is activated with addition of dexamethasone) must occur in the model in order for overgrowth of cells to occur. This paper describes a genetically tractable and modifiable system for studying the requirements for inducing cellular hyperplasia in a whole organism by combining overexpression of canonical genetic drivers of cancer (like Ras) with epigenetic modifiers (like specific transcription factors), which could be used to study an array of combinations and temporal relationships of these cancer drivers/modifiers.

Strengths:

The combination of Cre/lox inducible gene expression with potentially localized optogenetic induction (CreER and uncaging of tamoxifen analogues) of recombination as well as well inducible activation of a transcription factor expressed via mRNA injection (GR-fusion to the TF and dex induction) offers a flexible system for manipulating cell growth, identity, and transcriptional programs. With this system, the authors establish that Ras activation and at least transient Ventx overexpression are together required to induce a hyperproliferative phenotype in zebrafish tissues.

The ability to live image embryos over the course of days with inducible fluorophores indicating recombination events and transgene overexpression offers a tractable in vivo system for studying hyperplastic cells in the context of a whole organism.

The transplant experiments demonstrate the ability of the induced hyperplastic cells to grow upon transfer to new host.

Weaknesses:

There is minimal quantitation of key aspects of the system, most critically in the efficiency of activation of the Ras-TFP fusion (Fig 1) in, purportedly, a single cell. The authors note "On average the oncogene is then activated in a single cell, identified within ~1h by the blue fluorescence of its nuclear marker) but no additional quantitative information is provided. For a system that is aimed at "a statistically relevant single-cell<br /> tracking and characterization of the early stages of tumorigenesis", such information seems essential.

The authors indicate that a single cell is "initiated" (Fig 2) using the laser optogenetic technique, but without definitive genetic lineage tracing, it is not possible to conclude that cells expressing TFP distant from the target site near the ear are daughter cells of the claimed single "initiated" cell. A plausible alternative explanation is 1) that the optogenetic targeting is more diffuse (i.e. some of the light of the appropriate wavelength hits other cells nearby due to reflection/diffraction), so these adjacent cells are additional independent "initiated" cells or 2) that the uncaged tamoxifen analogue can diffuse to nearby cells and allow for CreER activation and recombination. In Fig 2B, the claim is made that "the activated cell has divided, giving rise to two cells" - unless continuously imaged or genetically traced, this is unproven. In addition, it appears that Figures S3 and S4 are showing that hyperplasica can arise in many different tissues (including intestine, pancreas, and liver, S4C) with broad Ras + Ventx activation (while unclear from the text, it appears these embryos were broadly activated and were not "single cell activated using the set-up in Fig 1E? This should be clarified in the manuscript). In Fig S7 where single cell activation and potential metastasis is discussed, similar gut tissues have TFP+ cells that are called metastatic, but this seems consistent with the possibility that multiple independent sites of initiation are occurring even when focal activation is attempted.

Although the hyperplastic cells are transplantable (Fig 4), the use of the term "cells of origin of cancer" or metastatic cells should be viewed with care in the experiments showing TFP+ cells (Fig 1, 2, 3) in embryos with targeted activation for the reasons noted above.

Reviewer #1 (Public Review):

Summary:

This is a well-conducted study about the mechanism of binding of a small molecule (fasudil) to a disordered protein (alpha-synuclein). Since this type of interaction has puzzled researchers for the last two decades, the results presented are welcome as they offer relevant insight into the physical principles underlying this interaction.

Strengths:

The results show convincingly that the mechanism of entropic expansion can explain the previously reported binding of fasudil to alpha-synuclein. In this context, the analysis of the changes in the entropy of the protein and of water is highly relevant. The combination use of machine learning for dimensional reduction and of Markov State Models could become a general procedure for the analysis of other systems where a compound binds a disordered protein.

Weaknesses:

It would be important to underscore the computational nature of the results, since the experimental evidence that fasudil binds alpha-synuclein is not entirely clear, at least to my knowledge.

Reviewer #2 (Public Review):

The manuscript by Menon et al describes a set of simulations of alpha-Synuclein (aSYN) and analyses of these and previous simulations in the presence of a small molecule.

While I agree with the authors that the questions addressed are interesting, I am not sure how much we learn from the present simulations and analyses. In parts, the manuscript reads more like an attempt to apply a whole range of tools rather than with a goal of answering any specific questions.

There's a lot going on in this paper, and I am not sure it is useful for the authors, readers or me to spell out all of my comments in detail. But here are at least some points that I found confusing/etc

Major concerns

p. 5 and elsewhere:<br /> I lack a serious discussion of convergence and the statistics of the differences between the two sets of simulations. On p. 5 it is described how the authors ran multiple simulations of the ligand-free system for a total of 62 µs; that is about 25 times less than for the ligand system. I acknowledge that running 1.5 ms is unfeasible, but at a bare minimum the authors should discuss and analyse the consequences for the relatively small amount of sampling. Here it is important to say that while 62 µs may sound like a lot it is probably not enough to sample the relevant properties of a 140-residue long disordered protein.

p. 7:<br /> The authors make it sound like a bad thing than some methods are deterministic. Why is that the case? What kind of uncertainty in the data do they mean? One can certainly have deterministic methods and still deal with uncertainty. Again, this seems like a somewhat ad hoc argument for the choice of the method used.

p. 8:<br /> The authors should make it clear (i) what the reconstruction loss and KL is calculated over and (ii) what the RMSD is calculated over.

p. 9/figure 1:<br /> The authors select a beta value that may be the minimum, but then is just below a big jump in the cross-validation error. Why does the error jump so much and isn't it slightly dangerous to pick a value close to such a large jump.

p. 10:<br /> Why was a 2-dimensional representation used in the VAE? What evidence do the authors have that the representation is meaningful? The authors state "The free energy landscape represents a large number of spatially close local minima representative of energetically competitive conformations inherent in αS" but they do not say what they mean by "spatially close". In the original space? If so, where is the evidence.

p. 10:<br /> It is not clear from the text whether the VAEs are the same for both aSYN and aSYN-Fasudil. I assume they are. Given that the Fasudil dataset is 25x larger, presumably the VAE is mostly driven by that system. Is the VAE an equally good representation of both systems?

p. 10/11:<br /> Do the authors have any evidence that the latent space representation preserves relevant kinetic properties? This is a key point because the entire analysis is built on this. The choice of using z1 and z2 to build the MSM seems somewhat ad hoc. What does the auto-correlation functions of Z1 and Z2 look like? Are the related to dynamics of some key structural properties like Rg or transient helical structure.

p. 11:<br /> What's the argument for not building an MSM with states shared for aSYN +- Fasudil?

p. 12:<br /> Fig. 3b/c show quite clearly that the implied timescales are not converged at the chosen lag time (incidentally, it would have been useful with showing the timescales in physical time). The CK test is stated to be validated with "reasonable accuracy", though it is unclear what that means.

p. 12:<br /> In Fig. 3d, what are the authors bootstrapping over? What are the errors if the authors analyse sampling noise (e.g. bootstrap over simulation blocks)?

p. 13:<br /> I appreciate that the authors build an MSM using only a subset of the fasudil simulations. Here, it would be important that this analysis includes the entire workflow so that the VAE is also rebuilt from scratch. Is that the case?

p. 18:<br /> I don't understand the goal of building the CVAE and DCVAE. Am I correct that the authors are building a complex ML model using only 3/6 input images? What is the goal of this analysis. As it stands, it reads a bit like simply wanting to apply some ML method to the data. Incidentally, the table in Fig. 6C is somewhat intransparent.

p. 22:<br /> "Our results indicate that the interaction of fasudil with αS residues governs the structural features of the protein."<br /> What results indicate this?

p. 23:<br /> The authors should add some (realistic) errors to the entropy values quoted. Fig. 8 have some error bars, though they seem unrealistically small. Also, is the water value quoted from the same force field and conditions as for the simulations?

p. 23:<br /> Has PDB2ENTROPY been validated for use with disordered proteins?

p. 23/24:<br /> It would be useful to compare (i) the free energies of the states (from their populations), (ii) the entropies (as calculated) and (iii) the enthalpies (as calculated e.g. as the average force field energy). Do they match up?

p. 31:<br /> It is unclear which previous simulation the new aSYN simulations were launched from. What is the size of the box used?

Reviewer #3 (Public Review):

Summary:

In this manuscript Menon, Adhikari, and Mondal analyze explicit solvent molecular dynamics (MD) computer simulations of the intrinsically disordered protein (IDP) alpha-synuclein in the presence and absence of a small molecule ligand, Fasudil, previously demonstrated to bind alpha-synuclein by NMR spectroscopy without inducing folding into more ordered structures. In order to provide insight into the binding mechanism of Fasudil the authors analyze an unbiased 1500us MD simulation of alpha-synuclein in the presence of Fasudil previously reported by Robustelli et.al. (Journal of the American Chemical Society, 144(6), pp.2501-2510). The authors compare this simulation to a very different set of apo simulations: 23 separate1-4us simulations of alpha-synuclein seeded from different apo conformations taken from another previously reported by Robustelli et. al. (PNAS, 115 (21), E4758-E4766), for a total of ~62us.

To analyze the conformational space of alpha-synuclein - the authors employ a variational auto-encoder (VAE) to reduce the dimensionality of Ca-Ca pairwise distances to 2 dimensions, and use the latent space projection of the VAE to build Markov state Models. The authors utilize k-means clustering to cluster the sampled states of alpha-synuclein in each condition into 180 microstates on the VAE latent space. They then coarse grain these 180 microstates into a 3-macrostate model for apo alpha-synuclein and a 6-macrostate model for alpha-synuclein in the presence of fasudil using the PCCA+ course graining method. Few details are provided to explain the hyperparameters used for PCCA+ coarse graining and the rationale for selecting the final number of macrostates.

The authors analyze the properties of each of the alpha-synuclein macrostates from their final MSMs - examining intramolecular contacts, secondary structure propensities, and in the case of alpha-synuclein:Fasudil holo simulations - the contact probabilities between Fasudil and alpha-synuclein residues.

The authors utilize an additional variational autoencoder (a denoising convolutional VAE) to compare denoised contact maps of each macrostate, and project onto an additional latent space. The authors conclude that their apo and holo simulations are sampling distinct regions of the conformational space of alpha-synuclein projected on the denoising convolutional VAE latent space.

