Reviewer #2 (Public review):

In this manuscript, Epiney et al., present a single-nucleus sequencing analysis of Drosophila adult central brain neurons and glia. By employing an ingenious permanent labeling technique, they trace the progeny of T2 neuroblasts, which play a key role in the formation of the central complex. This transcriptomic dataset is poised to become a valuable resource for future research on neurogenesis, neuron morphology, and behavior.

The authors further delve into this dataset with several analyses, including the characterization of neurotransmitter expression profiles in T2-derived neurons. While some of the bioinformatic analyses are preliminary, they would benefit from additional experimental validation in future studies.

Comments on revisions:

We appreciate the authors' efforts to address some of the comments. While these revisions have improved the clarity of certain sections, some of the larger concerns remain unaddressed. Specifically, the manuscript still lacks the additional analyses that would allow for more specific conclusions, rather than the general observations currently presented. Although the revisions have certainly made the text clearer, the core issue of needing more detailed analysis to draw more concrete conclusions still stands.

Reviewer #2 (Public review):

Summary:

The authors performed a series of population genetic analyses in Lantana camara using 19,008 genome-wide SNPs data from 359 individuals in India. They found a clear population structure that did not show a geographical pattern, and that flower color was rather associated with population structure. Excess of homozygosity indicates a high selfing rate, which may lead to fixation of alleles in local populations and explain the presence of population structure without a clear geographic pattern. The authors also performed a forward simulation analysis, theoretically confirming that selfing promotes fixation of alleles (higher Fst) and reduction in genetic diversity (lower heterozygosity).

Strengths:

Biological invasion is a critical driver of biodiversity loss, and it is important to understand how invasive species adapt to novel environments despite limited genetic diversity (genetic paradox of biological invasion). Lantana camara is one of the hundred most invasive species in the world (IUCN 2000), and the authors collected 359 plants from a wide geographical range in India, where L. camara has invaded. The scale of the dataset and the importance of the target species are the strengths of the present study.

Weaknesses:

One of the most critical weaknesses of this study would be that the output modelling analysis is largely qualitative, which cannot be directly comparable to the empirical data. The main findings of the SLiM-based simulation were that selfing promotes the fixation of alleles and the reduction of genetic diversity. These are theoretically well-reported knowledge, and such findings themselves are not novel, although it may have become interesting these findings are quantitatively integrated with their empirical findings in the studied species. In that sense, a coalescent-based analysis such as an Approximate Bayesian Computation method (e.g. DIY-ABC) utilizing their SNPs data would be more interesting. For example, by ABC-based methods, authors can infer the split time between subpopulations identified in this study. If such split time is older than the recorded invasion date, the result supports the scenario that multiple introductions may have contributed to the population structure of this species. In the current form of the manuscript, multiple introductions were implicated but not formally tested.

I also have several concerns regarding the authors' population genetic analyses. First, the authors removed SNPs that were not in Hardy-Weinberg equilibrium (HWE), but the studied populations would not satisfy the assumption of HWE, i.e., random mating, because of a high level of inbreeding. Thus, the first screening of the SNPs would be biased strongly, which may have led to spurious outputs in a series of downstream analyses. Second, in the genetic simulation, it is not clear how a set of parameters such as mutation rate, recombination rate, and growth rate were determined and how they are appropriate. Importantly, while authors assume the selfing rate in the simulation, selfing can also strongly influence the effective mutation rate (e.g. Nordborg & Donnelly 1997 Genetics, Nordborg 2000 Genetics). It is not clear how this effect is incorporated in the simulation. Third, while the authors argue the association between flower color and population structure, their statistical associations were not formally tested. Also, it is not mentioned how flower color polymorphisms are defined. Could it be possible to distinguish many flower color morphs shown in Figure 1b objectively? I am concerned particularly because the authors also mentioned that flower color may change temporally and that a single inflorescence can have flowers of different colors (L160).

Reviewer #3 (Public review):

Summary:

The authors utilized ChIP-seq on strains containing tagged transcription factor (TF)-overexpression plasmids to identify binding sites for 172 transcription factors in P. aeruginosa. High-quality binding site data provides a rich resource for understanding regulation in this critical pathogen. These TFs were selected to fill gaps in prior studies measuring TF binding sites in P. aeruginosa. The authors further perform a structured analysis of the resulting transcriptional regulatory network, focusing on regulators of virulence and metabolism, in addition to performing a pangenomic analysis of the TFs. The resulting dataset has been made available through an online database. While the implemented approach to determining functional TF binding sites has limitations, the resulting dataset still has substantial value to P. aeruginosa research.

Strengths:

The generated TF binding site database fills an important gap in regulatory data in the key pathogen P. aeruginosa. Key analyses of this dataset presented include an analysis of TF interactions and regulators of virulence and metabolism, which should provide important context for future studies into these processes. The online database containing this data is well organized and easy to access. As a data resource, this work should be of significant value to the infectious disease community.

Weaknesses:

Drawbacks of the study include 1) challenges interpreting binding site data obtained from TF overexpression due to unknown activity state of the TFs on the measured conditions, 2) limited practical value of the presented TRN topological analysis, and 3) lack of independent experimental validation of the proposed master regulators of virulence and metabolism.

Reviewer #2 (Public review):

Summary:

Earlier behavioral data in the budgerigar have suggested frequency selectivity that was different from that in many other avian species, showing particularly good selectivity at around 3-4 kHz. It was unknown whether this unusual selectivity was determined in the inner ear, or whether it was a more central adaptation. The results using direct auditory-nerve tuning curves and less invasive stimulus-frequency otoacoustic emissions, suggest fairly normal-looking cochlear tuning in the budgerigar, implying that any behavioral/perceptual differences in frequency selectivity are likely more central in original.

Strengths:

- The study presents novel data in budgerigar, comparing the bandwidths of auditory-nerve tuning curves with the latencies of stimulus-frequency otoacoustic emissions (SFOAEs), which are thought to reflect the sharpness of cochlear tuning.<br /> - Using a conversion factor taken from previous data in the chicken to avoid circularity of reasoning, the study shows quite good correspondence between the non-invasive estimates obtained from SFOAEs and the tuning obtained from auditory-nerve fibers. Similarity between budgerigar and chicken are harder to ascertain with the way the data are presented.

Weaknesses:

- The comparison of SFOAEs and auditory-nerve tuning curves in the most interesting regions (beyond 3.5 kHz, where some perceptual anomalies seem to occur in some previous data), relies on an extrapolation of the data from the chicken.<br /> - No new behavioral data are presented, so the comparisons made in the paper are between studies separated by decades. None of the behavioral studies cited used the more current techniques that have been claimed to provide a behavioral estimate of cochlear tuning.

Reviewer #3 (Public review):

In this study, the authors employ both computational and experimental methods to reveal functional conservation of RIP family kinases and associated proteins in animals, with particular focus on mammals and other major groups of vertebrates. The bionformatic part of the work involves genomic data from diverse animal groups, providing insightful data on loss and duplications patterns for RIP and other necroptosis-related genes, and positive selection signals for RIPK1/3 genes in certain mammalian clades. These findings are then extensively used for selecting species and RHIM tetrad candidates for further experiments, in which the authors demonstrate different modes of functional conservation for RIPK proteins in necroptosis and NF-kB signaling across vertebrate species.

As an only major drawback, I would mention several important findings which the authors make in the course of their research but do not pursue further in the experimental part of the paper. These include:

• An additional copy for RIPK2 (RIPK2B) found in monotremes and non-mammalian vertebrates and its functions;<br /> • The entire diversity of RHIM functional tetrad variants; of particular interest here are IQFG and IQLG tetrads specific for bats, which are known to harbor human-affecting viruses and were demonstrated to have their RIPK1/3 genes under positive selection in this study;<br /> • Functions and involvement of RIPK3 protein in NF-kB pathway in lampreys;<br /> • The mode of NF-kB activation in non-mammalian species retaining ZBP1 copies.

Further elucidation of some or all of these points in the experimental part would facilitate conceptualizing the paper's numerous findings, which otherwise might appear insufficiently scrutinized. On the other hand, I agree that at least some of them require separate studies to be elucidated in. Given the importance of the results presented in this paper, I believe these points will be further addressed in future works.

Reviewer #2 (Public review):

Summary:

The revised manuscript by Zhao and colleagues presents a novel and compelling investigation into the p53 isoforms, Δ133p53 and Δ160p53, which are implicated in aggressive cancer phenotypes. The primary goal of this study was to elucidate how these isoforms exert a dominant-negative impact on the activity of full-length p53 (FLp53). The authors demonstrate that the Δ133p53 and Δ160p53 isoforms display impaired binding to p53-regulated promoters. Their findings suggest that the dominant-negative effects observed are primarily due to the co-aggregation of FLp53 with Δ133p53 and Δ160p53.

Overall, the study is innovative, thoroughly executed, and supported by robust data analysis. The authors have effectively addressed the reviewers' criticisms and incorporated their suggestions in this revised manuscript.

Significance:

The manuscript by Zhao and colleagues presents a novel and compelling study on the p53 isoforms, Δ133p53 and Δ160p53, which are associated with aggressive cancer types. The main objective of the study was to understand how these isoforms exert a dominant negative effect on full-length p53 (FLp53). The authors discovered that the Δ133p53 and Δ160p53 proteins exhibit impaired binding to p53-regulated promoters. The data suggest that the predominant mechanism driving the dominant-negative effect is the co-aggregation of FLp53 with Δ133p53 and Δ160p53.

Reviewer #2 (Public review):

Summary:

Prével et al. present an in vivo study in which they reveal an interesting aspect of β-glucan, a known inducer of enhanced immune responses termed trained immunity in sterile inflammation. The authors can show that β-glucan's can reprogram alveolar macrophages (AMs) in the lungs through neutrophils and IFNγ signaling and independent of Dectin1. This reprogramming occurs at both transcriptional and metabolic levels. After β-glucan training, LPS induced sterile inflammation exacerbated acute lung injury via enhanced immunopathology. These findings highlight a new aspect of β-glucan's role in trained immunity and its potential detrimental effects when enhanced pathogen clearance is not required.

Strengths:

- This manuscript is well-written and effectively conveys its message.

- The authors provide important evidence that β-glucan training is not solely beneficial but depending on the context can also enhance immunopathology. This will be important to the field for two reasons. It shows again that trained immunity can also be harmful. Jentho et al. 2021 had already provided further evidence for this aspect. And it highlights anew that LPS application is an insufficient infection model.

Original weaknesses noted:

- Only a little physiological data from the in vivo models is provided.

- Effects in histology appear to be rather weak.

Comments on latest version:

The authors have revised the new version according to my suggestions or responded in a sufficient manner to my requests, with one exception. I recommend to rename TNF as explained by Grimstad in JAMA Dermatol. 2016;152(5):557.

Reviewer #2 (Public review):

Summary:

Kunkel et al aim to answer a fundamental question: Do placebo and nocebo effects differ in magnitude or longevity? To address this question, they used a powerful within-participants design, with a very large sample size (n=104), in which they compared placebo and nocebo effects - within the same individuals - across verbal expectations, conditioning, testing phase, and a 1-week follow-up. With elegant analyses, they establish that different mechanisms underlie the learning of placebo vs nocebo effects, with the latter being acquired faster and extinguished slower. This is an important finding for both the basic understanding of learning mechanisms in humans and for potential clinical applications to improve human health.

Strengths:

Beyond the above - the paper is well-written and very clear. It lays out nicely the need for the current investigation and what implications it holds. The design is elegant, and the analyses are rich, thoughtful, and interesting. The sample size is large which is highly appreciated, considering the longitudinal, in-lab study design. The question is super important and well-investigated, and the entire manuscript is very thoughtful with analyses closely examining the underlying mechanisms of placebo versus nocebo effects.

Weaknesses:

There were two highly addressable weaknesses in my opinion:

(1) I could not find the preregistration - this is crucial to verify what analyses the authors have committed to prior to writing the manuscript. Please provide a link leading directly to the preregistration - searching for the specified number in the suggested website yielded no results.

(2) There is a recurring issue which is easy to address: because the Methods are located after the Results, many of the constructs used, analyses conducted, and even the main placebo and nocebo inductions are unclear, making it hard to appreciate the results in full. I recommend finding a way to detail at the beginning of the results section how placebo and nocebo effects have been induced. While my background means I am familiar with these methods, other readers will lack that knowledge. Even a short paragraph or a figure (like Figure 4) could help clarify the results substantially. For example, a significant portion of the results is devoted to the conditioning part of the experiment, while it is unknown which part was involved (e.g., were temperatures lowered/increased in all trials or only in the beginning).

Reviewer #2 (Public review):

Summary:

In this work, Monroe JG and colleagues show a compelling case of convergent evolution in the fusion between an important mismatch repair protein (MSH6) and histone reader domains across the tree of life. These fused MSH6 readers have been shown to be important for the recruitment of MSH6 to exon-rich genome locations, therefore improving the efficiency of reducing mutation rates in coding regions.

Comparative genomic analyses here performed revealed independent instances of MSH6 fusion with histone readers in plants and metazoa with several instances of putative loss (or gain) across the phylogeny. The work also unveiled instances of MSH6 fusion putatively interesting domains in fungi which might be worth exploring in the future.

The authors also show potential signatures of purifying selection in functional amino acids MSH6 histone readers.

Overall the approach is adequate for the questions proposed to be answered, the analyses are rigorous and support the authors' claims.

DNA repair genes are essential to maintain genome stability and fidelity, and alterations in these pathways have been associated with hypermutation phenotypes in the context for instance of cancer in humans, with sometimes implications in treatment resistance. This is an important work that contributes to our understanding of the evolutionary consequences of the evolution of epigenome-targeted DNA repair.

Strengths:

The methods used are adequate for the questions and support the results. The search for MSH6 fusions was rigorous and conservative, which strengthens the significance of the claims on the evolutionary history of these fusion events.

Weaknesses:

I did not identify any major weaknesses, but please see my suggestions/recommendations.

Reviewer #2 (Public review):

Summary:

The authors used a visual flash discrimination task in which two flashes are presented one after another with different inter-stimulus intervals. Participants either perceive one flash or two flashes. The authors show that the simultaneous presence of an auditory input extends the temporal window of integration, meaning that two flashes presented shortly after one another are more likely to be perceived as a single flash. Auditory inputs are accompanied by a reduction in alpha frequency over visual areas. Prestimulus alpha frequency predicts perceptual outcomes in the absence of auditory stimuli, whereas prestimulus alpha phase becomes the dominant predictor when auditory input is present. A computational model based on phase-resetting theory supports these findings. Additionally, a transcranial stimulation experiment confirms the causal role of alpha frequency in unimodal visual perception but not in cross-modal contexts.

Strengths:

The authors elegantly combined several approaches-from behavior to computational modeling and EEG-to provide a comprehensive overview of the mechanisms involved in visual integration in the presence or absence of auditory input. The methods used are state-of-the-art, and the authors attempted to address possible pitfalls.

Weaknesses:

The use of Bayesian statistics could further strengthen the paper, especially given that a few p-values are close to the significance threshold (lines 162 & 258), but they are interpreted differently in different cases (absence of effect vs. trend).

Overall, these results provide new insights into the role of alpha oscillations in visual processing and offer an interesting perspective on the current debate regarding the roles of alpha phase and frequency in visual perception. More generally, they contribute to our understanding of the neural dynamics of multisensory integration.

Reviewer #2 (Public Review):

Summary:

In a 1.5m diameter, 0.8m high circular arena bumblebees were accustomed to exiting the entrance to their nest on the floor surrounded by an array of identical cylindrical landmarks and to forage in an adjacent compartment which they could reach through an exit tube in the arena wall at a height of 28cm. The movements of one group of bees were restricted to a height of 30cm, the height of the landmark array, while the other group was able to move up to heights of 80cm, thus being able to see the landmark array from above.

During one series of tests, the flights of bees returning from the foraging compartment were recorded as they tried to reach the nest entrance on the floor of the arena with the landmark array shifted to various positions away from the true nest entrance location. The results of these tests showed that the bees searched for the net entrance in the location that was defined by the landmark array.

In a second series of tests, access to the landmark array was prevented from the side, but not from the top, by a transparent screen surrounding the landmark array. These tests showed that the bees of both groups rarely entered the array from above, but kept trying to enter it from the side.<br /> The authors express surprise at this result because modelling the navigational information supplied by panoramic snapshots in this arena had indicated that the most robust information about the location of the nest entrance within the landmark array was supplied by views of the array from above, leading to the following strong conclusions:<br /> line 51: "Snapshot models perform best with bird's eye views";<br /> line 188: "Overall, our model analysis could show that snapshot models are not able to find home with views within a cluttered environment but only with views from above it.";<br /> line 231: "Our study underscores the limitations inherent in snapshot models, revealing their inability to provide precise positional estimates within densely cluttered environments, especially when compared to the navigational abilities of bees using frog's-eye views."

Strengths:

The experimental set-up allows for the recording of flight behaviour in bees, in great spatial and temporal detail. In principle, it also allows for the reconstruction of the visual information available to the bees throughout the arena.

Weaknesses:

Modelling:<br /> Modelling left out information potentially available to the bees from the arena wall and in particular from the top edge of the arena and cues such as cameras outside the arena. For instance, modelled IDF gradients within the landmark array degrade so rapidly in this environment, because distant visual features, which are available to bees, are lacking in the modelling. Modelling furthermore did not consider catchment volumes, but only horizontal slices through these volumes.

Behavioural analysis:<br /> The full potential of the set-up was not used to understand how the bees' navigation behaviour develops over time in this arena and what opportunities the bees have had to learn the location of the nest entrance during repeated learning flights and return flights.

Without a detailed analysis of the bees' behaviour during 'training', including learning flights and return flights, it is very hard to follow the authors' conclusions. The behaviour that is observed in the tests may be the result of the bees' extended experience shuttling between the nest and the entry to the foraging arena at 28cm height in the arena wall. For instance, it would have been important to see the return flights of bees following the learning flights shown in Figure 17.

Basically, both groups of bees (constrained to fly below the height of landmarks (F) or throughout the height of the arena (B)) had ample opportunities to learn that the nest entrance lies on the floor of the landmark array. The only reason why B-bees may not have entered the array from above when access from the side was prevented, may simply be that bumblebees, because they bumble, find it hard to perform a hovering descent into the array.

General:

The most serious weakness of the set-up is that it is spatially and visually constrained, in particular lacking a distant visual panorama, which under natural conditions is crucial for the range over which rotational image difference functions provide navigational guidance. In addition, the array of identical landmarks is not representative of natural clutter and, because it is visually repetitive, poses un-natural problems for view-based homing algorithms. This is the reason why the functions degrade so quickly from one position to the next (Figures 9-12), although it is not clear what these positions are (memory0-memory7).<br /> In conclusion, I do not feel that I have learnt anything useful from this experiment; it does suggest, however, that to fully appreciate and understand the homing abilities of insects, there is no alternative but to investigate these abilities in the natural conditions in which they have evolved.

Reviewer #2 (Public review):

Summary:

At a high level, the reviewers demonstrate that there is an explanation for pre-word-onset predictivity in neural responses that does not invoke a theory of predictive coding or processing. The paper does this by demonstrating that this predictivity can be explained solely as a property of the local mutual information statistics of natural language. That is, the reason that pre-word onset predictivity exists could simply boil down to the common prevalence of redundant bigram or skip-gram information in natural language.

Strengths:

The paper addresses a problem of significance and uses methods from modern NeuroAI encoding model literature to do so. The arguments, both around stimulus dependencies and the problems of residualization, are compellingly motivated and point out major holes in the reasoning behind several influential papers in the field, most notably Goldstein et al. This result, together with other papers that have pointed out other serious problems in this body of work, should provoke a reconsideration of papers from encoding model literature that have promoted predictive coding. The paper also brings to the forefront issues in extremely common methods like residualization that are good to raise for those who might be tempted to use or interpret these methods incorrectly.

Weaknesses:

The authors don't completely settle the problem of whether pre-word onset predictivity is entirely explainable by stimulus dependencies, instead opting to show why naive attempts at resolving this problem (like residualization) don't work. The paper could certainly be better if the authors had managed to fully punch a hole in this.

Reviewer #2 (Public review):

Summary:

The authors show, through rigorous and extensive analyses, that the visual cortex in both congenitally blind and sighted participants represented differences between individual words presented across sensory modalities. In both groups, the activation patterns for words in the visual cortex reflected physical, but not conceptual similarity between word referents. This suggests a similar representation for both groups of words, one derived from vision-oriented mechanisms, and does not reflect significant functional reorganization in blindness.

Strengths:

The theoretical question is sound, as is the analysis approach. The authors' literature discussion is thorough, and the writing is clear.

Weaknesses:

I have only minor concerns left open.

(1) In the representational connectivity analysis, what is the average value across the brain? The authors compare the representational correlation across brain regions to the average value, but the average itself is not reported.

(2) Can the authors add a map showing the representational connectivity values across the brain in addition to the bar plot? It would make it easier to see what networks show similar neural representation to the visual cortex.

(3) Are the participants in the behavioral experiment from which the physical and conceptual similarity between word referents were collected matching in age or education with the fMRI participants?

(4) Although there are no group differences in the correlation of the physical similarity, I think it is important to acknowledge that the effect is only significant at the searchlight level in the blind early visual cortex (Figure S6).

Reviewer #2 (Public review):

Summary:

This is a well-written manuscript that clearly demonstrates that the nrp encoded diisonitrile chalkophore is necessary for function of the bcc-aa3 oxidase supercomplex under low copper conditions. In addition, the study demonstrates the chlakophore is important early during infection when copper sequestration is employed by the host as a method of nutritional immunity.

Strengths:

The authors use genetic approaches, including single and double mutants of chalkophore biosynthesis, and both the Mtb oxidases. Use a copper chelators to restrict copper in vitro. A strength of the work was the use of a synthesized a Mtb chalkophore analogue to show chemical complementation of the mutant nrp locus. Oxphos metabolic activity was measured by oxygen consumption and ATP levels. Importantly, the study demonstrated that chalkophore, especially in a strain lacking the secondary oxidase, was necessary for early infection and ruled out a role for adaptive immunity in the chalkophore lacking Mtb by use of SCID mice. It is interesting that after two weeks of infection and onset of adaptive immunity the chalkophore is not required, which is consistent with the host environment switching from a copper restricted to copper overload in phagosomes.

Weaknesses:

None noted

Reviewer #2 (Public review):

In this revised version of the study, the authors investigate the role of caspases in neuronal modulation through non-lethal activation. They analyze proximal proteins of executioner caspases using a variety of techniques, including TurboID and a newly developed monitoring system based on Gal4 manipulation, called MASCaT. They demonstrate that overexpression of Fas3G promotes the non-lethal activation of caspase Dronc in olfactory receptor neurons. In addition, they investigate the regulatory mechanisms of non-lethal function of caspase by performing a comprehensive analysis of proximal proteins of executioner caspase Drice. It is important to point out that the authors use an array of techniques from western blot to behavioral experiments and also that the generated several reagents, from fly lines to antibodies. In this revised version of the manuscript the authors addressed the concerns raised by this reviewer in a very thorough way. This is an interesting work that would appeal to readers of multiple disciplines. As a whole these findings suggest that overexpression of Fas3G enhances a non-lethal caspase activation in ORNs, providing a novel experimental model that will allow for exploration of molecular processes that facilitate caspase activation without leading to cell death.

