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 #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 #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 #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 #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.

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.

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 #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 #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 #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.

"Forced to call I want", implies societal pressure to put labels on feelings... What does Elio think of this? Assigning definitions based on symptoms. Based on others telling you -- this is the transition that Elio takes to become Oliver.

Will he eventually not want him? And how does this prove identity is contradictory!?

Motif of skin introduced in CMBYN, where Oliver's duality of skin, tanned, and pink and untouched represents the multidimensionality of identity, and the contradictions that exist within him -- which is what fascinates Elio. The coexistence of both contradictions in such a beautiful, whole, masterpiece who has affinities leaping out of him is enlightening for Elio. Elio may see Oliver as an Elio who he wishes to mature into.

Does this foreshadow the duality and complexity of their relationship? Because there is a period of time when Elio is in an internal conflict with his desire and lack of desire for Oliver.

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:

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 #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 #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):

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 #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 #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 #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 #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 #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 #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.

DOI: 10.7554/eLife.89516

Resource: (ZFIN Cat# ZDB-GENO-070316-1,RRID:ZFIN_ZDB-GENO-070316-1)

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-070316-1

What is this?

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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):

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

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 #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 #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 #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 #1 (Public Review):

Summary:

Shakhawat et al., investigated how enhancement of plasticity and impairment could result in the same behavioral phenotype. The authors tested the hypothesis that learning impairments result from saturation of plasticity mechanisms and had previously tested this hypothesis using mice lacking two class I major histocompatibility molecules. The current study extends this work by testing the saturation hypothesis in a Purkinje-cell (L7) specific Fmr1 knockout mouse mice, which have enhanced parallel fiber-Purkinje cell LTD. The authors found that L7-Fmr1 knockout mice are impaired on an oculomotor learning task and both pre-training, to reverse LTD, and diazepam, to suppress neural activity, eliminated the deficit when compared to controls.

Strengths:

This study tests the "saturation hypothesis" to understand plasticity in learning using a well-known behavior task, VOR, and an additional genetic mouse line with a cerebellar cell-specific target, L7-Fmr1 KO. This hypothesis is of interest to the community as it evokes novel inquisition into LTD that has not been examined previously.

Utilizing a cell-specific mouse line that has been previously used as a genetic model to study Fragile X syndrome is a unique way to study the role of Purkinje cells and the Fmr1 gene. This increases the understanding in the field in regards to Fragile X syndrome and LTD.

The VOR task is a classic behavior task that is well understood, therefore using this metric is very reliable for testing new animal models and treatment strategies. The effects of pretraining are clearly robust and this analysis technique could be applied across different behavior data sets.

The rescue shown using diazepam is very interesting as this is a therapeutic that could be used in clinical populations as it is already approved.

All previous comments have been addressed with additional studies, explanations, or analyses. These additions strengthen a very impactful study.

The authors achieved their study objectives and the results strongly support their conclusion and proposed hypothesis. This work will be impactful on the field as it uses a new Purkinje-cell specific mouse model to study a classic cerebellar task. The use of diazepam could be further analyzed in other genetic models of neurodevelopmental disorders to understand if effects on LTD can rescue other pathways and behavior outcomes.

Reviewer #1 (Public Review):

In this paper, the effects of two sensory stimuli (visual and somatosensory) on fMRI responsiveness during absence seizures were investigated in GEARS rats with concurrent EEG recordings. SPM analysis of fMRI showed a significant reduction in whole-brain responsiveness during the ictal period compared to the interictal period under both stimuli, and this phenomenon was replicated in a structurally constrained whole-brain computational model of rat brains.

The conclusion of this paper is that whole-brain responsiveness to both sensory stimuli is inhibited and spatially impeded during seizures.

The authors have revised this paper with a lot of detail.

Reviewer #1 (Public Review):

Summary:

Human Abeta42 inhibits gamma-secretase activity in biochemical assays.

Strengths:

Determination of inhibitory concentration human Abeta42 on gamma-secretase activity in biochemical assays.

Reviewer #1 (Public Review):

Summary:

The paper investigates the physiological and neural processes that relate to infants' attention allocation in a naturalistic setting. Contrary to experimental paradigms that are usually employed in developmental research, this study investigates attention processes while letting the infants free to play with three toys in the vicinity of their caregiver, which is closer to a common, everyday life context. The paper focuses on infants at 5 and 10 months of age and finds differences in what predicts attention allocation. At 5 months, attention episodes are shorter and their duration is predicted by autonomic arousal. At 10 months, attention episodes are longer, and their duration can be predicted by theta power. Moreover, theta power predicted the proportion of looking at the toys, as well as a decrease in arousal (heart rate). Overall, the authors conclude that attentional systems change across development, becoming more driven by cortical processes.

Strengths:

I enjoyed reading the paper, I am impressed with the level of detail of the analyses, and I am strongly in favour of the overall approach, which tries to move beyond in-lab settings. The collection of multiple sources of data (EEG, heart rate, looking behaviour) at two different ages (5 and 10 months) is a key strength of this paper. The original analyses, which build onto robust EEG preprocessing, are an additional feat that improves the overall value of the paper. The careful consideration of how theta power might change before, during, and in the prediction of attention episodes is especially remarkable.

Weaknesses:

The levels of EEG noise across age groups and periods of attention allocation are not controlled for. I appreciate the analysis of noise reported in supplementary materials. The analysis focuses on a broad level (average noise in 5-month-olds vs 10-month-olds) but variations might be more fine-grained (for example, noise in 5mos might be due to fussiness and crying, while at 10 months it might be due to increased movements). More importantly, noise might even be the same across age groups, but correlated to other aspects of their behaviour (head or eye movements) that are directly related to the measures of interest. Is it possible that noise might co-vary with some of the behaviours of interest, thus leading to either spurious effects or false negatives? One way to address this issue would be for example to check if noise in the signal can predict attention episodes. If this is the case, noise should be added as a covariate in many of the analyses of this paper.

Concerning cross-correlation analyses, the authors state that "Interpreting the exact time intervals over which a cross-correlation is significant is challenging". Then, they say that asymmetry is enough to conclude that attention forward predicted theta power more than vice versa. I think it could be useful to add a bit more of explanation before reaching this conclusion, explaining why such statement is correct, and how it is supported by previous work in statistics.

Finally, the cognitive process under investigation (e.g., attention) and its operationalization (e.g., duration of consecutive looking toward a toy) are not fully distinguished, but conflated instead (e.g., "attention durations"). This does not impact the quality of the work or analyses, but it slightly reduces clarity.

General Remarks<br /> In general, the authors achieved their aim in that they successfully showed the relationship between looking behaviour (as a proxy of attention), autonomic arousal, and electrophysiology. Two aspects are especially interesting. First, the fact that at 5 months, autonomic arousal predicts the duration of subsequent attention episodes, but at 10 months this effect is not present. Conversely, at 10 months, theta power predicts the duration of looking episodes, but this effect is not present in 5-month-old infants. This pattern of results suggests that younger infants have less control over their attention, which mostly depends on their current state of arousal, but older infants have gained cortical control of their attention, which in turn impacts their looking behaviour and arousal.

Reviewer #1 (Public Review):

Summary:

Peterson et al., present a series of experiments in which the Pavlovian performance (i.e. time spent at a food cup/port) of male and female rats is assessed in various tasks in which context/cue/outcome relationships are altered. The authors find no sex differences in context-irrelevant tasks, and no such differences in tasks in which the context signals that different cues will earn different outcomes. They do find sex differences, however, when a single outcome is given and context cues must be used to ascertain which cue will be rewarded with that outcome (Ctx-dep O1 task). Specifically, they find that males acquired the task faster, but that once acquired, performance of the task was more resilient in female rats against exposures to a stressor. Finally, they show that these sex differences are reflected in differential rates of c-fos expression in all three subregions of rat OFC, medial, lateral and ventral, in the sense that it is higher in females than males, and only in the animals subject to the Ctx-dep O1 task in which sex differences were observed.

Strengths:

• Well written<br /> • Experiments elegantly designed<br /> • Robust statistics<br /> • Behaviour is the main feature of this manuscript, rather than any flashy techniques or fashionable lab methodologies, and luckily the behaviour is done really well.<br /> • For the most part I think the conclusions were well supported, although I do have some slightly different interpretations to the authors in places.

Weaknesses:

The authors have done an excellent job of addressing all previous weaknesses. I have no further comments.

Reviewer #1 (Public Review):

Summary:

This study examines a hypothesized link between autism symptomatology and efference copy mechanisms. This is an important question for a number of reasons. Efference copy is both a critical brain mechanism that is key to rapid sensorimotor behaviors, and one that has important implications for autism given recent empirical and theoretical work implicating atypical prediction mechanisms and atypical reliance on priors in ASD.<br /> The authors test this relationship in two different experiments, both of which show larger errors/biases in spatial updating for those with heightened autistic traits (as measured by AQ in neurotypical (NT) individuals).

Strengths:

The empirical results are convincing - effects are strong, sample sizes are sufficient, and the authors also rule out alternative explanations (ruling out differences in motor behavior or perceptual processing per se).

Weaknesses:

My main residual concern is that the paper should be more transparent about both (1) that this study does not include individuals with autism, and (2) acknowledging the limitations of the AQ.<br /> On the first point, and I don't think this is intentional, there are several instances where the line between heightened autistic traits in the NT population and ASD is blurred or absent. For example, in the second sentence of the abstract, the authors state "Here, we examine the idea that sensory overload in ASD may be linked to issues with efference copy mechanisms". I would say this is not correct because the authors did not test individuals with ASD. I don't see a problem with using ASD to motivate and discuss this work, but it should be clear in key places that this was done using AQ in NT individuals.<br /> For the second issue, the AQ measure itself has some problems. For example, reference 38 in the paper (a key AQ paper) also shows that the AQ is skewed more male than modern estimates of ASD, suggesting that the AQ may not fully capture the full spectrum of ASD symptomatology.<br /> Of course, this does not mean that the AQ is not a useful measure (the present data clearly show that it captures something important about spatial updating during eye movements), but it should not be confused with ASD, and its limitations need to be acknowledged. My recommendation would be to do this in the title as well - e.g. note impaired visuomotor updating in individuals with "heightened autistic traits".

Suggestions for improvement:<br /> - Figure 5 is really interesting. I think it should be highlighted a bit more, perhaps even with a model that uses the results of both tasks to predict AQ scores.<br /> - Some discussion of the memory demands of the tasks will be helpful. The authors argue that memory is not a factor, but some support for this is needed.<br /> - With 3 sessions for each experiment, the authors also have data to look at learning. Did people with high AQ get better over time, or did the observed errors/biases persist throughout the experiment?

Reviewer #2 (Public Review):

Summary:

The authors generated a DNA methylation score in cord blood for detecting exposure to cigarette smoke during pregnancy. They then asked if it could be used to predict height, weight, BMI, adiposity and WHR throughout early childhood.

Strengths:

The study included two cohorts of European ancestry and one of South Asian ancestry.

Weaknesses:

(1) Numbers of mothers who self-reported any smoking was very low likely resulting in underpowered analyses.

(2) Although it was likely that some mothers were exposed to second-hand smoke and/or pollution, data on this was not available.