Finally, the authors calculate water entropy and protein conformational entropy for each microstate. To facilitate water entropy calculations - the author's take a single structure from each macrostate - and ran a 20ps simulation at a finer timestep (4 femtoseconds) using a previously published method (DoSPT), which computes thermodynamic properties of water from MD simulations using autocorrelation functions of water velocities. The authors report that water entropy calculated from these individual 20ps simulations is very similar.

For each macrostate the authors compute protein conformational entropy using a previously published Maximum Information Spanning tree approach based on torsion angle distributions - and observe that the estimated protein conformational entropy is substantially more negative for the macrostates of the holo ensemble.

The authors calculate mean first passage times from their Markov state models and report a strong correlation between the protein conformational entropy of each state and the mean first passage time from each state to the highest populated state.

As the authors observe the conformational entropy estimated from macrostates of the holo alpha-synuclein:Fasudil is greater than those estimated from macrostates of the apo holo alpha-synuclein macrostates - they suggest that the driving force of Fasudil binding is an increase in the conformational entropy of alpha-synuclein. No consideration/quantification of the enthalpy of alpha-synuclein Fasudil binding is presented.

Strengths:

The author's utilize MD simulations run with an appropriate force field for IDPs (a99SB-disp and a99SB-disp water (Robustelli et. al, PNAS, 115 (21), E4758-E4766) - which has previously been used to perform MD simulations of alpha-synuclein that have been validated with extensive NMR data.

The contact probability between Fasudil and each alpha-synuclein residue observed in the previously performed 1500us MD simulation of alpha-synuclein in the presence of Fasudil (Robustelli et. al., Journal of the American Chemical Society, 144(6), pp.2501-2510) was previously found to be in good agreement with experimental NMR chemical shift perturbations upon Fasudil binding - suggesting that this simulation is a reasonable choice for understanding IDP:small molecule interactions.

Weaknesses:

Major Weakness 1: Simulations of apo alpha-synuclein and holo simulations of alpha-synuclein and fasudil are not comparable.

The most robust way to determine how presence of Fasudil affects the conformational ensemble of alpha-synuclein conclusions is to run apo and holo simulations of the same length from the same starting structures using the same simulation parameters.

The 23 1-4 us independent simulations of apo alpha-synuclein and the long unbiased 1500us alpha-synuclein in the presence of fasudil are not directly comparable. The starting structures of simulations used to build a Markov state model to describe apo alpha-synuclein were taken from a previously reported 73us MD simulation of alpha-synuclein run with the a99SB-disp force field and water model) with 100mM NaCl, (Robustelli et. al, PNAS, 115 (21), E4758-E4766). As the holo simulation of alpha-synuclein and Fasudil was run in 50mM NaCl, snapshots from the original apo alpha-synuclein simulation were resolvated with 50mM NaCl - and new simulations were run.

No justification is offered for how starting structures were selected. We have no sense of the conformational variability of the starting structures selected and no sense of how these conformations compare to the alpha-synuclein conformations sampled in the holo simulation in terms of standard structural descriptors such as tertiary contacts, secondary structure, radius of gyration (Rg), solvent exposed surface area etc. (we only see a comparison of projections on an uninterpretable non-linear latent-space and average contact maps). Additionally, 1-4 us is a relatively short timescale for a simulation of a 140 residue IDP- and one is unlikely to see substantial evolution for many structural properties of interest (ie. secondary structure, radius of gyration, tertiary contacts) in simulations this short. Without any information about the conformational space sample in the 23 apo simulations (aside from a projection on an uninterpretable latent space)- we have no way to determine if we observe transitions between distinct states in these short simulations, and therefore if it is possible the construct a meaningful MSM from these simulations.

If the structures used for apo simulations are on average more compact or contain more tertiary contacts - then it is unsurprising that in short independent simulations they sample a smaller region of conformational space. Similarly, if the starting structures have similar dimensions - but we only observe extremely local sampling around starting structures in apo simulations in the short simulation times - it would also not be surprising that we sample a smaller amount of conformational space. By only presenting comparisons of conformational states on an uninformative VAE latent space - it is not possible for a reader to ask simple questions about how the conformational ensembles compare.

It is noted that the authors attempt to address questions about sampling by building an MSM of single contiguous 60us portion of the holo simulation of alpha-synuclein and Fasudil - noting that:

"the MSM built using lesser data (and same amount of data as in water) also indicated the presence of six states of alphaS in presence of fasudil, as was observed in the MSM of the full trajectory. Together, this exercise invalidates the sampling argument and suggests that the increase in the number of metastable macrostates of alphaS in fasudil solution relative to that in water is a direct outcome of the interaction of alphaS with the small molecule."

However, the authors present no data to support this assertion - and readers have no sense of how the conformational space sampled in this portion of the trajectory compares to the conformational space sampled in the independent apo simulations or the full holo simulation. As the analyzed 60us portion of the holo trajectory may have no overlap with conformational space sampled in the independent apo simulations - it is unclear if this control provides any information. There is no quantification of the conformational entropy of the 6 states obtained from this portion of the holo trajectory or the full conformational space sampled. No information is presented to determine if we observe similar states in the shorter portion of the holo trajectory. Furthermore - as the authors provide almost no justification for the criteria used to select of the final number of macrostates for any of the MSMs reported in this work- and the number of macrostates is effectively a free parameter in the PCCA+ method, arriving at an MSM with 6 macrostates does not convey any information about the conformational entropy of alpha-synuclein in the presence or absence of ligands. Indeed - the implied timescale plot for 60us holo MSM (Figure S2) - shows that at least 10 processes are resolved in the 120 microstate model - and there is no information to provided explaining/justifying how a final 6-macrostate model was determined. The authors also do not project the conformations sampled in this sub- trajectory onto the latent space of the final VAE.

One certainly expects that an MSM built with 1/20th of the simulation data should have substantial differences from an MSM built from the full trajectory - so failing additional information and hyperparameter justification - one wonders if the emergence of a 6-state model could be the direct result of hardcoded VAE and MSM construction hyperparameter choices.

Required Controls For Supporting the Conclusions of the Study: The authors should initiate apo and holo simulations from the same starting structures - using the same simulation software and parameters. This could be done by adding a Fasudil ligand to the apo structures - or by removing the Fasudil ligand from a subset of holo structures. This would enable them to make apples-to-apples comparisons about the effect of Fasudil on alpha-synuclein conformational space.

Failing to add direct apples-to-apples comparisons, which would be required to truly support the studies conclusions, the authors should at least compare the conformational space sampled in the independent apo simulations and holo simulations using standard interpretable IDP order parameters (ie. Rg, end-to-end distance, secondary structure order parameters) and/or principal components from PCA or tICA obtained from the holo simulation. The authors should quantify the number of transitions observed between conformational states in their apo simulations. The authors could also perform more appropriate holo controls, without additional calculations, by taking batches of a similar number of short 1-4us segments of simulations used to compute the apo MSMs and examining how the parameters/macrostates of the holo MSMs vary with the input with random selections.

Major Weakness 2: There is little justification of how the hyperparameters MSMs were selected. It is unclear if the results of the study depend on arbitrary hyperparameter selections such as the final number of macrostates in each model.

It is unclear what criteria were used to determine the appropriate number of microstates and macrostates for each MSM. Most importantly - as all analyses of water entropy and conformational entropy are restricted to the final macrostates - the criteria used to select the final number of macrostates with the PCCA+ are extremely important to the results of the conclusions of the study. From examining the ITS plots in Figure 3 - it seems both MSMs show the same number of resolved processes (at least 11) - suggesting that a 10-state model could be apropraite for both systems. If one were to simply select a large number of macrostates for the 20x longer holo simulation - do these states converge to the same conformational entropy as the states seen in the short apo simulations? Is there some MSM quality metric used to determine what number of macrostates is more appropriate?

Required Controls For Supporting the Conclusions of the Study: The authors should specify the criteria used to determine the appropriate number of microstates and macrostates for their MSMs and present controls that demonstrate that the conformational entropies calculated for their final states are not simply a function of the ratio of the number macrostates chosen to represent very disparate amounts of conformational sampling.

Major Weakness 3: The use of variational autoencoders (VAEs) obscures insights into the underlying conformational ensembles of apo and holo alpha-synuclein rather than providing new ones.

No rationale is offered for the selection of the VAE architecture or hyperparameters used to reduce the dimensionality of alpha-synuclein conformational space.

It is not clear the VAEs employed in this study are providing any new insight into the conformational ensembles and binding mechanisms of Fasudil to alpha-synuclein, or if the underlying latent space of the VAEs are more informative or kinetically meaningful than standard linear dimensionality reduction techniques like PCA and tICA. The initial VAE is used to reduce the dimensionality of alpha-synuclein conformational ensembles to 2 degrees of freedom - but it is unclear if this projection is structurally or kinetically meaningful. It is not clear why the authors choice to use a 2-dimeinsional projection instead of a higher number of dimensions to build their MSMs. Can they produce a more kinetically and structurally meaningful model using a higher dimensional VAE latent space?

Additionally - it is not clear what insights are provided by the Denoising Convolutional Variational Autoencoder. The authors appear to be noising-and-denoising the contact maps of each macrostate, and then projecting the denoised values onto a new latent space - and commenting that they are different. Does this provide additional insight that looking at the contact maps in Figures 4&5 does not? Is this more informative than examining the distribution of the Radii of gyration or the secondary structure propensities of each ensemble? It is not clear what insight this analysis adds to the manuscript.

Suggested controls to improve the study: The authors should project interpretable IDP structural descriptors (ie. secondary structure, radius of gyration, secondary structure content, # of intramolecular contacts, # of intermolecular contacts between alpha-synuclein and Fasudil ) onto this latent space to illustrate if any of these properties are meaningful separated by the VAE projection. The authors should compare these projections, and MSMs built from these projections, to projections and MSMs built from projections using standard linear dimensionality projection techniques like PCA and tICA.

Major Weakness 4: The MSMs produced in this study have large discrepancies with MSMs previously produced on the same dataset by the same authors that are not discussed.

