Reviewer #2 (Public review):

In this study, the authors aim to forecast the evolution of viral proteins by simulating sequence changes under a constraint of folding stability. The central idea is that proteins must retain a certain level of structural stability (quantified by folding free energy, ΔG) to remain functional, and that this constraint can shape and restrict the space of viable evolutionary trajectories. The authors integrate a birth-death population model with a structurally constrained substitution (SCS) model and apply this simulation framework to several viral proteins from HIV-1, SARS-CoV-2, and influenza.

The motivation to incorporate biophysical constraints into evolutionary models is scientifically sound, and the general approach aligns with a growing interest in bridging molecular evolution and structural biology. The authors focus on proteins where immune pressure is limited and stability is likely to be a dominant constraint, which is conceptually appropriate. The method generates sequence variants that preserve folding stability, suggesting that stability-based filtering may capture certain evolutionary patterns.

However, the study does not substantiate its central claim of forecasting. The model does not predict future sequences with measurable accuracy, nor does it reproduce observed evolutionary paths. Validation is limited to endpoint comparisons in a few datasets. While KL divergence is used to compare amino acid distributions, this analysis is only applied to a single protein (HIV-1 MA), and there is no assessment of mutation-level predictive accuracy or quantification of how well simulated sequences recapitulate real evolutionary paths. No comparison is made to real intermediate variants available from extensive viral sequencing datasets which gather thousands of sequences with detailed collection date annotation (SARS-CoV-2, Influenza, RSV).

The selection of proteins is narrow and the rationale for including or excluding specific proteins is not clearly justified.

The analyzed datasets are also under-characterized: we are not given insight into how variable the sequences are or how surprising the simulated sequences might be relative to natural diversity. Furthermore, the use of consensus sequences to represent timepoints is problematic, particularly in the context of viral evolution, where divergent subclades often coexist - a consensus sequence may not accurately reflect the underlying population structure.

The fitness function used in the main simulations is based on absolute ΔG and rewards increased stability without testing whether real evolutionary trajectories tend to maintain, increase, or reduce folding stability over time for the particular systems (proteins) that are studied. While a variant of the model does attempt to center selection around empirical ΔG values, this more biologically plausible version is underutilized and not well validated.

Ultimately, the model constrains sequence evolution to stability-compatible trajectories but does not forecast which of these trajectories are likely to occur. It is better understood as a filter of biophysically plausible outcomes than as a predictive tool. The distinction between constraint-based plausibility and sequence-level forecasting should be made clearer. Despite these limitations, the work may be of interest to researchers developing simulation frameworks or exploring the role of protein stability in viral evolution, and it raises interesting questions about how biophysical constraints shape sequence space over time.

Reviewer #2 (Public review):

Summary:

Endo et al. investigate the novel role of ubiquitin response upon lysosomal damage in activating cellular signaling for cell survival. The authors provide a comprehensive transcriptome and proteome analysis of aging-related cells experiencing lysosomal damage, identifying transcription factors involved in transcriptome and proteome remodeling with a focus on the NF-κB signaling pathway. They further characterized the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis in controlling gene expression, inflammatory responses, and apoptotic processes.

Strengths:

In the aging-related model, the authors provide a comprehensive transcriptome and characterize the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis. Through compelling experiments and advanced tools, they elucidate its critical role in controlling gene expression, inflammatory responses, and apoptotic processes.

Weaknesses:

The study lacks deeper connections with previous research, particularly:

• The established role of TAB-TAK1 in AMPK activation during lysosomal damage

• The potential significance of TBK1 in NF-κB signaling pathways

Comments on revisions:

The authors have successfully addressed all the raised questions and the manuscript is now significantly improved.

Reviewer #2 (Public review):

Summary:

Bushey et al. investigate the feasibility of using RubyACRs, specifically A1ACR1 and HfACR1 (described previously in (Govorunova et al., 2020)) as red-shifted inhibitory opsins in Drosophila melanogaster. The study employs a wide range of techniques to demonstrate successful neuronal inhibition. Electrophysiology experiments established that HfACR1 was most effective at hyperpolarizing cells, compared to A1ACR1 and GtACR1; both RubyACRs also appeared to be more effective than GtACR1 when the latter was actuated by green light. The authors further demonstrate successful neuronal inhibition using calcium imaging. RubyACRs were also shown to be useful in in vivo behavioral setups, specifically in spontaneous locomotion, associative learning, and courtship paradigms. In the courtship assay, in particular, the authors test multiple wavelengths of light at various light intensities, thus providing a rigorous analysis of the RubyACRs' efficacy under different light conditions.

Strengths:

The work provides the Drosophila field with a promising new tool. Red-shifted opsins are particularly advantageous in behavioral assays as red light penetrates the cuticle better than green or blue light, and provides less visual stimulation to the fly. It is also ideal for imaging as it allows for simultaneous optogenetic stimulation and GCamp imaging. A particular strength of the paper is the direct demonstration of RubyACR's capacity to inhibit neurons via electrophysiology and calcium imaging. Furthermore, inhibition effects in the three behavioral assays are strong and convincing. Given the apparent efficacy of RubyACRs and the advantages of a red-sensitive anion channelrhodopsin, this tool has great potential.

Weaknesses:

This work convincingly demonstrates the efficacy and potential utility of RubyACRs in Drosophila for imaging and behavior. However, the lethality/toxicity of RubyACRs is a relevant concern that should be addressed in-depth rather than glossed over, as it may pose a major obstacle to use. Discussing this issue in the present study will also help guide potential users and will set the stage for potential future efforts to ameliorate RubyACRs as optogenetic inhibitors.

Major concerns:

(1) Table 1 demonstrates high lethality in the RubyACRs compared to GtACR1. For example, in the MI04979-VGlut driver, GtACR1 expression resulted in 32.9% lethality, while HfACR1 expression resulted in 98.7% lethality. This lethality presents an obstacle to the potential adoption of this tool, and should be discussed in detail, rather than in passing. The authors might like to present "% lethality" rather than "% survived", as the former is more relevant when discussing the relative yield and health of flies that can be used in experiments.

(2) In Figure 3D, driver>opsin flies have lower locomotion during the baseline (i.e., dark) phase, compared to opsin-only controls or GtACR1 flies. For some comparisons, flies are walking around 10-fold slower. For example, in the case of VGlut-GAL4>HfACR1, test flies are walking at <1 mm/s, while "Empty" test flies are walking at ~10 mm/s. This suggests that, for these drivers, neuronal and/or network function is affected. It opens the possibility that the lethality and locomotor defects could be due to cell-autonomous toxicity. We ask the authors to provide a description of this effect in the Results and to discuss it in the Discussion. Relatedly, VGlut-GAL4>GtACR1 flies in red light exhibit a locomotion increase, but this data is not mentioned in the text. The use of differing scales for the Y-axes in these panels can be confusing when the reader is expected to compare velocity across different panels. It would be best if the y-axes were set to a single range, e.g., 0 to 12 mm/s.

(3) Lethality in broad drivers could result from cell-autonomous toxicity or neuronal dysfunction resulting from RubyACR expression. Ideally, the authors would address or even investigate the possible mechanisms of toxicity of the RubyACRs. Do cells and/or synapses expressing RubyACRs have normal morphology and function? For example, the authors could compare cell survival between flies with RubyACR expression and flies with a fluorescent protein with no opsin. The authors may also want to present lethality data for other, less broad drivers (such as MB320C, which was used for the associative memory assay) in order to demonstrate whether this problem is confined to broad drivers such as VGlut-GAL4, or if this is a problem with narrow drivers as well. If new experiments are not possible, these issues should at least be mentioned in the Discussion.

Minor concerns

(1) The specific method used for quantifying lethality is mentioned briefly in Table 1 but is not detailed in the Methods. The authors derive lethality by comparing to a sibling control group with either the opsin or the driver alone, but the opsin alone or driver alone may cause some lethality by themselves. We suggest the use of a viability assay, e.g. (Rockwell et al., 2019), which would give potential users a clearer picture of which developmental stage is most affected by opsin expression, as well as allow opsin-only, driver-only and experimental groups to be assessed separately (lethality would then be reported as the % of embryos that reach each stage of development, and eventually enclosure).

(2) For the calcium imaging analysis in Figure 2, the U-shaped curve observed for mean ΔF/F0 for A1ACR1 and HfACR1 may not be due to actual desensitization for the channels, as the authors suggest (lines 143-145), but may be due simply to a shifting baseline. The authors use the 5-s period preceding stimulation onset as F0, but in some cases (e.g., HfACR1 at 250 uW/mm2), calcium fluorescence rises above baseline and remains high post-stimulation (ΔF/F0 of +0.5, which we observe is the same magnitude as the ΔF/F0 of -0.5 observed during inhibition), thus affecting the ΔF/F0 for subsequent trials. The authors should discuss this incomplete recovery in the text, or (if available) use a static channel instead to provide a stable F0 for calculating ΔF/F0. Alternatively, if the authors wish to rigorously test the hypothesis that high light intensity indeed results in desensitization of these channels, they may consider using different flies for each light intensity or longer inter-stimulus intervals.

(3) For Figure 3C (Flybowl assay), the authors mention that "simply expressing the opsins decreased baseline locomotor activity compared to empty driver lines". However, the "Empty" controls in 3C appear to refer to opsin-only controls, not driver-only controls. The driver-only controls are not presented in the figure. The use of "empty" differs between the text and the figure, as the text refers to "empty" driver lines, while the figure uses "empty" to apparently refer to opsin-only controls. We recommend changing the terminology across all figures to be unambiguous, e.g., by using "opsin-only" or "driver-only" as opposed to the ambiguous "empty". In addition, the fact that opsin-only controls move less than driver-only controls may suggest some toxicity as a result of the opsin-only construct; this should be discussed further.

(4) Figures 4 and 5 lack the reporting of driver-only controls.

(5) Figures 3 and 4 lack positive controls; that is, the benchmarking of the efficacy of RubyACRs in their respective behavioral paradigms against a known inhibitor, e.g., GtACR1 with green light. To confirm that this GtACR1 transgene is functional, the authors could include GtACR1 with green light as a positive control for these two figures, as they have done for Figure 5-supplement 2 and 3.

(6) Several citations are missing. In their discussion, the authors highlight that shorter wavelengths of light are more attenuated by tissue (lines 278-281); this should be accompanied by the relevant citations (Inagaki et al., 2014). Similarly, the claim that behavioral experiments exhibit greater sensitivity to shorter wavelengths should be substantiated (lines 281-283).

References:

Govorunova EG, Sineshchekov OA, Li H, Wang Y, Brown LS, Spudich JL. 2020. RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption. Proc Natl Acad Sci U S A 117:22833-22840.

Inagaki HK, Jung Y, Hoopfer ED, Wong AM, Mishra N, Lin JY, Tsien RY, Anderson DJ. 2014. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat Methods 11:325-332.

Rockwell AL, Beaver I, Hongay CF. 2019. A direct and simple method to assess Drosophila melanogaster's viability from embryo to adult. J Vis Exp e59996.

Reviewer #2 (Public review):

Summary:

This manuscript proposes that the use of a latent cause model for assessment of memory-based tasks may provide improved early detection in Alzheimer's Disease as well as more differentiated mapping of behavior to underlying causes. To test the validity of this model, the authors use a previously described knock-in mouse model of AD and subject the mice to several behaviors to determine whether the latent cause model may provide informative predictions regarding changes in the observed behaviors. They include a well-established fear learning paradigm in which distinct memories are believed to compete for control of behavior. More specifically, it's been observed that animals undergoing fear learning and subsequent fear extinction develop two separate memories for the acquisition phase and the extinction phase, such that the extinction does not simply 'erase' the previously acquired memory. Many models of learning require the addition of a separate context or state to be added during the extinction phase and are typically modeled by assuming the existence of a new state at the time of extinction. The Niv research group, Gershman et al. 2017, have shown that the use of a latent cause model applied to this behavior can elegantly predict the formation of latent states based on a Bayesian approach, and that these latent states can facilitate the persistence of the acquisition and extinction memory independently. The authors of this manuscript leverage this approach to test whether deficits in production of the internal states, or the inference and learning of those states, may be disrupted in knock-in mice that show both a build-up of amyloid-beta plaques and a deterioration in memory as the mice age.

Strengths:

I think the authors' proposal to leverage the latent cause model and test whether it can lead to improved assessments in an animal model of AD is a promising approach for bridging the gap between clinical and basic research. The authors use a promising mouse model and apply this to a paradigm in which the behavior and neurobiology are relatively well understood - an ideal situation for assessing how a disease state may impact both the neurobiology and behavior. The latent cause model has the potential to better connect observed behavior to underlying causes and may pave a road for improved mapping of changes in behavior to neurobiological mechanisms in diseases such as AD.<br /> The authors also compare the latent cause model to the Rescorla-Wagner model and a latent state model allowing for better assessment of the latent cause model as a strong model for assessing reinstatement.

Weaknesses:

I have several substantial concerns which I've detailed below. These include important details on how the behavior was analyzed, how the model was used to assess the behavior, and the interpretations that have been made based on the model.<br /> (1) There is substantial data to suggest that during fear learning in mice separate memories develop for the acquisition and extinction phases, with the acquisition memory becoming more strongly retrieved during spontaneous recovery and reinstatement. The Gershman paper, cited by the authors, shows how the latent causal model can predict this shift in latent causes by allowing for the priors to decay over time, thereby increasing the posterior of the acquisition memory at the time of spontaneous recovery. In this manuscript, the authors suggest a similar mechanism of action for reinstatement, yet the model does not appear to return to the acquisition memory after reinstatement, at least based on the simulation and examples shown in figures 1 and 3. More specifically, in figure 1, the authors indicate that the posterior probability of the latent cause, zA (the putative acquisition memory), increases, partially leading to reinstatement. This does not appear to be the case as test 3 (day 36) appears to have similar posterior probabilities for zA as well as similar weights for the CS as compared to the last days of extinction. Rather, the model appears to mainly modify the weights in the most recent latent cause, zB - the putative the 'extinction state', during reinstatement. The authors suggest that previous experimental data have indicated that spontaneous recovery or reinstatement effects are due to an interaction of the acquisition and extinction memory. These studies have shown that conditioned responding at a later time point after extinction is likely due to a balance between the acquisition memory and the extinction memory, and that this balance can shift towards the acquisition memory naturally during spontaneous recovery, or through artificial activation of the acquisition memory or inhibition of the extinction memory (see Lacagnina et al. for example). Here the authors show that the same latent cause learned during extinction, zB, appears to dominate during the learning phase of reinstatement, with rapid learning to the context - the weight for the context goes up substantially on day 35 - in zB. This latent cause, zB, dominates at the reinstatement test, and due to the increased associative strength between the context and shock, there is a strong CR. For the simulation shown in figure 1, it's not clear why a latent cause model is necessary for this behavior. This leads to the next point.

(2) The authors compared the latent cause model to the Rescorla-Wagner model. This is very commendable, particularly since the latent cause model builds upon the RW model, so it can serve as an ideal test for whether a more simplified model can adequately predict the behavior. The authors show that the RW model cannot successfully predict the increased CR during reinstatement (Appendix figure 1). Yet there are some issues with the way the authors have implemented this comparison:<br /> (2A) The RW model is a simplified version of the latent cause model and so should be treated as a nested model when testing, or at a minimum, the number of parameters should be taken into account when comparing the models using a method such as the Bayesian Information Criterion, BIC.<br /> (2B) The RW model provides the associative strength between stimuli and does not necessarily require a linear relationship between V and the CR. This is the case in the original RW model as well as in the LCM. To allow for better comparison between the models, the authors should be modeling the CR in the same manner (using the same probit function) in both models. In fact, there are many instances in which a sigmoid has been applied to RW associative strengths to predict CRs. I would recommend modeling CRs in the RW as if there is just one latent cause. Or perhaps run the analysis for the LCM with just one latent cause - this would effectively reduce the LCM to RW and keep any other assumptions identical across the models.<br /> (2C) In the paper, the model fits for the alphas in the RW model are the same across the groups. Were the alphas for the two models kept as free variables? This is an important question as it gets back to the first point raised. Because the modeling of the reinstatement behavior with the LCM appears to be mainly driven by latent cause zB, the extinction memory, it may be possible to replicate the pattern of results without requiring a latent cause model. For example, the 12-month-old App NL-G-F mice behavior may have a deficit in learning about the context. Within the RW model, if the alpha for context is set to zero for those mice, but kept higher for the other groups, say alpha_context = 0.8, the authors could potentially observe the same pattern of discrimination indices in figure 2G and 2H at test. Because the authors don't explicitly state which parameters might be driving the change in the DI, the authors should show in some way that their results cannot simply be due to poor contextual learning in the 12 month old App NL-G-F mice, as this can presumably be predicted by the RW model. The authors' model fits using RW don't show this, but this is because they don't consider this possibility that the alpha for context might be disrupted in the 12-month-old App NL-G-F mice. Of course, using the RW model with these alphas won't lead to as nice of fits of the behavior across acquisition, extinction, and reinstatement as the authors' LCM, the number of parameters are substantially reduced in the RW model. Yet the important pattern of the DI would be replicated with the RW model (if I'm not mistaken), which is the important test for assessment of reinstatement.

(3) As stated by the authors in the introduction, the advantage of the fear learning approach is that the memory is modified across the acquisition-extinction-reinstatement phases. Although perhaps not explicitly stated by the authors, the post-reinstatement test (test 3) is the crucial test for whether there is reactivation of a previously stored memory, with the general argument being that the reinvigorated response to the CS can't simply be explained by relearning the CS-US pairing, because re-exposure the US alone leads to increase response to the CS at test. Of course there are several explanations for why this may occur, particularly when also considering the context as a stimulus. This is what I understood to be the justification for the use of a model, such as the latent cause model, that may better capture and compare these possibilities within a single framework. As such, it is critical to look at the level of responding to both the context alone and to the CS. It appears that the authors only look at the percent freezing during the CS, and it is not clear whether this is due to the contextual-US learning during the US re-exposure or to increased responding to the CS - presumably caused by reactivation of the acquisition memory. The authors do perform a comparison between the preCS and CS period, but it is not clear whether this is taken into account in the LCM. For example, the instance of the model shown in figure 1 indicates that the 'extinction cause', or cause z6, develops a strong weight for the context during the reinstatement phase of presenting the shock alone. This state then leads to increased freezing during the final CS probe test as shown in the figure. If they haven't already, I think the authors must somehow incorporate these different phases (CS vs ITI) into their model, particularly since this type of memory retrieval that depends on assessing latent states is specifically why the authors justified using the latent causal model. In more precise terms, it's not clear whether the authors incorporate a preCS/ITI period each day the cue is presented as a vector of just the context in addition to the CS period in which the vector contains both the context and the CS. Based on the description, it seemed to me that they only model the CRs during the CS period on days when the CS is presented, and thereby the context is only ever modeled on its own (as just the context by itself in the vector) on extinction days when the CS is not presented. If they are modeling both timepoints each day that the CS I presented, then I would recommend explicitly stating this in the methods section.

(4) The authors fit the model using all data points across acquisition and learning. As one of the other reviewers has highlighted, it appears that there is a high chance for overfitting the data with the LCM. Of course, this would result in much better fits than models with substantially fewer free parameters, such as the RW model. As mentioned above, the authors should use a method that takes into account the number of parameters, such as the BIC.

(5) The authors have stated that they do not think the Barnes maze task can be modeled with the LCM. Whether or not this is the case, if the authors do not model this data with the LCM, the Barnes maze data doesn't appear valuable to the main hypothesis. The authors suggest that more sophisticated models such as the LCM may be beneficial for early detection of diseases such as Alzheimer's, so the Barnes maze data is not valuable for providing evidence of this hypothesis. Rather, the authors make an argument that the memory deficits in the Barnes maze mimic the reinstatement effects providing support that memory is disrupted similarly in these mice. Although, the authors state that the deficits in memory retrieval are similar across the two tasks, the authors are not explicit as to the precise deficits in memory retrieval in the reinstatement task - it's a combination of overgeneralizing latent causes during acquisition, poor learning rate, over differentiation of the stimuli.

Reviewer #2 (Public review):

Summary:

In this paper, the authors have characterized Rv2577 as a Fe3+/Zn2+ -dependent metallophosphatase and a nucleomodulin protein. The authors have also identified His348 and Asn359 as critical residues for Fe3+ coordination. The authors show that the proteins encode for two nuclease localization signals. Using C-terminal Flag expression constructs, the authors have shown that the MmpE protein is secretory. The authors have prepared genetic deletion strains and show that MmpE is essential for intracellular survival of M. bovis BCG in THP-1 macrophages, RAW264.7 macrophages, and a mouse model of infection. The authors have also performed RNA-seq analysis to compare the transcriptional profiles of macrophages infected with wild-type and MmpE mutant strains. The relative levels of ~ 175 transcripts were altered in MmpE mutant-infected macrophages and the majority of these were associated with various immune and inflammatory signalling pathways. Using these deletion strains, the authors proposed that MmpE inhibits inflammatory gene expression by binding to the promoter region of a vitamin D receptor. The authors also showed that MmpE arrests phagosome maturation by regulating the expression of several lysosome-associated genes such as TFEB, LAMP1, LAMP2, etc. These findings reveal a sophisticated mechanism by which a bacterial effector protein manipulates gene transcription and promotes intracellular survival.

Strength:

The authors have used a combination of cell biology, microbiology, and transcriptomics to elucidate the mechanisms by which Rv2577 contributes to intracellular survival.

Weakness:

The authors should thoroughly check the mice data and show individual replicate values in bar graphs.

Reviewer #2 (Public review):

Summary:

The authors decomposed response times into component processes and manipulated the duration of these processes in opposing directions by varying contrast, and overall by manipulating speed-accuracy tradeoffs. They identify different processes and their durations by identifying neural states in time and validate their functional significance by showing that their properties vary selectively as expected with the predicted effects of the contrast manipulation. They identify 3 processes: stimulus encoding, attention orienting, and decision. These map onto classical event-related potentials. The decision-making component matched the CPP, and its properties varied with contrast and predicted decision-accuracy, while also exhibiting a burst not characteristic of evidence accumulation.

Strengths:

The design of the experiment is remarkable and offers crucial insights. The analysis techniques are beyond state-of-the-art, and the analyses are well motivated and offer clear insights.

Weaknesses:

It is not clear to me that the results confirm that there are only 3 processes, since e.g., motor preparation and execution were not captured. While the authors discuss this, this is a clear weakness of the approach, as other components may also have been missed. It is also unclear to what extent topographies map onto processes, since, e.g., different combinations of sources can lead to the same scalp topography.

Reviewer #3 (Public review):

Summary:

This manuscript by McDougal et al, demonstrates species-specific activities of diverse IFIT1 orthologs, and seeks to utilize evolutionary analysis to identify key amino acids under positive selection that contribute to antiviral activity of this host factor. While the authors identify amino acid residues important for antiviral activity of some orthologs, and propose a possible mechanism by which these residues may function, the significance or applicability of these findings to other orthologs is unclear. However, the subject matter is of interest to the field, and these findings contribute to the body of knowledge regarding IFIT1 evolution.

Strengths:

Assessment of multiple IFIT1 orthologs shows the wide variety of antiviral activity of IFIT1, and identification of residues outside of the known RNA binding pocket in the protein suggests additional novel mechanisms which may regulate IFIT1 activity.

Weaknesses:

Given that there appears to be very little overlap observed in orthologs that inhibited the viruses tested, it's possible that other amino acids may be key drivers of antiviral activity in these other orthologs. Thus, it's difficult to conclude whether the findings that residues 362/4/6 are important for IFIT1 activity can be broadly applied to other orthologs, or whether these are unique to human and chimpanzee IFIT1. While additional molecular studies of the impact of these mutations on IFIT1 function (e.g. impact on IFIT complex formation) would lend further insight, as it stands, these findings demonstrate a role for these residues in IFIT1 activity.

Reviewer #2 (Public review):

Summary:

The authors present a software package "aTrack" for identification of motion types and parameter estimation in single-particle tracking data. The software is based on maximum likelihood estimation of the time-series data given an assumed motion model and likelihood ratio tests for model selection. They characterized the performance of the software mostly on simulated data and showed that it is applicable to experimental data.

Strengths:

Although many tools exist in the single-particle tracking (SPT) field, this particular software package is developed using an innovative mathematical model and a probabilistic approach. It also provide inference of motion types, which are critical to answer biological questions in SPT experiments.

(1) The authors adopt a novel mathematical framework, which is unique in the SPT field.

(2) The authors have validated their method extensively using simulated tracks and compared to existing methods when appropriate.

(3) The code is freely available

Weaknesses:

The authors did a good job during the revision to address most of the weaknesses in my (as well as other reviewer's) first round of review. Nevertheless, the following issue is still not fully addressed.<br /> The hypothesis testing method presented here lacks rigorous statistical foundation. The authors improved on this point after the revision, but in their newly added SI section "Statistical Test", only justified their choices using "hand-waving" arguments (i.e. there is not a single reference to proper statistical textbooks or earlier works in this important section). I understand that sometimes mathematical rigor comes later after some intuition-guided choices of critical parameters seems to work, but nevertheless need to point it out as a remaining weakness.

Reviewer #2 (Public review):

Summary:

This manuscript presents the development and characterization of iGABASnFR2, a genetically encoded GABA sensor with markedly improved performance over its predecessor, iGABASnFR1. The study is comprehensive and methodologically rigorous, integrating high-throughput mutagenesis, functional screening, structural analysis, biophysical characterization, and in vivo validation. iGABASnFR2 represents a significant advancement in GABA sensor engineering and application in imaging GABA transmission in slice and in vivo. This is a timely and technically strong contribution to the molecular toolkit for neuroscience.

Strengths:

The authors apply a well-established sensor optimization pipeline and iterative engineering strategy from single-site to combinatorial mutants to engineer iGABASnFR2. The development of both positive and negative going variants (iGABASnFR2 and iGABASnFR2n) offers experimental flexibility. The structure and interpretation of the key mutations provide insights into the working mechanism of the sensor, which also suggest optimization strategies. Although individual improvements in intrinsic properties are incremental, their combined effect yields clear functional gains, enabling detection of direction-selective GABA release in the retina and volume-transmitted GABA signaling in somatosensory cortex, which were challenging or missed using iGABASnFR1.

Weaknesses:

With minor revisions and clarifications, especially regarding membrane trafficking, this manuscript will be a valuable resource for probing inhibitory transmission.

Reviewer #2 (Public review):

Summary:

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

The study's main findings are that:

• A majority of neurons in MIo and a (somewhat smaller) percentage of SIo modulated their firing rates during tongue movements, with different modulation depending on the direction of movement (i.e., exhibited directional tuning). Examining the statistics of tuning across neurons, there was anisotropy (e.g., more neurons preferring anterior movement) and a lateral bias in which tongue direction neurons preferred that was consistent with the innervation patterns of tongue control muscles (although with some inconsistency between monkeys).<br /> • Consistent with this encoding, tongue position could be decoded with moderate accuracy even from small ensembles of ~28 neurons.<br /> • There were differences observed in the proportion and extent of directional tuning between the feeding and licking behaviors, with stronger tuning overall during licking. This potentially suggests behavioral context-dependent encoding.<br /> • The authors then went one step further and used a bilateral nerve block to the sensory inputs (trigeminal nerve) from the tongue. This impaired the precision of tongue movements and resulted in an apparent reduction and change in neural tuning in Mio and SIo.

Strengths:

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

Weaknesses:

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

One weakness of the current study is that there is substantial variability in results between monkeys.

This study focuses on describing directional tuning using the preferred direction (PD) / cosine tuning model popularized by Georgopoulous and colleagues for understanding neural control of arm reaching in the 1980s. This is a reasonable starting point and a decent first order description of neural tuning. However, the arm motor control field has moved far past that viewpoint, and in some ways an over-fixation on static representational encoding models and PDs held that field back for many years. The manuscript benefit from drawing the readers' attention (perhaps in their Discussion) that PDs are a very simple starting point for characterizing how cortical activity relates to kinematics, but that there is likely much richer population-level dynamical structure and that a more mechanistic, control-focused analytical framework may be fruitful. A good review of this evolution in the arm field can be found in Vyas S, Golub MD, Sussillo D, Shenoy K. 2020. Computation Through Neural Population Dynamics. Annual Review of Neuroscience. 43(1):249-75. A revised version of the manuscript incorporates more population-level analyses, but with inconsistent use of quantifications/statistics and without sufficient contextualization of what the reader is to make of these results.