Comments on revisions:

I would like to thank the authors for fully addressing my concerns.

Reviewer #2 (Public review):

Summary:

Using a suite of techniques (e.g., RNA seq, proteomics, and functional experiments ex vivo) this paper extensively focuses on the role of PIM1/2 kinases during CD8 T-cell activation and cytokine-driven (i.e., IL-2 or IL-15) differentiation. The authors key finding is that PIM1/2 enhance protein synthesis in response to IL-2 stimulation, but not IL-15, in CD8+ T cells. Loss of PIM1/2 made T cells less 'effector-like', with lower granzyme and cytokine production, and a surface profile that maintained homing towards secondary lymphoid tissue. The cytokines the authors focus on are IL-15 and Il-2, which drive naïve CD8 T cells towards memory or effector states, respectively. Although PIM1/2 are upregulated in response to T-cell activation and cytokine stimulation (e.g., IL-15, and to a greater extent, IL-2), using T cells isolated from a global mouse genetic knockout background of PIM1/2, the authors find that PIM1/2 did not significantly influence T-cell activation, proliferation, or expression of anything in the proteome under anti-CD3/CD28 driven activation with/without cytokine (i.e., IL-15) stimulation ex vivo. This is perhaps somewhat surprising given PIM1/2 are upregulated, albeit to a small degree, in response to IL-15, and yet PIM1/2 did not seem to influence CD8+ T cell differentiation towards a memory state. Even more surprising is that IL-15 was previously shown to influence the metabolic programming of intestinal intraepithelial lymphocytes, suggesting cell-type specific effects from PIM kinases. What the authors went on to show, however, is that PIM1/2 KO altered CD8 T cell proteomes in response to IL-2. Using proteomics, they saw increased expression of homing receptors (i.e., L-selectin, CCR7), but reduced expression of metabolism-related proteins (e.g., GLUT1/3 & cholesterol biosynthesis) and effector-function related proteins (e.g., IFNy and granzymes). Rather neatly, by performing both RNA-seq and proteomics on the same IL-2 stimulated WT vs. PIM1/2 KO cells, the authors found that changes at the proteome level were not corroborated by differences in RNA uncovering that PIM1/2 predominantly influence protein synthesis/translation. Effectively, PIM1/2 knockout reduced the differentiation of CD8+ T cells towards an effector state. In vivo adoptive transfer experiments showed that PIM1/2KO cells homed better to secondary lymphoid tissue, presumably owing to their heightened L-selectin expression (although this was not directly examined).

Strengths:

Overall, I think the paper is scientifically good, and I have no major qualms with the paper. The paper as it stands is solid, and while the experimental aim of this paper was quite specific/niche, it is overall a nice addition to our understanding of how serine/threonine kinases impact T cell state, tissue homing, and functionality. Of note, they hint towards a more general finding that kinases may have distinct behaviour in different T-cell subtypes/states. I particularly liked their use of matched RNA-seq and proteomics to first suggest that PIM1/2 kinases may predominantly influence translation (then going on to verify this via their protein translation experiment - although I must add this was only done using PIM kinase inhibitors not the PIM1/2KO cells). I also liked that they used small molecule inhibitors to acutely reduce PIM1/2 activity, which corroborated some of their mouse knockout findings - this experiment helps resolve any findings resulting from potential adaptation issues from the PIM1/2 global knockout in mice but also gives it a more translational link given the potential use of PIM kinase inhibitors in the clinic. The proteomics and RNA seq dataset may be of general use to the community, particularly for analysis of IL-15 or IL-2 stimulated CD8+ T cells.

Weaknesses:

None. My comments here have been addressed in the previous review.

Reviewer #3 (Public review):

This study aimed to examine the impact of seasonality on the population genetics of malaria parasites. To achieve this, the researchers conducted a longitudinal study in a region with seasonal malaria transmission. Over a 2.5-year period, blood samples were collected from 1,516 participants across four villages in the Upper River Region of The Gambia. These samples were tested for malaria parasite infection, and the parasites from positive samples were genotyped using a genetic barcode and/or whole genome sequencing. Genetic relatedness analysis was then performed to explore the findings

The study identified three key findings:

(1) The malaria parasite population undergoes continuous recombination, with no single genotype predominating, in contrast to viral populations;

(2) Parasite relatedness is influenced by both spatial and temporal factors; and

(3) The lowest genetic relatedness among parasites occurs during the transition from the low to high transmission seasons, which the authors linked to increased recombination during sexual reproduction in mosquitoes.

The results section is well-structured, and the figures are clear and self-explanatory. The methods are adequately described, providing a solid foundation for the findings. While there are no unexpected results, it is reassuring to see the anticipated outcomes supported by actual data. The conclusions are generally well-supported and the recommendation to target asymptomatic infections is logical and relevant.

Reviewer #2 (Public review):

Summary:

Min et al. attempt to demonstrate a mechanism whereby magnetic resonance imaging (MRI) can reflect changes in neuronal membrane potentials. They approach this goal by studying how MRI contrast and cellular potentials together respond to treatment of cultured cells with ionic solutions that are known to depolarize or hyperpolarize excitable cells. The authors specifically examine two MRI-based measurements: (A) the transverse (T2) relaxation rate, which reflects microscopic magnetic fields caused by solutes and biological structures; and (B) the fraction or "pool size ratio" (PSR) of water molecules estimated to be bound to macromolecules, using an MRI technique called magnetization transfer (MT) imaging. They see that depolarizing K+ and Ba2+ concentrations lead to T2 increases and PSR decreases that vary approximately linearly with parallel measurements of voltage in a neuroblastoma cell line and that change similarly in a second cell type. They also show that depolarizing potassium concentrations evoke T2 increases in rat brains, and that these changes are reversed when potassium is renormalized. Min et al. argue that their results suggest a basis for noninvasive functional imaging of cellular voltage signals. If this were true, it would help validate a recent paper published by some of the authors (Toi et al., Science 378:160-8, 2022), in which they claimed to be able to detect millisecond-scale neuronal responses by MRI.

Strengths:

The discovery of a mechanism for relating cellular membrane potential to MRI contrast could yield an important means for studying functions of the nervous system. Achieving this has been a longstanding goal in the MRI community, but previous strategies have proven insufficient for neuroscientific or clinical applications. The current paper suggests that one of the simplest and most widely used MRI contrast mechanisms-T2 weighted imaging-may indicate correlates of membrane potential if measured in the absence of the hemodynamic signals that most functional MRI (fMRI) experiments rely on. The authors make their case using quantitative tests that include some controls for ion and cell type-specificity of their in vitro results and reversibility of MRI changes observed in vivo.

Weaknesses:

The major weakness of the paper is that it uses only slow correlational experiments to probe the relationship between MRI contrast and membrane potential. The authors do not examine effects on the subsecond time scale that is of greatest interest, and they do not adequately consider how biophysical factors with only loose relationship to electrophysiological variables could explain their imaging results. Notably, depolarizing ionic solutions that perturb membrane potential can also induce changes in cellular volume and tissue structure that in turn alter MRI contrast properties similarly to the results shown here. For example, a study by Stroman et al. (Magn Reson Med 59:700-6, 2008) reported reversible potassium-dependent T2 increases in neural tissue that correlate closely with light scattering-based indications of cell swelling. Phi Van et al. (Sci Adv 10:eadl2034, 2024) showed that potassium addition to one of the cell lines used here likewise leads to cell size increases and T2 increases. In their revised manuscript, the authors acknowledge that cell swelling might contribute to the MRI signals they report, but they do nothing to probe the contributions or characteristics of such effects. If cell swelling accounted for the author's MRI results, it would likely operate on a time scale far too slow to yield useful indications of membrane potential. Given these considerations and the absence of data demonstrating correspondence of electrophysiological measures with MRI readouts on a fast time scale, the paper fails to provide evidence that membrane potential changes can be meaningfully detected by MRI.

Reviewer #3 (Public review):

Summary:

This study investigates evidence for a hypothesised, causal relationship between education, specifically the number of years spent in school, and brain structure as measured by common brain phenotypes such as surface area, cortical thickness, total volume and diffusivity.

To test their hypothesis, the authors rely on a "natural" intervention, that is, the 1972 ROSLA act that mandated an extra year of education for all 15-year olds. The study's aim is to determine potential discontinuities in the outcomes of interest at the time of the policy change, which would indicate a causal dependence. Naturalistic experiments of this kind are akin to randomised controlled trials, the gold standard for answering questions of causality.

Using two complementary, regression-based approaches, the authors find no discernible effect of spending an extra year in primary education on brain structure. The authors further demonstrate that observational studies showing an effect between education and brain structure may be confounded and thus unreliable when assessing causal relationships.

Strengths:

- A clear strength of this study is the large sample size totalling up to 30k participants from the UK Biobank. Although sample sizes for individual analyses are an order of magnitude smaller, most neuroimaging studies usually have to rely on much smaller samples.<br /> - This study has been preregistered in advance, detailing the authors' scientific question, planned method of inquiry and intended analyses, with only minor, justifiable changes in the final analysis.<br /> - The analyses look at both global and local brain measures used as outcomes, thereby assessing a diverse range of brain phenotypes that could be implicated in a causal relationship with a person's level of education.<br /> - The authors use multiple methodological approaches, including validation and sensitivity analyses, to investigate the robustness of their findings and, in the case of correlational analysis, highlight differences with related work by others.<br /> - The extensive discussion of findings and how they relate to the existing, somewhat contradictory literature gives a comprehensive overview of the current state of research in this area.

Weaknesses:

- This study investigates a well-posed but necessarily narrow question in a specific setting: 15-year old British students born around 1957 who also participate in the UKB imaging study roughly 60 years later. Thus conclusions about the existence or absence of any general effect of the number of years of education on the brain's structure are limited to this specific scenario.<br /> - The modelling approach used in this study requires that all covariates of no interest are equal before and after the cut-off, something that is impossible to test. However, other studies have not found specific issues that would invalidate ROSLA as a natural experiment.

Reviewer #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterised the cardiac phenotype associated to cardiac conduction system (CCS)-specific combined deletion of Tbx5 and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen inducible Cre recombination (MinK-creERT) at 6 weeks after birth. Their analysis of 8-9 weeks old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterising VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analysed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutant and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lays in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Linage tracing analysis using the reporter lines included in the crosses described in the study, will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot will demonstrate that indeed the cells that were specified as VCS in the adult heart become working myocardium in the absence of Tbx3 and Tbx5 function.

Comments on revisions:

I would like to thank the authors for their revised manuscript and for their corrections based on the suggestions from the 3 reviewers. Although I would have preferred to see some of the additional experiments suggested by any of the reviewers to improve the robustness and depth of the study integrated in the revised version of the manuscript, I acknowledge that the authors may prefer to develop them as follow-up studies. So, looking forward to seeing the follow-up study unravelling the detailed molecular regulation controlled by Tbx3/Tbx5 during the formation and maintenance of the ventricular cardiac conduction system.

Reviewer #2 (Public review):

Summary:

The study aims to probe the neural correlates of visual serial dependence - the phenomenon that estimates of a visual feature (here motion direction) are attracted towards the recent history of encoded and reported stimuli. The authors utilize an established retro-cue working memory task together with magnetoencephalography, which allows to probe neural representations of motion direction during encoding and retrieval (retro-cue) periods of each trial. The main finding is that neural representations of motion direction are not systematically biased during the encoding of motion stimuli, but are attracted towards the motion direction of the previous trial's target during the retrieval (retro-cue period), just prior to the behavioral response. By demonstrating a neural signature of attractive biases in working memory representations, which align with attractive behavioral biases, this study highlights the importance of post-encoding memory processes in visual serial dependence.

Strengths:

The main strength of the study is its elegant use of a retro-cue working memory task together with high temporal resolution MEG, enabling to probe neural representations related to stimulus encoding and working memory. The behavioral task elicits robust behavioral serial dependence and replicates previous behavioral findings by the same research group. The careful neural decoding analysis benefits from a large number of trials per participant, considering the slow-paced nature of the working memory paradigm. This is crucial in a paradigm with considerable trial-by-trial behavioral variability (serial dependence biases are typically small, relative to the overall variability in response errors). While the current study is broadly consistent with previous studies showing that attractive biases in neural responses are absent during stimulus encoding (prev. studies reported repulsive biases), to my knowledge, it is the first study showing attractive biases in current stimulus representations during working memory. The study also connects to previous literature showing reactivations of previous stimulus representations, although the link between reactivations and biases remains somewhat vague in the current manuscript. Together, the study reveals an interesting avenue for future studies investigating the neural basis of visual serial dependence.

Weaknesses:

The main weakness of the current manuscript is that the authors could have done more analyses to address the concern that their neural decoding results are driven by signals related to eye movements. The authors show that participants' gaze position systematically depended on the current stimuli's motion directions, which, together with previous studies on eye movement-related confounds in neural decoding, justifies such a concern. The authors seek to rule out this confound by showing that the consistency of stimulus-dependent gaze position does not correlate with (a) the neural reconstruction fidelity and (b) the attractive shift in reconstructed motion direction. However, the authors' approach of quantifying stimulus-dependent eye movements only considers gaze angle and not gaze amplitude, and thus potentially misses important features of eye movements that could manifest in the MEG data. Moreover, it is unclear whether the gaze consistency metric should correlate with attractive history biases in neural decoding, if there were a confound. These two concerns could be potentially addressed by (1) directly decoding stimulus motion direction from x-y gaze coordinates and relating this decoding performance to neural reconstruction fidelity, and (2) investigating whether gaze coordinates themselves are history-dependent and are attracted to the average gaze position associated with the previous trials' target stimulus. If the authors could show that (2) is not the case, I would be much more convinced that their main finding is not driven by eye movement confounds.

The sample size (n = 10) is definitely at the lower end of sample sizes in this field. The authors collected two sessions per participant, which partly alleviates the concern. However, given that serial dependencies can be very variable across participants, I believe that future studies should aim for larger sample sizes.

It would have been great to see an analysis in source space. As the authors mention in their introduction, different brain areas, such as PPC, mPFC and dlPFC have been implicated in serial biases. This begs the question which brain areas contribute to the serial dependencies observed in the current study? For instance, it would be interesting to see whether attractive shifts in current representations and pre-stimulus reactivations of previous stimuli are evident in the same or different brain areas.

Reviewer #2 (Public review):

Summary:

Egawa et al describe the developmental timeline of the assembly of nodes of Ranvier in the chick brainstem auditory circuit. In this unique system, the spacing between nodes varies significantly in different regions of the same axon from early stages, which the authors suggest is critical for accurate sound localization. Egawa et al set out to determine which factors regulate this differential node spacing. They do this by using immunohistological analyses to test the correlation of node spacing with morphological properties of the axons, and properties of oligodendrocytes, glial cells that wrap axons with the myelin sheaths that flank the nodes of Ranvier. They find that axonal structure does not vary significantly, but that oligodendrocyte density and morphology varies in the different regions traversed by these axons, which suggests this is a key determinant of the region-specific differences in node density and myelin sheath length. They also find that differential oligodendrocyte density is partly determined by secreted neuronal signals, as (presumed) blockage of vesicle fusion with tetanus toxin reduced oligodendrocyte density in the region where it is normally higher. Based on these findings, the authors propose that oligodendrocyte morphology, myelin sheath length, and consequently nodal distribution are primarily determined by intrinsic oligodendrocyte properties rather than neuronal factors such as activity.

Major comments:

(1) It is essential that the authors validate the efficiency of TeNT to prove that vesicular release is indeed inhibited, to be able to make any claims about the effect of vesicular release on oligodendrogenesis/myelination.

(2) Related to 1, can the authors clarify if their TeNT expression system results in the whole tract being silenced? It appears from Fig. 6 that their approach leads to sparse expression of TeNT in individual neurons, which enables them to measure myelination parameters. Can the authors discuss how silencing a single axon can lead to a regional effect in oligodendrocyte number?

(3) The authors need to fully revise their statistical analyses throughout and supply additional information that is needed to assess if their analyses are adequate:<br /> (3.1) the authors use a variety of statistical tests and it is not always obvious why they chose a particular test. For example, in Fig. 2G they chose a Kruskal-Wallis test instead of a two-way ANOVA or Mann-Whitney U test, which are much more common in the field. What is the rationale for the test choice?<br /> (3.2) in some cases, the choice of test appears wholly inappropriate. For example, in Fig. 3H-K, an unpaired t-test is inappropriate if the two regions were analysed in the same samples. In Fig. 5, was a t-test used for comparisons between multiple groups in the same dataset? If so, an ANOVA may be more appropriate.<br /> (3.3) in some cases, the authors do not mention which test was used (Fig 3: E-G no test indicated, despite asterisks; G/L/M - which regression test that was used? What does r indicate?)<br /> (3.4) more concerningly, throughout the results, data may have been pseudo-replicated. t-tests and ANOVAs assume that each observation in a dataset is independent of the other observations. In figures 1-4 and 6 there is a very large "n" number, but the authors do not indicate what this corresponds to. This leaves it open to interpretation, and the large values suggest that the number of nodes, internodal segments, or cells may have been used. These are not independent experimental units, and should be averaged per independent biological replicate - i.e. per animal (N).<br /> (3.5) related to the pseudo-replication issue, can the authors include individual datapoints in graphs for full transparency, per biological replicates, in addition or in alternative to bar-graphs (e.g. Fig. 5 and 6).

(4) The main finding of the study is that the density of nodes differs between two regions of the chicken auditory circuit, probably due to morphological differences in the respective oligodendrocytes. Can the authors discuss if this finding is likely to be specific to the bird auditory circuit?

(5) Provided the authors amend their statistical analyses, and assuming significant differences remain as shown, the study shows a correlation (but not causation) between node spacing and oligodendrocyte density, but the authors did not manipulate oligodendrocyte density per se (i.e. cell-autonomously). Therefore, the authors should either include such experiments, or revise some of their phrasing to soften their claims and conclusions. For example, the word "determine" in the title could be replaced by "correlate with" for a more accurate representation of the work. Similar sentences throughout the main text should be amended.

(6) The authors fail to introduce, or discuss, very pertinent prior studies, in particular to contextualize their findings with:<br /> (6.1) known neuron-autonomous modes of node formation prior to myelination, e.g. Zonta et al (PMID 18573915); Vagionitis et al (PMID 35172135); Freeman et al (PMID 25561543)<br /> (6.2) known effects of vesicular fusion directly on myelinating capacity and oligodendrogenesis, e.g. Mensch et al (PMID 25849985)<br /> (6.3) known correlation of myelin length and thickness with axonal diameter, e.g. Murray & Blakemore (PMID 7012280); Ibrahim et al (PMID 8583214); Hildebrand et al (PMID 8441812).<br /> (6.4) regional heterogeneity in the oligodendrocyte transcriptome (page 9, studies summarized in PMID 36313617)

Significance:

In our view the study tackles a fundamental question likely to be of interest to a specialized audience of cellular neuroscientists. This descriptive study is suggestive that in the studied system, oligodendrocyte density determines the spacing between nodes of Ranvier, but further manipulations of oligodendrocyte density per se are needed to test this convincingly.

Reviewer #3 (Public review):

Summary:

Salmonella is interesting due to its life within a compact compartment, which we call SCV or Salmonella containing vacuole in the field of Salmonella. SCV is a tight-fitting vacuole where the acquisition of nutrients is a key factor by Salmonella. The authors among many nutrients, focussed on beta-alanine. It is also known that Salmonella requires beta-alanine from many other studies. The authors have done in vitro RAW macrophage infection assays and In vivo mouse infection assays to see the life of Salmonella in the presence of beta-alanine. They concluded by comprehending that beta-alanine modulates the expression of many genes including zinc transporters which is required for pathogenesis.

Strengths:

Made a couple of knockouts in Salmonella and did transcriptomic to understand the global gene expression pattern

Weaknesses:

Transport of Beta-alanine to SCV is not yet elucidated. Is it possible to determine whether the Zn transporter is involved in B-alanine transport?

Beta-alanine can also be shuttled to form carnosine along with histidine. If beta-alanine is channelled to make more carnosine, then the virulence phenotypes may be very different.

Some amino acid transporters can be knocked out to see if beta-alanine uptake is perturbed. Like ArgT transport Arginine, and its mutation perturbs the uptake of beta-alanine. What is the beta-alanine concentration in the SCV? SCVS can be purified at different time points, and the Beta-alanine concentration can be measured

Reviewer #2 (Public review):

Summary:

In this work, the authors developed a model of tumour-immune dynamics, incorporating stochastic antigenic mutation accumulation and escape within the cancer cell population. They then used this model to investigate how tumour-immune interactions influence tumour outcome and summary statistics of sequencing data.

Strengths:

This novel modeling framework addresses an important and timely topic. The authors consider the useful question of how bulk and single-cell sequencing may provide insights into the tumour-immune interactions and selection processes.

Weaknesses:

One set of conclusions presented in the paper is the presence of cyclic dynamics between effector/cancer cells, antigenicity, and immunogenicity. However, these conclusions are supported in the manuscript by two sample trajectories of stochastic simulations, and these provide mixed support for the conclusions (i.e. the phasing asynchrony described in the text does not seem to apply to Figure 2C). Similarly, the authors also find immune selection effects on the shape of the mutational burden in Figure 5 D/H using a qualitative comparison between the distributions and theoretical predictions in the absence of immune response. However the discrepancy appears quite small in panel D, and there are no quantitative comparisons provided to evaluate the significance. An analysis of the robustness of all the conclusions to parameter variation is missing. Lastly, the role of the Appendix results in the main messages of the paper is unclear.

Reviewer #2 (Public review):

Summary:

Meier et al. explore the variability of locomotion-related modulations in mouse area V1. They present 4 major findings: V1 L2/3 neurons beneath M2- interpatches are more strongly locomotion-modulated than those beneath M2+ patches, while V1 L2/3 neurons are more strongly orientation tuned. They then use viral tracing to examine the relationship of M2- interpatches and M2+ patches with inputs from and outputs to HVOs, MO, RSP, and LP, and find evidence for different closed-loop subnetworks within L1; these relationships, however, are more complicated for cell bodies in L2/3. Finally, they also describe an overlap between M2- interpatches and SOM+ dendrites/axons.

Strengths:

The strength of the manuscript is the detailed anatomical quantification of closed-loop connectivity, and the description of the organizing principles of M2- interpatches and M2+ patches.

Weaknesses:

The major weakness of the manuscript is the lack of a direct connection between the functional and the anatomical data, and the somewhat puzzling effects observed in the analysis of noise correlations. The former issue might be alleviated by modelling, where the authors could explore the space of possibilities that could explain the functional data based on the anatomical connectivity. Some control analyses could be done, for the comparison of noise correlations.

Reviewer #2 (Public review):

Summary:

The paper investigates social-decision making, and how this changes after observing the behaviour of other people, in borderline personality disorder. The paper employs a task including three phases, the first where participants make decision on how to allocate rewards to oneself and to a virtual partner, the second where they observe the same task performed by someone else, and a third phase equivalent to phase one, but with a new partner. Using sophisticated computational modelling to analyse choice data, the study reports that borderline participants (versus controls) are more certain about their preferences in phase one, used more neutral priors and are less flexible during phase two, and are less influenced by partners in phase three.