Previously - two of the authors of this manuscript (Menon and Mondal) authored a preprint titled "Small molecule modulates α-synuclein conformation and its oligomerization via Entropy Expansion" (https://www.biorxiv.org/content/10.1101/2022.10.20.513005v1.full) that analyzed the same 1500us holo simulation of alpha-synuclein binding Fasudil. In this study - they utilized the variational approach to Markov processes (VAMP) to build an MSM using a 1D order parameter as input (the radius of gyration), first discretizing the conformational space into 300 microstates before similarly building a 6 macrostate model. From examining the contact maps and secondary structure propensities of the holo MSMs from the current study and the previous study- some of the macrostates appear similar, however there appear to be orders of magnitude differences in the timescales of conformational transitions between the two models. The timescales of conformational transitions in the previous MSM are on the order of 10s of microseconds, while the timescales of transitions in this manuscript are 100s-1000s microseconds. In the previous manuscript, a 3 state MSM is built from an apo α-synuclein obtained from a continuous 73ms unbiased MD simulation of alpha-synuclein run at a different salt concentration (100mM) and an additional 33 ms of shorter simulations. The apo MSM from the previous study similarly reports very fast timescales of transitions between apo states (on the order ~1ms) - while the MSM reported in the current study (Figure 9) are on the order of 10s-100s of microseconds).

These discrepancies raise further concerns that the properties of the MSMs built on these systems are extremely sensitive to the chosen projection methods and MSM modeling choices and hyperparameters, and that neither model may be an accurate description of the true underlying dynamics

Suggestions to improve the study: The authors should discuss the discrepancies with the MSMs reported in their previous studies.

Reviewer #1 (Public Review):

The manuscript from Chang et al. presents a new technique to track chromatin locus mobility in live cells, by specifically tracking Alu rich sequences using a CRISPR based technique. The experiments in Fig. 1-2 provide extensive validation of the reagent, and the experiments in Figs. 3-4 yield new insights into chromatin dynamics and its relationship to transcription. While the findings in this manuscript are interesting, some points need to be addressed to support the central claims.

One item of consideration is the use of bulk PIV methods to monitor chromatin mobility. While these whole genome methods certainly are useful for studying chromatin mobility at a diffraction limited (or higher scale) as well as tracking correlations at the micron scale, these methods obscure dynamics at the TAD/nucleosomal level (~200 nm). Since the studies use fluorescently labeled H2B to study chromatin dynamics, some consideration should be given to using Halo-tagged variants of H2B to get a single molecule view within specific chromatin contexts. A few recent studies (Saxton et al. 2023, Daugird et al. 2023) have used these methods to show how histone dynamics at the single molecule level depends on the chromatin context.

Secondly, there should be additional discussion of how the mean-squared network displacement relates to single locus and histone mobility at the sub-diffraction level. While it is reassuring to see that MSND and single particle tracking MSD exponents roughly agree at the sub-second time scale, how these relate at longer time scales is not clear. Figure S5A shows MSD for individual loci, but only timelags upto 1s are shown. It should be possible to track loci considerably longer than that. MSD exponents in the literature are quite varied beyond the second time-scale, and the authors have an excellent system to shed light on this question.

Finally, some additional discussion about why the transcriptional inhibition results shown here differ from other studies in the literature (e.g. Daugird et al. 2023) would better place these findings in context.

Reviewer #2 (Public Review):

Summary:

Chromatin organization and dynamics are critical for eukaryotic genome functions, but how are they related to each other? To address this question, Chang et al. developed a euchromatic labeling method using CRISPR/dCAS9 targeting Alu elements. These elements are highly enriched in the A compartment, which is closely associated with transcriptionally active and gene-rich regions. Labeling Alu elements allowed live-cell imaging of the gene-rich A compartment (euchromatin). Using the developed system, Chang et al. found while Alu-rich chromatin is depleted in regions with high chromatin density (putative heterochromatin), Alu density and chromatin density are not correlated in the euchromatin. Combining the live-cell imaging of Alu elements with bulk chromatin labeling (fluorescent histone H2B), the authors showed that transcriptionally active chromatin (A compartment) has an increased mobility. Transcription inhibitors flavopiridol and 𝛼-amanitin treatments increased the mobility of Alu-rich chromatin, and ActD had the opposite effect on chromatin mobility.

Strengths:

Alu labeling is a valuable euchromatin labeling method, and measuring its mobility would contribute to a comprehensive understanding of the relationship between chromatin dynamics and transcriptional activity.

Weaknesses:

Some of the findings are consistent with the previous reports and not new. There are some issues to be addressed. My specific comments are the following:

Line 58. "these methods generally lack information regarding the local chromatin environment (e.g., epigenetic state) and genomic context (e.g., A/B compartments and TADs)." This description is not accurate because Nozaki et al. (2023) performed euchromatin-specific nucleosome labeling/imaging (Hi-C contact domains with active histone marks, A-compartment). More recently, Semeigazin et al. (2024)(https://www.researchsquare.com/article/rs-3953132/v1) also did euchromatic-specific nucleosome labeling/imaging in living cells.

Line 154. "we defined the euchromatin regions in our images by excluding heterochromatin (top 5% pixel intensity) and nucleolar areas."<br /> I am not so sure that this definition is reasonable. How were the top 5% H2B intensity regions distributed? Did they include the nuclear periphery region, which is also heterochromatin-rich? Could the authors show the ΔPCC between whole H2B (including both euchromatin and heterochromatin) and dCas9-sgAlu?

Line 214. "our data suggests that Alu-rich (gene-rich) regions have increased chromatin mobility compared to Alu-poor (gene-poor) regions." A similar finding on nucleosome motion has already been published by Nozaki et al. 2023 and Semeigazin et al. 2024 (described above).

Line 282. A recent important paper on the relationship between histone acetylation, transcription initiation, and nucleosome mobility (PMID: 37792937) is missing and should be discussed.

Line 303. "Alu-rich chromatin may be more sensitive upon flavopiridol and 𝛼-amanitin treatments compared to Alu-poor chromatin (Figure 5)." Nagashima et al. (2019) also revealed that 𝛼-amanitin treatment did not increase the chromatin dynamics in heterochromatin-rich nuclear periphery regions.

Reviewer #3 (Public Review):

The manuscript by Chang, Quinodoz and Brangwynne describes the results of live cell imaging of fluorescently labeled Alu element genomic sites in combination with H2B-GFP marked chromatin in human cancer cells. The study includes dCas9 based genomic engineering for Suntag enhanced Alu element labeling. The motion of Alu elements and chromatin was analyzed in real time at 500 ms intervals over 1 min at high resolution. Advanced image analysis algorithms were developed.

The main objective of the study is to understand how motion of euchromatin or active chromatin relates to chromatin density. Alu elements, which are spread throughout the genome are used as a proxy for euchromatin or also A compartments. The study finds that Alu-rich chromatin is more mobile than Alu poor one and that actinomycin but not flavopyridol or alpha amanitin cause some decrease in the determined mobility. The authors emphasize the heterogeneity of motion, Alu clustering and chromatin density underscoring the complexity of the problem.

Although the topic is important and the imaging well performed, the study lacks depth and does not provide any truly new insights into our understanding of the link between genome activity and mobility nor diffusive behavior of the chromatin fiber in situ. Although the approach to record context dependent dynamics based on segmentation of pixels of varying intensity is elegant, the analysis of the trajectories requires further explanation and justification to be able to interpret the results. Important information on the biology of the engineered cell lines is lacking. Presented results are not discussed with respect to existing literature and knowledge.

Major concerns:<br /> - Are Alu elements a good proxy for A compartments? What consequences do massive dCas9 tags have on the genome and the engineered cells? How does the bulky dCas9-Suntag label impact mobility and transcription of Alu elements themselves? How many off target sites are potentially labeled?

(1) The authors should state the size of the dCas9-Suntag construct and perform FRAP analysis to compare the tag's behavior to the one of H2B-GFP<br /> (2) dCas9 locally unwinds DNA and is strongly bound to its target sequence impeding polymerase progression.<br /> (3) The authors need to check if DNA breaks are induced. An immunofluorescence using a gH2AX antibody is a minimum in all conditions tested. DNA breaks largely affect chromatin mobility which is a topic of major debate (see PMC5769766, PMID33061931).<br /> (4) The authors need to confirm that in dCas/sgAlu cells Alu elements are still transcribed similarly to wt cells (transcriptome or at least some qPCR).<br /> (5) Please compare H2B-GFP mobility of sgAlu tagged and untagged cells.<br /> (6) Figure 1D shows significant background in the Cut&run sgAlu line compared to H3K4me3 line. Are these off target sites? Was the H3K4me3 Cut&run performed in the engineered cell line? Did the authors test another guide RNA? Non-specific binding could also contribute to the observed heterogeneity in the measured dynamics.<br /> (7) Figure 3G shows that H2B MSND at tau=5s is high for high H2B density independently of Alu density questioning the value of using Alu sg tagging as a proxy for euchromatin.

- What are the physical principles of the measured motion? What is the rationale for the MSND analyses deployed in this study?<br /> (1) Please provide the equation used for MSND (seems to be different from the standard MSD one).<br /> (2) Figure 3: all MSD curves have a slope suggesting an alpha exponent significantly smaller than 0.5 reminiscent of subdiffusion (example panels A and E compare thick line to slope of the triangle bottom right). Please explain. Is it gaussian noise? Confinement? This was seen before for faster acquisition rates, but still requires explanation and interpretation.<br /> (3) What is the rationale for choosing the value at τ =5 s? Figure 3 panel E shows large variations in the MSND at all time points, curves do not start at the same lag time.<br /> (4) Figure S5 shows that for Alu elements, alpha is close to 0.5 at τ =<1 s but lower for larger tau, the relationship to intensity is inverse as well. Please explain.<br /> (5) It would be important to show the D values of your estimations. Plots for MSD curves in non log scale are important to be presented to show if there are different diffusion regimes (such as in Figure 4).<br /> (6) It is mentioned that the "Our measurements of total chromatin dynamics at lag time τ = 5 s are typically on the order of 10-2 μm2 (Figure 3 A, B), in agreement with past studies (Shaban et al., 2020; Zidovska et al., 2013)". This is inaccurate as both cited studies were performed at different time lags 0.2 sec. Change in time lag is supposed to show different diffusion behaviour. For consistency, the comparison should be done at the same time lag and the same number of analyzed video frames.<br /> (7) The study applies the MSND analysis for different time lags starting from 0.5 s to 11 s for videos of 60 s. Change in the number of data points affects the accuracy to calculate the diffusion coefficient. What is the impact of this uncertainty on the results and conclusions?