The described changes in tuning after nerve block could also be explained by changes in kinematics between these conditions, which temper the interpretation of these interesting results.

I am not convinced of the claim that tongue directional encoding fundamentally changes between drinking and feeding given the dramatically different kinematics and the involvement of other body parts like the jaw (e.g., the reference to Laurence-Chasen et al. 2023 just shows that there is tongue information independent of jaw kinematics, not that jaw movements don't affect these neurons' activities). I also find the nerve block results inconsistent (more tuning in one monkey, less in the other?) and difficult to really learn something fundamental from, besides that neural activity and behavior both change - in various ways - after nerve block (not at all surprising but still good to see measurements of).

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

Reviewer #2 (Public review):

Summary:

This study investigated how listeners adapt to and utilize statistical properties of different acoustic spaces to improve speech perception. The researchers used repetitive TMS to perturb neural activity in DLPFC, inhibiting statistical learning compared to sham conditions. The authors also identified the most effective room types for the effective use of reverberations in speech in noise perception, with regular human-built environments bringing greater benefits than modified rooms with lower or higher reverberation times.

Strengths:

The introduction and discussion sections of the paper are very interesting and highlight the importance of the current study, particularly with regard to the use of ecologically valid stimuli in investigating statistical learning. However, they could be condensed into parts. TMS parameters and task conditions were well-considered and clearly explained.

Weaknesses

(1) The Results section is difficult to follow and includes a lot of detail, which could be removed. As such, it presents as confusing and speculative at times.

(2) The hypotheses for the study are not clearly stated.

(3) Multiple statistical models are implemented without correcting the alpha value. This leaves the analyses vulnerable to Type I errors.

(4) It is confusing to understand how many discrete experiments are included in the study as a whole, and how many participants are involved in each experiment.

(5) The TMS study is significantly underpowered and not robust. Sample size calculations need further explanation (effect sizes appear to be based on behavioural studies?). I would caution an exploratory presentation of these data, and calculate a posteriori the full sample size based on effect sizes observed in the TMS data.

Reviewer #2 (Public review):

Summary:

The study by Rowley and Sedigh-Sarvestani presents modeling data suggesting that map reversals in mouse lateral extrastriate visual cortex do not coincide with areal borders, but instead represent borders between subregions within a single area V2. The authors propose that such an organization explains the partial coverage in higher-order areas reported by Zhuang et al., (2017). The scheme revisits an organization proposed by Kaas et al., (1989), who interpreted the multiple projection patches traced from V1 in the squirrel lateral extrastriate cortex as subregions within a single area V2. Kaas et al's interpretation was challenged by Wang and Burkhalter (2007), who used a combination of topographic mapping of V1 connections and receptive field recordings in mice. Their findings supported a different partitioning scheme in which each projection patch mapped a specific topographic location within single areas, each containing a complete representation of the visual field. The area map of mouse visual cortex by Wang and Burkhalter (2007) has been reproduced by hundreds of studies and has been widely accepted as ground truth (CCF) (Wang et al., 2020) of the layout of rodent cortex. In the meantime, topographic mappings in marmoset and tree shew visual cortex made a strong case for map reversals in lateral extrastriate cortex, which represent borders between functionally diverse subregions within a single area V2. These findings from non-rodent species raised doubts about whether during evolution, different mammalian branches have developed diverse partitioning schemes of the cerebral cortex. Rowley and Sedigh-Sarvestani favor a single master plan in which, across evolution, all mammalian species have used a similar blueprint for subdividing the cortex.

Strengths:

The story illustrates the enduring strength of science in search of definitive answers.

Weaknesses:

To me, it remains an open question whether Rowley and Sedigh-Sarvestani have written the final chapter of the saga. A key reason for my reservation is that the areas the maps used in their model are cherry-picked. The article disregards published complementary maps, which show that the entire visual field is represented in multiple areas (i.e. LM, AL) of lateral extrastriate cortex and that the map reversal between LM and AL coincides precisely with the transition in m2AChR expression and cytoarchitecture (Wang and Burkhalter, 2007; Wang et al., 2011). Evidence from experiments in rats supports the gist of the findings in the mouse visual cortex (Coogan and Burkhalter, 1993).

(1) The selective use of published evidence, such as the complete visual field representation in higher visual areas of lateral extrastriate cortex (Wang and Burkhalter, 2007; Wang et al., 2011) makes the report more of an opinion piece than an original research article that systematically analyzes the area map of mouse visual cortex we have proposed. No direct evidence is presented for a single area V2 with functionally distinct subregions.

(2) The article misrepresents evidence by commenting that m2AChR expression is mainly associated with the lower field. This is counter to published findings showing that m2AChR spans across the entire visual field (Gamanut et al., 2018; Meier et al., 2021). The utility of markers for delineating areal boundaries is discounted, without any evidence, in disregard of evidence for distinct areal patterns in early development (Wang et al., 2011). Pointing out that markers can be distributed non-uniformly within an area is well-familiar. m2AChR is non-uniformly expressed in mouse V1, LM and LI (Ji et al., 2015; D'Souza et al., 2019; Meier et al., 2021). Recently, it has been found that the patchy organization within V1 plays a role in the organization of thalamocortical and intracortical networks (Meier et al., 2025). m2AChR-positive patches and m2AChR-negative interpatches organize the functionally distinct ventral and dorsal networks, notably without obvious bias for upper and lower parts of the visual field.

(3) The study has adopted an area partitioning scheme, which is said to be based on anatomically defined boundaries of V2 (Zhuang et al., 2017). The only anatomical borders used by Zhuang et al. (2017) are those of V1 and barrel cortex, identified by cytochrome oxidase staining. In reality, the partitioning of the visual cortex was based on field sign maps, which are reproduced from Zhuang et al., (2017) in Figure 1A. It is unclear why the maps shown in Figures 2E and 2F differ from those in Figure 1A. It is possible that this is an oversight. But maintaining consistent areal boundaries across experimental conditions that are referenced to the underlying brain structure is critical for assigning modeled projections to areas or sub-regions. This problem is evident in Figure 2F, which is presented as evidence that the modeling approach recapitulates the tracings shown in Figure 3 of Wang and Burkhalter (2007). The dissimilarities between the modeling and tracing results are striking, unlike what is stated in the legend of Figure 2F.

(4) The Rowley and Sedigh-Sarvestani find that the partial coverage of the visual field in higher order areas shown by Zhuang et al (2017) is recreated by the model. It is important to caution that Zhuang et al's (2017) maps were derived from incomplete mappings of the visual field, which was confined to -25-35 deg of elevation. This underestimates the coverage we have found in LM and AL. Receptive field mappings show that LM covers 0-90 deg of azimuth and -30-80 elevation (Wang and Burkhalter, 2007). AL covers at least 0-90 deg of azimuth and -30-50 deg of elevation (Wang and Burkhalter, 2007; Wang et al., 2011). These are important differences. Partial coverage in LM and AL underestimates the size of these areas and may map two projection patches as inputs to subregions of a single area rather than inputs to two separate areas. Complete, or nearly complete, visual representations in LM and AL support that each is a single area. Importantly, both areas are included in a callosal-free zone (Wang and Burkhalter, 2007). The surrounding callosal connections align with the vertical meridian representation. The single map reversal is marked by a transition in m2AChR expression and cytoarchitecture (Wang et al., 2011).

(5) The statement that the "lack of visual field overlap across areas is suggestive of a lack of hierarchical processing" is predicated on the full acceptance of the mappings by Zhuang et al (2017). Based on the evidence reviewed above, the reclassification of visual areas proposed in Figure 1C seems premature.

(6) The existence of lateral connections is not unique to rodent cortex and has been described in primates (Felleman and Van Essen, 1991).

(7) Why the mouse and rat extrastriate visual cortex differ from those of many other mammals is unclear. One reason may be that mammals with V2 subregions are strongly binocular.

Reviewer #2 (Public review):

Summary:

Building on previous models of multisensory integration (including their earlier correlation-detection framework used for non-spatial signals), the author introduces a population-level Multisensory Correlation Detector (MCD) that processes raw auditory and visual data. Crucially, it does not rely on abstracted parameters, as is common in normative Bayesian models," but rather works directly on the stimulus itself (i.e., individual pixels and audio samples). By systematically testing the model against a range of experiments spanning human, monkey, and rat data - the authors show that their MCD population approach robustly predicts perception and behavior across species with a relatively small (0-4) number of free parameters.

Strengths:

(1) Unlike prior Bayesian models that used simplified or parameterized inputs, the model here is explicitly computable from full natural stimuli. This resolves a key gap in understanding how the brain might extract "time offsets" or "disparities" from continuously changing audio-visual streams.

(2) The same population MCD architecture captures a remarkable range of multisensory phenomena, from classical illusions (McGurk, ventriloquism) and synchrony judgments, to attentional/gaze behavior driven by audio-visual salience. This generality strongly supports the idea that a single low-level computation (correlation detection) can underlie many distinct multisensory effects.

(3) By tuning model parameters to different temporal rhythms (e.g., faster in rodents, slower in humans), the MCD explains cross-species perceptual data without reconfiguring the underlying architecture.

(4) The authors frame their model as a plausible algorithmic account of the Bayesian multisensory-integration models in Marr's levels of hierarchy.

Weaknesses:

What remains unclear is how the parameters themselves relate to stimulus quantities (like stimulus uncertainty), as is often straightforward in Bayesian models. A theoretical missing link is the explicit relationship between the parameters of the MCD models and those of a cue combination model, thereby bridging Marr's levels of hierarchy.

Likely Impact and Usefulness

The work offers a compelling unification of multiple multisensory tasks-temporal order judgments, illusions, Bayesian causal inference, and overt visual attention-under a single, fully stimulus-driven framework. Its success with natural stimuli should interest computational neuroscientists, systems neuroscientists, and machine learning scientists. This paper thus makes an important contribution to the field by moving beyond minimalistic lab stimuli, illustrating how raw audio and video can be integrated using elementary correlation analyses.

Reviewer #2 (Public review):

Summary:

This manuscript is a technical report on a new model of early neurogenesis, coupled to a novel platform for genetic screens. The model is more faithful than others published to date, and the screening platform is an advance over existing ones in terms of speed and throughput.

Strengths:

It is novel and useful.

Weaknesses:

The novelty of the results is limited in terms of biology, mainly a proof of concept of the platform and a very good demonstration of the hierarchical interactions of the top regulators of GRNs.

The value of the manuscript could be enhanced in two ways:

(1) by showing its versatility and transforming the level of neural tube to midbrain and hindbrain, and looking at the transcriptional hierarchies there.

(2) by relating the patterning of the organoids to the situation in vivo, in particular with the information in reference 49. The authors make a statement "To compare our findings with in vivo gene expression patterns, we applied the same approach to published scRNA-seq data from 4-week-old human embryos at the neurula stage" but it would be good to have a more nuanced reference: what stage, what genes are missing, what do they add to the information in that reference?

Reviewer #2 (Public review):

Summary:

The manuscript by Gitanjali Roy et al. applies deep transfer learning (DEGAS) to assign patient-level disease attributes (metadata) to single cells of T2D and non-diabetic patients, including obese patients. This led to the identification of a singular cluster of T2D-associated β-cells; and two subpopulations of obese- β-cells derived from either non-diabetic or T2D donors. The objective was to identify novel and established genes implicated in T2D and obesity. Their final goal is to validate their findings at the protein level using immunohistochemistry of pancreas tissue from non-diabetic and T2D organ donors.

Strengths:

This paper is well-written, and the findings are relevant for β-cell heterogeneity in T2D and obesity.

Weaknesses:

The validation they provide is not sufficiently strong: no DLK1 immunohistochemistry is shown of obese patient-derived sections. Additional presumptive relevant candidates from this transcriptomic analysis should be screened for, at the protein level.

Comments on revisions:

The authors have largely addressed my comments. No further experiments are requested.

Reviewer #2 (Public review):

Summary:

The authors are trying to understand why certain mutants of O-GlcNAc transferase (OGT) appear to cause developmental disorders in humans. As an important step towards that goal, the authors generated a mouse model with one of these mutations that disrupts OGT activity. They then go on to test these mice for behavioral differences, finding that the mutant mice exhibit some signs of hyperactivity and differences in learning and memory. They then examine alterations to the structure of the brain and skull, and again find changes in the mutant mice that have been associated with developmental disorders. Finally, they identify proteins that are up- or down-regulated between the two mice as potential mechanisms to explain the observations.

Strengths:

The major strength of this manuscript is the creation of this mouse model, as a key step in beginning to understand how OGT mutants cause developmental disorders. This line will prove important for not only the authors but other investigators as well, enabling the testing of various hypotheses and potentially treatments. The experiments are also rigorously performed, and the conclusions are well supported by the data.

Weaknesses:

The only weakness identified is a lack of mechanistic insight. However, this certainly may come in the future through more targeted experimentation using this mouse model.

Reviewer #2 (Public review):

Summary:

Zhou, Sajid et al. present a study investigating the STN involvement in signaled movement. They use fiber photometry, implantable lenses, and optogenetics during active avoidance experiments to evaluate this. The data are useful for the scientific community, and the overall evidence for their claims is solid, but many aspects of the findings are confusing and seemingly contradictory. For example, STN activity increases with contraversive turning in the fiber photometry experiments, but optogenetic stimulation of the STN evokes ipsiversive turning. While the authors present a huge collection of data, it is somewhat difficult to extract the key information and the meaningful implications resulting from this data.

Strengths:

The study is comprehensive in using many techniques, stimulation powers, frequencies, and configurations.

Weaknesses:

Here are the specific weaknesses of the paper.

(1) Vglut2 isn't a very selective promoter for the STN. Did the authors verify every injection across brain slices to ensure the para-subthalamic nucleus, thalamus, lateral hypothalamus, and other Vglut2-positive structures were never infected?

(2) The authors say in the methods that the high vs low power laser activation for optogenetic experiments was defined by the behavioral output. This is misleading, and the high vs low power should be objectively stated and the behavioral results divided according to the power used, not according to the behavioral outcome.

(3) In the fiber photometry experiments exposing mice to the range of tones, it is impossible to separate the STN response to the tone from the STN response to the movement evoked by the tone. The authors should expose the mouse to the tones in a condition that prevents movement, such as anesthetized or restrained, to separate out the two components.

(4) The claim 'STN activation is ideally suited to drive active avoids' needs more explanation. This claim comes after the fiber photometry experiments during active avoidance tasks, so there has been no causality established yet.

(5) The statistical comparisons in Figure 7E need some justification and/or clarification. The 9 neuron types are originally categorized based on their response during avoids, then statistics are run showing that they respond differently during avoids. It is no surprise that they would have significantly different responses, since that is how they were classified in the first place. The authors must explain this further and show that this is not a case of circular reasoning.

(6) The authors show that neurons that have strong responses to orientation show reduced activity during avoidance. What are the implications of this? The author should explain why this is interesting and important.

(7) It is not clear which conditions each mouse experienced in which order. This is critical to the interpretation of Figure 9 and the reduction of passive avoids during STN stimulation. Did these mice have the CS1+STN stimulation pairing or the STN+US pairing prior to this experiment? If they did, the stimulation of the STN could be strongly associated with either punishment or with the CS1 that predicts punishment. If that is the case, stimulating the STN during CS2 could be like presenting CS1+CS2 at the same time and could be confusing.

(8) The experiments in Figure 10 are used to say that STN stimulation is not aversive, but they only show that STN stimulation cannot be used as punishment in place of a shock. This doesn't mean that it is not aversive; it just means it is not as aversive as a shock. The authors should do a simpler aversion test, such as conditioned or real-time place preference, to claim that STN stimulation is not aversive. This is particularly surprising as previous work (Serra et al., 2023) does show that STN stimulation is aversive.

(9) In the discussion, the idea that the STN encodes 'moving away' from contralateral space is pretty vague and unsupported. It is puzzling that the STN activates more strongly to contraversive turns, but when stimulated, it evokes ipsiversive turns; however, it seems a stretch to speculate that this is related to avoidance. In the last experiments of the paper, the axons from the STN to the GPe and to the midbrain are selectively stimulated. Do these evoke ipsiversive turns similarly?

(10) In the discussion, the authors claim that the STN is essential for modulating action timing in response to demands, but their data really only show this in one direction. The STN stimulation reliably increases the speed of response in all conditions (except maximum speed conditions such as escapes). It seems to be over-interpreting the data to say this is an inability to modulate the speed of the task, especially as clear learning and speed modulation do occur under STN lesion conditions, as shown in Figure 12B. The mice learn to avoid and increase their latency in AA2 vs AA1, though the overall avoids and latency are different from controls. The more parsimonious conclusion would be that STN stimulation biases movement speed (increasing it) and that this is true in many different conditions.

(11) In the discussion, the authors claim that the STN projections to the midbrain tegmentum directly affect the active avoidance behavior, while the STN projections to the SNr do not affect it. This seems counter to their results, which show STN projections to either area can alter active avoidance behavior. What is the laser power used in these terminal experiments? If it is high (3mW), the authors may be causing antidromic action potentials in the STN somas, resulting in glutamate release in many brain areas, even when terminals are only stimulated in one area. The authors could use low (0.25mW) laser power in the terminals to reduce the chance of antidromic activation and spatially restrict the optical stimulation.

(12) Was normality tested for data prior to statistical testing?

(13) Why are there no error bars on Figure 5B, black circles and orange triangles?

Push

Push factor

Civilizing indigenous groups

Perhaps a blueprint for what woukd eventually happen in america?

Push

Reviewer #2 (Public review):

Summary:

Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels to those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one most significantly changing after treatment with 17α-Estradiol, suggesting an important role of these neurons on mediating its anti-ageing effects. This was also supported by an increase in circulating levels of oxytocin. It was also found that gene expression of corticotropin-releasing hormone neurons was significantly impacted by 17α-Estradiol even though it was not different between aged and young rats, suggesting that these neurons could be responsible for side effects related to this treatment. This article revealed some potential targets that should be further investigated in future studies regarding the role of 17α-Estradiol treatment in aged males.

Strengths:

• The single nucleus mRNA sequencing is a very powerful method for gene expression analysis and clustering. The supervised clustering of neurons was very helpful in revealing otherwise invisible differences between neuronal groups and helped identify specific neuronal populations as targets.

• There is a variety of functions used that allowed the differential analysis of a very complex type of data. This led to a better comparison between the different groups in many levels.

• There were some physiological parameters measured such as circulating hormone levels that helped the interpretation of the effects of the changes in hypothalamic gene expression.

Weaknesses:

• One main control group is missing from the study, the young males treated with 17α-Estradiol.

• Even though the technical approach is a sophisticated one, analyzing the whole rat hypothalamus instead of specific nuclei or subregions makes the study weaker.

• Although the authors claim to have several findings, the data fail to support these claims.

• The study is about improving ageing but no physiological data from the study demonstrated such claim with the exception of the testes histology which was not properly analyzed and was not even significantly different between the groups.

• Overall, the study remains descriptive with no physiological data to demonstrate that any of the effects on hypothalamic gene expression is related to metabolic, synaptic or other function.

Comments on revisions:

The authors revised part of the manuscript to address some of the reviewers' comments. This improved the language and the text flow to a certain extent. They also added an additional analysis including glial cells. However, they failed to address the main weaknesses brought up by the reviewers and did not add any experimental demonstration of their claims on lifespan expansion induced by 17α-estradiol in rats (the cited study does not include lifespan in rats). In addition, they insisted i keeping parts in the discussion that are not directly linked to any of the papers' findings.

Reviewer #2 (Public review):

Summary:

In this manuscript, Farber and colleagues have performed single cell RNAseq analysis on bone marrow derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach.

Strengths:

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting.

Weaknesses:

The manuscript is, in parts, hard to read due to the use of acronyms and there are some questions about data analysis that still need to be addressed.

Comments on revisions:

Dillard et al have made several improvements to their manuscript.

(1) We previously asked the authors to determine whether any cell types were enriched for BMD-related traits since the premise of the paper is that 'many genes impacting BMD do so by influencing osteogenic differentiation or ... adipogenic differentiation'. Given the potential for the cell culture method to skew the cell type distribution non-physiologically, it is important to establish which cell types in their assay are most closely associated with BMD traits. The new CELLECT analysis and Figure 1E address this point nicely. However, it would still be nice to see the correlations between these cell types and BMD traits in the mice as this would provide independent evidence to support their physiological importance more broadly.

(2) Shortening the introduction.

(3) Addressing limitations that arise from not accounting for founder genome SNPs when aligning scRNA-seq data.

(4) The main take-away of this paper is, to us, the development of a single cell approach to studying BMD-related traits. It is encouraging that the cells post-culture appear to be representative of those pre-culture (supplemental figure 3).

However, the authors seem to have neglected several comments made by both reviewers. While we share the authors' enthusiasm for the single cell analytical approach, we do not understand their reluctance to perform further statistical tests. We feel that the following comments have still not been addressed:

(1) The manuscript still contains the following:

"To provide further support that tradeSeq-identified genes are involved in differentiation, we performed a cell type-specific expression quantitative trait locus (eQTL) analysis for each mesenchymal cell type from the 80 DO mice. We identified 563 genes (eGenes) regulated by a significant cis-eQTL in specific cell types of the BMSC-OB scRNA-seq data (Supplementary Table S14). In total, 73 eGenes were also tradeSeq-identified genes in one or more cell type boundaries along their respective trajectories (Supplementary Table S9)."

The purpose of this paragraph is to convince readers that the eGenes approach aligns with the tradeSeq approach (and that their approach can therefore be trusted). It is essential that such claims are supported by statistical reasoning. Given that it would be very simple to perform permutation/enrichment analyses to address this point, and both reviewers requested similar analyses, we do not understand the author's reluctance here. Otherwise, this section should be rewritten so that it does not imply that the identification of these genes provides support for their approach.

(2) Given that a central purpose of this manuscript is to establish a systematic workflow for identifying candidate genes, the manuscript could still benefit from more explanation as to why the authors chose to highlight Tpx2 and Fgfrl1. Tpx2 does already have a role in bone physiology through the IMPC. The authors should comment on why they did not explore Kremen1, for instance, as this gene seems important for the transition to both OB1 and 2.

A final minor comment is that it would be very helpful if the authors could indicate if the DDGs in Table 1 are also eGenes for the relevant cell type. This is much more meaningful than looking through GTEx.

Reviewer #2 (Public review):

Summary:

This study introduces SemReps-8K, a large multimodal fMRI dataset collected while subjects viewed natural images and matched captions, and performed mental imagery based on textual cues. The authors aim to train modality-agnostic decoders--models that can predict neural representations independently of the input modality - and use these models to identify brain regions containing modality-agnostic information. They find that such decoders perform comparably or better than modality-specific decoders and generalize to imagery trials.

Strengths:

(1) The dataset is a substantial and well-controlled contribution, with >8,000 image-caption trials per subject and careful matching of stimuli across modalities - an essential resource for testing theories of abstract and amodal representation.

(2) The authors systematically compare unimodal, multimodal, and cross-modal decoders using a wide range of deep learning models, demonstrating thoughtful experimental design and thorough benchmarking.

(3) Their decoding pipeline is rigorous, with informative performance metrics and whole-brain searchlight analyses, offering valuable insights into the cortical distribution of shared representations.

(4) Extension to mental imagery decoding is a strong addition, aligning with theoretical predictions about the overlap between perception and imagery.

Weaknesses:

While the decoding results are robust, several critical limitations prevent the current findings from conclusively demonstrating truly modality-agnostic representations:

(1) Shared decoding ≠ abstraction: Successful decoding across modalities does not necessarily imply abstraction or modality-agnostic coding. Participants may engage in modality-specific processes (e.g., visual imagery when reading, inner speech when viewing images) that produce overlapping neural patterns. The analyses do not clearly disambiguate shared representational structure from genuinely modality-independent representations. Furthermore, in Figure 5, the modality-agnostic encoder did not perform better than the modality-specific decoder trained on images (in decoding images), but outperformed the modality-specific decoder trained on captions (in decoding captions). This asymmetry contradicts the premise of a truly "modality-agnostic" encoder. Additionally, given the similar performance between modality-agnostic decoders based on multimodal versus unimodal features, it remains unclear why neural representations did not preferentially align with multimodal features if they were truly modality-independent.

(2) The current analysis cannot definitively conclude that the decoder itself is modality-agnostic, making "Qualitative Decoding Results" difficult to interpret in this context. This section currently provides illustrative examples, but lacks systematic quantitative analyses.

(3) The use of mental imagery as evidence for modality-agnostic decoding is problematic. Imagery involves subjective, variable experiences and likely draws on semantic and perceptual networks in flexible ways. Strong decoding in imagery trials could reflect semantic overlap or task strategies rather than evidence of abstraction.

The manuscript presents a methodologically sophisticated and timely investigation into shared neural representations across modalities. However, the current evidence does not clearly distinguish between shared semantics, overlapping unimodal processes, and true modality-independent representations. A more cautious interpretation is warranted. Nonetheless, the dataset and methodological framework represent a valuable resource for the field.

Reviewer #2 (Public review):

Summary:

This study develops a joint epidemiological and population genetic model to infer variant-specific effective reproduction numbers Rt and growth advantages of SARS-CoV-2 variants using US case counts and sequence data (Jan 2021-Mar 2022). For this, they use the commonly used renewal equation framework, observation models (negative binomial with zero inflation and Dirichlet-multinomial likelihoods, both to account for overdispersion). For the parameterization of Rt, again, they used a classic cubic spline basis expansion. Additionally, they use Bayesian Inference, specifically SVI. I was reassured to see the sensitivity analysis on the generation time to check effects on Rt.

This is an incredibly robust study design. Integrating case and sequence data enables estimation of both absolute and relative variant fitness, overcoming limitations of frequency-only or case-only models. This reminds me of https://www.medrxiv.org/content/10.1101/2023.01.02.23284123v4.full

I also really appreciated the flexible and interpretable parameterization of the renewal equations with splines. But I may be biased since I really like splines!

The approach is justified, however, it has some big limitations. Specifically, there are some notable weaknesses, that I detail below.

(1) The model does not account for demographic stochasticity or transmission overdispersion (superspreading), which are known to affect SARS-CoV-2 dynamics and can bias Rt, especially in low incidence or early introduction phases.

(2) While the authors explore the sensitivity of generation time, the reliance on fixed generation time parameters (with some adjustments for Delta/Omicron) may still bias results

(3) There is no explicit adjustment for population immunity, which limits the ability to disentangle intrinsic variant fitness (even though the model allows for inclusion of covariates - this to me is one of two major flaws in the study.

(4) The second major flaw in my opinion is that there is no hierarchical pooling across states - each state is modeled independently. A hierarchical Bayesian model could borrow strength across states, improving estimates for states with sparse data and enabling more robust inference of shared variant effects.

I would strongly recommend the following things in order of priority, where the first two points I consider critical.

(1) Implement a hierarchical model for variant growth advantages and Rt across states.

(2) Include time-varying covariates for vaccination rates, prior infection, and non-pharmaceutical interventions directly. This would help disentangle intrinsic variant transmissibility from changes in population susceptibility and behavior.

(3) Extend the renewal model to a stochastic or branching process framework that explicitly models overdispersed transmission.

(4) It would be good to allow for multiple seeding events per variant and per state. This can be informed by phylogeography in a minimum effort way and would improve the accuracy of Rt.

(5) By now, I don't think it will be a surprise that addressing sampling bias is standard, reweighting sequence data or comparing results with independent surveillance data to assess the impact of non-representative sequencing.

Reviewer #2 (Public review):

This important paper studies the problem of learning from feedback given by sources of varying credibility. The convincing combination of experiment and computational modeling helps to pin down properties of learning, while opening unresolved questions for future research.