Strengths:

The topic is interesting and important, and the findings are potentially intriguing. The computational methods employed is clever and sophisticated, at the cutting edge of research in the field.

Weaknesses:

The paper is not based on specific empirical hypotheses formulated at the outset, but, rather, it uses an exploratory approach. Indeed, the task is not chosen in order to tackle specific empirical hypotheses. This, in my view, is a limitation since the introduction reads a bit vague and it is not always clear which gaps in the literature the paper aims to fill. As a further consequence, it is not always clear how the findings speak to previous theories on the topic.

Reviewer #2 (Public review):

Summary:

The reduction in a response to a specific stimuli after repeated exposures is called habituation. Alterations in habituation to noxious stimuli are associated with chronic pain in humans, however the underlying molecular mechanisms involved are not clear. This study uses the nematode C. elegans to study genes and mechanisms that underlie adaptation to a form of noxious stimuli based on heat, termed thermo-noxious stimuli. The authors previously showed that the Calcium/Calmodulin-dependent protein kinase (CMK-1) regulates thermo-nociceptive adaptation in the nematode C. elegans. Although CMK-1 is a kinase with many known substrates, the downstream targets relevant for thermo-nociceptive adaptation are not known. In this study, the authors use two different kinase screens to identify phosphorylation targets of CMK-1. One of the targets they identify is Calcineurin (TAX-6). The authors show that CMK-1 phosphorylates a regulatory domain of Calcineurin at a highly conserved site (S443). In a series of elegant experiments, the authors use genetic and pharmacological approaches to increase or decrease CMK-1 and Calcineurin signaling to study their effects on thermo-nociceptive adaptation in C. elegans. They also combine these various approaches to study the interactions between these two signaling proteins. The authors use specific promoters to determine in which neurons CMK-1 and Calcineurin function to regulate thermo-nociceptive adaptation. The authors propose a model based on their findings, illustrating that CMK-1 and Calcineurin act mostly in different neurons to antagonistically regulate adaptation to thermo-nociceptive stimuli in a complex manner.

Strengths:

- Given the conservation of adaptation across phylogeny, identifying genes and mechanisms that underlie nociceptive adaptation in C. elegans may be relevant for understanding chronic pain in humans.<br /> - The identification of canonical CaM Kinase phosphorylation motifs in the substrates identified in the CMK-1 substrate screen validates the screen.<br /> - The use of loss and gain of function approaches to study the effects of CMK-1 and Calcineurin on thermo-nociceptive responses and adaptation is elegant.<br /> - The ability to determine the cellular place of action of CMK-1 and Calcineurin using neuron specific promoters in the nematode is a clear strength of the genetic model system.

Weaknesses:

- The manuscript begins by identifying Calcineurin as a direct substrate of CMK-1 but ends by showing that CMK-1 and Calcineurin mostly act in different neurons to regulate nociceptive adaptation, thus the physiological relevance of CMK-1 phosphorylation of Calcineurin is not clear.

Reviewer #2 (Public review):

In their manuscript, Rijal and colleagues describe a 'loop grafting' strategy to enhance expression levels and stability of recombinant neuraminidase. The work is interesting and important.

Major points from first round of review:

(1) The authors overstress the importance of the epitopes covered by the loops they use and play down the importance of antibodies binding to the side, the edges, or the underside of the NA. A number of papers describing those mAbs are also not included.

(2) The rationale regarding the PR8 hybrid is not well described and should be described better.

(3) Figure 3B and 6C: This should be given as numbers (quantified), not as '+'.

(4) Figure 5A and 7A: Negative controls are missing.

(5) The authors claim that they generate stable tetramers. Judging from SDS-PAGE provided in Supplementary Figure 3B (BS3-crosslined), many different species are present including monomers, dimers, tetramers, and degradation products of tetramers. In line 7 for example there are at least 5 bands.

[Editors' note: the authors have appropriately responded to and addressed these points.]

Reviewer #2 (Public review):

Summary:

This study by Kremer et al. investigates the impact of modulation of expression of TFAM, a key protein involved in mitochondrial DNA (mtDNA) packaging and expression, in mtDNA mutator mice, which carry random mtDNA mutations. While previous research suggested that increasing TFAM could counteract the pathological effects of mtDNA mutations, this study reveals that the effects of TFAM modulation are tissue-specific. These findings highlight the complexity of mtDNA copy number regulation and gene expression, emphasizing that TFAM alone is not the sole determinant of mtDNA levels in contexts where oxidative phosphorylation is impaired. Other factors likely play a significant role, underscoring the need for nuanced approaches when targeting TFAM for therapeutic interventions.

Strengths:

The data presented in the manuscript are of high quality and support the major conclusions.

Comments on revisions:

The authors have thoroughly addressed all the points raised during the first round of review. Their revisions effectively clarify key aspects of the manuscript, and the additional data and explanations have significantly improved the overall quality of the work. I believe the manuscript is now well-prepared for publication.

Reviewer #2 (Public review):

Summary:

In this study, the authors report that Meioc is required to upregulate rRNA transcription and promote differentiation of spermatogonial stem cells in zebrafish. The authors show that upregulated protein synthesis is required to support spermatogonial stem cells' differentiation into multi-celled cysts of spermatogonia. Coiled coil protein Meioc is required for this upregulated protein synthesis and for increasing rRNA transcription, such that the Meioc knockout accumulates 1-2 cell spermatogonia and fails to produce cysts with more than 8 spermatogonia. The Meioc knockout exhibits continued transcriptional repression of rDNA. Meioc interacts with and sequesters Piwil1 to the cytoplasm. Loss of Meioc increases Piwil1 localization to the nucleolus, where Piwil1 interacts with transcriptional silencers that repress rRNA transcription.

Strengths:

This is fundamental study that expands our understanding of how ribosome biogenesis contributes to differentiation and demonstrates that zebrafish Meioc plays a role in this process during spermatogenesis. This work also expands our evolutionary understanding of Meioc and Ythdc2's molecular roles in germline differentiation. In mouse, the Meioc knockout phenocopies the Ythdc2 knockout, and studies thus far have indicated that Meioc and Ythdc2 act together to regulate germline differentiation. Here, in zebrafish, Meioc has acquired a Ythdc2-independent function. This study also identifies a new role for Piwil1 in directing transcriptional silencing of rDNA.

Comments on revisions:

Major and minor concerns were addressed in the revision.

Reviewer #2 (Public Review):

Summary:

The study by Ver Heul et al., investigates the consequences of RAG expression for type 2 innate lymphoid cell (ILC2) function. RAG expression is essential for the generation of the receptors expressed by B and T cells and their subsequent development. Innate lymphocytes, which arise from the same initial progenitor populations, are in part defined by their ability to develop in the absence of RAG expression. However, it has been described in multiple studies that a significant proportion of innate lymphocytes show a history of Rag expression. In compelling studies several years ago, members of this research team revealed that early Rag expression during the development of Natural Killer cells (Karo et al., Cell 2014), the first described innate lymphocyte, had functional consequences.

Here, the authors revisit this topic, a worthwhile endeavour given the broad history of Rag expression within all ILCs and the common use of RAG-deficient mice to specifically assess ILC function. Focusing on ILC2s and utilising state-of-the-art approaches, the authors sought to understand whether early expression of Rag during ILC2 development had consequences for activity, fitness, or function. Having identified cell-intrinsic effects in vivo, the authors investigated the causes of this, identifying epigenetic changes associated with the accessibility genes associated with core ILC2 functions.

The manuscript is well written and does an excellent job of supporting the reader through reasonably complex transcriptional and epigenetic analyses, with considerate use of explanatory diagrams. Overall I think that the conclusions are fair, the topic is thought-provoking, and the research is likely of broad immunological interest. I think that the extent of functional data and mechanistic insight is appropriate.

Strengths:

- The logical and stepwise use of mouse models to first demonstrate the impact on ILC2 function in vivo and a cell-intrinsic role. Initial analyses show enhanced cytokine production by ILC2 from RAG-deficient mice. Then through two different chimeric mice (including BM chimeras), the authors convincingly show this is cell intrinsic and not simply as a result of lymphopenia. This is important given other studies implicating enhanced ILC function in RAG-/- mice reflect altered competition for resources (e.g. cytokines).

- Use of Rag expression fate mapping to support analyses of how cells were impacted - this enables a robust platform supporting subsequent analyses of the consequences of Rag expression for ILC2.

- Use of snRNA-seq supports gene expression and chromatin accessibility studies - these reveal clear differences in the data sets consistent with altered ILC2 function.

- Convincing evidence of epigenetic changes associated with loci strongly linked to ILC2 function. This forms a detailed analysis that potentially helps explain some of the altered ILC2 functions observed in ex vivo stimulation assays.

- Provision of a wealth of expression data and bioinformatics analyses that can serve as valuable resources to the field.

Reviewer #2 (Public review):

Summary:

The Flower protein is expressed in various cell types, including neurons. Previous studies in flies have proposed that Flower plays a role in neuronal endocytosis by functioning as a Ca2+ channel. However, its precise physiological roles and molecular mechanisms in neurons remain largely unclear. This study employs C. elegans as a model to explore the function and mechanism of FLWR-1, the C. elegans homolog of Flower. This study offers intriguing observations that could potentially challenge or expand our current understanding of the Flower protein. Nevertheless, further clarification or additional experiments are required to substantiate the study's conclusions.

Strengths:

A range of approaches was employed, including the use of a flwr-1 knockout strain, assessment of cholinergic synaptic activity via analyzing aldicarb (a cholinesterase inhibitor) sensitivity, imaging Ca2+ dynamics with GCaMP3, analyzing pHluorin fluorescence, examination of presynaptic ultrastructure by EM, and recording postsynaptic currents at the neuromuscular junction. The findings include notable observations on the effects of flwr-1 knockout, such as increased Ca2+ levels in motor neurons, changes in endosome numbers in motor neurons, altered aldicarb sensitivity, and potential involvement of a Ca2+-ATPase and PIP2 binding in FLWR-1's function.

The authors have adequately addressed most of my previous concerns, however, I recommend minor revisions to further strengthen the study's rigor and interpretation:

Major suggestions

(1) This study relies heavily on aldicarb assays to support its conclusions. While these assays are valuable, their results may not fully align with direct assessment of neurotransmitter release from motor neurons. For instance, prior work has shown that two presynaptic modulators identified through aldicarb sensitivity assays exhibited no corresponding electrophysiological defects at the neuromuscular junction (Liu et al., J Neurosci 27: 10404-10413, 2007). Similarly, at least one study from the Kaplan lab has noted discrepancies between aldicarb assays and electrophysiological analyses. The authors should consider adding a few sentences in the Discussion to acknowledge this limitation and the potential caveats of using aldicarb assays, especially since some of the aldicarb assay results in this study are not easily interpretable.

(2) The manuscript states, "Elevated Ca2+ levels were not further enhanced in a flwr-1;mca-3 double mutant." (lines 549-550). However, Figure 7C does not include statistical comparisons between the single and double mutants of flwr-1 and mca-3. Please add the necessary statistical analysis to support this statement.

(3) The term "Ca2+ influx" should be avoided, as this study does not provide direct evidence (e.g. voltage-clamp recordings of Ca2+ inward currents in motor neurons) for an effect of the flwr-1 mutation of Ca2+ influx. The observed increase in neuronal GCaMP signals in response to optogenetic activation of ChR2 may result from, or be influenced by, Ca2+ mobilization from of intracellular stores. For example, optogenetic stimulation could trigger ryanodine receptor-mediated Ca2+ release from the ER via calcium-induced calcium release (CICR) or depolarization-induced calcium release (DICR). It would be more appropriate to describe the observed increase in Ca2+ signal as "Ca2+ elevation" rather than increased "Ca2+ influx".

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors proposed a method to quantitatively analyze 3D live imaging data of early developing embryos, using the ascidian development as an example. For this purpose, the previously proposed level set method was used to computationally track the temporal evolution of reference points introduced on the embryo surface. Then, from the obtained three-dimensional trajectories, the velocity field was obtained, from which the strain rate field was computed. The strain rate field was analyzed using spherical harmonics.

In this paper, the authors focused on the modes with lower order with real coefficients. The time evolution of these modes was analyzed using wavelet transforms. The results obtained by the pipeline reflected the developmental stages of ascidian embryos.

Strengths:

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

Weaknesses:

The mathematics behind this method is not straightforward to understand. The value of this method will be understood as analyses of real data using this method accumulate.

Comments on revised version:

I have reviewed the revised manuscript and the reply from the authors. All concerns have been addressed appropriately.

Reviewer #2 (Public review):

Summary:

Orientational symmetries of cells and tissues play an important role in describing processes in development and disease, and the methods used to investigate them rely on the detection of cell shape. In this interesting and very timely manuscript by Lea Happel et al., Minkowski tensors are introduced to study the orientational symmetries of cells and set in comparison to existing shape descriptors, such as the shape function introduced by Armengol-Collado et al., which captures the orientational symmetry by the vertex positions of the polygonal shape of the cell. As an advantage, the Minkowski tensors consider the real cell shape with its arbitrary curvature of the cortex. Using computational models, such as the active vertex model and the multiphase field model, as well as experimental support with MDCK monolayers, the authors find that the orientational symmetries are independent of one another, as well as that they are dependent on the activity and deformability of the cells, resulting in a monotonic trend. A trend that has not been observed for the hexatic symmetry using the shape function. Together with the lack of hexatic-nematic crossover at the tissue scale, the authors suggest a reconsideration of findings from other shape descriptors. Taken together, the Minkowski tensors set a framework to investigate orientational symmetries at a single cell scale and how they may interplay in biological tissues.

Strengths:

The authors introduce the Minkowski tensors, which capture the p-atic orders of cells in tissues, considering their real shape instead of a polygonal approximation as reported for other shape descriptors in the literature. Thus, they do not depend on the vertex positions of the cells nor on the number of neighboring cells. The Minkowski tensors capture the dependence of the p-atic orders on the cell activity and deformability in a monotonic manner, which makes them a robust tool for quantifying p-atic orders at a single-cell scale, especially for rounded cells. The robustness has been tested by comparing the results of two computational model systems that simulate cell monolayers and whose results have been extended with experimental data. The Minkowski tensors have been used to explore the role of cell-cell adhesion and density in epithelial cells and have shown similar results to the shape function, a polygonal shape descriptor.

Weaknesses:

The authors point out the importance of studying the orientational order in biological systems. However, the current version of the manuscript lacks statistical information, a description of analysis methods, and experimental support. This support is needed to strengthen (i) the results of the two computational models and (ii) give weight to the authors' strong claim against other widely accepted shape descriptors capturing p-atic orders. The Minkowski tensors, which consider the real cell shapes, are reported to be a better method to investigate the p-atic orders of cells than the shape function introduced by Armengol-Collado et al. While there may be differences in the reported results coming from the two different approaches, both approaches show similar trends. As it stands, there is substantiated discussion as to why one method would be better than the other. The shape function, γ₆, may not be monotonic for great changes in cell activity and deformability, hinting at a potential weakness. In contrast to the shape function and results by Armengol-Collado et al. and Eckert et al., the coarse-grained Minkowski tensors do not capture the hexatic-nematic crossover at the tissue scale, applied here only to computational models. The cells simulated in the computational models have a similar size and the monolayer has a nearly regular pattern, which does not reflect the density variance in biological tissues. To strengthen the author's claim that there is no crossover at the tissue scale, experimental verification is essential. Further, the robustness of the Minkowski tensors seems to rely on determining the p-atic orders on the shape of individual cells in the tissue. However, when applying the shape descriptor to experimental systems, the p-atic orders are very low, perhaps too low for comparisons between different p-atic orders with meaningful conclusions.

Reviewer #2 (Public review):

Summary:

In this study, dual-color super-resolution microscopy analysis was performed to study the co-operation between integrins and focal adhesion proteins in human fibroblast cells. The study focused on two integrins which have been previously found to be mainly responsible for focal adhesions, namely α5β1 and αvβ3.

Specifically, the study tried to shed light on the nanoclustering of integrins in focal adhesions.

In the current study, more integrin nanoclusters were observed in focal adhesions compared to other cell-matrix adhesion structures. The study revealed that both α5β1 and αvβ3 form nanoclusters, and those appear segregated from each other. While αvβ3 nanoclusters organize randomly inside focal adhesions regardless of their activation state, α5β1 nanoclusters, and particularly the nanoclusters containing β1-integrin in active conformation, preferentially organized at the edges of focal adhesions. The nanoclusters formed by each integrin were similar in size.

Cytoplasmic adapter proteins appeared less in nanocluster assemblies, suggesting that integrin nanoclusters are also forming without the studied cytoplasmic adapter proteins (talin, vinculin, paxillin). Active integrins were identified with the help of conformation-specific antibodies, and this enabled us to study the colocalization between integrins and their cytoplasmic adapter proteins. This analysis revealed that activated integrins are strongly engaged with adapter proteins

Strengths:

The study stems from the thorough computational modelling of the nanoclusters, which enables quantification of the behavior of the clusters, including their mesoscale distribution.

The study strengthens the view that α5β1 and αvβ3 have specific functions in focal adhesions, α5β1 nanoclusters localizing preferentially on focal adhesion edges. The study also revealed that nanoclusters localized at the edges of focal adhesion were enriched for talin and paxillin but not for vinculin.

Analysis of adaptor protein nanoclusters (paxillin, talin, and vinculin) revealed that all adapter protein nanoclusters studied here close to active β1 nanoclusters are enriched on the focal adhesion edge region, whereas integrin adaptor nanoclusters far from active β1 appear to be more uniformly distributed.

Importantly, the current study suggests that integrin subtype-specific nanoclusters are not only present at an early stage of adhesion formation, but integrin nanoclusters remain segregated from each other also in mature focal adhesions, maintaining their sizes and number of molecules.

Interestingly, the study revealed that selected cytoplasmic adaptors (paxillin, talin, and vinculin), also form nanoclusters of similar size and number of single molecule localizations as the integrins, regardless of whether they locate inside or outside focal adhesions. The adapter nanoclusters are enriched in the focal adhesion "belt", colocalizing with the active α5β1 integrin nanoclusters.

Weaknesses:

The current study is highly dependent on the antibodies. It is possible that antibodies containing two binding sites for antigen influence the nanoscale organization (and also activation) of the receptors. Control experiments to study the possible contribution of antibodies to the measured outcome should be performed to verify the main findings. One possible approach could be to use fluorescently tagged integrins available. Alternatively, integrins (or adapter proteins) could be tagged with a small ligand and detected using a monovalent binder.

Only a limited number of integrin adapter proteins were investigated. Given the high number of identified adapter proteins, this is an understandable choice. However, it would be fascinating to understand if the nanoclusters of inactive integrins are dominantly bound with a certain adapter protein, such as tensin.

Reviewer #2 (Public review):

Summary:

The authors sought to develop a rapid and non-invasive diagnostic method for primary amoebic meningoencephalitis (PAM), a highly fatal disease caused by Naegleria fowleri. Due to the challenges of early diagnosis, they investigated extracellular vesicles (EVs) from N. fowleri, identifying small RNA biomarkers. They developed an RT-qPCR assay to detect these biomarkers in various biofluids.

Strengths:

(1) This study has a clear methodological approach, which allows for the reproducibility of the experiments.

(2) Early and Non-Invasive Diagnosis - The identification of a small RNA biomarker that can be detected in urine, plasma, and cerebrospinal fluid (CSF) provides a non-invasive diagnostic approach, which is crucial for improving early detection of PAM.

(3) High Sensitivity and Rapid Detection - The RT-qPCR assay developed in the study is highly sensitive, detecting the biomarker in 100% of CSF samples from human PAM cases and in mouse urine as early as 24 hours post-infection. Additionally, the test can be completed in ~3 hours, making it feasible for clinical use.

(4) Potential for Disease Monitoring - Since the biomarker is detectable throughout the course of infection, it could be used not only for early diagnosis but also for tracking disease progression and monitoring treatment efficacy.

(5) Strong Experimental Validation - The study demonstrates biomarker detection across multiple sample types (CSF, urine, whole blood, plasma) in both animal models and human cases, providing robust evidence for its clinical relevance.

(6) Addresses a Critical Unmet Need - With a >97% case fatality rate, PAM urgently requires improved diagnostics. This study provides one of the first viable liquid biopsy-based diagnostic approaches, potentially transforming how PAM is detected and managed.

Weaknesses:

(1) Limited Human Sample Size - While the biomarker was detected in 100% of CSF samples from human PAM cases, the number of human samples analyzed (n=6 for CSF) is relatively small. A larger cohort is needed to validate its diagnostic reliability across diverse populations.

(2) Lack of Pre-Symptomatic or Early-Stage Human Data - Although the biomarker was detected in mouse urine as early as 24 hours post-infection, there is no data on whether it can be reliably detected before symptoms appear in humans, which is crucial for early diagnosis and treatment initiation.

(3) Plasma Detection Challenges - While the biomarker was detected in whole blood, it was not detected in human plasma, which could limit the ease of clinical implementation since plasma-based diagnostics are more common. Further investigation is needed to understand why it is absent in plasma and whether alternative blood-based approaches (e.g., whole blood assays) could be optimized.

Reviewer #2 (Public review):

Summary:

This study examines larval sleep patterns and compares them to sleep regulation in adult flies. The authors demonstrate hallmark sleep characteristics in larvae, including sleep rebound and increased arousal thresholds. Through genetic and behavioral analyses, they identify PK2-R1 as a key receptor involved in sleep modulation, likely via the HuginPC-IPC signaling pathway. Loss of PK2-R1 results in increased sleep, which aligns with previous findings in hugin knockout mutants. While the study presents significant contributions to the field, further investigation is needed to address discrepancies with earlier research and strengthen mechanistic claims.

Strengths:

(1) The study explores a relatively understudied aspect of sleep regulation, focusing on larval development.

(2) The use of an automated behavioral measurement system ensures precise quantification of sleep patterns.

(3) The findings provide strong genetic and behavioral evidence supporting the role of the HuginPC-IPC pathway in sleep regulation.

(4) The study has broader implications for understanding the evolution and functional divergence of sleep circuits.

Weaknesses:

(1) The manuscript does not sufficiently discuss previous studies, particularly concerning hugin mutants and their metabolic effects.

(2) The specificity of IPC secretion mechanisms is unclear, particularly regarding potential indirect effects on Dilp2.

(3) Alternative circuits, such as the HuginPC-DH44 pathway, require further consideration.

(4) Functional connectivity between HuginPC neurons and IPCs is not directly validated.

(5) Developmental differences in sleep regulatory mechanisms are not thoroughly examined.

Reviewer #2 (Public review):

Summary:

In this study, the authors propose an alternative platform for nanobody discovery using a phage-displayed synthetic library. The authors relied on DNA templates originally created by McMahon et al. (2018) to build the yeast-displayed synthetic library. To validate their platform, the authors screened for nanobodies against 8 Drosophila secreted proteins. Nanobody screening has been performed with phage-displayed nanobody libraries followed by an enzyme-linked immunosorbent assay (ELISA) to validate positive hits. Nanobodies with higher affinity have been tested for immunostaining and immunoblotting applications using Drosophila adult guts and hemolymph, respectively.