- Inhibition of polymerase 2 activity increases mobility as was shown before.<br /> (1) Figure 4: change in motion following alpha amanitin and Flavopiridol treatments recapitulate results from the Maeshima group (Nagashima 2019). Data shown for actinomycin treated cells appear extreme. A huge drop in H2B MSND (panel B and D). Please ensure that the cells are still alive after 4-6h exposure to ActD. ActD also affects cytoskeleton and replication, so different conclusion may be drawn if cells are still alive.<br /> (2) Treatment effects could also be enhanced should dCas9/ sgAlu induce massive DNA damage (see above). Check H2B-GFP motion in cells (both treated and not) not labeled with sgAlu.

- Positioning with respect to the literature:<br /> (1) The introduction, first paragraph is oversimplified, please review the literature citing work performed by many groups in the field using H2B-GFP, telomere or single site labeling in the past 10 years. Give details on the cell type used (mouse or human normal or cancer cells, amplified signals or single genes, same cell or cells at different stages of development, methodologies from whole genome to single particle tracking etc.).<br /> (2) The manuscript claims to introduce a novel mapping of the spatiotemporal dynamics of the A compartment in living cells. However, the authors did not discuss other previous approaches that were developed for the same purpose. The dynamic motion of active transcription chromatin domains/A compartment over the whole nucleus was investigated in different studies that used Mintbody labeling, please check PMCID: PMC7926250, PMCID: PMC8647360, PMID: 27534817, PMCID: PMC8491620<br /> (3) PIV applies a relatively large interrogation window size of micrometers to estimate the displacement vectors. Dynamic changes within the set window can include both A and B compartments, where the contribution of genomic processes to local chromatin motion, typically taking place at the nanometer scale, is missed. The Hi-D method ( PMCID: PMC7168861) introduced an Optical Flow approach to overcome this limitation of PIV (PMCID: PMC6061878 ). Could the authors test if Hi-D method to analyze the movies recorded in this study confirms their conclusions?

Heterogeneity of chromatin dynamics independent of chromatin density was shown by previous studies such as PMCID: PMC7775763 , and PMCID: PMC7168861 . Could the authors discuss their findings in the context of these studies?

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors use the model organism Drosophila to explore the sex and age impacts of a TBI method. They find age and sex differences: older age is susceptible to mild TBI and females are also more susceptible. In particular, they pursue a finding that virgin vs mated females show different responses: virgins are protected but mated females succumb to TBI with climbing deficits. In fact, virgin females compared to mated females are largely protected. They discover that this is associated with exposure of the females to Sex Peptides in the reproductive neurons of the female reproductive tract. When they extend to RNAseq of brains, they show that there are very few genes in common between males, mated females, virgins and females mated with males lacking Sex Peptide. The few chronic genes associated with mated females seem associated with the immune system. These findings suggest that mated females have a compromised immune system, which might make them more vulnerable.

Strengths:

This is an interesting paper that allows a detailed comparison of sex and age in TBI which is largely only possible in such a simple model, where large numbers and many variations can be addressed. Overall the findings are interesting.

Weaknesses:

Although the findings beyond Sex Peptide are observational, the work sets the stage for more detailed studies to pursue the role of the genes they find by RNAseq and whether for example, boosting the innate immune system would protect the mated females, among other experiments.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors use the Drosophila model system to study the impact of mild head trauma on sex-dependent brain deficits. They identify Sex Peptide as a modulator of greater negative outcome in female flies. Additionally, they observe that increased age at the time of injury results in worse outcomes, especially in females, and that this is due to chronic suppression of innate immune defense networks in mated females. The results demonstrate a novel signaling pathway that promotes age- and sex-dependent outcomes after head injury.

Strengths:

The authors have modified their previously reported TBI model in flies to mimic mild TBI, which is novel. Methods are explained in detail, allowing for reproducibility. Experiments are rigorous with appropriate statistics. A number of important controls are included. The work tells a complete mechanistic story and adds important data to increase our understanding of sex-dependent differences in recovery after TBI. The discussion is comprehensive and puts the work in the context of the field.

Weaknesses:

A very minor weakness is that exact n values should be included in the figure legends. There should also be confirmation of knockdown by RNAi in female flies either by immunohistochemistry or qRT-PCR if possible.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors used a Drosophila model to show that exposure to repetitive mild TBI causes neurodegenerative conditions that emerge late in life and disproportionately affect females. In addition to well-known age-dependent impact, the authors identified Sex Peptide (SP) signaling as a key factor in female susceptibility to post-injury brain deficits.

Strengths:

The authors have presented a compelling set of results showing that female Sex Peptide signaling adversely affects late-life neurodegeneration after early-life exposure to repetitive mild head injury in Drosophila. They have (1) compared the phenotypes of adult male and female flies sustaining TBI at different ages, and the phenotypes of virgin females and mated females, (2) compared the phenotypes of eliminating SP signaling in mating females and introducing SP-signaling into virgin females, (3) compared transcriptomic changes of different groups in response to TBI. The results are generally consistent and robust.

Weaknesses:

The authors have made their claims largely based on assaying climbing index and vacuole formation as the only indicators of late-life neurodegeneration after TBI. However, these phenotypes are not really specific to TBI-related neurodegeneration, and the significance and mechanisms of especially vacuole formation are not clear. The authors should perform additional analyses on TBI-related neurodegeneration in flies, which have been shown before (Genetics. 2015 Oct; 201(2): 377-402). Furthermore, it is also really surprising to see so few DEGs even in wild-type males and mated females, and to see that none of the DEGs overlapped among groups or are even related to the SP-signaling. This raises questions about the validity of the RNA-seq analysis. It is critical to independently verify their RNA-sequencing results and to add some more molecular evidence to support their conclusion. Finally, it is unknown what the implication of female fly mating and its associated Sex Peptide signaling would be to mammalians or humans, and what are the mechanisms underlying the sexual dimorphism.

Reviewer #1 (Public Review):

Summary:

In previously published work, the authors found that Transforming Growth Factor β Activated Kinase 1 (TAK1) may regulate esophageal squamous cell carcinoma (ESCC) tumor cell proliferation via the RAS/MEK/ERK axis. They explore the mechanisms for TAK1 as a possible tumor suppressor, demonstrating phospholipase C epsilon 1 as an effector of tumor cell migration, invasion and metastatic potential.

Strengths:

The authors show in vitro that TAK1 overexpression reduces tumor cell migration and invasion while TAK1 knockdown promotes a mesenchymal phenotype (epithelial-mesenchymal transition) and enhances migration and invasion. To explore possible mechanisms of action, the authors focused on phospholipase C epsilon 1 (PLCE1) as a potential effector, having identified this protein in co-immunoprecipitation experiments. Further, they demonstrate that TAK1-mediated phosphorylation of PLCE1 is inhibitory. Each of the observations is supported by different experimental strategies, e.g. use of different approaches for knockdown (pharmacologic, RNA inhibition, CRISPR/Cas). Xenograft experiments showed that suppression/loss of TAK1 is associated with more frequent metastases and conversely that PLCE1 is associated positively with xenograft metastases. A considerable amount of experimental data is presented for review, including supplemental data, that show that TAK1 regulation may be important in ESCC development.

Weaknesses:

As noted by the authors, immunoprecipitation (IP) experiments identified a number (24) of proteins as potential targets for the TAK1 ser/thr kinase. Prior work (cited as Shi et al, 2021) focused on a different phosphorylation target for TAK1, Ras association domain family 9 (RASSF9), but a more comprehensive discussion of the co-IP experiments would help place this work in a better context.

Reviewer #2 (Public Review):

Summary:

In this study, Ju Q et al performed both in vitro and in vivo experiments to test the effect of TAK1 on cancer metastasis. They demonstrated that TAK1 is capable of directly phosphorylating PLCE1 and this modification represses its enzyme activity, leading to suppression of PIP2 hydrolysis and subsequently signal transduction in the PKC/GSK-3β/β-Catenin axis.

Strengths:

The quality of data is good, and the presentation is well organized in a logical way.

Weaknesses:

The study missed some key link in connecting the effect of TAK1 on cancer metastasis via phosphorylating PLCE1.

Reviewer #3 (Public Review):

Summary:

The research by Qianqian Ju et al. found that the knockdown of TAK1 promoted ESCC migration and invasion, whereas overexpression of TAK1 resulted in the opposite outcome. These in vitro findings could be recapitulated in a xenograft metastasis mouse model.

Mechanistically, TAK1 phosphorylates PLCE1 S1060 in the cells, decreasing PLCE1 enzyme activity and repressing PIP2 hydrolysis. As a result, reducing DAG and inositol IP3, thereby suppressing signal transduction of PKC/GSK 3β/β Catenin. Consequently, cancer metastasis-related genes were impeded by TAK1.

Overall, this study offers some intriguing observations. Providing a potential druggable target for developing agents for dealing with ESCC.

The strengths of this research are:

(1) The research always uses different experimental approaches to address one question. The experiments are largely convincing and appear to be well executed.<br /> (2) The phenotypes were observed from different angles: at the mouse model, cellular level, and molecular level.<br /> (3) The molecular mechanism was down to a single amino acid modification on PLCE1.

The weaknesses part of this research are:

(1) Most of the phenotypes are only observed in the ECA-109 cell line. Whether TAK1-PLCE1 S1060 is a common pathway in other ESCC cells or just specific to the ECA-109 cell line is unclear. Using more cell lines to see whether this is a common mechanism of ESCC metastasis would greatly amplify the impact of this finding.<br /> (2) Most of the experiments were done in protein overexpression conditions, with the protein level increasing hundreds of folds in the cell, producing an artificial environment that would sometimes generate false positive results.<br /> (3) Whether TAK1 can directly phosphorylate PLCE1 S1060 needs more tests, especially the in vitro biochemical evidence.

Reviewer #1 (Public Review):

Summary:

In this study, López-Jiménez and colleagues demonstrated the utility of using high-content microscopy in dissecting host and bacterial determinants that play a role in the establishment of infection using Shigella flexneri as a model. The manuscript nicely identifies that infection with Shigella results in a block to DNA replication and protein synthesis. At the same time, the host responds, in part, via the entrapment of Shigella in septin cages.

Strengths:

The main strength of this manuscript is its technical aspects. They nicely demonstrate how an automated microscopy pipeline coupled with artificial intelligence can be used to gain new insights regarding elements of bacterial pathogenesis, using Shigella flexneri as a model system. Using this pipeline enabled the investigators to enhance the field's general understanding regarding the role of septin cages in responding to invading Shigella. This platform should be of interest to those who study a variety of intracellular microbial pathogens.