Summary:

This paper studies the problem of learning from feedback given by sources of varying credibility. Two bandit-style experiments are conducted in which feedback is provided with uncertainty, but from known sources. Bayesian benchmarks are provided to assess normative facets of learning, and alternative credit assignment models are fit for comparison. Some aspects of normativity appear, in addition to possible deviations such as asymmetric updating from positive and negative outcomes.

Strengths:

The paper tackles an important topic, with a relatively clean cognitive perspective. The construction of the experiment enables the use of computational modeling. This helps to pinpoint quantitatively the properties of learning and formally evaluate their impact and importance. The analyses are generally sensible, and advanced parameter recovery analyses (including cross-fitting procedure) provide confidence in the model estimation and comparison. The authors have very thoroughly revised the paper in response to previous comments.

Weaknesses:

The authors acknowledge the potential for cognitive load and the interleaved task structure to play a meaningful role in the results, though leave this for future work. This is entirely reasonable, but remains a limitation in our ability to generalize the results. Broadly, some of the results obtain in cases where the extent of generalization is not always addressed and remains uncertain.

Reviewer #2 (Public review):

Summary:

This article addresses a very pertinent question: what are the computational mechanisms underlying risky behaviour in patients who have attempted suicide? In particular, it is impressive how the authors find a broad behavioural effect whose mechanisms they can then explain and refine through computational modeling. This work is important because, currently, beyond previous suicide attempts, there has been a lack of predictive measures. This study is the first step towards that: understanding the cognition on a group level. This is before being able to include it in future predictive studies (based on the cross-sectional data, this study by itself cannot assess the predictive validity of the measure).

Strengths:

(1) Large sample size.

(2) Replication of their own findings.

(3) Well-controlled task with measures of behaviour and mood + precise and well-validated computational modeling.

Weaknesses:

I can't really see any major weakness, but I have a few questions:

(1) I can see from the parameter recovery that the parameters are very well identified. Is it surprising that this is the case, given how many parameters there are for 90 trials? Could the authors show cross-correlations? I.e., make a correlation matrix with all real parameters and all fitted parameters to show that not only the diagonal (i.e., same data is the scatter plots in S3) are high, but that the off-diagonals are low.

(2) Could the authors clarify the result in Figure 2B of a correlation between gambling rate and suicidal ideation score, is that a different result than they had before with the group main effect? I.e., is your analysis like this: gambling rate ~ suicide ideation + group assignment? (or a partial correlation)? I'm asking because BSI-C is also different between the groups. [same comment for later analyses, e.g. on approach parameter].

(3) The authors correlate the impact of certain rewards on mood with the % gambling variable. Could there not be a more direct analysis by including mood directly in the choice model?

(4) In the large online sample, you split all participants into S+ and S-. I would have imagined that instead, you would do analyses that control for other clinical traits. Or, for example, you have in the S- group only participants who also have high depression scores, but low suicide items.

Reviewer #2 (Public review):

Summary:

The function of neural circuits relies heavily on the balance of excitatory and inhibitory inputs. Particularly, inhibitory inputs are understudied when compared to their excitatory counterparts due to the diversity of inhibitory neurons, their synaptic molecular heterogeneity, and their elusive signature. Thus, insights into these aspects of inhibitory inputs can inform us largely on the functions of neural circuits and the brain.

Endophilin A1, an endocytic protein heavily expressed in neurons, has been implicated in numerous pre- and postsynaptic functions, however largely at excitatory synapses. Thus, whether this crucial protein plays any role in inhibitory synapse, and whether this regulates functions at the synaptic, circuit, or brain level remains to be determined.

The three remaining concerns are:

(1) The use of one-way ANOVA is not well justified.

(2) The use of superplots to show culture to culture variability would make it more transparent.

(3) Change EEN1 in Figure 8B to EndoA1.

Comments on revised version:

The authors addressed the concerns adequately.

Reviewer #2 (Public review):

The study by Chaguza et al. presents a novel perspective on pneumococcal growth kinetics, suggesting that the overall genetic background of Streptococcus pneumoniae, rather than specific loci, plays a more dominant role in determining growth dynamics. Through a genome-wide association study (GWAS) approach, the authors propose a shift in how we understand growth regulation, differing from earlier findings that pinpointed individual genes, such as wchA or cpsE, as key regulators of growth kinetics. This study highlights the importance of considering the cumulative impact of the entire genetic background rather than focusing solely on individual genetic loci.

The study emphasizes the cumulative effects of genetic variants, each contributing small individual impacts, as the key drivers of pneumococcal growth. This polygenic model moves away from the traditional focus on single-gene influences. Through rigorous statistical analyses, the authors persuasively advocate for a more holistic approach to understanding bacterial growth regulation, highlighting the complex interplay of genetic factors across the entire genome. Their findings open new avenues for investigating the intricate mechanisms underlying bacterial growth and adaptation, providing fresh insights into bacterial pathogenesis.

Strengths:

This study exemplifies a holistic approach to unraveling key factors in bacterial pathogenesis. By analyzing a large dataset of whole-genome sequences and employing robust statistical methodologies, the authors provide strong evidence to support their main findings. Which is a leap forward from previous studies focused on a relatively smaller number of strains. Their integration of genome-wide association studies (GWAS) highlights the cumulative, polygenic influences on pneumococcal growth kinetics, challenging the traditional focus on individual loci. This comprehensive strategy not only advances our understanding of bacterial growth regulation but also establishes a foundation for future research into the genetic underpinnings of bacterial pathogenesis and adaptation. The amount of data generated and corresponding approaches to analyze the data are impressive as well as convincing. The figures are convincing and comprehensible too. The revised version of the manuscript, after the addition and including explanations, is more convincing and acceptable.

Weaknesses:

This study suggests evidence that the genetic background significantly influences bacterial growth kinetics. However, the absence of experimental validation remains a critical limitation. Although the authors acknowledge in their response to reviewers that bench-experiments were beyond the scope of this work and are planned, this gap of experimental validation weakens the current conclusions. Demonstrable validation will be essential to corroborate the associations identified through the GWAS approach. Future experimental efforts will be critical to substantiate these findings and to deepen our understanding of the genetic determinants governing bacterial growth dynamics.

Reviewer #2 (Public review):

Summary:

Govorunova et al present three new anion opsins that have potential applications silencing neurons. They identify new opsins by scanning numerous databases for sequence homology to known opsins, focusing on anion opsins. The three opsin identified, are uncommonly fast, potent, and are able to silence neuronal activity. The authors characterize numerous parameters of the opsins and compare these opsins to the existing and widely used GtACR opsins.

Strengths:

This paper follows the tradition of the Spudich lab, presenting and rigorously characterizing potentially valuable opsins. Furthermore, they explore several mutations of the identified opsin that may make these opsins even more useful for the broader community. The opsins AnsACR and FtACR are particularly notable having extraordinarily fast onset kinetics that could have utility in many domains. Furthermore, the authors show AnsACR is useable in multiphoton experiments having a peak photocurrent in a commonly used wavelength. Overall, the author's detailed measurements and characterization make for an important resource - both presenting new opsins that may be important for future experiment, and providing characterizations to expand our understanding of opsin biophysics in general.

Reviewer #2 (Public review):

Summary:

This study makes a significant contribution to understanding the microenvironment of megakaryocytes (MKs) in the bone marrow, identifying an extracellular matrix (ECM) cage structure that influences MK localization and maturation. The authors provide compelling evidence for the presence of this ECM cage and its role in MK homeostasis, employing an array of sophisticated imaging techniques and molecular analyses.

The authors have addressed most of the concerns raised in the previous review, providing clarifications and additional data that strengthen their conclusions

More broadly, this work adds to a growing recognition of the ECM as an active participant in haematopoietic cell regulation in the bone marrow microenvironment. This work could pave the way to future studies investigating how the megakaryocytes' ECM cage affects their function as part of the haematopoietic stem cell niche, and by extension, influences global haematopoiesis.

Reviewer #2 (Public review):

Summary:

This manuscript addresses the gap in knowledge related to the cardiac function of the S-denitrosylase SNO-CoA Reductase 2 (SCoR2; product of the Akr1a1 gene). Genetic variants in SCoR2 have been linked to cardiovascular disease, yet its exact role in heart remains unclear. This paper demonstrates that mice deficient in SCoR2 show significant protection in a myocardial infarction (MI) model. SCoR2 influenced ketolytic energy production, antioxidant levels, and polyol balance through the S-nitrosylation of crucial metabolic regulators.

Strengths:

Addresses a well-defined gap in knowledge related to the cardiac function of SNO-CoA Reductase 2. Besides the in-depth case for this specific player, the manuscripts sheds more light on the links between S-nytrosylation and metabolic reprogramming in heart.

Rigorous proof of requirement through the combination of gene knockout and in vivo myocardial ischemia/reperfusion

Identification of precise Cys residue for SNO-modification of BDH1 as SCoR2 target in cardiac ketolysis

Weaknesses:

The experiments with BDH1 stability were performed in mutant 293 cells. Was there a difference in BDH1 stability in myocardial tissue or primary cardiomyocytes from SCoR2-null vs -WT mice? Same question extends to PKM2.

In the absence of tracing experiments, the cross-sectional changes in ketolysis, glycolysis or polyol intermediates presented in Figures 4 and 5 are suggestive at best. This needs to be stressed while describing and interpreting these results.

The findings from human samples with ischemic and non-ischemic cardiomyopathy do not seem immediately or linearly in line with each other and with the model proposed from the KO mice. While the correlation holds up in the non-ischemic cardiomyopathy (increased SNO-BDH1, SNO-PKM2 with decreased SCoR2 expression), how do the Authors explain the decreased SNO-BDH1 with preserved SCoR2 expression in ischemic cardiomyopathy? This seems counterintuitive as activation of ketolysis is a quite established myocardial response to the ischemic stress. It may help the overall message clarity to focus the human data part on only NICM patients.

(partially linked to the point above) an important proof that is lacking at present is the proof of sufficiency for SCoR2 in S-Nytrosylation of targets and cardiac remodeling. Does SCoR2 overexpression in heart or isolated cardiomyocytes reduce S-nitrosylation of BDH1 and other targets, undermining heart function at baseline or under stress?

Comments on revisions:

Some of my points have been addressed. However, the points related to 1) BDH1 stability effect in cardiomyocytes; 2) human relevance of SNO-BDH1; 3) SCoR2 sufficiency remain unclear. That said, this manuscript will provide useful information to the field as such.

Reviewer #2 (Public review):

Summary

This study provides new insights into organ morphogenesis using the Drosophila salivary gland (SG) as a model. The authors identify a requirement for sulfation in regulating lumen expansion, which correlates with several effects at the cellular level, including regulation of intracellular trafficking and the organization of Golgi, the aECM and the apical membrane. In addition, the authors show that the ZP proteins Dumpy (Dpy) and Pio form an aECM regulating lumen expansion. Previous reports already pointed to a role for Papss in sulfation in SG and the presence of Dpy and Pio in the SG. Now this work extends these previous analyses and provides more detailed descriptions that may be relevant to the fields of morphogenesis and cell biology (with particular focus on ECM research and tubulogenesis). This study nicely presents valuable information regarding the requirements of sulfation and the aECM in SG development.

Strengths:

- The results supporting a role for sulfation in SG development are strong. In addition, the results supporting the involvement of Dpy and Pio in the aECM of the SG, their role in lumen expansion, and their interactions, are also strong.

- The authors have made an excellent job in revising and clarifying the many different issues raised by the reviewers, particularly with the addition of new experiments and quantifications. I consider that the manuscript has improved considerably.

- The authors generated a catalytically inactive Papss enzyme, which is not able to rescue the defects in Papss mutants, in contrast to wild type Papss. This result clearly indicates that the sulfation activity of Papss is required for SG development.

Reviewer #2 (Public review):

Summary:

The study is well-conducted, revealing that NPY1, with previously less-characterized molecular functions, can suppress pid mutant phenotypes with a phosphorylation-based mechanism. Overexpression of NPY1 (NPY1-OE) results in PIN phosphorylation at unique sites and bypasses the requirement for PID for this event. Conversely, a C-terminal deleted form of NPY1 (NPY1-dC) fails to rescue pid despite promoting a certain phospho-profile in PIN proteins.

Strengths:

(1) The careful genetic analyses of pid suppression by NPY1-OE and the inability of NPY1dC to do the same.

(2) Phospho-proteomics approaches reveal that NPY1-OE induces phosphorylation of PINs at non-canonical sites, independent of PID.

Weaknesses:

(1) The native role of NPY1 is not tested by phospho-proteomics in loss-of-function npy1 mutants. Such analysis would be crucial to demonstrate that NPY1 is required for the observed phosphorylation events.

(2) The functional consequences of the newly identified phosphorylation sites in PINs remain speculative. Site-directed mutagenesis (phospho-defective and phospho-mimetic) would help clarify their physiological roles.

(3) The kinase responsible for NPY1-mediated phosphorylation remains unidentified. Since NPY1 is a non-kinase protein, a model involving recruitment of partner kinases (e.g., PIN-phosphorylating kinases other than PID) should be considered or discussed.

Reviewer #2 (Public Review):

This paper aims to dissect the relative importance of the various cues that establish PCP in the wing disc of Drosophila, which remains a prominent and relevant model for PCP. The authors suggest that one must consider cues at three scales (molecular, cell and tissue) and specifically design tests for the importance of cell-level cues, which they call non-local cell scale signalling. They develop clever experimental approaches that allow them to track complex stability and also to induce polarity at experimentally defined times. In a first set of experiments, they restore PCP after the global cues have disappeared (de novo polarisation) and conclude from the results that another (cell scale) cue must exist. In another set of experiments, they show that de novo repolarization is robust to the dosage of various components of core PCP, leading them to conclude that there must be an underlying cell scale polarity, which, apparently, has nothing to do with microtubule or cell shape polarity. They then describe nice evidence that de novo polarisation is relatively short range both in a polarised and unpolarised field. They conclude that there is a strong cell-intrinsic polarity that remains to be characterised.

Major concerns:

(1) The first set of repolarisation experiments is performed after the global cell rearrangements that have been shown to act as global signals. However, this approach does not exclude the possible contribution of an unknown diffusible global signal.

(2) The putative non-local cell scale signal must be more precisely defined (maybe also given a better name). It is not clear to me that one can separate cell-scale from molecular-scale signal. Local signals can redistribute within a cell (or membrane) so local signals are also cell-scale. Without a clear definition, it is difficult to interpret the results of the gene dosage experiments. The link between gene dosage and cell-scale signal is not rigorously stated. Related to this, the concluding statement of the introduction is too cryptic.

Critique:

The experiments described in this paper are of high quality with a sophisticated level of design and analysis. However, there needs to be some recalibration of the extent of the conclusions that can be drawn. Moreover, a limitation of this paper is that, despite the quality of their data, they cannot give a molecular hint about the nature of their proposed cell-scale signal.

Reviewer #2 (Public review):

Summary:

Using C. elegans as a model, the authors present an interesting story demonstrating a new regulatory connection between olfactory neurons and the digestive system. Mechanistically, they identified key factors (NSY-1, STR-130 et.al) in neurons, as well as critical 'signaling factors' (INS-23, DAF-2) that bridge different cells/tissues to execute the digestive shutdown induced by poor-quality food (Staphylococcus saprophyticus, SS).

Strengths:

The conclusions of this manuscript are mostly well supported by the experimental results shown.

Weaknesses:

The authors have done a nice job in addressing my comments.

Reviewer #2 (Public review):

Summary:

The authors aimed to uncover what role, if any, the UFD1/NPL4 complex might play in innate immune responses of the nematode C. elegans. The authors find that loss of the complex renders animals more sensitive to both pathogenic and non-pathogenic bacteria. However, there appears to be a complex interplay with known innate immune pathways since loss of UFD1/NPL4 actually results in increased survival of animals lacking the canonical innate immune pathways.

Strengths:

The authors perform robust genetic analysis to exclude and include possible mechanisms by which the UFD1/NPL4 pathway acts in the innate immune response.

Weaknesses:

The argument that the loss of the UFD1/NPL4 complex triggers a response that mimics that of an intracellular pathogen is not thoroughly investigated. Additionally, the finding of a role of the GATA transcription factor, ELT-2, in this response is suggestive, but experiments showing sufficiency in the context of loss of the UFD1/NPL4 complex need to be explored.

Comments on revisions:

The authors have performed several control experiments for their RNAi based experiments and also tested the requirement for xbp-1s in their paradigm. The findings and their interpretations are acceptable.

Reviewer #3 (Public review):

Summary:

This study investigates the role of the host protein RBMX2 in regulating the response to Mycobacterium bovis infection and its connection to epithelial-mesenchymal transition (EMT), a key pathway in cancer progression. Using bovine and human cell models, the authors have wisely shown that RBMX2 expression is upregulated following M. bovis infection and promotes bacterial adhesion, invasion, and survival by disrupting epithelial tight junctions via the p65/MMP-9 signaling pathway. They also demonstrate that RBMX2 facilitates EMT and is overexpressed in human lung cancers, suggesting a potential link between chronic infection and tumor progression. The study highlights RBMX2 as a novel host factor that could serve as a therapeutic target for both TB pathogenesis and infection-related cancer risk.

Strengths:

The major strengths lie in its multi-omics integration (transcriptomics, proteomics, metabolomics) to map RBMX2's impact on host pathways, combined with rigorous functional assays (knockout/knockdown, adhesion/invasion, barrier tests) that establish causality through the p65/MMP-9 axis. Validation across bovine and human cell models and in clinical tissue samples enhances translational relevance. Finally, identifying RBMX2 as a novel regulator linking mycobacterial infection to EMT and cancer progression opens exciting therapeutic avenues.

Weaknesses:

There are a few minor weaknesses like grammatical errors, spelling mistakes. Also, the manuscript is too dense; improving the narratives in the Results and Discussion section could help readers follow the logic of the experimental design and conclusions.

Reviewer #2 (Public review):

Aldridge et al. demonstrate the important role of IL-27 in limiting emergency myelopoiesis in response to Toxoplasma gondii infection. Interestingly, IL-27 acts specifically at the level of early haematopoietic progenitors, inducing STAT signalling, which, in this case, dampens proliferation and preserves HSC fitness.

They used different mouse genetic models such as HSC lineage tracing, IL27 and IL27R-deficient mice to show that :

HSCs actively participate in emergency myelopoiesis during Toxoplasma gondii infection.

The absence of IL27 and IL27R increases monocyte progenitors and monocytes, mainly inflammatory monocytes CCR2hi.

At steady state, loss of IL27 impairs HSC fitness as competitive transplantation shows long-term engraftment deficiency of IL27 BM cells. This impairment is exacerbated after infection.

IL27 is produced by various BM and other tissue cells at steady state and its expression increases with infection, mainly by increasing the number of monocytes producing it.

This article highlights a new mechanism that acts directly at the level of early hematopoietic cells to limit over-inflammation during infection.

Reviewer #3 (Public review):

Summary:

Chatzis et al showed that β-glucan trained macrophages have decreased phagocytic activity of apoptotic tumor cells and that is accompanied by lower levels of secreted IL-1β using mouse model.

Strengths:

This finding has potential impact on designing new cancer immunotherapeutic approaches by targeting macrophage efferocytosis.

The concerns have been addressed.

Reviewer #2 (Public review):

Hawes et al. investigated the role of striatal neurons in the patch compartment of the dorsal striatum. Using Sepw1-Cre line, the authors combined a modified version of the light/dark transition box test that allows them to examine locomotor activity in different environmental valence with a variety of approaches, including cell-type-specific ablation, miniscope calcium imaging, fiber photometry, and opto-/chemogenetics. First, they found ablation of patchy striatal neurons resulted in an increase in movement vigor when mice stayed in a safe area or when they moved back from more anxiogenic to safe environments. The following miniscope imaging experiment revealed that a larger fraction of striatal patchy neurons was negatively correlated with movement speed, particularly in an anxiogenic area. Next, the authors investigated differential activity patterns of patchy neurons' axon terminals, focusing on those in GPe, GPi, and SNr, showing that the patchy axons in SNr reflect movement speed/vigor. Chemogenetic and optogenetic activation of these patchy striatal neurons suppressed the locomotor vigor, thus demonstrating their causal role in the modulation of locomotor vigor when exposed to valence differentials. Unlike the activation of striatal patches, such a suppressive effect on locomotion was absent when optogenetically activating matrix neurons by using the Calb1-Cre line, indicating distinctive roles in the control of locomotor vigor by striatal patch and matrix neurons. Together, they have concluded that nigrostriatal neurons within striatal patches negatively regulate movement vigor, dependent on behavioral contexts where motivational valence differs.

The strengths of this work include the use of multiple experimental approaches, including genetic/viral ablation of patch neurons, miniscope single-cell imaging, as well as projection-specific recording of axonal activity by fiber photometry, and causal manipulation of the neurons by chemogenetic and optogenetics. Although similar findings were reported previously, the authors' results will be of value owing to multiple levels of investigation. In my view, this study will add to the important literature by demonstrating how patch (striosomal) neurons in the striatum controls movement vigor.

Reviewer #2 (Public review):

Summary:

Park et al. has made a tool for spatiotemporally restricted knockout of the astrocytic GABA transporter GAT3 leveraging CRISPR/Cas9 and viral transduction in adult mice, and evaluated the effects of GAT3 on neural encoding of visual stimulation.

Strengths:

This concise manuscript leverages state-of-the-art gene CRISPR/Cas9 technology for knocking out astrocytic genes. This has to a little degree been preformed previously in astrocytes and represents an important development in the field. Moreover they utilize in vivo two-photon imaging of neural responses to visual stimuli as a readout of neural activity, in addition to validating their data with ex vivo electrophysiology. Lastly, they use advanced statistical modeling to analyze the impact on GAT3 knockout. Overall, the study comes across as rigorous and convincing.

Weaknesses:

Adding the following experiments would potentially have strengthened the conclusions and helped interpret the findings, although may be considered outside the scope of this manuscript, and be pursued in future work:

(1) Neural activity is quite profoundly influenced by GAT3 knockout. Corroborating these relatively large changes to neural activity with in vivo electrophysiology of some sort as an additional readout would have strengthened the conclusions.

(2) Given the quite large effects on neural coding in visual cortex assessed with jRGECO imaging it would have been interesting the mouse groups could have been subjected to behavioral testing assessing the visual system.

Reviewer #2 (Public review):

This manuscript explores the mechanisms underlying cerebral cortical folding using a combination of physical modelling, computational simulations, and geometric morphometrics. The authors extend their prior work on human brain development (Tallinen et al., 2014; 2016) to a comparative framework involving three mammalian species: ferrets (Carnivora), macaques (Old World monkeys), and humans (Hominoidea). By integrating swelling gel experiments with mathematical differential growth models, they simulate sulcification instability and recapitulate key features of brain folding across species. The authors make commendable use of publicly available datasets to construct 3D models of fetal and neonatal brain surfaces: fetal macaque (ref. [26]), newborn ferret (ref. [11]), and fetal human (ref. [22]).

Using a combination of physical models and numerical simulations, the authors compare the resulting folding morphologies to real brain surfaces using morphometric analysis. Their results show qualitative and quantitative concordance with observed cortical folding patterns, supporting the view that differential tangential growth of the cortex relative to the subcortical substrate is sufficient to account for much of the diversity in cortical folding. This is a very important point in our field, and can be used in the teaching of medical students.

Brain folding remains a topic of ongoing debate. While some regard it as a critical specialization linked to higher cognitive function, others consider it an epiphenomenon of expansion and constrained geometry. This divergence was evident in discussions during the Strüngmann Forum on cortical development (Silver et al., 2019). Though folding abnormalities are reliable indicators of disrupted neurodevelopmental processes (e.g., neurogenesis, migration), their relationship to functional architecture remains unclear. Recent evidence suggests that the absolute number of neurons varies significantly with position-sulcus versus gyrus-with potential implications for local processing capacity (e.g., https://doi.org/10.1002/cne.25626). The field is thus in need of comparative, mechanistic studies like the present one.

This paper offers an elegant and timely contribution by combining gel-based morphogenesis, numerical modelling, and morphometric analysis to examine cortical folding across species. The experimental design - constructing two-layer PDMS models from 3D MRI data and immersing them in organic solvents to induce differential swelling - is well-established in prior literature. The authors further complement this with a continuum mechanics model simulating folding as a result of differential growth, as well as a comparative analysis of surface morphologies derived from in vivo, in vitro, and in silico brains.

I offer a few suggestions here for clarification and further exploration:

Major Comments

(1) Choice of Developmental Stages and Initial Conditions

The authors should provide a clearer justification for the specific developmental stages chosen (e.g., G85 for macaque, GW23 for human). How sensitive are the resulting folding patterns to the initial surface geometry of the gel models? Given that folding is a nonlinear process, early geometric perturbations may propagate into divergent morphologies. Exploring this sensitivity-either through simulations or reference to prior work-would enhance the robustness of the findings.

(2) Parameter Space and Breakdown Points

The numerical model assumes homogeneous growth profiles and simplifies several aspects of cortical mechanics. Parameters such as cortical thickness, modulus ratios, and growth ratios are described in Table II. It would be informative to discuss the range of parameter values for which the model remains valid, and under what conditions the physical and computational models diverge. This would help delineate the boundaries of the current modelling framework and indicate directions for refinement.

(3) Neglected Regional Features: The Occipital Pole of the Macaque

One conspicuous omission is the lack of attention to the occipital pole of the macaque, which is known to remain smooth even at later gestational stages and has an unusually high neuronal density (2.5× higher than adjacent cortex). This feature is not reproduced in the gel or numerical models, nor is it discussed. Acknowledging this discrepancy-and speculating on possible developmental or mechanical explanations-would add depth to the comparative analysis. The authors may wish to include this as a limitation or a target for future work.

(4) Spatio-Temporal Growth Rates and Available Human Data

The authors note that accurate, species-specific spatio-temporal growth data are lacking, limiting the ability to model inhomogeneous cortical expansion. While this may be true for ferret and macaque, there are high-quality datasets available for human fetal development, now extended through ultrasound imaging (e.g., https://doi.org/10.1038/s41586-023-06630-3). Incorporating or at least referencing such data could improve the fidelity of the human model and expand the applicability of the approach to clinical or pathological scenarios.

(5) Future Applications: The Inverse Problem and Fossil Brains

The authors suggest that their morphometric framework could be extended to solve the inverse growth problem-reconstructing fetal geometries from adult brains. This speculative but intriguing direction has implications for evolutionary neuroscience, particularly the interpretation of fossil endocasts. Although beyond the scope of this paper, I encourage the authors to elaborate briefly on how such a framework might be practically implemented and validated.

Conclusion

This is a well-executed and creative study that integrates diverse methodologies to address a longstanding question in developmental neurobiology. While a few aspects-such as regional folding peculiarities, sensitivity to initial conditions, and available human data-could be further elaborated, they do not detract from the overall quality and novelty of the work. I enthusiastically support this paper and believe that it will be of broad interest to the neuroscience, biomechanics, and developmental biology communities.

Note: The paper mentions a companion paper [reference 11] that explores the cellular and anatomical changes in the ferret cortex. I did not have access to this manuscript, but judging from the title, this paper might further strengthen the conclusions.

Reviewer #2 (Public review):

Summary:

The authors aim to quantify passive muscle forces in the legs of Drosophila, and test the hypothesis that these forces would be sufficient to support body weight in small insects. They take advantage of the genetic tools available in Drosophila, and use a combination of genetic silencing (optogenetic inactivation of motor neurons), kinematic measurements, and simulations using OpenSim. This integrative toolkit is used to examine the role of passive torques across multiple leg joints. They find that passive forces are weaker than expected - in particular, passive forces were found to be too weak to support the body weight of the fly. This challenges previous scaling assumptions derived from studies in larger insects and has potential implications for our understanding of motor control in small animals.

Strengths:

The primary strength of this work lies in its integration of multiple analyses. By pulling together simulations, kinematic measurements from high-resolution videos, and genetic manipulation, they are able to overcome limitations of past studies. In particular, optogenetic manipulation allowed for measurements to be made in whole animals, and the modeling component is valuable because it both validates experimental findings and elucidates the mechanism behind some of the observed dynamic consequences (e.g., the rapid fall after motor inactivation). The conclusions made in the study are well-supported by the data and could have an impact on a number of fields, including invertebrate neurobiology and bioinspired design.