(3) One of the European cohorts and half of the South Asian cohort had DNA methylation measured on only 2500 CpG sites including only 125 sites previously linked to prenatal smoking.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public Review):

Summary:

In this paper, Manley and Vaziri investigate whole-brain neural activity underlying behavioural variability in zebrafish larvae. They combine whole brain (single cell level) calcium imaging during the presentation of visual stimuli, triggering either approach or avoidance, and carry out whole brain population analyses to identify whole brain population patterns responsible for behavioural variability. They show that similar visual inputs can trigger large variability in behavioural responses. Though visual neurons are also variable across trials, they demonstrate that this neural variability does not degrade population stimulus decodability. Instead, they find that the neural variability across trials is in orthogonal population dimensions to stimulus encoding and is correlated with motor output (e.g. tail vigor). They then show that behavioural variability across trials is largely captured by a brain-wide population state prior to the trial beginning, which biases choice - especially on ambiguous stimulus trials. This study suggests that parts of stimulus-driven behaviour can be captured by brain-wide population states that bias choice, independently of stimulus encoding.

Strengths:

-The strength of the paper principally resides in the whole brain cellular level imaging in a well-known but variable behaviour.

- The analyses are reasonable and largely answer the questions the authors ask.

- Overall the conclusions are well warranted.

Weaknesses:

A more in-depth exploration of some of the findings could be provided, such as:

- Given that thousands of neurons are recorded across the brain a more detailed parcelation of where the neurons contribute to different population coding dimensions would be useful to better understand the circuits involved in different computations.

- Given that the behaviour on average can be predicted by stimulus type, how does the stimulus override the brain-wide choice bias on some trials? In other words, a better link between the findings in Figures 2 and 3 would be useful for better understanding how the behaviour ultimately arises.

- What other motor outputs do the noise dimensions correlate with?

The dataset that the authors have collected is immensely valuable to the field, and the initial insights they have drawn are interesting and provide a good starting ground for a more expanded understanding of why a particular action is determined outside of the parameters experimenters set for their subjects.

Reviewer #1 (Public Review):

Summary:

This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males and females is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.

Strengths:

• Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones.

• The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation.

• Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors'.

• Includes multiple follow-up experiments, which lead to tests of internal replication and an impactful mechanistic proposal.

• Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionarily ancient.

Weaknesses:

• As stated in the summary, the authors attribute the estrogen source to neurons and there isn't evidence this is the case. The impact of the findings doesn't rest on this either.

• The d4 versus d8 esr2a mutants showed different results for aggression. The meaning and implications of this finding are not discussed, leaving the reader wondering.

• Lack of attribution of previously published work from other research groups that would provide the proper context of the present study.

• There are a surprising number of citations not included; some of the ones not included argue against the authors' claims that their findings were "contrary to expectation".

• The experimental design for studying aggression in males has flaws. A standard test like a resident-intruder test should be used.

• While they investigate males and females, there are fewer experiments and explanations for the female results, making it feel like a small addition or an aside.

• The statistics comparing "experimental to experimental" and "control to experimental" aren't appropriate.

Reviewer #1 (Public Review):

Summary:

Recent years have seen spectacular and controversial claims that loss of function of the RNA splicing factor Ptbp1 can efficiently reprogram astrocytes into functional neurons that can rescue motor defects seen in 6-hydroxydopamine (6-OHDA)-induced mouse models of Parkinson's disease (PD). This latest study is one of a series that fails to reproduce these observations, but remarkably also reports that neuronal-specific loss of function of Ptbp1 both induces expression of dopaminergic neuronal markers in striatal neurons and rescues motor defects seen in 6-OHDA-treated mice. The claims, if replicated, are remarkable and identify a straightforward and potentially translationally relevant mechanism for treating motor defects seen in PD models. However, while the reported behavioral effects are strong and were collected without sample exclusion, other claims made here are less convincing. In particular, no evidence that Ptbp1 loss of function actually occurs in striatal neurons is provided, and the immunostaining data used to claim that dopaminergic markers are induced in striatal neurons is not convincing. Furthermore, no characterization of the molecular identity of Ptbp1-deficient striatal neurons is provided using single-cell RNA-Seq or spatial transcriptomics, making it difficult to conclude that these cells are indeed adopting a dopaminergic phenotype.

Overall, while the claims of behavioral rescue of 6-OHDA-treated mice appear compelling, it is essential that these be independently replicated as soon as possible before further studies on this topic are carried out. Insights into the molecular mechanisms by which neuronal-specific loss of function of Ptbp1 induces behavioral rescue are lacking, however. Moreover, the claims of induction of neuronal identity in striatal neurons by Ptbp1 require considerable additional work to be convincing.

Strengths of the study:

(1) The effect size of the behavioral rescue in the stepping and cylinder tests is strong and significant, essentially restoring 6-OHDA-lesioned mice to control levels.

(2) Since the neurotoxic effects of 6-OHDA treatment are highly variable, the fact that all behavioral data was collected blinded and that no samples were excluded from analysis increases confidence in the accuracy of the results reported here.

Weaknesses of the study:

(1) Neurons express relatively little Ptbp1. Indeed, cellular expression levels as measured by scRNA-Seq are substantially below those of astrocytes and other non-neuronal cell types, and Ptbp1 immunoreactivity has not been observed in either striatal or midbrain neurons (e.g. Hoang, et al. Nature 2023). This raises the question of whether any recovery of Th expression is indeed mediated by the loss of function of Ptbp1 rather than by off-target effects. AAV-mediated rescue of Ptbp1 expression could help clarify this.

(2) It is not clear why dopaminergic neurons, which are not normally found in the striatum, are observed following Ptbp1 knockout. This is very similar to the now-debunked claims made in Zhou, et al. Cell 2020, but here performed using the hSyn rather than GFAP mini promoter to control AAV expression. While this is the most dramatic and potentially translationally relevant claim of the study, this claim is extremely surprising and lacks any clear mechanistic explanation for why it might happen in the first place. This observation is even more surprising in light of reports that antisense oligonucleotide-mediated knockdown of Ptbp1, which should have affected both neuronal and glial Ptbp1 expression, failed to induce expression of dopaminergic neuronal markers in the striatum (Chen, et al. eLife 2022). Selective loss of function of Ptbp1 in striatal and midbrain astrocytes likewise results in only modest changes in gene expression It is critically important that this claim be independently replicated, and that additional data be provided to conclusively show that striatal neurons are indeed expressing dopaminergic markers.

(3) More generally, since multiple spectacular and irreproducible claims of single-step glial-to-neuron reprogramming have appeared in high-profile journals in recent years, a consensus has emerged that it is essential to comprehensively characterize the identity of "transformed" cells using either single-cell RNA-Seq or spatial transcriptomics (e.g. Qian, et al. FEBS J 2021; Wang and Zhang, Dev Neurobiol 2022). These concerns apply equally to claims of neuronal subtype conversion such as those advanced here, and it is essential to provide these same datasets.

(4) Low-power images are generally lacking for immunohistochemical data shown in Figures 3 and 4, which makes interpretation difficult. DAPI images in Figure 3C do not appear nuclear. Immunostaining for Th, DAT, and Dcx in Figure 4 shows a high background and is difficult to interpret.

(5) Insights into the mechanism by which neuronal-specific loss of Ptbp1 function induces either functional recovery, or dopaminergic markers in striatal neurons, is lacking.

Reviewer #1 (Public Review):

Summary:

In this study, Zhang et al., presented an electrophysiology method to identify the layers of macaque visual cortex with high density Neuropixels 1.0 electrode. They found several electrophysiology signal profiles for high-resolution laminar discrimination and described a set of signal metrics for fine cortical layer identification.

Strengths:

There are two major strengths. One is the use of high density electrodes. The Neuropixels 1.0 probe has 20 um spacing electrodes, which can provide high resolution for cortical laminar identification. The second strength is the analysis. They found multiple electrophysiology signal profiles which can be used for laminar discrimination. Using this new method, they could identify the most thin layer in macaque V1. The data support their conclusion.

Weaknesses:

While this electrophysiology strategy is much easier to perform even in awake animals compared to histological staining methods, it provides an indirect estimation of cortical layers. A parallel histological study can provide a direct matching between the electrode signal features and cortical laminar locations. However, there are technical challenges, for example the distortions in both electrode penetration and tissue preparation may prevent a precise matching between electrode locations and cortical layers. In this case, additional micro wires electrodes binding with Neuropixels probe can be used to inject current and mark the locations of different depths in cortical tissue after recording.

Reviewer #1 (Public Review):

Using multi-region two-photon calcium imaging, the manuscript meticulously explores the structure of noise correlations (NCs) across the mouse visual cortex and uses this information to make inferences about the organization of communication channels between primary visual cortex (V1) and higher visual areas (HVAs). Using visual responses to grating stimuli, the manuscript identifies 6 tuning groups of visual cortex neurons and finds that NCs are highest among neurons belonging to the same tuning group whether or not they are found in the same cortical area. The NCs depend on the similarity of tuning of the neurons (their signal correlations) but are preserved across different stimulus sets - noise correlations recorded using drifting gratings are highly correlated with those measured using naturalistic videos. Based on these findings, the manuscript concludes that populations of neurons with high NCs constitute discrete communication channels that convey visual signals within and across cortical areas.

Experiments and analyses are conducted to a high standard and the robustness of noise correlation measurements is carefully validated. However, the interpretation of noise correlation measurements as a proxy from network connectivity is fraught with challenges. While the data clearly indicates the existence of distributed functional ensembles, the notion of communication channels implies the existence of direct anatomical connections between them, which noise correlations cannot measure.

The traditional view of noise correlations is that they reflect direct connectivity or shared inputs between neurons. While it is valid in a broad sense, noise correlations may reflect shared top-down input as well as local or feedforward connectivity. This is particularly important since mouse cortical neurons are strongly modulated by spontaneous behavior (e.g. Stringer et al, Science, 2019). Therefore, noise correlation between a pair of neurons may reflect whether they are similarly modulated by behavioral state and overt spontaneous behaviors. Consequently, noise correlation alone cannot determine whether neurons belong to discrete communication channels.

Behavioral modulation can influence the gain of sensory-evoked responses (Niell and Stryker, Neuron, 2010). This can explain why signal correlation is one of the best predictors of noise correlations as reported in the manuscript. A pair of neurons that are similarly gain-modulated by spontaneous behavior (e.g. both active during whisking or locomotion) will have higher noise correlations if they respond to similar stimuli. Top-down modulation by the behavioral state is also consistent with the stability of noise correlations across stimuli. Therefore, it is important to determine to what extent noise correlations can be explained by shared behavioral modulation.

Reviewer #1 (Public Review):

In this study, Kim et al. investigated the mechanism by which uremic toxin indoxyl sulfate (IS) induces trained immunity, resulting in augmented pro-inflammatory cytokine production such as TNF and IL-6. The authors claim that IS treatment induced epigenetic and metabolic reprogramming, and the aryl hydrocarbon receptor (AhR)-mediated arachidonic acid pathway is required for establishing trained immunity in human monocytes. They also demonstrated that uremic sera from end-stage renal disease (ESRD) patients can generate trained immunity in healthy control-derived monocytes.