Another strength of the manuscript is the demonstration - using cell biology-based approaches- that infection with Shigella blocks DNA replication and protein synthesis. These observations nicely dovetail with the prior findings of other groups. Nevertheless, their clever click-chemistry-based approaches provide visual evidence of these phenomena and should interest many.

Weaknesses:

There are two main weaknesses of this work. First, the studies are limited to findings obtained using a single immortalized cell line. It is appreciated that HeLa cells serve as an excellent model for studying aspects of Shigella pathogenesis and host responses. However, it would be nice to see that similar observations are observed with an epithelial cell line of intestinal, preferably colonic origin, and eventually, with a non-immortalized cell line, although it is appreciated that the latter studies are beyond the scope of this work.

The other weakness is that the studies are minimally mechanistic. For example, the investigators have data to suggest that infection with Shigella leads to an arrest in DNA replication and protein synthesis; however, no follow-up studies have been conducted to determine how these host cell processes are disabled. Interestingly, Zhang and colleagues recently identified that the Shigella OspC effectors target eukaryotic translation initiation factor 3 to block host cell translation (PMID: 38368608). This paper should be discussed and cited in the discussion.

Reviewer #2 (Public Review):

Summary:

Septin caging has emerged as one of the innate immune responses of eukaryotic cells to infections by intracellular bacteria. This fascinating assembly of eukaryotic proteins into complex structures restricts bacteria motility within the cytoplasm of host cells, thereby facilitating recognition by cytosolic sensors and components of the autophagy machinery. Given the different types of septin caging that have been described thus far, a single-cell, unbiased approach to quantify and characterise septin recruitment at bacteria is important to fully grasp the role and function of caging. Thus, the authors have developed an automated image analysis pipeline allowing bacterial segmentation and classification of septin cages that will be very useful in the future, applied to study the role of host and bacterial factors, compare different bacterial strains, or even compare infections by clinical isolates.

Strengths:

The authors developed a solid pipeline that has been thoroughly validated. When tested on infected cells, automated analysis corroborated previous observations and allowed the unbiased quantification of the different types of septin cages as well as the correlation between caging and bacterial metabolic activity. This approach will prove an essential asset in the further characterisation of septin cages for future studies.

Weaknesses:

As the main aim of the manuscript is to describe the newly developed analysis pipeline, the results illustrated in the manuscript are essentially descriptive. The developed pipeline seems exceptionally efficient in recognising septin cages in infected cells but its application for a broader purpose or field of study remains limited.

Reviewer #3 (Public Review):

Summary:

The manuscript uses high-content imaging and advanced image-analysis tools to monitor the infection of epithelial cells by Shigella. They perform some analysis on the state of the cells (through measurements of DNA and protein synthesis), and then they focus on differential recruitment of Sept7 to the bacteria. They link this recruitment with the activity of the bacterial T3SS, which is a very interesting discovery. Overall, I found numerous exciting elements in this manuscript, and I have a couple of reservations. Please see below for more details on my reservations. Nevertheless, I think that these issues can be addressed by the authors, and doing so will help to make it a convincing and interesting piece for the community working on intracellular pathogens. The authors should also carefully re-edit their manuscript to avoid overselling their data (see below for issues I see there). I would consider taking out the first figure and starting with Figure 3 (Figure 2 could be re-organized in the later parts)- that could help to make the flow of the manuscript better.

Strengths:

The high-content analysis including the innovative analytical workflows are very promising and could be used by a large number of scientists working on intracellular bacteria.

The finding that Septins (through SEPT7) are differentially regulated through actively secreting bacteria is very exciting and can steer novel research directions.

Weaknesses:

The manuscript makes a connection between two research lines (1: Shigella infection and DNA/protein synthesis, 2: regulation of septins around invading Shigella) that are not fully developed - this makes it sometimes difficult to understand the take-home messages of the authors.

It is not clear whether the analysis that was done on projected images actually reflects the phenotypes of the original 3D data. This issue needs to be carefully addressed.

Reviewer #1 (Public Review):

Summary:

Soo-Yeon Hwang et al. synthesized and characterized a new set of small molecules targeting the interaction between ELF3-MED23, the transcription factor, and a coactivator for HER2 transcription, respectively. The authors used a combination of biochemical analysis, cell-based assays, and an in vivo xenograft model to prove that the lead compound 10 inhibits the HER2 transcription and protein expression levels, subsequently inducing anticancer activity in the gastric cancer cell line, the xenograft model, particularly in the trastuzumab-resistant cell line. The experiential data is solid and supports the model for the anticancer potency of the compound for the HER2+ gastric cancer model. Although the compound showed promising data for its potential antitumor activity for HER2+ cancers, it is a little bit narrow to the HER2+ cancer field since the most relevant HER2+ cancer model is HER2+ breast cancer and the Herceptin-resistance, indeed the author also discussed this point in the manuscript. Therefore, additional data with the breast cancer HER2+ cell model will help to impact the work in the field.

Strengths:

The current manuscript proposed a potential alternative strategy targeting HER2 overexpression cancers by attenuating HER2 transcription levels. The study provides solid evidence that the lead compound 10 can interrupt the binding of ELF3 to MED23, leading to the inhibition of HER2 transcription. Remarkably, the following cell-based assays and xenograft model revealed the promising antitumor activity of the compound in the gastric cancer model.

Weaknesses:

While the novel compound showed a promising potency to the HER2-positive gastric cancer cells and xenograft model, it would be great to also to be evaluated with the HER2-positive breast cancer cell models. The author did not compare the current compounds with other therapeutic strategies targeting HER2 expression at the genetic level. It is unclear whether the EGFR inhibitors gefitinib and canertinib but not HER2-specific inhibitors (i.e. tucatinib) were used as a control in the manuscript.

Reviewer #2 (Public Review):

Summary:

The findings highlight the importance of targeting the ELF3-MED23 protein-protein interaction (PPI) as a potential therapeutic strategy for HER2-overexpressing cancers, notably gastric cancers, as an alternative to trastuzumab. The evidence, including the strong potency of compound 10 in inhibiting ELF3-MED23 PPI, its capacity to lower HER2 levels, induce apoptosis, and impede proliferation both in laboratory settings and animal models, indicates that compound 10 holds promise as a novel therapeutic option, even for cases resistant to trastuzumab treatment.

Strengths:

The experiments conducted are robust and diverse enough to address the hypothesis posed.

Weaknesses:

The rationale behind the proposed structural modifications for the three groups of compounds is not clear.

Reviewer #3 (Public Review):

Summary:

The authors synthesized a compound which can inhibit ELF3 and MED23 interaction which leads to inhibition of HER2 expression in gastric cancer.

Strengths:

Enough evidence shows the potency of compound 10 in inhibiting ELF3 and MED23 interaction.

Weaknesses:

Compound 10 potency as PPI inhibitor has been shown in only one cell line NCI-N87.

Reviewer #1 (Public Review):

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

In this study, the authors build on their previously published protocols and develop a strategy that:

(1) allows for very high editing efficiency. The cell editing frequency of 1 edit per 70 cells reported in this study represents a 400-fold improvement over the previously published protocol,<br /> (2) reduces the negative effects of high sgRNA levels on parasite growth by using a weaker T7 promoter to drive sgRNA transcription.

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

Reviewer #2 (Public Review):

Summary:

Previously, the authors published a Leishmania cytosine base editor (CBE) genetic tool that enables the generation of functionally null mutants. This works by utilising a CAS9-cytidine deaminase variant that is targeted to a genetic locus by a small guide RNA (sgRNA) and causes cytosine to thymine conversion. This has the potential to generate a premature stop codon and therefore a loss of function mutant.

CBE has advantages over existing CAS-based knockout tools because it allows the targeting of multicopy gene families and, potentially, the easier generation of pooled loss of function mutants in complex population experiments. Although successful, the first generation of this genetic tool had several limitations that may have prevented its wider adoption, especially in complex genome-wide screens. These include nonspecific toxicity of the sgRNAs, low transfection efficiencies, low editing efficiencies, a proportion of transfectants that express multiple different sgRNAs, and insufficient effectivity in some Leishmania species.

Here, the authors set out to systematically solve each of these limitations. By trialling different transfection conditions and different CAS12a cut sites to promote sgRNA expression cassette integration, they increase the transfection efficiency 400-fold and ensure that only a single sgRNA expression cassette integrates that edits with high efficiencies. By trialling different T7 promoters, they significantly reduce the non-specific toxicity of sgRNA expression whilst retaining high editing efficiencies in several Leishmania species (Leishmania major, L. mexicana and L. donovani). By improving the sgRNA design, the authors predict that null mutants will be more efficiently produced after editing.

This tool will find adoption for producing null mutants of single-copy genes, multicopy gene families, and potentially genome-wide mutational analyses.

Strengths:

This is an impressive and thorough study that significantly improves the previous iteration of the CBE. The approach is careful and systematic and reflects the authors' excellent experience developing CRISPR tools. The quality of data and analysis is high and data are clearly presented.

Weaknesses:

Figure 4 shows that editing of PF16 is 'reversed' between day 6 and day 16 in L. mexicana WTpTB107 cells. The authors reasonably conclude that in drug-selected cells there is a mixed population of edited and non-edited cells, possibly due to mis-integration of the sgRNA expression construct, and non-edited cells outcompete edited cells due to a growth defect in PF16 loss of function mutants. However, this suggests that the CBE tool will not work well for producing mutants with strong fitness phenotypes without incorporating a limiting dilution cloning step (at least in L. mexicana and quite possibly other Leishmania species). Furthermore, it suggests it will not be possible to incorporate genes associated with a growth defect into a pooled drop-out screen as described in the paper. This issue is not well explored in the paper and the authors have not validated their tool on a gene associated with a severe growth defect, or shown that their tool works in a mixed population setting.

Although welcome, the improvements to the crRNA CBE design tool are hypothetical and untested.

The Sanger and Oxford Nanopore Technology analyses on integration sites of the sgRNA expression cassette integration will not detect the mis-integration of the sgRNA expression construct into an entirely different locus.