Weaknesses:

While (as mentioned above) the study's conclusions are well-supported by the results and modeling, limitations arise because of the assumptions made. For instance, using a linear approximation may not hold at larger joint angles, and future studies would benefit from accounting for nonlinearities. Future studies could also delve into the source of passive forces, which is important for more deeply understanding the anatomical and physical basis of the results in this study. For instance, assessments of muscle or joint properties to correlate stiffness values with physical structure might be an area of future consideration

Reviewer #2 (Public review):

Summary:

Based on MRI data of the ferret (a gyrencephalic non-primate animal, in whom folding happens postnatally), the authors create in vitro physical gel models and in silico numerical simulations of typical cortical gyrification. They then use genetic manipulations of animal models to demonstrate that cortical thickness and expansion rate are primary drivers of atypical morphogenesis. These observations are then used to explain cortical malformations in humans.

Strengths:

The paper is very interesting and original, and combines physical gel experiments, numerical simulations, as well as observations in MCD. The figures are informative, and the results appear to have good overall face validity.

Weaknesses:

On the other hand, I perceived some lack of quantitative analyses in the different experiments, and currently, there seems to be rather a visual/qualitative interpretation of the different processes and their similarities/differences.

Ideally, the authors also quantify local/pointwise surface expansion in the physical and simulation experiments, to more directly compare these processes. Time courses of eg, cortical curvature changes, could also be plotted and compared for those experiments.

I had a similar impression about the comparisons between simulation results and human MRI data. Again, face validity appears high, but the comparison appeared mainly qualitative.

I felt that MCDs could have been better contextualized in the introduction.

Reviewer #2 (Public review):

Summary:

Foik et al. studied the regulation of the fro operon in response to HOCl, an oxidant derived from immune cells, especially neutrophils. They use a transcriptional fusion of YFP to the froA promoter in an mCherry-expressing P. aeruginosa strain to determine fro-induction under the microscope. They use this system to study fro expression in medium, in the presence of neutrophils and macrophages, neutrophil-conditioned medium, and several chemical stimuli, including NaCl, HOCl, hydrogen peroxide, nitric acid, hydrochloric acid, and sodium hydroxide. They also use a corneal infection model to demonstrate that froA is upregulated in P. aeruginosa 20 h post-infection and perform transcriptional analyses in WT and a froR mutant in response to HOCl.

Strengths:

Their data clearly shows that HOCl is a strong inducer of the fro Operon. The addition of HOCl-quenching chemicals together with HOCl abrogates the response. They also show that a froR mutant is more susceptible to HOCl than WT. Their transcriptomic data reveal genes under control of the FroR/FroI sigma factor/anti sigma factor system.

Weaknesses:

Although the presented evidence is mostly solid, some of their findings need to be evaluated more carefully; explaining the rationale behind some of the experiments might enhance the article, and some of the models proposed by the authors seem far-fetched, as outlined below:

(1) In line 76 the authors claim "Relative to P. aeruginosa that were incubated in host cell-free media, P. aeruginosa in close proximity to human neutrophils or that were engulfed in mouse macrophages appeared to increase fro expression (Fig. 1C)". Counting bacterial cells in Figure 1C shows that 1 in 17 bacteria (5.8%) induce the froA-promotor in media in the absence of immune cells, while 4 in 72 bacteria (only 5.5%) do the same in the presence of neutrophils. Contrary to the authors' claims, it appears that P. aeruginosa actually decreases fro-expression in close proximity to neutrophils. There is a slight increase in fro-expression in bacteria co-incubated with macrophages (3 in 21, or 14.3%). A more rigorous statistical analysis might substantiate the authors' claim, but, as is, the claim "neutrophils increase fro expression" is untenable.

(2) The authors should explain the rationale behind some of the chemicals used. Why did they use nitric acid? Especially at these high concentrations, a strong acid such as nitric acid might have a significant influence on the medium pH. I understand that the medium is phosphate-buffered, but 25 mM nitric acid in an unbuffered medium would shift the pH well below 2. Similar considerations apply to hydrochloric acid and sodium hydroxide.

(3) In line 187, the authors state that "It is possible that oxidized methionine increases fro expression" and they suggest a model to that effect in Figure 5D. It is unclear why the authors singled out methionine sulfoxide, since a number of other things get oxidized by HOCl. In line 184, the authors state, in the same vein, that "HOCl oxidizes methionine residues 100-fold more rapidly than other cellular components". The authors should state which other cellular compounds they are referring to. Certainly not cysteine and other thiols, which react equally fast and are highly abundant in the cell: P. aeruginosa contains 340 µM GSH, 140 µM CoA-SH (https://doi.org/10.1074/jbc.RA119.009934) plus free cysteine and cysteines in proteins (based on codon usage, 1.34% of amino acids in proteins are cysteine, while methionine is only slightly more present at 2.10%, although a number of starting methionines are removed from mature proteins).

(4) Overall (and this is probably not addressable with the authors' data), some very interesting questions remain unanswered: what is the molecular mechanism of fro-induction? How is the FroR/FroI system modulated by HOCl? Does the system sense free or protein-bound methionine-sulfoxide? Are certain methionine residues in these proteins directly oxidized by HOCl? Many "HOCl-sensing" proteins are also modified at cysteine residues or amino groups; could those play a role? And lastly: what is the connection between shear/fluid flow and HOCl, or are these totally separate mechanisms of fro-induction?

Reviewer #2 (Public review):

Summary:

These studies investigate the phenotypic variability and roles of neutrophils in tuberculosis (TB) susceptibility by using a diverse collection of wild-derived inbred mouse lines. The authors aimed to identify new phenotypes during Mycobacterium tuberculosis infection by developing, infecting, and phenotyping 19 genetically diverse wild-derived inbred mouse lines originating from different geographic regions in North America and South America. The investigators achieved their main goals, which were to show that increasing genetic diversity increases the phenotypic spectrum observed in response to aerosolized M. tuberculosis, and further to provide insights into immune and/or inflammatory correlates of pulmonary TB. Briefly, investigators infected wild-derived mice with aerosolized M. tuberculosis and assessed early infection control at 21 days post-infection. The time point was specifically selected to correspond to the period after infection when acquired immunity and antigen-specific responses manifest strongly, and also early susceptibility (morbidity and mortality) due to M. tuberculosis infection has been observed in other highly susceptible wild-derived mouse strains, some Collaborative Cross inbred strains, and approximately 30% of individuals in the Diversity Outbred mouse population. Here, the investigators normalized bacterial burden across mice based on inoculum dose and determined the percent of immune cells using flow cytometry, primarily focused on macrophages, neutrophils, CD4 T cells, CD8 T cells, and B cells in the lungs. They also used single-cell RNA sequencing to identify neutrophil subpopulations and immune phenotypes, elegantly supplemented with in vitro macrophage infections and antibody depletion assays to confirm immune cell contributions to susceptibility. The main results from this study confirm that mouse strains show considerable variability to M. tuberculosis susceptibility. Authors observed that enhanced infection control correlated with higher percentages of CD4 and CD8 T cells, and B cells, but not necessarily with the percentage of interferon-gamma (IFN-γ) producing cells. High levels of neutrophils and immature neutrophils (band cells) were associated with increased susceptibility, and the mouse strain with the most neutrophils, the MANC line, exhibited a transcriptional signature indicative of a highly activated state, and containing potentially tissue-destructive, mediators that could contribute to the strain's increased susceptibility and be leveraged to understand how neutrophils drive lung tissue damage, cavitation, and granuloma necrosis in pulmonary TB.

Strengths:

The strengths are addressing a critically important consideration in the tuberculosis field - mouse model(s) of the human disease, and taking advantage of the novel phenotypes observed to determine potential mechanisms. Notable strengths include,

(1) Innovative generation and use of mouse models: Developing wild-derived inbred mice from diverse geographic locations is innovative, and this approach expands the range of phenotypic responses observed during M. tuberculosis infection. Additionally, the authors have deposited strains at The Jackson Laboratory making these valuable resources available to the scientific community.

(2) Potential for translational research: The findings have implications for human pulmonary TB, particularly the discovery of neutrophil-associated susceptibility in primary infection and/or neutrophil-mediated disease progression that could both inform the development of therapeutic targets and also be used to test the effectiveness of such therapies.

(3) Comprehensive experimental design: The investigators use many complementary approaches including in vivo M. tuberculosis infection, in vitro macrophage studies, neutrophil depletion experiments, flow cytometry, and a number of data mining, machine learning, and imaging to produce robust and comprehensive analyses of the wild-derives d strains and neutrophil subpopulations in 3 weeks after M. tuberculosis infection.

Weaknesses:

The manuscript and studies have considerable strengths and very few weaknesses. One minor consideration is that phenotyping is limited to a single limited-time point; however, this time point was carefully selected and has a strong biological rationale provided by investigators. This potential weakness does not diminish the overall findings, exciting results, or conclusions.

Reviewer #2 (Public review):

Pinon and colleagues have developed a Vessel-on-Chip model showcasing geometrical and physical properties similar to the murine vessels used in the study of systemic infections. The vessel was created via highly controllable laser photoablation in a collagen matrix, subsequent seeding of human endothelial cells, and flow perfusion to induce mechanical cues. This model could be infected with Neisseria meningitidis as a model of systemic infection. In this model, microcolony formation and dynamics, and effects on the host were very similar to those described for the human skin xenograft mouse model (the current gold standard for systemic studies) and were consistent with observations made in patients. The model could also recapitulate the neutrophil response upon N. meningitidis systemic infection.

The claims and the conclusions are supported by the data, the methods are properly presented, and the data is analyzed adequately. The most important strength of this manuscript is the technology developed to build this model, which is impressive and very innovative. The Vessel-on-Chip can be tuned to acquire complex shapes and, according to the authors, the process has been optimized to produce models very quickly. This is a great advancement compared with the technologies used to produce other equivalent models. This model proves to be equivalent to the most advanced model used to date (skin xenograft mouse model). The human skin xenograft mouse model requires complex surgical techniques and has the practical and ethical limitations associated with the use of animals. However, the Vessel-on-chip model is free of ethical concerns, can be produced quickly, and allows to precisely tune the vessel's geometry and to perform higher resolution microscopy. Both models were comparable in terms of the hallmarks defining the disease, suggesting that the presented model can be an effective replacement of the animal use in this area. In addition, the Vessel-on-Chip allows to perform microscopy with higher resolution and ease, which can in turn allow more complex and precise image-based analysis.

A limitation of this model is that it lacks the multicellularity that characterizes other similar models, which could be useful to research disease more extensively. However, the authors discuss the possibilities of adding other cells to the model, for example, fibroblasts. It is also not clear whether the technology presented in the current paper can be adopted by other labs. The methodology is complex and requires specialized equipment and personnel, which might hinder its widespread utilization of this model by researchers in the field.

This manuscript will be of interest for a specialized audience focusing on the development of microphysiological models. The technology presented here can be of great interest to researchers whose main area of interest is the endothelium and the blood vessels, for example, researchers on the study of systemic infections, atherosclerosis, angiogenesis, etc. This manuscript can have great applications for a broad audience and it can present an opportunity to begin collaborations, aimed at answering diverse research questions with the same model.

Reviewer #2 (Public review):

Summary:

Schmid & colleagues test an interesting hypothesis that V1 neurons might act as theta-tuned filters to incoming sensory information, and thereby influence downstream processing and detection performance.

Strengths:

The authors report that circular stimuli elicit theta oscillations in V1 single units and population activity. They also report that the phase of the theta oscillations influences performance in a change detection task.

Weaknesses:

The results are reported in terms of specific stimulus sizes. To truly reflect general-purpose spatial computations in the primary visual cortex, it will be important to establish a relationship between stimulus size and receptive field size.

I have several major concerns that I would like the authors to address:

(1) First paragraph of Results: The results are presented at very specific stimulus sizes: 0.3-degree, 1-degree, 4-degree, and so on. A key missing piece of information is the size of the receptive fields (RFs) that were recorded from. A related missing information is at what eccentricity these RFs were recorded from. Since there is nothing magical about a 1-degree stimulus, any general-purpose computation in the primary visual cortex has to establish a relationship between RF size and stimulus size.

(2) Second paragraph of Results: The authors state that "specific stimulus sizes consistently induced strong theta rhythmic activity: 1{degree sign} in MUA and 2{degree sign} in LFP". What is the interpretation of these specific sizes? Given that the LFP and MUAe reflect different aspects of neural activity, how does one interpret the discrepancy?

(3) Third paragraph of Results: Again related to (1), what is the relationship between the stimulus size that elicited the largest theta peaks and RF size at the population level? (1)-(3) taken together, there seems to be an opportunity to reveal something more fundamental about V1 processing that the authors might have missed here.

(4) Change detection task: It was not clear to me whether the timing of the luminance change, which varied from 500ms to 1500ms, was drawn from an exponential distribution or a uniform distribution. Only an exponential distribution has the property of a flat hazard function, which will be important to establish that the animal could not anticipate the timing of the upcoming change.

(5) Figure 3D: Have the authors tried to fit the data separately for each animal? There seems to be an inconsistency in the results between the 2 animals. The circular data points ('AL') seem positively correlated, similar to the overall trend, but the diamond data points ('DP') seem to have a negative slope.

Reviewer #2 (Public review):

Sasaki et al. use a combination of live-cell biosensors and patch-clamp electrophysiology to investigate the effect of membrane potential on the ERK MAPK signaling pathway, and probe associated effects on proliferation. This is an effect that has long been proposed, but a convincing demonstration has remained elusive, because it is difficult to perturb membrane potential without disturbing other aspects of cell physiology in complex ways. The time-resolved measurements here are a nice contribution to this question, and the perforated patch clamp experiments with an ERK biosensor are fantastic - they come closer to addressing the above difficulty of perturbing voltage than any prior work. It would have been difficult to obtain these observations with any other combination of tools.

However, there are still some concerns as detailed in specific comments below:

Specific comments:

(1) All the observations of ERK activation, by both high extracellular K+ and voltage clamp, could be explained by cell volume increase (more discussion in subsequent comments). There is a substantial literature on ERK activation by hypotonic cell swelling (e.g. https://doi.org/10.1042/bj3090013, https://doi.org/10.1002/j.1460-2075.1996.tb00938.x, among others). Here are some possible observations that could demonstrate that ERK activation by volume change is distinct from the effects reported here:

i) Does hypotonic shock activate ERK in U2OS cells?

ii) Can hypotonic shock activate ERK even after PS depletion, whereas extracellular K+ cannot?

iii) Does high extracellular K+ change cell volume in U2OS cells, measured via an accurate method such as fluorescence exclusion microscopy?

iv) It would be helpful to check the osmolality of all the extracellular solutions, even though they were nominally targeted to be iso-osmotic.

(2) Some more details about the experimental design and the results are needed from Figure 1:

i) For how long are the cells serum-starved? From the Methods section, it seems like the G1 release in different K+ concentration is done without serum, is this correct? Is the prior thymidine treatment also performed in the absence of serum?

ii) There is a question of whether depolarization constitutes a physiologically relevant mechanism to regulate proliferation, and how depolarization interacts with other extracellular signals that might be present in an in vivo context. Does depolarization only promote proliferation after extended serum starvation (in what is presumably a stressed cell state)? What fraction of total cells are observed to be mitotic (without normalization), and how does this compare to the proliferation of these cells growing in serum-supplemented media? Can K+ concentration tune proliferation rate even in serum-supplemented media?

(3) In Figure 2, there are some possible concerns with the perfusion experiment:

i) Is the buffer static in the period before perfusion with high K+, or is it perfused? This is not clear from the Methods. If it is static, how does the ERK activity change when perfused with 5 mM K+? In other words, how much of the response is due to flow/media exchange versus change in K+ concentration?

ii) Why do there appear to be population-average decreases in ERK activity in the period before perfusion with high K+ (especially in contrast to Fig. 3)? The imaging period does not seem frequent enough for photobleaching to be significant.

(4) Figure 3 contains important results on couplings between membrane potential and MAPK signaling. However, there are a few concerns:

i) Does cell volume change upon voltage clamping? Previous authors have shown that depolarizing voltage clamp can cause cells to swell, at least in the whole-cell configuration:

https://www.cell.com/biophysj/fulltext/S0006-3495(18)30441-7 . Could it be possible that the clamping protocol induces changes in ERK signaling due to changes in cell volume, and not by an independent mechanism?

ii) Does the -80 mV clamp begin at time 0 minutes? If so, one might expect a transient decrease in sensor FRET ratio, depending on the original resting potential of the cells. Typical estimates for resting potential in HEK293 cells range from -40 mV to -15 mV, which would reach the range that induces an ERK response by depolarizing clamp in Fig. 3B. What are the resting potentials of the cells before they are clamped to -80 mV, and why do we not see this downward transient?

(5) The activation of ERK by perforated voltage clamp and by high extracellular K+ are each convincing, but it is unclear whether they need to act purely through the same mechanism - while additional extracellular K+ does depolarize the cell, it could also be affecting function of voltage-independent transporters and cell volume regulatory mechanisms on the timescales studied. To more strongly show this, the following should be done with the HEK cells where there is already voltage clamp data:

i) Measure resting potential using the perforated patch in zero-current configuration in the high K+ medium. Ideally this should be done in the time window after high K+ addition where ERK activation is observed (10-20 minutes) to minimize the possibility of drift due to changes in transporter and channel activity due to post-translational regulation.

ii) Measure YFP/CFP ratio of the HEK cells in the high K+ medium (in contrast to the U2OS cells from Fig. 2 where there is no patch data).

iii) The assertion that high K+ is equivalent to changes in Vmem for ERK signaling would be supported if the YFP/CFP change from K+ addition is comparable to that induced by voltage clamp to the same potential. This would be particularly convincing if the experiment could be done with each of the 15 mM, 30 mM, and 145 mM conditions.

(6) Line 170: "ERK activity was reduced with a fast time course (within 1 minute) after repolarization to -80 mV." I don't see this in the data: in Fig. 3C, it looks like ERK remains elevated for > 10 min after the electrical stimulus has returned to -80 mV

Comments on revisions:

The authors have done a good job addressing the comments on the previous submission.

Reviewer #2 (Public review):

Summary:

Human histone H3K36 methyltransferase Setd2 has been previously shown to be a tumor suppressor in lung and pancreatic cancer. In this manuscript by Mack et al., the authors first use a mouse KRASG12D-driven lung cancer model to confirm in vivo that Setd2 depletion exacerbates tumorigenesis. They then investigate the enzymatic regulation of the Setd2 SET domain in vitro, demonstrating that H2A, H3, or H4 acetylation stimulates Setd2-SET activity, with specific enhancement by mono-acetylation at H3K14ac or H3K27ac. In contrast, histone ubiquitination has no effect. The authors propose that H3K27ac may regulate Setd2-SET activity by facilitating its binding to nucleosomes. This work provides insight into how cross-talk between histone modifications regulates Setd2 function.

Comments on revisions:

(1) Regarding New Figure 2F lane 1, please reference PMID: 33972509 Fig 4D bottom. Setd2-SET is a well-known robust K36 trimethylase. Why, under the authors' conditions, do WT nucleosomes show a significant amount of K36me1 and K36me2 accumulation, whereas K36me3 is not as pronounced? As a comparison, the authors should also report the evidence for the efficiency of each chemical modification that generates K36 methylation mimic.

(2) The bottom panel of Figure 2B does not match the top one; the number of repeats should be indicated in the figure legends.

(3) In Figure 4E, the differences between Setd2-bound WT and acetylated nucleosomes are minimal, as judged by both the decreasing trend of unbound nucleosomes and the increasing trend of bound fractions. This experiment needs to be quantified based on multiple repeats.

Reviewer #2 (Public review):

Summary:

In this manuscript by Wolfson et al., various adeno-associated viruses (AAVs) were delivered to mice to assess the cardiac-specificity, injury border-zone cardiomyocyte transduction rate, and temporal dynamics in the goal to find better AAVs for gene therapies targeting the heart. The authors delivered tissue regeneration enhancer elements (TREEs) controlling luciferase expression and used IVIS imaging to examine transduction in the heart and other organs. They found that luciferase expression increased in the first week after injury when using AAV9-TREE-Hsp68 promoter, waning to baseline levels by 7 weeks. However, AAV9 vectors transduced the liver, which was significantly reduced by using an AAV.cc84 liver de-targeting capsid. The authors then performed in vivo screening of AAV9 capsids and found AAV-IR41 to preferentially transduce injured myocardium when compared to AAV9. Finally, the authors combined TREEs with AAV-IR41 to show improved luciferase expression compared to AAV9-TREE at 7, 14 and 21 days after injury.

Overall, this manuscript provides insights into TREE expression dynamics when paired with various heart-targeting capsids, which can be useful for researchers studying ischemic injury of murine hearts. While the authors have shown the success of using AAV9-TREEs in porcine hearts, it is unknown whether the expression dynamics would be similar in pigs or humans, as mentioned in the limitations.

Strengths:

Important contribution to the AAV gene therapy literature.

Comments on revised version:

My concerns have been adequately addressed.

Reviewer #3 (Public review):

Summary:

In this work, Yamada, Brandani and Takada have developed a mesoscopic model of the interacting proteins in the postsynaptic density. They have performed simulations, based on this model and using the software ReaDDy, to study the phase separation in this system in 2D (on the membrane) and 3D (in the bulk). They have carefully investigated the reasons behind different morphologies observed in each case, and have looked at differences in valency, specific/non-specific interactions and interfacial tension.

Strengths:

The simulation model is developed very carefully, with strong reliance on binding valency and geometry, experimentally measured affinities, and physical considerations like the hydrodynamic radii. The presented analyses are also thorough, and great effort has been put into investigating different scenarios that might explain the observed effects.

Weaknesses:

The biggest weakness of the study, in my opinion, has been a lack of more in-depth and quantitative physical insights about phase separation theories. In the revised version, the authors have added text to point the interested reader to the respective theories, and have included a qualitative assessment of their findings in the light of said theories. This better positions their discussion. I still believe the role of entropic effects need more attention, which can be the subject of future studies.

The authors have revised their Introduction and added text to the Discussion, to enrich their view on the attractive and repulsive forces as well as mixing entropy. This version better covers the physics of phase separation.

I appreciate the added discussion about the different diffusive behavior in the membrane in contrast to the bulk (i.e. the Saffman-Delbrück model). This paves the way for future studies, including realistic kinetics of the studied system.

Reviewer #2 (Public review):

Summary:

In the study presented by Itani and colleagues it is shown that some strains of Aspergillus oryzae - especially those used industrially for the production of sake and soy sauce - develop hyphae with a significantly increased number of nuclei and cell volume over time. These thick hyphae are formed by branching from normal hyphae and grow faster and therefore dominate the colonies. The number of nuclei positively correlates with the thicker hyphae and also the amount of secreted enzymes. The addition of nutrients such as yeast extract or certain amino acids enhanced this effect. Genome and transcriptome analyses identified genes, including rseA, that are associated with the increased number of nuclei and enzyme production. The authors conclude from their data involvement of glycosyltransferases, calcium channels and the tor regulatory cascade in regulation of cell volume and number of nuclei. Thicker hyphae and an increased number of nuclei was also observed in high-production strains of other industrially used fungi such as Trichoderma reesei and Penicillium chrysogenum, leading to the hypothesis that the mentioned phenotypes are characteristic of production strains which is of significant interest for fungal biotechnology.

Strengths:

The study is very comprehensive and involves application of divers state-of-the-art cell biological, biochemical and genetical methods. Overall, the data are properly controlled and analyzed, figures and movies are of excellent quality.<br /> The results are particularly interesting with regard to the elucidation of molecular mechanisms that regulate the size of fungal hyphae and their number of nuclei. For this, the authors have discovered a very good model: (regular) strains with a low number of nuclei and strains with high number of nuclei. Also, the results can be expected to be of interest for the further optimization of industrially relevant filamentous fungi.

In the revision the authors addressed all my comments and as a result produced an even stronger study.

Reviewer #2 (Public review):

Summary:

The authors investigate the behavior of oncogenic cells in mammary and bronchial epithelia. They observe that individual oncogenic cells are preferentially excluded from the mammary epithelium, but they remain integrated in the bronchial epithelium. They also observe that clusters of oncogenic cells form a compact cluster in the mammary epithelium, but they disperse in the bronchial epithelium. The authors demonstrate experimentally and in the vertex model simulations that the difference in observed behavior is due to the differential tension between the mutant and wild-type cells due to a differential expression of actin and myosin.

Strengths:

(1) Very detailed analysis of experiments to systematically characterize and quantify differences between mammary and bronchial epithelia.

(2) Detailed comparison between the experiments and vertex model simulations to identify the differential cell line tension between the oncogenic and wild-type cells as one of the key parameters that are responsible for the different behavior of oncogenic cells in mammary and bronchial epithelia

Weaknesses:

(1) It is unclear what the mechanistic origin of the shape-tension coupling is, which is used in the vertex model, and how important that coupling is for the presented results. The authors claim that the shape-tension coupling is due to the anisotropic distribution of stress fibers when cells are under external stress. It is unclear why the stress fibers should affect an effective line tension on the cell boundaries and why the stress fibers should be sensitive to the magnitude of the internal isotropic cell pressure. In experiments, it makes sense that stress fibers form when cells are stretched. Similar stress fibers form when the cytoskeleton or polymer networks are stretched. It is unclear why the stress fibers should be sensitive to the magnitude of internal isotropic cell pressure. If all the surrounding cells have the same internal pressure, then the cell would not be significantly deformed due to that pressure, and stress fibers would not form. The authors should better justify the use of the shape-tension coupling in the model and also present simulation results without that coupling. I expect that most of the observed behavior is already captured by the differential tension, even if there is no shape-tension coupling.

(2) The observed difference of shape indices between the interfacial and bulk cells in simulations in the absence of differential line tension is concerning. This suggests that either there are not enough statistics from the simulations or that something is wrong with the simulations. For all presented simulation results, the authors should repeat multiple simulations and then present both averages and standard deviations. This way, it would be easier to determine whether the observed differences in simulations are statistically significant.

(3) The authors should also analyze the cell line tension data in simulations and make a comparison with experiments.

Reviewer #2 (Public review):

Summary:

The authors propose a mechanism to provide flexibility to learn new information while preserving stability in neural networks by combining structural plasticity and synaptic plasticity.

Strengths:

An intriguing idea, well embedded in experimental data.

Authors have done a great job addressing reviewers' concerns

Weaknesses:

None

Reviewer #2 (Public review):

In this study, Fontana et al. develop a paradigm for associative conditioning by pairing exposure to an alarm substance with a novel tank. Exposure to conspecific alarm substance (CAS) in the novel tank triggers freezing and what they characterize as evasive swimming behaviour, which is subsequently seen in a re-exposure to the novel tank without the CAS present. Importantly, these states are identified via automated processes, including postural tracking and a random forest classification process, which could be very useful tools for subsequent studies.

In their experiments, they focus on the differences in behaviour among strains of zebrafish (both males and females), and among individual zebrafish. For males and females of different strains, they find some differences, though the clearest message seems to be that the most robust measure of the behaviour in response to both the CAS and in the memory trials is the freezing behaviour, while evasive behaviour is more variable. and not always seen. This may relate to their observation of significant "evasiveness" in vehicle control experiments (discussed further below).

Moving on to individual variation from within this multi-strain male/female dataset, they first examine transition matrices between states and find tthat his is not dramatically altered by stimulus exposure. They then use clustering to identify 4 different "classes" of zebrafish that differ in their expression (or not) of two types of behaviour: freezing and/or evasive behaviour. They show that over the three exposure epochs of the experiment, this classification is somewhat stable in an individual fish, though many fish change their behaviour - e.g., evading + freezing -> only freezing.

In the final set of experiments, the authors move beyond behavioural analyses and perform whole-brain cFos mapping of these individual zebrafish. They perform analyses aimed at identifying correlations between individual behavioural expression and the number of cFos-positive cells in different brain regions. Using partial least squares analysis, they find areas associated with two types of behavioural contrasts, which differ in their weighting of different behavioural expression during the Memory trials. Covariation and network structure analysis within different classes of larvae also find some differences in covariation among brain areas, providing hypotheses as to underlying network effects that may govern the expression of freezing and/or evasive behavior in the memory trial phases.

Overall, I find this to be an interesting study that employs state of the are methods of behavioural analyses and whole-brain cFos analyses, but I am left a little bit confused as to what the take home message is and what can be concluded from this complex study that mixes in analyses of strain, sex, and individuality within a quite complex assay with multiple behavioural parameters.

My suggestions are as follows:

(1) My first concern relates to the claim in the abstract that "We found that fear memory behavior fell into four distinct groups: non-reactive, evaders, evading freezers, and freezers".

In my opinion, the "freezing" aspect is well supported as being both triggered by the CAS and for memory effect upon re-exposure to the tank, but I am less convinced about the "evasive" behaviour. In Figure 2, it appears that "evasiveness" is generally not increased in both the Exposure or Memory phases for many groups, and in Figure 5, it appears that "evasiveness" is expressed by nearly 50% of the fish in the pre-exposure condition before CAS addition and in all phases in the vehicle condition. Therefore, it appears that most of the expression of this behaviour is independent of any memory-based effect.

(2) My second concern relates to the claim in the abstract that "background strain and sex influenced how fish respond to CAS, with males more likely to increase evasive behaviors than females and the TU strain more likely to be non-reactive."

My understanding, based on the introduction and on the methods, is that it is likely important that the CAS be prepared from conspecifics of the same strain and sex, and for this reason, they prepared different CAS specific for each strain and each sex. Therefore, the "CAS" that is applied is necessarily different for each condition, and I am concerned about if the differences observed could relate more to variation in the quality, purity, concentration, etc. of the specific CAS samples for different groups, rather than their reactivity to the substance or their ability to form memories based on such experiences.

(3) My third concern relates to the interpretation of the cFos data.

As I mentioned above, I feel as though the behavioural analysis is perhaps more complex than is warranted via the inclusion of evasiveness, and I wonder if the conclusions from the experiments would be simpler if analyzed only from the perspective of freezing.

But considering the presented analyses: while I dont think there is anything wrong with the partial least squares approach and the network analyses, I am concerned that the simple messaging in the text does not reflect the complexity of this analysis combining different weightings of different behavioural characteristics in a behavioural contrast, or covariations among many regions and what such analyses mean at the level of brain function. For these reasons, I feel like statements along the lines of "Behavioral variation is driven by differences in the activity of brain regions outside the telencephalon, such as the cerebellum, preglomerular nuclei, preoptic area and hypothalamus" are not well supported.

Reviewer #2 (Public review):

Summary:

Overall, the work is exceptionally well done and controlled and the results properly and appropriately interpreted. While several of the approaches, while powerful, are somewhat indirect (i.e., following gene expression via ribosomal profiling) additional experiments utilizing traditional gene expression assays added in revision combine to ultimately provide a compelling answer to the main questions being asked.

The key finding from this work is the discovery that Apj1 regulates Hsf1 attenuation in a manner that includes Hsp70. That finding is strongly supported by the experimental data. While it would be ideal to also demonstrate Apj1-controlled differential binding of Ssa1/2 to Hsf1 at either the N- or C-terminal binding sites during attenuation, the Hsp70-Hsf1 interactions are difficult to reproducibly assess in cell extracts and are likely beyond the scope of this study. However, this work paves the way in the future for potential biochemical reconstitution assays that could elucidate both Hsp70-Hsf1 interactions as well as the distinct JDP-Hsf1 interactions reported here.

This discovery raises additional new questions about JDP specificity in HSR regulation and the role of JDPs in navigating protein aggregation and sensing of proteostatic challenge in the nucleus, thus advancing the field and opening new, exciting avenues for exploration.

Reviewer #2 (Public review):

This manuscript describes experiments characterising how malaria parasites respond to physiologically relevant heat-shock conditions. The authors show, quite convincingly, that moderate heat-shock appears to increase cytoadherance, likely by increasing trafficking of surface proteins involved in this process.

While generally of a high quality and including a lot of data, I have a few small questions and comments, mainly regarding data interpretation.

(1) The authors use sorbitol lysis as a proxy for trafficking of PSAC components. This is a very roundabout way of doing things and does not, I think, really show what they claim. There could be a myriad of other reasons for this increased activity (indeed, the authors note potential PSAC activation under these conditions). One further reason could be a difference in the membrane stability following heat shock, which may affect sorbitol uptake, or the fragility of the erythrocytes to hypotonic shock. I really suggest that the authors stick to what they show (increased PSAC) without trying to use this as evidence for increased trafficking of a number of non-specified proteins that they cannot follow directly.

(2) Supplementary Figure 6C/D: The KAHRP signal does not look like it should. In fact, it doesn't look like anything specific. The HSP70-X signal is also blurry and overexposed. These pictures cannot be used to justify the authors' statements about a lack of colocalisation in any way.

(3) Figure 6: This experiment confuses me. The authors purport to fractionate proteins using differential lysis, but the proteins they detect are supposed to be transmembrane proteins and thus should always be found associated with the pellet, whether lysis is done using equinatoxin or saponin. Have they discovered a currently unknown trafficking pathway to tell us about? Whilst there is a lot of discussion about the trafficking pathways for TM proteins through the host cell, a number of studies have shown that these proteins are generally found in a membrane-bound state. The authors should elaborate, or choose an experiment that is capable of showing compartment-specific localisation of membrane-bound proteins (protease protection, for example).

(4) The red blood cell contains, in addition to HSP70-X, a number of human HSPs (HSP70 and HSP90 are significant in this current case). As the name suggests, these proteins non-specifically shield exposed hydrophobic domains revealed upon partial protein unfolding following thermal insult. I would thus have expected to find significantly more enrichment following heat shock, but this is not the case. Is it possible that the physiological heat shock conditions used in this current study are not high enough to cause a real heat shock?

Reviewer #2 (Public review):

Summary:

The C-terminal region of EB1 is responsible for protein-protein interactions, thereby recruiting the binding partners of EB1 to microtubules; the coiled-coil region (EBH) and the acidic tail are critical for their binding partners. The authors demonstrated by using NMR that the binding mode of EBH with the SxIP motif, which is a two-step process termed "dock-and-lock". The ITC analysis supports the results obtained from NMR. The initial version of the manuscript contained ambiguities on the ITC data; however, the results of the revised manuscript are convincing and support the two-step binding model.

Strength:

The authors propose a novel model of "dock-and-lock" by using multiple methods of NMR, ITC and cell biology.

Reviewer #2 (Public review):

Summary:

This paper formulates an individual-based model to understand the evolution of division of labor in vertebrates. The model considers a population subdivided in groups, each group has a single asexually-reproducing breeder, other group members (subordinates) can perform two types of tasks called "work" or "defense", individuals have different ages, individuals can disperse between groups, each individual has a dominance rank that increases with age, and upon death of the breeder a new breeder is chosen among group members depending on their dominance. "Workers" pay a reproduction cost by having their dominance decreased, and "defenders" pay a survival cost. Every group member receives a survival benefit with increasing group size. There are 6 genetic traits, each controlled by a single locus, that control propensities to help and disperse, and how task choice and dispersal relate to dominance. To study the effect of group augmentation without kin selection, the authors cross-foster individuals to eliminate relatedness. The paper allows for the evolution of the 6 genetic traits under some different parameter values to study the conditions under which division of labour evolves, defined as the occurrence of different subordinates performing "work" and "defense" tasks. The authors envision the model as one of vertebrate division of labor.

The main conclusion of the paper is that group augmentation is the primary factor causing the evolution of vertebrate division of labor, rather than kin selection. This conclusion is drawn because, for the parameter values considered, when the benefit of group augmentation is set to zero, no division of labor evolves and all subordinates perform "work" tasks but no "defense" tasks.

Strengths:

The model incorporates various biologically realistic details, including the possibility to evolve age polytheism where individuals switch from "work" to "defence" tasks as they age or vice versa, as well as the possibility of comparing the action of group augmentation alone with that of kin selection alone.

Weaknesses:

The model and its analysis is limited, which makes the results insufficient to reach the main conclusion that group augmentation and not kin selection is the primary cause of the evolution of vertebrate division of labor. There are several reasons.

First, the model strongly restricts the possibility that kin selection is relevant. The two tasks considered essentially differ only by whether they are costly for reproduction or survival. "Work" tasks are those costly for reproduction and "defense" tasks are those costly for survival. The two tasks provide the same benefits for reproduction (eqs. 4, 5) and survival (through group augmentation, eq. 3.1). So, whether one, the other, or both tasks evolve presumably only depends on which task is less costly, not really on which benefits it provides. As the two tasks give the same benefits, there is no possibility that the two tasks act synergistically, where performing one task increases a benefit (e.g., increasing someone's survival) that is going to be compounded by someone else performing the other task (e.g., increasing that someone's reproduction). So, there is very little scope for kin selection to cause the evolution of labour in this model. Note synergy between tasks is not something unusual in division of labour models, but is in fact a basic element in them, so excluding it from the start in the model and then making general claims about division of labour is unwarranted. I made this same point in my first review, although phrased differently, but it was left unaddressed.

Second, the parameter space is very little explored. This is generally an issue when trying to make general claims from an individual-based model where only a very narrow parameter region has been explored of a necessarily particular model. However, in this paper, the issue is more evident. As in this model the two tasks ultimately only differ by their costs, the parameter values specifying their costs should be varied to determine their effects. Instead, the model sets a very low survival cost for work (yh=0.1) and a very high survival cost for defense (xh=3), the latter of which can be compensated by the benefit of group augmentation (xn=3). Some very limited variation of xh and xn is explored, always for very high values, effectively making defense unevolvable except if there is group augmentation. Hence, as I stated in my previous review, a more extensive parameter exploration addressing this should be included, but this has not been done. Consequently, the main conclusion that "division of labor" needs group augmentation is essentially enforced by the limited parameter exploration, in addition to the first reason above.

Third, what is called "division of labor" here is an overinterpretation. When the two tasks evolve, what exists in the model is some individuals that do reproduction-costly tasks (so-called "work") and survival-costly tasks (so-called "defense"). However, there are really no two tasks that are being completed, in the sense that completing both tasks (e.g., work and defense) is not necessary to achieve a goal (e.g., reproduction). In this model there is only one task (reproduction, equation 4,5) to which both "tasks" contribute equally and so one task doesn't need to be completed if the other task compensates for it. So, this model does not actually consider division of labor.

Reviewer #3 (Public review):

Summary:

The authors developed a new phenological lag metric and applied this analytical framework to a global dataset to synthesize shifts in spring phenology and assess how abiotic constraints influence spring phenology.

Strengths:

The dataset developed in this study is extensive, and the phenological lag metric is valuable.

Weaknesses:

The stability of the method used in this study needs improvement, particularly in the calculation of forcing requirements. In addition, the visualization of the results (such as Table 1) should be enhanced.

Reviewer #2 (Public review):

Summary:

Whole-brain network modeling is a common type of dynamical systems-based method to create individualized models of brain activity incorporating subject-specific structural connectome inferred from diffusion imaging data. This type of model has often been used to infer biophysical parameters of the individual brain that cannot be directly measured using neuroimaging but may be relevant to specific cognitive functions or diseases. Here, Ziaeemehr et al introduce a new toolkit, named "Virtual Brain Inference" (VBI), offering a new computational approach for estimating these parameters using Bayesian inference powered by artificial neural networks. The basic idea is to use simulated data, given known parameters, to train artificial neural networks to solve the inverse problem, namely, to infer the posterior distribution over the parameter space given data-derived features. The authors have demonstrated the utility of the toolkit using simulated data from several commonly used whole-brain network models in case studies.

Strength:

- Model inversion is an important problem in whole-brain network modeling. The toolkit presents a significant methodological step up from common practices, with the potential to broadly impact how the community infers model parameters.<br /> - Notably, the method allows the estimation of the posterior distribution of parameters instead of a point estimation, which provides information about the uncertainty of the estimation, which is generally lacking in existing methods.<br /> - The case studies were able to demonstrate the detection of degeneracy in the parameters, which is important. Degeneracy is quite common in this type of models. If not handled mindfully, they may lead to spurious or stable parameter estimation. Thus, the toolkit can potentially be used to improve feature selection or to simply indicate the uncertainty.<br /> - In principle, the posterior distribution can be directly computed given new data without doing any additional simulation, which could improve the efficiency of parameter inference on the artificial neural network is well-trained.

Weaknesses:

- The z-scores used to measure prediction error are generally between 1-3, which seems quite large to me. It would give readers a better sense of the utility of the method if comparisons to simpler methods, such as k-nearest neighbor methods, are provided in terms of accuracy.<br /> - A lot of simulations are required to train the posterior estimator, which is computationally more expensive than existing approaches. Inferring from Figure S1, at the required order of magnitudes of the number of simulations, the simulation time could range from days to years, depending on the hardware. The payoff is that once the estimator is well-trained, the parameter inversion will be very fast given new data. However, it is not clear to me how often such use cases would be encountered. It would be very helpful if the authors could provide a few more concrete examples of using trained models for hypothesis testing, e.g., in various disease conditions.

Reviewer #2 (Public review):

Summary:

The authors' initial goal was to demonstrate loss of PG during the slow sporulation process of Myxococcus xanthus, with examination of the PG degradation products in order to implicate possible enzymes involved. Upon finding a predominance of LGT products, they examined sporulation in strains lacking each of the 14 candidate LGTs encoded in the genome, leading to the identification of two sporulation-linked LGTs. An extensive characterization of the roles played by these LGTs. One LGT is responsible for the slow sporulation PG degradation, while another is required for the rapid sporulation process. Interestingly, the "slow" LGT seems to provide an important regulatory brake on the rapid enzyme. Single-molecule fluorescent tracking of these enzymes was used to develop a model for their interaction with PG that mimics their observed activity. The rate of PG synthesis activity was also shown to impact the rate of PG degradation, suggesting potential interplay between the synthetic and degradative enzymes.

Strengths:

The genetic analysis to identify sporulation-linked LGTs and their effects on growth, sporulation, and spore properties was well done and productive. The fluorescence microscopy to track LGT mobility, presumably tied to activity, produced a convincing argument about the mechanism of regulation of one LGT by another.

Weaknesses:

While the impact of LGTs on sporulation was clearly demonstrated, the PG analysis that resulted from the study of LGTs raised some important unanswered questions. The analyses suggest that the PG is degraded to quite small fragments, which would normally be lost during the purification of PG. How these small fragments were thus detected is unclear, and this suggests a more complex story concerning PG metabolism during sporulation. An anti-PG antibody is used to quantify PG in the spores, but it is not made clear what the specificity of this antibody is, and thus whether it would recognize the LGT-altered PG of the spore. The authors suggest a "new mechanism of sporulation" when they have actually simply identified an important factor (PG degradation by LGTs) within a complex "process of sporulation".

Reviewer #2 (Public review):

The authors of this paper have done much pioneering work to decipher and understand LRRK2 structure and function and uncover the mechanism by which LRRK2 binds to microtubules and to study the roles that this may play in biology. Their previous data demonstrated that LRRK2 in the active conformation (pathogenic mutation or Type I inhibitor complex) bound to microtubule filaments in an ordered helical arrangement. This they showed induced a "roadblock" in the microtubule impacting vesicular trafficking. The authors have postulated that this is a potentially serious flaw with Type 1 inhibitors and that companies should consider generating Type 2 inhibitors in which the LRRK2 is trapped in the inactive conformation. Indeed the authors have published much data that LRRK2 complexed to Type 2 inhibitors does not seem to associate with microtubules and cause roadblocks in parallel experiments to those undertaken with type 1 inhibitors published above.

In the current study the authors have undertaken an in vitro reconstitution of microtubule bound filaments of LRRK2 in the inactive conformation, which surprisingly revealed that inactive LRRK2 can also interact with microtubules in its auto-inhibited state. The authors' data shows that while the same interphases are seen with both the active LRRK2 and inactive microtubule bound forms of LRRK2, they identified a new interphase that involves the WD40-ARM-ANK- domains that reportedly contributes to the ability of the inactive form of LRRK2 to bind to microtubule filaments. The structures of the inactive LRRK2 complexed to microtubules are of medium resolution and do not allow visualisation of side chains.

This study is extremely well written and the figures incredibly clear and well presented. The finding that LRRK2 in the inactive autoinhibited form can associate with microtubules is an important observation that merits further investigation. This new observation makes an important contribution to the literature and builds upon the pioneering research that this team of researchers has contributed to the LRRK2 fields.

Comments on revised version:

The authors have adequately addressed my questions and those of the other Reviewers in my opinion.

Reviewer #2 (Public review):

Summary:

In this manuscript, Meijer and colleagues investigated the effects of inactivation (conditional silencing) of cortical layer 6b neurons on sleep-wake states and EEG spectral power under the following three conditions: during natural sleep-wake states, after sleep deprivation, or after intracerebroventricular administration of orexin A and B. The authors report that silencing of L6b neurons did not have a significant effect on the total time spent in sleep-wake states, duration, or number of state epochs, or the response to sleep deprivation. However, silencing of L6b neurons did slow down theta-frequency (6-9 Hz) during wake and REM sleep, and reduced the total EEG power during NREM sleep. Infusion of orexin A in the mice in which cortical layer 6b neurons were inactivated produced an increase in wakefulness. A similar effect was observed after infusion of orexin A in the mice in which these neurons were not silenced, but the effect (i.e., increase in wakefulness) was of a smaller magnitude. Silencing of cortical layer 6b neurons attenuated the effect of orexin B in increasing theta activity, as was observed in the control mice. The authors conclude that the cortical neurons in layer 6b play an essential role in state-dependent dynamics of brain activity, vigilance state control, and sleep regulation.

Strengths:

(1) A focus on cortical layer 6b neurons, which are an understudied neuronal population, especially in the context of brain and behavioral state transitions.

(2) The authors used a well-established mouse model to study the effect of inactivation of cortical layer 6b neurons.

Weaknesses:

(1) Although the authors used a highly selective approach to silence layer 6b neurons, the observed changes in EEG oscillations cannot be solely attributed to layer 6b neurons because of the ICV route for orexin administration.

(2) The rationale for using only male rats is not provided.

Reviewer #2 (Public review):

Summary:

This study uses single-cell RNA sequencing to explore how electroacupuncture (EA) stimulation alters the brain's cellular and molecular landscape after blood-brain barrier (BBB) opening. The authors aim to identify changes in gene expression and signaling pathways across brain cell types in response to EA stimulation using single-cell RNA sequencing. This direction holds promise for understanding the consequences of noninvasive methods of BBB opening for therapeutic drug delivery across the BBB.

Strengths:

(1) The study addresses an emerging and potentially important application of noninvasive stimulation methods to manipulate BBB permeability.

(2) The dataset provides broad transcriptional profiling across multiple brain cell types using single-cell resolution, which could serve as a valuable community resource.

(3) Analyses of receptor-ligand signaling and cell-cell communication are included and have the potential to offer mechanistic insight into BBB regulation.

Weaknesses:

(1) The work falls short in its current form. The experimental design lacks a clear justification, and readers are not provided with sufficient background information on the extent, timing, or regional specificity of BBB opening in this EA model. These details, established in prior work, are critical to understanding the rationale behind the current transcriptomic analyses.

(2) Further, the results are often presented with minimal context or interpretation. There is no model of intercellular or molecular coordination to explain the BBB-opening process, despite the stated goal of identifying such mechanisms. The statement that EA induces a "unique frontal cortex-specific transcriptome signature" is not supported, as no data from other brain regions are presented. Biological interpretation is at times unclear or inaccurate - for instance, attributing astrocyte migration effects to endothelial cell clusters or suggesting microglial tight junction changes without connecting them meaningfully to endothelial function.

(3) The study does include analyses of receptor-ligand signaling and cell-cell communication, which could be among its most biologically rich outputs. However, these are relegated to supplementary material and not shown in the leading figures. This choice limits the utility of the manuscript as a hypothesis-generating resource.

(4) Overall, while the dataset may be of interest to BBB researchers and those developing technologies for drug delivery across the BBB, the manuscript in its current form does not yet fulfill its interpretive goals. A more integrated and biologically grounded analysis would be beneficial.

Reviewer #2 (Public review):

Summary:

Chang et al. develop an RNN model of a BCI sequential learning task to examine the emergence of motor memory in the network. They use this system to quantify signatures of memory in continual learning, comparing their model with experimental observations from monkeys in prior publications. They show that the RNN model has signatures of shifts associated with sequential learning without any non-standard learning rules. This convincing study contributes to the knowledge of how motor memories are formed and shaped so that they are flexible in acquiring multiple behaviors.

Strengths:

This paper describes a well-designed numerical experiment that comes to a clear interpretation of a set of neural BCI experiments. The learning signatures the authors describe are interesting and well laid out, and the paper is well written. I find it insightful that the neural signature of motor learning emerges in a trained network without special learning rules.

Weaknesses:

The paper could be stronger if it made a stronger interpretation of how memory traces and uniform shifts are related. These two observations are taken from the BCI sequential learning literature and introduced by two different prior experimental papers on two different tasks, so it seems like there is an opportunity here to use the RNN model to unite these concepts, or define another metric for signatures of learning from a more normative approach.

Reviewer #2 (Public review):

Summary:

This paper describes a new approach for analyzing genome sequences.

Strengths:

The work was performed with great rigor and provides much greater insights than earlier classification systems.

Weaknesses:

A minor weakness is that the clinical application of LIN coding could be articulated in a more in-depth way. The LIN coding system is very impressive and is certainly superior to other protocols. My recommendation, although not necessary for this paper, is that the authors expand their analysis to noncoding sequences, especially those upstream of open reading frames. In this respect, important cis-acting regulatory mutations that might help to further distinguish strains could be identified.

Reviewer #2 (Public review):

Summary:

The authors investigated microtubule distribution and their possible post-translational modifications (PTM) in Plasmodium berghei during development of the liver stage, using either hepatocytes or HeLa cells as models. They used conventional immunofluorescence assays and expansion microscopy with various antibodies recognising tubulin and, in the second part of the work, its candidate PTMs, as well as markers of Plasmodium, in addition to live imaging with a fluorescent marker for tubulin. In the third part of the study, they generated 3 mutants deprived of either the last four residues or the last 11 residues, or where a candidate polyglutamylation site was substituted by an alanine residue.

Strengths:

In the first part, microtubules are monitored by a combination of two approaches (IFA and live), revealing nicely the evolution of the sporozoite subpellicular microtubules (SSPM, the sporozoite is the developmental stage present in salivary glands of the mosquitoes and that infects hepatocytes) into a different structure termed liver-stage parasite microtubule bundle (LSPMB). The LSPMB shrinks during the course of parasite development and finally disappears while hemi-spindles emerge over time. Contact points between these two structures are observed frequently in live cells and occasionally in fixed cells, suggesting the intriguing possibility that tubulin might be recycled from the LSPMB to contribute to hemi-spindle formation.

In the second part, antibodies recognising (1) the final tyrosine found at the C-terminal tail and (2) a stretch of 3 glutamate residues in a side chain are used to monitor these candidate PTMs. Signals are positive at the SSPM, and while it remains positive for polyglutamylation, it becomes negative for the final tyrosine at the LSPM, while a positive signal emerges at hemi-spindles at later stages of development.

In the last part, the three mutants are fed to mosquitoes, where they show reduced development, the one lacking the alpha-tubulin tail even failing to reach the salivary glands. However, the two other mutants infect HeLa cells normally, whereas sporozoites with the C-terminal tail deletion recovered from the haemolymph did not develop in these cells.

The first part provides convincing evidence that microtubules are extensively remodelled during the infection of hepatocytes and HeLa cells, in agreement with the spectacular Plasmodium morphogenetic changes accompanying massive and rapid proliferation. The third part brings further confirmation that the C-terminal tail of alpha-tubulin is essential for multiple stages of parasite development, in agreement with previous work (50). Since it is the region where several post-translational modifications take place in other organisms (detyrosination, polyglutamylation, glycylation), it makes sense to propose that the essential function is related to these PTMs also in Plasmodium.

Weaknesses:

The significance of tubulin PTM relies on two antibodies whose reactivity to Plasmodium tubulins is unclear (see below). The interpretation of the literature on detyrosination and polyglutamylation is confusing in several places, meaning that the statements about the possible role of these PTMs need to be carefully revisited.

The authors use the term "tyrosination" but the alpha1-tubulin studied here possesses the final tyrosine when it is synthesised, so it is "tyrosinated" by default. It could potentially be removed by a tyrosine carboxypeptidase of the vasoinhibin family (VASH) as reported in other species. After removal, this tyrosine can be added again by a tubulin-tyrosine ligase (TTL) enzyme. It is therefore more appropriate to talk about detyrosination-retyrosination rather than tyrosination (this confusion is unfortunately common in the literature, see Janke & Magiera, 2020).

The difficulty here is that there is so far no evidence that detyrosination takes place in Plasmodium. Neither VASH nor TTL could be identified in the Plasmodium genome (ref 31, something we can confirm with our unsuccessful BLAST analyses), and mass spectrometry studies of purified tubulin, albeit from blood stages, did not find evidence for detyrosination (reference 43). Western blots using an antibody against detyrosinated tubulin did not produce a positive signal, neither on purified tubulin, nor on whole parasites (43). Of course, the situation could be different in liver stages, but the question of the detyrosinating enzyme is still there. The existence of a unique Plasmodium system for detyrosination cannot be formally ruled out, but given the high degree of conservation of these PTMs and their associated enzymes, it sounds difficult to imagine.

The fact that the anti-tyrosinated antibody still produced a signal in the cell line where the final tyrosine is deleted raises issues about its specificity. A cross-reactivity with beta-tubulin is proposed, but the Plasmodium beta-tubulin does not carry a final tyrosine, further raising concerns about antibody specificity.

The interpretation of these results should therefore be considered carefully. There also seems to be some confusion in the function of detyrosination cited from the literature. It is said in line 229 that "tyrosination has been associated with stable microtubules" (33, 34, 50, 55). References 33 and 34 actually show that tyrosinated microtubules turn over faster in neurons or in epithelial cells, respectively, while references 50 and 55 do not study de/retyrosination. The general consensus is that tyrosinated microtubules are more dynamic (see reference 24).

The situation is a bit different for polyglutamylation since several candidate poly- or mono-glutamylases have been identified in the Plasmodium genome, and at least mono-glutamylation of beta-tubulin has been formally proven, still in bloodstream stages (ref 43). The authors propose that the residue E445 is the polyglutamylation site. To our knowledge, this has not been demonstrated for Plasmodium. This residue is indeed the favourite one in several organisms such as humans and trypanosomes (Eddé et al., Science 1990; Schneider et al., JCS, 1997), and it is tempting to propose it would be the same here. However, TTLLs bind the tubulin tails from their C-terminal end like a glove on a finger (Garnham et al., Cell, 2015), and the presence of two extra residues in Plasmodium tubulins would mean that the reactive glutamate might be in position E447 rather than E445. This is worth discussing.<br /> On the positive side, it is encouraging to see that signals for both anti-tyrosinated tail and poly-glutamylated side chain are going down in the various mutants, but this would need validation with a comparison for alpha-tubulin signal.

Line 316: polyglutamylation "is commonly associated with dynamic microtubule behavior (78-80)". Actually, references 78 and 79 show the impact of this PTM on interaction with spastin, and reference 80 discusses polyglutamylation as a marker of stable microtubules in the context of cilia and flagella. The consensus is that polyglutamylated microtubules tend to be more stable (ref24).

Conclusion:

The first and the third parts of this manuscript - evolution of microtubules and importance of the C-terminal tails for Plasmodium development - are convincing and well supported by data. However, the presence and role of tubulin PTM should be carefully reconsidered.

Plasmodium tubulins are more closely related to plant tubulins and are sensitive to inhibitors that do not affect mammalian microtubules. They therefore represent promising drug targets as several well-characterised compounds used as herbicides are available. The work produced here further defines the evolution of the microtubule network in sporozoites and liver stages, which are the initial and essential first steps of the infection. Moreover, Plasmodium has multiple specificities that make it a fascinating organism to study both for cell biology and evolution. The data reported here are elegant and will attract the attention of the community working on parasites but also on the cytoskeleton at large. It will be interesting to have the feedback of other people working on tubulin PTMs to figure out the significance of this part of the work.