These are interesting results that introduce the important new concept of trained immunity and its importance in showing endogenous inflammatory stimuli-induced innate immune memory. Additional evidence proposing that IS plays a critical role in the initiation of inflammatory immune responses in patients with CKD is also interesting and a potential advance of the field.

Comments on the revised version:

In the revised manuscripts, the authors have addressed essentially almost all of the points raised by the reviewers and have revised the manuscript accordingly. The additional comments improved the manuscript and strengthened the overall impact of the paper.

Reviewer #1 (Public Review):

Summary:

Thayer et al build upon their prior findings that ASAR long noncoding RNAs (lncRNAs) are chromatin-associated and are implicated in control of replication timing. To explore the mechanism of function of ASAR transcripts, they leveraged the ENCODE RNA binding protein eCLIP datasets to show that a 7kb region of ASAR6-141 is bound by multiple hnRNP proteins. Deletion of this 7kb region resulted in delayed chromosome 6 replication. Furthermore, ectopic integration of the ASAR6-141 7kb region into autosomes or the inactive X-chromosome also resulted in delayed chromosome replication. They then use RNA FISH experiments to show that knockdown of these hnRNP proteins disrupts ASAR6-141 localization to chromatin and in turn replication timing.

Strengths:

Given prior publications showing HNRNPU to be important for chromatin retention of XIST and Firre, this work expands upon our understanding on the role of hnRNP proteins in lncRNA function.

Weaknesses:

The work presented is mechanistically interesting, however, one must be careful with the over interpretation that hnRNP proteins can regular chromosome replication directly.

Joint Public Review:

Xie et al. propose that the asymmetric segregation of the NuRD complex is regulated in a V-ATPase-dependent manner, and plays a crucial role in determining the differential expression of the apoptosis activator egl-1 and thus critical for the life/death fate decision.

Remaining concerns are the following:

The authors should provide the point-by-point response to the following issues. In particular, authors should provide clear reasoning as to why they did not address some of the following comments in the previous revisions. The next response should be directly answering to the following concerns.

(1) Discussion should be added regarding the criticism that NuRD asymmetric segregation is simply a result of daughter cell size asymmetry. It is perfectly fine that the NuRD asymmetry is due to the daughter cell size difference (still the nucleus within the bigger daughter would have more NuRD, which can determine the fate of daughter cells). Once the authors add this clarification, some criticisms about 'control' may become irrelevant.

(2) ZEN-4 is a kinesin that predominantly associates with the midzone microtubules and a midbody during mitosis. Given that midbodies can be asymmetrically inherited during cell division, ZEN-4 is not a good control for monitoring the inheritance of cytoplasmic proteins during asymmetric cell division. Other control proteins, such as a transcriptional factor that predominantly localizes in the cytoplasm during mitosis and enters into nucleus during interphase, are needed to clarify the concern.

As for pHluorin experiments, symmetric inheritance of GFP and mCherry is not an appropriate evidence to estimate the level of pHluorin during asymmmetric Q cell division. This issue remains unsolved.

(3) Q-Q plot (quantile-quantile plot) in Figure S10 can be used for visually checking normality of the data, but it does not guarantee that the distribution of each sample is normal and has the standard deviation compared with the other samples. I recommend the authors to show the actual statistical comparison P-values for each case. The authors also need to show the number of replicate experiments for each figure panel.

The authors left inappropriate graphs in the revised manuscript. In Figure 3E, some error bars are disconnected and the other are stuck in the bars. In Figure S4C, LIN-53 in QR.a/p graph shows lines disconnected from error bars.

I am bit confused with the error bars in Figure 2B. Each dot represents a fluorescent intensity ratio of either HDA-1 or LIN-53 between the two daughter cells in a single animal. Plots are shown with mean and SEM, but several samples (for example, the left end) exhibit the SEM error bar very close to a range of min and max. I might misunderstand this graph but am concerned that Figure 2B may contain some errors in representing these data sets. I would like to ask the authors to provide all values in a table format so that the reviewers could verify the statistical tests and graph representation.

(4) The authors still do not provide evidence that the increase in sAnxV::GFP and Pegl-1gfp or the increase in H3K27ac at the egl-1 gene in hda-1(RNAi) and lin-53(RNAi) animals is not a consequence of global effects on development. Indeed, the images provided in Figure S7B demonstrate that there are global effects in these animals. no causal interactions have been demonstrated.

(5) Figure 4: Due to the lack of appropriate controls for the co-IP experiment (Fig. 4), I remain unconvinced of the claim that the NuRD complex and V-ATPase specifically interact. Concerning the co-IP, the authors now mention that the co-IP was performed three times: "Assay was performed using three biological replicates. Three independent biological replicates of the experiment were conducted with similar results." However, the authors did not use ACT-4::GFP or GFP alone as controls for their co-IP as previously suggested. This is critical considering that the evidence for a specific HDA-1::GFP - V-ATPase interaction is rather weak (compare interactions between HDA-1::GFP and V-ATPase subunits in Fig 4B with those of HDA-1::GFP and subunits of NuRD in Fig S8B).

(6) Based on Fig 5E, it appears that Bafilomycin treatment causes pleiotropic effects on animals (see differences in HDA-1::GFP signal in the three rows). The authors now state: "Although BafA1-mediated disruption of lysosomal pH homeostasis is recognized to elicit a wide array of intracellular abnormalities, we found no evidence of such pleiotropic effects at the organismal level with the dosage and duration of treatment employed in this study". However, the 'evidence' mentioned is not shown. It is critical that the authors provide this evidence.

Reviewer #2 (Public Review):

I have read the re-submission of the manuscript "The optimal clutch size revisited: separating individual quality from the parental survival costs of reproduction" by LA Winder and colleagues.

I have to say that I am quite disappointed not to see any formalisation of the mechanism that the authors have in mind to explain the results they have and to draw general conclusions from it. In my original review, I strongly recommended "improving the theoretical component of the analysis by providing a solid theoretical framework before, from it, drawing conclusions. This, at a minimum, requires [...] most importantly a mechanistic model describing the assumed relationships."

Without it, it is impossible to follow, agree or disagree with the authors and learn something from the meta-analysis other than: the clutch size-annual survival relationship has opposite slopes for manipulated and natural populations. Such a set of equations (would replace pages of verbose and) is not only necessary for the readers to be able to understand the authors' points and to clearly understand the simplifying assumptions, but also for the authors to ensure they conclusions are sound. For these reasons this is a central part of such studies, see, e.g. (Walker et al., 2008). This is supposedly replaced here by a figure (figure 5), which top-left part reads: "Parental survival costs of reproduction constrain intra-specific reproduction" - "no the effect size on fig 4 is too small". Figure 4 is the output of simulations where the authors have incorporated the mean effect on survival rate per egg from the manipulated populations into a model where they compute R0 for various increases in the annual fertility rate, and related decreases in annual survival rates, showing that along the slow-fast gradient, for balanced survival-reproduction (certainly not far from R0=1), R0 is not affected (or very little) by change in fertility-survival along the trade-off. Nowhere on this figure, do we have any information inferring that survival costs of reproduction do not constrain intra-specific reproduction. It is actually possible to build a simple mechanistic model with a trade-off mechanism that strongly affects the LRS and its variance between individuals and to would produce the exact same figure.

This is compounded in this manuscript by the constant verbose, imprecisions, outright mistakes, with a general confusion between magnitudes and variation of magnitudes, which makes it very hard to read. Let us just look at two examples illustrating my points. In the abstract, I read: " ... revealed that reproduction presented negligible costs, except when reproductive effort was forced beyond the level observed within species, to that seen between species" means nothing: what is the level of reproductive effort seen between species? I suppose the authors mean "forced beyond the maximum level observed within species, to that seen between species" or something like that. Caption figure 4:" Selection differentials (i.e., the difference in lifetime reproductive output between hypothetical control and brood-manipulated populations)" It cannot be how this was calculated however: the difference between equal things is 0, not 1. These errors and all the other imprecisions, lengthy definitions that are for some almost impossible to fathom are the direct result of trying at all costs not to use a single equation, the most important tool in the study of ecology and trade-offs in particular, in a paper on costs of reproduction.

Reviewer #1 (Public Review):

The manuscript of Davidsen and Sullivan describes an improved tRNA-seq protocol to determine aminoacyl-tRNA levels. The improvements include: (i) optimizing the Whitfeld or oxidation reaction to select aminoacyl-tRNAs from oxidation-sensitive non-acylated tRNAs; (ii) using a splint-assisted ligation to modify the tRNAs' ends for the following RT-PCR reaction; (iii) using an error-tolerating mapping algorithm to map the tRNA sequencing reads that contain mismatches at modified nucleotides.

The revised manuscript of Davidsen and Sullivan has addressed my concerns in the previous review. The authors performed a end-to-end comparison, which I requested - Fig. 2 and Fig S2. This is exactly what I meant, albeit the differences in each method to perform the comparison of the detectability. The manuscript is a strong methodological improvement of the tRNA quantification protocols!

Joint Public Review:

In this study, Kashio et al examined the role of TRPV4 in regulating perspiration in mice. They find coexpression of TRPV4 with the chloride channel ANO1 and aquaporin 5, which implies possible coupling of heat sensing through TRPV4 to ion and water excretion through the latter channels. Calcium imaging of eccrine gland cells revealed that the TRPV4 agonist GSK101 activates these cells in WT mice, but not in TRPV4 KO. This effect is reduced with cold-stimulating menthol treatment. Temperature-dependent perspiration in mouse skin, either with passive heating or with ACh stimulation, was reduced in TRPV4 KO mice. Functional studies in mice - correlating the ability to climb a slippery slope to properly regulate skin moisture levels - reveal potential dysregulation of foot pad perspiration in TRPV4 KO mice, which had fewer successful climbing attempts. Lastly, a correlation of TRPV4 to hypohydrosis in humans was shown, as anhidrotic skin showed reduced levels of TRPV4 expression compared to normohidrotic or control skin.

Overall this is an interesting study on how TRPV4 regulates perspiration.

(1) The functional relationship between TRPV3 and ANO1 remains correlative.

(2) Littermate controls were not used, but TRPV4ko were backcrossed onto the WT strain.

(3) In general, the results support the authors' claims that TRPV4 activity is a necessary component of sweat gland secretion, which may have important implications for controlling perspiration; secretion from other glands where TRPV4 may be expressed remains a possibility given the lack of us of exocrine-specific knockouts.

Reviewer #1 (Public Review):

In this study, Gu at al., investigated the role of the central noradrenaline system from LC to VLPO in the recovery of consciousness induced by midazolam. Combining pharmacology, optogenetics/chemogenetics, they found that the LC to VLPO NE circuits are essential for consciousness rebooting after midazolam, activation of this circuit strongly speeded up the recovery process, dependent on alpha1 adrenergic receptors in the VLPO neurons. The topic is important and their findings are of some interest.<br /> However, substantial improvements are needed in the language, for grammar, clarity, and layout. There are significant experimental errors (see below 1-2). Further experiments are required to support their main conclusions.