Reviewer #3 (Public Review):

Genetic manipulation of Leishmania has some challenges, including some limitations in the DNA repair strategies that are present in the organism and the absence of RNA interference in many species. The senior author has contributed significantly to expanding the available routes towards Leishmania genetic manipulation by developing and adapting CRISPR-Cas9 tools to allow gene manipulation via DNA double-strand break repair and, more recently, base modification. This work seeks to improve on some limitations in the tools previously described for the latter approach of base modification leading to base change.

The work in the paper is meticulously described, with solid evidence for most of the improvements that are claimed: Figure1 clearly describes reduced impairment in the growth of parasites expressing sgRNAs via changes in promoters; Figures 2 and 3 compellingly document the usefulness of using AsCas12a for integration after transformation; and Figures 1 and 4 demonstrate the capacity of the combined modifications to efficiently edit a gene in three different Leishmania species. There is little doubt these new tools will be adopted by the Leishmania community, adding to the growing arsenal of approaches for genetic manipulation.

There are two weaknesses the authors may wish to address, one smaller and one larger.

(1) The main advance claimed here is in this section title: 'Integration of CBE sgRNA expression cassettes via AsCas12a ultra-introduced DSBs increase editing rates', with the evidence for this presented in Figure 4. It is hard work in the submission to discern what direct evidence there is for editing rates being improved relative to earlier, Cas9-based approaches. Did they directly compare the editing by the new and old approach? If not, can they more clearly explain how they are able to make this claim, either by adding text or a new figure? A side-by-side comparison would emphasise the advance of the new approach more clearly.

(2) The ultimate, stated goal of this work is (abstract) to 'enable a variety of loss-of-function screens', as the older approach had some limitations. This goal is not tested for the new tools that have been developed here; the experiment in Figure 5 merely shows that they can, not unexpectedly, make a gene mutant, which was already possible with available tools. Thus, to what extent is this paper describing a step forward? Why have the authors not run an experiment - even the same one that was described previously in Engstler and Beneke (2023) - to show that the new approach improves on previous tools in such a screen, either in scale or accuracy?

Reviewer #1 (Public Review):

Summary:

Moir, Merheb et al. present an intriguing investigation into the pathogenesis of Pol III variants associated with neurodegeneration. They established an inducible mouse model to overcome developmental lethality, administering 5 doses of tamoxifen to initiate the knock-in of the mutant allele. Subsequent behavioral assessments and histological analyses revealed potential neurological deficits. Robust analyses of the tRNA transcriptome, conducted via northern blotting and RNA sequencing, suggested a selective deleterious effect of the variant on the cerebrum, in contrast to the cerebellum and non-cerebral tissues. Through this work, the authors identified molecular changes caused by Pol III mutations, particularly in the tRNA transcriptome, and demonstrated its relative progression and selectivity in brain tissue. Overall, this study provides valuable insights into the neurological manifestations of certain genetic disorders and sheds light on transcripts/products that are constitutively expressed in various tissues.

Strengths:

The authors utilize an innovative mouse model to constitutively knock in the gene, enhancing the study's robustness. Behavioral data collection using a spectrometer reduces experimenter bias and effectively complements the neurological disorder manifestations. Transcriptome analyses are extensive and informative, covering various tissue types and identifying stress response elements and mitochondrial transcriptome patterns. Additionally, metabolic studies involving pancreatic activity and glucose consumption were conducted to eliminate potential glucose dysfunction, strengthening the histological analyses.

Weaknesses:

The study could have explored identifying the extent of changes in the tRNA transcriptome among different cell types in the cerebrum. Although the authors attempted to show the temporal progression of tRNA transcriptome changes between P42 and P75 mice, the causal link was not established. A subsequent rescue experiment in the future could address this gap.

Nonetheless, the claims and conclusions are supported by the presented data.

Reviewer #2 (Public Review):

Summary:

The study "Molecular basis of neurodegeneration in a mouse model of Polr3 related disease" by Moir et.al. showed that how RNA Pol III mutation affects production, maturation and transport of tRNAs. Furthermore, their study suggested that RNA pol III mutation leads to behavioural deficits that are commonly observed in neurodegeneration. Although, this study used a mouse model to establish theses aspects, the study seems to lack a clear direction and mechanism as to how the altered level of tRNA affects locomotor behaviour. They should have used conditional mouse to delete the gene in specific brain area to test their hypothesis. Otherwise, this study shows a more generalized developmental effect rather than specific function of altered tRNA level. This is very evident from their bulk RNA sequencing study. This study provides some discrete information rather than a coherent story.

Strengths:

The study created a mouse model to investigate role of RNA PolIII transcription. Furthermore, the study provided RNA seq analysis of the mutant mice and highlighted expression specific transcripts affected by the RNA PolIII mutation.

Weaknesses:

1) The abstract is not clearly written. It is hard to interpret what is the objective of the study and why they are important to investigate. For example: "The molecular basis of disease pathogenesis is unknown." Which disease? 4H leukodystrophy? All neurodegenerative disease?

2) How cerebral pathology and exocrine pancreatic atrophy are related? How altered tRNA level connects these two axes?

3) Authors mentioned that previously observed reduction mature tRNA level also recapitulated in their study. Why this study is novel then?

4) It is very intuitive that deficit in Pol III transcription would severely affect protein synthesis in all brain areas as well as other organs. Hence, growth defect observed in Polr3a mutant mice is not very specific rather a general phenomenon.

5) Authors observed specific myelination defect in cortex and hippocampus but not in cerebellum. This is an interesting observation. It is important to find the link between tRNA removal and myelin depletion in hippocampus or cortex? Why is myelination not affected in cerebellum?

6) How was the locomotor activity measured? The detailed description is missing. Also, locomotion is primarily cerebellum dependent. There is no change in term of growth rate and myelination in cerebellar neurons. I do not understand why locomotor activity was measured.

7) The correlation with behavioural changes and RNA seq data is missing. There a number of transcripts are affected and mostly very general factors for cellular metabolism. Most of them are RNA Pol II transcribed. How a Pol III mutation influences RNA Pol II driven transcription? I did not find differential expression of any specific transcripts associated with behavioural changes. What is the motivation for transcriptomics analysis? None of these transcripts are very specific for myelination. It is rather a general cellular metabolism effect that indirectly influences myelination.

8) What genes identified by transcriptomics analysis regulates maturation of tRNA? Authors should at least perform RNAi study to identify possible factor and analyze their importance in maturation of tRNA.

9) What factors are influencing tRNA transport to cytoplasm? It may be possible that Polr3a mutation affect cytoplasmic transport of tRNA. Authors should study this aspect using an imaging experiment.

10) Does alteration of cytoplasmic level of tRNA affects translation? Author should perform translation assay using bio-orthoganal amino acid (AHA) labelling.

Reviewer #1 (Public Review):

In the manuscript "Cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and gates rapid phase shifts of the circadian clock", Brenna et al study the role of Cdk5 on circadian rhythms and they conclude that the CDK5 gates the activity of light on phase shifts at ZT by showing that the behavioural shifts to light as a result of CDK5 silencing only affect light-induced phase shifts at ZT/CT 14 but not at other times.

Further, they delineate the mechanism behind this phenotype and demonstrate that 1) CDK5 activity is downregulated following a light pulse via a loss of interaction with p35 and demonstrate this via an activity assay. 2) knock-down of CDK5: increases CREB, CAMK-ii/iv phosphorylation, likely via increasing calcium levels along with alterations to the localisation of Cav3.1, 3) reduces: light-induced response in vivo at ZT14 in the SCN.

They suggest this mechanism involves light 'silencing' CDK5-pathway (possibly by disrupting P35 interaction and dysregulating this pathway) which under basal conditions phosphorylates DARP32 leading to PKA inhibition and by extension reduction in activation of the calcium-calmodulin kinase activity and leading to reduced CREB activity. The authors finally evaluate gene expression changes of previously described light-responsive-genes in at ZT14 and the SCN.

This is an interesting piece of work that explains how circadian responses to light could be gated and is generally well supported by a wealth of data. Whilst I found the overall involvement of CDK5 in gating light response interesting and convincing, I have some concerns about their interpretation of the data surrounding the mechanism, which I have detailed below. I also think this manuscript could be improved with a slightly different structure and concise discussion for the benefit of a broader scientific audience.

Reviewer #2 (Public Review):

Summary:

Definition of the role of CdK5 in circadian locator activity and light induced neural activity in the mouse SCN in-vivo revealing its mode of action through PKA-CaMK-CREB signaling pathway.

Strengths:

The experimental approaches are carried from in-vivo, to cellular and molecular level and provide first evidence for the specific involvement of CdK5 in light-induced phase advance of the free-running rhythm.

Weaknesses:

The behavioral analyses are limited to some selected parameters.<br /> Downstream effects on circadian oscillation of gene expression and physiological functions in other brain regions, and organs is missing.

Reviewer #2 (Public Review):

This work provides a new tool (H3-Opt) for the prediction of antibody and nanobody structures, based on the combination of AlphaFold2 and a pre-trained protein language model, with a focus on predicting the challenging CDR-H3 loops with enhanced accuracy than previously developed approaches. This task is of high value for the development of new therapeutic antibodies. The paper provides an external validation consisting of 131 sequences, with further analysis of the results by segregating the test sets in three subsets of varying difficulty and comparison with other available methods. Furthermore, the approach was validated by comparing three experimentally solved 3D structures of anti-VEGF nanobodies with the H3-Opt predictions

Strengths:

The experimental design to train and validate the new approach has been clearly described, including the dataset compilation and its representative sampling into training, validation and test sets, and structure preparation. The results of the in silico validation are quite convincing and support the authors' conclusions.

The datasets used to train and validate the tool and the code are made available by the authors, which ensures transparency and reproducibility, and allows future benchmarking exercises with incoming new tools.

Compared to AlphaFold2, the authors' optimization seems to produce better results for the most challenging subsets of the test set.

Weaknesses:

None

Reviewer #3 (Public Review):

Summary:

The manuscript introduces a new computational framework for choosing 'the best method' according to the case for getting the best possible structural prediction for the CDR-H3 loop. The authors show their strategy improves on average the accuracy of the predictions on datasets of increasing difficulty in comparison to several state-of-the-art methods. They also show the benefits of improving the structural predictions of the CDR-H3 in the evaluation of different properties that may be relevant for drug discovery and therapeutic design.