Reviewer #2 (Public review):

Summary:

The authors sought to investigate the role of nociceptor neurons in the pathogenesis of pollution-mediated neutrophilic asthma. The authors overall achieved the aim of demonstrating that nociceptor neurons are important to the pathogenesis of pollution-exacerbated asthma. Their results support their conclusions overall, although there are ways the study findings can be strengthened. This work further evaluates how nociceptor neurons contribute to asthma pathogenesis important for consideration while proposing treatment strategies for under treated asthma endotypes.

Strengths:

The authors utilize TRPV1 ablated mice to confirm the effects of intranasally administered QX-314 utilized to block sodium currents.

Use of intravital microscopy to track alveolar macrophage and neutrophil motility in their model

The authors demonstrate that via artemin, which is upregulated in alveolar macrophages in response to pollution, sensitizes JNC neurons thereby increasing their responsiveness to pollution. Ablation or inactivity of nociceptor neurons prevented the pollution induced increase in inflammation.

Weaknesses:

While neutrophilic, unclear of the endotype of asthma represented by the model

Comments on revisions:

The authors have addressed or commented on all concerns.

Reviewer #2 (Public review):

This study focuses on the differential binding of the RNA-binding protein HuR to CCL2 transcript (genetic variants rs13900 T or C). The study explores how this interaction influences the stability and translation of CCL2 mRNA. Employing a combination of bioinformatics, reporter assays, binding assays, and modulation of HuR expression, the study proposes that the rs13900T allele confers increased binding to HuR, leading to greater mRNA stability and higher translational efficiency. These findings indicate that rs13900T allele might contribute to heightened disease susceptibility due to enhanced CCL2 expression mediated by HuR. The study is interesting and most results are convincing, however the interpretation relative to RNA transcription and/or stability must be modified, and some data need better presentation or interpretation.

Major Points

Figure 2C:<br /> The authors describe an experiment to assess mRNA stability by labeling nascent RNA with EU for 3 hours, followed by washout of EU, and then incubation with or without actinomycin D for an additional 4 hours before measuring the remaining EU-labeled RNA. While the approach to label nascent RNA with EU is appropriate for tracking RNA decay, I have concerns regarding the use and interpretation of actinomycin D in this context.<br /> After EU washout, the pool of EU-labeled RNA is fixed and no new EU incorporation can occur. Therefore, the addition of actinomycin D at this stage should not affect the decay rate of the already labeled RNA, as transcription of EU-labeled RNA has effectively ceased. In this design, measuring the decrease in EU-labeled RNA over time reflects mRNA stability (even in absence of actinomycin D) rather than transcriptional activity.<br /> Therefore, the authors' statement that the non-actinomycin D treatment group represents transcriptional changes is not accurate here. Since EU labeling was stopped prior to the 4-hour incubation, any changes in EU-labeled RNA levels during this period reflect RNA decay, not new transcription.

In summary:<br /> To assess transcriptional changes, one would compare the amount of EU-labeled RNA synthesized during the initial labeling period (the first 3 hours), before washout.<br /> If the authors wish to use actinomycin D to block transcription, this should be done in a separate decay assay without EU labeling.<br /> In the current experimental setup, actinomycin D is unnecessary after EU washout and does not influence the decay of the labeled RNA.<br /> I recommend the authors reconsider the interpretation of their data accordingly. I recommend to remove the data points relative to the presence of actinomycin D, as the non-actinomycin D samples are already representative of post-transcriptional changes given that EU was washed out. If Authors want to assess transcriptional changes, they would have to assess the levels during the initial labeling period (before the washout). Transcriptional differences were not assessed, therefore I would modify the text accordingly.<br /> In this context, any changes observed in the actinomycin D-treated samples are likely attributable to general cellular stress induced by actinomycin D, which is known to be highly stressful for cells. This stress could indirectly influence the decay rates of already-labeled EU-RNA.

Figure 4C and 4D:<br /> The Author provided an updated gel with relative quantification - which effectively show the enhanced binding of CCL2 mRNA carrying the T variant to HuR - but they only provided it as data for reviewers (Figure R1). I highly recommend to use these data in the final manuscript instead of the data currently presented in Figure 4C and 4D. This would be important in order not to not create confusion in the reader or concerns regarding probe degradation or saturation.

Minor points<br /> For the IP, I recommend to explain in the final version why the input was not provided (lack of material) and to clarify that the specific binding of Actin was used as a loading control in absence of input. This would be highly beneficial for the readers.

Reviewer #2 (Public review):

Summary

Calcium ions play a key role in synaptic transmission and plasticity. To improve calcium measurements at synaptic terminals, previous studies have targeted genetically encoded calcium indicators (GECIs) to pre- and postsynaptic locations. Here, Chen et al. improve these constructs by incorporating the latest GCaMP8 sensors and a stable red fluorescent protein to enable ratiometric measurements. In addition, they develop a new analysis platform, 'CaFire', to facilitate automated quantification. Using these tools, the authors demonstrate favorable properties of their sensors relative to earlier constructs. Impressively, by positioning postsynaptic GCaMP8m near glutamate receptors, they show that their sensors can report miniature synaptic events with speed and sensitivity approaching that of intracellular electrophysiological recordings. These new sensors and the analysis platform provide a valuable tool for resolving synaptic events using all-optical methods.

Strengths:

The authors present a rigorous characterization of their sensors using well-established assays. They employ immunostaining and super-resolution STED microscopy to confirm correct subcellular targeting. Additionally, they quantify response amplitude, rise and decay kinetics, and provide side-by-side comparisons with earlier-generation GECIs. Importantly, they show that the new sensors can reproduce known differences in evoked Ca²⁺ responses between distinct nerve terminals. Finally, they present what appears to be the first simultaneous calcium imaging and intracellular mEPSP recording to directly assess the sensitivity of different sensors in detecting individual miniature synaptic events.

Weaknesses:

Major points:

(1) While the authors rigorously compared the response amplitude, rise, and decay kinetics of several sensors, key parameters like brightness and photobleaching rates are not reported. I feel that including this information is important as synaptically tethered sensors, compared to freely diffusible cytosolic indicators, can be especially prone to photobleaching, particularly under the high-intensity illumination and high-magnification conditions required for synaptic imaging. Quantifying baseline brightness and photobleaching rates would add valuable information for researchers intending to adopt these tools, especially in the context of prolonged or high-speed imaging experiments.

(2) In several places, the authors compare the performance of their sensors with synthetic calcium dyes, but these comparisons are based on literature values rather than on side-by-side measurements in the same preparation. Given differences in imaging conditions across studies (e.g., illumination, camera sensitivity, and noise), parameters like indicator brightness, SNR, and photobleaching are difficult to compare meaningfully. Additionally, the limited frame rate used in the present study may preclude accurate assessment of rise times relative to fast chemical dyes. These issues weaken the claim made in the abstract that "...a ratiometric presynaptic GCaMP8m sensor accurately captures .. Ca²⁺ changes with superior sensitivity and similar kinetics compared to chemical dyes." The authors should clearly acknowledge these limitations and soften their conclusions. A direct comparison in the same system, if feasible, would greatly strengthen the manuscript.

(3) The authors state that their indicators can now achieve measurements previously attainable with chemical dyes and electrophysiology. I encourage the authors to also consider how their tools might enable new measurements beyond what these traditional techniques allow. For example, while electrophysiology can detect summed mEPSPs across synapses, imaging could go a step further by spatially resolving the synaptic origin of individual mEPSP events. One could, for instance, image MN-Ib and MN-Is simultaneously without silencing either input, and detect mEPSP events specific to each synapse. This would enable synapse-specific mapping of quantal events - something electrophysiology alone cannot provide. Demonstrating even a proof-of-principle along these lines could highlight the unique advantages of the new tools by showing that they not only match previous methods but also enable new types of measurements.

(4) For ratiometric measurements, it is important to estimate and subtract background signals in each channel. Without this correction, the computed ratio may be skewed, as background adds an offset to both channels and can distort the ratio. However, it is not clear from the Methods section whether, or how, background fluorescence was measured and subtracted.

(5) At line 212, the authors claim "... GCaMP8m showing 345.7% higher SNR over GCaMP6s....(Fig. 3D and E) ", yet the cited figure panels do not present any SNR quantification. Figures 3D and E only show response amplitudes and kinetics, which are distinct from SNR. The methods section also does not describe details for how SNR was defined or computed.

(6) Lines 285-287 "As expected, summed ΔF values scaled strongly and positively with AZ size (Fig. 5F), reflecting a greater number of Cav2 channels at larger AZs". I am not sure about this conclusion. A positive correlation between summed ΔF values and AZ size could simply reflect more GCaMP molecules in larger AZs, which would give rise to larger total fluorescence change even at a given level of calcium increase.

(7) Lines 313-314: "SynapGCaMP quantal signals appeared to qualitatively reflect the same events measured with electrophysiological recordings (Fig. 6D)." This statement is quite confusing. In Figure 6D, the corresponding calcium and ephys traces look completely different and appear to reflect distinct sets of events. It was only after reading Figure 7 that I realized the traces shown in Figure 6D might not have been recorded simultaneously. The authors should clarify this point.

(8) Lines 310-313: "SynapGCaMP8m .... striking an optimal balance between speed and sensitivity", and Lines 314-316: "We conclude that SynapGCaMP8m is an optimal indicator to measure quantal transmission events at the synapse." Statements like these are subjective. In the authors' own comparison, GCaMP8m is significantly slower than GCaMP8f (at least in terms of decay time), despite having a moderately higher response amplitude. It is therefore unclear why GCaMP8m is considered 'optimal'. The authors should clarify this point or explain their rationale for prioritizing response amplitude over speed in the context of their application.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the role of IκBα in regulating mouse embryonic stem cell (ESC) pluripotency and differentiation. The authors demonstrate that IκBα knockout impairs the exit from the naïve pluripotent state during embryoid body differentiation. Through mechanistic studies using various mutants, they show that IκBα regulates ESC differentiation through chromatin-related functions, independent of the canonical NF-κB pathway.

Strengths:

The authors nicely investigate the role of IκBα in pluripotency exit, using embryoid body formation and complementing the phenotypic analysis with a number of genome-wide approaches, including transcriptomic, histone marks deposition, and DNA methylation analyses. Moreover, they generate a first-of-its-kind mutant set that allows them to uncouple IκBα's function in chromatin regulation versus its NF-κB-related functions. This work contributes to our understanding of cellular plasticity and development, potentially interesting a broad audience including developmental biologists, chromatin biology researchers, and cell signaling experts.

Weaknesses:

Future experiments will likely help establish a more direct mechanistic link between IκBα activity and the chromatin remodeling events observed in pluripotent cells.

Reviewer #2 (Public review):

Summary:

This is an interesting theoretical exploration of how a flexible protein domain, which has multiple DNA-binding sites along it, affects the stability of the protein-DNA complex. It proposes a mechanism ("octopusing") for protein doing a random walk while bound to DNA which simultaneously enables exploration of the DNA strand and stability of the bound state.

Strengths:

Stability of the protein-DNA bound state and the ability of the protein to perform 1d diffusion along the DNA are two properties of a transcription factor that are usually seen as being in opposition of each other. The octopusing mechanism is an elegant resolution of the puzzle of how both could be accommodated. This mechanism has interesting biological implications for the functional role of intrinsically disordered domains in transcription factor (TF) proteins. They show theoretically how these domains, if flexible and able to make multiple weak contacts with the DNA, can enhance the ability of the TF to efficiently find their binding site on the DNA from which they exert control over the transcription of their target gene. The paper concludes with a comparison of model predictions with experimental data which gives further support to the proposed mechanism. Overall, this is an interesting and well-executed theoretical paper that proposes an interesting idea about the functional role for IDR domains in TFs.

Weaknesses:

It is not clear how ubiquitous among eukaryotic transcription factors are the DNA binding sites for multiple subdomains along the IDR, which are assumed by the model. These assumptions though, provide interesting points of departure for further experiments.

Reviewer #2 (Public review):

Summary:

This study establishes a platform for studying mosquito flight activity over the course of several weeks and demonstrates key applications of such a paradigm: the comparison of daily activity profiles across different Aedes aegypti populations and the quantification of responses to physiological and environmental perturbations.

Strengths:

(1) Overall, the authors succeed in setting up a low-cost, scalable tracking system that stably records mosquito flight activity for several weeks and uses it to demonstrate compelling use cases.

(2) The text is organized well, is easy to read, and is understandable for a broad audience.

(3) Instructions for constructing housing and for performing tracking with a dedicated GUI are available on an accompanying website, with open-source (and well-organized) code.

(4) A complementary pair of methods (one testing for activity signals at specific times of the day, and the other capturing broader daily patterns) is used effectively.

Weaknesses:

(1) In the interval-based GLMM results, since each time interval is tested independently, p-values should be corrected for multiple hypotheses (for instance, through controlling the false discovery rate).

(2) The accompanying GUI application needs some modifications to fully work out of the box on a sample video.

Reviewer #2 (Public review):

Summary:

In this manuscript, Michelson, Gupta, and Murphy use calcium imaging to map the distribution of neural activity across the cerebral cortex of grooming, head-restrained mice. Animals groomed spontaneously and in response to wetting of the face. Individual movement elements, such as bilateral strokes across the face, resembled those observed in freely-moving animals. Sequencing of movement elements was structured, but did not consist of full "syntactic grooming chains." Widefield imaging across the cortex revealed distinct patterns of activity for distinct movement elements. Individual neurons responded strongly during movement and had largely similar properties across cortical areas.

Strengths:

In my opinion, this is a solid paper that will be of interest to the mouse sensorimotor neuroscience community. The experiments are technically sound, the text is well-written, and the figures are clear. The activity maps are presented in standardized Allen Atlas coordinates, and I expect they will be very useful for future studies of orofacial and limb movement.

Weaknesses:

While the manuscript provides a valuable description of cortical activity during head-restrained grooming, I think it could engage a bit more with contemporary theories and debates in cortical physiology and motor control. The Abstract nicely highlights an apparent paradox: the motor cortex sends strong projections to the spinal cord, and is strongly modulated during behaviors like grooming. Nevertheless, blocking corticospinal traffic by inactivating or lesioning the motor cortex leaves such behaviors intact. There are several potential resolutions to this paradox. First, cortical activity during grooming could be confined to an "output-null" subspace that is responsible for monitoring sensorimotor events and preparing voluntary movements, but does not drive muscle activity (c.f. work in the macaque: Kaufman et al., Nature Neuroscience 2014; Churchland & Shenoy, Nature Reviews Neuroscience 2024). Second, cortical activity during grooming could be transmitted to lower centers, but gated out through inhibition. Third, it is possible that cortical activity in intact animals does contribute to muscle activation during grooming, but following a lesion or inactivation, other descending pathways compensate for the cortical deficit. The authors might wish to discuss their findings in light of these considerations.

In the first paragraph of the Introduction, it could be made clearer which results are specific to mice. The Niell & Stryker finding, for example, holds in mice, but not marmosets (Liska et al., eLife 2024).

The "hotspots" in Figure 3G appear to be more anterior during bilateral elliptical than unilateral elliptical movements. How do the authors interpret this finding?

The distribution of single-neuron responses looks relatively similar across cortical areas, including forelimb, hindlimb, and trunk somatosensory cortex, and primary and secondary forelimb motor cortex. What do the authors make of this?

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

For readers who are outside the field, some clarifications of the system and design would be helpful.

Reviewer #2 (Public review):

Summary

This study provides new insights into organ morphogenesis using the Drosophila salivary gland (SG) as a model. The authors identify a requirement for sulfation in regulating lumen expansion, which correlates with several effects at the cellular level, including regulation of intracellular trafficking and the organization of Golgi, the aECM and the apical membrane. In addition, the authors show that the ZP proteins Dumpy (Dpy) and Pio form an aECM regulating lumen expansion. Previous reports already pointed to a role for Papss in sulfation in SG and the presence of Dpy and Pio in the SG. Now this work extends these previous analyses and provides more detailed descriptions that may be relevant to the fields of morphogenesis and cell biology (with particular focus on ECM research and tubulogenesis). This study nicely presents valuable information regarding the requirements of sulfation and the aECM in SG development.

Strengths

-The results supporting a role for sulfation in SG development are strong. In addition, the results supporting the involvement of Dpy and Pio in the aECM of the SG, their role in lumen expansion, and their interactions, are also strong.

-The authors have made an excellent job in revising and clarifying the many different issues raised by the reviewers, particularly with the addition of new experiments and quantifications. I consider that the manuscript has improved considerably.

-The authors generated a catalytically inactive Papss enzyme, which is not able to rescue the defects in Papss mutants, in contrast to wild type Papss. This result clearly indicates that the sulfation activity of Papss is required for SG development.

Weaknesses

-The main concern is the lack of clear connection between sulfation and the phenotypes observed at the cellular level, and, importantly, the lack of connection between sulfation and the Pio-Dpy matrix. Indeed, the mechanism/s by which sulfation affects lumen expansion are not elucidated and no targets of this modification are identified or investigated. A direct (or instructive) role for sulfation in aECM organization is not clearly supported by the results, and the connection between sulfation and Pio/Dpy roles seems correlative rather than causative. As it is presented, the mechanisms by which sulfation regulates SG lumen expansion remains elusive in this study.

-In my opinion the authors overestimate their findings with several conclusions, as exemplified in the abstract:

"In the absence of Papss, Pio is gradually lost in the aECM, while the Dpy-positive aECM structure is condensed and dissociates from the apical membrane, leading to a thin lumen. Mutations in dpy or pio, or in Notopleural, which encodes a matriptase that cleaves Pio to form the luminal Pio pool, result in a SG lumen with alternating bulges and constrictions, with the loss of pio leading to the loss of Dpy in the lumen. Our findings underscore the essential role of sulfation in organizing the aECM during tubular organ formation and highlight the mechanical support provided by ZP domain proteins in maintaining luminal diameter."

The findings leading to conclude that sulfation organizes the aECM and that the absence of Papss leads to a thin lumen due to defects in Dpy/Pio are not strong. The authors certainly show that Papss is required for proper Pio and Dpy accumulation. They also show that Pio is required for Dpy accumulation, and that Pio and Dpy form an aECM required for lumen expansion. However, the absence of Pio and Dpy do not fully recapitulate Papss mutant defects (thin lumen). I wonder whether other hypothesis and models could account for the observed results. For instance, a role for Papss affecting secretion, in which case sulfation would have an indirect role in aECM organization. This study does not address the mechanical properties of Dpy in normal and mutant salivary glands.

-Minor issues relate to the genotype/phenotype analysis. It is surprising that the authors detect only mild effects on sulfation in Papss mutants using an anti-sulfoTyr antibody, as Papss is the only Papss synthathase. Generating germ line clones (which is a feasible experiment) would have helped to prove that this minor effect is due to the contribution of maternal product. The loss of function allele used in this study seems problematic, as it produces effects in heterozygous conditions difficult to interpret. Cleaning the chromosome or using an alternative loss of function condition (another allele, RNAi, etc...) would have helped to present a more reliable explanation.

Reviewer #2 (Public review):

Richevaux et al investigate how anterior thalamic (AD) and retrosplenial (RSC) inputs are integrated by single presubicular (PrS) layer 3 neurons. They show that these two inputs converge onto single PrS layer 3 principal cells. By performing dual wavelength photostimulation of these two inputs in horizontal slices, the authors show that in most layer 3 cells, these inputs summate supra-linearly. They extend the experiments by focusing on putative layer 4 PrS neurons and show that they do not receive direct anterior thalamic nor retrosplenial inputs; rather, they are (indirectly) driven to burst firing in response to strong activation of the PrS network.

This is a valuable study, which investigates an important question - how visual landmark information (possibly mediated by retrosplenial inputs) converges and integrates with HD information (conveyed by the AD nucleus of the thalamus) within PrS circuitry. The data indicate that near-coincident activation of retrosplenial and thalamic inputs leads to non-linear integration in target layer 3 neurons, thereby offering a potential biological basis for landmark + HD binding.

Main limitations relate to the anatomical annotation of 'putative' PrS L4 neurons, and to the presentation of retrosplenial / thalamic input modularity. Specifically, more evidence should be provided to convincingly demonstrate that the 'putative L4 neurons' of the PrS are not distal subicular neurons (as the authors' anatomy and physiology experiments seem to indicate). The modularity of thalamic and retrosplenial inputs could be better clarified in relation to the known PrS modularity.

Reviewer #2 (Public review):

In this paper, Hamid et al present 40 genomes from the Faroe Islands. They use these data (a pilot study for an anticipated larger-scale sequencing effort) to discuss the population genetic diversity and history of the sample, and the Faroes population. I think this is an overall solid paper; it is overall well-polished and well-written. It is somewhat descriptive (as might be expected for an explorative pilot study), but does make good use of the data.

The data processing and annotation follows a state-of-the-art protocol, and at least I could not find any evidence in the results that would pinpoint towards bioinformatic issues having substantially biased some of the results, and at least preliminary results lead to the identification of some candidate disease alleles, showing that small, isolated cohorts can be an efficient way to find populations with locally common, but globally rare disease alleles.

I also enjoyed the population structure analysis in the context of ancient samples, which gives some context to the genetic ancestry of Faroese, although it would have been nice if that could have been quantified, and it is unfortunate that the sampling scheme effectively precludes within-Faroes analyses.

I am unfortunately quite critical of the selection analysis, both on a statistical level and, more importantly, I do not believe it measures what the authors think it does.

Major comments:

(1) Admixture timing/genomic scaling/localization:<br /> As the authors lay out, the Faroes were likely colonized in the last 1,000-1,500 years, i.e., 40-60 generations ago. That means most genomic processes that have happened on the Faroese should have signatures that are on the order of ~1-2cM, whereas more local patterns likely indicate genetic history predating the colonization of the islands. Yet, the paper seems to be oblivious to this (to me) fascinating and somewhat unique premise. Maybe this thought is wrong, but I think the authors miss a chance here to explain why the reader should care beyond the fact that the small populations might have high-frequency risk alleles and the Faroes are intrinsically interesting, but more importantly, it also makes me think it leads to some misinterpretations in the selection analysis

(2) ROH:<br /> Would the sampling scheme impact ROH? How would it deal with individuals with known parental coancestry? As an example of what I mean by my previous comment, 1MB is short enough in that I would expect most/many 1MB ROH-tracts to come from pedigree loops predating the colonization of the Faroes. (i.e, I am actually quite surprised that there isn't much more long ROH, which makes me wonder if that would be impacted by the sampling scheme).

(3) Selection scan:

We are talking about a bottlenecked population that is recently admixed (Faroese), compared to a population (GBR) putatively more closely related to one of its sources. My guess would be that selection in such a scenario would be possibly very hard to detect, and even then, selection signals might not differentiate selection in Faroese vs. GBR, but rather selection/allele frequency differences between different source populations. I think it would be good to spell out why XP-EHH/iHS measures selection at the correct time scale, and how/if these statistics are expected to behave differently in an admixed population.

(4) Similarly, for the discussion of LCT, I am not convinced that the haplotypes depicted here are on the right scale to reflect processes happening on the Faroes. Given the admixture/population history, it at the very least should be discussed in the context of whether the 13910 allele frequency on the Faroes is at odds with what would be expected based on the admixture sources.

(5) I am lacking information to evaluate the procedure for turning the outliers into p-values. Both iHS and XP-EHH are ratio statistics, meaning they might be heavy-tailed if one is not careful, and the central limit theorem may not apply. It would be much easier (and probably sufficient for the points being made here) to reframe this analysis in terms of empirical outliers.

(6) Oldest individual predating gene flow: It seems impossible to make any statements based on a single individual. Why is it implausible that this person (or their parents), e.g., moved to the Faroes within their lifetime and died there?

Reviewer #2 (Public review):

This paper describes an analysis of a commercially available panel for a spatial transcriptomic approach and introduces a computational tool to predict potential off-target binding sites for the type of probe used in the aforementioned panel. The performance of the prediction tool was validated by examining a dataset that profiled the same cancer tissue with multiple modalities. Finally, a detailed analysis of the potential pitfalls in a published study communicated by the company that commercialized the spatial transcriptomic platform in question is provided, along with best practice guidelines for future studies to follow.

Strengths:

The manuscript is clearly written and easy to follow.

The authors provide clean, organized, and well-documented code in the associated GitHub repository.

Weaknesses:

The manuscript section on the software tool feels underdeveloped.

Reviewer #2 (Public review):

Summary:

The paper by Kim et al. investigates the potential of stimulating the dopaminergic A13 region to promote locomotor restoration in a Parkinson's mouse model. Using wild-type mice, 6-OHDA injection depletes dopaminergic neurons in the substantia nigra pars compacta, without impairing those of the A13 region and the ventral tegmentum area, as previously reported in humans. Moreover, photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region improves bradykinesia and akinetic symptoms after 6-OHDA injection. Whole-brain imaging with retrograde and anterograde tracers reveals that the A13 region undergoes substantial changes in the distribution of its afferents and projections after 6-OHDA injection, thus suggesting a remodeling of the A13 connectome. Whether this remodelling contributes to pro-locomotor effects of the photostimulation of the A13 region remains unknown as causality was not addressed.

Strengths:

Photostimulation of presumably excitatory (CAMKIIa) neurons in the vicinity of the A13 region promotes locomotion and locomotor recovery of wild-type mice 1 month after 6-OHDA injection in the medial forebrain bundle, thus identifying a new potential target for restoring motor functions in Parkinson's disease patients. The study also provides a description of the A13 region connectome pertaining to motor behaviors and how it changes after a dopaminergic lesion. Although there is no causal link between anatomical and behavioral data, it raises interesting questions for further studies.

Weaknesses:

Although CAMKIIa is a marker of presumably excitatory neurons and can be used as an alternative marker of dopaminergic neurons, some uncertainty remains regarding the phenotype of neurons underlying recovery of akinesia and improvement of bradykinesia.

Figure 4 is improved, but the results from the correlation analyses remain difficult to interpret, as they may reflect changes in various impaired brain regions independently of the A13 region. While the analysis offers a snapshot of correlated changes within the connectome, it does not identify which specific cell or axonal populations are actually increasing or decreasing. Although functional MRI connectome analyses are well-established, anatomical data seem less suitable for this purpose. How can one interpret correlated changes in anatomical inputs or outputs between two distinct regions?

Figure 5 is also improved, but there is room for further enhancement. As currently presented, it is difficult to distinguish the differences between the sham and 6-OHDA groups. The first column could compare afferents, while the second column could compare efferents. Given the small sample size, it would be more appropriate to present individual data rather than the mean and standard deviation.

Appraisal and impact

Although the behavioral experiments are convincing, the low number of animals in the anatomical studies is insufficient to make any relevant statistical conclusions due to extremely low statistical power.

Reviewer #2 (Public review):

Summary:

Bonnifet et al. sought to characterize the expression pattern of L1 ORF1p expression across the entire mouse brain, in young and aged animals and to corroborate their characterization with Western blotting for L1 ORF1p and L1 RNA expression data from human samples. They also queried L1 ORF1p interacting partners in the mouse brain by IP-MS.

Strengths:

A major strength of the study is the use of two approaches: a deep-learning detection method to distinguish neuronal vs. non-neuronal cells and ORF1p+ cells vs. ORF1p- cells across large-scale images encompassing multiple brain regions mapped by comparison to the Allen Brain Atlas, and confocal imaging to give higher resolution on specific brain regions. These results are also corroborated by Western blotting on six mouse brain regions. Extension of their analysis to post-mortem human samples, to the extent possible, is another strength of the paper. The identification of novel ORF1p interactors in brain is also a strength in that it provides a novel dataset for future studies.

Weaknesses:

The main weakness of the IP-MS portion of the study is that none of the interactors were individually validated or subjected to follow-up analyses. The list of interactors was compared to previously published datasets, but not to ORF1p interactors in any other mouse tissue.