(1) One major issue arises in Figure 4, the recording of VLPO Ca2+ activity. In Lines 211-215, they stated that they injected AAV2/9-DBH-GCaMP6m into the VLPO, while activating LC NE neurons. As they claimed in line 157, DBH is a specific promoter for NE neurons. This implies an attempt to label NE neurons in the VLPO, which is problematic because NE neurons are not present in the VLPO. This raises concerns about their viral infection strategy since Ca activity was observed in their photometry recording. This means that DBH promoter could randomly label some non-NE neurons. Is DBH promoter widely used? The authors should list references. Additionally, they should quantify the labeling efficiency of both DBH and TH-cre throughout the paper.<br /> (2) A similar issue arises with chemogenetic activation in Fig. 5 L-R, the authors used TH-cre and DIO-Gq virus to label VLPO neurons. Were they labelling VLPO NE or DA neurons for recording? The authors have to clarify this.<br /> (3) Another related question pertains to the specificity of LC NE downstream neurons in the VLPO. For example, do they preferentially modulate GABAergic or glutamatergic neurons?<br /> (4) In Figure 1A-D, in the measurement of the dosage-dependent effect of Mida in LORR, were they only performed one batch of testing? If more than one batch of mice were used, error bar should be presented in 1B. Also, the rationale of testing TH expression levels after Mid is not clear. Is TH expression level change related to NE activation specifically? If so, they should cite references.<br /> (5) Regarding the photometry recording of LC NE neurons during the entire process of midazolam injection in Fig. 2 and Fig. 4, it is unclear what time=0 stands for. If I understand correctly, the authors were comparing spontaneous activity during the four phases. Additionally, they only show traces lasting for 20s in Fig. 2F and Fig. 4L. How did the authors select data for analysis, and what criteria were used? The authors should also quantify the average Ca2+ activity and Ca2+ transient frequency during each stage instead of only quantifying Ca2+ peaks. In line 919, the legend for Figure 2D, they stated that it is the signal at the BLA; were they also recorded from the BLA?

Reviewer #1 (Public Review):

Summary:

In this MS, Muenker and colleagues, explore the intracellular mechanics of a range of animal adherent cells. The study is based on the use of an optical tweezer set up, which allows to apply oscillatory forces on endocytosed/phagocytosed glass beads with a large frequency range (from ~1 to 1000 Hz) , allowing to probe cytoplasm material properties at multiple time scales. By switching off the laser trap, the authors also record the positional fluctuations of beads, to extract passive rheological signatures. The combination of both methods allow to fit 6 parameters (from power law fits) that allow to characterize the viscous and elastic nature of the cytoplasm material as well as an effective active energy driven by cellular metabolism. Using these methodologies, the authors first establish/confirm, using HeLa cells, that the cytoplasm is more solid like at short frequencies, and more fluid like at higher frequencies, and that these material states depend on both microtubules and actin cytoskeleton. The manuscript then go on to explore how these parameters evolve in other 6 cell types including muscles, highly migratory and epithelial cells. These results show for instance that muscle cells are much stiffer, while migratory cells are more fluid like with an increased active energy. Finally using statistical methods and principal component analysis, the authors establish some mechanical fingerprints (activity, fluidity and resistance) that allow to distinguish cell's mechanical state and relate it to their particular functions.

Strengths:

Overall this is a very well-executed work, which provides a large body of rigorous numbers and data to understand the regulation of cytoplasm mechanics and its relation to cell state/function.

Weaknesses:

A limit of the paper is that the biological mechanisms by which intracellular mechanics is modulated (e.g. among cell types) remains unexplored and only briefly discussed. Yet this limit is greatly offset by the rigor of the approach.

Reviewer #1 (Public Review):

Summary:

In this study, Kroll et al. conduct an in-depth behavioral analysis of F0 knockouts of 4 genes associated with late-onset Alzheimer's Disease (AD), together with 3 genes associated with early-onset AD. Kroll and colleagues developed a web application (ZOLTAR) to compare sleep-associated traits between genetic mutants with those obtained from a panel of small molecules to promote the identification of affected pathways and potential therapeutic interventions. The authors make a set of potentially important findings vis-à-vis the relationship between AD-associated genes and sleep. First, they find that loss-of-function in late-onset AD genes universally results in nighttime sleep loss, consistent with the well-supported hypothesis that sleep disruption contributes to Alzheimer's-related pathologies. psen-1, an early-onset associated AD gene, which the authors find is principally responsible for the generation of AB40 and AB42 in zebrafish, also shows a slight increase in activity at night and slight decreases in nighttime sleep. Conversely, psen-2 mutations increase daytime sleep, while appa/appb mutations have no impact on sleep. Finally, using ZOLTAR, the authors identify serotonin receptor activity as potentially disrupted in sorl1 mutants, while betamethasone is identified as a potential therapeutic to promote reversal of psen2 knockout-associated phenotypes.

This is a highly innovative and thorough study, yet a handful of key questions remain. First, are nighttime sleep loss phenotypes observed in all knockouts for late-onset AD genes in the larval zebrafish a valid proxy for AD risk? For those mutants that cause nighttime sleep disturbances, do these phenotypes share a common underlying pathway? e.g. Do 5-HT reuptake inhibitors promote sleep across all 4 late-onset genes in addition to psen1? Can 5-HT reuptake inhibitors reverse other AD-related pathologies in zebrafish? Can compounds be identified that have a common behavioral fingerprint across all or multiple AD risk genes? Do these modify sleep phenotypes? Finally, the web-based platform presented could be expanded to facilitate comparison of other behavioral phenotypes, including stimulus-evoked behaviors. Finally, the authors propose but do not test the hypothesis that sorl1 might regulate localization/surface expression of 5-HT2 receptors. This could provide exciting / more convincing mechanistic support for the assertion that serotonin signaling is disrupted upon loss of AD-associated genes. Despite these important considerations, this study provides a valuable platform for high-throughput analysis of sleep phenotypes and correlation with small-molecule-induced sleep phenotypes.

Strengths:

- Provides a useful platform for comparison of sleep phenotypes across genotypes/drug manipulations.

- Presents convincing evidence that nighttime sleep is disrupted in mutants for multiple late-onset AD-related genes.

- Provides potential mechanistic insights for how AD-related genes might impact sleep and identifies a few drugs that modify their identified phenotypes

Weaknesses:

- Exploration of potential mechanisms for serotonin disruption in sorl1 mutants is limited.

- The pipeline developed can only be used to examine sleep-related / spontaneous movement phenotypes and stimulus-evoked behaviors are not examined.

- Comparisons between mutants/exploration of commonly affected pathways are limited.

Reviewer #1 (Public Review):

In this study, Hoops et al. showed that Netrin-1 and UNC5c can guide dopaminergic innervation from nucleus accumbens to cortex during adolescence in rodent models. They found that these dopamine axons project to the prefrontal cortex in a Netrin-1 dependent manner and knocking down Netrin-1 disrupted motor and learning behaviors in mice. Furthermore, the authors used hamsters, a seasonal model that is affected by the length of daylight, to demonstrate that the guidance of dopamine axons is mediated by the environmental factor such as daytime length and in sex dependent manner.

Regarding the cell type specificity of Netrin-1 expression, the authors began by stating "this question is not the focus of the study and we consider it irrelevant to the main issue we are addressing, which is where in the forebrain regions we examined Netrin-1+ cells are present." This statement contradicts the exact issue regarding the specificity issue I raised. They then went on to show the RNAscope data for Netriin-1 in Figure 2, which showed Netrin-1 mRNA was actually expressed quite ubiquitously in anterior cingulate cortex, dorsopeduncular cortex, infralimbic cortex, prelimbic cortex, etc. In addition, contrary to the authors' statement that Netrin-1 is a "secreted protein", the confocal images in Figure 1 in the rebuttal letter actually show Netrin-1 present in "granule-like" organelles inside the cytoplasm of neurons. Finally, the authors presented Figure 7 to indicate the location where virus expressing Netrin-1 shRNA might be located. Again, the brain region targeted was quite focal and most likely did not cover all the Netrin-1+ brain regions in Figure 2. Collectively, these results raised more questions regarding the specificity of Netrin-1 expression in brain regions that are behaviorally relevant to this study.

With respect to the effectiveness of Netrin-1 knockdown in the animals in this study, the authors cited data in HEK293 cells (Figure 5), which did not include any statistics, and previously published in vivo data in a separate, independent study (Figure 6). They do not provide any data regarding the effectiveness of Netrin-1 knockdown in THIS study.

Similar concerns regarding UNC5C knockdown (points #6, #7, and #8) were not adequately addressed.

In brief, while this study provides a potential role of Netrin-1-UNC5C in target innervation of dopaminergic neurons and its behavioral output in risk-taking, the data lack sufficient evidence to firmly establish the cause-effect relationship.

Reviewer #1 (Public Review):

Summary:

This study trained a CNN for visual word classification and supported a model that can explain key functional effects of the evoked MEG response during visual word recognition, providing an explicit computational account from detection and segmentation of letter shapes to final word-form identification.

Strengths:

This paper not only bridges an important gap in modeling visual word recognition, by establishing a direct link between computational processes and key findings in experimental neuroimaging studies, but also provides some conditions to enhance biological realism.

Weaknesses:

The interpretation of CNN results, especially the number of layers in the final model and its relationship with the processing of visual words in the human brain, needs to be further strengthened.

Reviewer #1 (Public Review):

Summary:

The authors are looking to assess fragmentomics effects using the Delfi method in exonic regions (Exome sequencing). They argue that this is to make the test more cost effective by extracting this information from exome sequencing.

Strengths:

Well written and explained. Different ML approaches tried.

Weaknesses:

To assess fragmentomics in WES, it doesn't seem valid to downsample WGS. WES is generated by a different library preparations so to answer this question, it would be necessary to try this in WES samples. The coverage of WES is generally done much higher because this is necessary to assess mutation calls therefore the approach of combining seems flawed because these were not generated by the same experiment.

The authors do not really show why they included longer fragment sizes in their model that had previously been excluded from the original Delfi publication

As a proof of concept this is a good idea but really needs a bit of a rethink on the utility and impact.

Reviewer #1 (Public Review):

Summary:

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

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

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

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

Afterwards, the authors analysed the cell-specific expression patterns of the theanine biosynthesis genes, combining the same molecular tools. They concluded that theanine biosynthesis is more enriched in cluster 8 "pericycle cells" than glutamine biosynthesis (Lines 271-272). However, the statement made in Line 250 states that the highest expression levels of genes responsible for glutamine biosynthesis were observed in Clusters 1, 3, 4, 6, and 8, leading to an unclear conclusion.

The regulation of theanine biosynthesis by the MYB transcription factor family is well-established. In particular, CsMYB6, a transcription factor expressed specifically in roots, has been found to promote theanine biosynthesis by binding to the promoter of the TSI gene responsible for theanine synthesis. However, their findings indicate that CsMYB6 expression is present in Cluster 3 (SCN), Cluster 6 (cambium cells), and Cluster 1 (xylem cells) but not in Cluster 8 (pericycle cells), which is known for its high expression of CsTSI. Similarly, their scRNA-seq data indicated that CsMYB40 and CsHHO3, which activate and repress CsAlaDC expression, respectively, did not show high expression in Cluster 1 (the cell cluster with high CsAlaDC expression). Based on these findings, the authors hypothesised that transcription factors and target genes are not necessarily always highly expressed in the same cells. Nonetheless, additional evidence is essential to substantiate this presumption.