Strengths:

The authors introduce a novel framework, which can be easily adapted and improved. Authors use a well-defined dataset to test their new method. A modest average accuracy gain is obtained in comparison to other state-of-the art methods for the same task, while avoiding testing different prediction approaches. Although the accuracy gain is mainly ascribed to easy cases, the accuracy and precision for moderate to challenging cases is comparable to the best PLM methods (see Fig. 4b and Extended Data Fig. 2), reflecting the present methodological limit in the field. The proposed method includes a confidence score for guiding users about the accuracy of the predictions.

Joint Public Review:

Identifying dietary biomarkers, in particular, has become a main focus of nutrition research in the drive to develop personalized nutrition.

The aim of this study was to determine the accuracy of using food composition databases to assess the association between dietary intake and health outcomes. The authors found that using food composition data to assess dietary intake of specific bioactives and the impact consumption has on systolic blood pressure provided vastly different outcomes depending on the method used. These findings demonstrate the difficulty in elucidating the relationship between diet and health outcomes and the need for more stringent research in the development of dietary biomarkers.

The primary strength of the study is the use of a large cohort in which dietary data and the measurement of three specific bioactives and blood pressure were collected on the same day. The bioactives selected have been extensively researched for their health effects. Another strength is that the authors controlled for as many variables as possible when running the simulations to get a more accurate account of how the variability in food composition can impact research findings that associate the intake of certain food components with health outcomes.

Reviewer #1 (Public Review):

Summary:

Gräßle et al. provide a series of four post-mortem cases of chimpanzees with PCR-proven Bacillus cereus biovar anthracis (Bcbva), who reportedly died of this infection. One control case is also provided. Compelling post-mortem Magnetic resonance imaging scans of the highest technical standards are presented. Last, the authors provide some histopathology of the brains aiming at showing the neuroinfective potential of Bcbva.

Strengths:

The merits of this study are highly acknowledged. This reviewer deems it very important to implement the latest methodology in such veterinary observational studies, in order to investigate what is going on in wildlife regarding zoonoses. The scans of five whole post-mortem chimpanzee brains with exquisite MRI technology (extremely good scan quality) represent such an implementation.

Weaknesses:

The conclusions from the necropsies are, unfortunately, on weak grounds:

(1) The authors claim that all 4 infected individuals have suffered from meningitis. However, I do not see evidence for that, neither in the gross macroscopical images provided in Figure 1. The authors claim congestion of superficial veins, at least in cases 1-3, and interpret this as pointing towards meningitis. I do not see major superficial vein congestion in any of the cases. Furthermore, vessel congestion here would rather indicate brain swelling and subsequent inhibition of venous blood outflow from the skull, which would relate to brain edema. Bacterial meningitis would itself display as clouding of the meninges, while the meninges presented in all 4 cases are perfectly translucent and gracile.

(2) The authors show a bacterial overgrowth, of brains, which was most severe in cases 1 and 2, less so in case 3, and least in case 4 (Table 1). This correlates very well with post-mortem intervals (Supplementary Table 1). The amount of bacteria is remarkable, while there is practically no brain inflammation, only moderate microglia activation. Also, the authors do not convincingly prove the proposed meningitis at the histological level, since Figure 6 does not show it in a convincing manner. Also, moderate superficial gliosis shown in Figure 6 g+h is for me not evidence of meningitis. I would expect masses of granulocytes and lymphocytes, given the amount of bacteria shown.

(3) The pattern of bacterial invasion, i.e. first confined to vessels as in case 4 with short post-mortem interval, and then overgrowing the brain with practically no glial or inflammatory reaction, is very typical of post-mortem putrefication. It is conceivable that the chimpanzees had severe bacteremia, which, after death, quickly led to bacterial invasion into the brain parenchyma. While authors state the post-mortem intervals in hours, they do not state whether bodies were immediately cooled after death.

(4) I find it difficult to see evidence of superficial siderosis in any of the images. In particular, case 2 in Figure 1 does not convincingly display leptomeningeal hemorrhage. Dark granules, e.g. shown in Figure 4 e, are very typical of so-called formalin pigment. If that would be hemosiderin or some other form of iron, it would be expected that it displays much stronger in the DAB-enhanced perls stain (Figure 4 c).

Reviewer #2 (Public Review):

In "Neuroinfectiology of an atypical anthrax-causing pathogen in wild chimpanzees" Tobias et.al. provide a detailed histologic characterization of B.cereus biovar anthracis in the brain of four wild chimpanzees in comparison to an uninfected age-matched chimp. The authors present a combination of special stains, radiography (MRI), bacterial culture, and immunohistochemistry including some quantitative image analysis to support the assessment of the neuropathogenicity of Bcbva. However, the study has major limitations that detract from the conclusions presented regarding the neurovirulence of this strain. Namely, there is a near complete lack of traditional histopathological and radiographic interpretation by qualified experts in which to frame the detailed tissue studies. The authors mention that facultative anaerobes are capable of post-mortem replication. Pathologists use comprehensive pathological assessments to determine the extent of disease caused by the primary infection, none of which is mentioned in this study (spleen, heart, lungs), which makes it difficult to determine if the findings in the brain align with the rest of the post-mortem assessment. If these were not included due to severe post-mortem autolysis, it heightens the risk of post-mortem bacterial replication in the CNS. The most important limitation is the fact that the meninges were removed and were not available for assessment therefore any comparisons with existing data on neuropathogenicity of B. anthracis is not possible. An advantage of the study is the inclusion of the control age-matched chimp, but the controls are not shown for many of the IHC and special stains - limiting interpretations. In general, the article is difficult to follow with the figures since many panels are only discussed and interpreted in the figure legends and not the text. In some cases, the results are overly technical with limited clinical insight which makes the article less easy to interpret next to human clinical reports.

Reviewer #1 (Public Review):

Summary:

The study explored the biomechanics of kangaroo hopping across both speed and animal size to try and explain the unique and remarkable energetics of kangaroo locomotion.

Strengths:

The study brings kangaroo locomotion biomechanics into the 21st century. It is a remarkably difficult project to accomplish. There is excellent attention to detail, supported by clear writing and figures.

Weaknesses:

The authors oversell their findings, but the mystery still persists. The manuscript lacks a big-picture summary with pointers to how one might resolve the big question.

General Comments

This is a very impressive tour de force by an all-star collaborative team of researchers. The study represents a tremendous leap forward (pun intended) in terms of our understanding of kangaroo locomotion. Some might wonder why such an unusual species is of much interest. But, in my opinion, the classic study by Dawson and Taylor in 1973 of kangaroos launched the modern era of running biomechanics/energetics and applies to varying degrees to all animals that use bouncing gaits (running, trotting, galloping and of course hopping). The puzzling metabolic energetics findings of Dawson & Taylor (little if any increase in metabolic power despite increasing forward speed) remain a giant unsolved problem in comparative locomotor biomechanics and energetics. It is our "dark matter problem".

This study is certainly a hop towards solving the problem. But, the title of the paper overpromises and the authors present little attempt to provide an overview of the remaining big issues. The study clearly shows that the ankle and to a lesser extent the mtp joint are where the action is. They clearly show in great detail by how much and by what means the ankle joint tendons experience increased stress at faster forward speeds. Since these were zoo animals, direct measures were not feasible, but the conclusion that the tendons are storing and returning more elastic energy per hop at faster speeds is solid. The conclusion that net muscle work per hop changes little from slow to fast forward speeds is also solid. Doing less muscle work can only be good if one is trying to minimize metabolic energy consumption. However, to achieve greater tendon stresses, there must be greater muscle forces. Unless one is willing to reject the premise of the cost of generating force hypothesis, that is an important issue to confront. Further, the present data support the Kram & Dawson finding of decreased contact times at faster forward speeds. Kram & Taylor and subsequent applications of (and challenges to) their approach supports the idea that shorter contact times (tc) require recruiting more expensive muscle fibers and hence greater metabolic costs. Therefore, I think that it is incumbent on the present authors to clarify that this study has still not tied up the metabolic energetics across speed problems and placed a bow atop the package. Fortunately, I am confident that the impressive collective brain power that comprises this author list can craft a paragraph or two that summarizes these ideas and points out how the group is now uniquely and enviably poised to explore the problem more using a dynamic SIMM model that incorporates muscle energetics (perhaps ala' Umberger et al.). Or perhaps they have other ideas about how they can really solve the problem.

I have a few issues with the other half of this study (i.e. animal size effects). I would enjoy reading a new paragraph by these authors in the Discussion that considers the evolutionary origins and implications of such small safety factors. Surely, it would need to be speculative, but that's OK.

Reviewer #2 (Public Review):

Summary

This is a fascinating topic that has intrigued scientists for decades. I applaud the authors for trying to tackle this enigma. In this manuscript, the authors primarily measured hopping biomechanics data from kangaroos and performed inverse dynamics. While these biomechanical analyses were thorough and impressively incorporated collected anatomical data and an Opensim model, I'm afraid that they did not satisfactorily address how kangaroos can hop faster and not consume more metabolic energy, unique from other animals. Noticeably, the authors did not collect metabolic data nor did they model metabolic rates using their modelling framework. Instead, they performed a somewhat traditional inverse dynamics analysis from multiple animals hopping at a self-selected speed. Within these analyses, the authors largely focused on ankle EMA, discussing its potential importance (because it affects tendon stress, which affects tendon strain energy, which affects muscle mechanics) on the metabolic cost of hopping. However, EMA was roughly estimated (CoP was fixed to the foot, not measured) and did not detectibly associate with hopping speed (see results). Yet, the authors interpret their EMA findings as though it systematically related with speed to explain their theory on how metabolic cost is unique in kangaroos vs. other animals. These speed vs. biomechanics relationships were limited by comparisons across different animals hopping at different speeds and could have been strengthened using repeated measures design. There are also multiple inconsistencies between the authors' theory on how mechanics affect energetics and the cited literature, which leaves me somewhat confused and wanting more clarification and information on how mechanics and energetics relate. My apologies for the less-than-favorable review, I think that this is a neat biomechanics study - but am unsure if it adds much to the literature on the topic of kangaroo hopping energetics in its current form.

Reviewer #3 (Public Review):

Summary:

The goal of this study is to understand how, unlike other mammals, kangaroos are able to increase hopping speed without a concomitant increase in metabolic cost. They use a biomechancial analysis of kangaroo hopping data across a range of speeds to investigate how posture, effective mechanical advantage, and tendon stress vary with speed and mass. The main finding is that a change in posture leads to increasing effective mechanical advantage with speed, which ultimately increases tendon elastic energy storage and returns via greater tendon strain. Thus kangaroos may be able to conserve energy with increasing speed by flexing more, which increases tendon strain.