Strengths:

The authors presented a detailed protocol with various and complementary approaches to select nanobodies and test their application for immunostaining and immunoblotting experiments. Data are convincing and the manuscript is well-written, clear, and easy to read.

Weaknesses:

On the eight Drosophila secreted proteins selected to screen for nanobodies, the authors failed to identify nanobodies for three of them. While the authors mentioned potential improvements of the protocol in the discussion, none of them have been tested in this manuscript.

The same comment applies to the experiments using membrane-tethered forms of the antigens to test the affinity of nanobodies identified by ELISA. Many nanobodies fail to recognize the antigens. While authors suggested a low affinity of these nanobodies for their antigens, this hypothesis has not been tested in the manuscript.

Improving the protocol at each step for nanobody selection would greatly increase the success rate for the discovery of nanobodies with high affinity.

Reviewer #2 (Public review):

Summary:

In this paper, Bhojappa et al. provide insights into the function of septin-related kinases Elm1, Gin4, Hsl1, and Kcc4 in septin organization and actomyosin ring (AMR) structure and constriction. Their findings are both corroborative of and complementary to previous related studies.

First, the authors provide a comparative analysis of the dynamic localization of these kinases at the bud neck, as well as a comparative analysis of defects in septin localization, splitting dynamics, AMR constriction rates, and cell morphology in kinase-deficient cells. They find that septin localization and splitting kinetics, as well as AMR constriction rates, are significantly perturbed in elm1∆ and gin4∆ mutants but remain largely unaffected in hsl1∆ and kcc4∆. A similar trend is observed in terms of cell morphology and viability.

Next, the authors focus on elm1∆ and gin4∆ cells, demonstrating that the residence time of the F-BAR protein Hof1 is significantly increased and defective in these mutants. Using yeast two-hybrid (Y2H) and in vitro binding assays, they show that the KA1 domain of Gin4 interacts with the F-BAR domain of Hof1, which may explain the cytokinesis-related functions of Elm1 and Gin4. Supporting this, they find that Gin4's role in septin localization, AMR constriction kinetics, and Hof1 bud neck localization is kinase-independent.

The authors then conduct a series of artificial tethering experiments given their bud neck localization is mostly interdependent. They first demonstrate that artificially tethering Gin4 to the bud neck rescues the morphology defects of elm1∆ cells, with the strongest rescue observed when Gin4 was forced to interact with Hsl1-an effect that was also kinase-independent. Additionally, artificial tethering of Hsl1 to the bud neck restores the morphology of elm1∆ cells in a KA1 domain-dependent manner, suggesting that Hsl1 functions downstream of Elm1 to maintain normal cell morphology. Consistently, artificial tethering of Elm1 to the bud neck in gin4∆ cells rescues morphology defects, as well as defects in Myo1 localization and AMR constriction, but only in the presence of full-length Hsl1. The rescue fails in the absence of Hsl1 or when using a version of Hsl1 lacking the KA1 domain, which supports the role of Hsl1 downstream to Elm1 in cytokinesis.

Strengths

Altogether, this study offers valuable insights into the mode of cytokinesis regulation mediated by the septin-related kinases, mainly Elm1, Gin4, and Hsl1, and would be an important contribution to the field of septins and cytokinesis after addressing current weaknesses.

Weaknesses

(1) When assessing rescue of the elm1∆ phenotype, it needs to become clearer whether only morphology or also cytokinesis and septin organization are rescued.

(2) The quantification of the microscopy data does not always match up with the example images, and it's not always clear how the authors quantitatively analyzed their data.

(3) The forced tethering data are key to the paper, but the lack of a summarizing table makes it difficult to grasp the full picture.

(4) Novel results and those confirming earlier results could be better distinguished.

Reviewer #2 (Public review):

Summary:

The authors investigate single-neuron activity in rhesus macaques during model-based (MB) and model-free (MF) reinforcement learning (RL). Using a well-established two-step choice task, they analyze neural correlates of MB and MF learning across four brain regions: the anterior cingulate cortex (ACC), dorsolateral PFC (DLPFC), caudate, and putamen. The study provides strong evidence that these regions encode distinct RL-related signals, with ACC playing a dominant role in MB learning and caudate updating value representations after rare transitions. The authors apply rigorous statistical analyses to characterize neural encoding at both population and single-neuron levels.

Strengths:

(1) The research fills a gap in the literature, which has been limited in directly dissociating MB vs. MF learning at the single unit level and across brain areas known to be involved in reinforcement learning. This study advances our understanding of how different brain regions are involved in RL computations.

(2) The study used a two-step choice task Miranda et al., (2020), which was previously established for distinguishing MB and MF reinforcement learning strategies.

(3) The use of multiple brain regions (ACC, DLPFC, caudate, and putamen) in the study enabled comparisons across cortical and subcortical structures.

(4) The study used multiple GLMs, population-level encoding analyses, and decoding approaches. With each analysis, they conducted the appropriate controls for multiple comparisons and described their methods clearly.

(5) They implemented control regressors to account for neural drift and temporal autocorrelation.

(6) The authors showed evidence for three main findings:<br /> a) ACC as the strongest encoder of MB variables from the four areas, which emphasizes its role in tracking transition structures and reward-based learning. The ACC also showed sustained representation of feedback that went into the next trial.<br /> b) ACC was the only area to represent both MB and MF value representations.<br /> c) The caudate selectively updates value representations when rare transitions occur, supporting its role in MB updating.

(7) The findings support the idea that MB and MF reinforcement learning operate in parallel rather than strictly competing.

(8) The paper also discusses how MB computations could be an extension of sophisticated MF strategies.

Weaknesses: o

(1) There is limited evidence for a causal relationship between neural activity and behavior. The authors cite previous lesion studies, but causality between neural encoding in ACC, caudate, and putamen and behavioral reliance on MB or MF learning is not established.

(2) There is a heavy emphasis on ACC versus other areas, but it is unclear how much of this signal drives behavior relative to the caudate.

(3) The role of the putamen is somewhat underexplored here.

(4) The authors mention the monkeys were overtrained before recording, which might have led to a bias in the MB versus MF strategy.

(5) The GLM3 model combines MB and MF value estimates but does not clearly mention how hyperparameters were optimized to prevent overfitting. While the hybrid model explains behavior well, it does not clarify whether MB/MF weighting changes dynamically over time.

(6) It was unclear from the task description whether the images used changed periodically or how the transition effect (e.g., in Figure 3) could be disambiguated from a visual response to the pair of cues.

Reviewer #2 (Public review):

Summary:

Photoreceptor neurons are crucial for vision, and discovering pathways necessary for photoreceptor health and survival can open new avenues for therapeutics. Studies have shown that metabolic dysfunction can cause photoreceptor degeneration and vision loss, but the metabolic pathways maintaining photoreceptor health are not well understood. This is a fundamental study that shows that glutamine catabolism is critical for photoreceptor cell health using in vivo model systems.

Strengths:

The data are compelling, and the consideration of potential confounding factors (such as glutaminase 2 expression) and additional experiments to examine the synaptic connectivity and inner retina added strength to this work. The authors were also careful not to overstate their claims, but to provide solid conclusions that fit the results and data provided in their study. The findings linking asparagine supplementation and the inhibition of the integrated stress response to glutamine catabolism within the rod photoreceptor cell are intriguing and innovative. Overall, the authors provide convincing data to highlight that photoreceptors utilize various fuel sources to meet their metabolic needs, and that glutamine is critical to these cells for their biomass, redox balance, function and survival.

Reviewer #2 (Public review):

In the manuscript entitled "VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent transcription and tumorigenesis by engaging p300", Gu et al. investigated two Hippo pathway-related gene fusion events (i.e., VGLL2-NCOA2, TEAD1-NCOA2) in spindle cell rhabdomyosarcoma (scRMS). They demonstrate that these fusion proteins activate Hippo downstream gene transcription independently of YAP/TAZ. Using BioID-based mass spectrometry analysis, the authors identify histone acetyltransferase CBP/p300 as a specific binding protein for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Pharmacologically targeting p300 inhibits the fusion proteins-induced Hippo downstream gene transcription and tumorigenesis.

Overall, this work provides novel mechanistic insights into scRMS-associated gene fusions in tumorigenesis and reveals potential therapeutic targets for cancer treatment. The manuscript is well-written and easy to follow. Below are a few comments based on the revised study.

(1) While the study majorly focuses on Hippo downstream gene transcription, a significant portion of genes regulated by the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins are non-Hippo downstream genes (Fig. 3). Further characterization of how both Hippo and non-Hippo downstream genes contribute to fusion proteins-induced oncogenesis would enhance our understanding of scRMS etiology.

(2) A potential limitation of this study is the reliance on overexpression approaches to investigate VGLL2-NCOA2 and TEAD1-NCOA2 fusion genes, which may not fully reflect pathological conditions in scRMS patients. Despite this, the significant study offers valuable mechanistic insights into fusion genes-induced scRMS and provides molecular foundation for developing targeted therapies.

Reviewer #2 (Public review):

Summary:

Giménez-Orenga carried out this study to assess whether human endogenous retroviruses (HERVs) could be used to improve the diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM). To this end, they used the HERV-V3 array developed previously, to characterize the genome-wide changes in expression of HERVs in patients suffering from ME/CFS, FM or both, compared to controls. In turn, they present a useful repertoire of HERVs that might characterize ME/CFS and FM. For most part, the paper is written in a manner that allows a natural understanding of the workflow and analyses carried out, making it compelling. The figures and additional tables presents solid support for the findings. However, some statements made by the authors seem incomplete and would benefit by a more thorough literature review. Overall, this work will be of interest to the medical community seeking in better understanding the co-occurrence of these pathologies, hinting at a novel angle by integrating HERVs, which are often overlooked, into their assessment.

Strengths:

- The work is well-presented, allowing the reader to understand the overall workflow and how the specific aims contribute to filling the knowledge gap in the field.

- The analyses carried out to understand the potential impact on gene expression mediated by HERVs are in line with previous works, making it solid and robust in the context of this study.

Weaknesses:

- The authors claim to obtain genome-wide HERV expression profiles. However, the array used was developed using hg19, while the genomic analysis of this work are carried out using a liftover to hg38. It would improve the statement and findings to include a comparation of the differences in HERVs available in hg38, and how this could impact the "genome-wide" findings.

- The authors in some points are not thorough with the cited literature. Two examples are:<br /> (1) Lines 396-397 the authors say "the MLT1, usually found enriched near DE genes (Bogdan et al., 2020)". I checked the work by Bogdan, and they studied bacterial infection. A single work in a specific topic is not sufficient to support the statement that MLT1 is "usually" in close vicinity to differentially expressed genes. More works are needed to support this.<br /> (2) After the previous statement, the authors go on to mention "contributing to the coding of conserved lncRNAs (Ramsay et al., 2017)". First, lnc = long non-coding, so this doesn't make sense. Second, in the work by Ramsay they mention "that contributed a significant amount of sequence to primate lncRNAs whose expression was conserved", which is different to what the authors in this study are trying to convey. Again, additional work and a rephrasing might help to support this idea.

- When presenting the clusters, the authors overlook the fact that cluster 4 is clearly control-specific, and fail to discuss what this means. Could this subset of HERV be used as bona fide markers of healthy individuals in the context of these diseases? Are they associated with DE genes? What could be the impact of such associations?

Appraisals on aims:

The authors set specific questions and presented the results to successfully answer them. The evidence is solid, with some weaknesses discussed above that will methodologically strengthen the work.

Likely impact of work on the field:<br /> This work will be of interest to the medical community looking for novel ways to improve clinical diagnosis. Although future works with a greater population size, and more robust techniques such as RNA-Seq, are needed, this is the first step in presenting a novel way to distinguish these pathologies.

It would be of great benefit to the community to provide a table/spreadsheet indicating the specific genomic locations of the HERVs specific to each condition. This will allow proper provenance for future researchers interesting in expanding on this knowledge, as these genomic coordinates will be independent of the technique used (as was the array used here).

Comments on revisions:

When addressing the comments made in the previous round, there are some answers that lack substance and don't seem to be incorporated in the manuscript. For example, the authors say:

Authors' response: This is an important point. However, the low number of probes (less than 100) that were excluded from our analysis by lack of correspondence with hg38 among the 1,290,800 probesets was interpreted as insignificant for "genome-wide" claims. An aspect that will be explained in the revised version of this manuscript.

I checked the revised manuscript with tracked changes, and there doesn't seem to be an updated explanation to this. In which lines is this explained?

For the other response:

Authors' response: Using control DE HERV as bona fide markers of healthy individuals seems like an interesting possibility worth exploring. Control DE HERV (cluster 4) associate with DE genes involved in apoptosis, T cell activation and cell-cell adhesion (modules 1 and 6). The impact of which deserves further study.

I couldn't find an updated mention of this in the discussion.

Another point that I raised was regarding the decision of using an FDR of 0.1 instead of 0.05. The authors only speculate about the impacts in their answer, while I believe that this could have been rigorously addressed. Since this was done in R, and DE analysis are relatively fast, I don't see a reason as to why this part was not repeated and discussed accordingly.

For other analyses, there doesn't seem to be a problem with using 0.05 as threshold. Examples of this are the "Overrepresentation functional analysis", or the "Statistical analysis" part of the methods they say "we used a Fisher exact test to calculate p-value, considering enriched in the provided list if an adjusted p-value (FDR) was less than 0.05".

Just to make this point clear: I'm not asking the authors to repeat all the work using the 0.05 FDR threshold, but rather that they are aware and conscious about the impact of this, and give an idea to the audience on how it would change the DE numbers. This would put in perspective the findings to any future reader.

I think that most of the other answers to both my previous concerns and the other reviewer's concerns are ok. My last outstanding concern is that the probe coordinates apparently can't be shared, which undermines a lot this study reproducibility, and its use by future researches which won't be able to compare their results to this study.

Reviewer #3 (Public review):

Summary

This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation to the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network, constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience.

Strengths

Adaptive optics imaging of murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is a benefit for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.

The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used, a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.

One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. This model would potentially allow for controlling the size, depth and severity of the laser injury opening interesting avenues for future study.

The time-course, 2D and 3D spatial activation pattern of microglial activation are striking and provide an unprecedented view of the retinal response to mild injury.

Weaknesses

Generalization of the (lack of) neutrophil response to photoreceptor loss - there is ample evidence in literature that neutrophils are heavily recruited in response to severe retinal damage that includes photoreceptor loss. Why the same was not observed here in this article remains an open question. One could hypothesize that neutrophil recruitment might indeed occur under conditions that are more in line with the more extreme damage models, for example, with a stronger and global ablation (substantially more photoreceptor loss over a larger area). This parameter space is unwieldy and sufficiently large to address the question conclusively in the current article, i.e. how much photoreceptor loss leads to neutrophil recruitment? By the same token, the strong and general conclusion in the title - Photoreceptor loss does not recruit neutrophils - cannot be made until an exhaustive exploration be made of the same parameter space. A scaling back may help here, to reflect the specific, mild form of laser damage explored here, for instance - Mild photoreceptor loss does not recruit neutrophils despite...

EIU model - The EIU model was used as a positive control for neutrophil extravasation. Prior work with flow cytometry has shown a substantial increase in neutrophil counts in the EIU model. Yet, in all, the entire article shows exactly 2 examples in vivo and 3 ex vivo (Figure 7) of extravasated neutrophils from the EIU model (n = 2 mice). The general conclusion made about neutrophil recruitment (or lack thereof) is built partly upon this positive control experiment. But these limited examples, especially in the case where literature reports a preponderance of extravasated neutrophils, raise a question on the paradigm(s) used to evaluate this effect in the mild laser damage model.

Overall, the strengths outweigh the weaknesses, provided the conclusions/interpretations are reconsidered.

Reviewer #2 (Public review):

The authors have now addressed the most important points, and they include more comprehensive evaluation of their method and comparisons to other approaches for multiple datasets.

Some points would benefit from clarification:

- Figure 1B now compares "Otsu thresholding", "WNet 3D - No artifacts" and "WNet 3d". Why don't you also report the score for "Otsu thresholding - No Artifacts"? To my understanding this is a post-processing operation to remove small and very large objects, so it could easily be applied to the Otsu thresholding. Given the good results for Otsu thresholding alone (quite close F1-score to WNet 3d), it seems like DL might not really be necessary at all for this dataset and including "Otsu thresholding - No artifacts" would enable evaluating this point.

- CellPose and StarDist perform poorly in all the experiments performed by the authors. In almost all cases they underperform Otsu thresholding, which is in most cases on par with the WNet results (except for "Mouse Skull Nuclei CBG"). This is surprising and contradicts the collective expertise of the community: good CellPose and StarDist models can be trained for the 3D instance segmentation tasks studied here. Perhaps these methods were not trained in an optimal way. Seems unlikely that it is not possible to get much better CellPose or StarDist models for these tasks (current versions are on par or much worse than Otsu!), as I have applied both of these models successfully in similar settings. Specifically, it seems unlikely that the developers of CellPose or StarDist would obtain similarly poor scores on the same data (note I am not one of the developers).

The current experiments still highlight an interesting aspect: the problem of training / fine-tuning these methods correctly on new data and the technical challenges associated with this. But the reported results should by no means be taken as a fair assessment of the capabilities of StarDist or CellPose.

Please note that I did not have time to test the Napari plugin again, so I did not evaluate whether it improved in usability.

Reviewer #2 (Public review):

Summary:

In this manuscript, Zhang et al. examine neural activity in motor cortex as monkeys make reaches in a novel target interception task. Zhang et al. begin by examining the single neuron tuning properties across different moving target conditions, finding several classes of neurons: those that shift their preferred direction, those that change their modulation gain, and those that shift their baseline firing rates. The authors go on to find an interesting, tilted ring structure of the neural population activity, depending on the target speed, and find that 1) the reach direction has consistent positioning around the ring, and 2) the tilt of the ring is highly predictive of the target movement speed. The authors then model the neural activity with a single neuron representational model and a recurrent neural network model, concluding that this population structure requires a mixture of the three types of single neurons described at the beginning of the manuscript.

Strengths:

I find the task the authors present here to be novel and exciting. It slots nicely into an overall trend to break away from a simple reach-to-static-target tasks to better characterize the breadth of how motor cortex generates movements. I also appreciate the movement from single neuron characterization to population activity exploration, which generally serves to anchor the results and make them concrete. Further, the orbital ring structure of population activity is fascinating, and the modeling work at the end serves as a useful baseline control to see how it might arise.

Weaknesses:

While I find the behavioral task presented here to be excitingly novel, I find the presented analyses and results to be far less interesting than they could be. Key to this, I think, is that the authors are examining this task and related neural activity primarily with a single-neuron representational lens. This would be fine as an initial analysis, since the population activity is of course composed of individual neurons, but the field seems to have largely moved towards a more abstract "computation through dynamics" framework that has, in the last several years, provided much more understanding of motor control than the representational framework has. As the manuscript stands now, I'm not entirely sure what interpretation to take away from the representational conclusions the authors made (i.e. the fact that the orbital population geometry arises from a mixture of different tuning types). As such, by the end of the manuscript, I'm not sure I understand any better how motor cortex or its neural geometry might be contributing to the execution of this novel task.

Main Comments:

My main suggestions to the authors revolve around bringing in the computation through a dynamics framework to strengthen their population results. The authors cite the Vyas et al. review paper on the subject, so I believe they are aware of this framework. I have three suggestions for improving or adding to the population results:

(1) Examination of delay period activity: one of the most interesting aspects of the task was the fact that the monkey had a random-length delay period before he could move to intercept the target. Presumably, the monkey had to prepare to intercept at any time between 400 and 800 ms, which means that there may be some interesting preparatory activity dynamics during this period. For example, after 400ms, does the preparatory activity rotate with the target such that once the go cue happens, the correct interception can be executed? There is some analysis of the delay period population activity in the supplement, but it doesn't quite get at the question of how the interception movement is prepared. This is perhaps the most interesting question that can be asked with this experiment, and it's one that I think may be quite novel for the field--it is a shame that it isn't discussed.

(2) Supervised examination of population structure via potent and null spaces: simply examining the first three principal components revealed an orbital structure, with a seemingly conserved motor output space and a dimension orthogonal to it that relates to the visual input. However, the authors don't push this insight any further. One way to do that would be to find the "potent space" of motor cortical activity by regression to the arm movement and examine how the tilted rings look in that space. Presumably, then, the null space should contain information about the target movement. The ring tilt will likely be evident if the authors look at the highest variance neural dimension orthogonal to the potent space (the "null space")--this is akin to PC3 in the current figures, but it would be nice to see what comes out when you look in the data for it.

The authors attempt this sort of analysis in the supplement, alongside their dPCA results, but the results seem misinterpreted. The authors do identify one kind of output-potent space using the reach direction components of dPCA, and the reach directions are indeed aligned here. However, they then go on to interpret the target-velocity space as the output-null space, orthogonal to the potent space. There are two problems with this. 1) The target-velocity space is not necessarily orthogonal to the reach-direction space. This is a key aspect of dPCA--while the individual components within a particular marginalization space are orthogonal, the marginalization spaces themselves are not necessarily orthogonal unless they are forced to be (which the authors don't mention doing). 2) Even if the target-velocity space were orthogonal to the reach-direction space, it would not comprise the whole output-null space--such a null space would also include dimensions of neural population activity that have target-velocity/reach-direction interaction, which the authors show is a major component of neural population variance. Incidentally, the dPCA analysis the authors present shows what I would expect from their unsupervised results, but as it is written, the dPCA results are interpreted in a strange or potentially misleading way.

(3) RNN perturbations: as it's currently written, the RNN modeling has promise, but the perturbations performed don't provide me with much insight. I think this is because the authors are trying to use the RNN to interpret the single neuron tuning, but it's unclear to me what was learned from perturbing the connectivity between what seems to me almost arbitrary groups of neurons. It seems to me that a better perturbation might be to move the neural state before the movement onset to see how it changes the output. For example, the authors could move the neural state from one tilted ring to another to see if the virtual hand then reaches a completely different (yet predictable) target. Moreover, if the authors can more clearly characterize the preparatory movement, perhaps perturbations in the delay period would provide even more insight into how the interception might be prepared.

Reviewer #3 (Public review):

This work describes the tandem linkage of influenza hemagglutinin (HA) receptor binding domains of diverse subtypes to create 'beads on a string' (BOAS) immunogens. They show that these immunogens elicit ELISA binding titers against full-length HA trimers in mice, as well as varying degrees of vaccine mismatched responses and neutralization titers. They also compare these to BOAS conjugated on ferritin nanoparticles and find that this did not largely improve immune responses. This work offers a new type of vaccine platform for influenza vaccines, and this could be useful for further studies on the effects of conformation and immunodominance on the resulting immune response. 

Overall, the central claims of immunogenicity in a murine model of the BOAS immunogens described here are supported by the data. 

Strengths included the adaptability of the approach to include several, diverse subtypes of HAs. The determination of an optimal composition of strains in the 5-BOAS that overall yielded the best immune responses was an interesting finding and one that could also be adapted to other vaccine platforms. Lastly, as the authors discuss, the ease of translation to an mRNA vaccine is indeed a strength of this platform. 

One interesting and counter-intuitive result is the high levels of neutralization titers seen to vaccine-mismatched, group 2 H7 in the 5-BOAS group that differs from the 4-BOAS with the addition of a group 1 H5 RBD. At the same time, no H5 neutralization titers were observed for any of the BOAS immunogens, yet they were seen for the BOAS-NP. Uncovering where these immune responses are being directed and why these discrepancies are being observed would be informative future work. 

There are a few caveats in the data that should be noted: 

(1) 20 ug is a pretty high dose for a mouse and the majority of the serology presented is after 3 doses at 20 ug. By comparison, 0.5-5 ug is a more typical range (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380945/, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9980174/). Also, the authors state that 20 ug per immunogen was used, including for the BOAS-NP group, which would mean that the BOAS-NP group was given a lower gram dose of HA RBD relative to the BOAS groups. 

(2) Serum was pooled from all animals per group for neutralization assays, instead of testing individual animals. This could mean that a single animal with higher immune responses than the rest in the group could dominate the signal and potentially skew the interpretation of this data. 

(3) In Figure S2, it looks like an apparent increase in MW by changing the order of strains here, which may be due to differences in glycosylation. Further analysis would be needed to determine if there are discrepancies in glycosylation amongst the BOAS immunogens and how those differ from native HAs. 

Comments on revisions:

The authors have addressed all concerns upon revision.

Reviewer #2 (Public review):

Recent findings in the field of motor learning have pointed to the combined action of multiple mechanisms that potentially contribute to changes in motor output during adaptation. A nearly ubiquitous motor learning process occurs via the trial-by-trial compensation of motor errors, often attributed to cerebellar-dependent updating. This error-based learning process is slow and largely unconscious. Additional learning processes that are rapid (e.g., explicit strategy-based compensation) have been described in discrete movements like goal-directed reaching adaptation. However, the role of rapid motor updating during continuous movements such as walking has been either under explored or inconsistent with those found during adaptation of discrete movements. Indeed, previous results have largely discounted the role of explicit strategy-based mechanisms for locomotor learning. In the current manuscript, Rossi et al. provide convincing evidence for a previously unknown rapid updating mechanism for locomotor adaptation. Unlike the now well-studied explicit strategies employed during reaching movements, the authors demonstrate that this stimulus-response mapping process is largely unconscious. The authors show that in approximately half of subjects, the mapping process appears to be memory based while the remainder of subjects appear to perform structural learning of the task design. The participants that learned using a structural approach had the capability to rapidly generalize to previously unexplored regions of the perturbation space.

One result that will likely be particularly important to the field of motor learning is the authors' quite convincing correlation between the magnitude of proprioceptive recalibration and the magnitude error-based updating. This result beautifully parallels results in other motor learning tasks and appears to provide a robust marker for the magnitude of the mapping process (by means of subtracting off the contribution of error-based motor learning). This is a fascinating result with implications for the motor learning field well beyond the current study.

A major strength of this manuscript is the large sample size across experiments and the extent of replication performed by the authors in multiple control experiments.

Finally, I commend the authors on extending their original observations via Experiment 2. While it seems that participants use a range of mapping mechanisms (or indeed a combination of multiple mapping mechanisms), future experiments may be able to tease apart why some subjects use memory versus structural mapping. A future ability to push subjects to learn structurally-based mapping rules has the potential to inform rehabilitation strategies.

Overall, the manuscript is well written, the results are clear, and the data and analyses are convincing.

Strengths:

(1) Convincing behavioral data supporting the existence of multiple learning processes during split-belt adaptation. Further convincing correlations typing the extent of forward-model based adaptation with proprioceptive recalibration.<br /> (2) The authors test a veritable "zoo" of prior motor learning models to show that these models do not account for their behavioral results.<br /> (3) The authors develop a convincing alternative model (PM-ReMap) that appears to account for their behavioral results by explicitly modeling forward-model based adaptation in parallel with goal remapping.

Reviewer #2 (Public Review):

Summary:

Naïve CD4 T cells in CD11c-Cre p28-floxed mice express highly elevated levels of proinflammatory IFNg and the transcription factor T-bet. This phenotype turned out to be imposed by thymic dendritic cells (DCs) during CD4SP T cell development in the thymus [PMID: 23175475]. The current study affirms these observations, first, by developmentally mapping the IFNg dysregulation to newly generated thymic CD4SP cells [PMID: 23175475], second, by demonstrating increased STAT1 activation being associated with increased T-bet expression in CD11c-Cre p28-floxed CD4 T cells [PMID: 36109504], and lastly, by confirming IL-27 as the key cytokine in this process [PMID: 27469302]. The authors further demonstrate that such dysregulated cytokine expression is specific to the Th1 cytokine IFNg, without affecting the expression of the Th2 cytokine IL-4, thus proposing a role for thymic DC-derived p28 in shaping the cytokine response of newly generated CD4 helper T cells. Mechanistically, CD4SP cells of CD11c-Cre p28-floxed mice were found to display epigenetic changes in the Ifng and Tbx21 gene loci that were consistent with increased transcriptional activities of IFNg and T-bet mRNA expression. Moreover, in autoimmune Aire-deficiency settings, CD11c-Cre p28-floxed CD4 T cells still expressed significantly increased amounts of IFNg, exacerbating the autoimmune response and disease severity. Based on these results, the investigators propose a model where thymic DC-derived IL-27 is necessary to suppress IFNg expression by CD4SP cells and thus would impose a Th2-skewed predisposition of newly generated CD4 T cells in the thymus, potentially relevant in autoimmunity.

Strengths:

Experiments are well-designed and executed. The conclusions are convincing and supported by the experimental results.

Weaknesses from the original round of review:

The premise of the current study is confusing as it tries to use the CD11c-p28 floxed mouse model to explain the Th2-prone immune profile of newly generated CD4SP thymocytes. Instead, it would be more helpful to (1) give full credit to the original study which already described the proinflammatory IFNg+ phenotype of CD4 T cells in CD11c-p28 floxed mice to be mediated by thymic dendritic cells [PMID: 23175475], and then, (2) build on that to explain that this study is aimed to understand the molecular basis of the original finding.

In its essence, this study mostly rediscovers and reaffirms previously reported findings, but with different tools. While the mapping of epigenetic changes in the IFNg and T-bet gene loci and the STAT1 gene signature in CD4SP cells are interesting, these are expected results, and they only reaffirm what would be assumed from the literature. Thus, there is only incremental gain in new insights and information on the role of DC-derived IL-27 in driving the Th1 phenotype of CD4SP cells in CD11c-p28 floxed mice.

Altogether, the major issues of this study remain unresolved:

(1) It is still unclear why the p28-deficiency in thymic dendritic cells would result in increased STAT1 activation in CD4SP cells. Based on their in vitro experiments with blocking anti-IFNg antibodies, the authors conclude that it is unlikely that the constitutive activation of STAT1 would be a secondary effect due to autocrine IFNg production by CD4SP cells. However, this possibility should be further tested with in vivo models, such as Ifng-deficient CD11c-p28 floxed mice. Alternatively, is this an indirect effect by other IFNg producers in the thymus, such as iNKT cells? It is necessary to explain what drives the STAT1 activation in CD11c-p28 floxed CD4SP cells in the first place.

(2) It is also unclear whether CD4SP cells are the direct targets of IL-27 p28. The cell-intrinsic effects of IL-27 p28 signaling in CD4SP cells should be assessed and demonstrated, ideally by CD4SP-specific deletion of IL-27Ra, or by establishing bone marrow chimeras of IL-27Ra germline KO mice.

[Editors' note: The resubmitted paper was minimally revised, and many of the initial concerns remain unresolved.]

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

IDR domains are assumed flexible which I believe is not always the case. Also, I'm not sure how ubiquitous are the assumed binding sites on the DNA for multiple subdomains along the IDR. These assumptions though seem like interesting points of departure for further experiments.

Reviewer #2 (Public review):

In this manuscript, Hes et al. present a comprehensive multi-species atlas of the dorsal vagal complex (DVC) using single-nucleus RNA sequencing, identifying over 180,000 cells and 123 cell types across five levels of granularity in mice and rats. Intriguingly, the analysis uncovered previously uncharacterized cell populations, including Kcnj3-expressing astrocytes, neurons co-expressing Th and Cck, and a population of leptin receptor-expressing neurons in the rat area postrema, which also express the progenitor marker Pdgfra. These findings suggest species-specific differences in appetite regulation. This study provides a valuable resource for investigating the intricate cellular landscape of the DVC and its role in metabolic control, with potential implications for refining obesity treatments targeting this hindbrain region.

In line with previous work published by the PI, the topic is of clear scientific relevance, and the data presented in this manuscript are both novel and compelling. Additionally, the manuscript is well-structured, and the conclusions are robust and supported by the data. Overall, this study significantly enhances our understanding of the DVC and sheds light on key differences between rats and mice.

I applaud the authors for the depth of their analysis. However, I have a few major concerns, comments, and suggestions that should be addressed.

(1) If I understand the methodology correctly, mice were fasted overnight and then re-fed for 2 hours before being sacrificed (lines 91-92), which occurred 4 hours after the onset of the light phase (line 111). This means that the re-fed animals had access and consequently consumed food when they typically would not. While I completely recognize that every timepoint has its limitations, the strong influence of the circadian rhythm on the DVC gene expression (highlighted by the work published by Lukasz Chrobok), and the fact that timing of food/eating is a potent Zeitgeber, might have an impact on the analysis and should be mentioned as a potential limitation in the discussion (along with citing Dr Chrobok's work). Could this (i.e., eating during a time when the animals are not "primed by their own circadian clock to eat" potentially explain why the meal-related changes in gene expression were relatively small?

(2) In the Materials and Methods section, LiCl is mentioned as one of the treatment conditions; however, very little corresponding data are presented or discussed. Please include these results and elaborate on the rationale for selecting LiCl over other anorectic compounds.

(3) The number of animals used differs significantly between species, which the authors acknowledge as a limitation in the discussion. Since the authors took advantage of previously published mouse data sets (Ludwig and Dowsett data sets), I wonder if the authors could compare/integrate any rat data set currently available in rats as well to partially address the sample size disparity.

(4) Dividing cells in AP vs NTS vs DMX clusters and analyzing potential species differences would significantly enhance the quality of the manuscript, given the partially diverse functions of these regions. This could be done by leveraging existing published datasets that employed spatial transcriptomics or more classical methodologies (e.g., PMID: 39171288, PMID: 39629676, PMID: 38092916). I would be interested to hear the authors' perspective on the feasibility of such an analysis.

(5) Given the manuscript's focus on feeding and metabolism, I believe a more detailed description and comparison of the transcription profile of known receptors, neurotransmitters, and neuropeptides involved in food intake and energy homeostasis between mice and rats would add value. Adding a curated list of key genes related to feeding regulation would be particularly informative.

Reviewer #2 (Public review):

This work aims to study the evolution of nitrogenanses, understanding how their structure and function adapted to changes in the environment, including oxygen levels and changes in metal availability.

The study predicts > 5000 structures of nitrogenases, corresponding to extant, ancestral, and alternative ancestral sequences. It is observed that structural variations in the nitrogenases correlate with phylogenetic relationships. The amount of data generated in this study represents a massive undertaking that is certain to be a resource for the community. The study also provides strong insight into how structural evolution correlates with environmental and biological phenotypes.

The challenge with this study is that all (or nearly all) of the quantitative analyses presented are based on RMSD calculations, many of which are under 2 angstroms. For all intents and purposes, two structures with RMSD < 2 angstroms could be considered 'structurally identical'. A lot of insight generated is based on minuscule differences in RMSD, for which it is not clear that they are significantly different. The suggestion would be to find a way to evaluate the RMSD metric and determine whether these values, as obtained for structures being compared, are reliable. Some options are provided in earlier studies: PMID: 11514933, PMID: 17218333, PMID: 11420449, PMID: 8289285 (and others).

It could also be valuable to focus more on site-specific RMSDs rather than Global RMSDs. The high conservation in the nitrogenases likely ensures that the global RMSDs will remain low across the family. Focusing on specific regions might reveal interesting differences between clades that are more informative regarding the evolution of structure in tandem with environment/time.

Reviewer #2 (Public review):

As remains obvious from my previous reviews, I still consider this to be an important paper and that is final and publishable in its current state.

In that previous review, I revealed my identity to help reassure the authors that I was doing my best to remain unbiased because I work in this area and some of the authors' results directly impact my prior research. I was genuinely excited to see the earlier preprint version of this paper when it first appeared. I get a lot of joy out of trying to - collectively, as a field - really understand the nature of our data, and I continue to commend the authors here for pushing at the sources of aperiodic activity!

In their manuscript, Schmidt and colleagues provide a very compelling, convincing, thorough, and measured set of analyses. Previously I recommended that the push even further, and they added the current Figure 5 analysis of event-related changes in the ECG during working memory. In my opinion this result practically warrants a separate paper its own!

The literature analysis is very clever, and expanded upon from any other prior version I've seen.

In my previous review, the broadest, most high-level comment I wanted to make was that authors are correct. We (in my lab) have tried to be measured in our approach to talking about aperiodic analyses - including adopting measuring ECG when possible now - because there are so many sources of aperiodic activity: neural, ECG, respiration, skin conductance, muscle activity, electrode impedances, room noise, electronics noise, etc. The authors discuss this all very clearly, and I commend them on that. We, as a field, should move more toward a model where we can account for all of those sources of noise together. (This was less of an action item, and more of an inclusion of a comment for the record.)

I also very much appreciate the authors' excellent commentary regarding the physiological effects that pharmacological challenges such as propofol and ketamine also have on non-neural (autonomic) functions such as ECG. Previously I also asked them to discuss the possibility that, while their manuscript focuses on aperiodic activity, it is possible that the wealth of literature regarding age-related changes in "oscillatory" activity might be driven partly by age-related changes in neural (or non-neural, ECG-related) changes in aperiodic activity. They have included a nice discussion on this, and I'm excited about the possibilities for cognitive neuroscience as we move more in this direction.

Finally, I previously asked for recommendations on how to proceed. The authors convinced me that we should care about how the ECG might impact our field potential measures, but how do I, as a relative novice, proceed. They now include three strong recommendations at the end of their manuscript that I find to be very helpful.

As was obvious from previous review, I consider this to be an important and impactful cautionary report, that is incredibly well supported by multiple thorough analyses. The authors have done an excellent job responding to all my previous comments and concerns and, in my estimation, those of the previous reviewers as well.

Reviewer #2 (Public review):

Summary:

In this work the authors show that dopaminergic neurons (DANs) from the DL1 cluster in Drosophila larvae are required for the formation of aversive memories. DL1 DANs complement pPAM cluster neurons which are required for the formation of attractive memories. This shows the compartmentalized network organization of how an insect learning center (the mushroom body) encodes memory by integrating olfactory stimuli with aversive or attractive teaching signals. Interestingly, the authors found that the 4 main dopaminergic DL1 neurons act partially redundant, and that single cell ablation did not result in aversive memory defects. However, ablation or silencing of a specific DL1 subset (DAN-f1,g1) resulted in reduced salt aversion learning, which was specific to salt but no other aversive teaching stimuli tested. Importantly, activation of these DANs using an optogenetic approach was also sufficient to induce aversive learning in the presence of high salt. Together with the functional imaging of salt and fructose responses of the individual DANs and the implemented connectome analysis of sensory (and other) inputs to DL1/pPAM DANs this represents a very comprehensive study linking the structural, functional and behavioral role of DL1 DANs. This provides fundamental insight into the function of a simple yet efficiently organized learning center which displays highly conserved features of integrating teaching signals with other sensory cues via dopaminergic signaling.

Strengths:

This is a very careful, precise and meticulous study identifying the main larval DANs involved in aversive learning using high salt as a teaching signal. This is highly interesting because it allows to define the cellular substrates and pathways of aversive learning down to the single cell level in a system without much redundancy. It therefore sets the basis to conduct even more sophisticated experiments and together with the neat connectome analysis opens the possibility to unravel different sensory processing pathways within the DL1 cluster and integration with the higher order circuit elements (Kenyon cells and MBONs). The authors' claims are well substantiated by the data and balanced, putting their data in the appropriate context. The authors also implemented neat pathway analyses using the larval connectome data to its full advantage, thus providing network pathways that contribute towards explaining the obtained results.

Weaknesses:

Previous comments were fully addressed by the authors.

Reviewer #2 (Public Review):

Summary:

In this work, Yuasa et al. aimed to study the spatial resolution of modulations in alpha frequency oscillations (~10Hz) within the human occipital lobe. Specifically, the authors examined the receptive field (RF) tuning properties of alpha oscillations, using retinotopic mapping and invasive electroencephalogram (iEEG) recordings. The authors employ established approaches for population RF mapping, together with a careful approach to isolating and dissociating overlapping, but distinct, activities in the frequency domain. Whereby, the authors dissociate genuine changes in alpha oscillation amplitude from other superimposed changes occurring over a broadband range of the power spectrum. Together, the authors used this approach to test how spatially tuned estimated RFs were when based on alpha range activity, vs. broadband activities (focused on 70-180Hz). Consistent with a large body of work, the authors report clear evidence of spatially precise RFs based on changes in alpha range activity. However, the size of these RFs were far larger than those reliably estimated using broadband range activity at the same recording site. Overall, the work reflects a rigorous approach to a previously examined question, for which improved characterization leads to improved consistency in findings and some advance of prior work.

Strengths:

Overall, the authors take a careful and well-motivated approach to data analyses. The authors successfully test a clear question with a rigorous approach and provide strong supportive findings. Firstly, well-established methods are used for modeling population RFs. Secondly, the authors employ contemporary methods for dissociating unique changes in alpha power from superimposed and concomitant broadband frequency range changes. This is an important confound in estimating changes in alpha power not employed in prior studies. The authors show this approach produces more consistent and robust findings than standard band-filtering approaches. As noted below, this approach may also account for more subtle differences when compared to prior work studying similar effects.

Original Weaknesses:

- Theoretical framing: The authors frame their study as testing between two alternative views on the organization, and putative functions, of occipital alpha oscillations: i) alpha oscillation amplitude reflects broad shifts in arousal state, with large spatial coherence and uniformity across cortex; ii) alpha oscillation amplitude reflects more specific perceptual processes and can be modulated at local spatial scales. However, in the introduction this framing seems mostly focused on comparing some of the first observations of alpha with more contemporary observations. Therefore, I read their introduction to more reflect the progress in studying alpha oscillations from Berger's initial observations to the present. I am not aware of a modern alternative in the literature that posits alpha to lack spatially specific modulations. I also note this framing isn't particularly returned to in the discussion. A second important variable here is the spatial scale of measurement. It follows that EEG based studies will capture changes in alpha activity up to the limits of spatial resolution of the method (i.e. limited in ability to map RFs). This methodological distinction isn't as clearly mentioned in the introduction, but is part of the author's motivation. Finally, as noted below, there are several studies in the literature specifically addressing the authors question, but they are not discussed in the introduction.

- Prior studies: There are important findings in the literature preceding the author's work that are not sufficiently highlighted or cited. In general terms, the spatio-temporal properties of the EEG/iEEG spectrum are well known (i.e. that changes in high frequency activity are more focal than changes in lower frequencies). Therefore, the observations of spatially larger RFs for alpha activities is highly predicted. Specifically, prior work has examined the impact of using different frequency ranges to estimate RF properties, for example ECoG studies in the macaque by Takura et al. NeuroImage (2016) [PubMed: 26363347], as well as prior ECoG work by the author's team of collaborators (Harvey et al., NeuroImage (2013) [PubMed: 23085107]), as well as more recent findings from other groups (Luo et al., (2022) BioRxiv: https://doi.org/10.1101/2022.08.28.505627). Also, a related literature exists for invasively examining RF mapping in the time-voltage domain, which provides some insight into the author's findings (as this signal will be dominated by low-frequency effects). The authors should provide a more modern framing of our current understanding of the spatial organization of the EEG/iEEG spectrum, including prior studies examining these properties within the context of visual cortex and RF mapping. Finally, I do note that the author's approach to these questions do reflect an important test of prior findings, via an improved approach to RF characterization and iEEG frequency isolation, which suggests some important differences with prior work.

- Statistical testing: The authors employ many important controls in their processing of data. However, for many results there is only a qualitative description or summary metric. It appears very little statistical testing was performed to establish reported differences. Related to this point, the iEEG data is highly nested, with multiple electrodes (observations) coming from each subject, how was this nesting addressed to avoid bias?

[Editors' note: the authors have addressed the original concerns.]

Reviewer #2 (Public review):

In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin (Nlgn) family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapses, prompting Golf et al. to further investigate astrocytic Neuroligin function in the current study. Using postnatal conditional deletion of Nlgn1-3 specifically from astrocytes in mice, Golf et al. show that virtually no changes in the expression of synaptic proteins or in the properties of synaptic transmission at either excitatory or inhibitory synapses are observed. Moreover, no alterations in the morphology of astrocytes themselves were found. To further extend this finding, the authors additionally analyzed human neurons co-cultured with mouse glia lacking expression of Nlgn1-4. No difference in excitatory synaptic transmission was observed between neurons cultured in the present of wildtype vs. Nlgn1-4 conditional knockout glia. The authors conclude that while Neuroligins are indeed expressed in astrocytes and are hence likely to play some role there, this role does not include any direct consequences on synaptic structure and function, in direct contrast to the model proposed by Stogsdill et al.

Overall, this is a strong study that addresses a fundamental and highly relevant question in the field of synaptic neuroscience. Neuroligins are not only key regulators of synaptic function, they have also been linked to numerous psychiatric and neurodevelopmental disorders, highlighting the need to precisely define their mechanisms of action. The authors take a wide range of approaches to convincingly demonstrate that under their experimental conditions, Nlgn1-3 are efficiently deleted from astrocytes in vivo, and that this deletion does not lead to major alterations in the levels of synaptic proteins or in synaptic transmission at excitatory or inhibitory synapses, or in the morphology of astrocytes. While the co-culture experiments are somewhat more difficult to interpret due to lack of a control for the effect of wildtype mouse astrocytes on human neurons, they are also consistent with the notion that deletion of Nlgn1-4 from astrocytes has no consequences for the function of excitatory synapses. Together, the data from this study provide compelling and important evidence that, whatever the role of astrocytic Neuroligins may be, they do not contribute substantially to synapse formation or function under the conditions investigated.

Reviewer #2 (Public review):

This study by Tardiff, Kang & Gold seeks to i) develop a normative account of how observers should adapt their decision-making across environments with different levels of correlation between successive pairs of observations, and ii) assess whether human decisions in such environments are consistent with this normative model. The authors first demonstrate that, in the range of environments under consideration here, an observer with full knowledge of the generative statistics should take both the magnitude and sign of the underlying correlation into account when assigning weight in their decisions to new observations: stronger negative correlations should translate into stronger weighting (due to the greater information furnished by an anticorrelated generative source), while stronger positive correlations should translate into weaker weighting (due to the greater redundancy of information provided by a positively correlated generative source). The authors then report an empirical study in which human participants performed a perceptual decision-making task requiring accumulation of information provided by pairs of perceptual samples, under different levels of pairwise correlation. They describe a nuanced pattern of results with effects of correlation being largely restricted to response times and not choice accuracy, which could be captured through fits of their normative model (in this implementation, an extension of the well-known drift diffusion model) to the participants' behaviour while allowing for mis-estimation of the underlying correlations. An intriguing result is that the observed pattern of behavioural effects is best explained by a model in which observers marginally underestimated the level of correlation between the generative sources, and that this bias affects behaviour through effects on stimulus encoding that then shape how the evidence furnished by each stimulus sample is weighted in decision formation.

As the authors point out in their very well-written paper, appropriate weighting of information gathered in correlated environments has important consequences for real-world decision-making. Yet, while this function has been well studied for 'high-level' (e.g. economic) decisions, how we account for correlations when making simple perceptual decisions on well-controlled behavioural tasks has not been investigated. As such, this study addresses an important and timely question that will be of broad interest to psychologists and neuroscientists. The computational approach to arrive at normative principles for evidence weighting across environments with different levels of correlation is elegant, makes strong connections with prior work in different decision-making contexts, and should serve as a valuable reference point for future studies in this domain. The empirical study is well designed and executed, and the modelling approach applied to these data showcases an impressively deep understanding of relationships between different parameters of the drift diffusion model and its novel application to this setting. Another strength of the study is that it is preregistered.

In my view, any major weaknesses of the study have been well addressed by the authors during review. An outstanding question that arises from the current work and remains unanswered here is around the (normative?) origin of the correlation underestimates, and the present work lays a strong foundation from which to pursue this question in the future.

Reviewer #2 (Public review):

Summary:

The authors aimed to elucidate the role of Ctla-4 in maintaining intestinal immune homeostasis by using a novel Ctla-4-deficient zebrafish model. This study addresses the challenge of linking CTLA-4 to inflammatory bowel disease (IBD) due to the early lethality of CTLA-4 knockout mice. Four lines of evidence were shown to show that Ctla-4-deficient zebrafish exhibited hallmarks of IBD in mammals: 1) impaired epithelial integrity and infiltration of inflammatory cells; 2) enrichment of inflammation-related pathways and the imbalance between pro- and anti-inflammatory cytokines; 3) abnormal composition of immune cell populations; and 4) reduced diversity and altered microbiota composition. By employing various molecular and cellular analyses, the authors established ctla-4-deficient zebrafish as a convincing model of human IBD.

Strengths:

The characterization of the mutant phenotype is very thorough, from anatomical to histological and molecular levels. The finding effectively established ctla-4 mutants as a novel zebrafish model for investigating human IBD. Evidence from the histopathological and transcriptome analysis was very strong and supports a severe interruption of immune system homeostasis in the zebrafish intestine. Additional characterization using sCtla-4-Ig further probed the molecular mechanism of the inflammatory response, and provided a potential treatment plan for targeting Ctla-4 in IBD models.

Weaknesses:

To probe the molecular mechanism of Ctla-4, the authors used a spectrum of antibodies that target Ctla-4 or its receptors. The phenotype assayed was lymphocyte proliferation, while it was the composition rather than number of immune cells that was observed to be different in the scRNASeq assay. Although sCtla-4 has an effect of alleviating the IBD-like phenotypes, I found this explanation a bit oversimplified.

Comments on revised version:

The authors have sufficiently addressed all my concerns and I don't have further suggestions.

Reviewer #2 (Public review):

Summary:

Chakravarty et al. propose a 'synchronized framework' for studying perceptual decision-making (DM) across species -namely humans, rats, and mice. Although all species shared hallmarks of evidence accumulation, the results highlighted species-specific differences. Humans were the slowest and most accurate, rats optimized the speed-accuracy tradeoff to maximize the reward rate and mice were the fastest but least accurate. In addition, while humans were better fit by a classic DDM with fixed bounds, rodents were better fit by a DDM with collapsing bounds. While comparing behavioral strategies in evidence accumulation tasks across species is an important and timely question, some of the presented differences across species lack a clear interpretation and could be simply caused by differences in the task design. There is important information and analyses missing about the DDM and the other models used, which lowers the confidence and enthusiasm about the results.

Strengths:

The comparison of behavior across species, including humans and commonly used laboratory species like rats and mice, is a fundamental step in neuroscience to establish more informed links between animal experiments and human cognition. In this work, Chakravarty et al. analyze and model the behavior of three species during the same evidence accumulation task. They draw conclusions about the different strategies used in each case.

Weaknesses:

Novelty:<br /> While quite relevant, some parts of the work presented are more novel than others. That EA drives choice behavior and these choices can be described with a DDM have been shown before (see e.g. (Kane et al. 2023; Brunton et al. in 2013; Pinto et al 2018)). The novelty here mostly lies in the comparison of three species in the same task and in fitting the same exact model (close quantitative comparison of behavioral strategies). However, some of the differences lack a clear interpretation. For instance, the values of some of the DDM fitted parameters between the three species are not ordered "as expected" (e.g. non-decision time or DDM BIC). Other comparison results completely lack an explanation (e.g. rats' RT are near optimal while humans and mice are not). The aspect that I found most novel and exciting is the application of HMMs to each of the species. However, this part comes at the end of the paper and has been done without sufficient depth. There is almost no explanation for the results. I would suggest the authors bring up this part and move back to other aspects which are, in my opinion, less novel or interpretable (e.g. results around the optimality of RT).

Task design:<br /> Since there is no fixation, the response time (RT) reflects both the evidence integration time plus the motor time (stimuli are played until a response is given). This design makes it hard to compare RTs between species. While humans just had to press a button, rodents had to move their whole bodies from a central port to a side port. When comparing rats and mice, their difference in size relative to port distance could explain different RTs. This could for example explain the large difference in non-decision time (ndt) in Figure 3F between mice and rats. Are the measurements of the rat and the mouse boxes comparable? The authors should explain this difference more openly and discuss its implications when interpreting the results. The Methods should also provide information about the distance between ports for each species. I also strongly recommend including a few videos of rats and mice performing the task to have a sense of the movements involved in the task in each species.

(1) DDM

Goodness of fit:<br /> The authors conclude that the three species use an accumulation of evidence strategy because they can fit a DDM. However, there is little information about the goodness of these fits. They only show the RT distributions for one example subject (too small to distinguish whether the fit of the histograms is good or not). We suggest they make a figure showing in more detail the match of the RT distributions across subjects (e.g. they can compare RT quartiles for data and model for the entire group of subjects). Then they provide BIC which is a measure that depends on the number of trials. Were the number of trials matched across subjects/species? Could the authors provide a measure independent of the number of trials (e.g. cross-validated log-likelihood per trial)? Moreover, is this BIC computed only on the RTs, mouse responses, or both?

Overparameterization:<br /> The authors chose to include as DDM parameters the variability of the initial offset, the variability in non-decision time, and the variability of the drift rate. Having so many parameters with just one stimulus condition (80:20 ratio of flashes) may lead to unidentifiability problems as recognized previously (e.g. see M. Jones (2021) here osf.io/preprints/psyarxiv/gja3u). Their parameter recovery Supplementary Figure 3 shows that at least two of these variability parameters can not be recovered. I also couldn't find the values of these parameters for the fitted DDM. So I was wondering the extent to which adding these parameters improves the fits and is overall necessary.

Tachometric curves:<br /> The authors show increasing tachometric curves (i.e. Accuracy vs RT) and use this finding as proof of accumulation. They fit these curves using a GAAM with little justification or detail (in fact the GAAM seems to over-fit the data a bit). The authors do not say, however, that the other model used, i.e. the DDM, may not reproduce these increasing tachometric curves because "in its basic form", the DDM gives flat tachometric curves. Does the DDM fitted to the individual RT and choice data capture the monotonic increase observed in the tachometric curves?

Correct vs Error trials:<br /> In a similar line, the authors do not test the fitted DDM separately in correct vs error trials, which is a classical distinction that most DDMs can't capture. It would be good to know if: (1) the RT in the data of correct vs error responses are similar (quantified in panel Figure 2B because in 2E it is not clear) and (2) the same trend between correct and error RTs are observed in the fitted DDMs.

Urgency model:<br /> It is not clear how the urgency model used works. The authors cite Ditterich (2006), but in that paper, the urgency signal was applied to a race model with two decision variables: the urgency signal "accelerated" both DVs equally and sped up the race without favoring one DV versus the other. In a one-dimensional DDM, it is not clear where the urgency is applied. We assume it is applied in the direction of the stimulus, but then it is unclear how the urgency knows about the stimulus, which is what the DDM is trying to estimate in the first place. The authors should explain this model in greater detail and try to resolve this question.

Despite finding differences between species, the analyses seem mostly exploratory instead of hypothesis-driven. There is little justification for why differences in some DDM parameters across species would be expected.

(2) GLM and HMM

The GLM fits show nicely that humans, rats, and mice weigh differently the total provided evidence (Figures 6C-D). This may be because the internal noise in the accumulation of evidence is higher but also it could simply be because animals do not weigh the evidence that is presented when they are already moving towards the side ports. A parsimonious alternative to the "more noisy" species is simply that they only consider the first part of the stimulus. Extending the GLM to capture the differential weighting of each sequential sample (what is called the Psychophysical kernel, PK) should be straightforward and would provide a more fair comparison between species (i.e. perhaps the slope of the psychometric curves is not that different, once evidence is weighted in each species with its corresponding PK.

Choice Bias:<br /> Panel 3G (DDM starting point) shows that both rats and mice are slightly but systematically biased to the Left (x0 < 0.5). Panel 6D "Bias" seems to be showing the absolute value of the GLM bias parameter. It would be nice to (i) show the signed GLM bias parameter. (ii) Compare that the biases computed in the DDM and GLM are comparable across species and subjects; it looks like from the GLM they are comparable in magnitude across species whereas the in DDM they weren't (mice had a much bigger |x0| in the DDM), (iii) explain (or at least comment) on why animals show a systematic bias to one side.

Reviewer #2 (Public review):

Summary:

This is an excellent paper that demonstrates Computational Modeling at its best. The authors propose a mechanism to provide flexibility to learn new information while preserving stability in neural networks by combining structural plasticity and synaptic plasticity.

Strengths:

An intriguing idea, that is well embedded in experimental data.

The problem posed is real, the model uses data to be designed and implemented yet adds to the data novel and useful insight. The project proposes a parsimonious explanation for why neurogenesis can be better than classical plasticity and how stability versus flexibility can be solved with this approach.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #2 (Public review):

Summary:

Shrews go through winter by shrinking their brain and most organs, then regrow them in the spring. The gene expression changes underlying this unusual brain size plasticity were unknown. Here, the authors looked for potential adaptations underlying this trait by looking at differential expression in the hypothalamus. They found enrichments for DE in genes related to the blood brain barrier and calcium signaling, as well as used comparative data to look at gene expression differences that are unique in shrews. This study leverages a fascinating organismal trait to understand plasticity and what might be driving it at the level of gene expression. This manuscript also lays the groundwork for further developing this interesting system.

Strengths:

One strength is that the authors used OU models to look for adaptation in gene expression. The authors also added cell culture work to bolster their findings.

Comments on revised version:

I think that the authors have made a strong revision. No other comments.

Reviewer #2 (Public review):

Summary:

This valuable work aims to infer, from microbiome data, microbial species interaction patterns associated with healthy and unhealthy human gut microbiomes. Using solid techniques from statistical physics, the authors propose that healthy and unhealthy microbiome interaction patterns substantially differ. Unhealthy microbiomes are closer to instability and single-strain dominance; whereas healthy microbiomes showcase near-neutral dynamics, mostly driven by demographic noise and immigration.

Strengths:

A well-written article, relatively easy to follow and transparent despite the high degree of technicality of the underlying theory. The authors provide a powerful inferring procedure, which bypasses the issue of having only compositional data.

Weaknesses:

(1) This sentence in the introduction seems key to me: "Focusing on single species properties as species abundance distribution (SAD), fail to characterise altered states of microbiome." Yet it is not explained what is meant by 'fail', and thus what the proposed approach 'solves'.

(2) Lack of validation, following arbitrary modelling choices made (symmetry of interactions, weak-interaction limit, uniform carrying capacity).<br /> Inconsistent interpretation of instability. Here, instability is associated with the transition to the marginal phase, which becomes chaotic when interaction symmetry is broken. But as the authors acknowledge, the weak interaction limit does not reproduce fat-tailed abundance distributions found in data. On the other hand, strong interaction regimes, where chaos prevails, tend to do so (Mallmin et al, PNAS 2024). Thus, the nature of the instability towards which unhealthy microbiomes approach is unclear.

(3) Three technical points about the methodology and interpretation.<br /> a) How can order parameters h and q0 can be inferred, if in the compositional data they are fixed by definition?<br /> b) How is it possible that weaker interaction variance is associated with approach to instability, when the opposite is usually true?<br /> c) Having an idea of what the empirical data compares to the theoretical fits would be valuable.

Implications:

As the authors say, this is a proof of concept. They point at limits and ways to go forward, in particular pointing at ways in which species abundance distributions could be better reproduced by the predicted dynamical models. One implication that is missing, in my opinion, is the interpretability of the results, and what this work achieves that was missing from other approaches (see weaknesses section above): what do we learn from the fact that changes in microbial interactions characterise healthy from unhealthy microbiota? For instance, what does this mean for medical research?

Reviewer #2 (Public review):

Summary:

This study shows that transcription factor Sp1 is required for correct ventral vs. dorsal targeting of limb-innervating LMC motor neurons using mouse and chick as model systems. In a wild-type embryo, lateral LMC axons specifically target dorsal muscles while medial LMC axons target ventral muscles. The authors convincingly show that this specificity is lost when Sp1 is knocked down or knocked out - axons of both lateral and medial LMC motor neurons project to both dorsal and ventral muscles in mutant conditions. The authors then conduct RNA-seq and ChIP experiments to show that Sp1 loss of function disrupts Ephrin-Epha receptor signaling pathway genes. These molecules are known to provide attractive or repulsive cues to guide LMC axons to their targets. The authors show that attraction/repulsion properties of medial and lateral LMC axons to specific Ephrin/Epha molecules are in fact disrupted in Sp1 mutants using ex vivo explant studies. Finally, the authors show that behaviors like coordinated movement and grip strength are also affected in Sp1 mutant mice. This study convincingly shows that Sp1 is important for correct circuit wiring of LMC neurons, and moves the field forward by elucidating a new level of transcriptional regulation required in this process. However, the claims made by the authors that the mode of Sp1-mediated regulation is through cis-attenuation of Epha activity is not well supported. These and additional strengths and weaknesses in approach and in data interpretation are discussed below.

Strengths:

(1) The study convincingly shows that wildtype levels of Sp1 are necessary for LMC axon targeting specificity. The combination of the following approaches is a strength:<br /> a) Both loss of function and gain of function experiments are performed for Sp1 and show complementary effects on the axon targeting phenotype.<br /> b) Retrograde labeling of LMC neurons from dorsal and ventral muscles shows that Sp1 mutants clearly lose the specificity of LMC axon targeting.<br /> c) The authors also use explant experiments to show that both loss of Sp1 and gain of Sp1 show clear changes in attraction and repulsion to specific ephrin and epha receptor molecules.<br /> d) The Sp1 loss and gain of function experiments are well controlled to show that the changes in axon wiring observed are not due to cell death, cell fate switches, or due to unequal numbers of medial and lateral LMC neurons being labeled in the experiments.

(2) It is also convincing that Sp1 requires cofactors p300 and CBP for its function. In the absence of these cofactors, the gain of function phenotypes of Sp1 are subdued.

Weaknesses:

(1) The robustness of RNAseq and ChIP experiments is difficult to judge as methods are not described. For example, it is unclear if RNAseq is performed on purified motor neurons or on whole spinal cords. This is an important consideration as Sp1 is a broadly expressed protein.

(2) The authors state that expression of Ephrin A5 and Ephrin B2 is reduced based on RNAseq data, however, it is not shown that this reduction occurs specifically in LMC neurons.

(3) The authors show Sp1 ChIP peaks at Ephrin B2 promoter, but nothing is mentioned about peaks at Eprin A5 or other types of signaling molecules like Sema7a, which are also differentially expressed in Sp1 mutants. There is also no mention of the correlation between changes in gene expression seen in RNAseq data and the binding profile of Sp1 seen in ChIP data, which could help establish the robustness of these datasets.

(4) The authors conclude that Sp1 functions by activating Ephrin A5 in medial LMC and Ephrin B2 in lateral LMC. The argument, as I understand it, is that this activation leads to cis attenuation of their respective Epha receptors and therefore targeting the correct muscle. Though none of the data presented go against this hypothesis, this hypothesis is also not fully supported. Specifically:<br /> a) It would be important to know that modulation of Sp1 expression leads to changes in EphrinA5 and B2 in LMC lateral/medial neurons.<br /> b) It would also be important to show that none of the other changes caused by Sp1 are responsible for axon mistargeting by performing rescue experiments with Ephrin A5 and Ephrin B2.<br /> c) To make the most convincing case, experiments showing increased or decreased cis-binding of Ephrin molecules with Epha receptors would be necessary. This study would still be compelling without this last experiment, but the language in the abstract would need to be modulated.

(5) All behavior experiments are done in a pan-neuronal knockout of Sp1. As Sp1 is broadly expressed in neurons, a statement describing whether and why the authors think the phenotypes arise from Sp1's function in LMC motor neurons would be helpful. Experimentally, rescue experiments in which Sp1 is restored in LMC neurons or motor neurons would also make this claim more convincing.

Reviewer #2 (Public review):

Summary:

The manuscript presents findings that indicate a role in controlling Drosophila heart rate for a conserved miRNA (miR-238 in flies). Further, the manuscript localizes the relevant tissue for the function of this miRNA to a subset of neurons that are heavily involved in circadian regulation, thus presenting an interesting mechanistic link between the circadian system and heart rate. Either ubiquitous knockout or ubiquitous overexpression negatively impacts several aspects of heart performance, with a pronounced effect on heart rate. Interestingly, knockdowns in the heart itself are innocuous, but knockdown in LNvS neurons recapitulates the effect on heart rate. Authors use bioinformatics to identify the clockwork orange (cwo) gene as a potential target and validate that cwo expression is reduced when miR-238 is knocked down in LNvS neurons in vivo and also validate that cwo is regulated by miR-238 in cell culture luciferase assays. Exercise shows a modest ability to restore normal cwo expression and a trend toward an effect on survival, but shows a much stronger rescue of the heart rate phenotype.

Strengths:

Evidence is strong for the effect of miR-238 in pdf-positive neurons on the control of heart rate and for cwo as a downstream effector of miR-238.

Work to identify specific targets of miR-283 is well-done and successfully identified a key downstream regulator in cwo.

The potential mechanism using miR-238 to link circadian neurons to heart rate regulation is novel and exciting.

Weaknesses:

The evidence that this is related to normal aging is rather weak, and the effect of exercise on the observed parameters is small and not necessarily working through the miR-238/cwo mechanism.

The authors seem to be conflating two hypotheses in their interpretations. Is miR-283 working through circadian mechanisms or age-related mechanisms? While it is true that aging tends to reduce heart rate, I don't think that means that any intervention that reduces heart rate is causing "senescence". Similarly, reduced survival in miR-283 knockdown flies does not prove that miR-283 promotes healthy aging per se, just that miR-283 is required for health regardless of age.

Survival reduction is quite modest which does not necessarily support the idea that the bradycardia is causing major health issues or premature senescence for the flies. The interpretation of the longevity experiments throughout the manuscript seems overstated.

The study would benefit greatly from a direct test of the author's proposed pathway for exercise to improve bradycardia.

The statement in the discussion "inducing endurance exercise of anti gravity climbing in flies with miR-283 knockdown in LNvs can improve bradyarrhythmic features by decreasing brain miR-283 expression" is not fully supported by data in the paper. There is an association there, but it cannot be said to be the full cause (or even required) without doing more experiments

The summary figure includes both data-supported mechanistic relationships and mechanisms that are inferred or assumed.

Reviewer #2 (Public review):

Summary:

The goal of the study was to uncover the mechanisms mediating tactile-context-dependent locomotion modulation in C. elegans, which represents an interesting model of behavioral plasticity. Starting from a candidate genetic screen focusing on guanylate cyclase (GCY) mutants, the authors identified the AFD-specific gcy-18 gene as essential for tactile-context-dependent locomotion modulation. AFD is primarily characterized as a thermo-sensory neuron. However, key thermosensory transduction genes and the sensory ending structure of AFD were shown here to be dispensable for tactile-context locomotion modulation. AFD actuates tactile-context locomotion modulation via the cell-autonomous actions of GCY-18 and the CNG-3 cyclic nucleotide-gated channel, and via AFD's connection with AIB interneurons through electrical synapses. This represents a potentially relevant synaptic connection linking AFD to the mechanosensory-behavior circuit.

Strengths:

(1) The fact that AFD mediates tactile-context locomotion modulation is new, rather surprising, and interesting.

(2) The authors have combined a very clever microfluidic-based behavioral assay with a large set of genetic manipulations to dissect the molecular and cellular pathways involved. Rescue experiments with single-copy transgenes are very convincing.

(3) The study is very clearly written, and figures are nicely illustrated with diagrams that effectively convey the authors' interpretation.

Weaknesses:

(1) Whereas GCY-18 in AFD and the AFD-AIB synaptic connection clearly play a role in tactile-context locomotion modulation, whether and how they actually modulate the mechanosensory circuit and/or locomotion circuit remains unclear. The possibility of non-synaptic communication linking mechanosensory neurons and AFD (in either direction) was not explored. Thus, in the end, we have not learned much about what GCY-18 and the AFD-AIB module are doing to actuate tactile context-dependent locomotion modulation.

(2) The authors only focused on speed readout, and we don't know if the many behavioral parameters that are modulated by tactile context are also under the control of AFD-mediated modulation.

(3) The AFD-AIB gap junction reconstruction experiment was conducted in an innexin double mutant background, in which the whole nervous system's functioning might be severely impaired, and its results should be interpreted with this limitation in mind.

Reviewer #2 (Public review):

Summary:

This study presents a valuable characterization of the effects of intracranial theta-burst stimulation of the basolateral amygdala on single units spiking activity in several areas in the human brain, associated with memory processing. It is written clearly and concisely, allowing readers to fully understand the analysis used.

The authors used a visual recognition memory task previously employed by their group to characterize the effects of basolateral amygdala stimulation upon memory consolidation (Inman et al, 2018). This current report is an interesting analysis to complement the results reported in the 2018 paper.

Strengths:

Rare combination of human neurophysiology and behavior -<br /> The type of experiment performed in the manuscript, which contains both neurophysiological data, behavior, and a deep brain stimulation intervention (DBS), is incredibly rare, takes many years to accomplish with tight collaboration between clinical and research teams. Our understanding of spiking dynamics of human neurons is very limited, and this report is an important piece in the puzzle that allows DBS to be used in future interventions that will benefit patients' health.

Multiple brain areas included -<br /> It's important to note that the report analyzes brain areas with which the Amygdala has extensive connections (Fig. 1A) - Hippocampus, OFC, Amygdala, ACC. It seems that neurons in all these areas were modulated by the stimulation, except the ACC, in which firing rates were so low, that only a handful of neurons were included in the analysis. This is an important demonstration that low amplitude stimulation (even when reduced to 0.5mA) can travel far and wide across the human brain.

The experiment is cleverly designed to tease apart responses due to visual stimuli (image presentation) and electrical stimulation. Authors suggest that the units modulated by stimulation are largely distinct from those responsive to image offset during trials without stimulation. The subpopulation that responds strongly also tends to have a higher baseline of firing rate. It's important to add that the chosen modulation index is more likely to be significant in neurons with higher firing rates.

Weaknesses:

Readers can benefit from understanding with more details the locations chosen for stimulation - in light of previous studies that found differences between effects based on proximity to white matter (For example - PMID 32446925, Mohan et al, Brain Stimul. 2020 and PMID 33279717 Mankin et al Brain Stimul. 2021).

Reviewer #2 (Public review):

Summary:

This study from the CenGEN consortium addresses several limitations of single-cell RNA (scRNA) and bulk RNA sequencing in C. elegans with a focus on cells in the nervous system. scRNA datasets can give very specific expression profiles, but detecting rare and non-polyA transcripts is difficult. In contrast, bulk RNA sequencing on isolated cells can be sequenced to high depth to identify rare and non-polyA transcripts but frequently suffers from RNA contamination from other cell types. In this study, the authors generate a comprehensive set of bulk RNA datasets from 53 individual neurons isolated by fluorescence-activated cell sorting (FACS). The authors combine these datasets with a previously published scRNA dataset (Taylor et al., 2021) to develop a novel method, called LittleBites, to estimate and subtract contamination from the bulk RNA data. The authors validate the method by comparing detected transcripts against gold-standard datasets on neuron-specific and non-neuronal transcripts. The authors generate an "integrated" list of protein-coding expression profiles for the 53 neuron sub-types, with fewer but higher confidence genes compared to expression profiles based only on scRNA. Also, the authors identify putative novel pan-neuronal and cell-type specific non-coding RNAs based on the bulk RNA data. LittleBites should be generally useful for extracting higher confidence data from bulk RNA-seq data in organisms where extensive scRNA datasets are available. The additional confidence in neuron-specific expression and non-coding RNA expands the already great utility of the neuronal expression reference atlas generated by the CenGEN consortium.

Strengths:

The study generates and analyzes a very comprehensive set of bulk RNA datasets from individual fluorescently tagged transgenic strains. These datasets are technically challenging to generate and significantly expand our knowledge of gene expression, particularly in cells that were poorly represented in the initial scRNA-seq datasets. Additionally, all transgenic strains are made available as a resource from the Caenorhabditis Elegans Genetics Center (CGC).

The study uses the authors' extensive experience with neuronal expression to benchmark their method for reducing contamination utilizing a set of gold-standard validated neuronal and non-neuronal genes. These gold-standard genes will be helpful for benchmarking any C. elegans gene expression study.

Weaknesses:

The bulk RNA-seq data collected by the authors has high levels of contamination and, in some cases, is based on very few cells. The methodology to remove contamination partly makes up for this shortcoming, but the high background levels of contaminating RNA in the FACS-isolated neurons limit the confidence in cell-specific transcripts.

The study does not experimentally validate any of the refined gene expression predictions, which was one of the main strengths of the initial CenGEN publication (Taylor et al, 2021). No validation experiments (e.g., fluorescence reporters or single molecule FISH) were performed for protein-coding or non-coding genes, which makes it difficult for the reader to assess how much gene predictions are improved, other than for the gold standard set, which may have specific characteristics (e.g., bias toward high expression as they were primarily identified in fluorescence reporter experiments).

The study notes that bulk RNA-seq data, in contrast to scRNA-seq data, can be used to identify which isoforms are expressed in a given cell. However, no analysis or genome browser tracks were supplied in the study to take advantage of this important information. For the community, isoform-specific expression could guide the design of cell-specific expression constructs or for predictive modeling of gene expression based on machine learning.

Reviewer #2 (Public review):

Summary:

This study provides valuable context for ongoing research on the role of dopamine in memory and locomotion. DANs have been a fascinating area of study due to their complexity, and this work dissects specific DANs, exploring their roles in different memory-related behaviors while offering some explanations. The discussions provided by the authors effectively situates the study in the broader field of learning, memory, DAN circuitry and behavioral computation in insect brains. The study achieves what it sets out to and it does so unequivocally. The experiments were elegantly designed, leaving little room for doubt in the study's claims. However, the study lacks context regarding the molecular pathways underlying these results. While it strengthens current knowledge by providing robust evidence, it does little to explore the molecular mechanisms behind these effects.

Strengths:

(1) Experiment design is one of the strengths of this study. The experiments are thorough and cover the length and breadth of the core findings of the study. Although a lot of work has already been done in studying the role of dopamine in memory and locomotion, the dissection of the functions of distinct DANs in larvae has been done meticulously with well-structured experiments.<br /> (2) This study fits quite nicely into the puzzle of memory, especially in the context of Dopamine. Previous studies in *Drosophila* adults have shown the opposing roles of DANs in locomotion depending on the context of DAN activation. This study drives that point home for larvae, providing conclusive evidence in that regard.<br /> (3) The use of clear figures and simple language is one of the strengths of this paper. The figures are comprehensive, complete and manage to narrate the story by themselves. The flow of information is smooth. The simple and effective language used maintains scientific rigor while remaining accessible to those new to the field. A pleasant read.

Weaknesses:<br /> (1) The authors have done a great job at structuring the figures. But some main figures would benefit from including the controls instead of placing them in supplementary.<br /> (2) The paper would benefit from a deeper discussion regarding molecular mechanisms underlying their results. It would be interesting to see what the authors think about different Dopamine receptors and how they relate to the findings of this paper.<br /> (3) Throughout the paper, the authors have been clear and comprehensive, but in some cases, further explanation of their choices were missing. For example, the choice to compare bending and tail velocity over other parameters within the same clusters is unclear.

Reviewer #2 (Public review):

Summary:

The authors aimed to develop a novel and efficient method for SV detection, utilizing data from the 1000 Genomes Project (1KGP) for modeling and calibration. This method was subsequently validated using UK population data and applied to identify structural variants associated with specific disease phenotypes.

Strengths:

Third-generation single-molecule sequencing data offers several advantages over traditional high-throughput sequencing methods, particularly due to its long-read lengths, which provide valuable insights into significant forms of genomic variation. The authors have developed an efficient method for detecting structural variations and optimizing the utilization of genomic data. We hope that this method will continue to be refined, enabling researchers to more effectively leverage long-read data, high-throughput data, or even a synergistic combination of both.

Weaknesses:

Although this research contributes to our ability to more effectively utilize long-length and high-throughput data, there are some key issues that need to be addressed in terms of analyzing the specific results as well as writing the article.

Reviewer #3 (Public review):

Summary:

The goal of this study was to carry out an in-depth granular and unbiased phenotyping of peripheral blood circulating Tfh specific to two malaria vaccine candidates, PfSEA-1A and PfGARP, and correlate these with age (children vs adults) and protection from malaria (antibody titers against Plasmodium antigens.) Authors further attempted to identify any specific differences of the Tfh responses to these two distinct malaria antigens.

Strengths:

The authors had access to peripheral blood samples from children and adults living in a malaria-endemic region of Kenya. The authors studied these samples using in vitro restimulation in the presence of specific malaria antigens. Authors generated a very rich data set from these valuable samples using cutting-edge spectral flow cytometry and a 21-plex panel that included a variety of surface markers, cytokines and transcription factors.

Update following first revision (R1) of the manuscript:

The authors have made a great effort to comprehensively address comments raised by the reviewers. In particular, clearly showing expression of ICOS and Bcl6 on CXCR5+ cells greatly strengthens the case for defining these cells as Tfh-like circulatory lymphocytes (cTfh).

Weaknesses:

Update following first revision (R1) of the manuscript:

Unfortunately, my main concern remains. As it stands, the study is not really on antigen-specific T cells, but rather on the overall CD4 T cell compartment plus or minus antigenic stimulation. Although authors used an in vitro restimulation strategy with malaria antigens, they do not focus on cells de-novo expressing activation markers as a result of restimulation, neither they use tetramers to detect antigen-specific T cells. Moreover, their data shows that the number of CXCR5+ CD4 T cells de-novo expressing activation markers and/or cytokines as a result of their in vitro restimulation is negligible, even when using a prototypic superantigen (SEB).

Thus, no antigen-specific CXCR5+ CD4 T cells could be analysed with the data that the authors provide in this manuscript.

Reviewer #2 (Public review):

Summary:

Kapoor et. al. investigated the role of the mycobacterial protein Wag31 in lipid and peptidoglycan synthesis and sought to delineate the role of the N- and C- terminal domains of Wag31. They demonstrated that modulating Wag31 levels influences lipid homeostasis in M. smegmatis and cardiolipin (CL) localisation in cells. Wag31 was found to preferentially bind CL-containing liposomes, and deleting the N-terminus of the protein significantly decreased this interaction. Novel interactions between Wag31 and proteins involved in lipid metabolism and cell wall synthesis were identified, suggesting that Wag31 recruits proteins to the intracellular membrane domain by direct interaction.

Strengths:

(1) The importance of Wag31 in maintaining lipid homeostasis is supported by several lines of evidence.<br /> (2) The interaction between Wag31 and cardiolipin, and the role of the N-terminus in this interaction was convincingly demonstrated.

Weakness:

(1) Interactome analysis with truncated versions of the proteins could not be performed in M. smegmatis due to protein instability.

Reviewer #2 (Public review):

Summary:

In this work, Degutis and colleagues addressed an interesting issue related to the concurrent coding of sensory percepts and visual working memory contents in visual cortices. They used generalization analyses to test whether working memory representations change over time, diverge from sensory percepts, and vary across distraction conditions. Temporal generalization analysis demonstrated that off-diagonal decoding accuracies were lower than on-diagonal decoding accuracies, regardless of the presence of intervening distractions, implying that working memory representations can change over time. They further showed that the coding space for working memory contents showed subtle but statistically significant changes over time, potentially explaining the impaired off-diagonal decoding performance. The neural coding of sensory distractions instead remained largely stable. Generalization analyses between target and distractor codes showed overlaps but were not identical. Cross-condition decodings had lower accuracies compared to within-condition decodings. Finally, within-condition decoding revealed more reliable working memory representations in the condition with intervening random noises compared to cross-condition decoding using a trained classifier on data from the no-distraction condition, indicating a change in the VWM format between the noise distractor and no-distractor trials.

Strengths:

This paper demonstrates a clever use of generalization analysis to show changes in the neural codes of working memory contents across time and distraction conditions. It provides some insights into the differences between representations of working memory and sensory percepts, and how they can potentially coexist in overlapping brain regions.

Comments on revisions:

I appreciate the authors' efforts in addressing my previous concerns. The inclusion of additional analyses and data has strengthened the paper. I have no further concerns.

Reviewer #2 (Public review):

The study by Deganutti and co-workers is a methodological report on an adaptive sampling approach, multiple walker supervised molecular dynamics (mwSuMD), which represents an improved version of the previous SuMD.<br /> Case-studies concern complex conformational transitions in a number of G protein Coupled Receptors (GPCRs) involving long time-scale motions such as binding-unbinding and collective motions of domains or portions. GPCRs are specialized GEFs (guanine nucleotide exchange factors) of heterotrimeric Gα proteins of the Ras GTPase superfamily. They constitute the largest superfamily of membrane proteins and are of central biomedical relevance as privileged targets of currently marketed drugs.<br /> MwSuMD was exploited to address:

a) binding and unbinding of the arginine-vasopressin (AVP) cyclic peptide agonist to the V2 vasopressin receptor (V2R);<br /> b) molecular recognition of the β2-adrenergic receptor (β2-AR) and heterotrimeric GDP-bound Gs protein;<br /> c) molecular recognition of the A1-adenosine receptor (A1R) and palmotoylated and geranylgeranylated membrane-anchored heterotrimeric GDP-bound Gi protein;<br /> d) the whole process of GDP release from membrane-anchored heterotrimeric Gs following interaction with the glucagon-like peptide 1 receptor (GLP1R), converted to the active state following interaction with the orthosteric non-peptide agonist danuglipron.

The revised version has improved clarity and rigor compared to the original also thanks to the reduction in the number of complex case studies treated superficially.<br /> The mwSuMD method is solid and valuable, has wide applicability and is compatible with the most world-widely used MD engines. It may be of interest to the computational structural biology community.<br /> The huge amount of high-resolution data on GPCRs makes those systems suitable, although challenging, for method validation and development.<br /> While the approach is less energy-biased than other enhanced sampling methods, knowledge, at the atomic detail, of binding sites/interfaces and conformational states is needed to define the supervised metrics, the higher the resolution of such metrics is the more accurate the outcome is expected to be. Definition of the metrics is a user- and system-dependent process.

Reviewer #2 (Public review):

Zeeshan et al. investigate the function of the protein SUN1, a proposed nuclear envelope protein linking nuclear and cytoplasmic cytoskeleton, during the rapid male gametogenesis of the rodent malaria parasite Plasmodium berghei. They reveal that SUN1 localises to the nuclear envelope (NE) in male and female gametes and show that the male NE has unexpectedly high dynamics during the rapid process of gametogenesis. Using expansion microscopy, the authors find that SUN1 is enriched at the neck of the bipartite MTOC that links the intranuclear spindle to the basal bodies of the cytoplasmic axonemes. Upon deletion of SUN1, the basal bodies of the eight axonemes fail to segregate, no spindle is formed, and emerging gametes are anucleated, leading to a complete block in transmission. By interactomics the authors identify a divergent allantoicase-like protein, ALLAN, as a main interaction partner of SUN1 and further show that ALLAN deletion largely phenocopies the effect of SUN1.

Overall, the authors use an extensive array of fluorescence and electron microscopy techniques as well as interactomics to convincingly demonstrate that SUN1 and ALLAN play a role in maintaining the structural integrity of the bipartite MTOC during the rapid rounds of endomitosis in male gametogenesis.

Two suggestions for improvement of the work remain:

(1) Lipidomic analysis of WT and SUN1-knockout gametocytes before and after activation resulted in only minor changes in some lipid species. Without statistical analysis, it remains unclear if these changes are statistically significant and not rather due to expected biological variability. While the authors clearly toned down their conclusions in the revised manuscript, some phrasings in the results and the discussion still suggest that gametocyte activation and/or SUN1-knockout affects lipid composition. Similarly, some phrases suggest that SUN1 is responsible for the observed loops and folds in the NE and that SUN1 KO affects the NE dynamics. Currently, I do not think that the data supports these statements.

(2) It is interesting to note that ALLAN has a much more specific localisation to basal bodies than SUN1, which is located to the entire nuclear envelope. Knock out of ALLAN also exhibits a milder (but still striking) phenotype than knockout of SUN1. These observations suggest that SUN1 has additional roles in male gametogenesis besides its interaction with ALLAN, which could be discussed a bit more.

This study uses extensive microscopy and genetics to characterise an unusual SUN1-ALLAN complex, thus providing new insights into the molecular events during Plasmodium male gametogenesis, especially how the intranuclear events (spindle formation and mitosis) are linked to the cytoplasmic separation of the axonemes. The characterisation of the mutants reveals an interesting phenotype, showing that SUN1 and ALLAN are localised to and maintain the neck region of the bipartite MTOC. The authors here confirm and expand the previous knowledge about SUN1 in P. berghei, adding more detail to its localisation and dynamics, and further characterise the interaction partner ALLAN. Given the evolutionary divergence of Plasmodium, these results are interesting not only for parasitologists, but also for more general cell biologists.

Reviewer #2 (Public review):

Summary:

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

Strengths:

The authors provide compelling evidence that ornithine enhances the palatability of several chemical stimuli (i.e., IMP, MSG, MPG, Intralipos, sucrose, NaCl, quinine). Ornithine also increases CT nerve responses to MSG. Additionally, the authors provide evidence that the effects of ornithine are mediated by GPRC6A, a G-protein-coupled receptor family C group 6 subtype A, and that this receptor is expressed primarily in fungiform taste buds. Taken together, these results indicate that ornithine enhances the palatability of multiple taste stimuli in rats, and that the enhancement is mediated, at least in part, within fungiform taste buds. This finding could stand on its own. The question of whether ornithine produces these effects by eliciting kokumi-like perceptions (see below) should be presented as speculation in the Discussion section.

Weaknesses:

I am still unconvinced that the measurements in rats reflect the "kokumi" taste percept described in humans. The authors conducted long-term preference tests, 10-min avidity tests and whole chorda tympani (CT) nerve recordings. None of these procedures specifically model features of "kokumi" perception in humans, which (according to the authors) include increasing "intensity of whole complex tastes (rich flavor with complex tastes), mouthfulness (spread of taste and flavor throughout the oral cavity), and persistence of taste (lingering flavor)." While it may be possible to develop behavioral assays in rats (or mice) that effectively model kokumi taste perception in humans, the authors have not made any effort to do so. As a result, I do not think that the rat data provide support for the main conclusion of the study--that "ornithine is a kokumi substance and GPRC6A is a novel kokumi receptor."

Why are the authors hypothesizing that the primary impacts of ornithine are on the peripheral taste system? While the CT recordings provide support for peripheral taste enhancement, they do not rule out the possibility of additional central enhancement. Indeed, based on the definition of human kokumi described above, it is likely that the effects of kokumi stimuli in humans are mediated at least in part by the central flavor system.

The authors include (in the supplemental data section) a pilot study that examined the impact of ornithine on variety of subjective measures of flavor perception in humans. The presence of this pilot study within the larger rat study does not really make sense. If the human studies are so important, as the authors state, then why did the authors relegate them to the supplemental data section? Usually one places background and negative findings in this section of a paper. Accordingly, I recommend that the human data be published in a separate article.

Reviewer #2 (Public review):

Summary:

Consensus-independent component analysis and closely related methods have previously been used to reveal components of transcriptomic data which are not captured by principal component or gene-gene coexpression analyses.

Here, the authors asked whether applying consensus-independent component analysis (c-ICA) to published high-grade serous ovarian cancer (HGSOC) microarray-based transcriptomes would reveal subtle transcriptional patterns which are not captured by existing molecular omics classifications of HGSOC.

Statistical associations of these (hitherto masked) transcriptional components with prognostic outcomes in HGSOC would lead to additional insights into underlying mechanisms and, coupled with corroborating evidence from spatial transcriptomics, are proposed for further investigation.

This approach is complementary to existing transcriptomics classifications of HGSOC.

The authors have previously applied the same approach in colorectal carcinoma (for example, Knapen et al. (2024) Commun. Med).

Strengths:

Overall, this study describes a solid data-driven description of c-ICA-derived transcriptional components that the authors identified in HGSOC microarray transcriptomics data, supported by detailed methods and supplementary documentation.

The biological interpretation of transcriptional components is convincing based on (data-driven) permutation analysis and a suite of analyses of association with copy-number, gene sets, and prognostic outcomes.<br /> The resulting annotated transcriptional components have been made available in a searchable online format.

For the highlighted transcriptional component which has been annotated as related to synaptic signalling, the detection of the transcriptional component among 11 published spatial transcriptomics samples from ovarian cancers is compelling and supports the need for further mechanistic follow-up.

Further comments:

This revised version includes a suite of comparisons between the c-ICA-derived components and existing published transcriptomic/genomic-based classifications of ovarian cancers. Newly described components will require experimental validation, as acknowledged by the authors.

Here, the authors primarily interpret the c-ICA transcriptional components as a deconvolution of bulk transcriptomics due to the presence of cells from tumour cells and the tumour microenvironment.<br /> In this revised version, the authors additionally investigate their TC scores in single cells from a published HGSOC single-cell RNAseq dataset, highlighting examples of TC scores within and between cell types.

c-ICA is not explicitly a deconvolution method with respect to cell types: the transcriptional components do not necessarily correspond to distinct cell types, and may reflect differential dysregulation within a cell type. This application of c-ICA for the purpose of data-driven deconvolution of cell populations is distinct from other deconvolution methods which explicitly use a prior cell signature matrix.

Reviewer #2 (Public review):

Summary:

While the phylogenetic position of Acoels (and Xenacoelomorpha) remains still debated, investigations of various representative species are critical to understanding their overall biology.

Hofstenia is an Acoels species that can be maintained in laboratory conditions and for which several critical techniques are available. The current manuscript provides a comprehensive and widely descriptive investigation of the productive system of Hofstenia miamia.

Strengths:

(1) Xenacoelomorpha is a wide group of animals comprising three major clades and several hundred species, yet they are widely understudied. A comprehensive state-of-the-art analysis on the reprodutive system of Hofstenia as representative is thus highly relevant.

(2) The investigations are overall very thorough, well documented, and nicely visualised in an array of figures. In some way, I particularly enjoyed seeing data displayed in a visually appealing quantitative or semi-quantitative fashion.

(3) The data provided is diverse and rich. For instance, the behavioral investigations open up new avenues for further in-depth projects.

Weaknesses:

While the analyses are extensive, they appear in some way a little uni-dimensional. For instance the two markers used were characterized in a recent scRNAseq data-set of the Srivastava lab. One might have expected slightly deeper molecular analyses. Along the same line, particularly the modes of spermatogenesis or oogenesis have not been further analysed, nor the proposed mode of sperm-storage.

Reviewer #2 (Public review):

Summary:

The authors recently uncovered a novel nested gene, Aff3ir, and this work sets out to study its function in endothelial cells further. Based on differences in expression correlating with areas of altered shear stress, they investigate a role for the isoform Aff3ir-ORF2 in endothelial activation and development of atherosclerosis downstream of disturbed shear stress. Using a knockout mouse model and in vivo overexpression experiments, they demonstrate a strong potential for Aff3ir-ORF2 to alleviate atherosclerosis. They find that Aff3ir-ORF2 interacts with the pro-inflammatory transcription factor IRF5 and retains it in the cytoplasm, hence preventing upregulation of inflammation-associated genes. The data expands our knowledge of IRF5 regulation which could be relevant to researchers studying various inflammatory diseases as well as adding to our understand of atherosclerosis development.

Strengths:

The in vivo data is convincing using immunofluorescence staining to assess AFF3ir-ORF2 expression, a knockout mouse model, overexpression and knockdown studies and rescue experiments in combination with two atherosclerotic models to demonstrate that Aff3ir-ORF2 can lessen atherosclerotic plaque formation in ApoE-/- mice.

Weaknesses:

The effect on atherosclerosis is clear and there is sufficient evidence to conclude that this is the result of reduced endothelial cell activation. However, other cell types such as smooth muscle cells or macrophages could be contributing to the effects observed. The mouse model is a global knockout and the shRNA knockdowns (Fig. 5) and overexpression data in Figure 2 are not cell type-specific. Only the overexpression construct in Figure 6 uses an ICAM-2 promoter construct, which drives expression in endothelial cells, though leaky expression of this promoter has been reported in the literature.

The in vitro experiments are solidly executed, but most experiments are performed in mouse embryonic fibroblasts (MEFs) and results extrapolated to endothelial cell responses. However, several key experiments are repeated in HUVEC, thereby making a solid case that Aff3ir-ORF2 can regulate IRF5 in both MEFs and HUVEC. It is important to note that the sequence of AFF3ir-ORF2 is not conserved in humans and lacks an initiation codon, hence the regulatory pathway is not conserved. However, the overexpression studies in HUVEC suggest that mouse AFF3ir-ORF2 can also regulate human IRF5 and hence the mechanism retains relevance for possible human health interventions.