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

Additionally, the available literature suggests that the transcription factors belonging to the Lateral Organ Boundaries Domain (LBD) family play a crucial role in regulating the development of lateral roots and secondary root growth. Considering this, they confirmed that pericycle cells exhibit a higher expression of CsLBD37. A recent experiment revealed that overexpression of CsLBD37 in transgenic Arabidopsis thaliana plants led to fewer lateral roots than the wild type. From this observation, the researchers concluded that CsLBD37 regulates lateral root development in tea plants. I respectfully submit that the current conclusion may require additional research before it can be considered definitive.

Further efforts should be made to investigate the signalling mechanisms that govern CsLBD37 expression to arrive at a more comprehensive understanding of this process. In the context of Arabidopsis lateral root founder cells, the establishment of asymmetry is regulated by LBD16/ASL18 and other related LBD/ASL proteins, as well as the AUXIN RESPONSE FACTORs (ARF7 and ARF19). This is achieved by activating plant-specific transcriptional regulators such as LBD16/ASL18 (Go et al., 2012, https://doi.org/10.1242/dev.071928). On the other hand, other downstream homologues of LBD genes regulated by cytokinin signalling play a role in secondary root growth (Ye et al., 2021, https://doi.org/10.1016/j.cub.2021.05.036). It is imperative to shed light on the hormonal regulation of CsLBD37 expression in order to gain a comprehensive understanding of its involvement in the morphogenic process.

Strength:

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

Weaknesses:

In its current form, the article presents certain weaknesses that need to be addressed to improve its overall quality. Specifically, the authors' conclusions appear to have been drawn in haste without sufficient experimental data and a comprehensive discussion of the entire plant. It is strongly advised that the authors devote additional effort to resolving the abovementioned issues to bolster the article's credibility and dependability. This will ensure that the article is of the highest quality, providing readers with reliable and trustworthy information.

Reviewer #1 (Public Review):

In this study, Sarver and colleagues carried out an exhaustive analysis of the functioning of various components (Complex I/II/IV) of the mitochondrial electron transport chain (ETC) using a real-time cell metabolic analysis technique (commonly referred as Seahorse oxygen consumption rate (OCR) assay). The authors aimed to generate an atlas of ETC function in about 3 dozen tissue types isolated from all major mammalian organ systems. They used a recently published improvised method by which ETC function can be quantified in freshly frozen tissues. This method enabled them to collect data from almost all organ systems from the same mouse and use many biological replicates (10 mice/experiment) required for an unbiased and statistically robust analysis. Moreover, they studied the influence of sex (male and female) and aging (young adult and old age) on ETC function in these organ systems. The main findings of this study are (1) cells in the heart and kidneys have very active ETC complexes compared to other organ systems, (2) the sex of the mice has little influence on the ETC function, and (3) aging undermined the mitochondrial function in most tissue, but surprisingly in some tissue aging promoted the activity of ETC complexes (e.g., Quadriceps, plantaris muscle, and Diaphragm). Although this study provides a comprehensive outlook on the ETC function in various tissues, the main caveat is that it's too technical and descriptive. The authors didn't invest much effort in putting their findings in the context of the biological function of the tissue analyzed, i.e., some tissues might be more glycolytic than others and have low ETC activity. Also, it is unclear what slight changes in the activity of one or the other ETC complex mean in terms of mitochondrial ATP production. Likely, these small changes reported do not affect the mitochondrial respiration. With such a detailed dataset, the study falls short of deriving more functionally relevant conclusions about the heterogeneity of mitochondrial function in various tissues. In the current format, the readers get lost in the large amount of data presented in a technical manner. Also, it is highly recommended that all the raw data and the values be made available as an Excel sheet (or other user-friendly formats) as a resource to the community.

Major concerns

(1) In this study, the authors used the method developed by Acin-Perez and colleagues (EMBO J, 2020) to analyze ETC complex activities in mitochondria derived from the snap-frozen tissue samples. However, the preservation of cellular/mitochondrial integrity in different types of tissues after being snap-frozen was not validated. Additionally, the conservation of mitochondrial respiration in snap-frozen tissues might differ, especially in those derived from old mice. For example, quadriceps (young male/female), plantaris (young male/female), intestinal segments (duodenum), and pancreas preparations show almost no activity (nearly flat OCR in Seahorse assays). For such a comprehensive study, the author must at least validate those tissues where the OCR plots looked suboptimal with the mitochondrial preparations derived from the fresh tissue. Since aging has been identified as the most important effector in this study, it is essential to validate how aging affects respiration in various fresh frozen tissues. Such analysis will ensure that the results presented are not due to the differential preservation of the mitochondrial respiration in the frozen tissue. In addition, such validations will further strengthen the conclusions and promote the broad usability of this "new" method.

(2) In this study, the authors sampled the maximal activity of ETC complex I, II, and IV, but throughout the manuscript, they discussed the data in the context of mitochondrial function. However, it is unclear how the changes in CI, CII, and CIV activity affect overall mitochondrial function (if at all) and how small changes seen in the maximal activity of one or more complexes affect the efficiency and efficacy of ATP production (OxPhos). The authors report huge variability between the activity of different complexes - in some tissues all three complexes (CI, CII, and CIV) and often in others, just one complex was affected. For example, as presented in Figure 4, there is no difference in CI activity in the hippocampus and cerebellum, but there is a slight change in CII and CIV activity. In contrast, in heart atria, there is a change in the activity of CI but not in CII and CIV. However, the authors still suggest that there is a significant difference in mitochondrial activity (e.g., "Old males showed a striking increase in mitochondrial activity via CI in the heart atria....reduced mitochondrial respiration in the brain cortex..." - Lines 5-7, Page 9). Until and unless a clear justification is provided, the authors should not make these broad claims on mitochondrial respiration based on small changes in the activity of one or more complexes (CI/CII/CIV). With such a data-heavy and descriptive study, it is confusing to track what is relevant and what is not for the functioning of mitochondria.

(3) What do differences in the ETC complex CI, CII, and CIV activity in the same tissue mean? What role does the differential activity of these complexes (CI, CII, and CIV) play in mitochondrial function? What do changes in Oxphos mean for different tissues? Does that mean the tissue (cells involved) shift more towards glycolysis to derive their energy? In the best world, a few experiments related to the glycolytic state of the cells would have been ideal to solidify their finding further. The authors could have easily used ECAR measurements for some tissues to support their key conclusions.

(4) The authors further analyzed parameters that significantly changed across their study (Figure 7, 98 data points analyzed). The main caveat of such analysis is that some tissue types would be represented three or even more times (due to changes in the activity of all three complexes - CI, CII, and CIV, and across different ages and sexes), and some just once. Such a method of analysis will skew the interpretation towards a few over-represented organ/tissue systems. Perhaps the authors should separately analyze tissue where all three complexes are affected from those with just one affected complex.

(5) The current protocol does not provide cell-type-specific resolution and will be unable to identify the cellular source of mitochondrial respiration. This becomes important, especially for those organ systems with tremendous cellular heterogeneity, such as the brain. The authors should discuss whether the observed changes result from an altered mitochondria respiratory capacity or if changes in proportions of cell types in the different conditions studied (young vs. aged) might also contribute to differential mitochondrial respiration.

(6) Another critical concern of this study is that the same datasets were repeatedly analyzed and reanalyzed throughout the study with almost the same conclusion - namely, aging affects mitochondrial function, and sex-specific differences are limited to very few organs. Although this study has considerable potential, the authors missed the chance to add new insights into the distinct characteristics of mitochondrial activity in various tissue and organ systems. The author should invest significant efforts in putting their data in the context of mitochondrial function.

Reviewer #1 (Public Review):

Summary

Type 1 diabetes mellitus (T1DM) progression is accelerated by oxidative stress and apoptosis. Eugenol (EUG) is a natural compound previously documented as anti-inflammatory, anti-oxidative, and anti-apoptotic. In this manuscript by Jiang et al., the authors study the effects of EUG on T1DM in MIN6 insulinoma cells and a mouse model of chemically induced T1DM. The authors show that EUG increases nuclear factor E2-related factor 2 (Nrf2) levels. This results in a reduction of pancreatic beta-cell damage, apoptosis, oxidative stress markers, and a recovery of insulin secretion. The authors highlight these effects as indicative of the therapeutic potential of EUG in managing T1DM.

Strengths

Relevant, timely, and addresses an interesting question in the field. The authors consistently observe enhanced beta cell functionality following EUG treatment, which makes the compound a promising candidate for T1DM therapy.

Weaknesses

The in vivo experiments have too few biological replicates. With an n=3 (as all figure legends indicate) in complex mouse studies such as these, drawing robust conclusions becomes challenging. It is important to reproduce these results in a larger cohort, to validate the conclusions of the authors. Another big concern is the lack of quantifications and statistical analysis throughout the manuscript. Although the authors claim statistical significance in various experiments, the limited information provided makes it difficult to verify. The authors use vague and minimal descriptions of their experiments, which further reduces the reader's comprehension and the reproducibility of the experiments. Finally, the use of Min6 cells as a model for pancreatic beta cells is a strong limitation of this study. Future studies should seek to reproduce these findings in a more translational model and use more relevant in vitro cell systems (eg. Islets).

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors used a coarse-grained DNA model (cgNA+) to explore how DNA sequences and CpG methylation/hydroxymethylation influence nucleosome wrapping energy and the probability density of optimal nucleosomal configuration. Their findings indicate that both methylated and hydroxymethylated cytosines lead to increased nucleosome wrapping energy. Additionally, the study demonstrates that methylation of CpG islands increases the probability of nucleosome formation.

Strengths:

The major strength of this method is that the model explicitly includes elastic constraints on the positions of phosphate groups facing a histone octamer, as DNA-histone binding site constraints. The authors claim that their model enhances the accuracy and computational efficiency and allows comprehensive calculations of DNA mechanical properties and deformation energies.

Weaknesses:

A significant limitation of this study is that the parameter sets for the methylated and hydroxymethylated CpG steps in the cgNA+ model are derived from all-atom molecular dynamics (MD) simulations that suggest that both methylated and hydroxymethylated cytosines increase DNA stiffness and nucleosome wrapping energy (Pérez A, et al. Biophys J. 2012; Battistini, et al. PLOS Comput Biol. 2021). It could predispose the coarse-grained model to replicate these findings. Notably, conflicting results from other all-atom MD simulations, such as those by Ngo T in Nat. Commun. 2016, shows that hydroxymethylated cytosines increase DNA flexibility, contrary to methylated cytosines. If the cgNA+ model was trained on these later parameters or other all-atom force fields, different conclusions might be obtained regarding the effects of methylated and hydroxymethylation on nucleosome formation.

Despite the training parameters of the cgNA+ model, the results presented in the manuscript indicate that methylated cytosines increase both DNA stiffness and nucleosome wrapping energy. However, when comparing nucleosome occupancy scores with predicted nucleosome wrapping energies and optimal configurations, the authors find that methylated CGIs exhibit higher nucleosome occupancies than unmethylated ones, which seems to contradict their findings from the same paper which showed that increased stiffness should reduce nucleosome formation affinity. In the manuscript, the authors also admit that these conclusions "apparently runs counter to the (perhaps naive) intuition that high nucleosome forming affinity should arise for fragments with low wrapping energy". Previous all-atom MD simulations (Pérez A, et al. Biophys J. 2012; Battistini, et al. PLOS Comput Biol. 202; Ngo T, et al. Nat. Commun. 20161) show that the stiffer DNA upon CpG methylation reduces the affinity of DNA to assemble into nucleosomes or destabilizes nucleosomes. Given these findings, the authors need to address and reconcile these seemingly contradictory results, as the influence of epigenetic modifications on DNA mechanical properties and nucleosome formation are critical aspects of their study.<br /> Understanding the influence of sequence-dependent and epigenetic modifications of DNA on mechanical properties and nucleosome formation is crucial for comprehending various cellular processes. The authors' study, focusing on these aspects, will definitely garner interest from the DNA methylation research community.

Reviewer #1 (Public Review):

Summary:

HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase1 is predicted to be involved in Acetyl-CoA metabolic process and mevalonate-cholesterol pathway. To induce diet-induced diabetes, they fed wild-type littermates either a standard chow (Control) or a high fat-high sucrose (HFHG) diet, where the diet composition consisted of 60% fat, 20% protein, and 20% carbohydrate (H10060, Hfkbio, China). The dietary regimen was maintained for 14 weeks. Throughout this period, body weight and fasting blood glucose (FBG) levels were measured on a weekly basis. Although the authors induced diabetes with a diet also rich in fat, the cholesterol concentration or metabolism was not investigated. After the treatment, were the animals with endothelial dysfunction? How was the blood pressure of the animals?

Strengths:

To explore the potential role of circHMGCS1 in regulating endothelial cell function, the authors cloned exons 2-7 of HMGCS1 into lentiviral vectors for ectopic overexpression of circHMGCS1 (Figure S2). The authors could use this experiment as a concept proof and investigate the glucose concentration in the cell culture medium. Is the pLV-circ HMGCS1 transduction in HUVEC increasing the glucose release? (Line 163)

Weaknesses:

(1) Pg 20. The cells were transfected with miR-4521 mimics, miR-inhibitor, or miR-NC and incubated for 24 hours. Subsequently, the cells were treated with PAHG for another 24 hours.

Were the cells transfected with lipofectanine? The protocol or the lipofectamine kit used should be described. The lipofectamine protocol suggests using an incubation time of 72 hours. Why did the authors incubate for only 24 hours?

If the authors did the mimic and inhibitor curves, these should be added to the supplementary figures. Please, describe the miRNA mimic and antagomir concentration used in cell culture.

(2) Pg 20, line 507. What was the miR-4521 agomiR used to treatment of the animals?

(3) Figure 1B. The results are showing the RT-qPCR for only 5 circRNA, however, the results show 48 circRNAs were upregulated, and 18 were downregulated (Figure S1D). Why were the other cicRNAs not confirmed? The circRNAs upregulated with high expression are not necessarily with the best differential expression comparing control vs. PAHG groups. Furthermore, Figure 1A and S1D show circRNAs downregulated also with high expression. Why were these circRNAs not confirmed?

(4) Figure 1B shows the relative circRNAs expression. Were host genes expressed in the same direction?

(5) Line 128. The circRNA RT-qPCR methodology was not described. The methodology should be described in detail in the Methods Session.

(6) Line 699. The relative gene expression was calculated using the 2-ΔΔCt method. This is not correct, the expression for miRNA and gene expression are represented in percentage of control.

(7) Line 630. Detection of ROS for tissue and cells. The methodology for tissue was described, but not for cells.

(8) Line 796. RNA Fluorescent In Situ Hybridization (RNA-FISH). Figure 1F shows that the RNA-Fluorescence in situ hybridization (RNA-FISH) confirmed the robust expression of cytoplasmic circHMGCS1 in HUVECs (Figure 1F). However, in the methods, lines 804 and 805 described the probes targeting circMAP3K5 and miR-4521 were applied to the sections. Hybridization was performed in a humid chamber at 37{degree sign}C overnight. Is it correct?

(9) Line 14. Fig 1-H. The authors discuss qRT-PCR demonstrated that circHMGCS1 displayed a stable half-life exceeding 24 h, whereas the linear transcript HMGCS1 mRNA had a half-life less than 8 h (Figure 1H).<br /> Several of the antibodies may contain trace amounts of RNases that could degrade target RNA and could result in loss of RNA hybridization signal or gene expression. Thus, all of the solutions should contain RNase inhibitors. The HMGCS1 mRNA expression could be degraded over the incubation time (0-24hs) leading to incorrect results. Moreover, in the methods is not mentioned if the RNAse inhibitor was used. Please, could the authors discuss and provide information?

(10) Further experiments demonstrated that the overexpression of circHMGCS1 stimulated the expression of adhesion molecules (VCAM1, ICAM1, and ET-1) (Figures 2B and 2C), suggesting that circHMGCS1 is involved in VED. How were these genes expressed in the RNA-seq?

(11) Line 256. By contrast, the combined treatment of circHMGCS1 and miR-4521 agomir did not significantly affect the body weight and blood glucose levels. OGTT and ITT experiments demonstrated that miR-4521 agomir considerably enhanced glucose tolerance and insulin resistance in diabetic mice (Figures 5C, 5D, and Figures S5B and S5C). Why didi the miR-4521 agomir treatment considerably enhance glucose tolerance and insulin resistance in diabetic mice, but not the blood glucose levels?

(12) In the experiments related to pull-down, the authors performed Biotin-coupled miR-4521 or its mutant probe, which was employed for circHMGCS1 pull-down. This result only confirms the Luciferase experiments shown in Figure 4A. The experiment that the authors need to perform is pull-down using a biotin-labeled antisense oligo (ASO) targeting the circHMGCS1 backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads to capture the associated miRNAs and RNA binding proteins (RBPs). Also, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA.

(13) In Figure 5, the authors showed that the results suggest that miR-4521 can inhibit the occurrence of diabetes, whereas circHMGCS1 specifically dampens the function of miR-4521, weakening its protective effect against diabetes. In this context, what are the endogenous target genes for the miR-4521 that could be regulating diabetes?

(14) In the western blot of Figure 5, the β-actin band appears to be different from the genes analyzed. Was the same membrane used for the four proteins? The Ponceau S membrane should be provided.

(15) Why did the authors use AAV9, since the AAV9 has a tropism for the liver, heart, skeletal muscle, and not to endothelial vessels?

Reviewer #1 (Public Review):

Summary:

This is a well-written and detailed manuscript showing important results on the molecular profile of 4 different cohorts of female patients with lung cancer.

The authors conducted comprehensive multi-omic profiling of air-pollution-associated LUAD to study the roles of the air pollutant BaP. Utilizing multi-omic clustering and mutation-informed interface analysis, potential novel therapeutic strategies were identified.

Strengths:

The authors used several different methods to identify potential novel targets for therapeutic interventions.

Weaknesses:

Statistical test results need to be provided in comparisons between cohorts.

Reviewer #1 (Public Review):

Summary:

This manuscript reports the substrate-bound structure of SiaQM from F. nucleatum, which is the membrane component of a Neu5Ac-specific Tripartite ATP-dependent Periplasmic (TRAP) transporter. Until recently, there was no experimentally derived structural information regarding the membrane components of the TRAP transporter, limiting our understanding of the transport mechanism. Since 2022, there have been 3 different studies reporting the structures of the membrane components of Neu5Ac-specific TRAP transporters. While it was possible to narrow down the binding site location by comparing the structures to proteins of the same fold, a structure with substrate bound has been missing. In this work, the authors report the Na+-bound state and the Na+ plus Neu5Ac state of FnSiaQM, revealing information regarding substrate coordination. In previous studies, 2 Na+ ion sites were identified. Here, the authors also tentatively assign a 3rd Na+ site. The authors reconstitute the transporter to assess the effects of mutating the binding site residues they identified in their structures. Of the 2 positions tested, only one of them appears to be critical to substrate binding.

Strengths:

The main strength of this work is the capture of the substrate-bound state of SiaQM, which provides insight into an important part of the transport cycle.

Weaknesses:

The main weakness is the lack of experimental validation of the structural findings. The authors identified the Neu5Ac binding site, but only tested 2 residues for their involvement in substrate interactions, which was very limited. The authors tentatively identified a 3rd Na+ binding site, which if true would be an impactful finding, but this site was not tested for its contribution to Na+ dependent transport, and the authors themselves report that the structural evidence is not wholly convincing. This lack of experimental validation undermines the confidence of the findings. However, the reporting of these new data is important as it will facilitate follow-up studies by the authors or other researchers.

Reviewer #1 (Public Review):

In this manuscript by Wu et al., the authors present the high resolution cryoEM structures of the WT Kv1.2 voltage-gated potassium channel. Along with this structure the authors have solved several structures of mutants or experimental conditions relevant to the slow inactivation process that these channels undergo and which is not yet completely understood.

One of the main findings is the determination of the structure of a mutant (W366F) that is thought to correspond to the slow inactivated state. These experiments confirm results in similar mutants in different channels from Kv1.2 that indicate that inactivation is associated with an enlarged selectivity filter.

Another interesting structure is the complex of Kv1.2 with the pore blocking toxin Dendrotoxin 1. The results shown in the revised version indicate that the mechanism of block is similar to that of related blocking-toxins, in which a lysine residue penetrates in the pore. Surprisingly, in these new structures, the bound toxin results in a pore with empty external potassium binding sites.

The quality of the structural data presented in this revised manuscript is very high and allows for unambiguous assignment of side chains. The conclusions are supported by the data. This is an important contribution that should further our understanding of voltage-dependent potassium channel gating. In the revised version, the authors have addressed my previous specific comments, which are appended below.

(1) In the main text's reference to Figure 2d residues W18' and S22' are mentioned but are not labeled in the insets.

(2) On page 8 there is a discussion of how the two remaining K+ ions in binding sites S3 and S4 prevent permeation K+ in molecular dynamics. However, in Shaker, inactivated W434F channels can sporadically allow K+ permeation with normal single-channel conductance but very reduced open times and open probability at not very high voltages.

(3) The structures of WT in the absence of K+ shows a narrower selectivity filter, however Figure 4 does not convey this finding. In fact, the structure in Figure 4B is constructed in such an angle that it looks as if the carbonyl distances are increased, perhaps this should be fixed. Also, it is not clear how the distances between carbonyls given in the text on page 12 are measured. Is it between adjacent or kitty-corner subunits?

(4) It would be really interesting to know the authors opinion on the driving forces behind slow inactivation. For example, potassium flux seems to be necessary for channels to inactivate, which might indicate a local conformational change is the trigger for the main twisting events proposed here.

Taming The Tinny Typing Sound of a Brother JP-1 Typewriter (Part 3 – the Finale?) by [[Theodore Munk]]

Adjusting Ring & Cylinder on a Brother JP-1 by [[Theodore Munk]]

Reviewer #1 (Public Review):

General comments:

This paper investigates the pH-specific enzymatic activity of mouse acidic mammalian chitinase (AMCase) and aims to elucidate its function's underlying mechanisms. The authors employ a comprehensive approach, including hydrolysis assays, X-ray crystallography, theoretical calculations of pKa values, and molecular dynamics simulations to observe the behavior of mouse AMCase and explore the structural features influencing its pH-dependent activity.

The study's key findings include determining kinetic parameters (Kcat and Km) under a broad range of pH conditions, spanning from strong acid to neutral. The results reveal pH-dependent changes in enzymatic activity, suggesting that mouse AMCase employs different mechanisms for protonation of the catalytic glutamic acid residue and the neighboring two aspartic acids at the catalytic motif under distinct pH conditions.<br /> The novelty of this research lies in the observation of structural rearrangements and the identification of pH-dependent mechanisms in mouse AMCase, offering a unique perspective on its enzymatic activity compared to other enzymes. By investigating the distinct protonation mechanisms and their relationship to pH, the authors reveal the adaptive nature of mouse AMCase, highlighting its ability to adjust its catalytic behavior in response to varying pH conditions. These insights contribute to our understanding of the pH-specific enzymatic activity of mouse AMCase and provide valuable information about its adaptation to different physiological conditions.<br /> Overall, the study enhances our understanding of the pH-dependent activity and catalytic properties of mouse AMCase and sheds light on its adaptation to different physiological pH environments.

Comments on revised version:

In their revised manuscript, the authors have made significant efforts to address the reviewers' comments.

Reviewer #1 (Public Review):

This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.

However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts. Additionally, it is noted that the main claims put forth in the manuscript are only partially supported by the data presented.

Reviewer #1 (Public Review):

This manuscript presents a pipeline incorporating a deep generative model and peptide property predictors for the de novo design of peptide sequences with dual antimicrobial/antiviral functions. The authors synthesized and experimentally validated three peptides designed by the pipeline, demonstrating antimicrobial and antiviral activities, with one leading peptide exhibiting antimicrobial efficacy in animal models. However, the manuscript as it stands, has several major limitations on the computational side.

Major issues:

(1) The choice of GAN as the generative model. There are multiple deep generative frameworks (e.g., language models, VAEs, and diffusion models), and GANs are known for their training difficulty and mode collapse. Could the authors elaborate on the specific rationale behind choosing GANs for this task?

(2) The pipeline is supposed to generate peptides showing dual properties. Why were antiviral peptides not used to train the GAN? Would adding antiviral peptides into the training lead to a higher chance of getting antiviral generations?

(3) For the antimicrobial peptide predictor, where were the contact maps of peptides sourced from?

(4) Morgan fingerprint can be used to generate amino acid features. Would it be better to concatenate ESM features with amino acid-level fingerprints and use them as node features of GNN?

(5) Although the number of labeled antiviral peptides may be limited, the input features (ESM embeddings) should be predictive enough when coupled with shallow neural networks. Have the authors tried simple GNNs on antiviral prediction and compared the prediction performance to those of existing tools?

(6) Instead of using global alignment to get match scores, the authors should use local alignment.

(7) How novel are the validated peptides? The authors should run a sequence alignment to get the most similar known AMP for each validated peptide, and analyze whether they are similar.

(8) Only three peptides were synthesized and experimentally validated. This is too few and unacceptable in this field currently. The standard is to synthesize and characterize several dozens of peptides at the very least to have a robust study.

Reviewer #1 (Public Review):

Summary:

This work shows, based on basic laboratory investigations of in vitro grown bacteria as well as human bone samples, that conventional bacterial culture can substantially underrepresent the quantity of bacteria in infected tissues. This has often been mentioned in the literature, however, relatively limited data has been provided to date. This manuscript compares culture to a digital droplet PCR approach, which consistently showed greater levels of bacteria across the experiments (and for two different strains).

Strengths:

Consistency of findings across in vitro experiments and clinical biopsies. There are real-world clinical implications for the findings of this study.

Weaknesses:<br /> No major weaknesses. Only 3 human samples were analyzed, although the results are compelling.

Reviewer #1 (Public Review):

Summary:

This manuscript by Vuong and colleagues reports a study that pooled data from 3 separate longitudinal study that collectively spanned an observation period of over 15 years. The authors examined for correlation between viraemia measured at various days from illness onset with thrombocytopaenia and severe dengue, according to the WHO 2009 classification scheme. The motivation for this study is both to support the use of viraemia measurement as a prognostic indicator of dengue and also to, when an antiviral drug becomes licensed for use, guide the selection of patients for antiviral therapy. They found that the four DENVs show differences in peak and duration of viraemia and that viraemia levels before day 5 but not those after from illness onset correlated with platelet count and plasma leakage at day 7 onwards. They concluded that the viraemia kinetics call for early measurement of viraemia levels in the early febrile phase of illness.

Strengths:

This is a unique study due to the large sample size and longitudinal viraemia measurements in the study subjects. The data addresses a gap in information in the literature, where although it has been widely indicated that viraemia levels are useful when collected early in the course of illness, this is the first time anyone has systematically examined this notion. The inclusion of correlation between rate of viraemia decline and risk of severe dengue/plasma leakage further strengthens the relevance of this paper to those interested in anti-dengue therapeutic research and development.

Weaknesses:

The study only analysed data from dengue patients in Vietnam. Moreover, the majority of these patients had DENV-1 infection; few had DENV-4 infection. The data could thus be skewed by the imbalance in the prevalence of the different types of DENV during the period of observation. The use of patient-reported time of symptom onset as a reference point for viraemia measurement is pragmatic although there is subjectivity and thus noise in the data.

Reviewer #1 (Public Review):

Summary:

Authors previously demonstrated that species-specific variation in primate CD4 impacts its ability to serve as a functional receptor for diverse SIVs. Here, Warren and Barbachano-Guerrero et al. perform population genetics analyses and functional characterization of great ape CD4 with a particular focus on gorillas, which are natural hosts of SIVgor. They first used ancestral reconstruction to derive the ancestral hominin and hominid CD4. Using pseudotyped viruses representing a panel of envelopes from SIVcpz and HIV strains, they find that these ancestral reconstructions of CD4 are more similar to human CD4 in terms of being a broadly susceptible entry receptor (in the context of mediating entry into Cf2Th cells stably expressing human CCR5). In contrast, extant gorilla and chimpanzee CD4 are functional entry receptors for a narrower range of HIV and SIVcpz isolates. Based on these differences, authors next surveyed gorilla sequences and identified several CD4 haplotypes, specifically in the region encoding the CD4 D1 domain, which directly contacts the viral glycoprotein and thus may impact the interaction. Consistent with this possibility, authors demonstrated that gorilla CD4 haplotypes are, on average, less capable of supporting entry than human CD4, and that some are largely unable to function as SIV entry receptors. Interestingly, individual residues found at key positions in the gorilla CD4 D1 when tested in the context of human CD4 reduce entry of some virions pseudotyped with diverse SIVcpz envelopes, suggesting that individual amino acids can in part explain the observed differences across gorilla CD4 haplotypes. Finally, the authors perform statistical tests to infer that CD4 from great apes with endemic SIV (i.e., chimpanzees and gorillas) but not non-reservoirs (i.e., orangutans, bonobos) or recent spillover hosts (i.e., humans), have been subject to selection as a result of pressure from endemic SIV.

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

Strengths:

(1) The functional assays are appropriate to test the stated hypothesis, and the authors use a broad diversity of envelopes from HIV and SIVcpz strains. Authors also partially characterize one potential mechanism of gorilla CD4 resistance - receptor glycosylation at the derived N15 found in 5/6 gorilla haplotypes.

(2) Ancestral reconstruction provides a particularly interesting aspect of the study, allowing authors to infer the ancestral state of hominid CD4 relative to modern CD4 from gorillas and chimpanzees. This, coupled with evidence supporting SIV-driven selection of gorilla CD4 diversity and the characterization of functional diversity of extant haplotypes provides several interesting findings.

Weaknesses:

(3). The major inference of the work is that SIV infection of gorillas drove the observed diversity in gorilla CD4. This is supported by the majority of SNPs being localized to the CD4 D1, which directly interacts with envelope, and the demonstrated functional consequences of that diversity for viral entry. However, SIVgor (to the best of my knowledge) only infects Western lowland gorillas (Gorilla gorilla gorilla), and one Gorilla gorilla diehli and three Gorilla beringei graueri individuals were included in the haplotype and allele frequency analyses. The presence of these haplotypes or the presence of similar allele frequencies in Eastern lowland and mountain gorillas would impact this conclusion. It would be helpful for the authors to clarify this point.

(4) The authors appear to use a somewhat atypical approach to assess intra-population selection to compensate for relatively small numbers of NHP sequences (Fig. 6). However, they do not cite precedence for the robustness of the approach or the practice of grouping sequences from multiple species for the endemic vs other comparison. They also state in the methods that some genes encoded in the locus were removed from the analysis "because they have previously been shown to directly interact with a viral protein." This seems to undercut the analysis, and prevents alternative explanations for the observed diversity in CD4 (e.g., passenger mutations from selection at a neighboring locus).

(5) Data in Figure 5 is graphed as % infected cells instead of virus titer (TDU/mL). It's unclear why this is the case, and prevents a comparison to data in Figure 2 and Figure 4.

(6) The lack of pseudotyping with SIVgor envelope is a surprising omission from this study, that would help to contextualize the findings. Similarly, building gorilla CD4 haplotype SNPs onto the hominin ancestor (as opposed to extant human CD4) may provide additional insights that are meaningful towards understanding the evolutionary trajectory of gorilla CD4.

Comments on revised version:

In the revised manuscript, the authors more appropriately contextualize conclusions that can be made based on their data versus inferences, which are now much more clearly described in the discussion. The authors also included more references to substantiate claims, additional description of methodology, and provided well-reasoned responses to the weaknesses described in my primary review.

Re: #3. As the authors point out, we do not know if eastern gorillas were at one time exposed to SIV. The authors use a variety of phylogenetic and functional approaches to infer that SIVcpz is the selective pressure-shaping gorilla CD4. While I agree this is a highly likely scenario, the allelic diversity of CD4 across gorilla subpopulations raises multiple evolutionary scenarios consistent with the data.

Re: #4. The explanation provided by the authors is reasonable. However, a demonstration that this approach is robust to potential factors that might skew the data (e.g., recombination) is argued but not tested. Part of the concern here is that the study is limited by very small sample sizes, and to the best of my knowledge, grouping sequences from multiple species to make claims about selection is not an established practice. The authors note in their response that they confirmed the existence of CD4 alleles in this study with those identified in 100 gorilla individuals from Russell et al. 2021 (unavailable to the authors at the time of submission) - a re-analysis that includes that data from Russell et al. 2021 would have strengthened the analyses.

Joint Public Review:

The premise of this work carries great potential. Namely, developing a humanized mouse system in which features of adaptive immunity that contribute to inflammatory demyelination can be interrogated will allow for traction into therapeutics currently unavailable to the field. Immediate questions stemming from the current study include the potential effect of ex vivo activation of PBMCs (or individual T and B cells) in vitro prior to transfer as well as the TCR and BCR repertoire of CNS vs peripheral lymphocytes before and after immunization. This group has been thoughtful and clever about their approach (e.g. use of subjects treated with natalizumab), which gives hope that fundamental aspects of pathogenesis will be uncovered by this form of modeling MS disease.

Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system where immune cells play an important role in disease pathobiology. Increased incidence of disease in individuals carrying certain HLA class-II genes plus studies in animal models suggests that HLA-DRB1*15 restricted CD4 T cells might be responsible for disease initiation, and other immune cells such as B cells, CD8 T cells, monocytes/macrophages, and dendritic cells (DC) also contribute to disease pathology. However, a direct role of human immune cells in disease is lacking to a lag between immune activation and the first sign of clinical disease. Therefore, there is an emphasis on understanding whether immune cells from HLA-DR15+ MS patients differ from HLA-DR15+ healthy controls in their phenotype and pro-inflammatory capacity. To overcome this, authors have used severely immunodeficient B2m-NOG mice that lack B, T cells and NK cells and have defective innate immune responses and engrafted PBMCs from 3 human donors (HLA-DR15+ MS and HI donors, HLA-DR13+ MS donor) in these B2m-NOG mice to determine whether they can induce CNS inflammation and demyelination like MS.

The study's strength is the use of PBMCs from HLADRB1-typed MS subjects and healthy control, the use of NOG mice, the characterization of immune subsets (revealing some interesting observations), CNS pathology etc. Weaknesses are lack of phenotype in mice and no disease phenotype even in humanized mice immunized for disease using standard disease induction protocol employed in an animal model of MS, and lack of mechanistic data on why CD8 T cells are more enriched than CD4+ T cells. The last point is important as postmortem human MS patients' brain tissue had been shown to have more CD8+ T cells than CD4+ T cells.

Thus, this work is an important step in the right direction as previous humanized studies have not used HLA-DRB1 typed PBMCs however the weaknesses as highlighted above are limitations in the model.

Reviewer #1 (Public Review):

I'll begin by summarizing what I understand from the results presented, and where relevant how my understanding seems to differ from the authors' claims. I'll then make specific comments with respect to points raised in my previous review (below), using the same numbering. Because this is a revision I'll try to restrict comments here to the changes made, which provide some clarification, but leave many issues incompletely addressed.

As I understand it the main new result here is that certain recurrent network architectures promote emergence of coordinated grid firing patterns in a model previously introduced by Kropff and Treves (Hippocampus, 2008). The previous work very nicely showed that single neurons that receive stable spatial input could 'learn' to generate grid representations by combining a plasticity rule with firing rate adaptation. The previous study also showed that when multiple neurons were synaptically connected their grid representations could develop a shared orientation, although with the recurrent connectivity previously used this substantially reduced the grid scores of many of the neurons. The advance here is to show that if the initial recurrent connectivity is consistent with that of a line attractor then the network does a much better job of establishing grid firing patterns with shared orientation.

Beyond this point, things become potentially confusing. As I understand it now, the important influence of the recurrent dynamics is in establishing the shared orientation and not in its online generation. This is clear from Figure S3, but not from an initial read of the abstract or main text. This result is consistent with Kropff and Treves' initial suggestion that 'a strong collateral connection... from neuron A to neuron B... favors the two neurons to have close-by fields... Summing all possible contributions would result in a field for neuron B that is a ring around the field of neuron A.' This should be the case for the recurrent connections now considered, but the evidence provided doesn't convincingly show that attractor dynamics of the circuit are a necessary condition for this to arise. My general suggestion for the authors is to remove these kind of claims and to keep their interpretations more closely aligned with what the results show.

Major (numbered according to previous review)

(1) Does the network maintain attractor dynamics after training? Results now show that 'in a trained network without feedforward Hebbian learning the removal of recurrent collaterals results in a slight increase in gridness and spacing'. This clearly implies that the recurrent collaterals are not required for online generation of the grid patterns. This point needs to be abundantly clear in the abstract and main text so the reader can appreciate that the recurrent dynamics are important specifically during learning.<br /> (2) Additional controls for Figure 2 to test that it is connectivity rather than attractor dynamics (e.g. drawing weights from Gaussian or exponential distributions). The authors provide one additional control based on shuffling weights. However, this is far from exhaustive and it seems difficult on this basis to conclude that it is specifically the attractor dynamics that drive the emergence of coordinated grid firing.<br /> (3) What happens if recurrent connections are turned off? The new data clearly show that the recurrent connections are not required for online grid firing, but this is not clear from the abstract and is hard to appreciate from the main text.<br /> (4) This is addressed, although the legend to Fig. S2D could provide an explanation / definition for the y-axis values.<br /> (5) Given the 2D structure of the network input it perhaps isn't surprising that the network generates 2D representations and this may have little to do with its 1D connectivity. The finding that the networks maintain coordinated grids when recurrent connections are switched off supports my initial concern and the authors explanation, to me at least, remain confusing. I think it would be helpful to consider that the connectivity is specifically important for establishing the coordinated grid firing, but that the online network does not require attractor dynamics to generate coordinated grid firing.<br /> (6) Clarity of the introduction. This is somewhat clearer, but I wonder if it would be hard for someone not familiar with the literature to accurately appreciate the key points.<br /> (7) Remapping. I'm not sure why this is ill posed. It seems the proposed model can not account for remapping results (e.g. Fyhn et al. 2007). Perhaps the authors could just clearly state this as a limitation of the model (or show that it can do this).

Previous review:

This study investigates the impact of recurrent connections on grid fields generated in networks trained by adjusting the strength of feedforward spatial inputs. The main result is that if the recurrent connections in the network are given a 1D continuous attractor architecture, then aligned grid firing patterns emerge in the network following training. Detailed analyses of the low dimensional dynamics of the resulting networks are then presented. The simulations and analyses appear carefully carried out.

The feedforward model investigated by the authors (previously introduced by Kropff & Treves, 2008) is an interesting and important alternative to models that generate grid firing patterns through 2-dimensional continuous attractor network (CAN) dynamics. However, while both classes of model generate grid fields, in making comparisons the manuscript is insufficiently clear about their differences. In particular, in the CAN models grid firing is a direct result of their 2-D architecture, either a torus structure with a single activity bump (e.g. Guanella et al. 2007, Pastoll et al. 2013), or sheet with multiple local activity bumps (Fuhs & Touretzky, Burak & Fiete, 2009). In these models, spatial input can anchor the grid representations but is not necessary for grid firing. By contrast, in the feedforward models neurons transform existing spatial inputs into a grid representation. Thus, the two classes of model implement different computations; CANs path integrate, while the feedforward models transform spatial representations. A demonstration that a 1D CAN generates coordinated 2D grid fields would be surprising and important, but its less clear why coordination between grids generated by the feedforward mechanism would be surprising. As written, it's unclear which of these claims the study is trying to make. If the former, then the conclusion doesn't appear well supported by the data as presented, if the latter then the results are perhaps not so unexpected, and the imposed attractor dynamics may still not be relevant.

Whichever claim is being made, it could be helpful to more carefully evaluate the model dynamics given predictions expected for the different classes of model. Key questions that are not answered by the manuscript include:

- At what point is the 1D attractor architecture playing a role in the models presented here? Is it important specifically for training or is it also contributing to computation in the fully trained network?

- Is an attractor architecture required at all for emergence of population alignment and gridness? Key controls missing from Figure 2 include training on networks with other architectures. For example, one might consider various architectures with randomly structured connectivity (e.g. drawing weights from exponential or Gaussian distributions).

- In the trained models do the recurrent connections substantially influence activity in the test conditions? Or after training are the 1D dynamics drowned out by feedforward inputs?

- What is the low dimensional structure of the input to the network? Can the apparent discrepancy between dimensionality of architecture and representation be resolved by considering structure of the inputs, e.g. if the input is a 2 dimensional representation of location then is it surprising that the output is too?

- What happens to representations in the trained networks presented when place cells remap? Is the 1D manifold maintained as expected for CAN models, or does it reorganise?

Reviewer #1 (Public Review):

Summary:

This study examined the role of statistical learning in pain perception, suggesting that individuals' expectations about a sequence of events influence their perception of pain intensity. They incorporated the components of volatility and stochasticity into their experimental design and asked participants (n = 27) to rate the pain intensity, their prediction, and their confidence level. They compared two different inference strategies: Bayesian inference vs. heuristic-employing Kalman filters and model-free reinforcement learning. They showed that the expectation-weighted Kalman filter best explained the temporal pattern of participants' ratings. These results provide evidence for a Bayesian inference perspective on pain, supported by a computational model that elucidates the underlying process.

Strengths:

- Their experimental design included a wide range of input intensities and the levels of volatility and stochasticity. With elaborated computational models, they provide solid evidence that statistical learning shapes pain.

Weaknesses:

- Relevance to clinical pain: While the authors underscore the relevance of their findings to chronic pain, they did not include data pertaining to clinical pain.

Reviewer #1 (Public Review):

Summary:

This paper investigates the neural population activity patterns of the medial frontal cortex in rats performing a nose poking timing task using in vivo calcium imaging. The results showed neurons that were active at the beginning and end of the nose poking and neurons that formed sequential patterns of activation that covaried with the timed interval during nose poking on a trial-by-trial basis. The former were not stable across sessions, while the latter tended to remain stable over weeks. The analysis on incorrect trials suggests the shorter non-rewarded intervals were due to errors in the scaling of the sequential pattern of activity.

Strengths:

This study measured stable signals using in vivo calcium imaging during experimental sessions that were separated by many days in animals performing a nose poking timing task. The correlation analysis on the activation profile to separate the cells in the three groups was effective and the functional dissociation between beginning and end, and duration cells was revealing. The analysis on the stability of decoding of both the nose poking state and poking time was very informative. Hence, this study dissected a neural population that formed sequential patterns of activation that encoded timed intervals.

Weaknesses:

It is not clear whether animals had enough simultaneously recorded cells to perform the analyzes of Figures 2-4. In fact, rat 3 had 18 responsive neurons which probably is not enough to get robust neural sequences for the trial-by-trial analysis and the correct and incorrect trial analysis. In addition, the analysis of behavioral errors could be improved. The analysis in Figure 4A could be replaced by a detailed analysis on the speed, and the geometry of neural population trajectories for correct and incorrect trials. In the case of Figure 4G is not clear why the density of errors formed two clusters instead of having a linear relation with the produce duration. I would be recommendable to compute the scaling factor on neuronal population trajectories and single cell activity or the computation of the center of mass to test the type III errors.

Due to the slow time resolution of calcium imaging, it is difficult to perform robust analysis on ramping activity. Therefore, I recommend downplaying the conclusion that: "Together, our data suggest that sequential activity might be a more relevant coding regime than the ramping activity in representing time under physiological conditions."

Reviewer #1 (Public Review):

Summary:

This work studies spatio-temporal patterns of structure-function coupling in developing brains, using a large set of imaging data acquired from children aged 5-22. Magnetic resonance imaging data of brain structure and function were obtained from a publicly available database, from which structural and functional features and measures were derived. The authors examined the spatial patterns of structure-function coupling and how they evolve with brain development. This work further sought correlations of brain structure-function coupling with behavior and explored evolutionary, microarchitectural and genetic bases that could potentially account for the observed patterns.

Strength:

The strength of this work is the use of currently available state-of-the-art analysis methods, along with a large set of high-quality imaging data, and comprehensive examinations of structure-function coupling in developing brains. The results are comprehensive and illuminating.

Weakness:

As with most other studies, transcriptomic and cellular architectures of structure-function coupling were characterized only on the basis of a common atlas in this work.

The authors have achieved their aims in this study, and the findings provide mechanistic insights into brain development, which will inspire further basic and clinical studies along this line.