Comments on revisions:

The co-staining of Orf1p with Parvalbumin (PV) presented in Supplemental Figure S5 is a welcome addition exploring the cell type-specificity of Orf1p staining, and broadly corroborates the work of Bodea et al. while revealing that Orf1p also is expressed in non-PV+ cells, consistent with L1 activity across a range of neuronal subtypes. The authors also have strengthened their findings regarding the increased intensity of ORF1p staining in aged compared to young animals, and the newly presented results are indeed more convincing. The prospect of increased neuronal L1 activity with age is exciting, and the results in this paper have provided the groundwork for ongoing discoveries in this area. While it is disappointing that no Orf1p interactors were followed up, this is understandable and the data are nonetheless valuable and will likely prove useful to future studies.

Reviewer #2 (Public review):

In this paper, Biswas et al. describe the role of acetylcholine (ACh) signaling in protection against chronic oxidative stress in C. elegans. They showed that disruption of ACh signaling in either unc-17 mutants or gar-3 mutants led to sensitivity to toxicity caused by chronic paraquat (PQ) treatment. Using RNA seq, they found that approximately 70% of the genes induced by chronic PQ exposure in wild type failed to upregulate in these mutants. The overexpression of gar-3 selectively in cholinergic neurons was sufficient to promote protection against chronic PQ exposure in an ACh-dependent manner. The study points to a previously undescribed role for ACh signaling in providing organism-wide protection from chronic oxidative stress, likely through the transcriptional regulation of numerous oxidative stress-response genes. The paper is well-written, and the data are robust, though some conclusions seem preliminary and do not fully support the current data. While the study identifies the muscarinic ACh receptor gar-3 as an important regulator of the response to PQ, the specific neurons in which gar-3 functions were not unambiguously identified, and the sources of ACh that regulate GAR-3 signaling and the identities of the tissues targeted by gar-3 were not addressed, limiting the scope of the study.

Major Comments:

(1) The site of action of cholinergic signaling for protection from PQ was not adequately explored. The authors' conclusion that cholinergic motor neurons are protective is based on studies using overexpression of gar-3 and an unc-17 allele that may selectively disrupt ACh in cholinergic motor neurons (Figure 9F), but these approaches are indirect. To more directly address the site of action, the authors should conduct rescue experiments using well-defined heterologous promoters. Figure 7G shows that gar-3 expressed under a 7.5 kb promoter fragment fully rescues the defect of gar-3 mutants, but the authors did not report where this promoter fragment is expressed, nor did they conduct rescue experiments of the specific tissues where gar-3 is known to be expressed (cholinergic neurons, GABAergic neurons, pharynx, or muscles). UNC-17 rescue experiments could also be useful to address the site of action. Does expression of unc-17 selectively in cholinergic motor neurons rescue the stress sensitivity of unc-17 mutants (or restore resistance to gar-3(OE); unc-17 mutants)? These experiments may also address whether ACh acts in an autocrine or paracrine manner to activate gar-3, which would be an important mechanistic insight to this study that is currently lacking.

(2) The genetic pan-neuronal silencing experiments presented in Figure 1 motivated the subsequent experiments, but the authors did not relate these observations to ACh/gar-3 signaling. For example, the authors did not address whether silencing just the cholinergic motor neurons at the different times tested has the same effects on survival as pan-neuronal silencing.

(3) It is assumed that protection occurs through inter-tissue signaling of ACh to target tissues, where it impacts gene expression. While this is a reasonable assumption, it has not been directly shown here. It is recommended that the authors examine GFP reporter expression of a sampling of the genes identified in this study (including proteasomal genes that the authors highlight) that are regulated by unc-17 and gar-3. This would serve to independently confirm the RNAseq data and to identify target tissues that are subject to gene expression regulation by ACh, which would significantly strengthen the study.

Reviewer #2 (Public review):

Summary:

Lesser et al. present an atlas of Drosophila wing sensory neurons. They proofread the axons of all sensory neurons in the wing nerve of an existing electron microscopy dataset, the female adult fly nerve cord (FANC) connectome. These reconstructed sensory axons were linked with light microscopy images of full-scale morphology to identify their origin in the periphery of the wing and encoded sensory modalities. The authors described the morphology and postsynaptic targets of proprioceptive neurons as well as previously unknown sensory neurons.

Strengths:

The authors present a valuable catalogue of wing sensory neurons, including previously undescribed sensory axons in the Drosophila wing. By providing both connectivity information with linked genetic drive lines, this research facilitates future work on the wing motor-sensory network and applications relating to Drosophila flight. The findings were linked to previous research as well as their putative role in the proprioceptive and nerve cord circuitry, providing testable hypotheses for future studies.

Weaknesses:

(1) With future use as an atlas, it should be noted that the evidence is based on sensory neurons on only one side of the nerve cord. Fruit flies have stereotyped left/right hemispheres in the brain and left/right hemisegments in the nerve cord. The comparison of left and right neurons of the nervous system can give a sense of how robust the morphological and connectivity findings are. Here, the authors have not compared the left and right side sensory axons from the wing nerve, leaving potential for developmental variability across samples and left/right hemisegments.

(2) Not all links between the EM reconstructions and driver lines are convincing. To strengthen these, for all EM-LM matches in Figures 3-7, rotated views of the driver line (matching the rotated EM views) should be shown to provide a clearer comparison of the data. In particular, Figure 3G and Figure 7B are not very convincing based on the images shown. MCFO imaging of the driver lines in Figure 3G and 7B would make this position stronger if a clone that matches the EM reconstruction could be identified.

(3) Figure 7B looks like the driver line might have stochastic expression in the sensory neuron, which further reduces confidence in the result shown in Figure 7C. Is this expression pattern in the wing consistently seen? Many split-GAL4s have stochastic expressions. The evidence would be strengthened if the authors presented multiple examples (~4-5) of each driver line's expression pattern in the supplement.

(4) Certain claims in this work lack quantitative evidence. On line 128, for instance, "Overall, our comprehensive reconstruction revealed many morphological subgroups with overlapping postsynaptic partners, suggesting a high degree of integration within wing sensorimotor circuits." If a claim of subgroups having shared postsynaptic partners is being made, there should have been quantitative evidence. For example, cosine similar amongst members of each group compared to the cosine similarity of shuffled/randomised sets of axons from different groups. The heat map of cosine similarity in Figure 2B alone is not sufficient.

(5) Similarly, claims about putative electrical connections to b1 motor neurons are very speculative. The authors state that "their terminals contain very densely packed mitochondria compared to other cells", without providing a quantitative comparison to other sensory axons. There is also no quantitative comparison to the one example of another putative electrical connection from the literature. Further, it should be noted that this connection from Trimarchi and Murphey, 1997, is also stated as putative on line 167, which further weakens this evidence. Quantification would strongly strengthen this position. Identification of an example of high mitochondrial density at a confirmed electrical connection would be even better. In the related discussion section "A potential metabolic specialization for flight circuitry", it should be more clearly noted that the dense mitochondria could be unrelated to a putative electrical connection. If the authors have an alternative hypothesis about the mitochondria density, this should be stated as well.

(6) It would be appropriate to cite previous work using a similar strategy to match sensory axons to their cell bodies/dendrites at the periphery using driver lines and connectomics (see Figure 5 for example in the following paper: https://doi.org/10.7554/eLife.40247 ).

The methods section is very sparse. For the sake of replicability, all sections should be expanded upon.

Reviewer #2 (Public review):

Summary:

The authors aimed to identify the specific neurons, neurotransmitters, and neuropeptides that mediate the longevity effects of the hypoxic response in C. elegans. By genetically dissecting the pathway downstream of HIF-1, they define a neural circuit involving ADF serotonergic neurons, the SER-7 receptor in the RIS interneuron, tyraminergic signaling from RIM, and neuropeptide NLP-17, ultimately linking neuronal hypoxic sensing to pro-longevity signaling in the intestine.

Strengths:

The study employs a diverse genetic toolkit, including neuron-specific transgenes, tissue-specific knockouts and rescues, RNAi knockdowns, allowing the authors to pinpoint causality, sufficiency, and necessity with high resolution. The comprehensive mapping of cell-nonautonomous signaling adds depth to our understanding of how HIF and serotonin signaling interface with aging pathways. The conclusions are supported by consistent survival assays and fmo-2 gene expression analyses.

Weaknesses:

A key limitation is the lack of clear evidence showing epistasis of so many identified molecular/neuronal components downstream of HIF-1 and serotonin. Thus, the mechanisms of how a diverse set of molecules/neurons coordinate and mediate neuronal HIF-1 effects on intestinal fmo-2 and longevity remain murky. Some rescue strategies may inadvertently cause non-physiological expression. Additionally, environmental hypoxia was not tested in parallel, so the claim on "hypoxia respone" throughout the manuscript is not justified by genetic manipulation alone, and the translational relevance of the genetic manipulations remains somewhat uncertain.

Reviewer #2 (Public review):

Summary:

Fu and colleagues have shown that VALOR, a model of multimodal and dynamic stimulus features, better predicts brain responses compared to unimodal or static models such as AlexNet, WordNet, or CLIP. The authors demonstrated the robustness of their findings by generalizing encoding results to an external dataset. They demonstrated the models' practical benefit by showing that semantic mappings were comparable to another model that required labor-intensive manual annotation. Finally, the authors showed that the model reveals predictive coding mechanisms of the brain, which held a meaningful relationship with individuals' fluid intelligence measures.

Strengths:

Recent advances in neural network models that extract visual, linguistic, and semantic features from real-world stimuli have enabled neuroscientists to build encoding models that predict brain responses from these features. Higher prediction accuracy indicates greater explained variance in neural activity, and therefore a better model of brain function. Commonly used models include AlexNet for visual features, WordNet for audio-semantic features, and CLIP for visuo-semantic features; these served as comparison models in the study. Building on this line of work, the authors developed an encoding model using VALOR, which captures the multimodal and dynamic nature of real-world stimuli. VALOR outperformed the comparison models in predicting brain responses. It also recapitulated known semantic mappings and revealed evidence of predictive processing in the brain. These findings support VALOR as a strong candidate model of brain function.

Weaknesses:

The authors argue that this modeling contributes to a better understanding of how the brain works. However, upon reading, I am less convinced about how VALOR's superior performance over other models tells us more about the brain. VALOR is a better model of the audiovisual stimulus because it processes multimodal and dynamic stimuli compared to other unimodal or static models. If the model better captures real-world stimuli, then I almost feel that it has to better capture brain responses, assuming that the brain is a system that is optimized to process multimodal and dynamic inputs from the real world. The authors could strengthen the manuscript if the significance of their encoding model findings were better explained.

In Study 3, the authors show high alignment between WordNet and VALOR feature PCs. Upon reading the method together with Figure 3, I suspect that the alignment almost has to be high, given that the authors projected VALOR features to the Huth et al.'s PC space. Could the authors conduct non-parametric permutation tests, such as shuffling the VALOR features prior to mapping onto Huth et al.'s PC space, and then calculating the Jaccard scores? I imagine that the null distribution would be positively shifted. Still, I would be convinced if the alignment is higher than this shifted null distribution for each PC. If my understanding of this is incorrect, I suggest editing the relevant Method section (line 508) because this analysis was not easy to understand.

In Study 4, the authors show that individuals whose superior parietal gyrus (SPG) exhibited high prediction distance had high fluid cognitive scores (Figure 4C). I had a hard time believing that this was a hypothesis-driven analysis. The authors motivate the analysis that "SPG and PCu have been strongly linked to fluid intelligence (line 304)". Did the authors conduct two analyses only-SPG-fluid intelligence and PCu-fluid intelligence-without relating other brain regions to other individual differences measures? Even if so, the authors should have reported the same r-value and p-value for PCu-fluid intelligence. If SPG-fluid intelligence indeed holds specificity in terms of statistical significance compared to all possible scenarios that were tested, is this rationally an expected result, and could the authors explain the specificity? Also, the authors should explain why they considered fluid intelligence to be the proxy of one's ability to anticipate upcoming scenes during movie watching. I would have understood the rationale better if the authors had at least aggregated predictive scores for all brain regions that held significance into one summary statistic and found a significant correlation with the fluid intelligence measure.

Reviewer #2 (Public review):

Summary:

This paper continues the authors' research on the roles of the basolateral amygdala (BLA) and the perirhinal cortex (PRh) in sensory preconditioning (SPC) and second-order conditioning (SOC). In this manuscript, the authors explore how prior exposure to stimuli may influence which regions are necessary for conditioning to the second-order cue (S2). The authors perform a series of experiments which first confirm prior results shown by the author - that NMDA receptors in the PRh are necessary in SPC during conditioning of the first-order cue (S1) with shock to allow for freezing to S2 at test; and that NMDA receptors in the BLA are necessary for S1 conditioning during the S1-shock pairings. The authors then set out to test the hypothesis that the PRh encodes associations in a peripheral state of attention, whereas the BLA encodes associations in a focal state of attention, similar to the A1 and A2 states in Wagner's theory of SOP. To do this, they show that BLA is necessary for conditioning to S2 when the S2 is first exposed during a serial compound procedure - S2-S1-shock. To determine whether pre-exposure of S2 will shift S2 to a peripheral focal state, the authors run a design in which S2-S1 presentations are given prior to the serial compound phase. The authors show that this restores NMDA receptor activity within the PRh as necessary for the fear response to S2 at test. They then test whether the presence of S1 during the serial compound conditioning allows the PRh to support the fear responses to S2 by introducing a delay conditioning paradigm in which S1 is no longer present. The authors find that PRh is no longer required and suggest that this is due to S2 remaining in the primary focal state.

Strengths:

As with their earlier work, the authors have performed a rigorous series of experiments to better understand the roles of the BLA and PRh in the learning of first- and second-order stimuli. The experiments are well-designed and clearly presented, and the results show definitive differences in functionality between the PRh and BLA. The first experiment confirms earlier findings from the lab (and others), and the authors then build on their previous work to more deeply reveal how these regions differ in how they encode associations between stimuli. The authors have done a commendable job of pursuing these questions.

Table 1 is an excellent way to highlight the results and provide the reader with a quick look-up table of the findings.

Weaknesses:

The authors have attempted to resolve the question of the roles of the PRh and BLA in SPC and SOC, which the authors have explored in previous papers. Laudably, the authors have produced substantial results indicating how these two regions function in the learning of first- and second-order cues, providing an opportunity to narrow in on possible theories for their functionality. Yet the authors have framed this experiment in terms of an attentional framework and have argued that the results support this particular framework and hypothesis - that the PRh encodes peripheral and the BLA encodes focal states of learning. This certainly seems like a viable and exciting hypothesis, yet I don't see why the results have been completely framed and interpreted this way. It seems to me that there are still some alternative interpretations that are plausible and should be included in the paper.

Name - Tariq Siddiqui Designation - Founder at Adswom

Make the description word count under two lines

Reviewer #2 (Public review):

Summary:

This manuscript addresses an important impediment in the field of Alzheimer's disease (AD) and tauapathy research by showing that 12 specific phosphomimetic mutations in full-length tau allow the protein to aggregate into fibrils with the AD fold and the fold of chronic traumatic encephalopathy fibrils in vitro. The paper presents comprehensive structural and cell based seeding data indicating the improvement of their approach over previous in vitro attempts on non-full-length tau constructs. The main weaknesses of this work results from the fact that only up to 70% of the tau fibrils form the desired fibril polymorphs. In addition, some of the figures are of low quality and confusing.

Strengths:

This study provides significant progress towards a very important and timely topic in the amyloid community, namely the in vitro production of tau fibrils found in patients.

The 12 specific phosphomimetic mutations presented in this work will have an immediate impact in the field since they can be easily reproduced.

Multiple high-resolution structures support the success of the phosphomimetic mutation approach.

Additional data show the seeding efficiency of the resulting fibrils, their reduced tendency to bundle, and their ability to be labeled without affecting core structure or seeding capability.

Comments on revised version:

Generally, I am satisfied with the revisions. Specifically, the new results showing 100% formation of PHF is a significant improvement.

Reviewer #2 (Public review):

Summary:

Moret et al. present an engineered family of fluorescent calcium indicators based on HaloCamp, a HaloTag-based sensor system that utilizes Janelia Fluorophores (JF dyes) to report calcium dynamics. By introducing single or multiple amino acid substitutions, the authors reduce HaloCamp's calcium affinity, making these low-affinity variants well-suited for imaging calcium transients in high-calcium environments such as the endoplasmic reticulum (ER) and mitochondria. The study validates the sensors' dissociation constants (Kd), spectra, and multiplex capabilities. It demonstrates improved performance compared to existing tools when targeted to subcellular compartments in mammalian cells and cultured neurons. The sensors can be tuned across the red-to-far-red spectrum via JF585 and JF635 labeling, enabling flexible multiplexed imaging. For example, the authors show that HaloCamp can be targeted to mitochondria and used alongside other green and red sensors, allowing simultaneous imaging of calcium dynamics in the cytosol, ER, and mitochondria. Overall, they achieve their goals, and the data demonstrate that HaloCamp variants are effective for detecting ER and mitochondrial calcium changes under physiological conditions. The presented experiments support the conclusions. However, some key aspects, such as sensor kinetics and axonal validation, would benefit from further analysis.

This work is likely to have an important impact on the fields of calcium imaging and organelle physiology. The modular design of HaloCamp and its compatibility with a wide range of fluorophores offer a broad application range for cell biologists and neuroscientists.

Strengths:

(1) The authors introduce the first tunable, dye-based, low-affinity HaloTag calcium sensors for subcellular imaging, addressing a significant unmet need for ER and mitochondrial calcium detection.

(2) The ability to pair HaloCamp with JF585 and JF635 extends the spectral range, facilitating multiplexed imaging with existing calcium indicators.

(3) The sensors are validated in a range of subcellular compartments (ER, mitochondria, cytosol) in both mammalian cells and neurons.

(4) The authors successfully demonstrate simultaneous imaging of three compartments using orthogonal sensors, a technically impressive feat.

(5) Kd values are measured, and fluorescent responses are tested under physiologically relevant stimulation.

Weaknesses:

(1) The authors do not quantify the kinetics (e.g., decay tau or off-rate) of the fluorescent signals, particularly after stimulation. For example, in the ER imaging experiments in neurons, the decay of the HaloCamp fluorescence after field stimulation (20 APs @ 20 Hz) is not analyzed or compared to ER-GCaMP6-210 or R-CEPIer.

(2) It remains unclear whether the observed decay represents the sensor's off-kinetics or actual physiological calcium clearance from the ER. A comparison between sensors or an independent measurement of ER clearance rates in vitro would clarify this.

(3) The choice of 20 APs at 20 Hz is not justified. Specifically, single APs or low-frequency stimulations are not tested, leaving unclear what the detection threshold of the new sensors is.

(4) In neuron experiments, the authors report measuring ER calcium in axons based presumably on morphology, but no specific justification for selection, markers, or post hoc labeling is described.

(5) Figure 5 assumes that all three indicators (cytosolic, ER, and mitochondrial) are fast enough to report calcium dynamics in response to histamine. This assumption is not fully validated. Cross-controls (e.g., expressing GCaMP6-210 in mitochondria and HaloCamp in the ER) would strengthen confidence that the sensors are correctly reporting dynamic changes.

(6) It is not clear why Thapsigargin leads to depletion in HeLa cells and neurons in experiments shown in Figure 1E, but not in 2B upon field stimulation.

Reviewer #2 (Public review):

Summary:

This study presents valuable findings on the molecular mechanisms driving the female-to-male transformation in the ricefield eel (Monopterus albus) during aging. The authors explore the role of temperature-activated TRPV4 signaling in promoting testicular differentiation, proposing a TRPV4-Ca²⁺-pSTAT3-Kdm6b axis that facilitates this gonadal shift.

Strengths:

The manuscript describes an interesting mechanism potentially underlying sex differentiation in M. albus.

Weaknesses:

The current data are insufficient to fully support the central claims, and the study would benefit from more rigorous experimental approaches.

(1) Overstated Title and Claims:

The title "TRPV4 mediates temperature-induced sex change" overstates the evidence. No histological confirmation of gonadal transformation (e.g., formation of testicular structures) is presented. Conclusions are based solely on molecular markers such as dmrt1 and sox9a, which, although suggestive, are not definitive indicators of functional sex reversal.

(2) Temperature vs Growth Rate Confounding (Figure 1E):

The conclusion that warm temperature directly induces gonadal transformation is confounded by potential growth rate effects. The authors state that body size was "comparable" between 25{degree sign}C and 33{degree sign}C groups, but fail to provide supporting data. In ectotherms, growth is intrinsically temperature-dependent. Given the known correlation between size and sex change in M. albus, growth rate-rather than temperature per se-may underlie the observed sex ratio shifts. Controlled growth-matched comparisons or inclusion of growth rate metrics are needed.

(3) TRPV4 as a Thermosensor-Insufficient Evidence:

The characterisation of TRPV4 as a direct thermosensor lacks biophysical validation. The observed transcriptional upregulation of Trpv4 under heat (Figure 2) reflects downstream responses rather than primary sensor function. Functional thermosensors, including TRPV4, respond to heat via immediate ion channel activity-typically measurable within seconds-not mRNA expression over hours. No patch-clamp or electrophysiological data are provided to confirm TRPV4 activation thresholds in eel gonadal cells. Additionally, the Ca²⁺ imaging assay (Figure 2F) lacks essential details: the timing of GSK1016790A/RN1734 administration relative to imaging is unclear, making it difficult to distinguish direct channel activity from indirect transcriptional effects.

(4) Cellular Context of TRPV4 Activity Is Unclear:

In situ hybridisation suggests TRPV4 expression shifts from interstitial to somatic domains under heat (Figures. 2H, S2C), implying potential cell-type-specific roles. However, the study does not clarify: (i) whether TRPV4 plays the same role across these cell types, (ii) why somatic cells show stronger signal amplification, or (iii) the cellular composition of explants used in in vitro assays. Without this resolution, conclusions from pharmacological manipulation (e.g., GSK1016790A effects) cannot be definitively linked to specific cell populations.

(5) Rapid Trpv4 mRNA Elevation and Channel Function:

The authors report a dramatic increase in Trpv4 mRNA within one day of heat exposure (Figures 4D, S2B). Given that TRPV4 is a membrane channel, not a transcription factor, its rapid transcriptional sensitivity to temperature raises mechanistic questions. This finding, while intriguing, seems more correlational than functional. A clearer explanation of how TRPV4 senses temperature at the molecular level is needed.

(6) Inconclusive Evidence for the Ca²⁺ -pSTAT3-Kdm6b Axis:

Although the authors propose a TRPV4-Ca²⁺ -pSTAT3-Kdm6b-dmrt1 pathway, intermediate steps remain poorly supported. For example, western blot data (Figures 3C, 4B) do not convincingly demonstrate significant pSTAT3 elevation at 34{degree sign}C. Higher-resolution and properly quantified blots are essential. The inferred signalling cascade is based largely on temporal correlation and pharmacological inhibition, which are insufficient to establish direct regulatory relationships.

(7) Species-Specific STAT3-Kdm6b Regulation Is Unresolved:

The proposed activation of Kdm6b by pSTAT3 contrasts with findings in the red-eared slider turtle (Trachemys scripta), where pSTAT3 represses Kdm6b. This divergence in regulatory direction between the two TSD species is surprising and demands further justification. Cross-species differences in binding motifs or epigenetic context should be explored. Additional evidence, such as luciferase reporter assays (using wild-type and mutant pSTAT3 binding motifs in the Kdm6b promoter) is needed to confirm direct activation. A rescue experiment-testing whether Kdm6b overexpression can compensate for pSTAT3 inhibition-would also greatly strengthen the model.

(8) Immunofluorescence-Lack of Structural Markers:

All immunofluorescence images should include structural markers to delineate gonadal boundaries. Furthermore, image descriptions in the figure legends and main text lack detail and should be significantly expanded for clarity.

(9) Pharmacological Reagents-Mechanisms and References:

The manuscript lacks proper references and mechanistic descriptions for the pharmacological agents used (e.g., GSK1016790A, RN1734, Stattic). Established literature on their specificity and usage context should be cited to support their application and interpretation in this study.

(10) Efficiency of Experimental Interventions:

The percentage of gonads exhibiting sex reversal following pharmacological or RNAi treatments should be reported in the Results. This is critical for evaluating the strength and reproducibility of the interventions.

Reviewer #2 (Public review):

5-methylcytosine (5mC) is a key epigenetic mark in DNA and plays a crucial role in regulating gene expression in many eukaryotes including humans. The DNA methyltransferases (DNMTs) that establish and maintain 5mC, are conserved in many species across eukaryotes, including animals, plants, and fungi, mainly in a CpG context. Interestingly, 5mC levels and distributions are quite variable across phylogenies with some species even appearing to have no such DNA methylation.

This interesting and well-written paper discusses continuation of some of the authors' work published several years ago. In that previous paper, the laboratory demonstrated that DNA methylation pathways coevolved with DNA repair mechanisms, specifically with the alkylation repair system. Specifically, they discovered that DNMTs can introduce alkylation damage into DNA, specifically in the form of 3-methylcytosine (3mC). (This appears to be an error in the DNMT enzymatic mechanism where the generation 3mC as opposed to its preferred product 5-methylcytosine (5mC), is caused by the flipped target cytosine binding to the active site pocket of the DNMT in an inverted orientation.) The presence of 3mC is potentially toxic and can cause replication stress, which this paper suggests may explain the loss of DNA methylation in different species. They further showed that the ALKB2 enzyme plays a crucial role in repairing this alkylation damage, further emphasizing the link between DNA methylation and DNA repair.

The co-evolution of DNMTs with DNA repair mechanisms suggest there can be distinct advantages and disadvantages of DNA methylation to different species which might depend on their environmental niche. In environments that expose species to high levels of DNA damage, high levels of 5mC in their genome may be disadvantageous. This present paper sets out to examine the sensitivity of an organism to genotoxic stresses such as alkylation and oxidation agents as the consequence of DNMT activity. Since such a study in eukaryotes would be complicated by DNA methylation controlling gene regulation, these authors cleverly utilize Escherichia coli (E.coli) and incorporate into it the DNMTs from other bacteria that methylate the cytosines of DNA in a CpG context like that observed in eukaryotes; the active sites of these enzymes are very similar to eukaryotic DNMTs and basically utilize the same catalytic mechanism (also this strain of E.coli does not specifically degrade this methylated DNA) .

The experiments in this paper more than adequately show that E. coli expression of these DNMTs (comparing to the same strain without the DNMTS) do indeed show increased sensitivity to alkylating agents and this sensitivity was even greater than expected when a DNA repair mechanism was inactivated. Moreover, they show that this E. coli expressing this DNMT is more sensitive to oxidizing agents such as H2O2 and has exacerbated sensitivity when a DNA repair glycosylase is inactivated. Both propensities suggest that DNMT activity itself may generate additional genotoxic stress. Intrigued that DNMT expression itself might induce sensitivity to oxidative stress, the experimenters used a fluorescent sensor to show that H2O2 induced reactive oxygen species (ROS) are markedly enhanced with DNMT expression. Importantly, they show that DNMT expression alone gave rise to increased ROS amounts and both H2O2 addition and DNMT expression has greater effect that the linear combination of the two separately. They also carefully checked that the increased sensitivity to H2O2 was not potentially caused by some effect on gene expression of detoxification genes by DNMT expression and activity. Finally, by using mass spectroscopy, they show that DNMT expression led to production of the 5mC oxidation derivatives 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) in DNA. 5fC is a substrate for base excision repair while 5hmC is not; more 5fC was observed. Introduction of non-bacterial enzymes that produce 5hmC and 5fC into the DNMT expressing bacteria again showed a greater sensitivity than expected. Remarkedly, in their assay with addition of H2O2, bacteria showed no growth with this dual expression of DNMT and these enzymes.

Overall, the authors conduct well thought-out and simple experiments to show that a disadvantageous consequence of DNMT expression leading to 5mC in DNA is increased sensitivity to oxidative stress as well as alkylating agents.

Again, the paper is well-written and organized. The hypotheses are well-examined by simple experiments. The results are interesting and can impact many scientific areas such as our understanding of evolutionary pressures on an organism by environment to impacting our understanding about how environment of a malignant cell in the human body may lead to cancer.

In a new revised version of the paper, the authors have adequately addressed issues put forth by other reviewers.

Reviewer #2 (Public review):

This paper presents interesting and fresh approach as it investigates whether female moths utilize plant-emitted ultrasounds, particularly those associated with dehydration stress, in their egg-laying decision-making process. It provides the first empirical evidence suggesting that acoustic information may contribute to insect-plant interactions.

The revised version is significantly strengthened by the addition of supplementary data and improved explanations. The authors present robust results across multiple experiments, enhancing the credibility of their conclusions.

Female moths showed a preference for moist, fresh plants over dehydrated ones in experiments using actual plants. Additionally, when both plants were fresh but ultrasonic sounds specific to dehydrated plants were presented from one side, the moths chose the silent plant. However, in experiments without plants, contrary to the hypothesis derived from the above results, the moths preferred to oviposit near ultrasonic playback mimicking the sounds of dehydrated plants.　

These results clearly indicate that moths can perceive plant presence through sound.　The findings also highlight the need for future investigation into the multi-modal nature of moth decision-making, as acoustic cues alone may not fully explain the behavioral choices observed across different contexts.

Overall, the results are intriguing, and I think the experiments are very well designed. The authors successfully demonstrate that plant-derived acoustic signals influence oviposition behavior in female moths, thereby achieving the study's objectives. The experimental design and analysis protocols are reproducible and well suited for adaptation to other species.

Reviewer #2 (Public review):

Summary:

The authors were to investigate functional role of IL10 on mucosal immunity in chickens. CRISPR technology was employed to generate IL10 knock out chickens in both exon and putative enhancer regions. IL10 expressions were either abolished (knockout in exon) or reduced (enhancer knock-out). IL-10 play an important role in the composition of the caecal microbiome. Through various enteric pathogens challenge, deficient IL10 expression was associated with enhanced pathogen clearance, but with more severe lesion score and body weight loss.

Strengths:

Both in vitro and in vivo knock-out in abolished and reduced IL10 expression and broad enteric pathogens were challenged in vivo and various parameters were examined to evaluate the functional role of IL10 on mucosal immunity.

Weaknesses:

Overexpression of IL10 either in vitro or in vivo may further support the findings from this study.

Comments on revised version:

The authors' response and justifications are appropriate.

Reviewer #3 (Public review):

The authors have made considerable efforts to conduct functional analyses to the fullest extent possible in this study; however, it is understandable that meaningful results have not yet been obtained. In the revised version, they have appropriately framed their claims within the limits of the current data and have adjusted their statements as needed in response to the reviewers' comments.

Reviewer #3 (Public review):

Summary:

Yamada et al. build on classic and more recent studies (Chen et al., 2023; Lemmon et al., 1992; Nichol et al., 2016; Zheng et al., 1994; Schense and Hubbell, 2000) to better understand the relationship between substrate adhesion and neurite outgrowth.

Strengths:

The primary strength of the manuscript lies in developing a method for investigating the role of adhesion in axon outgrowth and traction force generation using a femtosecond laser technique. The most exciting finding is that both outgrowth and traction force generation have a biphasic relationship with laminin concentration.

Weaknesses:

The primary weaknesses, as written, are a lack of discussion of prior studies that have directly measured the strength of growth cone adhesions to the substrate (Zheng et al., 1994) and traction forces (Koch et al., 2012), the inverse correlation between retrograde flow rate and outgrowth (Nichol et al., 2016), and prior studies noting a biphasic effect of substrate concentration of neurite outgrowth (Schense and Hubbell, 2000).

Overall, the claims and conclusions are well justified by the data. The main exception is that the data is more relevant to how the rate of neurite outgrowth is controlled rather than axonal guidance.

This manuscript will help foster interest in the interrelationship between neurite outgrowth, traction forces, and substrate adhesion, and the use of a novel method to study this problem.

The authors did an excellent job in addressing my original concerns in the revision.

Reviewer #2 (Public review):

Summary:

In their manuscript, the authors reveal that the spectraplakin Shot, which can bind both microtubules and actin, is essential for the proper pruning of dendrites in a developing Drosophila model. A molecular basis for the coordination of these two cytoskeletons during neuronal development has been elusive, and the authors' data point to the role of Shot in regulating microtubule polarity and growth through one of its actin-binding domains. The authors also propose an intriguing new activity for a spectraplakin: functioning as part of a microtubule-organizing center (MTOC).

Strengths:

(1) A strength of the manuscript is the authors' data supporting the idea that Shot regulates dendrite pruning via its actin-binding CH1 domain and that this domain is also implicated in Shot's ability to regulate microtubule polarity and growth (although see comments below); these data are consistent with the authors' model that Shot acts through both the actin and microtubule cytoskeletons to regulate neuronal development.

(2) Another strength of the manuscript is the data in support of Rab11 functioning as an MTOC in young larvae but not older larvae; this is an important finding that may resolve some debates in the literature. The finding that Rab11 and Msps coimmunoprecipitate is nice evidence in support of the idea that Rab11(+) endosomes serve as MTOCs.

Weaknesses:

(1) A significant, major concern is that most of the authors' main conclusions are not (well) supported, in particular, the model that Shot functions as part of an MTOC. The story has many interesting components, but lacks the experimental depth to support the authors' claims.

(2) One of the authors' central claims is that Shot functions as part of a non-centrosomal MTOC, presumably a MTOC anchored on Rab11(+) endosomes. For example, in the Introduction, last paragraph, the authors summarize their model: "Shot localizes to dendrite tips in an actin-dependent manner where it recruits factors cooperating with an early-acting, Rab11-dependent MTOC." This statement is not supported. The authors do not show any data that Shot localizes with Rab11 or that Rab11 localization or its MTOC activity is affected by the loss of Shot (or otherwise manipulating Shot). A genetic interaction between Shot and Rab11 is not sufficient to support this claim, which relies on the proteins functioning together at a certain place and time. On a related note, the claim that Shot localization to dendrite tips is actin-dependent is not well supported: the authors show that the CH1 domain is needed to enrich Shot at dendrite tips, but they do not directly manipulate actin (it would be helpful if the authors showed the overexpression of Mical disrupted actin, as they predict).

(3) The authors show an image that Shot colocalizes with the EB1-mScarlet3 comet initiation sites and use this representative image to generate a model that Shot functions as part of an MTOC. However, this conclusion needs additional support: the authors should quantify the frequency of EB1 comets that originate from Shot-GFP aggregates, report the orientation of EB1 comets that originate from Shot-GFP aggregates (e.g., do the Shot-GFP aggregates correlate with anterogradely or retrogradely moving EB1 comets), and characterize the developmental timing of these events. The genetic interaction tests revealing ability of shot dsRNA to enhance the loss of microtubule-interacting proteins (Msps, Patronin, EB1) and Rab11 are consistent with the idea that Shot regulates microtubules, but it does not provide any spatial information on where Shot is interacting with these proteins, which is critical to the model that Shot is acting as part of a dendritic MTOC.

(4) It is unclear whether the authors are proposing that dendrite pruning defects are due to an early function of Shot in regulating microtubule polarity in young neurons (during 1st instar larval stages) or whether Shot is acting in another way to affect dendrite pruning. It would be helpful for the authors to present and discuss a specific model regarding Shot's regulation of dendrite pruning in the Discussion.

(5) The authors argue that a change in microtubule polarity contributes to dendrite pruning defects. For example, in the Introduction, last paragraph, the authors state: "Loss of Shot causes pruning defects caused by mixed orientation of dendritic microtubules." The authors show a correlative relationship, not a causal one. In Figure 4, C and E, the authors show that overexpression of Mical disrupts microtubule polarity but not dendrite pruning, raising the question of whether disrupting microtubule polarity is sufficient to cause dendrite pruning defects. The lack of an association between a disruption in microtubule polarity and dendrite pruning in neurons overexpressing Mical is an important finding.

(6) The authors show that a truncated Shot construct with the microtubule-binding domain, but no actin-binding domain (Shot-C-term), can rescue dendrite pruning defects and Khc-lacZ localization, whereas the longer Shot construct that lacks just one actin-binding domain ("delta-CH1") cannot. Have the authors confirmed that both proteins are expressed at equivalent levels? Based on these results and their finding that over-expression of Shot-delta-CH1 disrupts dendrite pruning, it seems possible that Shot-delta-CH1 may function as a dominant-negative rather than a loss-of-function. Regardless, the authors should develop a model that takes into account their findings that Shot, without any actin-binding domains and only a microtubule-binding domain, shows robust rescue.

(7) The authors state that: "The fact that Shot variants lacking the CH1 domain cannot rescue the pruning defects of shot[3] mutants suggested that dendrite tip localization of Shot was important for its function." (pages 10-11). This statement is not accurate: the Shot C-term construct, which lacks the CH1 domain (as well as other domains), is able to rescue dendrite pruning defects.

(8) The authors state that: "In further support of non-functionality, overexpression of Shot[deltaCH1] caused strong pruning defects (Fig. S3)." (page 8). Presumably, these results indicate that Shot-delta-CH1 is functioning as a dominant-negative since a loss-of-function protein would have no effect. The authors should revise how they interpret these results. This comment is related to another comment about the ability of Shot constructs to rescue the shot[3] mutant.

Reviewer #2 (Public review):

Summary:

In this work, the authors applied a range of computational methods to probe the translocation of cholesterol through the Smoothened receptor. They test whether cholesterol is more likely to enter the receptor straight from the outer leaflet of the membrane or via a binding pathway in the inner leaflet first. Their data reveal that both pathways are plausible but that the free energy barriers of pathway 1 are lower, suggesting this route is preferable. They also probe the pathway of cholesterol transport from the transmembrane region to the cysteine-rich domain (CRD).

Strengths:

(1) A wide range of computational techniques is used, including potential of mean force calculations, adaptive sampling, dimensionality reduction using tICA, and MSM modelling. These are all applied in a rigorous manner, and the data are very convincing. The computational work is an exemplar of a well-carried out study.

(2) The computational predictions are experimentally supported using mutagenesis, with an excellent agreement between their PMF and mRNA fold change data.

(3) The data are described clearly and coherently, with excellent use of figures. They combine their findings into a mechanism for cholesterol transport, which on the whole seems sound.

(4) The methods are described well, and many of their analysis methods have been made available via GitHub, which is an additional strength.

Weaknesses:

(1) Some of the data could be presented a little more clearly. In particular, Figure 7 needs additional annotation to be interpretable. Can the position of the cholesterol be shown on the graph so that we can see the diameter change more clearly?

(2) In Figure 3C, it doesn't look like the Met is constricting the tunnel at all. What residue is constricting the tunnel here? Can we see the Ala and Met panels from the same angle to compare the landscapes? Or does the mutation significantly change the tunnel? Why not A283 to a bulkier residue? Finally, the legend says that the figure shows that cholesterol can still pass this residue, but it doesn't really show this. Perhaps if the HOLE graph was plotted, we could see the narrowest point of the tunnel and compare it to the size of cholesterol.

(3) The PMF axis in 3b and d confused me for a bit. Looking at the Supplementary data, it's clear that, e.g., the F455I change increases the energy barrier for chol entering the receptor. But in 3d this is shown as a -ve change, i.e., favourable. This seems the wrong way around for me. Either switch the sign or make this clearer in the legend, please.

(4) The impact of G280V is put down to a decrease in flexibility, but it could also be a steric hindrance. This should be discussed.

(5) Are the reported energy barriers of the two pathways (5.8{plus minus}0.7 and 6.5{plus minus}0.8 kcal/mol) significantly and/or substantially different enough to favour one over the other? This could be discussed in the manuscript.

(6) Are the energy barriers consistent with a passive diffusion-driven process? It feels like, without a source of free energy input (e.g., ion or ATP), these barriers would be difficult to overcome. This could be discussed.

(7) Regarding the kinetics from MSM, it is stated that the values seen here are similar to MFS transporters, but this then references another MSM study. A comparison to experimental values would support this section a lot.

Reviewer #2 (Public review):

Summary:

This study identifies the outer‑mitochondrial GTPase MIRO1 as a central regulator of vascular smooth muscle cell (VSMC) proliferation and neointima formation after carotid injury in vivo and PDGF-stimulation ex vivo. Using smooth muscle-specific knockout male mice, complementary in vitro murine and human VSMC cell models, and analyses of mitochondrial positioning, cristae architecture, and respirometry, the authors provide solid evidence that MIRO1 couples mitochondrial motility with ATP production to meet the energetic demands of the G1/S cell cycle transition. However, a component of the metabolic analyses is suboptimal and would benefit from more robust methodologies. The work is valuable because it links mitochondrial dynamics to vascular remodelling and suggests MIRO1 as a therapeutic target for vasoproliferative diseases, although whether pharmacological targeting of MIRO1 in vivo can effectively reduce neointima after carotid injury has not been explored. This paper will be of interest to those working on VSMCs and mitochondrial biology.

Strengths:

The strength of the study lies in its comprehensive approach, assessing the role of MIRO1 in VSMC proliferation in vivo, ex vivo, and importantly in human cells. The subject provides mechanistic links between MIRO1-mediated regulation of mitochondrial mobility and optimal respiratory chain function to cell cycle progression and proliferation. Finally, the findings are potentially clinically relevant given the presence of MIRO1 in human atherosclerotic plaques and the available small molecule MIRO1.

Weaknesses:

(1) There is a consistent lack of reporting across figure legends, including group sizes, n numbers, how many independent experiments were performed, or whether the data is mean +/- SD or SEM, etc. This needs to be corrected.

(2) The in vivo carotid injury experiments are in male mice fed a high-fat diet; this should be explicitly stated in the abstract, as it's unclear if there are any sex- or diet-dependent differences. Is VSMC proliferation/neointima formation different in chow-fed mice after carotid injury?

(3) The main body of the methods section is thin, and it's unclear why the majority of the methods are in the supplemental file. The authors should consider moving these to the main article, especially in an online-only journal.

(4) Certain metabolic analyses are suboptimal, including ATP concentration and Complex I activity measurements. The measurement of ATP/ADP and ATP/AMP ratios for energy charge status (luminometer or mass spectrometry), while high-resolution respirometry (Oroboros) to determine mitochondrial complex I activity in permeabilized VSMCs would be more informative.

(5) The statement that 'mitochondrial mobility is not required for optimal ATP production' is poorly supported. XF Seahorse analysis should be performed with nocodazole and also following MIRO1 reconstitution +/- EF hands.

(6) The authors should consider moving MIRO1 small molecule data into the main figures. A lot of value would be added to the study if the authors could demonstrate that therapeutic targeting of MIRO1 could prevent neointima formation in vivo.

Reviewer #2 (Public review):

This study explores the dynamic association between malate dehydrogenase (MDH1) and citrate synthase (CIT1) in Saccharomyces cerevisiae, with the aim of linking this interaction to respiratory metabolism. Utilizing a NanoBiT split-luciferase system, the authors monitor protein-protein interactions in vivo under various metabolic conditions.

Major Concerns:

(1) NanoBiT Signal May Reflect Protein Abundance Rather Than Interaction Strength

In Figure 1C, the authors report increased MDH1-CIT1 interaction under respiratory (acetate) conditions and decreased interaction during fermentation (glucose), as indicated by NanoBiT luminescence. However, this signal appears to correlate strongly with the expression levels of MDH1 and CIT1, raising the possibility that the observed luminescence reflects protein abundance rather than specific interaction dynamics. To resolve this, NanoBiT signals should be normalized to the expression levels of both proteins to distinguish between abundance-driven and interaction-driven changes.

(2) Lack of Causal Evidence

The study presents a series of metabolic perturbation experiments (e.g., arsenite, AOA, antimycin A, malonate) and correlates changes in metabolite levels with NanoBiT signals. However, these data are correlative and do not establish a functional role for the MDH1-CIT1 interaction in metabolic regulation. To demonstrate causality, the authors should implement approaches to specifically disrupt the MDH1-CIT1 interaction. One strategy could involve using a 15-residue peptide (Pept1) derived from the Pro354-Pro366 region of CIT1, previously shown to mediate the interaction, or introducing the cit1Δ3 (Arg362Glu) mutation, which perturbs binding. Metabolic flux analysis using ^13C-labeled glucose and mitochondrial respiration assays (e.g., Seahorse) could then assess functional consequences.

(3) Absence of Protein Expression Controls Under Perturbation Conditions

In experiments involving acetate, arsenite, AOA, antimycin A, and malonate, the authors infer changes in MDH1-CIT1 association based solely on NanoBiT signals. However, no accompanying data are provided on MDH1 and CIT1 protein levels under these conditions. This omission weakens the conclusions, as altered expression rather than interaction strength could underlie the observed luminescence changes. Immunoblotting or quantitative proteomics should be used to confirm constant protein expression across conditions.

Conclusion:

Although the central question is compelling and the use of NanoBiT in live cells is a strength, the manuscript requires additional experimental rigor. Specifically, normalization of interaction signals, introduction of causative perturbations, and validation of protein expression are essential to substantiate the study's claims.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors focus on the identification of the mechanisms involved in the acquired resistance to Sotorasib in non-small lung KRASG12C mutant cells. To perform this study, the authors generate different clones of cell lines, cell-derived xenografts, patient-derived xenograft organoids, and patient-derived xenografts. In all these models, the authors generate resistant forms (i.e., resistant cell lines PDXs and organoids) and the genetic and molecular changes were characterised using whole-exome sequencing, proteomics, and phospho-proteomics. This analysis led to the identification of an important role of the PI3K/AKT/mTORC1/2 signalling network in the acquisition of resistance in several of the models tested. Molecular characterisation identified changes in the expression of some of the proteins in this network as key changes for the acquisition of resistance, and in particular, the authors show that changes in 4E-BP1 are common to some of the cells downstream of PI3K. Using pharmacological testing, they show that different drugs targeting PI3K, AKT, and MTORC1/2 sensitise some of the resistant models to Sotorasib. The analyses showed that the PI3K inhibitor copanlisib has an effect in NSCLC cells that, in some cases, seems to be synergistic with Sotorasib. Based on the work performed, the authors conclude that the PI3K/mTORC1/2 mediated 4E-BP1 phosphorylation is one of the mechanisms associated with the acquisition of resistance to Sotorasib and that targeting this signalling module could result in effective treatments for NSCLC patients.

The work as presented in the current manuscript is very interesting, provides cell models that benefit the community, and can be used to expand our knowledge of the mechanism of resistance to KRAS targeting therapies. Overall, the techniques and methodology seem to be performed in agreement with standard practice, and the results support most of the conclusions made by the authors. However, there are some points that, if addressed, would increase the value and relevance of the findings and further extend the impact of this work. Some of the recommendations for changes relate to the way things are explained and presented, which need some work. Other changes might require the performance of additional experiments or reanalysis of the existing data.

Strengths:

(1) One of the stronger contributions of this article is the different models used to study the acquisition of resistance to Sotorasib. The resistant cell lines, PDXs and PDXOs, and the fact that the authors have different clones for each, made this collection especially relevant, as they seem to show different mechanisms that the cells used to become resistant to Sotorasib. Although logically, the authors focus on one of these mechanisms, the differential responses of the different clones and models to the treatments used in this work show that some of the clones used additional mechanisms of resistance that can be explored in other studies. Importantly, as they use in vitro and in vivo models, the results also consider the tumour microenvironment and other factors in the response to the treatments.

(2) Another strength is the molecular characterisation of the different Sotorasib-resistant tumour cells by WES, which shows that these cells do not seem to acquire secondary mutations.

(3) The use of MS-based proteomics also identifies proteome signatures that are associated with the acquisition of resistance, including PI3K/mTORC1/2. The combination of proteomics and phospho-proteomics results should allow the identification of several mechanisms that are deregulated in Sotorasib-resistant cells.

(4) The results show a strong response of the NSCLC cells and PDXs to copanlisib, a drug for which there is limited information in this cancer type.

(5) The way they develop the PDX-resistant and the PDXO seems to be appropriate.

Weaknesses:

In general, the data is of good quality, but due to the sheer amount of data included and the way it is presented and discussed, several of the claims or conclusions are not clear.

(1) The abstract is rather long and gives details that are not usually included in one. This makes it very complicated to identify the most relevant findings of the work. The use of acronyms PDX, PDXO, and CDX without defining them makes it complicated for the non-specialist to know what the models are. Rewriting and reorganisation of the abstract would benefit the manuscript.

(2) Expression, presentation, and grammar should be reviewed in all sections of the manuscript.

(3) In the different parts of the result section where the models shown in Figure 2 are described the authors indicate "Whole-exome sequencing (WES) confirmed that XXX model retained the KRASG12C mutation with no additional KRAS mutations detected" however, it is not indicated where this data is shown and in not all the cases there is explanation to other possible modifications that might relate to mechanisms of resistance. This information should be included in the manuscript, and the WES made publicly available.

(4) The way the proteomics analysis of the TC303 and TC314 parental and resistant PDX is described in the text is confusing. The addition of an experimental layout figure would facilitate the understanding. As it is written, it is not obvious that the parental PDX were also analysed. For instance, the authors say, "The global and phosphoproteomic analyses identified over 8,000 and 4,000 gene protein products (GPPs), respectively". Is this comparing only resistant cells, or from the comparison of the parental and resistant pairs? And where are these numbers presented in the figures? Also, there is information that seems more adequate for the materials and methods sections, i.e., "Samples were analyzed using label-free nanoscale liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) on a Thermo Fusion Mass Spectrometer. The resulting data were processed and quantified using the Proteome Discoverer 2.5 interface with the Mascot search engine, referencing the NCBI RefSeq protein database (Saltzman, Ruprecht). Two-component analysis is better named principal component analysis."

(5) While the presentation of the proteomics data could be done in different ways, the way the data is presented in Figure 3 does not allow the reader to get an idea of many of the findings from this experiment. Although it is indicated that a table with the data will be made available, this should be central to the way the data is presented and explained. A table (ie, Excel doc) where the raw data and all the analysis are presented should be included and referenced. Additionally, heat maps for the whole proteomes identified should be included. In the text, it is said, "Global proteomic heatmap analysis revealed unique protein profiles in TC303AR and TC314AR PDXs compared to their sensitive counterparts (Figure 3C)." However, this figure only shows the histogram of the differentially regulated cells. Inclusion of the histogram showing all the cells is necessary, and it might be informative to include the histogram comparing the two isogenic pairs, which could identify common mechanisms and differences between both sets. In Figure 3C, the protein names should be readable, or a reference to tables where the proteins are listed should be included.

(6) In Figure 3, the pathway enrichment tool and GO used should be mentioned in the text. The tables with all significant tables should also be provided. The proteomics data seems to convincingly identify mTOR as one of the pathways deregulated in resistant cells, but there is little explanation of what is considered a significant FDR value and if there are other pathways or networks that are also modified, which might not be common to both isogenic models. In MS-based Phosphoproteome could help with the identification of differentially regulated pathways, but it is not really presented in the current manuscript. Most of the analysis of phospho-proteomics comes from the RPPA analysis, which is targeted proteomics. With the way the data is presented, the authors show evidence for a role of mTOR in the acquisition of resistance, but unfortunately, they do not discuss or allow the reader to explore if other pathways might also contribute to this change.

(7) Where is the proteomics data going to be deposited, and will it be made public to comply with FAIR principles?

(8) The authors claim that the resistance shown for H23AR and H353AR cells is due to reactivation of KRAS signalling. This is done by looking to phosphorylation of ERK as a surrogate, as they claim, "KRAS inhibition is commonly assessed by evaluating the inhibition of ERK phosphorylation (p-ERK)". While this might be true in many cases, the data presented does not demonstrate that the increase in p-ERK is due to reactivation of KRAS. To make this claim, the authors should measure activation of KRAS (and possibly H- and NRAS) using GST-pull down or an image-based method.

(9) The experiments in Figure 4 are very confusing, and some controls are missing. There is no blot where they show the effect of Sotorasib treatment in H23 and H358 parental cells. Is the increase shown in resistant cells shown in parental or is it exclusive for resistant cells only (and therefore acquired)? Experiment 4B should include this control. What is clear is that there is an increase in the expression of AKT and PI3K.

(10) The main point here is whether this is acquired resistance or the sensitivity to the drug is already there, and there was no need to do an omics experiment to find this. In some cases, it seems that the single treatment with PI3K inhibitors is as effective as Sotorasib treatment, promoting the death of the parental cells. This is in line with previous data in H23 and H353 that show sensitivity to PI3K inhibition ( i.e., H358 10.1016/j.jtcvs.2005.06.051 ; 10.1016/j.jtcvs.2005.06.051H23 10.20892/j.issn.2095-3941.2018.0361). The data is clear, especially for copanlisib, but would it be the case that this treatment could be used for the treatment of NSCLC alone or directly in combination with Sotorasib and prevent resistance? The results shown in Figure 4C strongly support that a single treatment might be effective in cases that do not respond to Sotorasib. The data in figure 4D-F (please correct typo "inhibition" in labels) seem to support that PI3K treatment of parental cells is as effective as in the resistant cells.

(11) The experiments presented in Figure 7 show synergy between Sotorasib and copanlisib treatment in some of the resistant cells. But in Figure 7G, the single treatment of H23AR is as effective as the combination. Did the authors check the effect of this drug on the parental cells? As they do not include this control, it is not possible to know if this is acquired sensitivity to PI3K inhibition or if the parental cells were already sensitive (as indicated by the Figure 4 results).

Reviewer #2 (Public review):

Summary:

In this manuscript, Jiang et al. developed a robust workflow for identifying proline hydroxylation sites in proteins. They identified proline hydroxylation sites in HEK293 and RCC4 cells, respectively. The authors found that the more hydrophilic HILIC fractions were enriched in peptides containing hydroxylated proline residues. These peptides showed differences in charge and mass distribution compared to unmodified or oxidized peptides. The intensity of the diagnostic hydroxyproline iminium ion depended on parameters including MS collision energy, parent peptide concentration, and the sequence of amino acids adjacent to the modified proline residue. Additionally, they demonstrate that a combination of retention time in LC and optimized MS parameter settings reliably identifies proline hydroxylation sites in peptides, even when multiple proline residues are present

Strengths:

Overall, the manuscript presents an advanced, standardized protocol for identifying proline hydroxylation. The experiments were well designed, and the developed protocol is straightforward, which may help resolve confusion in the field.

Weaknesses:

(1) The authors should provide a summary of the standard protocol for identifying proline hydroxylation sites in proteins that can easily be followed by others.

(2) Cockman et al. proposed that HIF-α is the only physiologically relevant target for PHDs. Their approach is considered the gold standard for identifying PHD targets. Therefore, the authors should discuss the major progress they made in this manuscript that challenges Cockman's conclusion.

Reviewer #2 (Public review):

Summary:

The field of protein translation has long sought the structure of a Type 2 Internal Ribosome Entry Site (IRES). In this work, Das and Hussain pair cryo-EM with algorithmic RNA structure prediction to present a structure of the Type 2 IRES found in Encephalomyocarditis virus (EMCV). Using medium to low resolution cryo-EM maps, they resolve the overall shape of a critical domain of this Type 2 IRES. They use algorithmic RNA prediction to model this domain onto their maps and attempt to explain previous results using this model.

Strengths:

(1) This study reveals a previously unknown/unseen binding modality used by IRESes: a direct interaction of the IRES with the initiator tRNA.

(2) Use of an IRES-associated factor to assemble and pull down an IRES bound to the small subunit of the ribosome from cellular extracts is innovative.

(3) Algorithmic modeling of RNA structure to complement medium to low resolution cryo-EM maps, as employed here, can be implemented for other RNA structures.

Weaknesses:

(1) Maps at the resolution presented prevent unambiguous modelling of the EMCV-IRES. This, combined with the lack of any biochemical data, calls into question any inferences made at the level of individual nucleotides, such as the GNRA loop and CAAA loop (Figure 4).

(2) The EMCV IRES contains an upstream AUG at position 826, where the PIC can assemble (Pestova et al 1996; PMID 8943341). It is unclear if this start codon was mutated in this study. If it were not mutated, placement of AUG-834 over AUG-826 in the P-site is unexplained.

(3) The claims the authors make about (i) the general overall shape and binding site of the IRES, (ii) its gross interaction with the two ribosomal proteins, (iii) the P-in state of the 48S, (iv) the rearrangement of the ternary complex are all warranted. Their claims about individual nucleotides or smaller stretches of the IRES-without any supporting biochemical data-is not warranted by the data.