Strengths:

The approach and effort invested into collecting this valuable dataset of kangaroo locomotion is impressive. The dataset alone is a valuable contribution.

Weaknesses:

Despite these strengths, I have concerns regarding the strength of the results and the overall clarity of the paper and methods used (which likely influences how convincingly the main results come across).

(1) The paper seems to hinge on the finding that EMA decreases with increasing speed and that this contributes significantly to greater tendon strain estimated with increasing speed. It is very difficult to be convinced by this result for a number of reasons:<br /> • It appears that kangaroos hopped at their preferred speed. Thus the variability observed is across individuals not within. Is this large enough of a range (either within or across subjects) to make conclusions about the effect of speed, without results being susceptible to differences between subjects? In the literature cited, what was the range of speeds measured, and was it within or between subjects?<br /> • Assuming that there is a compelling relationship between EMA and velocity, how reasonable is it to extrapolate to the conclusion that this increases tendon strain and ultimately saves metabolic cost? They correlate EMA with tendon strain, but this would still not suggest a causal relationship (incidentally the p-value for the correlation is not reported). Tendon strain could be increasing with ground reaction force, independent of EMA. Even if there is a correlation between strain and EMA, is it not a mathematical necessity in their model that all else being equal, tendon stress will increase as ema decreases? I may be missing something, but nonetheless, it would be helpful for the authors to clarify the strength of the evidence supporting their conclusions.<br /> • The statistical approach is not well-described. It is not clear what the form of the statistical model used was and whether the analysis treated each trial individually or grouped trials by the kangaroo. There is also no mention of how many trials per kangaroo, or the range of speeds (or masses) tested. Related to this, there is no mention of how different speeds were obtained. It seems that kangaroos hopped at a self-selected pace, thus it appears that not much variation was observed. I appreciate the difficulty of conducting these experiments in a controlled manner, but this doesn't exempt the authors from providing the details of their approach.<br /> • Some figures (Figure 2 for example) present means for one of three speeds, yet the speeds are not reported (except in the legend) nor how these bins were determined, nor how many trials or kangaroos fit in each bin. A similar comment applies to the mass categories. It would be more convincing if the authors plotted the main metrics vs. speed to illustrate the significant trends they are reporting.

(2) The significance of the effects of mass is not clear. The introduction and abstract suggest that the paper is focused on the effect of speed, yet the effects of mass are reported throughout as well, without a clear understanding of the significance. This weakness is further exaggerated by the fact that the details of the subject masses are not reported.

(3) The paper needs to be significantly re-written to better incorporate the methods into the results section. Since the results come before the methods, some of the methods must necessarily be described such that the study can be understood at some level without turning to the dedicated methods section. As written, it is very difficult to understand the basis of the approach, analysis, and metrics without turning to the methods.

Reviewer #1 (Public Review):

Summary:

Major findings or outcomes include a genome for the wasp, characterization of the venom constituents and teratocyte and ovipositor expression profiles, as well as information about Trichopria ecology and parasitism strategies. It was found that Trichopria cannot discriminate among hosts by age, but can identify previously parasitized hosts. The authors also investigated whether superparasitism by Trichopria wasps improved parasitism outcomes (it did), presumably by increasing venom and teratocyte concentrations/densities. Elegant use of Drosophila ectopic expression tools allowed for functional characterization of venom components (Timps), and showed that these proteins are responsible for parasitoid-induced delays in host development. After finding that teratocytes produce a large number of proteases, experiments showed that these contribute to digestion of host tissues for parasite consumption.<br /> The discussion ties these elements together by suggesting that genes used for aiding in parasitism via different parts of the parasitism arsenal arise from gene duplication and shifts in tissue of expression (to venom glands or teratocytes).

Strengths:

The strength of this manuscript is that it describes the parasitism strategies used by Trichopria wasps at a molecular and behavioral level with broad strokes. It represents a large amount of work that in previous decades might have been published in several different papers. Including all of these data in a manuscript together makes for a comprehensive and interesting study.

Weaknesses:

The weakness is that the breadth of the study results in fairly shallow mechanistic or functional results for any given facet of Trichopria's biology. Although none of the findings are especially novel given results from other parasitoid species in previous publications, integrating results together provides significant information about Trichopria biology.

Reviewer #2 (Public Review):

Summary:

Key findings of this research include the sequencing of the wasp's genome, identification of venom constituents and teratocytes, and examination of Trichopria drosophilae (Td)'s ecology and parasitic strategies. It was observed that Td doesn't distinguish between hosts based on age but can recognize previously parasitized hosts. The study also explored whether multiple parasitisms by Td improved outcomes, which indeed it did, possibly by increasing venom and teratocyte levels. Utilizing Drosophila ectopic expression tools, the authors functionally characterized venom components, specifically tissue inhibitors of metalloproteinases (Timps), which were found to cause delays in host development. Additionally, experiments revealed that teratocytes produce numerous proteases, aiding in the digestion of host tissues for parasite consumption. The discussion suggests that genes involved in different aspects of parasitism may arise from gene duplication and shifts in tissue expression to venom glands or teratocytes.

Strengths:

This manuscript provides an in-depth and detailed depiction of the parasitic strategies employed by Td wasps, spanning both molecular and behavioral aspects. It consolidates a significant amount of research that, in the past, might have been distributed across multiple papers. By presenting all this data in a single manuscript, it delivers a comprehensive and engaging study that could help future developments in the field of biological control against a major insect pest.

Weaknesses:

While none of the findings are particularly groundbreaking, as similar results have been reported for other parasitoid species in prior research, the integration of these results into one comprehensive overview offers valuable biological insights into an interesting new potential biocontrol species.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Huang et al have investigated the exercise mimetic role of Eugenol (a natural product) in skeletal muscle and whole-body fitness. The authors report that Eugenol facilitates skeletal muscle remodeling to a slower/oxidative phenotype typically associated with endurance. Eugenol also remodels the fat driving browning the WAT. In both skeletal muscle and fat Eugenol promotes oxidative capacity and mitochondrial biogenesis markers. Eugenol also improves exercise tolerance in a swimming test. Through a series of in vitro studies the authors demonstrate that eugenol may function through the trpv1 channel, Ca mobilization, and activation of CaN/NFAT signaling in the skeletal muscle to regulate slow-twitch phenotype. In addition, Eugenol also induces several myokines but mainly IL-15 through which it may exert its exercise mimetic effects. Overall, the manuscript is well-written, but there are several mechanistic gaps, physiological characterization is limited, and some data are mostly co-relative without vigorous testing (e.g. link between Eugenol, IL15 induction, and endurance). Specific major concerns are listed below.

Strengths:

A natural product activator of the TRPV1 channel that could elicit exercise-like effects through skeletal muscle remodeling. Potential for discovering other mechanisms of action of Eugenol.

Weaknesses:

(1) Figure 1: Histomorphological analysis using immunostaining for type I, IIA, IIX, and IIB should be performed and quantified across different muscle groups and also in the soleus. Fiber type switch measured based on qPCR and Westerns does not sufficiently indicate the extent of fiber type switch. Better images for Fig. 1c should be provided.

(2) Figure 2: Histomorphological analysis for SDH and NADH-TR should be performed and quantified in different muscle groups. Seahorse or oroborous respirometry experiments should be performed to determine the actually increase in mitochondrial respiratory capacity either in isolated mitochondria or single fibers from vehicle and Eugenol-treated mice. Em for mitochondrial should be added to determine the extent of mitochondrial remodeling. The current data is insufficient to indicate the extent of mitochondrial or oxidative remodeling.

(3) Figure 2: Gene expression analysis is limited to a few transcriptional factors. A thorough analysis of gene expression through RNA-seq should be performed to get an unbiased effect of Eugenol on muscle transcriptome. This is especially important because eugenol is proposed to work through CaN/NFAT signaling, major transcriptional regulators of muscle phenotype.

(4) I suggest the inclusion of additional exercise or performance testing including treadmill running, wheel running, and tensiometry. Quantification with a swimming test and measurement of the exact intensity of exercise, etc. is limited.

(5) In addition to muscle performance, whole-body metabolic/energy homeostatic effects should also be measured to determine a potential increase in aerobic metabolism over anaerobic metabolism.

(6) For the swimming test and other measurements, only 4 weeks of vehicle vs. Eugenol treatment was used. For this type of pharmacological study, a time course should be performed to determine the saturation point of the effect. Does exercise tolerance progressively increase with time?

(7) The authors should also consider measuring adaptation to exercise training with or without Eugenol.

(8) Histomorphological analysis of Wat is also lacking. EchoMRI would give a better picture of lean and fat mass.

(9) The experiments performed to demonstrate that Eugenol functions through trpv1 are mostly correlational. Some experiments are needed with trpv1 KO or KD instead of inhibitor. Similarly, KD for other trpv channels should be tested (at least 1-4 that seem to be expressed in the muscle). Triple KO or trpv null cells should be considered to demonstrate that eugenol does not have another biological target.

(10) Eugenol + trpv1 inhibition studies are performed in c2c12 cells and only looks at myofiber genes expression. This is incomplete. Some studies in mitochondrial and oxsphos genes should be done.

(11) The experiments linking Eugenol to ca handling, and calcineurin/nfat activation are all performed in c2c12 cells. There seems to be a link between Eugenol activation and CaN/NFAT activation and fiber type regulation in cells, however, this needs to be tested in mouse studies at the functional level using some of the parameters measured in aims 1 and 2.

(12) The myokine studies are incomplete. The authors show a link between Eugenol treatment and myokines/IL-15 induction. However, this is purely co-relational, without any experiments performed to show whether IL-15 mediates any of the effects of eugenol in mice.

(13) An additional major concern is that it cannot be ruled out that Engenol is uniquely mediating its effects through trpv1. Ideally, muscle-specific trpv1 mice should be used to perform some experiments with Eugenol to confirm that this ion channel is involved in the physiological effects of eugenol.

Comments on revised version:

Unfortunately, in the revision the authors have not addressed any of my comments with new experimental data. For example, some of the histological experiments I suggested are quite easy to do or standardize. Other in vitro experiments could also be conducted to show direct mechanistic link. The current revision does not further improve the manuscript from the 1st submission.

Reviewer #2 (Public Review):

Summary: