6,922 Matching Annotations
  1. Last 7 days
    1. Reviewer #2 (Public Review):

      Summary:

      The authors present the Perceptual Error Adaptation (PEA) model, a computational approach offering a unified explanation for behavioral results that are inconsistent with standard state-space models. Beginning with the conventional state-space framework, the paper introduces two innovative concepts. Firstly, errors are calculated based on the perceived hand position, determined through Bayesian integration of visual, proprioceptive, and predictive cues. Secondly, the model accounts for the eccentricity of vision, proposing that the uncertainty of cursor position increases with distance from the fixation point. This elegantly simple model, with minimal free parameters, effectively explains the observed plateau in motor adaptation under the implicit motor adaptation paradigm using the error-clamp method. Furthermore, the authors experimentally manipulate visual cursor uncertainty, a method established in visuomotor studies, to provide causal evidence. Their results show that the adaptation rate correlates with perturbation sizes and visual noise, uniquely explained by the PEA model and not by previous models. Therefore, the study convincingly demonstrates that implicit motor adaptation is a process of Bayesian cue integration

      Strengths:

      In the past decade, numerous perplexing results in visuomotor rotation tasks have questioned their underlying mechanisms. Prior models have individually addressed aspects like aiming strategies, motor adaptation plateaus, and sensory recalibration effects. However, a unified model encapsulating these phenomena with a simple computational principle was lacking. This paper addresses this gap with a robust Bayesian integration-based model. Its strength lies in two fundamental assumptions: motor adaptation's influence by visual eccentricity, a well-established vision science concept, and sensory estimation through Bayesian integration. By merging these well-founded principles, the authors elucidate previously incongruent and diverse results with an error-based update model. The incorporation of cursor feedback noise manipulation provides causal evidence for their model. The use of eye-tracking in their experimental design, and the analysis of adaptation studies based on estimated eccentricity, are particularly elegant. This paper makes a significant contribution to visuomotor learning research.

      The authors discussed in the revised version that the proposed model can capture the general implicit motor learning process in addition to the visuomotor rotation task. In the discussion, they emphasize two main principles: the automatic tracking of effector position and the combination of movement cues using Bayesian integration. These principles are suggested as key to understanding and modeling various motor adaptations and skill learning. The proposed model could potentially become a basis for creating new computational models for skill acquisition, especially where current models fall short.

      Weaknesses:

      The proposed model is described as elegant. In this paper, the authors test the model within a limited example condition, demonstrating its relevance to the sensorimotor adaptation mechanisms of the human brain. However, the scope of the model's applicability remains unclear. It has shown the capacity to explain prior data, thereby surpassing previous models that rely on elementary mathematics. To solidify its credibility in the field, the authors must gather more supporting evidence.

    1. Reviewer #2 (Public Review):

      Weng et al. perform a comprehensive study of gene expression changes in young and old animals, in wild-type and daf-2 insulin receptor mutants, in the whole animal and specifically in the nervous system. Using this data, they identify gene families that are correlated with neuronal ageing, as well as a distinct set of genes that are upregulated in neurons of aged daf-2 mutants. This is particularly interesting as daf-2 mutants show both extended lifespan and healthier neurons in aged animals, reflected by better learning/memory in older animals compared with wild-type controls. Indeed, knockdown of several of these upregulated genes resulted in poorer learning and memory. In addition, the authors showed that several genes upregulated during ageing in wild-type neurons also contribute to learning and memory; specifically, knockdown of these genes in young animals resulted in improved memory. This indicates that (at least in this small number of cases), genes that show increased transcript levels with age in the nervous system somehow suppress memory, potentially by having damaging effects on neuronal health.

      Finally, from a resource perspective, the neuronal transcriptome provided here will be very useful for C. elegans researchers as it adds to other existing datasets by providing the transcriptome of older animals (animals at day 8 of adulthood) and demonstrating the benefits of performing tissue-specific RNAseq instead of whole-animal sequencing.

      The work presented here is of high quality and the authors present convincing evidence supporting their conclusions.

    1. Reviewer #2 (Public Review):

      Summary:

      The study explores how single striatal projection neurons (SPNs) utilize dendritic nonlinearities to solve complex integration tasks. It introduces a calcium-based synaptic learning rule that incorporates local calcium dynamics and dopaminergic signals, along with metaplasticity to ensure stability for synaptic weights. Results show SPNs can solve the nonlinear feature binding problem and enhance computational efficiency through inhibitory plasticity in dendrites, emphasizing the significant computational potential of individual neurons. In summary, the study provides a more biologically plausible solution to single-neuron learning and gives further mechanical insights into complex computations at the single-neuron level.

      Strengths:

      The paper introduces a novel learning rule for training a single multicompartmental neuron model to perform nonlinear feature binding tasks (NFBP), highlighting two main strengths: the learning rule is local, calcium-based, and requires only sparse reward signals, making it highly biologically plausible, and it applies to detailed neuron models that effectively preserve dendritic nonlinearities, contrasting with many previous studies that use simplified models.

      Weaknesses:

      I am concerned that the manuscript was submitted too hastily, as evidenced by the quality and logic of the writing and the presentation of the figures. These issues may compromise the integrity of the work. I would recommend a substantial revision of the manuscript to improve the clarity of the writing, incorporate more experiments, and better define the goals of the study.

      Major Points:

      (1) Quality of Scientific Writing: The current draft does not meet the expected standards. Key issues include:

      i. Mathematical and Implementation Details: The manuscript lacks comprehensive mathematical descriptions and implementation details for the plasticity models (LTP/LTD/Meta) and the SPN model. Given the complexity of the biophysically detailed multicompartment model and the associated learning rules, the inclusion of only nine abstract equations (Eq. 1-9) in the Methods section is insufficient. I was surprised to find no supplementary material providing these crucial details. What parameters were used for the SPN model? What are the mathematical specifics for the extra-synaptic NMDA receptors utilized in this study? For instance, Eq. 3 references [Ca2+]-does this refer to calcium ions influenced by extra-synaptic NMDARs, or does it apply to other standard NMDARs? I also suggest the authors provide pseudocodes for the entire learning process to further clarify the learning rules.

      ii. Figure quality. The authors seem not to carefully typeset the images, resulting in overcrowding and varying font sizes in the figures. Some of the fonts are too small and hard to read. The text in many of the diagrams is confusing. For example, in Panel A of Figure 3, two flattened images are combined, leading to small, distorted font sizes. In Panels C and D of Figure 7, the inconsistent use of terminology such as "kernels" further complicates the clarity of the presentation. I recommend that the authors thoroughly review all figures and accompanying text to ensure they meet the expected standards of clarity and quality.

      iii. Writing clarity. The manuscript often includes excessive and irrelevant details, particularly in the mathematical discussions. On page 24, within the "Metaplasticity" section, the authors introduce the biological background to support the proposed metaplasticity equation (Eq. 5). However, much of this biological detail is hypothesized rather than experimentally verified. For instance, the claim that "a pause in dopamine triggers a shift towards higher calcium concentrations while a peak in dopamine pushes the LTP kernel in the opposite direction" lacks cited experimental evidence. If evidence exists, it should be clearly referenced; otherwise, these assertions should be presented as theoretical hypotheses. Generally, Eq. 5 and related discussions should be described more concisely, with only a loose connection to dopamine effects until more experimental findings are available.

      (2) Goals of the Study: The authors need to clearly define the primary objective of their research. Is it to showcase the computational advantages of the local learning rule, or to elucidate biological functions?

      i. Computational Advantage: If the intent is to demonstrate computational advantages, the current experimental results appear inadequate. The learning rule introduced in this work can only solve for four features, whereas previous research (e.g., Bicknell and Hausser, 2021) has shown capability with over 100 features. It is crucial for the authors to extend their demonstrations to prove that their learning rule can handle more than just three features. Furthermore, the requirement to fine-tune the midpoint of the synapse function indicates that the rule modifies the "activation function" of the synapses, as opposed to merely adjusting synaptic weights. In machine learning, modifying weights directly is typically more efficient than altering activation functions during learning tasks. This might account for why the current learning rule is restricted to a limited number of tasks. The authors should critically evaluate whether the proposed local learning rule, including meta-plasticity, actually offers any computational advantage. This evaluation is essential to understand the practical implications and effectiveness of the proposed learning rule.

      ii. Biological Significance: If the goal is to interpret biological functions, the authors should dig deeper into the model behaviors to uncover their biological significance. This exploration should aim to link the observed computational features of the model more directly with biological mechanisms and outcomes.

    1. Reviewer #2 (Public Review):

      The authors investigate the properties of the transcriptional co-activator Taiman in regulating tissue growth. In previously published work they had shown that cells that overexpress Tiaman in the pupal wing can cause the death of thoracic cells adjacent to the wing tip to die and thus allow the wing to invade the thorax. This was mediated by the secretion of Spz ligands. Here, they investigate the properties of cells that are homozygous for a hypomorphic allele of taiman (tai). They show that homozygous mutant clones are much smaller than their wild-type twin spots and that cells in the clones are dying by apoptosis which is inferred from elevated levels of anti-Dcp1 staining (Figure 1).

      By generating clones during eye development, the authors screen for dominant modifiers that increase the representation of homozygous tai tissue in the adult eye (Figure 2). They find that reducing the levels of hid, the entire rpr/hid/grim locus and Apc (and/or Apc2) each increase the representation of tai clones. They then show that the survival of tissue to the adult stage correlates with the size of lones in the third-instar larval wing disc (Figure 3). The rest of the study derives from the modification of the phenotype by Apc and investigates the interaction between Wnt signaling and tai clone survival.

      The authors then investigate interactions between tai and the wingless (wg) pathway. First, they show that increasing tai expression increases the expression of a wg reporter (nkd-lacZ) while reducing tai levels decreases its expression (Figure 4) indicating that wg signaling is likely reduced when tai levels are decreased. This finding is strengthened by examining wg-lacZ expression since the expression of this reporter is normally restricted to the D/V boundary in the wing disc by feedback inhibition via Wg signaling. Expression of the reporter is increased when tai expression is reduced and decreased when tai expression is increased (Figure 5).

      The authors then look at Wg protein away from the DV boundary. They find increased levels when tai expression is increased and decreased levels when tai is decreased. They conclude that tai activity increased Wg protein in cells (Figure 6). They suggest that this could be the result of the regulation of expression of Dally-like protein (Dlp). Consistent with this idea, increasing tai expression increases Dlp levels, and decreasing tai decreases Dlp levels (Figure 7). They then show that increasing Dlp levels when tai is reduced increases Wg levels which presumably means that Dlp is epistatic to tai. Puzzlingly, increasing both tai and Dlp decreases Wg.

      The authors then examine the effect of reducing Dlp in the cells that secrete Wg. They find that increasing tai results in the diffusion of Wg further from its source while reducing tai reduces its spread (Figure 8). They then show that in clones with reduced tai, there is increased cytoplasmic Dlp (Figure 9). They therefore propose that tai clones fail to survive because they do not secrete enough Dlp which results in reduced capture of the Wg for those cells and hence decreased Wg signaling.

      Evaluation

      While the authors present good evidence in support of most of their conclusions, there are alternative explanations in many cases that have not been excluded.

      From the results in Figure 1 (and Figure 3), the authors conclude that "The data indicate the existence of an extracellular competition mechanism that allows normal tai[wt] cells to kill tai[k15101] neighbors" (line 127). However, the experiments have been done with a single allele, and these experiments do not exclude the possibility that there is another mutation on the same chromosome arm that is responsible for the observed phenotype. Since the authors have a UAS-tai stock, they could strengthen their results using a MARCM experiment where they could test whether the expression of UAS-tai rescues the elimination of tai mutant clones. Alternatively, they could use a second (independent) allele to demonstrate that the phenotype can be attributed to a reduction in tai activity.

      By screening for dominant modifiers of a phenotype one would not expect to identify all interacting genes - only those that are haploinsufficient in this situation. The authors have screened a total of 21 chromosomes for modification and have not really explained which alleles are nulls and which are hypomorphs. The nature of each of the alleles screened needs to be explained better. Also, the absence of a dominant modification does not necessarily exclude a function of that gene or pathway in the process. This is especially relevant for the Spz/Toll pathway which the authors have previously implicated in the ability of tai-overexpressing cells to kill wild-type cells. The most important discovery from this screen is the modification by the Apc alleles. This part of the paper would be strengthened by testing for modification by other components of the Wingless pathway. The authors show modification by Apc[MI01007] and the double mutant Apc[Q8] Apc2[N175A]. Without showing the Apc[Q8] and Apc2[N175A] alleles separately, it is hard to know if the effect of the double mutant is due to Apc, Apc2,` or the combination.

      RNAi of tai seems to block the formation of the Wg gradient. If so, one might expect a reduction in wing size. Indeed, this could explain why the wings of tai/Df flies are smaller. The authors mention briefly that the posterior compartment size is reduced when tai-RNAi is expressed in that compartment. However, this observation merits more emphasis since it could explain why tai/Df flies are smaller (Are their wings smaller?).

      In Figure 7, the authors show the effect of manipulating Tai levels alone or in combination with increasing Dlp levels. However, they do not include images of Wg protein distribution upon increasing Dlp levels alone.

      In Figure 8, there is more Wg protein both at the DV boundary and spreading when tai is overexpressed in the source cells using bbg-Gal4. However, in an earlier experiment (Figure 5C) they show that the wg-lacZ reporter is downregulated at the DV boundary when tai is overexpressed using en-Gal4. They therefore conclude that wg is not transcriptionally upregulated but is, instead secreted at higher levels when tai is expressed in the source cells. Wg protein is reduced in the DV stripe with tai is overexpressed using the en-Gal4 driver (Figure 6B') and is increased at the same location when tai is overexpressed with the bbg-Gal4 driver. (Figure 8) I don't know how to reconcile these observations.

      In Figure 9, the tai-low clones have elevated levels of Dlp. How can this be reconciled with the tai-RNAi knockdown shown in Figure 7C' where reducing tai levels causes a strong reduction in Dlp levels?

    1. Reviewer #2 (Public Review):

      Summary:

      This manuscript uses cell lines representative of germ line cells, somatic cells and pluripotent cells to address the question of how the endocrine disrupting compound BPS affects these various cells with respect to gene expression and DNA methylation. They find a relationship between the presence of estrogen receptor gene expression and the number of DNA methylation and gene expression changes. Notably, PGCLCs do not express estrogen receptors and although they do have fewer changes, changes are nevertheless detected, suggesting a nonconical pathway for BPS-induced perturbations. Additionally, there was a significant increase in the occurrence of BPS-induced epimutations near EREs in somatic and pluripotent cell types compared to germ cells. Epimutations in the somatic and pluripotent cell types were predominantly in enhancer regions whereas that in the germ cell type was predominantly in gene promoters.

      Strengths:

      The strengths of the paper include the use of various cell types to address sensitivity of the lineages to BPS as well as the observed relationship between the presence of estrogen receptors and changes in gene expression and DNA methylation.

      Weaknesses:

      The weakness includes the fact that exposures are more complicated in a whole organism than in an isolated cell line.

    1. Reviewer #2 (Public Review):

      Summary:

      Scx is a well-established marker for tenocytes, but the expression in myogenic-lineage cells was unexplored. In this study, the authors performed lineage-trace and scRNA-seq analyses and demonstrated that Scx is expressed in activated SCs. Further, the authors showed that Scx is essential for muscle regeneration using conditional KO mice and identified the target genes of Scx in myogenic cells, which differ from those of tendons.

      Strengths:

      Sometimes, lineage-trace experiments cause mis-expression and do not reflect the endogenous expression of the target gene. In this study, the authors carefully analyzed the unexpected expression of Scx in myogenic cells using some mouse lines and scRNA-seq data.

      Weaknesses:

      Scx protein expression has not been verified.

    1. Reviewer #2 (Public Review):

      This study compares the activity of neural populations in the primary and non-primary auditory cortex of ferrets while the animals actively behaved or passively listened to a sound discrimination task. Using a variety of methods, the authors convincingly show differential effects of task engagement on population neural activity in primary vs non-primary auditory cortex; notably that in the primary auditory cortex, task-engagement (1) improves discriminability for both task-relevant and non-task relevant dimensions, and (2) improves the alignment between covariability and sound discrimination axes; whereas in the non-primary auditory cortex, task-engagement (1) improves discriminability for only task-relevant dimensions, and (2) does not affect the alignment between covariability and sound discrimination axes. They additionally show that task-engagement changes in gain can account for the selectivity noted in the discriminability of non-primary auditory neurons. They also admirably attempt to isolate task-engagement from arousal fluctuations, by using fluctuations in pupil size as a proxy for physiological arousal. This is a well-carried out study with thoughtful analyses which in large part achieves its aims to evaluate how task-engagement changes neural activity across multiple auditory regions . As with all work, there are several caveats or areas for future study/analysis. First, the sounds used here (tones, and narrow-band noise) are relatively simple sounds; previous work suggests that exactly what activity is observed within each region (e.g., sensory only, decision-related, etc) may depend in part upon what stimuli are used. Therefore, while the current study adds importance to the literature, future work may consider the use of more varied stimuli. Second, the animals here were engaged in a behavioral task; but apart from an initial calculation of behavioral d', the task performance (and its effect on neural activity) is largely unaddressed.

    1. Reviewer #3 (Public Review):

      Summary:<br /> The study conducted by Pisanski et al investigates the role of the lateral parafacial area (pFL) in controlling active expiration. Stereotactic injections of bicuculline were utilized to map various pFL sites and their impact on respiration. The results indicate that injections at more rostral pFL locations induce the most robust changes in tidal volume, minute ventilation, and combined respiratory responses. The study indicates that the rostro-caudal organization of the pFL and its influence on breathing is not simple and uniform.

      Strengths:<br /> The data provide novel insights into the importance of rostral locations in controlling active expiration. The authors use innovative analytic methods to characterize the respiratory effects of bicuculline injections into various areas of the pFL.

      Weaknesses:<br /> Bicuculline injections increase the excitability of neurons. Aside of blocking GABA receptors, bicuculline also inhibits calcium-activated potassium currents and potentiates NMDA currents, thus insights into the role of GABAergic inhibition are limited.<br /> Increasing the excitability of neurons provides little insights into the activity pattern and function of the activated neurons. Without recording from the activated neurons, it is impossible to know whether an effect on active expiration or any other respiratory phase is caused by bicuculline acting on rhythmogenic neurons or tonic neurons that modulate respiration. While this approach is inappropriate to study the functional extent of the conditional "oscillator" for active expiration, it still provides valuable insights into this region's complex role in controlling breathing .

    1. Reviewer #2 (Public Review):

      This article describes a novel mechanism of host defense in the gut of Drosophila larvae. Pathogenic bacteria trigger the activation of a valve that blocks them in the anterior midgut where they are subjected to the action of antimicrobial peptides. In contrast, beneficial symbiotic bacteria do not activate the contraction of this sphincter, and can access the posterior midgut, a compartment more favorable to bacterial growth.

      Strengths:

      The authors decipher the underlying mechanism of sphincter contraction, revealing that ROS production by Duox activates the release of DH31 by enteroendocrine cells that stimulate visceral muscle contractions. The use of mutations affecting the Imd pathway or lacking antimicrobial peptides reveals their contribution to pathogen elimination in the anterior midgut.

      Weaknesses:

      - The mechanism allowing the discrimination between commensal and pathogenic bacteria remains unclear.

      - The use of only two pathogens and one symbiotic species may not be sufficient to draw a conclusion on the difference in treatment between pathogenic and symbiotic species.

      - We can also wonder how the process of sphincter contraction is affected by the procedure used in this study, where larvae are starved. Does the sphincter contraction occur in continuous feeding conditions? Since larvae are continuously feeding, is this process physiologically relevant?

    1. Reviewer #2 (Public Review):

      In the present study, Boffi et al. investigate the manner in which the dorsal cortex of the of the inferior colliculus (DCIC), an auditory midbrain area, encodes sound location azimuth in awake, passively listening mice. By employing volumetric calcium imaging (scanned temporal focusing or s-TeFo), complemented with high-density electrode electrophysiological recordings (neuropixels probes), they show that sound-evoked responses are exquisitely noisy, with only a small portion of neurons (units) exhibiting spatial sensitivity. Nevertheless, a naïve Bayesian classifier was able to predict the presented azimuth based on the responses from small populations of these spatially sensitive units. A portion of the spatial information was provided by correlated trial-to-trial response variability between individual units (noise correlations). The study presents a novel characterization of spatial auditory coding in a non-canonical structure, representing a noteworthy contribution specifically to the auditory field and generally to systems neuroscience, due to its implementation of state-of-the-art techniques in an experimentally challenging brain region. However, nuances in the calcium imaging dataset and the naïve Bayesian classifier warrant caution when interpreting some of the results.

      Strengths:<br /> The primary strength of the study lies in its methodological achievements, which allowed the authors to collect a comprehensive and novel dataset. While the DCIC is a dorsal structure, it extends up to a millimetre in depth, making it optically challenging to access in its entirety. It is also more highly myelinated and vascularised compared to e.g., the cerebral cortex, compounding the problem. The authors successfully overcame these challenges and present an impressive volumetric calcium imaging dataset. Furthermore, they corroborated this dataset with electrophysiological recordings, which produced overlapping results. This methodological combination ameliorates the natural concerns that arise from inferring neuronal activity from calcium signals alone, which are in essence an indirect measurement thereof.

      Another strength of the study is its interdisciplinary relevance. For the auditory field, it represents a significant contribution to the question of how auditory space is represented in the mammalian brain. "Space" per se is not mapped onto the basilar membrane of the cochlea and must be computed entirely within the brain. For azimuth, this requires the comparison between miniscule differences between the timing and intensity of sounds arriving at each ear. It is now generally thought that azimuth is initially encoded in two, opposing hemispheric channels, but the extent to which this initial arrangement is maintained throughout the auditory system remains an open question. The authors observe only a slight contralateral bias in their data, suggesting that sound source azimuth in the DCIC is encoded in a more nuanced manner compared to earlier processing stages of the auditory hindbrain. This is interesting, because it is also known to be an auditory structure to receive more descending inputs from the cortex.

      Systems neuroscience continues to strive for the perfection of imaging novel, less accessible brain regions. Volumetric calcium imaging is a promising emerging technique, allowing the simultaneous measurement of large populations of neurons in three dimensions. But this necessitates corroboration with other methods, such as electrophysiological recordings, which the authors achieve. The dataset moreover highlights the distinctive characteristics of neuronal auditory representations in the brain. Its signals can be exceptionally sparse and noisy, which provide an additional layer of complexity in the processing and analysis of such datasets. This will be undoubtedly useful for future studies of other less accessible structures with sparse responsiveness.

      Weaknesses:<br /> Although the primary finding that small populations of neurons carry enough spatial information for a naïve Bayesian classifier to reasonably decode the presented stimulus is not called into question, certain idiosyncrasies, in particular the calcium imaging dataset and model, complicate specific interpretations of the model output, and the readership is urged to interpret these aspects of the study's conclusions with caution.

      I remain in favour of volumetric calcium imaging as a suitable technique for the study, but the presently constrained spatial resolution is insufficient to unequivocally identify regions of interest as cell bodies (and are instead referred to as "units" akin to those of electrophysiological recordings). It remains possible that the imaging set is inadvertently influenced by non-somatic structures (including neuropil), which could report neuronal activity differently than cell bodies. Due to the lack of a comprehensive ground-truth comparison in this regard (which to my knowledge is impossible to achieve with current technology), it is difficult to imagine how many informative such units might have been missed because their signals were influenced by spurious, non-somatic signals, which could have subsequently misled the models. The authors reference the original Nature Methods article (Prevedel et al., 2016) throughout the manuscript, presumably in order to avoid having to repeat previously published experimental metrics. But the DCIC is neither the cortex nor hippocampus (for which the method was originally developed) and may not have the same light scattering properties (not to mention neuronal noise levels). Although the corroborative electrophysiology data largely eleviates these concerns for this particular study, the readership should be cognisant of such caveats, in particular those who are interested in implementing the technique for their own research.

      A related technical limitation of the calcium imaging dataset is the relatively low number of trials (14) given the inherently high level of noise (both neuronal and imaging). Volumetric calcium imaging, while offering a uniquely expansive field of view, requires relatively high average excitation laser power (in this case nearly 200 mW), a level of exposure the authors may have wanted to minimise by maintaining a low the number of repetitions, but I yield to them to explain. Calcium imaging is also inherently slow, requiring relatively long inter-stimulus intervals (in this case 5 s). This unfortunately renders any model designed to predict a stimulus (in this case sound azimuth) from particularly noisy population neuronal data like these as highly prone to overfitting, to which the authors correctly admit after a model trained on the entire raw dataset failed to perform significantly above chance level. This prompted them to feed the model only with data from neurons with the highest spatial sensitivity. This ultimately produced reasonable performance (and was implemented throughout the rest of the study), but it remains possible that if the model was fed with more repetitions of imaging data, its performance would have been more stable across the number of units used to train it. (All models trained with imaging data eventually failed to converge.) However, I also see these limitations as an opportunity to improve the technology further, which I reiterate will be generally important for volume imaging of other sparse or noisy calcium signals in the brain.

      Transitioning to the naïve Bayesian classifier itself, I first openly ask the authors to justify their choice of this specific model. There are countless types of classifiers for these data, each with their own pros and cons. Did they actually try other models (such as support vector machines), which ultimately failed? If so, these negative results (even if mentioned en passant) would be extremely valuable to the community, in my view. I ask this specifically because different methods assume correspondingly different statistical properties of the input data, and to my knowledge naïve Bayesian classifiers assume that predictors (neuronal responses) are assumed to be independent within a class (azimuth). As the authors show that noise correlations are informative in predicting azimuth, I wonder why they chose a model that doesn't take advantage of these statistical regularities. It could be because of technical considerations (they mention computing efficiency), but I am left generally uncertain about the specific logic that was used to guide the authors through their analytical journey.

      That aside, there remain other peculiarities in model performance that warrant further investigation. For example, what spurious features (or lack of informative features) in these additional units prevented the models of imaging data from converging? In an orthogonal question, did the most spatially sensitive units share any detectable tuning features? A different model trained with electrophysiology data in contrast did not collapse in the range of top-ranked units plotted. Did this model collapse at some point after adding enough units, and how well did that correlate with the model for the imaging data? How well did the form (and diversity) of the spatial tuning functions as recorded with electrophysiology resemble their calcium imaging counterparts? These fundamental questions could be addressed with more basic, but transparent analyses of the data (e.g., the diversity of spatial tuning functions of their recorded units across the population). Even if the model extracts features that are not obvious to the human eye in traditional visualisations, I would still find this interesting.

      Finally, the readership is encouraged to interpret certain statements by the authors in the current version conservatively. How the brain ultimately extracts spatial neuronal data for perception is anyone's guess, but it is important to remember that this study only shows that a naïve Bayesian classifier could decode this information, and it remains entirely unclear whether the brain does this as well. For example, the model is able to achieve a prediction error that corresponds to the psychophysical threshold in mice performing a discrimination task (~30 {degree sign}). Although this is an interesting coincidental observation, it does not mean that the two metrics are necessarily related. The authors correctly do not explicitly claim this, but the manner in which the prose flows may lead a non-expert into drawing that conclusion. Moreover, the concept of redundancy (of spatial information carried by units throughout the DCIC) is difficult for me to disentangle. One interpretation of this formulation could be that there are non-overlapping populations of neurons distributed across the DCIC that each could predict azimuth independently of each other, which is unlikely what the authors meant. If the authors meant generally that multiple neurons in the DCIC carry sufficient spatial information, then a single neuron would have been able to predict sound source azimuth, which was not the case. I have the feeling that they actually mean "complimentary", but I leave it to the authors to clarify my confusion, should they wish.

      In summary, the present study represents a significant body of work that contributes substantially to the field of spatial auditory coding and systems neuroscience. However, limitations of the imaging dataset and model as applied in the study muddles concrete conclusions about how the DCIC precisely encodes sound source azimuth and even more so to sound localisation in a behaving animal. Nevertheless, it presents a novel and unique dataset, which, regardless of secondary interpretation, corroborates the general notion that auditory space is encoded in an extraordinarily complex manner in the mammalian brain.

    1. Reviewer #2 (Public Review):

      Sharp wave ripples are transient oscillations occurring in the hippocampus that are thought to play an important role in organising temporal sequences during the reactivation of neuronal activity. This study addresses the mechanism by which these temporal sequences are generated in the CA3 region focusing on two different subtypes of pyramidal neurons, thorny and athorny. Using high-quality electrophysiological recordings from up to 8 pyramidal neurons at a time the authors measure the connectivity rates between these pyramidal cell subtypes in a large dataset of 348 cells. This is a significant achievement and provides important data. The most striking finding is how similar connection characteristics are between cell types. There are no differences in synaptic strength or failure rates and some small differences in connectivity rates and short-term plasticity. Using model simulations, the authors explore the implications of the differences in connectivity rates for the temporal specificity of pyramidal cell firing within sharp-wave ripple events. The simulations show that the experimentally observed connectivity rates may contribute to the previously observed temporal sequence of pyramidal cell firing during sharp wave ripples.

      The conclusions drawn from the simulations are not experimentally tested so remain theoretical. In the simple network model, the authors include basket cell and anti-SWR interneurons but the connectivity of these cell types is not measured experimentally and variations in interneuron parameters may also influence temporal specificity of firing. In addition, the influence of short-term plasticity measured in their experiments is not tested in the model. Interestingly, the experimental data reveal a large variability in many of the measured parameters. This may strongly influence the firing of pyramidal cells during SWRs but it is not represented within the model which uses the averaged data.

    1. Reviewer #2 (Public Review):

      Summary:

      This is an interesting manuscript that describes a series of molecular dynamics studies on the peptide transporter PepT2 (SLC15A2). They examine, in particular, the effect on the transport cycle of protonation of various charged amino acids within the protein. They then validate their conclusions by mutating two of the residues that they predict to be critical for transport in cell-based transport assays. The study suggests a series of protonation steps that are necessary for transport to occur in Petp2. Comparison with bacterial proteins from the same family show that while the overall architecture of the proteins and likely mechanism are similar, the residues involved in the mechanism may differ.

      Strengths:

      This is an interesting and rigorous study that uses various state of the art molecular dynamics techniques to dissect the transport cycle of PepT2 with nearly 1ms of sampling. It gives insight into the transport mechanism, investigating how protonation of selected residues can alter the energetic barriers between various states of the transport cycle. The authors have, in general, been very careful in their interpretation of the data.

      Weaknesses:

      Interestingly, they suggest that there is an additional protonation event that may take place as the protein goes from occluded to inward-facing (clear from Figure 8) but as the authors comment they have not identified this residue(s).

    1. Reviewer #2 (Public Review):

      Summary:

      This study presents a significant finding that enhances our understanding of spermatogenesis. TMC7 belongs to a family of transmembrane channel-like proteins (TMC1-8), primarily known for their role in the ear. Mutations to TMC1/2 are linked to deafness in humans and mice and were originally characterized as auditory mechanosensitive ion channels. However, the function of the other TMC family members remains poorly characterized. In this study, the authors begin to elucidate the function of TMC7 in acrosome biogenesis during spermatogenesis. Through analysis of transcriptomics datasets, they elevated levels of TMC7 in round spermatids in both mouse and human testis. They then generate Tmc7-/- mice and find that male mice exhibit smaller testes and complete infertility. Examination of different developmental stages reveals spermatogenesis defects, including with reduced sperm count, elongated spermatids and large vacuoles. Additionally, abnormal acrosome morphology are observed beginning at the early-stage Golgi phase, indicating TMC7's involvement in proacrosomal vesicle trafficking and fusion. They observed localization of TMC7 in the cis-Golgi and suggest that its presence is required for maintaining Golgi integrity, with Tmc7-/- leading to reduced intracellular Ca2+, elevated pH and increased ROS levels, likely resulting in spermatid apoptosis. Overall, the work delineates a new function of TMC7 in spermatogenesis and the authors propose that that its ion channel and/or scramblase activity is likely important for Golgi homeostasis. This work is of significant interest to the community and is of high quality.

      Strengths:

      The biggest strength of the paper is the phenotypic characterization of the TMC7-/- mouse model, which has clear acrosome biogenesis/spermatogenesis defects. This is the main claim of the paper and it is supported with the data that are presented.

      Weaknesses:

      It isn't clear whether TMC7 functions as an ion channel from the current data presented in this paper, but the authors are careful in their interpretation and present this merely as a hypothesis supporting this idea.

    1. Reviewer #2 (Public Review):

      Summary:

      This is an interesting paper that delves into the post-translational modifications of the yeast Srs2 helicase and proteins with which it interacts in coping with DNA damage. The authors use mutants in some interaction domains with RPA and Srs2 to argue for a model in which there is a balance between RPA binding to ssDNA and Srs2's removal of RPA. The idea that a checkpoint is being regulated is based on observing Rad53 and Rad9 phosphorylation (so there are the attributes of a checkpoint), but evidence of cell cycle arrest is lacking. The only apparent delay in the cell cycle is the re-entry into the second S phase (but it could be an exit from G2/M); but in any case, the wild-type cells enter the next cell cycle most rapidly. No direct measurement of RPA residence is presented.

      Strengths:

      Data concern viability assays in the presence of camptothecin and in the post-translational modifications of Srs2 and other proteins.

      Weaknesses:

      There are a couple of overriding questions about the results, which appear technically excellent. Clearly, there is an Srs2-dependent repair process here, in the presence of camptothecin, but is it a consequence of replication fork stalling or chromosome breakage? Is repair Rad51-dependent, and if so, is Srs2 displacing RPA or removing Rad51 or both? If RPA is removed quickly what takes its place, and will the removal of RPA result in lower DDC1-MEC1 signaling?

      Moreover, It is worth noting that in single-strand annealing, which is ostensibly Rad51 independent, a defect in completing repair and assuring viability is Srs2-dependent, but this defect is suppressed by deleting Rad51. Does deleting Rad51 have an effect here?

      Neither this paper nor the preceding one makes clear what really is the consequence of having a weaker-binding Rfa1 mutant. Is DSB repair altered? Neither CPT nor MMS are necessarily good substitutes for some true DSB assay.

      With camptothecin, in the absence of site-specific damage, it is difficult to test these questions directly. (Perhaps there is a way to assess the total amount of RPA bound, but ongoing replication may obscure such a measurement). It should be possible to assess how CPT treatment in various genetic backgrounds affects the duration of Mec1/Rad53-dependent checkpoint arrest, but more than a FACS profile would be required.

      It is also notable that MMS treatment does not seem to yield similar results (Fig. S1).

    1. Reviewer #3 (Public Review):

      The authors presented point light displays of human walkers to children (mean = 9 years) with and without ADHD to compare their biological motion perception abilities, and relate them to IQ, social responsiveness scale (SRS) scores and age. They report that children with ADHD were worse at all three biological motion tasks, but that those loading more heavily on local processing related to social interaction skills and global processing to age. The valuable and solid findings are informative for understanding this complex condition, as well as biological motion processing mechanisms in general. However, the correlations present a pattern that needs further examination in future studies because many of the differences between correlations are not significant.

      Strengths:

      The authors present differences between ADHD and TD children in biological motion processing, and this question has not received as much attention as equivalent processing capabilities in autism. They use a task that appears well controlled. They raise some interesting mechanistic possibilities for differences in local and global motion processing, which are distinctions worth exploring. The group differences will therefore be of interest to those studying ADHD, as well as other developmental conditions, and those examining biological motion processing mechanisms in general.

      Weaknesses:

      The data are not strong enough to support claims about differences between global and lobal processing wrt social communication skills and age. The mechanistic possibilities for why these abilities may dissociate in such a way are interesting, but the crucial tests of differences between correlations do not present a clear picture. Further empirical work would be needed to test this further. Specifics:

      The authors state frequently that it was the local BM task that related to social communication skills (SRS) and not the global tasks. However, the results section shows a correlation between SRS and all three tasks. The only difference is that when looking specifically within the ADHD group, the correlation is only significant for the local task. The supplementary materials demonstrate that tests of differences between correlations present an incomplete picture. Currently they have small samples for correlations, so this is unsurprising.

      Theoretical assumptions. The authors make some statements about local vs global biological motion processing that may have been made in previous studies, but would appear controversial and not definitive. E.g., that local BM processing does not improve with age.

    1. Reviewer #3 (Public Review):

      It is well established that there is extensive post-transcriptional gene regulation in nervous systems, including the fly brain. For example, dynamic regulation of hundreds of genes during photoreceptor development could only be observed at the level of translated mRNAs, but not the entire transcriptomes. The present study instead addresses the role of differential translational regulation between cell types (or rather classes: neurons and glia, as both are still highly heterogenous groups) in the adult fly brain. By performing bulk RNA-seq and Ribo-seq on the same lysates, the authors are able to compare translation efficiency (TE) of all transcripts between neurons and glia. Many genes display differential TE, but interestingly, they tend to be the genes that already show strong differences at their mRNA level. The most striking observation is the finding that neuronal transcripts in glia display increased ribosome stalling at their 5' UTR, and in particular at the start codons of short "upstream ORFs". This could suggest that glia specifically employ a mechanism to upregulate upstream ORF translation, enabling them to better suppress the expression of the genes that have them. And neuronal genes tend to have longer 5' UTRs, perhaps to facilitate this type of regulation.

      However, it is difficult to evaluate the functional significance of these differences because the authors provide only one follow-up experiment to their RNA-seq analysis. Venus expressed with the Rh1 UTR sequences may be displaying differential levels between glia and neurons, but I find this image (Fig. 5C) rather unconvincing to support that conclusion. There are no quantifications of colocalization, or even sample size information provided for this experiment. And if there is indeed a difference, it would still be difficult argue this is because of the 5' stalling phenomenon authors observe with Rh1, because they switched both the 5' and 3' UTRs.

      I also find it puzzling that the TE differences between the groups are mostly among the transcripts that are already strongly differentially expressed at the transcriptional level. The authors would like to frame this as a mechanism of 'contrast sharpening'; but it is unclear why that would be needed. Rh1, for instance, is not just differentially expressed between neurons and glia, but it is actually only expressed by a very specific neuronal type (photoreceptors). Thus it's not clear to me why the glia would need this 5' stalling mechanism to fully suppress Rh1 expression, while all the other neurons can apparently do so without it.

      Response to authors' revisions:

      The authors have addressed most of the technical points in their revised manuscript. However, it is still rather unclear whether this mechanism would have any significant impact on differential gene expression between cell types in vivo. Considering that it's mostly occurring on genes that are already strongly differentially transcribed, that doesn't appear very likely.

    1. Reviewer #2 (Public Review):

      Summary:

      In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that localization of USP8 to early endosomes are disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although that Rabex5 are accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

      Strengths:

      The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidences to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members in the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

      Weaknesses:

      - The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell that whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion.<br /> - The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient of both factors displayed similar defects in late endosomes/lysosomes. But the authors didn't confirm whether and/or to which extent that USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model.

    1. Reviewer #2 (Public Review):

      Summary:

      Golluscio et al. addresses one of the mechanisms of IKs (KCNQ1/KCNE1) channel upregulation by a polyunsaturated fatty acid (PUFA). PUFAs are known to upregulate KCNQ1 and KCNQ1/KCNE1 channels by two mechanisms: one shifts the voltage dependence to the negative direction, and the other increases the maximum conductance (Gmax). While the first mechanism is known to affect the voltage sensor equilibrium by charge effect, the second mechanism is less known. By applying the single-channel recordings and mutagenesis on the putative binding sites (most of them related to the selectivity filter), they concluded that the selectivity filter is stabilized to a conductive state by PUFA binding.

      Strengths:

      The manuscript employed single-channel recordings and directly assessed the behavior of the selectivity filter. The method is straightforward and convincing enough to support the claims.

      Weaknesses:

      Although the analysis using selectivity filter mutants supports the hypothesis that PUFA binding stabilizes the conducting state of the filter, it may be somewhat speculative how PUFAs bind to the KCNQ1 channel in the presence of KCNE1.

    1. Reviewer #2 (Public Review):

      Summary:

      The primary goal of this study was to identify the transport pathway that is responsible for the release of dietary citrate from enterocytes into blood across the basolateral membrane.

      Strengths:

      The transport pathway responsible for the entry of dietary citrate into enterocytes was already known, but the transporter responsible for the second step remained unidentified. The studies presented in this manuscript identify SLC35G1 as the most likely transporter that mediates the release of absorbed citrate from intestinal cells into the serosal side. This fills an important gap in our current knowledge of the transcellular absorption of dietary citrate. The exclusive localization of the transporter in the basolateral membrane of human intestinal cells and the human intestinal cell line Caco-2 and the inhibition of the transporter function by chloride support this conclusion.

      Weaknesses:

      (i) The substrate specificity experiments have been done with relatively low concentrations of potential competing substrates, considering the relatively low affinity of the transporter for citrate. Given that NaDC1 brings in not only citrate as a divalent anion but also other divalent anions such as succinate, it is possible that SLC35G1 is responsible for the release of not only citrate but also other dicarboxylates. But the substrate specificity studies show that the dicarboxylates tested did not compete with citrate, meaning that SLc35G1 is selective for the citrate (2-), but this conclusion might be flawed because of the low concentration of the competing substrates used in the experiment.

      (ii) The authors have used MDCK cells for assessment of the transcellular transfer of citrate via SLC35G1, but it is not clear whether this cell line expresses NaDC1 in the apical membrane as the enterocytes do. Even though the authors expressed SLC35G1 ectopically in MDCK cells and showed that the transporter localizes to the basolateral membrane, the question as to how citrate actually enters the apical membrane for SLC35G1 in the other membrane to work remains unanswered.

      (iii) There is one other transporter that has already been identified for the efflux of citrate in some cell types in the literature (SLC62A1, PLoS Genetics; 10.1371/journal.pgen.1008884), but no mention of this transporter has been made in the current manuscript.

    1. Reviewer #2 (Public Review):

      Summary:

      This work is of great significance in revealing the regulatory mechanisms of pathogenic fungi in toxin production, pathogenicity, and in its prevention and pollution control. Overall, this is generally an excellent manuscript.

      Strengths:

      The data in this manuscript is robust and the experiments conducted are appropriate.

      Weaknesses:

      (1) The authors found that SntB played key roles in oxidative stress response of A. flavus by ChIP-seq and RNA sequencing. To confirm the role of SntB in oxidative stress, authors have better to measure the ROS levels in the ΔsntB and WT strains, besides the ΔcatC strain.<br /> (2) Why the authors only studied the function of catC among the 7 genes related to oxidative response listed in Table S14.

    1. Reviewer #2 (Public Review):

      In this manuscript, the authors first developed a new small molecular inhibitor that could target specifically the M1 metalloproteases of both important malaria parasite species Plasmodium falciparum and P. vivax. This was done by a chemical modification of a previously developed molecule that targets PfM1 as well as PfM17 and possibly other Plasmodial metalloproteases. After the successful chemical synthesis, the authors showed that the derived inhibitor, named MIPS2673, has a strong antiparasitic activity with IC50 342 nM and it is highly specific for M1. With this in mind, the authors first carried out two large-scale proteomics to confirm the MIPS2673 interaction with PfM1 in the context of the total P. falciparum protein lysate. This was done first by using thermal shift profiling and subsequently limited proteolysis. While the first demonstrated overall interaction, the latter (limited proteolysis) could map more specifically the site of MIPS2673-PfM1 interaction, presumably the active site. Subsequent metabolomics analysis showed that MIPS2673 cytotoxic inhibitory effect leads to the accumulation of short peptides many of which originate from hemoglobin. Based on that the authors argue that the MIPS2673 mode of action (MOA) involves inhibition of hemoglobin digestion that in turn inhibits the parasite growth and development.

      Comments on the revised version:

      The authors addressed all my comments from the previous round of reviews.

    1. Reviewer #2 (Public Review):

      Summary:

      Bone resorption by osteoclasts plays an important role in bone modeling and homeostasis. The multinucleated mature osteoclasts have higher bone-resorbing capacity than their mononuclear precursors. The previous work by authors has identified that increased cell-surface level of La protein promotes the fusion of mononuclear osteoclast precursor cells to form fully active multinucleated osteoclasts. In the present study, the authors further provided convincing data obtained from cellular and biochemical experiments to demonstrate that the nuclear-localized La protein where it regulates RNA metabolism was oxidized by redox signaling during osteoclast differentiation and the modified La protein was translocated to the osteoclast surface where it associated with other proteins and phospholipids to trigger cell-cell fusion process. The work provides novel mechanistic insights into osteoclast biology and provides a potential therapeutic target to suppress excessive bone resorption in metabolic bone diseases such as osteoporosis and arthritis.

      Strengths:

      Increased intracellular ROS induced by osteoclast differentiation cytokine RANKL has been widely studied in enhancing RANKL signaling during osteoclast differentiation. The work provides novel evidence that redox signaling can post-translationally modify proteins to alter the translocation and functions of critical regulators in the late stage of osteoclastogenesis. The results and conclusions are mostly supported by the convincing cellular and biochemical assays,

      Weaknesses:

      The lack of in vivo studies in animal models of bone diseases such as postmenopausal osteoporosis, inflammatory arthritis, and osteoarthritis reduces the translational potential of this work.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors studied the sequence determinants of C-terminal tags that govern protein degradation in bacteria. They introduce a new strategy to determine degron sequences: Detox (Degron Enrichment by toxin). This unbiased approach links degron efficiency to cell growth as degrons are C-terminally fused to the toxin VapC, which inhibits protein translation. Selecting for bacterial growth and thus toxin degradation enabled the identification of potent degron derived from a randomized library of pentapeptides. Remarkably, most degrons show sequence similarity to the SsrA-tag, which is fused to incomplete polypeptides at stalled ribosomes by the tmRNA-tagging system. These findings underline the extraordinary efficiency of the SsrA-tag and the ClpXP protease in removing incomplete polypeptides and demonstrate that most proteins are spared from degradation by harboring different C-termini. The introduced method will be highly useful to determine degron sequences in other positions and other bacterial species.

      Strengths:

      The work introduces an innovative and powerful strategy to identify degron sequences in bacteria. The study is well-controlled and results have been thoroughly analyzed. It will now become important to broaden the technology, making it also accessible for more complex degrons.

      Weaknesses:

      The approach is efficient in identifying strong degron sequences that are predominantly recognized by the ClpXP protease. The sequence specificity of other proteolytic systems, however, is not efficiently addressed, pointing to a potential limitation of this technology. The GS-rich linker sequence connecting the degron and the toxin might also impact proteolysis and thus outcome.

    1. Reviewer #2 (Public Review):

      Summary:

      Shimin Wang et al. investigated the role of Sertoli cells in mediating spermatogenesis disorders in non-obstructive azoospermia (NOA) through stage-specific communications. The authors utilized scRNA-seq and scATAC-seq to analyze the molecular and epigenetic profiles of germ cells and Sertoli cells at different stages of spermatogenesis.

      Strengths:

      By understanding the gene expression patterns and chromatin accessibility changes in Sertoli cells, the authors sought to uncover key regulatory mechanisms underlying male infertility and identify potential targets for therapeutic interventions. They emphasized that the absence of the SC3 subtype would be a major factor contributing to NOA.

      Weaknesses:

      Although the authors used cutting-edge techniques to support their arguments, it is difficult to find conceptual and scientific advances compared to Zeng S et al.'s paper (Zeng S, Chen L, Liu X, Tang H, Wu H, and Liu C (2023) Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia. Front. Endocrinol. 14:1138386.). Overall, the authors need to improve their manuscript to demonstrate the novelty of their findings in a more logical way.

    1. Reviewer #2 (Public Review):

      Summary:

      In the manuscript "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans", Xu and colleagues examine the role of cholinergic signaling by C. elegans motor neurons in modulating lifespan. The authors show that manipulating motor neuronal activity using genetic techniques can be beneficial or detrimental to lifespan, depending on when motor neuron activity is modulated.

      Strengths:

      A large body of data showing the effects of knockdown of cholinergic receptors and neurotransmitters on lifespan is presented. This would be of value to the community.

      Weaknesses:

      However, the studies are incomplete. More rigorous approaches would be needed to support the key conclusions, and substantiate the main findings and pathway components.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors explored the importance of data quality and representation for ligand-based virtual screening approaches. I believe the results could be of potential benefit to the drug discovery community, especially to those scientists working in the field of machine learning applied to drug research. The in silico design is comprehensive and adequate for the proposed comparisons.

      This manuscript by Chong A. et al describes that it is not necessary to resort to the use of sophisticated deep learning algorithms for virtual screening, since based on their results considering conventional ML may perform exceptionally well if fed by the right data and molecular representations.

      The article is interesting and well-written. The overview of the field and the warning about dataset composition are very well thought-out and should be of interest to a broad segment of the AI ​​in drug discovery readership. This article further highlights some of the considerations that need to be taken into consideration for the implementation of data-centric AI for computer-aided drug design methods.

      Strengths:

      This study contributes significantly to the field of machine learning and data curation in drug discovery. The paper is, in general, well-written and structured. However, in my opinion, there are some suggestions regarding certain aspects of the data analyses.

      Weaknesses:

      The conclusions drawn in the study are based on the analysis of a single dataset, and I am not sure they can be generalized. Therefore, in my opinion, the conclusions are only partially supported by the data. To generalize the conclusions, it is imperative to conduct a benchmark with diverse datasets, for different molecular targets.<br /> The conclusion cannot be immediately extended to molecular descriptors or features different from the ones used in this study<br /> It is advisable to present statistical analyses to ascertain whether the observed differences in metrics hold statistical significance.

    1. Reviewer #2 (Public Review):

      Summary

      Patrícia Graça et al., examined the role of the putative oxidoreductase MftG in regeneration of redox cofactors from the mycofactocin family in Mycolicibacerium smegmatis. The authors show that the mftG is often co-encoded with genes from the mycofactocin synthesis pathway in M. smegmatis genomes. Using a mftG deletion mutant, the authors show that mftG is critical for growth when ethanol is the only available carbon source, and this phenotype can be complemented in trans. The authors demonstrate the ethanol associated growth defect is not due to ethanol induced cell death, but is likely a result of carbon starvation, which was supported by multiple lines of evidence (imaging, transcriptomics, ATP/ADP measurement and respirometry using whole cells and cell membranes). The authors next used LC-MS to show that the mftG deletion mutant has much lower oxidised mycofactocin (MFFT-8 vs MMFT-8H2) compared to WT, suggesting an impaired ability to regenerate myofactocin redox cofactors during ethanol metabolism. These striking results were further supported by mycofactocin oxidation assays after over-expression of MftG in the native host, but also with recombinantly produced partially purified MftG from E. coli. The results showed that MftG is able to partially oxidise mycofactocin species, finally respirometry measurements with M. smegmatis membrane preparations from WT and mftG mutant cells show that the activity of MftG is indispensable for coupling of mycofactocin electron transfer to the respiratory chain. Overall, I find this study to be comprehensive and the conclusions of the paper are well supported by multiple complementary lines of evidence that are clearly presented.

      Strengths

      The major strengths of the paper are that it is clearly written and presented and contains multiple, complementary lines of experimental evidence that support the hypothesis that MftG is involved in the regeneration of mycofactocin cofactors, and assists with coupling of electrons derived from ethanol metabolism to the aerobic respiratory chain. The data appear to support the the authors hypotheses.

      Weaknesses

      No major weaknesses were identified, only minor weaknesses mostly surrounding presentation of data in some figures.

    1. Reviewer #2 (Public Review):

      Summary: Blocking a weak base compound's protonation increased intracellular diffusion and fractional recovery in the cytoplasm, which may improve the intracellular availability and distribution of weakly basic, small molecule drugs and be impactful in future drug development.

      Strengths:

      1) The intracellular distribution of drugs and the chemical properties that drive their distribution are much needed in the literature. Thus, the idea behind this paper is of relevance.

      2) The study used common compounds that were relevant to others.

      3) Altering a compound's pKa value and measuring cytosolic diffusion rates certainly is inciteful on how weak base drugs and their relatively high pKa values affect distribution and pharmacokinetics. This particular experiment demonstrated relevance to drug targeting and drug development.

      4) The manuscript was fairly well written.

      Weaknesses:

      1) Small sample sizes. 2 acids and 1 neutral compound vs 6 weak bases (Figure 1).

      2) A comparison between the percentage of neutral and weak base drug accumulation in lysosomes would have helped indicate weak base ion trapping. Such a comparison would have strengthened this study.

      3) When cytosolic diffusion rates of compounds were measured, were the lysosomes extracted from the image using Imaris to determine a realistic cytosolic value? In real-time, lysosomes move through the cytosol at different rates. Because weak base drugs get trapped, it is likely the movement of a weak base in the lysosome being measured rather than the movement of a weak base itself throughout the cytosol. This was unclear in the methods. Please explain.

      4) Because weak base drugs can be protonated in the cytoplasm, the authors need to elaborate on why they thought that inhibiting lysosome accumulation of weak bases would increase cytosolic diffusion rates. Ion trapping is different than "micrometers per second" in the cytosol. Moreover, treating cells with sodium azide de-acidifies lysosomes and acidifies the cytosol; thus, more protons in the cytosol means more protonation of weak base drugs. The diffusion rates were slowed down in the presence of lysosome inhibition (Figure 7), which is more fitting of the story about blocking protonation increases diffusion rates, but in this case, increasing cytosolic protonation via lysosome de-acidification agents decreases diffusion rates. Please elaborate.

      A discussion of the likely impact:<br /> The manuscript certainly adds another dimension to the field of intracellular drug distribution, but the manuscript needs to be strengthened in its current form. Additional experiments need to be included, and there are clarifications in the manuscript that need to be addressed. Once these issues are resolved, then the manuscript, if the conclusions are further strengthened, is much needed and would be inciteful to drug development.

    1. Reviewer #2 (Public Review):

      Summary:

      Glioblastoma is a common primary brain cancer, that is difficult to treat and has a low survival rate. The lack of genetically tractable and immunocompetent vertebrate animal model has prevented discovery of new therapeutic targets and limited efforts for screening of pharmaceutical agents for the treatment of the disease. Here Weiss et al., express oncogenic variants frequently observed in human glioblastoma within zebrafish lacking the tumor suppressor TP53 to generate a patient-relevant in vivo model. The authors demonstrate that loss of TP53 and overexpression of EGFR, PI3KCA, and mScarlet (p53EPS) in neural progenitors and radial glia leads to visible fluorescent brain lesions in live zebrafish. The authors performed RNA expression analysis that uncovered a molecular signature consistent with human mesenchymal glioblastoma and identified gene expression patterns associated with inflammation. Live imaging revealed high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. To define functional roles for regulators of inflammation on specific immune-related responses during tumorigenesis, transient CRISPR/Cas9 gene targeting was used to disrupt interferon regulator factor proteins and showed Inflammation-associated irf7 and irf8 are required to inhibit p53EPS tumor formation. Further, experiments to deplete the macrophages using clodronate liposomes suggest that macrophages contribute to the suppression of tumor engraftment following transplantation. The authors' conclusions are supported by the data and the experiments are thoroughly controlled throughout. Taken together, these results provide new insights into the regulation of glioblastoma initiation and growth by the surrounding microenvironment and provide a novel in vivo platform for the discovery of new molecular mechanisms and testing of therapeutics.

      Strengths/Weaknesses:

      The authors convincingly show that co-injection of activated human EGFRviii, PI3KCAH1047R, and mScarlet into TP53 null zebrafish promotes formation of fluorescent brain lesions and glioblastoma-like tumor formation. The authors include histological characterization of the tumors, as well as quantifications of p-ERK and p-AKT staining to highlight increased activation of the MAPK/AKT signaling pathways in their tumor model.

      The authors use a transplantation assay to further test the tumorigenic potential of dissociated cells from glial-derived tumors in the context of specific manipulations of the tumour microenvironment.

      The authors nicely show high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. Quantification of the emergence of macrophages over time in relation to tumor initiation and growth is provided and supports the observations of tumor suppressive activity of the phagocytes. The authors also attempt to delineate if other leukocyte populations are involved and observe tumor formation without significant infiltration of neutrophils.

      The authors provide evidence for key genetic regulators of the local microenvironment, showing increased p53EPS tumor initiation following Ifr7 gene knock-down and loss of irf7 expression in the TME.

    1. Reviewer #2 (Public Review):

      Summary:

      Transmembrane signaling in plants is crucial for homeostasis. In this study, the authors set out to understand to what extent catalytic activity in the EFR tyrosine kinase is required in order to transmit a signal. This work was driven by mounting data that suggest many eukaryotic kinases do not rely on catalysis for signal transduction, relying instead on conformational switching to relay information. The crucial findings reported here involve the realisation that a kinase-inactive EFR can still activate (ie lead to downstream phosphorylation) of its partner protein BAK1. Using a convincing set of biochemical, mass spectrometric (HD-exchange) and in vivo assays, the team suggests a model in which EFR is likely phosphorylated in the canonical activation segment (where two Ser residues are present), which is sufficient to generate a conformation that can activate BAK1 through dimersation. A model is put forward involving C-helix positioning in BAK1, and the model is extended to other 'non-RD' kinases in Arabidopsis kinases that likely do not require kinase activity for signaling.

      Strengths:

      The work uses logical and well-controlled approaches throughout, and is clear and convincing in most areas, linking data from IPs, kinase assays (including clear 32P-based biochemistry), HD-MX data (from non-phosphorylated EFR) structural biology, oxidative burst data and infectivity assays. Repetitions and statistical analysis all appear appropriate.

      Overall, the work builds a convincing story and the discussion does a clear job of explaining the potential impact of these findings (and perhaps an explanation of why so many Arabidopsis kinases are 'pseudokinases', including XPS1 and XIIa6, where this is shown explicitly).

      Impact:

      The work is an important new step in the huge amount of follow-up work needed to examine how kinases and pseudokinases 'talk' to each other in (especially) the plant kingdom, where significant genetic expansions have occurred. The broader impact is that we might understand better how to manipulate signaling for the benefit of plants and mankind; as the authors suggest, their study is a natural progression both of their own work and the kingdom-wide study of the Kannan group.

    1. Reviewer #2 (Public Review):

      Diaphorina citri is the primary vector of Candidatus Liberibacter asiaticus (CLas), but the mechanism of how D. citri maintains a balance between lipid metabolism and increased fecundity after infection with CLas remains unknown. In their study, Li et al. presented convincing methodology and data to demonstrate that CLas exploits AKH/AKHR-miR-34-JH signaling to enhance D. citri lipid metabolism and fecundity, while simultaneously promoting CLas replication. These findings are both novel and valuable, not only have theoretical implications for expanding our understanding of the interaction between insect vectors and pathogenic microorganisms but also provide new targets for controlling D. citri and HLB in practical implications. The conclusions of this paper are well supported by data.

    1. Reviewer #2 (Public Review):

      Summary:

      This paper investigates a rare and severe brain disease called Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids (HDLS). The authors aimed to understand how mutations in the gene CSF-1R affect microglia, the resident immune cells in the brain, and which alterations and factors lead to the specific pathophysiology. To model the human brain with the pathophysiology of HDLS, they used the human-specific model system of induced pluripotent stem cell (iPSC)-derived forebrain organoids with integrated iPSC-derived microglia (iMicro) from patients with the HDLS-causing mutation and an isogenic cell line with the corrected genome. They found that iPSC-derived macrophages (iMac) with HDLS mutations showed changes in their response, including increased inflammation and altered metabolism. Additionally, they studied these iMacs in forebrain organoids, where they differentiate into iMicro, and showed transcriptional differences in isolated iMicro when carrying the HDLS mutation. In addition, the authors described the influence of the mutation within iMicro on the transcriptional level of neurons and neural progenitor cells (NPCs) in the organoid. They observed that the one mutation showed implications for impaired development of neurons, possibly contributing to the progression of the disease. Overall, this study provides valuable insights into the mechanisms underlying HDLS and emphasizes the importance of studying diseases like these with a suitable model system. These findings, while promising, represent only an initial step towards understanding HDLS and similar neurodegenerative diseases, and thus, their direct translation into new treatment options remains uncertain.

      Strengths:

      The strength of the work lies in the successful reprogramming of two HDLS patient-derived induced pluripotent stem cells (iPSCs) with different mutations, which is crucial for the study of HDLS using human forebrain organoid models. The use of corrected isogenic iPSC lines as controls increases the validity of the mutation-specific observations. In addition, the model effectively mimics HDLS, particularly concerning deficits in the frontal lobe, mirroring observations in the human brain. Obtaining iPSCs from patients with different CSF1R mutations is particularly valuable given the limitations of rodent and zebrafish models when studying adult-onset neurodegenerative diseases. The study also highlights significant metabolic changes associated with the CSF1R mutation, particularly in the HD2 mutant line, which is confirmed by the HD1 line. In addition, the work shows transcriptional upregulation of the proinflammatory cytokine IL-1beta in cells carrying the mutation, particularly when they phagocytose apoptotic cells, providing further insight into disease mechanisms.

      Weaknesses:

      Most of the points have been addressed in the revision, but some points remain (see below) and are well within the scope of the current manuscript in this reviewer's opinion.

      (1) The characterization of iMicros is incomplete, with limited protein-level analysis (e.g. validate RNA-seq data via flow cytometry, ELISA etc.).

      (2) Additionally, the claim of microglial-like morphology lacks adequate evidence, as the provided image is insufficient for such an assessment (also the newly provided Supp. Fig. 3C is insufficient and looks rather like background). Show single channels for each staining. Show examples for both cell lines.

      (3) RNA-seq experiments are still difficult to read. A combination of data from both lines into one big analysis would be advantageous. E.g. showing overlapping GO terms for both lines. What is common, what is different in both lines?

      (4) Statistical test information is missing in the legends.

    1. Reviewer #2 (Public Review):

      Summary:

      This work presents new genetic tools for enhanced Cre-mediated gene deletion and genetic lineage tracing. The authors optimise and generate mouse models that convert temporally controlled CreER or DreER activity to constitutive Cre expression, coupled with the expression of tdT reporter for the visualizing and tracing of gene-deleted cells. This was achieved by inserting a stop cassette into the coding region of Cre, splitting it into N- and C-terminal segments. Removal of the stop cassette by Cre-lox or Dre-rox recombination results in the generation of modified Cre that is shown to exhibit similar activity to native Cre. The authors further demonstrate efficient gene knockout in cells marked by the reporter using these tools, including intersectional genetic targeting of pericentral hepatocytes.

      Strengths:

      The new models offer several important advantages. They enable tightly controlled and highly effective genetic deletion of even alleles that are difficult to recombine. By coupling Cre expression to reporter expression, these models reliably report Cre-expressing i.e. gene-targeted cells, and circumvent false positives that can complicate analyses in genetic mutants relying on separate reporter alleles. Moreover, the combinatorial use of Dre/Cre permits intersectional genetic targeting, allowing for more precise fate mapping.

      Weaknesses:

      The scenario where the lines would demonstrate their full potential compared to existing models has not been tested. Mosaic genetics is increasingly recognized as a key methodology for assessing cell-autonomous gene functions. The challenge lies in performing such experiments, as low doses of tamoxifen needed for inducing mosaic gene deletion may not be sufficient to efficiently recombine multiple alleles in individual cells while at the same time accurately reporting gene deletion. Therefore, a demonstration of the efficient deletion of multiple floxed alleles in a mosaic fashion would be a valuable addition.

      In addition, a drawback of this line is the constitutive expression of Cre. When combined with the confetti line, the reporter cassette will continue flipping, potentially leading to misleading lineage tracing results. Constitutive expression of Cre is also associated with toxicity, as discussed by the authors in the introduction. These drawbacks should be acknowledged.

    1. Reviewer #2 (Public Review):

      In this study, the authors investigate how diverse bacterial species influence Orsay virus transmission and host susceptibility in C. elegans. They find that Ochrobactrum species increase infection rates, while Pseudomonas species decrease infection rates, and they identify regulators of quorum sensing and the gacA two-component system as genetic factors in the effects of Pseudomonas on infection. These findings provide important insights into the species-specific effects that bacteria can have on viral infection in C. elegans, and they may have relevance for the impact of bacterial species on viral infection in other systems. Overall the manuscript has high rigor. However, a few minor concerns are listed below.

      (1) The authors state that the amount of bacteria added to each plate was standardized by seeding plates with equivalent volumes of overnight culture. This approach does not account for differences in bacterial growth rate. A more rigorous approach would be to standardize based on OD600 measurements or CFU's. Alternatively, the authors could include bacterial growth curves to demonstrate that each strain/species has reached a similar growth phase (i.e. late log) at the time of plating, as bacterial physiology and virulence is dependent on the stage of growth. At the least, if it is not possible to perform these experiments, it would be useful to include a statement that potential differences in bacterial growth rate may influence their conclusions.

      (2) Line 314-315: The claim "We did not observe any potent effect on host susceptibility to infection by Orsay virus from any supernatant (Supp. Fig. 9)" is not fully supported by the data, as the data in Fig S9 only show pals-5p::GFP levels. To confirm that host susceptibility is not affected, the authors would also measure the viral infection rate and/or viral load. Otherwise, the authors should rephrase the conclusion to increase accuracy. For example, "We did not observe any potent effect on pals-5p::GFP activation upon Orsay virus infection when animals were exposed to bacterial culture supernatant".

      (3) The Ct values shown in Fig 3B-F should be normalized to a reference gene (i.e. Ct values for snb-1).

    1. Reviewer #3 (Public Review):

      The work by Ghasemahmad et al. has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the basolateral amygdala (BLA) while an animal listens to social vocalizations.

      Ghasemahmad et al. made changes to the manuscript that have significantly improved the work. In particular, the transparency in showing the underlying levels of Ach, DA, and 5HIAA is excellent. My previous concerns have been adequately addressed.

    1. Reviewer #2 (Public Review):

      Cerebellar diseases can manifest as various behavioral phenotypes, such as ataxia, dystonia, and tremor. In this study, van der Heijden and colleagues aim to understand whether these differing behavioral phenotypes are associated with disease-specific changes in the firing patterns of cerebellar output neurons in the cerebellar nuclei (CN). The authors effectively demonstrate that across different mouse models of cerebellar disease, there are distinct changes in the firing properties of CN neurons. They take a crucial step further by attempting to replicate disease-specific firing patterns in the cerebellar output neurons of healthy (control) mice using optogenetics. When Purkinje cells are stimulated in a manner that results in similar firing properties in CN neurons, the authors observe a variety of atypical behavioral responses, many of which align with the behavioral phenotypes observed in mouse models of the respective diseases.

      Overall, the primary results are quite convincing. Specifically, they show that (1) different mouse models of cerebellar disease exhibit different statistics of firing in CN neurons, and (2) driving CN neurons in a time-varying manner that mimics the statistics measured in disease models results in behavioral phenomena reminiscent of the disease states. These findings suggest that aberrant activity in the CN can originate from various sources (e.g., developmental circuit deficits, abnormal plasticity, insult), but ultimately, these changes are funneled through the CN neurons, whose firing rates are affected, and this, in turn, drives some portion of the aberrant behavior. This is a noteworthy observation that underscores the potential of targeting these output neurons in the treatment of cerebellar disease. Moreover, this manuscript provides valuable insights into the firing patterns associated with the most common cerebellar-dependent disease phenotypes.

      However, the applicability of the classifier for identifying mice cerebellar behavioral phenotypes directly from the spiking activity of neurons in the cerebellar nuclei remains this paper's weak point. Cross-validated performance of the model on a single mouse model of tremor is, for instance, only 54%. However, a benefit of this classifier is its overall simplicity; only three parameters are required to achieve average classifier performance of 76%. While more sophisticated models might provide improved classifier performance and enhanced generalization, such models would suffer from a lack of interpretability. This paper, therefore, represents a reasonable starting point for understanding the parameter space of cerebellar nuclei firing and its relationship to behavioral phenotypes during disease.

    1. Reviewer #2 (Public Review):

      Reward and punishment learning have long been seen as emerging from separate networks of frontal and subcortical areas, often studied separately. Nevertheless, both systems are complimentary and distributed representations of reward and punishments have been repeatedly observed within multiple areas. This raised the unsolved question of the possible mechanisms by which both systems might interact, which this manuscript went after. The authors skillfully leveraged intracranial recordings in epileptic patients performing a probabilistic learning task combined with model-based information theoretical analyses of gamma activities to reveal that information about reward and punishment was not only distributed across multiple prefrontal and insular regions, but that each system showed specific redundant interactions. The reward subsystem was characterized by redundant interactions between orbitofrontal and ventromedial prefrontal cortex, while the punishment subsystem relied on insular and dorsolateral redundant interactions. Finally, the authors revealed a way by which the two systems might interact, through synergistic interaction between ventromedial and dorsolateral prefrontal cortex.

      Here, the authors performed an excellent reanalysis of a unique dataset using innovative approaches, pushing our understanding on the interaction at play between prefrontal and insular cortex regions during learning. Importantly, the description of the methods and results is truly made accessible, making it an excellent resource to the community. The authors also carefully report individual subjects' data, which brings confidence in the reproducibility of their observations.

      This manuscript goes beyond what is classically performed using intracranial EEG dataset, by not only reporting where a given information, like reward and punishment prediction errors, is represented but also by characterizing the functional interactions that might underlie such representations. The authors highlight the distributed nature of frontal cortex representations and proposed new ways by which the information specifically flows between nodes. This work is well placed to unify our understanding of the complementarity and specificity of the reward and punishment learning systems.

    1. Reviewer #2 (Public Review):

      Summary:

      In the current manuscript, Tresenrider et al., present their recent study focusing on screening of small molecules to enhance the conversion from Müller cells (MG) to retina neurons induced by ectopic Ascl1 expression.

      Strengths:

      To analyze results from multiple treatment conditions in a single experiment, the authors employed a method called sci-Plex to perform scRNA-seq on mixed samples to investigate the effects of different durations of Ascl1 expression and screen for potential small molecules to promote reprogramming. Ultimately, they identified two compounds with intended activities on mouse retina. The findings may aid in future development of a cell replacement strategy for treating retinal degeneration.

      Weaknesses:

      The mechanistic insights are limited. Certain claims are confusing or superficial at this point.

    1. Reviewer #2 (Public Review):

      In this study, Leighton et al performed remarkable experiments by combining in-vivo patch-clamp recording with two-photon dendritic Ca2+ imaging. The voltage-clamp mode is a major improvement over the pioneer versions of this combinatorial experiment that had led to major breakthroughs in the neuroscience field for visualizing and understanding synaptic input activities in single cells in-vivo (sharp electrodes: Svoboda et al, Nature 1997, Helmchen et al, Nature Neurosci 1999; whole-cell current-clamp: Jia et al, Nature 2010, Chen et al, Nature 2011. I suggest that these papers would be cited). This is because in voltage-clamp mode, despite a full control of membrane voltage in-vivo is not realistic, is nevertheless most effective in preventing back-propagation action potentials, which would severely confound the measurement of individual synaptically-induced Ca2+ influx events. Furthermore, clamping the cell body at a strongly depolarized potential (here the authors did -30mV) also facilitates the detection of synaptically-induced Ca2+ influx. As a result, the authors successfully recorded high-quality Ca2+ imaging data that can be used for precise analysis. To date, even in view of the rapid progress of voltage-sensitive indicators and relevant imaging technologies in the recent years, this very old 'art' of combining single-cell electrophysiology and two-photon imaging (ordinary, raster-scanned, video-rate imaging) of Ca2+ signals still enable measurements of the best-level precision.

      On the other hand, the interpretation of data in this study is a bit narrow-minded and lacks a comprehensive picture. Some suggestions to improve the manuscript are as follows:

      (1) The authors made a segregation of 'spine synapse' and 'shaft synapse' based solely on the two-photon images in-vivo. However, caution shall be taken here, because the optical resolution under in-vivo imaging conditions like this cannot reliably tell apart whether a bright spot within or partially overlapping a segment of dendrite is a spine on top (or below) of it. Therefore, what the authors consider as a 'shaft synapse' (by detecting Ca2+ hotspots) has an unknown probability to be just a spine on top or below the dendrite. If there is other imaging data of higher axial resolution to validate or calibrate, the authors shall take some further considerations or analysis to check the consistency of their data, as the authors do need such a segregation between spine and shaft synapses to show how they evolve over the brain development stages.<br /> (2) The use of terminology 'bursts of spontaneous inputs' for describing voltage-clamp data seems improper. Conventionally, 'burst' refers to suprathreshold spike firing events, but here, the authors use 'burst' to refer to inward synaptic currents collected at the cell body. It is obvious that not every excitatory synaptic input (or ensemble of inputs) activation will lead to spike firing under naturalistic conditions, therefore, these two concepts are not equivalent. It is recommended to use 'barrage of inputs' instead of 'burst of inputs'. Imagine a full picture of the entire dendritic tree, the fact that the authors could always capture spontaneous Ca2+ events here and there within a few pieces of dendrites within an arbitrary field-of-view suggest that, the whole dendritic tree must have many more such events going on as a barrage while the author's patch electrode picks up the summed current flow from the whole dendritic tree.<br /> (3) Following the above issue, an analysis of the temporal correlation between synaptic (not segregating 'spine' or 'shaft') Ca2+ events and EPSCs is absent. Again, the authors drew arbitrary time windows to clump the events for statistical analysis. However, the demonstrated example data already show that the onset times of individual synaptic Ca2+ events do not necessarily align with the beginning of a 'barrage' inward current event.<br /> (4) The authors claim that "these observations indicate that the activity patterns investigated here are not or only slightly affected by low-level anesthesia". It would be nice to show some of the recordings in this work without any anesthesia to support this claim.<br /> (5) I suggest the authors should provide the number of cells and mice recorded in the figure legends.<br /> (6) Instead of showing only cartoon illustrations of dendrites in Figure 3-6, I suggest showing the two-photon images as well together with the cartoon.

      The authors have addressed most of my issues, but I miss the responses to my points 5 and 6. I have no additional comments.

    1. Reviewer #2 (Public Review):

      Summary:

      Shahshahani and colleagues used a combination of statistical modelling and whole-brain fMRI data in an attempt to separate the contributions of cortical and cerebellar regions in different cognitive contexts.

      Strengths:

      * The manuscript uses a sophisticated integration of statistical methods, cognitive neuroscience and systems neurobiology.<br /> * The authors use multiple statistical approaches to ensure robustness in their conclusions.<br /> * The consideration of the cerebellum as not a purely 'motor' structure is excellent and important.

      Weaknesses:

      * The assumption that cortical BOLD responses in cognitive tasks should be matched irrespective of cerebellar involvement does not cohere directly with the notion of 'forcing functions' introduced by Houk and Wise, suggesting the need for future work.

    1. Reviewer #2 (Public Review):

      In an image-computable model of speeded decision-making, the authors introduce and fit a combined CCN-EAM (a 'VAM') to flanker-task-like data. They show that the VAM can fit mean RTs and accuracies as well as the congruency effect that is present in the data, and subsequently analyze the VAM in terms of where in the network congruency effects arise.

      Overall, combining DNNs and EAMs appears to be a promising avenue to seriously model the visual system in decision-making tasks compared to the current practice in EAMs. Some variants have been proposed or used before (e.g., doi.org/10.1016/j.neuroimage.2017.12.078 , doi.org/10.1007/s42113-019-00042-1), but always in the context of using task-trained models, rather than models trained on behavioral data. However, I was surprised to read that the authors developed their model in the context of a conflict task, rather than a simpler perceptual decision-making task. Conflict effects in human behavior are particularly complex, and thereby, the authors set a high goal for themselves in terms of the to-be-explained human behavior. Unfortunately, the proposed VAM does not appear to provide a great account of conflict effects that are considered fundamental features of human behavior, like the shape of response time distributions, and specifically, delta plots (doi.org/10.1037/0096-1523.20.4.731). The authors argue that it is beyond the scope of the presented paper to analyze delta plots, but as these are central to studies of human conflict behavior, models that aim to explain conflict behavior will need to be able to fit and explain delta plots.

      Theories on conflict often suggest that negative/positive-trending delta plots arise through the relative timing of response activation related to relevant and irrelevant information. Accumulation for relevant and irrelevant information would, as a result, either start at different points in time or the rates vary over time. The current VAM, as a feedforward neural network model, does not appear to be able to capture such effects, and perhaps fundamentally not so: accumulation for each choice option is forced to start at the same time, and rates are a static output of the CNN.

      The proposed solution of fitting five separate VAMs (one for each of five RT quantiles) is not satisfactory: it does not explain how delta plots result from the model, for the same reason that fitting five evidence accumulation models (one per RT quantile) does not explain how response time distributions arise. If, for example, one would want to make a prediction about someone's response time and choice based on a given stimulus, one would first have to decide which of the five VAMs to use, which is circular. But more importantly, this way of fitting multiple models does not explain the latent mechanism that underlies the shape of the delta plots.

      As such, the extensive analyses on the VAM layers and the resulting conclusions that conflict effects arise due to changing representations across layers (e.g., "the selection of task-relevant information occurs through the orthogonalization of relevant and irrelevant representations") - while inspiring, they remain hard to weigh, as they are contingent on the assumption that the VAM can capture human behavior in the conflict task, which it struggles with. That said, the promise of combining CNNs and EAMs is clearly there. A way forward could be to either adjust the proposed model so that it can explain delta plots, which would potentially require temporal dynamics and time-varying evidence accumulation rates, or perhaps to start simpler and combine CCNs-EAMs that are able to fit more standard perceptual decision-making tasks without conflict effects.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors of this manuscript aim to develop a novel animal model to accurately simulate the retinal ischemic process in retinal artery occlusion (RAO). A unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mouse model was established using silicone wire embolization combined with carotid artery ligation. This manuscript provided data to show the changes in major classes of retinal neural cells and visual dysfunction following various durations of ischemia (30 minutes and 60 minutes) and reperfusion (3 days and 7 days) after UPOAO. Additionally, transcriptomics was utilized to investigate the transcriptional changes and elucidate changes in the pathophysiological process in the UPOAO model post-ischemia and reperfusion. Furthermore, the authors compared transcriptomic differences between the UPOAO model and other retinal ischemic-reperfusion models, including HIOP and UCCAO, and revealed unique pathological processes.

      Strengths:

      The UPOAO model represents a novel approach to studying retinal artery occlusion. The study is very comprehensive.

      Weaknesses:

      Some statements are incorrect and confusing. It would be helpful to review and clarify these to ensure accuracy and improve readability.

    1. Reviewer #2 (Public Review):

      Aybar-Torres and colleagues utilize common human STING alleles to dissect the mechanism of SAVI inflammatory disease. The authors demonstrate that these common alleles alleviate SAVI pathology in mice, and perhaps more importantly use the differing functionality of these alleles to provide insight into requirements of SAVI disease induction. Their findings suggest that it is residue A230 and/or Q293 that are required for SAVI induction, while the ability to induce an interferon-dependent inflammatory response is not. This is nicely exemplified by the AQ/SAVI mice that have an intact inflammatory response to STING activation, yet minimal disease progression. As both mutants seem to be resistant STING-dependent cell death, this manuscript also alludes to the importance of STING-dependent cell death, rather than STING-dependent inflammation, in the progression of SAVI pathology. While I have some concerns, I believe this manuscript makes some important connections between STING pathology mouse models and human genetics that would contribute to the field.

      Some points to consider:

      (1) While the CD4+ T cell counts from HAQ/SAVI and AQ/SAVI mice suggest that these T cells are protected from STING-dependent cell death, an assay that explores this more directly would strengthen the manuscript. This is also supported by Fig 2C, but I believe a strength of this manuscript is the comparison between the two alleles. Therefore, if possible, I would recommend the isolation of T cells from these mice and direct stimulation with diABZI or other STING agonist with a cell death readout.<br /> (2) Related to the above point - further exemplifying that the Q293 locus is essential to disease, even in human cells, would also strengthen the paper. It seems that CD4 T cell loss is a major component of human SAVI. While not completely necessary, repeating the THP1 cell death experiments from Fig 2 with a human T cell line would round out the study nicely.<br /> (3) While I found the myeloid cell counts and BMDM data interesting, I think some more context is needed to fully loop this data into the story. Is myeloid cell expansion exemplified by SAVI patients? Do we know if myeloid cells are the major contributors to the inflammation these patients experience? Why should the SAVI community care about the Q293 locus in myeloid cells?<br /> (4) The functional assays in Figure 4 are exciting and really connect the alleles to disease progression. To strengthen the manuscript and connect all the data, I would recommend additional readouts from these mice that address the inflammatory phenotype shown in vitro in Figure 5. For example, measuring cytokines from these mice via ELISA or perhaps even Western blots looking for NFkB or STING activation would be supportive of the story. This would also allow for some tissue specificity. I believe looking for evidence of inflammation and STING activation in the lungs of these mice, for example, would further connect the data to human SAVI pathology.

    1. Reviewer #2 (Public Review):

      Summary:

      This manuscript presents data demonstrating NopT's interaction with Nod Factor Receptors NFR1 and NFR5 and its impact on cell death inhibition and rhizobial infection. The identification of a truncated NopT variant in certain Sinorhizobium species adds an interesting dimension to the study. These data try to bridge the gaps between classical Nod-factor-dependent nodulation and T3SS NopT effector-dependent nodulation in legume-rhizobium symbiosis. Overall, the research provides interesting insights into the molecular mechanisms underlying symbiotic interactions between rhizobia and legumes.

      Strengths:

      The manuscript nicely demonstrates NopT's proteolytic cleavage of NFR5, regulated by NFR1 phosphorylation, promoting rhizobial infection in L. japonicus. Intriguingly, authors also identify a truncated NopT variant in certain Sinorhizobium species, maintaining NFR5 cleavage but lacking NFR1 interaction. These findings bridge the T3SS effector with the classical Nod-factor-dependent nodulation pathway, offering novel insights into symbiotic interactions.

      Weaknesses:

      (1) In the previous study, when transiently expressed NopT alone in Nicotiana tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. However, this phenotype was not observed when expressing the same NopT in Nicotiana benthamiana (Figure 1A). Conversely, cell death and a hypersensitive reaction were observed in Figure S8. This raises questions about the suitability of the exogenous expression system for studying NopT proteolysis specificity.

      (2)NFR5 Loss-of-function mutants do not produce nodules in the presence of rhizobia in lotus roots, and overexpression of NFR1 and NFR5 produces spontaneous nodules. In this regard, if the direct proteolysis target of NopT is NFR5, one could expect the NGR234's infection will not be very successful because of the Native NopT's specific proteolysis function of NFR5 and NFR1. Conversely, in Figure 5, authors observed the different results.

      (3) In Figure 6E, the model illustrates how NopT digests NFR5 to regulate rhizobia infection. However, it raises the question of whether it is reasonable for NGR234 to produce an effector that restricts its own colonization in host plants.

      (4) The failure to generate stable transgenic plants expressing NopT in Lotus japonicus is surprising, considering the manuscript's claim that NopT specifically proteolyzes NFR5, a major player in the response to nodule symbiosis, without being essential for plant development.

    1. Reviewer #2 (Public Review):

      This paper analyzes the effect of axon de-myelination and re-myelination on action potential speed, and propagation failure. Next, the findings are then incorporated in a standard spiking ring attractor model of working memory.

      I think the results are not very surprising or solid and there are issues with method and presentation.<br /> The authors did many simulations with random parameters, then averaged the result, and found for instance that the Conduction Velocity drops in demyelination. It gives the reader little insight into what is really going on. My personal preference is for a well understood simple model rather than a poorly understood complex model. The link between the model outcome of WM and data remains qualitative and is further weakened by the existence of known other age-related effects in PFC circuits.

      Comments on revised version:

      The paper has improved in the revision, although I still think a reduced model would have been nice.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors introduce a simple Self Returning Excluded Volume (SR-EV) model to investigate the 3D organization of chromatin. This is a random walk with a probability to self-return accounting for the excluded volume effects. The authors use this method to study the statistical properties of chromatin organization in 3D. They compute contact probabilities, 3D distances, and packing properties of chromatin and compare them with a set of experimental data.

      Strengths:

      (1) Typically, to generate a polymer with excluded volume interactions, one needs to run long simulations with computationally expensive repulsive potentials like the Weeks-Chanlder-Anderson potential. However, here, instead of performing long simulations, the authors have devised a method where they can grow polymer, enabling quick generation of configurations.

      (2) Authors show that the chromatin configurations generated from their models do satisfy many of the experimentally known statistical properties of chromatin. Contact probability scalings and packing properties are comparable with Chromatin Scanning Transmission Electron Microscopy (ChromSTEM)  experimental data from some of the cell types.

      Weaknesses:

      This can only generate broad statistical distributions. This method cannot generate sequence-dependent effects, specific TAD structures, or compartments without a prior model for the folding parameter alpha. It cannot generate a 3D distance between specific sets of genes. This is an interesting soft-matter physics study. However, the output is only as good as the alpha value one provides as input.

    1. Reviewer #2 (Public Review):

      Summary:

      Generating biophysically detailed computational models that capture the characteristic physiological properties of biological neurons for diverse cell types is an important and difficult problem in computational neuroscience. One major challenge lies in determining the large number of parameters of such models, which are notoriously difficult to fit into experimental data. Thereby, the computational and energy costs can be significant. The study 'ElectroPhysiomeGAN: Generation of Biophysical Neuron Model Parameters from Recorded Electrophysiological Responses' by Kim et al. describes a computationally efficient approach for predicting model parameters of Hodgkin-Huxley neuron models using Generative Adversarial Networks (GANs) trained on simulation data. The method is applied to generate models for 9 non-spiking neurons in C. elegans based on electrophysiological recordings. While the generated models capture the responses of these neurons to some degree, they generally show significant deviations from the empirically observed responses in important features. While interesting, in its current form, the method has not been demonstrated to generate models that faithfully capture empirically observed responses.

      Strengths:

      The authors work on an important and difficult problem. A noteworthy strength of their approach is that once trained, the GANs can generate models from new empirical data with very little computational effort. The generated models reproduce the average voltage during current injections reasonably well.

      Weaknesses:

      Major 1: While the models generated with EP-GAN reproduce the average voltage during current injections reasonably well, the dynamics of the response are not well captured. For example, for the neuron labeled RIM (Figure 2), the most depolarized voltage traces show an initial 'overshoot' of depolarization, i.e. they depolarize strongly within the first few hundred milliseconds but then fall back to a less depolarized membrane potential. In contrast, the empirical recording shows no such overshoot. Similarly, for the neuron labeled AFD, all empirically recorded traces slowly ramp up over time. In contrast, the simulated traces are mostly flat. Furthermore, all empirical traces return to the pre-stimulus membrane potential, but many of the simulated voltage traces remain significantly depolarized, far outside of the ranges of empirically observed membrane potentials. While these deviations may appear small in the Root mean Square Error (RMSE), the only metric used in the study to assess the quality of the models, they likely indicate a large mismatch between the model and the electrophysiological properties of the biological neuron.

      Major 2: Other metrics than the RMSE should be incorporated to validate simulated responses against electrophysiological data. A common approach is to extract multiple biologically meaningful features from the voltage traces before, during and after the stimulus, and compare the simulated responses to the experimentally observed distribution of these features. Typically, a model is only accepted if all features fall within the empirically observed ranges (see e.g. https://doi.org/10.1371/journal.pcbi.1002107). However, based on the deviations in resting membrane potential and the return to the resting membrane potential alone, most if not all the models shown in this study would not be accepted.

      Major 3: Abstract and introduction imply that the 'ElectroPhysiome' refers to models that incorporate both the connectome and individual neuron physiology. However, the work presented in this study does not make use of any connectomics data. To make the claim that ElectroPhysiomeGAN can jointly capture both 'network interaction and cellular dynamics', the generated models would need to be evaluated for network inputs, for example by exposing them to naturalistic stimuli of synaptic inputs. It seems likely that dynamics that are currently poorly captured, like slow ramps, or the ability of the neuron to return to its resting membrane potential, will critically affect network computations.

    1. Reviewer #2 (Public Review):

      Summary:

      In this manuscript, the authors recorded activity in the posterior parietal cortex (PPC) of monkeys performing a perceptual decision-making task. The monkeys were first shown two choice dots of two different colors. Then, they saw a random dot motion stimulus. They had to learn to categorize the direction of motion as referring to either the right or left dot. However, the rule was based on the color of the dot and not its location. So, the red dot could either be to the right or left, but the rule itself remained the same. It is known from past work that PPC neurons would code the learned categorization. Here, the authors showed that the categorization signal depended on whether the executed saccade was in the same hemifield as the recorded PPC neuron or in the opposite one. That is, if a neuron categorized the two motion directions such that it responded stronger for one than the other, then this differential motion direction coding effect was amplified if the subsequent choice saccade was in the same hemifield. The authors then built a computational RNN to replicate the results and make further tests by simulated "lesions".

      Strengths:

      Linking the results to RNN simulations and simulated lesions.

      Weaknesses:

      Potential interpretational issues due to a lack of evidence on what happens at the time of the saccades.

    1. Reviewer #2 (Public Review):

      Summary:

      In short, the paper presents a theoretical framework that predicts how resources should be optimally distributed between receptors and optics in eyes.

      Strengths:

      The authors build on the principle of resource allocation within an organism and develop a formal theory for optimal distribution of resources within an eye between the receptor array and the optics. Because the two parts of eyes, receptor arrays and optics, share the same role of providing visual information to the animal it is possible to isolate these from resource allocation in the rest of the animal. This allows for a novel and powerful way of exploring the principles that govern eye design. By clever and thoughtful assumptions/constraints, the authors have built a formal theory of resource allocation between the receptor array and the optics for two major types of compound eye as well as for camera-type eyes. The theory is formalized with variables that are well characterized in a number of different animal eyes, resulting in testable predictions.

      The authors use the theory to explain a number of design features that depend on different optimal distribution of resources between the receptor array and the optics in different types of eyes. As an example, they successfully explain why eye regions with different spatial resolution should be built in different ways. They also explain differences between different types of eyes, such as long photoreceptors in apposition compound eyes and much shorter receptors in camera type eyes. The predictive power in the theory is impressive.

      To keep the number of parameters at a minimum, the theory was developed for two types of compound eye (neural superposition, and apposition) and for camera-type eyes. It is possible to extend the theory to other types of eyes, although it would likely require more variables and assumptions/constraints to the theory. It is thus good to introduce the conceptual ideas without overdoing the applications of the theory.

      The paper extends a previous theory, developed by the senior author, that develops performance surfaces for optimal cost/benefit design of eyes. By combining this with resource allocation between receptors and optics, the theoretical understanding of eye design takes a major leap and provides entirely new sets of predictions and explanations for why eyes are built the way they are.

      The paper is well written and even though the theory development in the Results may be difficult to take in for many biologists, the Discussion very nicely lists all the major predictions under separate headings, and here the text is more tuned for readers that are not entirely comfortable with the formalism of the Results section. I must point out though that the Results section is kept exemplary concise. The figures are excellent and help explain concepts that otherwise may go above the head of many biologists.

    1. Reviewer #2 (Public Review):

      Summary:

      The study combines computational modeling of choice behavior with an economic, effort-based decision-making task to assess how willingness to exert physical effort for a reward varies as a function of individual differences in apathy and anhedonia, or depression, as well as chronotype. They find an overall reduction in effort selection that scales with apathy and anhedonia and depression. They also find that later chronotypes are less likely to choose effort than earlier chronotypes and, interestingly, an interaction whereby later chronotypes are especially unwilling to exert effort in the morning versus the evening.

      Strengths:

      This study uses state-of-the-art tools for model fitting and validation and regression methods which rule out multicollinearity among symptom measures and Bayesian methods which estimate effects and uncertainty about those estimates. The replication of results across two different kinds of samples is another strength. Finally, the study provides new information about the effects not only of chronotype but also chronotype by timepoint interactions which are previously unknown in the subfield of effort-based decision-making.

      Weaknesses:

      The study has few weaknesses. One potential concern is that the range of models which were tested was narrow, and other models might have been considered. For example, the Authors might have also tried to fit models with an overall inverse temperature parameter to capture decision noise. One reason for doing so is that some variance in the bias parameter might be attributed to noise, which was not modeled here. Another concern is that the manuscripts discuss effort-based choice as a transdiagnostic feature - and there is evidence in other studies that effort deficits are a transdiagnostic feature of multiple disorders. However, because the present study does not investigate multiple diagnostic categories, it doesn't provide evidence for transdiagnosticity, per se.

    1. Reviewer #2 (Public Review):

      Summary:

      The goal of the paper was to trace the transitions hippocampal microglia undergo along aging. ScRNA-seq analysis allowed the authors to predict a trajectory and hypothesize about possible molecular checkpoints, which keep the pace of microglial aging. E.g. TGF1b was predicted as a molecule slowing down the microglial aging path and indeed, loss of TGF1 in microglia led to premature microglia aging, which was associated with premature loss of cognitive ability. The authors also used the parabiosis model to show how peripheral, blood-derived signals from the old organism can "push" microglia forward on the aging path.

      Strengths:

      A major strength and uniqueness of this work is the in-depth single-cell dataset, which may be a useful resource for the community, as well as the data showing what happens to young microglia in heterochronic parabiosis setting and upon loss of TGFb in their environment.

      Weaknesses:

      That said, given what we recently learned about microglia isolation for RNA-seq analysis, there is a danger that some of the observations are a result of not age, but cell stress from sample preparation (enzymatic digestion 10min at 37C; e.g. PMID: 35260865). Changes in cell state distribution along aging were made based on scRNA-seq and were not corroborated by any other method, such as imaging of cluster-specific marker expression in microglia at different ages. This analysis would allow confirming the scRNA-seq data and would also give us an idea of where the subsets are present within the hippocampus, and whether there is any interesting distribution of cell states (e.g. some are present closer to stem cells?). Since TGFb is thought to be crucial to microglia biology, it would be valuable to include more analysis of the mice with microglia-specific Tgfb deletion e.g. what was the efficiency of recombination in microglia? Did their numbers change after induction of Tgfb deletion in Cx3cr1-creERT2::Tgfb-flox mice.

      Overall:

      In general, I think the authors did a good job following the initial observations and devised clever ways to test the emerging hypotheses. The resulting data are an important addition to what we know about microglial aging and can be fruitfully used by other researchers, e.g. those working on microglia in a disease context.

    1. Reviewer #2 (Public Review):

      This study significantly advances our understanding of the metabolic reprogramming underlying astrocyte activation in neurological diseases such as multiple sclerosis. By employing an experimental autoimmune encephalomyelitis (EAE) mouse model, the authors discovered a notable nuclear translocation of PKM2, a key enzyme in glycolysis, within astrocytes.

      Preventing this nuclear import via DASA 58 substantially attenuated primary astrocyte activation, characterized by reduced proliferation, glycolysis, and inflammatory cytokine secretion.<br /> Moreover, the authors uncovered a novel regulatory mechanism involving the ubiquitin ligase TRIM21, which mediates PKM2 nuclear import. TRIM21 interaction with PKM2 facilitated its nuclear translocation, enhancing its activity in phosphorylating STAT3, NFκB, and c-myc. Single-cell RNA sequencing and immunofluorescence staining further supported the upregulation of TRIM21 expression in astrocytes during EAE.

      Manipulating this pathway, either through TRIM21 overexpression in primary astrocytes or knockdown of TRIM21 in vivo, had profound effects on disease severity, CNS inflammation, and demyelination in EAE mice. This comprehensive study provides invaluable insights into the pathological role of nuclear PKM2 and the ubiquitination-mediated regulatory mechanism driving astrocyte activation.

      The author's use of diverse techniques, including single-cell RNA sequencing, immunofluorescence staining, and lentiviral vector knockdown, underscores the robustness of their findings and interpretations. Ultimately, targeting this PKM2-TRIM21 axis emerges as a promising therapeutic strategy for neurological diseases involving astrocyte dysfunction.

      While the strengths of this piece of work are undeniable, some concerns could be addressed to refine its impact and clarity further; as outlined in the recommendations for the authors.

    1. Reviewer #2 (Public Review):

      Summary:

      Here Vogt et al., provide new insights into the need for sleep and the molecular and physiological response to sleep loss. The authors expand on their previously published work (Bjorness et al., 2020) and draw from recent advances in the field to propose a neuron-centric molecular model for the accumulation and resolution of sleep need and the basis of restorative sleep function. While speculative, the proposed model successfully links important observations in the field and provides a framework to stimulate further research and advances on the molecular basis of sleep function. In my review, I highlight the important advances of this current work, and the clear merits of the proposed model, and indicate areas of the model that can serve to stimulate further investigation.

      Strengths:

      Reviewer comment on new data in Vogt et al., 2024<br /> Using classic slice electrophysiology, the authors conclude that wakefulness (sleep deprivation (SD)) drives a potentiation of excitatory glutamate synapses, mediated in large part by "un-silencing" of NMDAR-active synapses to AMPAR-active synapses. Using a modern single nuclear RNAseq approach the authors conclude that SD drives changes in gene expression primarily occurring in glutamatergic neurons. The two experiments combined highlight the accumulation and resolution of sleep need centered on the strength of excitatory synapses onto excitatory neurons. This view is entirely consistent with a large body of extant and emerging literature and provides important direction for future research.

      Consistent with prior work, wakefulness/SD drives an LTP-type potentiation of excitatory synaptic strength on principle cortical neurons. It has been proposed that LTP associated with wake, leads to the accumulation of sleep need by increasing neuronal excitability, and by the "saturation" of LTP capacity. This saturation subsequently impairs the capacity for further ongoing learning. This new data provides a satisfying mechanism of this saturation phenomenon by introducing the concept of silent synapses. The new data show that in mice well rested, a substantial number of synapses are "silent", containing an NMDAR component but not AMPARs. Silent synapses provide a type of reservoir for learning in that activity can drive the un-silencing, increasing the number of functional synapses. SD depletes this reservoir of silent synapses to essentially zero, explaining how SD can exhaust learning capacity. Recovery sleep led to restoration of silent synapses, explaining how recovery sleep can renew learning capacity. In their prior work (Bjorness et al., 2020) this group showed that SD drives an increase in mEPSC frequency onto these same cortical neurons, but without a clear change in pre-synaptic release probability, implying a change in the number of functional synapses. This prediction is now born out in this new dataset.

      The new snRNAseq dataset indicates the sleep need is primarily seen (at the transcriptional level) in excitatory neurons, consistent with a number of other studies. First, this conclusion is corroborated by an independent, contemporary snRNAseq analysis recently available as a pre-print (Ford et al., 2023 BioRxiv https://doi.org/10.1101/2023.11.28.569011). A recently published analysis on the effects of SD in drosophila imaged synapses in every brain region in a cell-type dependent manner (Weiss et al., PNAS 2024), concluding that SD drives brain wide increases in synaptic strength almost exclusively in excitatory neurons. Further, Kim et al., Nature 2022, heavily cited in this work, show that the newly described SIK3-HDAC4/5 pathway promotes sleep depth via excitatory neurons and not inhibitory neurons.

      The new experiments provided in Fig1-3 are expertly conducted and presented. This reviewer has no comments of concern regarding the execution and conclusions of these experiments.

      Reviewer comment on the model in Vogt et al., 2024

      In the view of this reviewer the new model proposed by Vogt et al., is an important contribution. The model is not definitively supported by new data, and in this regard should be viewed as a perspective, providing mechanistic links between recent molecular advances, while still leaving areas that need to be addressed in future work. New snRNAseq analysis indicates that SD drives the expression of synaptic shaping components (SSCs) consistent with the excitatory synapse as a major target for the restorative basis of sleep function. SD-induced gene expression is also enriched for autism spectrum disorder (ASD) risk genes. As pointed out by the authors, sleep problems are commonly reported in ASD, but the emphasis has been on sleep amount. This new analysis highlights the need to understand the impact on sleep's functional output (synapses) to fully understand the role of sleep problems in ASD.

      Importantly, SD-induced gene expression in excitatory neurons overlaps with genes regulated by the transcription factor MEF2C and HDAC4/5 (Figure 4). In their prior work, the authors show loss of MEF2C in excitatory neurons abolished the SD transcriptional response and the functional recovery of synapses from SD by recovery sleep. Recent advances identified HDAC4/5 as major regulators of sleep depth and duration (in excitatory neurons) downstream of the recently identified sleep-promoting kinase SIK3. In Zhou et al., and Kim et al., Nature 2022, both groups propose a model whereby "sleep-need" signals from the synapse activate SIK3, which phosphorylates HDAC4/5, driving cytoplasmic targeting, allowing for the de-repression and transcriptional activation of "sleep genes". Prior work shows that HDAC4/5 are repressors of MEF2C. Therefore, the "sleep genes" derepressed by HDAC4/5 may be the same genes activated in response to SD by MEF2C. The new model thereby extends the signaling of sleep need at synapses (through SIK3-HDAC4/5) to the functional output of synaptic recovery by expression of synaptic/sleep genes by MEF2C. The model thereby links aspects of the expression of sleep need with the resolution of sleep need by mediating sleep function: synapse renormalization.

      Weaknesses:

      Areas for further investigation

      In the discussion section Vogt et al., explore the links between excitatory synapse strength, arguably the major target of "sleep function", and NREM slow-wave activity (SWA), the most established marker of sleep need. SIK3-HDAC4/5 have major effects on the "depth" of sleep by regulating NREM-SWA. The effects of MEF2C loss of function on NREM SWA activity are less obvious, but clearly impact the recovery of glutamatergic synapses from SD. The authors point out how adenosine signaling is well established as a mediator of SWA, but the links between adenosine and glutamatergic strength are far from clear. The mechanistic links between SIK3/HDAC4/5, adenosine signaling, and MEF2C, are far from understood. Therefore, the molecular/mechanistic links between a synaptic basis of sleep need and resolution with NREM-SWA activity require further investigation.

      Additional work is also needed to understand the mechanistic links between SIK3-HDAC4/5 signaling and MEF2C activity. The authors point out that constitutively nuclear (cn) HDAC4/5 (acting as a repressor) will mimic MEF2C loss of function. This is reasonable, however, there are notable differences in the reported phenotypes of each. Notably, cnHDAC4/5 suppresses NREM amount and NREM SWA but had no effect on the NREM-SWA increase following SD (Zhou et al., Nature 2022). Loss of MEF2C in CaMKII neurons had no effect on NREM amount and suppressed the increase in NREM-SWA following SD (Bjorness et al., 2020). These instances indicate that cnHDAC4/5 and loss of MEF2C do not exactly match suggesting additional factors are relevant in these phenotypes. Likely HDAC4/5 have functionally important interactions with other transcription factors, and likewise for MEF2C, suggesting areas for future analysis.

      One emerging theme may be that the SIK3-HDAC4/5 axis is a major regulator of the sleep state, perhaps stabilizing the NREM state once the transition from wakefulness occurs. MEF2C is less involved in regulating sleep per se, and more involved in executing sleep function, by promoting restorative synaptic modifications to resolve sleep need.

      Finally, advances in the roles of the respective SIK3-HDAC4/5 and MEF2C pathways point towards transcription of "sleep genes", as clearly indicated in the model of Figure 4. Clearly, more work is needed to understand how the expression of such genes ultimately leads to the resolution of sleep need by functional changes at synapses. What are these sleep genes and how do they mechanistically resolve sleep need? Thus, the current work provides a mechanistic framework to stimulate further advances in understanding the molecular basis for sleep need and the restorative basis of sleep function.

    1. Reviewer #2 (Public Review):

      This study uses single-unit recordings in the monkey STN to examine the evidence for three theoretical models that propose distinct roles for the STN in perceptual decision-making. Importantly, the proposed functional roles are predictive of unique patterns of neural activity. Using k-means clustering with seeds informed by each model's predictions, the current study identified three neural clusters with activity dynamics that resembled those predicted by the described theoretical models. The authors are thorough and transparent in reporting the analyses used to validate the clustering procedure and the stability of the clustering results. To further establish a causal role for the STN in decision-making, the researchers applied microstimulation to the STN and found effects on response times, choice preferences, and latent decision parameters estimated with a drift diffusion model. Overall, the study provides strong evidence for a functionally diverse population of STN neurons that could indeed support multiple roles involved in perceptual decision-making. The manuscript would benefit from stronger evidence linking each neural cluster to specific decision roles in order to strengthen the overall conclusions.

      The interpretation of the results, and specifically, the degree to which the identified clusters support each model, is largely dependent on whether the artificial vectors used as model-based clustering seeds adequately capture the expected behavior under each theoretical model. The manuscript would benefit from providing further justification for the specific model predictions summarized in Figure 1B. Further, although each cluster's activity can be described in the context of the discussed models, these same neural dynamics could also reflect other processes not specific to the models. That is, while a model attributing the STN's role to assessing evidence accumulation may predict a ramping up of neural activity, activity ramping is not a selective correlate of evidence accumulation and could be indicative of a number of processes, e.g., uncertainty, the passage of time, etc. This lack of specificity makes it challenging to infer the functional relevance of cluster activity and should be acknowledged in the discussion.

      Additionally, although the effects of STN microstimulation on behavior provide important causal evidence linking the STN to decision processes, the stimulation results are highly variable and difficult to interpret. The authors provide a reasonable explanation for the variability, showing that neurons from unique clusters are anatomically intermingled such that stimulation likely affects neurons across several clusters. It is worth noting, however, that a substantial body of literature suggests that neural populations in the STN are topographically organized in a manner that is crucial for its role in action selection, providing "channels" that guide action execution. The authors should comment on how the current results, indicative of little anatomical clustering amongst the functional clusters, relate to other reports showing topographical organization.

      Overall, the association between the identified clusters and the function ascribed to the STN by each of the models is largely descriptive and should be interpreted accordingly. For example, Figure 3 is referenced when describing which cluster activity is choice/coherence dependent, yet it is unclear what specific criteria and measures are being used to determine whether activity is choice/coherence "dependent." Visually, coherence activity seems to largely overlap in panel B (top row). Is there a statistically significant distinction between low and high coherence in this plot? The interpretation of these plots and the methods used to determine choice/coherence "dependence" needs further explanation.

      In general, the association between cluster activity and each model could be more directly tested. At least two of the models assume coordination with other brain regions. Does the current dataset include recordings from any of these regions (e.g., mPFC or GPe) that could be used to bolster claims about the functional relevance of specific subpopulations? For example, one would expect coordinated activity between neural activity in mPFC and Cluster 2 according to the Ratcliff and Frank model. Additionally, the reported drift-diffusion model (DDM) results are difficult to interpret as microstimulation appears to have broad and varied effects across almost all the DDM model parameters. The DDM framework could, however, be used to more specifically test the relationships between each neural cluster and specific decision functions described in each model. Several studies have successfully shown that neural activity tracks specific latent decision parameters estimated by the DDM by including neural activity as a predictor in the model. Using this approach, the current study could examine whether each cluster's activity is predictive of specific decision parameters (e.g., evidence accumulation, decision thresholds, etc.). For example, according to the Ratcliff and Frank model, activity in cluster 2 might track decision thresholds.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors did not find an increased representation of CS+ throughout reinforcement learning in the tuft dendrites of Rbp4-positive neurons from layer 5B of the barrel cortex, as previously reported for soma from layer 2/3 of the visual cortex.

      Alternatively, the authors observed an increased selectivity to both stimuli (CS+ and CS-) during reinforcement learning. This feature:

      (1) was not present in repeated exposures (without reinforcement),<br /> (2) was not explained by the animal's behaviour (choice, licking, and whisking), and<br /> (3) was long-lasting, being present even when the mice disengaged from the task.

      Importantly, increased selectivity was correlated with learning (% correct choices), and neural discriminability between stimuli increased with learning.

      In conclusion, the authors show that tuft dendrites from layer 5B of the barrel cortex increase the representation of conditioned (CS+) and unconditioned stimuli (CS-) applied to the whiskers, during reinforcement learning.

      Strengths:

      The results presented are very consistent throughout the entire study, and therefore very convincing:

      (1) The results observed are very similar using two different imaging techniques (2-photon -planar imaging- and SCAPE-volumetric imaging). Figure 3 and Figure 4 respectively.

      (2) The results are similar using "different groups" of tuft dendrites for the analysis (e.g. initially unresponsive and responsive pre- and post-learning). Figure 5.

      (3) The results are similar from a specific set of trials (with the same sensory input, but different choices). Figure 7.

      (4) Additionally, the selectivity of tuft dendrites from layer 5B of the barrel cortex was higher in the mice that exclusively used the whisker to respond to the stimuli (CS+ and CS-).<br /> The results presented are controlled against a group of mice that received the same stimuli presentation, except for the reinforcement (reward).

      Additionally, the behaviour outputs, such as choice, whisking, and licking could not account for the results observed.

      Although there are no causal experiments, the correlation between selectivity and learning (percentage of correct choices), as well as the increased neural discriminability with learning, but not in repeated exposure, are very convincing.

      Weaknesses:

      The biggest weakness is the absence of causality experiments. Although inhibiting specifically tuft dendritic activity in layer 1 from layer 5 pyramidal neurons is very challenging, tuft dendritic activity in layer 1 could be silenced through optogenetic experiments as in Abs et al. 2018. By manipulating NDNF-positive neurons the authors could specifically modify tuft dendritic activity in the barrel cortex during CS presentations, and test if silencing tuft dendritic activity in layer 1 would lead to the lack of selectivity and an impairment of reinforcement learning. Additionally, this experiment will test if the selectivity observed during reinforcement learning is due to changes in the local network, namely changes in local synaptic connectivity, or solely due to changes in the long-range inputs.

    1. Reviewer #2 (Public Review):

      Summary:

      Cold-induced lipid metabolism is well-established in adipose tissues. The authors set out to determine whether cold could alter brain lipid metabolism. By QPCR analysis of brain punches after acute cold, they found that mRNA expressions of several lipolysis-related genes were upregulated compared to RT controls. By combining fluorescent sensors and in vivo fiberphotometry, they observed cold-induced lipid peroxidation/lipolysis, which could be blocked by pharmacological inhibitors of neuronal activity (muscimol and kynurenic acid). The brain is not traditionally considered an organ with high lipid metabolism (vs carbohydrate); therefore, the observation and hypothesis proposed by the authors are unexpected and can be interesting. However, the experiments and data were rather preliminary and superficial and did not support the authors' conclusions. In addition, the main hypothesis, in relationship to the role of cold/temperature, remains incoherent and needs a major update.

      Strengths:

      A set of relatively novel and interesting observations.

      Creative use of several in vivo sensors and techniques.

      Weaknesses:

      (1) The physiological relevance of lipolysis and thermogenesis genes in the PVH. The authors need to provide quantitative and substantial characterizations of lipid metabolism in the brain beyond a panel of qPCRs, especially considering these genes are likely expressed at very low levels. mRNA and protein level quantification of genes in Fig 1, in direct comparison to BAT/iWAT, should be provided. Besides bulk mRNA/protein, IHC/ISH-based characterization should be added to confirm to cellular expression of these genes.

      (2) The fiberphotometry work they cited (Chen 2022, Andersen 2023, Sun 2018) used well-established, genetically encoded neuropeptide sensors (e.g., GRABs). The authors need to first quantitatively demonstrate that adapting BD-C11 and EnzCheck for in vivo brain FP could effectively and accurately report peroxidation and lipolysis. For example, the sensitivity, dynamic range, and off-time should all be calibrated with mass spectrometry measurements before any conclusions can be made based on plots in Figures 4, 5, and 6. This is particularly important because the main hypothesis heavily relies on this unvalidated technique.

      (3) Generally, the histology data need significant improvement. It was not convincing, for example, in Figure 3, how the Fos+ neurons can be quantified based on the poor IF images where most red signals were not in the neurons.

      (4) The hypothesis regarding the direct role of brain temperature in cold-induced lipid metabolism is puzzling. From the introduction and discussion, the authors seem to suggest that there are direct brain temperature changes in responses to cold, which could be quite striking. However, this was not supported by any data or experiments. The authors should consolidate their ideas and update a coherent hypothesis based on the actual data presented in the manuscript.

    1. Reviewer #2 (Public Review):

      Summary:

      The study has demonstrated how two neurotransmitters and neuromodulators from the same neurons can be regulated and utilized in thermoregulation.

      The study utilized electrophysiological methods to examine the characteristics and thermoregulation of Neurotensin (Nts)-expressing neurons in the medial preoptic area (MPO). It was discovered that GABA and Nts may be co-released by neurons in MPO when communicating with their target neurons.

      Strengths:

      The study has leveraged optogenetic, chemogenetic, knockout, and pharmacological inhibitors to investigate the release process of Nts and GABA in controlling body temperature.

      The findings are relevant to those interested in the various functions of specific neuron populations and their distinct regulatory mechanisms on neurotransmitter/neuromodulator activities

      Weaknesses:

      Key points for consideration include:

      (1) The co-release of GABA and Nts is primarily inferred rather than directly proven. Providing more direct evidence for the release of GABA and the co-release of GABA and Nts would strengthen the argument. Further in vitro analysis could strengthen the conclusion regarding this co-releasing process.

      (2) The differences between optogenetic and chemogenetic methods were not thoroughly investigated. A comparison of in vitro results and direct observation of release patterns could clarify the mechanisms of GABA release alone or in conjunction with Nts under different stimulation techniques.

      (3) Neuronal transcripts were mainly identified through PCR, and alternative methods like single-cell sequencing could be explored.

      (4) In Figure 6, the impact of GABA released from Nts neurons in MPO on CBT regulation appears to vary with ambient temperatures, requiring a more detailed explanation for better comprehension.

      (5) The model should emphasize the key findings of the study.

    1. Reviewer #2 (Public Review):

      Summary:

      In this study, the authors aim to combine automated whole-cell patch clamp recording simultaneously from multiple cells. Using a 2-electrode approach, they are able to sample as many cells (and connections) from one slice, as would be achieved with a more technically demanding and materially expensive 8-electrode patch clamp system. They provide data to show that this approach is able to successfully record from 52% of attempted cells, which was able to detect 3 pairs in 71 screened neurons. The authors state that this is a step forward in our ability to record from randomly connected ensembles of neurons.

      Strengths:

      The conceptual approach of recording multiple partner cells from another in a stepwise manner indeed increases the number of tested connections. An approach that is widely applicable to both automated and manual approaches. Such a method could be adopted for many connectivity studies using dual recording electrodes.

      The implementation of automated robotic whole-cell patch-clamp techniques from multiple cells simultaneously is a useful addition to the multiple techniques available to ex vivo slice electrophysiologists.

      The approach using 2 electrodes, which are washed between cells is economically favourable, as this reduces equipment costs for recording multiple cells, and limits the wastage of capillary glass that would otherwise be used once.

      Weaknesses:

      (1) The premise of this article is based upon the fact that even a "skilled" whole-cell electrophysiologist is only capable of recording ~10 cells per day are flawed. Many studies have shown that capable electrophysiologists can record upwards of 50 cells a day, given adequate slice quality and reliable recording conditions with multiple electrodes (e.g. Pastoll et al., 2020 eLife, Booker et al., 2014, JoVE, Peng et al., 2017); often with over 80% success rates for recording. It is not convincing that this approach is a dramatic improvement on such approaches - except when a less skilled researcher is beginning recordings.

      Importantly, could the patch walk protocol not be alternatively implemented using manual recording approaches? Yes, the use of a semi-automated robotic system aids recording from many cells by a less experienced colleague, but the inferences about the number of connections tested are common to the approach, not the technique used. This seems like a crucial conceptual point to include.

      (2) A key omission of this study is the absence of brain area, cell type, and layer recorded from. It is mentioned in Figure 2 that this is the somatosensory and visual cortices. Which were these, and how were they confirmed?

      (3) A comparison of measurements shown in Figure 2 to other methods - e.g. conventional dual patch, 8-electrode patch, single electrode. How do the values obtained for cell quality measurements compare to those expected for the cell population recorded (which is unclear - see point 2)?

      (4) What is the reliability of performing outside-out patch configuration to obtain sealed and biocytin-filled cells under these conditions? A key tenet of performing high-throughput paired recordings is the ability to identify the cell types involved in the local microcircuit, and if their axon has been preserved in the slice configuration (which varies between cell types). Not having confirmation of morphological identity and integrity likely leads to a dramatic underestimation of connection probability, given that main axon collaterals could be severed during acute brain slice preparation.

      (5) The quality control criteria used in this manuscript require further clarification. An upper limit of 50 MΩ access resistance is extremely high (i.e. 20-30 MΩ is a more typical and stringent cut-off), which is worsened as no real information is given to the degree of resistance change that could be accepted. This is simply listed as "If the seal quality decreased during recording, the cell is excluded from analysis". Indeed, the range of access resistances plotted in Figure 2 is from 10-100 MΩ, which implies that some neurons included in this data did not meet recording criteria. Also, it is widely accepted in the field that a 10-20% change in access during recording is acceptable - within a more defined range. I would consider re-assessing the recorded cells to only include cells with access resistances <30MΩ and those that did not fluctuate by more than 20%.

      Appraisal of aims:

      The authors certainly established a system that is useful for interrogating synaptic connectivity in an automated manner. However, it remains unclear how widely used this would be in the field, and whether this truly represents an advancement from manual recordings or >4 electrode recordings.

      Discussion of impact:

      This approach, particularly the conceptual approach to paired testing, is of use to the field. However, in practice, many researchers using conventional dual-electrode paired recording likely implement similar approaches - especially when targeting specific cell types (see Booker et al., 2014 JoVE, Qi et al., 2020 Front Synaptic Neurosci.). This may pave the way for greater implementation of dual and multi-electrode recordings using robotic patch-clamp techniques.

    1. Reviewer #2 (Public Review):

      Summary:

      In this work, the authors attempt to advance our capacity to image the intact spinal cord in living mice, with the ultimate goal of allowing optical access to all spinal layers, from the dorsal (sensory-related) to the ventral (motor-related) laminae. They demonstrate the potency of 3-photon excited fluorescence imaging (3PEF) to collect fluorescent signals in anesthetized adult mice to depths of up to 450 µm from the dorsal surface.

      Strengths:

      • 3PEF is convincingly demonstrated as a significant improvement over previously used 2-photon imaging.

      • The images show very good spatial resolution and stable signal-to-noise ratio up to 450 µm from the dorsal surface, providing unprecedented access to intermediate ventral laminae.

      Weaknesses:

      • The paper in its current form lacks a detailed description of the experimental apparatus used, including its invasiveness (removal of vertebrae and muscles) and its impact on animal behavior. One can hope that, in the future, a similar implantation chamber may be used for awake, freely-moving animals.

      • In general, non-optic specialists may find it difficult to appreciate some of the findings due to technical writing at times, and minimally described metrics.

      • The possibility that the 3-photon illumination may cause tissue damage, notably by heat induction, is not evaluated or discussed.

      • At this stage, no attempt has been made to image cellular activity. The reader should keep in mind that motor neurons, as well as most of their upstream circuits, are located between 500 and 900 µm from the dorsal surface. Hence, although the method is a significant advancement, it still does not allow for the evaluation of morphological (or possibly, activity) changes in the whole spinal cord, particularly excluding motor-related laminae."

    1. Reviewer #2 (Public Review):

      Summary:

      This important work by Meisner et al., developed an automated apparatus (MarmoAPP) to collect a wide array of behavioral data (lever pulling, gaze direction, vocalizations) in marmoset monkeys, with the goal of modernizing collection of behavioral data to coincide with the investigation of neurological mechanisms governing behavioral decision making in an important primate neuroscience model. The authors show a variety of "proof-of-principle" concepts that this apparatus can collect a wide range of behavioral data, with higher behavioral resolution than traditional methods. For example, the authors highlight that typical behavioral experiments on primate cooperation provide around 10 trials per session, while using their approach the authors were able to collect over 100 trials per 20-minute session with the MarmoAAP.

      Overall the authors argue that this approach has a few notable advantages:<br /> (1) it enhances behavioral output which is important for measuring small or nuanced effects/changes in behavior;<br /> (2) allows for more advanced analyses given the higher number of trials per session;<br /> (3) significantly reduces the human labor of manually coding behavioral outcomes and experimenter interventions such as reloading apparatuses for food or position;<br /> (4) allows for more flexibility and experimental rigor in measuring behavior and neural activity simultaneously.

      Strengths:

      The paper is well-written and the MarmoAPP appears to be highly successful at integrating behavioral data across many important contexts (cooperation, gaze, vocalizations), with the ability to measure significantly many more behavioral contexts (many of which the authors make suggestions for).

      The authors provide substantive information about the design of the apparatus, how the apparatus can be obtained via a long list of information Apparatus parts and information, and provide data outcomes from a wide number of behavioral and neurological outcomes. The significance of the findings is important for the field of social neuroscience and the strength of evidence is solid in terms of the ability of the apparatus to perform as described, at least in marmoset monkeys. The advantage of collecting neural and freely-behaving behavioral data concurrently is a significant advantage.

      Weaknesses:

      While this paper has many significant strengths, there are a few notable weaknesses in that many of the advantages are not explicitly demonstrated within the evidence presented in the paper. There are data reported (as shown in Figures 2 and 3), but in many cases, it is unclear if the data is referenced in other published work, as the data analysis is not described and/or self-contained within the manuscript, which it should be for readers to understand the nature of the data shown in Figures 2 and 3.

      (1) There is no data in the paper or reference demonstrating training performance in the marmosets. For example, how many sessions are required to reach a pre-determined criterion of acceptable demonstration of task competence? The authors reference reliably performing the self-reward task, but this was not objectively stated in terms of what level of reliability was used. Moreover, in the Mutual Cooperation paradigm, while there is data reported on performance between self-reward vs mutual cooperation tasks, it is unclear how the authors measured individual understanding of mutual cooperation in this paradigm (cooperation performance in the mutual cooperation paradigm in the presence or absence of a partner; and how, if at all, this performance varied across social context). What positive or negative control is used to discern gained advantages between deliberate cooperation vs two individuals succeeding at self-reward simultaneously?

      (2) One of the notable strengths of this approach argued by the authors is the improved ability to utilize trials for data analysis, but this is not presented or supported in the manuscript. For example, the paper would be improved by explicitly showing a significant improvement in the analytical outcome associated with a comparison of cooperation performance in the context of ~150 trials using MarmoAAP vs 10-12 trials using conventional behavioral approaches beyond the general principle of sample size. The authors highlight the dissection of intricacies of behavioral dynamics, but more could be demonstrated to specifically show these intricacies compared to conventional approaches. Given the cost and expertise required to build and operate the MarmoAAP, it is critical to provide an important advantage gained on this front. The addition of data analysis and explicit description(s) of other analytical advantages would likely strengthen this paper and the advantages of MarmoAAP over other behavioral techniques.

    1. Reviewer #2 (Public Review):

      Summary:

      This manuscript explores the role of Nrn1 in T cell tolerance. A previous study has demonstrated that Nrn1 is up-regulated in the Tfr fraction of Foxp3+ T regulatory cells. These authors now confirm the expression of Nrn1 in Tregs as well as report here that Nrn1 is also greatly over-expressed in anergic CD4 T cells, and this is the stepping-off point for this investigation.

      Most remarkably, experiments show that anergy induction is defective when T cells cannot express Nrn1. Furthermore, differentiation to a Foxp3+ Treg phenotype is inhibited in the absence of Nrn1, and the Tregs that do develop appear functionally defective. With such defects in the anergy induction and Treg differentiation and function, auto-reactive effector T cell activation is unrestrained, and Nrn1-/- mice are more susceptible to severe EAE development.

      Strengths:

      The characterizations of T cell Nrn1 expression both in vitro and in vivo are comprehensive and convincing. The in vivo functional studies of anergy development, Treg suppression, and EAE development are also well done to strengthen the notion that Nrn1 is an important regulator of CD4 responsiveness.

      Weaknesses:

      The major weakness of this study stems from a lack of a clear molecular mechanism involving Nrn1. Previous studies of Nrn1 have suggested its role as a soluble molecule involved in intracellular communication, perhaps influencing cellular ion channel function and/or triggering downstream NFAT and mTOR activation. However, a unique receptor for Nrn1 has not been discovered and it remains unclear whether it acts in a cell-intrinsic or cell-extrinsic fashion for any particular cell type.

      Data shown here provide evidence of alterations in the electrical and metabolic state of T cells when the Nrn1 gene is deleted. Nrn1-/- Tregs and Teffector cells each express a unique pattern of genes associated with Neurotransmitter receptor, Metal ion transmembrane transport, Amino acid transport, and mTORC1 signaling activities, different than that seen in wild-type mice. Although the biochemical and informatics studies are well-performed, it is my opinion that these results are inconclusive in part due to the absence of key "naive" control groups. This limits my ability to understand the significance of these data.

      Specifically, studies of the electrical and metabolic state of Nrn1-/- inducible Treg cells (iTregs) would benefit from similar data collected from wild-type and Nrn1-/- naive CD4 T cells. Even though naive T cells don't express Nrn1, they may be positively influenced by soluble Nrn1. Does deletion of Nrn1 lead to changes in metabolic and electrical state in naive T cells? Is that why Nrn1 deletion in mice blocks naive T cell activation?

      Since the loss of Nrn1 inhibits the activation of T cells, are Nrn1-/- iTregs transcriptionally, electrically, and metabolically similar to naive T cells due to their suboptimal activation? Does this account for their persistent functional defects? Or is up-regulation of Nrn1 (and cell-intrinsic Nrn1 signaling) necessary to complete Treg differentiation and to promote T regulatory function (similar to how cell-intrinsic Nrn1 facilitates anergy induction)? The study of naive cells in parallel with iTregs would address these possibilities.

      A comparison of Nrn1-/- naive cells to Teffector cells should also be undertaken to reveal how it is that Nrn1-/- Teffector cells regain the capacity to respond effectively to stimulation (e.g. increased mTOR activation) despite their early activation defects.

    1. Reviewer #2 (Public Review):

      Summary

      The authors developed new tools for isolating PI3K activity and for labeling newly made membrane proteins for monitoring membrane trafficking. They found that PI3K activity alone was able to explain the increased presence of TRPV1 on the membrane independent of other cascades induced by NGF signaling. They also showed an interesting feedback between PI3K and the insulin receptor trafficking to the membrane.

      Strengths:

      A major strength of the paper is the innovative combination of techniques. The first technique used the optogenetic PhyB/PIF system. They anchored PhyB to the membrane and fused PIF with the interSH2 domain from PI3K. This allowed them to use 650nm light to induce an interaction between the PhyB and PIF resulting in a recruitment of the endogenous PI3K to the membrane through the iSH2 domain without actual activation of an RTK. This allowed them to dissect out one function, just PI3K recruitment/activation from the vast number of RTK downstream cascades.

      The second technique was the development of a new non-canonical amino acid that is cell-impermeant. The authors synthesized the sTSO-sulfa-Cy5 compound that will react with the Tet3 ncAA through click chemistry. They showed that the sulfa-Cy5 did not cross the membrane and would be used to track protein production over time, though the reaction rates were slow as noted by the authors. The comparison of the sulfa-Cy5 data with the standard GFP with TIRF showed a clear difference indicating the useful information that is gained with the ncAA.

      Another strength comes from the discovery that an isolated PI3K is responsible for increasing TRPV1 and InR trafficking to the plasma membrane.

      Weakness:

      The discussion does not go into much detail regarding the importance of their discovery of TRPV1 and InR increases trafficking due to PI3K activation. It also jumps to the limitations of in vivo implementation prematurely. These weaknesses are minor however.

      The authors achieved their goal of creating the tools needed to separate out one of the many RTK signals and give a strong proof of concept implementation of their tools. Their results support their conclusions and will help understand how TRPV1 is regulated by signals other than the traditional channel activators. The tools developed in the article will be of use to the broader cell biology and biophysics community, not just the channel community. The opto control of the PhyB/PIF system makes it more convenient than other systems since it does not take the typical wavelengths needed for fluorescence. The cell-impermeant ncAA will also be a great tool for those studying membrane proteins, protein trafficking and protein dynamics.

    1. Reviewer #2 (Public Review):

      Summary:

      The study tries to connect energy metabolism with immune tolerance during bacterial infection. The mechanism details the role of pyruvate transporter expression via ERRalpha-PGC1 axis, resulting in pro-inflammatory TNF alpha signalling responsible for acquired infection tolerance.

      Strengths:

      Overall, the study is an excellent addition to the role of energy metabolism during bacterial infection. The mechanism-based approach in dissecting the roles of metabolic coactivator, transcription factor, mitochondrial transporter, and pro-inflammatory cytokine during acquired tolerance towards infections indicates a detailed and well-written study. The in vivo studies in mice nicely corroborate with the cell line-based data, indicating the requirement for further studies in human infections with another bacterial model system.

      Weaknesses:

      The authors have involved various mechanisms to justify their findings. However, they have missed out on certain aspects which connect the mechanism throughout the paper. For example, they measured ATP and acetyl COA production linked with bacterial re-exposures and added various targets like MCP1, EER alpha, PGC1 alpha and TNF alpha. However, they skipped PGC1 alpha levels, ATP and acetyl COA in various parts of the paper. Including the details would make the work more comprehensive.

      The use of public data sets to support their claim on immune tolerance is missing. Including various data sets of similar studies will strengthen the findings independently.

    1. Reviewer #2 (Public Review):

      Summary:

      In the article, "Untargeted Pixel-by-Pixel Imaging of Metabolite Ratio Pairs as a Novel Tool for Biomedical Discovery in Mass Spectrometry Imaging" the authors describe their software package in R for visualizing metabolite ratio pairs. I think the novelty of this manuscript is overstated and there are several notable issues with the figures that prevent detailed assessment but the work would be of interest to the mass spectrometry community.

      Strengths:

      The authors describe a software that would be of use to those performing MALDI MSI. This software would certainly add to the understanding of metabolomics data and enhance the identification of critical metabolites.

      Weaknesses:

      The authors are missing several references and discussion points, particularly about SIMS MSI, where ratio imaging has been previously performed.

      There are several misleading sentences about the novelty of the approach and the limitations of metabolite imaging.

      Several sentences lack rigor and are not quantitative enough.

      The figures are difficult to interpret/ analyze in their current state and lack some critical components, including labels and scale bars.

    1. Reviewer #2 (Public Review):

      This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is compelling and goes beyond the current state of the art. The results are of interest to neuroscientists working on motor control and reward-based learning.

      Comments on revised version:

      The revision has produced a stronger manuscript. Thank you for your thorough responses to the comments and concerns.

    1. Reviewer #2 (Public Review):

      This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is highly compelling and goes beyond the current state-of-the-art, but it is incomplete with respect to examining different signatures of learning, clarifying probed learning processes, and investigating changes in all relevant subcortical structures is incomplete and would benefit from more rigorous approaches. With the rationale and data presentation strengthened this paper would be of interest to neuroscientists working on motor control and reward-based learning.

    1. Reviewer #2 (Public Review):

      In this manuscript, the authors reported the biological role of RBM7 deficiency in promoting metastasis of breast cancer. They further used a combination of genomic and molecular biology approaches to discover a novel role of RBM7 in controlling alternative splicing of many genes in cell migration and invasion, which is responsible for the RBM7 activity in suppressing metastasis. They conducted an in-depth mechanistic study on one of the main targets of RBM7, MFGE8, and established a regulatory pathway between RBM7, MFGE8-L/MFGE8-S splicing switch, and NF-κB signaling cascade. This link between RBM7 and cancer pathology was further supported by analysis of clinical data.

      Overall, this is a very comprehensive study with lots of data, and the evidence is consistent and convincing. Their main conclusion was supported by many lines of evidence, and the results in animal models are pretty impressive.

    1. Reviewer #2 (Public Review):

      In this study, authors identified TOR, HOG and CWI signaling network genes as modulators of the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation. They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, especially that the conserved site K19 of FKBP3 plays a key role in regulating aflatoxin biosynthesis. In general, the study involved a heavy workload and the findings are potentially interesting and important for understanding or controlling the aflatoxin biosynthesis. However, the findings have not been deeply explored and the conclusions mostly are based on parallel phenotypic observations.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors have utilised deep profiling methods to generate deeper insights into the features of the TME that drive responsiveness to PD-1 therapy in NSCLC.

      Strengths:

      The main strengths of this work lie in the methodology of integrating single-cell sequencing, genetic data, and TCRseq data to generate hypotheses regarding determinants of IO responsiveness.

      Some of the findings in this study are not surprising and well precedented eg. association of Treg, STAT3, and NFkB with ICI resistance and CD8+ activation in ICI responders and thus act as an additional dataset to add weight to this prior body of evidence. Whilst the role of Th17 in PD-1 resistance has been previously reported (eg. Cancer Immunol Immunother 2023 Apr;72(4):1047-1058, Cancer Immunol Immunother 2024 Feb 13;73(3):47, Nat Commun. 2021; 12: 2606 ) these studies have used non-clinical models or peripheral blood readouts. Here the authors have supplemented current knowledge by characterization of the TME of the tumor itself.

      Weaknesses:

      Unfortunately, the study is hampered by the small sample size and heterogeneous population and whilst the authors have attempted to bring in an additional dataset to demonstrate the robustness of their approach, the small sample size has limited their ability to draw statistically supported conclusions. There is also limited validation of signatures/methods in independent cohorts, no functional characterisation of the findings, and the discussion section does not include discussion around the relevance/interpretation of key findings that were highlighted in the abstract (eg. role of Th17, TRM, STAT3, and NFKb). Because of these factors, this work (as it stands) does have value to the field but will likely have a relatively low overall impact.

      Related to the absence of discussion around prior TRM findings, the association between TRM involvement in response to IO therapy in this manuscript is counter to what has been previously demonstrated (Cell Rep Med. 2020;1(7):100127, Nat Immunol. 2017;18(8):940-950., J Immunol. 2015;194(7):3475-3486.). However, it should be noted that the authors in this manuscript chose to employ alternative markers of TRM characterisation when defining their clusters and this could indicate a potential rationale for differences in these findings. TRM population is generally characterised through the inclusion of the classical TRM markers CD69 (tissue retention marker) and CD103 (TCR experienced integrin that supports epithelial adhesion), which are both absent from the TRM definition in this study. Additional markers often used are CD44, CXCR6, and CD49a, of which only CXCR6 has been included by the authors. Conversely, the majority of markers used by the authors in the cell type clustering are not specific to TRM (eg. CD6, which is included in the TRM cluster but is expressed at its lowest in cluster 3 which the authors have highlighted as the CD8+ TRM population). Therefore, whilst there is an interesting finding of this particular cell cluster being associated with resistance to ICI, its annotation as a TRM cluster should be interpreted with caution.

    1. Reviewer #3 (Public Review):

      Summary:

      The authors try to elucidate the molecular mechanisms underlying the intra-organ crosstalks that perpetuate intestinal permeability and inflammation.

      Strengths:

      This study identifies a hepatocyte-specific rela/stat3 network as a potential therapeutic target for intestinal diseases via the gut liver axis using both murine models and human samples.

      Weaknesses:

      (1) The mechanism by which DSS administration induces the activation of the Rela and Stat3 pathways and subsequent modification of the bile acid pathway remains clear. As the authors state, intestinal bacteria are one candidate, and this needs to be clarified. I recommend the authors investigate whether gut sterilization by administration of antibiotics or germ free condition affects 1. the activation of the Rela and Stat3 pathway in the liver by DSS-treated WT mice and 2. the reduction of colitis in DSS-treated relaΔhepstat3Δhep mice.

      (2) It has not been shown whether DSS administration causes an increase in primary bile acids, represented by CDCA, in the colon of WT mice following activation of the Rela and Stat3 pathways, as demonstrated in Figure 6.

      (3) The implications of these results for IBD treatment, especially in what ways they may lead to therapeutic intervention, need to be discussed.

      The above weakness points have been resolved by the revision and additional experiments.

    1. Reviewer #3 (Public Review):

      This work compares transcriptional responses of shoots and roots harvested from four plate-based assays that aim to simulate drought and from plants subjected to water deficit in pots using the model plant Arabidopsis thaliana with the goal to select a plate-based assay that best recapitulates transcriptional changes that are observed during water-deficit in pots. For the plate-based assays polyethylene glycol (PEG), mannitol, and sodium chloride (salt) treatments were used as well as a 'hard agar' assay which was newly developed by the authors. In the 'hard agar' assay, less water was added to the solid components of the media leading to an increase in agar strength and nutrient concentration. Plants in pots were grown on vermiculite with the same nutrient mix as used in the plates and drought was induced by withholding watering for five days.

      The authors observed a good directional agreement of differential expressed genes for shoots between the plate assays on the vermiculite drying experiment. However, less directional agreement was observed for differential expressed genes of roots, except for their newly developed 'hard agar' assay which had good directional agreement. Testing whether the increase in agar strength or more concentrated nutrients are attributed to this, they found that both factors contributed to the effect of the 'hard agar'. Arabidopsis ecotypes that showed a stronger reduction in shoot size when grown on the 'hard agar' tended to have a lower fitness according to an external study which may indicate that the 'hard agar' assay simulates physiological relevant conditions.

      The work highlights that transcriptional responses for simulated drought on plates and drought caused by water deficit are highly variable and dependent on the tissues that are observed. The authors demonstrate that transcriptomics can be used to select a suitable plate assay that most closely recapitulates drought through water deficit for plants grown in pots. Interestingly their newly developed 'hard agar' assay provides an alternative to traditional plate-based assays with improved directional agreement of differential expressed genes in roots in comparison to plants experiencing water deficit in vermiculite. It is promising that the impact of 'hard agar' on the shoot size of 20 diverse Arabidopsis accessions shows some association with plant fitness under drought in the field. Their methodology could be powerful in identifying a better substitute for plate-based high-throughput drought assays that have an emphasis on gene expression changes.

    1. Reviewer #2 (Public Review):

      Summary

      In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

      Strengths:

      The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

      Weaknesses:

      (1) I suspect that there may be variation in survivorship after long-term exposure to cold conditions (10ºC, 35 days), which could also be quantified and mapped using genome-wide association studies (GWAS). Since blocking Ir21a neuronal transmission prevented flies from exiting diapause, it is possible that natural genetic variation could have a similar effect, influencing the success rate of exiting diapause and post-diapause mortality. If there is variation in this trait, could it affect post-diapause fecundity? I am concerned that this could be a confounding factor in the analysis of post-diapause fecundity. However, I also believe that understanding phenotypic variation in this trait itself could be significant in regulating adult diapause.

      (2) On p.10, the authors conclude that "Dip-𝛾 and sbb are required in neurons for successful diapause, consistent with the enrichment of this gene class in the diapause GWAS." While I acknowledge that the results support their neuronal functions, I remain unconvinced that these genes are required for "successful diapause". According to the RNAi scheme (Figure 4I), Dip-γ and sbb are downregulated only during the post-diapause period, but still show a significant effect, comparable to that seen in the nSyb Gal4 RNAi lines (Figure 4K). In addition, two other RNAi lines (SH330386, 80461) that did not show lethality did not affect post-diapause fecundity. Notably, RNAi (27049, KK104056) substantially reduced non-diapause fecundity, suggesting impairment of these genes affects fecundity in general regardless of diapause experience. Therefore, the reduced post-diapause fecundity observed may be a result of this broader effect on fecundity, particularly in a more "sensitized" state during the post-diapause period, rather than a direct regulation of adult diapause by these genes.

      (3) The authors characterized 546 genetic variants and 291 genes associated with phenotypic variation across DGRP lines but did not prioritize them by significance. They did prioritize candidate genes with multiple associated variants (p.9 "Genes with multiple SNPs are good candidates for influencing diapause traits."), but this is not a valid argument, likely due to a misunderstanding of LD among variants in the same gene. A gene with one highly significantly associated variant may be more likely to be the causal gene in a QTL than a gene with many weakly associated variants in LD. I recommend taking significance into account in the analysis.

    1. Reviewer #2 (Public Review):

      Summary:

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

      Strengths:

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

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

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

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

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

      Weaknesses:

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

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

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

    1. Reviewer #2 (Public Review):

      The authors performed a Multi-Omics Factor Analysis (MOFA) on analysis of two published MDS patient cohorts-1 from bone marrow mononuclear cells (BMMNCs) and CD34 cells (ref 17) and another from CD34+ cells (ref 15) --with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, Retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to attempt to identify the latent factors with significant impact on MDS prognosis.

      SF3B1 was found to be the only mutation among 13 mutations in the BMMNC cohort that indicated a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34+ cohort. The MOFA factor representing inflammation showed a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases showed a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Also, MOFA identified RTE expression as a risk factor for MDS. They proposed that this work showed the efficacy of their integrative approach to assess MDS prognostic risk that 'goes beyond all the scoring systems described thus far for MDS'.

      Several issues need clarification and response:

      (1) The authors do not provide adequate known clinical and molecular information which demonstrates prognostic risk of their sample cohorts in order to determine whether their data and approach 'goes 'beyond all the scoring systems described thus far for MDS'. For example, what data have the authors that their features provide prognostic data independent of the prior known factors related to prognosis (eg, marrow blasts, mutational, cytogenetic features, ring sideroblasts, IPSS-R, IPSS-M, MDA-SS)?

      (2) A major issue in analyzing this paper relates to the specific patient composition from whom the samples and data were obtained. The cells from the Shiozawa paper (ref 17) is comprised of a substantial number of CMML patients. Thus, what evidence have the authors that much of the data from the BMMNCs from these patients and mutant SRSF2 related predominantly to their monocytic differentiation state?

      (3) In addition, as the majority of patients in the Shiozawa paper have ring sideroblasts (n=59), thus potentially skewing the data toward consideration mainly of these patients, for whom better outcomes are well known.

      (4) Further, regarding this patient subset, what evidence have the authors that the importance of the SF3B1 mutation was merely related to the preponderance of sideroblastic patients from whom the samples were analyzed?

      (5) An Erratum was reported for the Shiozawa paper (Shiozawa Y, Malcovati L, Gallì A, et al. Gene expression and risk of leukemic transformation in myelodysplasia. Blood. 2018 Aug 23;132(8):869-875. doi: 10.1182/blood-2018-07-863134) that resulted from a coding error in the construction of the logistic regression model for subgroup prediction based on the gene expression profiles of BMMNCs. This coding error was identified after the publication of the article. The authors should indicate the effect this error may have had on the data they now report.

      (6) What information have the authors as to whether the differing RTE findings were not predominantly related to the differentiation state of the cell population analyzed (ie higher in BM MNCs vs CD34, Fig 1)? What control data have the authors regarding these values from normal (non-malignant) cell populations?

      (7) The statement in the Discussion regarding the effects of SRSF2 mutation is speculative and should be avoided. Many other somatic gene mutations have known stronger effects on prognosis for MDS.

    1. Reviewer #2 (Public Review):

      Summary:

      This manuscript investigates the role of the abundant NK cells that are observed in colon cancer liver metastasis using sequencing and spatial approaches in an effort to clarify the pro and anti-tumourigenic properties of NK cells. This descriptive study characterises different categories of NK cells in tumour and tumour-adjacent tissues and some correlations. An attempt has been made using pseudotime trajectory analysis but no models around how these NK cells might be regulated are provided.

      Strengths:

      This study integrates multiomics data to attempt to resolve correlates of protection that might be useful in understanding NK cell diversity and activation.

      Weaknesses:

      While this work is interesting, the power of such studies is in taking the discovered information and applying this to other cohorts to determine the strength and predictive power of the genes identified. It is also clear that these 'snapshots' analysed poorly take account of the dynamic temporal changes that occur within a tumour. It would have been good to see a proposed model of NK cell regulation as it might occur in the tumour (accounting for turnover and recruitment) beyond the static data.

    1. Reviewer #2 (Public Review):

      The fledgling field of epitranscriptomics has encountered various technical roadblocks with implications for the validity of early epitranscriptomics mapping data. As a prime example, the low specificity of (supposedly) modification-specific antibodies for the enrichment of modified RNAs, has been ignored for quite some time and is only now recognized for its dismal reproducibility (between different labs), which necessitates the development of alternative methods for modification detection. Furthermore, early attempts to map individual epitranscriptomes using sequencing-based techniques are largely characterized by the deliberate avoidance of orthogonal approaches aimed at confirming the existence of RNA modifications that have been originally identified.

      Improved methodology, the inclusion of various controls, and better mapping algorithms as well as the application of robust statistics for the identification of false-positive RNA modification calls have allowed revisiting original (seminal) publications whose early mapping data allowed making hyperbolic claims about the number, localization and importance of RNA modifications, especially in mRNA. Besides the existence of m6A in mRNA, the detectable incidence of RNA modifications in mRNAs has drastically dropped.

      As for m5C, the subject of the manuscript submitted by Zhou et al., its identification in mRNA goes back to Squires et al., 2012 reporting on >10.000 sites in mRNA of a human cancer cell line, followed by intermittent findings reporting on pretty much every number between 0 to > 100.000 m5C sites in different human cell-derived mRNA transcriptomes. The reason for such discrepancy is most likely of a technical nature. Importantly, all studies reporting on actual transcript numbers that were m5C-modified relied on RNA bisulfite sequencing, an NGS-based method, that can discriminate between methylated and non-methylated Cs after chemical deamination of C but not m5C. RNA bisulfite sequencing has a notoriously high background due to deamination artifacts, which occur largely due to incomplete denaturation of double-stranded regions (denaturing-resistant) of RNA molecules. Furthermore, m5C sites in mRNAs have now been mapped to regions that have not only sequence identity but also structural features of tRNAs. Various studies revealed that the highly conserved m5C RNA methyltransferases NSUN2 and NSUN6 do not only accept tRNAs but also other RNAs (including mRNAs) as methylation substrates, which in combination account for most of the RNA bisulfite-mapped m5C sites in human mRNA transcriptomes. Is m5C in mRNA only a result of the Star activity of tRNA or rRNA modification enzymes, or is their low stoichiometry biologically relevant?

      In light of the short-comings of existing tools to robustly determine m5C in transcriptomes, other methods - like DRAM-seq, that allow the mapping of m5C independently of ex-situ RNA treatment with chemicals - are needed to arrive at a more solid "ground state", from which it will be possible to state and test various hypotheses as to the biological function of m5C, especially in lowly abundant RNAs such as mRNA.

      Importantly, the identification of >10.000 sites containing m5C increases through DRAM-Seq, increases the number of potential m5C marks in human cancer cells from a couple of 100 (after rigorous post-hoc analysis of RNA bisulfite sequencing data) by orders of magnitude. This begs the question of whether or not the application of these editing tools results in editing artefacts overstating the number of actual m5C sites in the human cancer transcriptome.

      Comments:

      (1) The use of two m5C reader proteins is likely a reason for the high number of edits introduced by the DRAM-Seq method. Both ALYREF and YBX1 are ubiquitous proteins with multiple roles in RNA metabolism including splicing and mRNA export. It is reasonable to assume that both ALYREF and YBX1 bind to many mRNAs that do not contain m5C.

      To substantiate the author's claim that ALYREF or YBX1 binds m5C-modified RNAs to an extent that would allow distinguishing its binding to non-modified RNAs from binding to m5C-modified RNAs, it would be recommended to provide data on the affinity of these, supposedly proven, m5C readers to non-modified versus m5C-modified RNAs. To do so, this reviewer suggests performing experiments as described in Slama et al., 2020 (doi: 10.1016/j.ymeth.2018.10.020). However, using dot blots like in so many published studies to show modification of a specific antibody or protein binding, is insufficient as an argument because no antibody, nor protein, encounters nanograms to micrograms of a specific RNA identity in a cell. This issue remains a major caveat in all studies using so-called RNA modification reader proteins as bait for detecting RNA modifications in epitranscriptomics research. It becomes a pertinent problem if used as a platform for base editing similar to the work presented in this manuscript.

      (2) Since the authors use a system that results in transient overexpression of base editor fusion proteins, they might introduce advantageous binding of these proteins to RNAs. It is unclear, which promotor is driving construct expression but it stands to reason that part of the data is based on artifacts caused by overexpression. Could the authors attempt testing whether manipulating expression levels of these fusion proteins results in different editing levels at the same RNA substrate?

      (3) Using sodium arsenite treatment of cells as a means to change the m5C status of transcripts through the downregulation of the two major m5C writer proteins NSUN2 and NSUN6 is problematic and the conclusions from these experiments are not warranted. Sodium arsenite is a chemical that poisons every protein containing thiol groups. Not only do NSUN proteins contain cysteines but also the base editor fusion proteins. Arsenite will inactivate these proteins, hence the editing frequency will drop, as observed in the experiments shown in Figure 5, which the authors explain with fewer m5C sites to be detected by the fusion proteins.

      (4) The authors should move high-confidence editing site data contained in Supplementary Tables 2 and 3 into one of the main Figures to substantiate what is discussed in Figure 4A. However, the data needs to be visualized in another way than an Excel format. Furthermore, Supplementary Table 2 does not contain a description of the columns, while Supplementary Table 3 contains a single row with letters and numbers.

      (5) The authors state that "plotting the distribution of DRAM-seq editing sites in mRNA segments (5'UTR, CDS, and 3'UTR) highlighted a significant enrichment near the initiation codon (Figure 3F).", which is not true when this reviewer looks at Figure 3F.

      (6) The authors state that "In contrast, cells expressing the deaminase exhibited a distinct distribution pattern of editing sites, characterized by a prevalence throughout the 5'UTR.", which is not true when this reviewer looks at Figure 3F.

      (7) The authors claim in the final conclusion: "In summary, we developed a novel deaminase and reader protein assisted RNA m5C methylation approach...", which is not what the method entails. The authors deaminate As or Us close to 5mC sites based on the binding of a deaminase-containing protein.

      (8) The authors claim that "The data supporting the findings of this study are available within the article and its Supplementary Information." However, no single accession number for the deposited sequencing data can be found in the text or the supplementary data. Without the primary data, none of the claims can be verified.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors showed the applicability and usefulness of a new AlphaFold2 pipeline called PabFold, which can predict linear antibody epitopes (B-cell epitopes) that can be helpful for the selection of reagents to be applied in competitive ELISA assay.

      Strengths:

      The authors showed the accuracy of the pipeline to identify correctly the binding epitope for three different antibody-antigen systems (Myc, HA, and Sars-Cov2 nucleocapsid protein). The design of scFvs from Fab of the three antibodies to speed up the analysis time is extremely interesting.

      Weaknesses:

      The article justifies correctly the findings and no great weaknesses are present. However, it could be useful for a broader audience to show in detail how pLDDT was calculated for both Simple-Max approach (per residue-pLDDT) and Consensus analysis ( average pLDDT for each peptide), with associated equations.

    1. Reviewer #2 (Public Review):

      Lalwani et al. measured BOLD variability during the viewing of houses and faces in groups of young and old healthy adults and measured ventrovisual cortex GABA+ at rest using MR spectroscopy. The influence of the GABA-A agonist lorazepam on BOLD variability during task performance was also assessed, and baseline GABA+ levels were considered as a mediating variable. The relationship of local GABA to changes in variability in BOLD signal, and how both properties change with age, are important and interesting questions. The authors feature the following results: 1) younger adults exhibit greater task-dependent changes in BOLD variability and higher resting visual cortical GABA+ content than older adults, 2) greater BOLD variability scales with GABA+ levels across the combined age groups, 3) administration of a GABA-A agonist increased condition differences in BOLD variability in individuals with lower baseline GABA+ levels but decreased condition differences in BOLD variability in individuals with higher baseline GABA+ levels, and 4) resting GABA+ levels correlated with a measure of visual sensory ability derived from a set of discrimination tasks that incorporated a variety of stimulus categories.

      Strengths of the study design include the pharmacological manipulation for gauging a possible causal relationship between GABA activity and task-related adjustments in BOLD variability. The consideration of baseline GABA+ levels for interpreting this relationship is particularly valuable. The assessment of feature-richness across multiple visual stimulus categories provided support for the use of a single visual sensory factor score to examine individual differences in behavioral performance relative to age, GABA, and BOLD measurements. Weaknesses of the study include the absence of an interpretation of the physiological mechanisms that contribute to variability in BOLD signal, particularly for the chosen contrast that compared viewing houses with viewing faces. Whether any of the observed effects can be explained by patterns in mean BOLD signal, independent of variability would be useful to know. The positive correlation between resting GABA+ levels and the task-condition effect on BOLD variability reaches significance at the total group level, when the young and old groups are combined, but not separately within each group. This correlation may be explained by age-related differences since younger adults had higher values than older adults for both types of measurements. This is not to suggest that the relationship is not meaningful or interesting, but that it may be conceptualized differently than presented. Two separate dosages of lorazepam were used across individuals, but the details of why and how this was done are not provided, and the possible effects of the dose are not considered. The observation of greater BOLD variability during the viewing of houses than faces may be specific to these two behavioral conditions, and lingering questions about whether these effects generalize to other types of visual stimuli, or other non-visual behaviors, in old and young adults, limit the generalizability of the immediate findings.

      The observed age-related differences in patterns of BOLD activity and ventrovisual cortex GABA+ levels along with the investigation of GABA-agonist effects in the context of baseline GABA+ levels are particularly valuable to the field, and merit follow-up. Assessing background neurochemical levels is generally important for understanding individualized drug effects. Therefore, the data are particularly useful in the fields of aging, neuroimaging, and vision research.

    1. Reviewer #2 (Public Review):

      Grid cells - originally discovered in single-cell recordings from the rodent entorhinal cortex, and subsequently identified in single-cell recordings from the human brain - are believed to contribute to a range of cognitive functions including spatial navigation, long-term memory function, and inferential reasoning. Following a landmark study by Doeller et al. (Nature, 2010), a plethora of human neuroimaging studies have hypothesised that grid cell population activity might also be reflected in the six-fold (or 'hexadirectional') modulation of the BOLD signal (following the six-fold rotational symmetry exhibited by individual grid cell firing patterns), or in the amplitude of oscillatory activity recorded using MEG or intracranial EEG. The mechanism by which these network-level dynamics might arise from the firing patterns of individual grid cells remains unclear, however.

      In this study, Khalid and colleagues use a combination of computational modelling and mathematical analysis to evaluate three competing hypotheses that describe how the hexadirectional modulation of population firing rates (taken as a simple proxy for the BOLD, MEG, or iEEG signal) might arise from the firing patterns of individual grid cells. They demonstrate that all three mechanisms could account for these network-level dynamics if a specific set of conditions relating to the agent's movement trajectory and the underlying properties of grid cell firing patterns are satisfied.

      The computational modelling and mathematic analyses presented here are rigorous, clearly motivated, and intuitively described. In addition, these results are important both for the interpretation of hexadirectional modulation in existing data sets and for the design of future experiments and analyses that aim to probe grid cell population activity. As such, this study is likely to have a significant impact on the field by providing a firmer theoretical basis for the interpretation of neuroimaging data. To my mind, the only weakness is the relatively limited focus on the known properties of grid cells in rodent entorhinal cortex, and the network level activity that these firing patterns might be expected to produce under each hypothesis. Strengthening the link with existing neurobiology would further enhance the importance of these results for those hoping to assay grid cell firing patterns in recordings of ensemble-level neural activity.

    1. Reviewer #2 (Public Review):

      In this study, Notartomaso et al. used optical activation of systemic JF-NP-26, a caged, baseline inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices, thalamus, and BLA to investigate the roles of these receptors in various brain regions in pain processing. They found that alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, but this analgesic effect was reversed by light-induced drug inactivation in the prelimbic and infralimbic cortices, and thalamus. In contrast, these pharmacological effects were reversed in the BLA. They further found that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. They thus concluded that mGlu5 receptors had differential effects in distinct brain regions. mGlu5 receptors are important receptors in pain processing, and their regional specificity has not been studied in detail. Further, this is an interesting study regarding the use of optical activation of pro-drugs, and the findings are timely. The combination of in vivo pharmacology, biochemistry, and slice EP provides complementary results.

    1. Reviewer #2 (Public Review):

      Plaza-Alanso et al. characterize synaptic properties across human medial entorhinal cortex across layers and, importantly, across multiple individuals. Using an impressive collection of post-mortem autopsy samples, they generate high resolution 3d FIB-SEM volumes across layers and MEC subregions and measure features such as synapse density, spatial distribution, size, shape and target location. The use of volumes permits a richer local context to synaptic reconstructions, and the methods used to count and quantify synapses appear thorough and convincing, although with limited descriptions at times. The core findings suggest few differences in most properties across either layers or individuals, with some modest exceptions in layers 1 and 6. A particular strength of the dataset is the large number of high quality synaptic contact reconstructions.<br /> However, because the volumes have no specific labels and are too small to associate axons or dendrites with individual cells or cell types, it is not clear how to extrapolate these findings to new insights toward the stated goal of a better understanding of the networks and connectivity characteristics of the MEC. Broadly speaking, the paper would benefit from a better explanation of why these specific parameters were chosen and what the authors hoped to gain from them. It might be useful to think of what would need to be the case to see something substantially different. Many of the measures here reflect the properties of dendrites passing through a small volume, which depends on the number of cells of different cell types, the length and thickness of their dendritic arbors, synapse density distributions, local and long range afferents, and more. One interpretation of these results is that these neuropil volumes across layers and individuals are effectively fully packed with dendrites, with a similar ratio of excitatory and inhibitory neurons, dendrites with roughly similar thickness and synaptic input density and local E/I balance. Can the authors disentangle these cellular-scale contributions or constrain their inter-individual variability across individuals? The lack of variability is perhaps the main observation here, and understanding this more clearly could be useful for thinking about larger volumes where fewer replicates are currently possible.

    1. Reviewer #2 (Public Review):

      This prospective study advances our understanding of the predictive value of preoperative serum CA125, CA19-9, CA72-4, CEA, and AFP in endometrial cancer. The evidence supporting the conclusions is convincing with rigorous analysis of the association between prognostic values of several serum markers with the clinical data of endometrial cancer patients. However, there are a few areas in which the article may be improved through further validation of the prognostic value of the risk score in patients with endometrial cancer at different stages. Moreover, the authors should provide a more detailed explanation of the choice of statistical methods in the manuscript. The work will be of broad interest to clinicians, medical researchers and scientists working in endometrial cancer.

      (1) The groups of patients with endometrial cancer in the manuscript are classified according to age greater than/less than 60. Please explain why 60 years old is chosen as the boundary value of age.<br /> (2) Among the patients with endometrial cancer selected in the manuscript, AFP outliers accounted for a relatively small proportion. The authors chose the clinical detection outliers of CA-125, CA19-9, AFP and CEA as the dividing line, instead of re-selecting the optimal cut-off value in this population, which should be classified and the prognostic value explored.<br /> (3) In cancer research, stage is an important prognostic factor to guide the treatment of patients in clinical work. Patients with different stages of endometrial cancer have obvious prognostic differences. The authors constructed a new prognostic risk score based on serum level of AFP, CEA and CA125, the prognostic value of the risk score should be validated in patients with endometrial cancer at different stages。

    1. Reviewer #2 (Public Review):

      This study focuses on the role of miR221/222 in the pathogenesis of rheumatoid arthritis. Through the use of different murine models and genome-wide techniques, the authors individuate a miR221/222 elicited mechanism leading to synovial fibroblast hyperproliferation. These discoveries may provide a rationale for future targeted therapies for RA treatment.

      miR-221 and miR-222 have been linked with arthritis in previous studies from this and other laboratories: miR-221 and miR-222 have been found upregulated in SFs derived from the huTNFtg mouse model and RA patients, where their expression correlates with disease activity. The novelty of the present study resides in the analysis of the role of miR-221/miR-222 in an in vivo system and provides insight into cellular and molecular mechanisms linking miR-221/222 to RA progression.

    1. Reviewer #2 (Public Review):

      How and why nutritional requirements and intake targets change over development and differ between species are significant questions with wide-ranging implications spanning ecology to health. In this manuscript, Talal et al. set out to address these questions in laboratory and field experiments with grasshoppers and in a comparative analysis of different species.

      The authors conclude that the target intake of protein to non-protein energy (in this case carbohydrate) (P:C) falls over developmental stages and that this occurs because of a decline in mass-specific intake of protein whereas mass-specific carbohydrate intake remains more constant. The decrease in mass-specific protein consumption rate is tightly correlated with a decline in specific growth rate. Hence, protein consumption directly reflects requirements for growth, with hypometric scaling of protein intake serving as a useful relationship in nutritional ecology.

      The laboratory experiments on the locust, Schistocerca cancellata, provide an elegant dataset in which different instars have been provided with one of two nutritionally complementary food pairings differing in protein to carbohydrate (P: C) content, and their self-selected protein to carbohydrate "intake target" measured.

      These lab locust results were then compared with independently collected field data for late instar nymphs of the same locust species, and the conclusion is drawn that field insects ingested similar protein but 50-90% more carbohydrate (with only 23% increased mass-specific resting oxygen consumption rates). Numerous uncontrolled variables between the lab and field studies make meaningful conclusions difficult to draw from this observation.

      A graph is then provided showing comparative data across a selection of species, making the case that protein consumption scales similarly both developmentally and across taxa. Questions need to be addressed for this to be convincing, including which criteria were used to select the examples in the graph and how comprehensively do these represent the available literature.

    1. Reviewer #2 (Public Review):

      Strengths are that the topic is of significant interest and understudied and the combination of both genetic and pharmacological approaches. However, while there is great enthusiasm for the need to better understand mGluR5 roles in striatal circuitry, in its present form, there are three overarching concerns that significantly limit the impact of this study. First, while a Jaccard method is used to measure the spatiotemporal dynamics of dSPN activity, collectively the data herein do not support the authors' interpretation of the data that mGluR5 is a modulator of spatiotemporal dSPN dynamics. Specifically, pharmacological and genetic manipulations of mGluR5 do not differentially/preferentially modulate the activity of proximal vs distal dSPNs, therefore, it could also be interpreted that mGluR5 is blanketly boosting/suppressing all dSPN activity as opposed to differential proximal/distal spatial relationships. While this is acknowledged in the manuscript (Figure 2i), it leaves open for question the extent to which mGluR5 is modulating other aspects of dSPN activity independent of the spatiotemporal relationship across dSPNs (i.e. amplitude, firing probability, etc.). Second, while it is a strength that mGluR5 NAM, PAM, and D1 Cre mGluR5-cKO were used to bidirectionally manipulate mGluR5 signaling, the manuscript lacks a clear model of where mGluR5 is acting to affect dSPN activity. This concern can be readily addressed by treating D1 Cre mGluR5-cKO mice with the mGluR5 NAM (as described in Ln. 413-416) to determine the extent to which other sources of mGluR5 are contributing to dSPN activity. The authors' working model predicts that the NAM would have no significant effects on the D1 Grm5 cKO model. Third, there are some concerns about the statistical basis for conclusions that are drawn detailed below that when addressed will strengthen the rigor of the conclusions. Addressing these suggestions should strengthen the mechanistic understanding and further allow the authors to present a more clear working model for their findings.

  2. Jun 2024
    1. Reviewer #2 (Public Review):

      This paper uses a novel maze design to explore mouse navigation behaviour in an automated analogue of the Barnes maze. A major strength is the novel and clever experimental design which rotates the floor and intramaze cues before the start of each new trial, allowing the previous goal location to become the next starting position. The modelling sampling a Markov chain of navigation strategies is elegant, appropriate and solid, appearing to capture the behavioural data well. This work provides a valuable contribution and I'm excited to see further developments, such as neural correlates of the different strategies and switches between them.

    1. Reviewer #2 (Public Review):

      Summary:

      This paper describes evolution experiments performed on yeast amino acid transporters aiming at the enlargement of the substrate range of these proteins. Yeast cells lacking 10 endogenous amino acid transporters and thus being strongly impaired to feed on amino acids were again complemented with amino acid transporters from yeast and grown on media with amino acids as the sole nitrogen source.

      In the first set of experiments, complementation was done with seven different yeast amino acid transporters, followed by measuring growth rates. Despite most of them having been described before in other experimental contexts, the authors show that many of them have a broader substrate range than initially thought.

      Moving to the evolution experiments, the authors used the OrthoRep system to perform random mutagenesis of the transporter gene while it is actively expressed in yeast. The evolution experiments were conducted such that the medium would allow for poor/slow growth of cells expressing the wt transporters, but much better/faster growth if the amino acid transporter would mutate to efficiently take up a poorly transported (as in case of citrulline and AGP1) or non-transported (as in case of Asp/Glu and PUT4) amino acid.

      This way and using Sanger sequencing of plasmids isolated from faster-growing clones, the authors identified a number of mutations that were repeatedly present in biological replicates. When these mutations were re-introduced into the transporter using site-directed mutagenesis, faster growth on the said amino acids was confirmed. Growth phenotype were confirmed by uptake experiments using radioactive amino acids; corresponding correlation plots show that the assays based on growth rates versus radioactive uptake assays indeed can explain the effect of the mutations to a large extent.

      When mapped to Alphafold prediction models on the transporters, the mutations mapped to the substrate permeation site, which suggests that the changes allow for more favorable molecular interactions with the newly transported amino acids.<br /> Finally, the authors compared growth rates of the evolved transporter variants with those of the wt transporter and found that some variants exhibit a somewhat diminished capacity to transport its original range of amino acids, while other variants were as fit as the wt transporter in terms of uptake of its original range of amino acids.<br /> Based on these findings, the author conclude that transporters can evolve novel substrates through generalist intermediates, either by increasing a weak activity or by establishing a new one.

      Strengths:

      The study provides evidence in favour of an evolutionary model, wherein a transporter can "learn" to translocate novel substrates without "forgetting" what it used to transport before. This evolutionary concept has been proposed for enzymes before, and this study shows that it also can apply to transporters. The concept behind the study is easy to understand, i.e. improving growth by uptake of more amino acids as nitrogen source. In addition, the study contains a large and extensive characterization of the transporter variants, including growth assays and radioactive uptake measurements. The authors performed experiments as part of the revision to show that the studied mutations do not greatly change surface expression of the transporters. Further they showed that in the absence of the evolutionary pressure, overexpression of the mutants versus the wildtype transporters does not affect growth rates, which is important to assess. Finally, the authors make careful conclusions saying that in real life, the evolutionary landscape is way more complex than under these "reductive" laboratory conditions with a strain lacking ten natively expressed amino acid transporters and being selected on a single amino acid in a defined medium.

      Weaknesses:

      The authors took a genetic gain-of-function approach based on random mutagenesis of the transporter. While this experimental approach is suited to find some gain-of-function variants for some of the amino acids, it has also its inherent limitations, the most important being that loss-of-function mutants are not sampled (though they might be interesting) and that mutagenesis is entirely random, thus not targeted. These weaknesses cannot be easily overcome other than by restarting the entire study and conducting for example deep mutational scanning experiments. The authors have done what they could do within the scope of this study to make this manuscript as complete and rigorous as possible.

    1. Reviewer #2 (Public Review):

      Neuromodulators are important for circuit function, but their roles in the retinal circuitry are poorly understood. This study by Gonschorek and colleagues aims to determine the modulatory effect of nitric oxide on the response properties of retinal ganglion cells. The authors used two photon calcium imaging and multi-electrode arrays to classify and compare cell responses before and after applying a NO donor DETA-NO. The authors found that DETA-NO selectively increases activity in a subset of contrast-suppressed RGC types. In addition, the authors found cell-type specific changes in light response in the absence of pharmacological manipulation in their calcium imaging paradigm. While this study focuses on an important question and the results are interesting, the following issues need further clarification for better interpretation of the data.

      (1) Design of the calcium imaging experiments: the control-control pair has a different time course from the control-drug pair (Fig 1e). First, the control-control pair has a 10 minute interval while the control-drug pair has a 25 minute interval. Second, Control 1 Field 2 was imaged 10 min later than Control 1 Field 1 since the start of the calcium imaging paradigm.

      Given that the control dataset is used to control for time-dependent adaptational changes throughout the experiment, I wonder why the authors did not use the same absolute starting time of imaging and the same interval between the first and second round of imaging for both the control-control and the control-drug pairs. This can be readily done in one of the two ways: 1. In a set of experiment, add DETA/NO between "Control 1 Field 1 and "Control 2 Field 1" in Fig. 1e as the drug group; or 2. Omit DETA/NO in the Fig. 1e protocol as the control group to monitor the time course of adaptational changes.

      Related to the concern above, to determine NO-specific effect, the authors used the criterion that "the response changes observed for control (ΔR(Ctrl2−Ctrl1)) and NO (ΔR(NO−Ctrl1)) were significantly different". This criterion assumes that without DETA-NO, imaging data obtained at the time points of "Control 1 Field 2" and "DETA/NO Field 2" would give the same value of ΔR as ΔR(Ctrl2−Ctrl1) for all RGC types. It is not obvious to me why this should be the case, because of the unknown time-dependent trajectory of the adaptational change for each RGC type. For example, a RGC type could show stable response in the first 30 min and then change significantly in the following 30 min. DETA/NO may counteract this adaptational change, leading to the same ΔR as the control condition (false negative). Alternatively, DETA/NO may have no effect, but the nonlinear time-dependent response drift can give false positive results.

      I also wonder why washing-out, a standard protocol for pharmacological experiments, was not done for the calcium protocol since it was done in the MEA experiments. A reversible effect by washing in and out DETA/NO in the calcium protocol would provide a much stronger support that the observed NO modulation is due to NO and not to other adaptive changes.

      (2) Effects of Strychnine: In lines 215-219, " In the light-adapted retina, On-cone BCs boost light-Off responses in Off-cone BCs through cross-over inhibition (83, 84) and hence, strychnine affects Off-response components in RGCs - in line with our observations (Fig. S2)" However, Fig. S2 doesn't seem to show a difference in the Off-response components. Rather, the On response is enhanced with strychnine. In addition, suppressed-by-contrast cells are known to receive glycinergic inhibition from VGluT3 amacrine cells (Tien et al., 2016). However, the G32 cluster in Fig. S2 doesn't seem to show a change with strychnine. More explanation on these discrepancies will be helpful.

      (3) This study uses DETA-NO as an NO donor for enhancing NO release. However, a previous study by Thompson et al., Br J Pharmacol. 2009 reported that DETA-NO can rapidly and reversible induce a cation current independent of NO release at the 100 uM used in the current study, which could potentially cause the observed effect in G32 cluster such as reduced contrast suppression and increased activity. This potential caveat should at least be discussed, and ideally excluded by showing the absence of DETA-NO effects in nNOS knockout mice, and/or by using another pharmacological reagent such as the NO donor SNAP or the nNOS inhibitor l-NAME.

      (4) Clarification of methods: In the Methods, lines 1119-1127, the authors describe the detrending, baseline subtraction, and averaging. Then, line 1129, " the mean activity r(t) was computed and then traces were normalized such that: max t(|r(t)|) = 1. How is the normalization done? Is it over the entire recording (control and wash in) for each ROI? Or is it normalized based on the mean trace under each imaging session (i.e. twice for each imaging field)?

      As for the clustering of RGC types, I assume that each ROI's cluster identity remains unchanged through the comparison. If so, it may be helpful to emphasize this in the text.

    1. Reviewer #2 (Public Review):

      Summary:

      This manuscript by Liu et al. uses Confetti labeling of hematopoietic stem and progenitor cells in situ to infer the clonal dynamics of adult hematopoiesis. The authors apply a new mathematical framework to analyze the data, allowing them to increase the range of applicability of this tool up to tens of thousands of precursors. With this tool, they (1) provide evidence for the large polyclonality of adult hematopoiesis, (2) offer insights on the expansion dynamics in the fetal liver stage, (3) assess the clonal dynamics in a Fanconi anemia model (Fancc), which has engraftment defects during transplantation.

      Strengths:

      The manuscript is well written, with beautiful and clear figures, and both methods and mathematical models are clear and easy to understand.

      Since 2017, Mikel Ganuza and Shannon McKinney-Freeman have been using these Confetti approaches that rely on calculating the variance across independent biological replicates as a way to infer clonal dynamics. This is a powerful tool and it is a pleasure to see it being implemented in more labs around the world. One of the cool novelties of the current manuscript is using a mathematical model (based on a binomial distribution) to avoid directly regressing the Confetti labeling variance with the number of clones (which only has linearity for a small range of clone numbers). As a result, this current manuscript of Liu et al. methodologically extends the usability of the Confetti approach, allowing them more precise and robust quantification.

      They then use this model to revisit some questions from various Ganuza et al. papers, validating most of their conclusions. The application to the clonal dynamics of hematopoiesis in a model of Fanconi anemia (Fancc mice) is very much another novel aspect, and shows the surprising result that clonal dynamics are remarkably similar to the wild-type (in spite of the defect that these Fancc HSCs have during engraftment).<br /> Overall, the manuscript succeeds at what it proposes to do, stretching out the possibilities of this Confetti model, which I believe will be useful for the entire community of stem cell biologists, and possibly make these assays available to other stem cell regenerating systems.

      Weaknesses:

      My main concern with this work is the choice of CreER driver line, which then relates to some of the conclusions made. Scl-CreER succeeds at being as homogenous as possible in labeling HSC/MPPs... however it is clear that it also labels a subcompartment of HSC clones that become dominant with time... This is seen as the percentage of Confetti-recombined cells never ceases to increase during the 9-month chase of labeled cells, suggesting that non-labeled cells are being replaced by labeled cells. The reason why this is important is that then one cannot really make conclusions about the clonal dynamics of the unlabeled cells (e.g. for estimating the total number of clones, etc.).

      I am not sure about the claims that the data shows little precursor expansion from E11 to E14. First, these experiments are done with fewer than 5 replicates, and thus they have much higher error, which is particularly concerning for distinguishing differences of such a small number of clones. Second, the authors do see a ~0.5-1 log difference between E11 and E14 (when looking at months 2-3). When looking at months 5+, there is already a clear decline in the total number of clones in both adult-labeled and embryonic-labeled, so these time points are not as good for estimating the embryonic expansion. In any case, the number of precursors at E11 (which in the end defines the degree of expansion) is always overestimated (and thus, the expansion underestimated) due to the effects of lingering tamoxifen after injection (which continues to cause Confetti allele recombination as stem cell divide). Thus, I think these results are still compatible with expansion in the fetal liver (the degree of which still remains uncertain to me).

    1. Reviewer #2 (Public Review):

      Summary:

      The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).

      Strengths:

      The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.

      Weaknesses:

      The final section of the results - concerning the weighting of polarised light cues into the path integrator - lacks clarity and should be reworked and expanded in both the Methods and the Results (also possibly with an extra methods figure). I was really unsure of what these experiments were trying to show or what the meaning of the results actually are.

      Impact:

      The authors have discovered that nocturnal bull ants while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this is the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.

    1. Bloomington stock 4559

      DOI: 10.1038/s41598-022-26530-2

      Resource: RRID:BDSC_4559

      Curator: @DavidDeutsch

      SciCrunch record: RRID:BDSC_4559


      What is this?

    2. Bloomington Drosophila Stock Center

      DOI: 10.1038/s41598-022-26530-2

      Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

      Curator: @DavidDeutsch

      SciCrunch record: RRID:SCR_006457


      What is this?

    1. Reviewer #2 (Public Review):

      Summary:

      In the manuscript, Hartman et al. present a detailed comparison of 6 distinct multiplexed in situ gene expression profiling technologies, including both academic and commercial systems.

      The main concept of the study is to evaluate publicly accessible mouse brain datasets provided by the platforms' developers, where optimal performance in showcasing their technologies is expected. The authors stress the difficulty of making a comparison with standard metrics, e.g., the count of total molecules per cell, considering the differences in gene panel sizes across platforms. To make a fair comparison, the authors conceived a metric of specificity performance, which is called "MECR", an average of mutually exclusive gene co-expression rates in the sample. The authors found that the rate mainly depends on the choice of cell segmentation method, thus reanalyzed 5 of these datasets (excluding STARmap PLUS, due to the lack of molecule location information) with an independent cell segmentation algorithm (i.e., Baysor). Based on the reanalysis, the authors clearly suggest the best-performing platform at the end of the manuscript.

      Strengths:

      I consider that the paper is a valuable contribution to the community, for the following two reasons:

      (1) As the authors mentioned, I fully agree that the spatial transcriptomics community indeed needs better metrics in terms of comparison across technologies, rather than traditional metrics, e.g., molecule counts per cell. In that regard, I believe introducing a new metric, MECR, is quite valuable.

      (2) This work highlights the differences in results based on the choice of cell segmentation used for each platform, which suggests a need for trying out different segmentation algorithms to derive the right results. I believe this is an urgent warning that should be widespread in the community as soon as possible.

      Weaknesses:

      I disagree with the conclusion of the manuscript where the authors compare the technologies and suggest the best-performing ones, because of the following major points:

      (1) As the authors mentioned, MECR is a measure of "specificity" not "sensitivity". Still, the comparison of sensitivity was done with the mean counts per cell (Figure 3e). However, I strongly disagree with using the mean counts per cell as a measure of sensitivity because the comparison was done with different gene panels. The counts per cell can be highly dependent on the choice of genes, especially due to optical crowding.

      (2) The authors compared sensitivity based on the Baysor cell segmentation, but in fact, Baysor uses spatial gene expression for cell segmentation, which depends on the sensitivity of the platform. Thus, a comparison of sensitivity based on an algorithm that is based on sensitivity seems to be nonsensical.

    1. Reviewer #2 (Public Review):

      Summary:

      This study takes a new approach to studying the role of corticofugal projections from auditory cortex to inferior colliculus. The authors performed two-photon imaging of cortico-recipient IC neurons during a click detection task in mice with and without lesions of auditory cortex. In both groups of animals, they observed similar task performance and relatively small differences in the encoding of task-response variables in the IC population. They conclude that non-cortical inputs to the IC can provide substantial task-related modulation, at least when AC is absent.

      Strengths:

      This study provides valuable new insight into big and challenging questions around top-down modulation of activity in the IC. The approach here is novel and appears to have been executed thoughtfully. Thus, it should be of interest to the community.

      Weaknesses:

      Analysis of single unit activity is limited in its scope.

    1. Reviewer #2 (Public Review):

      Summary:

      Hwang, Ran-Der et al utilized a CRISPR-Cas9 knockout in human retinal pigment epithelium (RPE1) cells to evaluate for suppressors of toxicity by the proteasome inhibitor MG132 and identified that knockout of dihydrolipoamide branched chain transacylase E2 (DBT) suppressed cell death. They show that DBT knockout in RPE1 cells does not alter proteasome or autophagy function at baseline. However, with MG132 treatment, they show a reduction in ubiquitinated proteins but with no change in proteasome function. Instead, they show that DBT knockout cells treated with MG132 have improved autophagy flux compared to wildtype cells treated with MG132. They show that MG132 treatment decreases ATP/ADP ratios to a greater extent in DBT knockout cells, and in accordance causes activation of AMPK. They then show downstream altered autophagy signaling in DBT knockout cells treated with MG132 compared to wild-type cells treated with MG132. Then they express the ALS mutant TDP43 M337 or expanded polyglutamine repeats to model Huntington's disease and show that knockdown of DBT improves cell survival in RPE1 cells with improved autophagic flux. They also utilize a Drosophila models and show that utilizing either a RNAi or CRISPR-Cas9 knockout of DBT improves eye pigment in TDP43M337V and polyglutamine repeat-expressing transgenic flies. Finally, they show evidence for increased DBT in postmortem spinal cord tissue from patients with ALS via both immunoblotting and immunofluorescence.

      Strengths:

      This is a mechanistic and well-designed paper that identifies DBT as a novel regulator of proteotoxicity via activating autophagy in the setting of proteasome inhibition. Major strengths include careful delineation of a mechanistic pathway to define how DBT is protective. These conclusions are well-justified.

      Weaknesses:

      None

    1. Reviewer #2 (Public Review):

      Summary:

      A new toolbox is presented that builds on previous toolboxes to distinguish between real and spurious oscillatory activity, which can be induced by non-sinusoidal waveshapes. Whilst there are many toolboxes that help to distinguish between 1/f noise and oscillations, not many tools are available that help to distinguish true oscillatory activity from spurious oscillatory activity induced in harmonics of the fundamental frequency by non-sinusoidal waveshapes. The authors present a new algorithm which is based on autocorrelation to separate real from spurious oscillatory activity. The algorithm is extensively validated using synthetic (simulated) data, and various empirical datasets from EEG, and intracranial EEG in various locations and domains (i.e. auditory cortex, hippocampus, etc.).

      Strengths:

      Distinguishing real from spurious oscillatory activity due to non-sinusoidal waveshapes is an issue that has plagued the field for quite a long time. The presented toolbox addresses this fundamental problem which will be of great use for the community. The paper is written in a very accessible and clear way so that readers less familiar with the intricacies of Fourier transform and signal processing will also be able to follow it. A particular strength is the broad validation of the toolbox, using synthetic, scalp EEG, EcoG, and stereotactic EEG in various locations and paradigms.

      Weaknesses:

      A weakness is that the algorithm seems to be quite conservative in identifying oscillatory activity which may render it only useful for analyzing very strong oscillatory signals (i.e. alpha), but less suitable for weaker oscillatory signals (i.e. gamma).

    1. Reviewer #2 (Public Review):

      Summary:

      The authors set out to non-invasively track neuronal development in rat neonates, which they achieved with notable success. However, the direct relationship between the results and broader conclusions regarding developmental biology and potential human implications is somewhat overstretched without further validation.

      Strengths:

      If adequately revised and validated, this work could have a significant impact on the field, providing a non-invasive tool for longitudinal studies of brain development and neurodevelopmental disorders in preclinical settings.

      Weaknesses:

      (1) Consistency and Logical Flow:

      - The manuscript suffers from a lack of strategic flow in some sections. Specifically, transitions between major findings and methodological discussions need refinement to ensure a logical progression of ideas. For example, the jump from the introduction of developmental trajectories and the technicalities of MRS (Magnetic Resonance Spectroscopy) processing on page 3 could benefit from a bridging paragraph that explicitly states the study's hypotheses based on existing literature gaps.

      (2) Scientific Rigour:

      - While the novel application of diffusion-weighted MRS is commendable, there's a notable gap in the rigorous validation of this approach against gold-standard histological or molecular techniques. Particularly, the assertions regarding the sphere fraction and morphological changes inferred from biophysical modelling mandates direct validation to solidify the claims made. A study comparing these in vivo findings with ex vivo confirmation in at least a subset of samples would significantly enhance the reliability of these conclusions.

      (3) Clarity and Novelty:

      - The manuscript often delves deeply into technical specifics at the expense of accessibility to readers not deeply familiar with MRS technology. The introduction and discussions would benefit from a clearer elucidation of why these specific metabolite markers were chosen and their known relevance to neuronal and glial cells, placing this in the context of what is novel compared to existing literature.<br /> - The novelty aspect could be reinforced by a more structured discussion on how this method could change the current understanding or practices within neurodevelopmental research, compared to the current state of the art.

      (4) Completeness:

      - The Discussion section requires expansion to offer a more comprehensive interpretation of how these findings impact the broader field of neurodevelopment and psychiatric disorders. Specifically, the implications for human studies or clinical translation are touched upon but not fully explored.<br /> - Further, while supplementary material provides necessary detail on methodology, key findings from these analyses should be summarized and discussed in the main text to ensure the manuscript stands complete on its own.

      (5) Grammar, Style, Orthography:

      - There are sporadic grammatical and typographical errors throughout the text which, while minor, detract from the overall readability. For example, inconsistencies in metabolite abbreviations (e.g., tCr vs Cr+PCr) should be standardized.

      (6) References and Additional Context:

      - The current reference list is extensive but lacks integration into the narrative. Direct comparisons with existing studies, especially those with conflicting or supportive findings, are scant. More dedicated effort to contextualize this work within the existing body of knowledge would be beneficial.