Overall, the paper succeeds in demonstrating a link between Aff3ir-ORF2 and atherosclerosis. The study shows a functional interaction between Aff3ir-ORF2 and IRF5 in embryonic fibroblasts, but makes a solid case that this mechanism is relevant for atherosclerosis development via endothelial cell activation.

Reviewer #2 (Public review):

Summary:

Sakagiannis et al. propose a hierarchically layer architecture to larval locomotion and foraging. They go from exploration to chemotaxis and odour preference test after associative learning.

Strengths:

A new locomotion model based on two oscillators that also incorporates peristaltic strides.

Weaknesses:

• The model is not always clearly or sufficiently explained (chemotaxis and odour test).

• Data analysis of the model movement is not very thorough.

• Comparisons with locomotion of behaving animals missing in chemotaxis and odour preference test after associative learning.

• Overall it is hard to judge the descriptive and predictive value of the model.

Reviewer #2 (Public review):

This is an interesting and important work from Shihabeddin et al, to identify master regulators for rod photoreceptor regenerations in a zebrafish model of Retinitis Pigmentosa. Building on their scRNA-seq data, Shihabeddin et al dissected the progenitor cell types and performed trajectory analyses to predict transcription factors that apparently drive the progenitor proliferation and differentiation into rod photoreceptors. Their analyses predicted e2f1, e2f2, and e2f3 as critical drivers of progenitor proliferation, Prdm1a as a driver of rod photoreceptor differentiation, and SP1 as a driver of rod photoreceptor maturation. Genetic experiments provide clear support for the roles of e2fs in progenitor proliferation. It's also apparent from Figure 8 that prdm1 knockdown appears to cause a decrease in rhodopsin expression. By colocalizing BrdU and Retp1, the authors inferred that the apparent "new rods" (which exhibit mixed BrdU and Retp1 signal) are decreased with prdm1, providing further support. Overall I found the work to be interesting, rigorous, and informative for the community.

I have a few suggestions for the authors to consider:

(1) Perhaps the authors can consider explaining why the Prdm1a knock-down cells would have a higher Retp1 signal per cell in Fig 9B. Is this a representative picture? This appears to contradict Figure 8's conclusion, although I could tell that the number of Retp1+ cells in the ONL appears to be lower.

(2) The authors noted "Surprisingly, the knockdown of prdm1a resulted in a significantly higher number of rhodopsin-positive cells in the INL (p=0.0293)", while it appears in Figure 9B, 9C that the difference is 2 cells vs 0 in a rightly broader field. It seems to be too strong of a statement for this effect.

(3) It appears to this reviewer that the proteomic data didn't reveal much in line with the overall hypothesis or the mechanism, and it's unclear why the authors went for proteomics rather than bulk RNA-seq or ChIP-seq for a transcription factor knock-down experiment. Overall this is a minor point.

Reviewer #2 (Public review):

Summary:

In the manuscript, the authors seek to discover putative gene regulatory interactions underlying the lineage bifurcation process of neural progenitor cells in the embryonic mouse anterior brainstem into GABAergic and glutamatergic neuronal subtypes. The authors analyze single-cell RNA-seq and single-cell ATAC-seq datasets derived from the ventral rhombomere 1 of embryonic mouse brainstems to annotate cell types and make predictions or where TFs bind upstream and downstream of the effector TFs using computational methods. They add data on the genomic distributions of some of the key transcription factors and layer these onto the single-cell data to get a sense of the transcriptional dynamics.

Strengths:

The authors use a well-defined fate decision point from brainstem progenitors that can make two very different kinds of neurons. They already know the key TFs for selecting the neuronal type from genetic studies, so they focus their gene regulatory analysis squarely on the mechanisms that are immediately upstream and downstream of these key factors. The authors use a combination of single-cell and bulk sequencing data, prediction and validation, and computation.

Weaknesses:

The study generates a lot of data about transcription factor binding sites, both predicted and validated, but the data are substantially descriptive. It remains challenging to understand how the integration of all these different TFs works together to switch terminal programs on and off.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors were providing a proof of concept that they can identify and mutate a cholesterol-binding site of a high-interest class B receptor, the GLP-1R, and functionally characterize the impact of this mutation on receptor behavior in the membrane and downstream signaling with the intent that similar methods can be useful to optimize small molecules that as ligands or allosteric modulators of GLP-1R can improve the therapeutic tools targeting this signaling system.

Strengths:

The majority of results on receptor behavior are elucidated in INS-1 cells expressing the wt or mutant GLP-1R, with one experiment translating the findings to primary mouse beta-cells. I think this paper lays a very strong foundation to characterize this mutation and does a good job discussing how complex cholesterol-receptor interactions can be (ie lower cholesterol binding to V229A GLP-1R, yet increased segregation to lipid rafts). Table 1 and Figure 9 are very beneficial to summarize the findings. The lower interaction with cholesterol and lower membrane diffusion in V229A GLP-1R resembles the reduced diffusion of wt GLP-1R with simv-induced cholesterol reductions, by presumably decreasing the cholesterol available to interact with wt GLP-1R. The effects of this mutation are not due to differences in Ex-4:recepotor affinity. I think this paper will be of interest to many physiologists who may not be familiar with many of the techniques used in this paper and the authors largely do a good job explaining the goals of using each method in the results section. While not necessary for this paper, a comparison of islet cholesterol content after this cholesterol diet vs the more typical 60% HFD used in obesity research would be beneficial for GLP-1 physiology research broadly to take these findings into consideration with model choice.

Weaknesses:

There are no obvious weaknesses in this manuscript and overall, I believe the authors achieved their aims and have demonstrated the importance of cholesterol interactions on GLP-1R functioning in beta-cells.

Certainly many follow-up experiments are possible from these initial findings and of primary interest is how this mutation affects insulin homeostasis in vivo under different physiological conditions. One of the biggest pathologies in insulin homeostasis in obesity/t2d is an elevation of baseline insulin release (as modeled in Fig 1E) that renders the fold-change in glucose stimulated insulin levels lower and physiologically less effective. Future work by the authors may determine the effects of the GLP-1R V229A mutation on insulin secretion responses under diet-induced metabolic stress conditions. Furthermore, the authors may additionally investigate if V229A would have the same impact in a different cell type, especially in neurons, with implications in the regulation of satiation, gut motility, and especially nausea, which are of high translational interest.

The comparison is drawn in the discussion between this mutation and ex4-phe1 to have biased agonism towards Gs over beta-arrestin signaling. Ex4-phe1 lowered pica behavior (a proxy for nausea) in the authors previously co-authored paper on ex4-phe1 (PMID 29686402) and drawing a parallel for this mutation or modification of cholesterol binding to potentially mitigate nausea is a novel direction.

Reviewer #2 (Public review):

Summary:

In this work, Walker and collaborators study the evolution of hepatitis C virus (HCV) in a cohort of 14 subjects with recent HCV infections. They focus in particular on the interplay between HCV and the immune system, including the accumulation of mutations in CD8+ T cell epitopes to evade immunity. Using a computational method to estimate the fitness effects of HCV mutations, they find that intrinsic viral fitness declines as the virus mutates to escape T cell responses. In long-term infections, they found that viral fitness can rebound later in infection as HCV accumulates additional mutations.

Strengths:

This work is especially interesting for several reasons. Individuals who developed chronic infections were followed over fairly long times and, in most cases, samples of the viral population were obtained frequently. At the same time, the authors also measured CD8+ T cell and antibody responses to infection. The analysis of HCV evolution focused not only on variation within particular CD8+ T cell epitopes, but also the surrounding proteins. Overall, this work is notable for integrating information about HCV sequence evolution, host immune responses, and computational metrics of fitness and sequence variation. The evidence presented by the authors supports the main conclusions of the paper described above.

Weaknesses:

After revision, this paper has no outstanding weaknesses. Points where further investigation is needed have been clearly identified.

Reviewer #2 (Public review):

Summary

The manuscript presents valuable findings, particularly in the crystal structure of the Sld3CBD-Cdc45 interaction and the identification of additional sequences involved in their binding. The modeling of the Sld7-Sld3CBD-CDC45 subcomplex is novel, and the results provide insights into potential conformational changes that occur upon interaction. Although the single-stranded DNA binding data from Sld3 of different species is a minor weakness, the experiments support a model in which the release of Sld3 from the complex may be promoted by its binding to origin single-stranded DNA exposed by the helicase.

Strengths

• The Sld3CBD-Cdc45 structure is a novel contribution, revealing critical residues involved in the interaction.<br /> • The model structures generated from the crystal data are well presented and provide valuable insights into the interaction sequences between Sld3 and Cdc45.<br /> • The experiments testing the requirements for interaction sequences are thorough and conducted well, with clear figures supporting the conclusions.<br /> • The conformational changes observed in Sld3 and Cdc45 upon binding are interesting and enhance our understanding of the interaction.<br /> • The modeling of the Sld7-Sld3CBD-CDC45 subcomplex is a new and valuable addition to the field.<br /> • The proposed model of Sld3 release from the complex through binding to single stranded DNA at the origin is intriguing.

Weaknesses

• The section on the binding of Sld3 complexes to origin single-stranded DNA is somewhat weakened by the use of Sld3 proteins from different species. The comparisons between Sld3-CBD, Sld3CBD-Cdc45, and Sld7-Sld3CBD-Cdc45 involve complexes from different species, limiting the comparisons' value.<br /> • Although the study reveals that Sld3 binds to different residues of Cdc45 than those previously shown to bind Mcm or GINS, the data in the paper do not shed any additional light on how GINS and Sld3 binding to Cdc45 or Mcms. would affect each other. Other previous research has suggested that the binding of GINS and Sld3 to Mcm or Cdc45 may be mutually exclusive. The authors acknowledge that a structural investigation of Sld3, Sld7, Cdc45, and MCM during the stage of GINS recruitment will be a significant goal for future research.

Reviewer #2 (Public review):

Summary:

This work investigates the roles of TANGO2 orthologs in different model systems and suggests bioenergetic dysfunction and oxidative stress (and not heme metabolism) as crucial pathways in TANGO2 deficiency disorders (TDD). Specifically, studies in C. elegans showed that the lack of TANGO2 ortholog activity (i) does not provide a survival benefit upon toxic heme exposure; (ii) results in a series of defects related to energy levels (reduced pharyngeal pumping, lawn avoidance, poor motility, and low brood size); (iii) reduces the fluorescence of the heme analog ZnMP in the intestine. Furthermore, upon oxidative stress, one TANGO2 ortholog, hrg-9, is upregulated compared to control conditions. Additional studies on yeast and zebrafish models failed to replicate prior findings on heme distribution and muscle integrity.

These findings have a clear therapeutic impact, as TDD currently has no cure but only symptom-managing treatments. Identifying the correct pathway to correct the disease is pivotal to finding a cure.

Although compelling, the authors' primary claim is based on indirect evidence that only hints toward it. Unfortunately, I do not see any direct and convincing evidence linking TANGO2 orthologs to bioenergetic and oxidative stress pathways.

Strengths:

(1) The study refutes and extends previous findings, highlighting new aspects of TANGO2's roles in cell physiology.

(2) The use of different model systems to address the main research questions is useful.

(3) The results suggest a broader impact than previously described, somewhat supporting the novelty of the study.

Weaknesses:

(1) The manuscript is written mainly as a criticism of a previously published paper. Although reproducibility in science is an issue that needs to be acknowledged, a manuscript should focus on the new data and the experiments that can better prove and strengthen the new claims.

(2) The current presentation of the logic of the study and its results does not help the authors deliver their message, although they possess great potential.

(3) The study is missing experiments to link hrg-9 and hrg-10 more directly to bioenergetic and oxidative stress pathways.

Reviewer #2 (Public review):

Summary:

The Activity-by-Contact (ABC) model is a relatively widespread model of enhancer-gene regulation. This model leverages CRISPRi data to predict whether a gene is regulated by a given enhancer. To make this possible, this model accounts for the activity of an enhancer and its contact frequency with a target promoter in order to produce an "ABC score". However, while quantitative in its ability to predict enhancer-promoter regulation, this model is mostly phenomenological and does not commit to specific molecular mechanisms.

In this manuscript, the authors formalize the molecular and mathematical assumptions made by the ABC model. Specifically, they demonstrate a basic set of assumptions that can be made to arrive at the ABC model's mathematical structure. The resulting default model (basically, a null model) places particular emphasis on the requirement that gene activation and enhancer-gene communication must be independent and at a steady state. The authors leverage and extend a graph-based formalism they have previously spearheaded to show the generality of their conclusions with respect to different molecular realizations of the process by which enhancers interact with their promoters.

Previously published works have found that specific models of how multiple enhancers communicate with the same gene can result in additive mRNA production rates. Here, the authors demonstrate that steady-state mRNA levels are additive regardless of the specific Markovian model for how any individual enhancer communicates with the gene, as long as the model follows the basic assumptions of their default model.

By coarse-graining, both gene activation and enhancer-gene communication to simple two-state models, the authors then clearly demonstrate that the mathematical structure of the ABC model emerges. This mathematical structure implies that the ABC score summed over all the enhancers regulating a given gene must equal 1. However, experimental measurements show values ranging from 0 to 3. The authors show that, in order to explain these experimental deviations with respect to the theory, at least one of the assumptions of the default model must be broken. They demonstrate that either invoking enhancer cooperativity in mRNA production rates or breaking the assumption that individual enhancers communicate with the gene independently can explain existing experimental data.

Strengths:

By demonstrating that the mathematical structure of the ABC model emerges from a set of basic assumptions including the independence of gene activation and enhancer-gene communication, the authors succeeded in their aim to put the ABC model on a formal and molecular footing. Since some experimental results do not agree with the ABC model, the authors importantly demonstrated which assumptions of the model can be broken to explain such data. The theoretical work in this manuscript is written in a reasonably accessible manner that features how a graph theory-based approach to modeling biochemical networks can result in general statements about biological phenomena.

Weaknesses:

While the authors discuss a number of experimental techniques that can be used to test the validity of their model, a more specific discussion of proposed experiments could have strengthened the impact of the paper by providing explicit opportunities for dialogue with experimentalists.

Reviewer #2 (Public review):

Summary:

This study profiles the genome-wide distribution of DNA methylation using methylation capture sequencing in four stages of male germ cells: Thy1+ (undifferentiated spermatogonia), Kit+ (differentiated spermatogonia), pachytene spermatocytes, and round spermatids. These analyses revealed site-specific loss of DNA methylation in pachytene cells compared with differentiating spermatogonia. Integrated analysis using published datasets indicates that hypomethylated sites correlate with nucleosome retention sites and bivalent histone methylation sites in sperm.

Strengths:

The methyl-seq approach provides a comprehensive profile of DNA methylation in male germ cells. The concept that DNA hypomethylation in meiotic cells precedes histone modification and histone retention in sperm is interesting.

Weaknesses:

(1) In the title, the word "presets" should be changed to "precedes" or "correlates with". Preset means a causal relationship, which is not the case. This needs to be changed throughout the manuscript. For example, in the abstract, "predetermine" needs to be changed to "precede".

(2) The statement that "Based on these results, we propose that meiosis is a process of epigenetic reprogramming that sets up embryonic gene regulation" (lines 94-95) is a speculation that in the opinion of this reviewer should be removed from the text. It is too broad and not supported by the data presented.

(3) Figure 1B: details are missing. How many cells were analyzed/used? How many times was this experiment done [(The number of experiments (n)]? Were the changes statistically significant (Lines 109-111)?

(4) Figure 1A and Figure 1D: These seem to be contradictory. According to Figure 1D, leptotene/zygotene spermatocytes show bright 5mC staining. However, the diagram in 1A shows delayed recovery of DNA methylation. The authors should clarify this. It appears that 5mC was high in Kit+ spermatogonia and leptotene/zygotene spermatocytes, and then decreased in pachytene spermatocytes.

(5) L121-122: Statement: These results suggest that 5mC levels change dynamically during spermatogenesis before and after the transient reduction of DNA methylation in the premeiotic S phase. In order to make this claim about the premeiotic S phase, I suggest performing 5mC staining in premeiotic S phase cells, which can be pulse-labelled with BrdU or cite a reference if available.

Reviewer #2 (Public review):

Summary:

This important study by Turner et al. examines the functional role of a sparse but unique population of neurons in the cortex that express Nitric oxide synthase (Nos1). To do this, they pharmacologically ablate these neurons in the focal region of whisker-related primary somatosensory (S1) cortex using a saponin-substance P conjugate. Using widefield and 2-photon microscopy, as well as field recordings, they examine the impact of this cell-specific lesion on blood flow dynamics and neuronal population activity. Locally within the S1 cortex, they find changes in neural activity patterns, decreased delta band power, and reduced sensory-evoked changes in blood flow (specifically eliminating the sustained blood flow change after stimulation). Surprisingly, given the tiny fraction of cortical neurons removed by the lesion, they also find far-reaching effects on neural activity patterns and blood volume oscillations between the cerebral hemispheres.

Strengths:

This was a technically challenging study and the experiments were executed in an expert manner. The manuscript was well written and I appreciated the cartoon summary diagrams included in each figure. The analysis was rigorous and appropriate. Their discovery that Nos1 neurons can have far-reaching effects on blood flow dynamics and neural activity is quite novel and surprising (to me at least) and should seed many follow-up, mechanistic experiments to explain this phenomenon. The conclusions were justified by the convincing data presented.

Weaknesses:

I did not find any major flaws in the study. I have noted some potential issues with the authors' characterization of the lesion and its extent. The authors may want to re-analyse some of their data to further strengthen their conclusions. Lastly, some methodological information was missing, which should be addressed.

Reviewer #2 (Public review):

Summary:

The authors present an interesting paper where they test the antagonistic pleiotropy theory. Based on this theory they hypothesize that genetic variants associated with later onset of age at menarche and age at first birth may have a positive effect on a multitude of health outcomes later in life, such as epigenetic aging and prevalence of chronic diseases. Using a mendelian randomization and colocalization approach, the authors show that SNPs associated with later age at menarche are associated with delayed aging measurements, such as slower epigenetic aging and reduced facial aging and a lower risk of chronic diseases, such as type 2 diabetes and hypertension. Moreover, they identify 128 fertility-related SNPs that associate with age-related outcomes and they identified BMI as a mediating factor for disease risk, discussing this finding in the context of evolutionary theory.

Strengths:

The major strength of this manuscript is that it addresses the antagonistic pleiotropy theory in aging. Aging theories are not frequently empirically tested although this is highly necessary. The work is therefore relevant for the aging field as well as beyond this field, as the antagonistic pleiotropy theory addresses the link between fitness (early life health and reproduction) and aging.

The authors addressed the remarks on the previous version very well. Addressing the two points below would further increase the quality of the manuscript.

(1) In the previous version the authors mentioned that their results are also consistent with the disposable soma theory: "These results are also consistent with the disposable soma theory that suggests aging as an outcome tradeoff between an organism's investment in reproduction and somatic maintenance and repair."

Although the antagonistic pleiotropy and disposable soma theories describe different mechanisms, both provide frameworks for understanding how genes linked to fertility influence health. The antagonistic pleiotropy theory posits that genes enhancing fertility early in life may have detrimental effects later. In contrast, the disposable soma theory suggests that energy allocation involves a trade-off, where investment in fertility comes at the expense of somatic maintenance, potentially leading to poorer health in later life.

To strengthen the manuscript, a discussion section should be added to clarify the overlap and distinctions between these two evolutionary theories and suggest directions for future research in disentangling their specific mechanisms.

(2) In response to the question why the authors did not include age at menopause in addition to the already included age at first child and age at menarche the following explanation was provided: "Our manuscript focuses on the antagonistic pleiotropy theory, which posits that inherent trade-off in natural selection, where genes beneficial for early survival and reproduction (like menarche and childbirth) may have costly consequences later. So, we only included age at menarche and age at first childbirth as exposures in our research."

It remains, however, unclear why genes beneficial for early survival and reproduction would be reflected only in age at menarche and age at first childbirth, but not in age at menopause. While age at menarche marks the onset of fertility, age at menopause signifies its end. Since evolutionary selection acts directly until reproduction is no longer possible (though indirect evolutionary pressures persist beyond this point), the inclusion of additional fertility-related measures could have strengthened the analysis. A more detailed justification for focusing exclusively on age at menarche and first childbirth would enhance the clarity and rigor of the manuscript.

Reviewer #2 (Public review):

Summary:

The manuscript by Hathaway et al. describes a set of elegant behavioral experiments designed to understand which aspects of cue-reward contingencies drive risky choice behavior. The authors developed several clever variants of the well-established rodent gambling task (also developed by this group) to understand how audiovisual cues alter learning, choice behavior, and risk. Computational and sophisticated statistical approaches were used to provide evidence that: (1) audiovisual cues drive risky choice if they are paired with rewards and decrease risk if only paired with loss, (2) pairing cues with rewards reduces learning from punishment, and (3) differences in risk-taking seem to be present early on in training.

Strengths:

The paper is well-written, the experiments are well-designed, and the results are highly interesting, particularly for understanding how cues can motivate and invigorate normal and abnormal behavior.

Weaknesses:

Additional support and evidence are needed for the claims made by the authors. Some of the statements are inconsistent with the data and/or analyses or are only weakly supportive of the claims.

Reviewer #2 (Public review):

Summary:

This work addresses the question of whether artificial deep neural network models of the brain could be improved by incorporating top-down feedback, inspired by the architecture of the neocortex.

In line with known biological features of cortical top-down feedback, the authors model such feedback connections with both, a typical driving effect and a purely modulatory effect on the activation of units in the network.

To assess the functional impact of these top-down connections, they compare different architectures of feedforward and feedback connections in a model that mimics the ventral visual and auditory pathways in the cortex on an audiovisual integration task.

Notably, one architecture is inspired by human anatomical data, where higher visual and auditory layers possess modulatory top-down connections to all lower-level layers of the same modality, and visual areas provide feedforward input to auditory layers, whereas auditory areas provide modulatory feedback to visual areas.

First, the authors find that this brain-like architecture imparts the models with a light visual bias similar to what is seen in human data, which is the opposite in a reversed architecture, where auditory areas provide a feedforward drive to the visual areas.

Second, they find that, in their model, modulatory feedback should be complemented by a driving component to enable effective audiovisual integration, similar to what is observed in neural data.

Last, they find that the brain-like architecture with modulatory feedback learns a bit faster in some audiovisual switching tasks compared to a feedforward-only model.

Overall, the study shows some possible functional implications when adding feedback connections in a deep artificial neural network that mimics some functional aspects of visual perception in humans.

Strengths:

The study contains innovative ideas, such as incorporating an anatomically inspired architecture into a deep ANN, and comparing its impact on a relevant task to alternative architectures.

Moreover, the simplicity of the model allows it to draw conclusions on how features of the architecture and functional aspects of the top-down feedback affect the performance of the network.

This could be a helpful resource for future studies of the impact of top-down connections in deep artificial neural network models of the neocortex.

Weaknesses:

Overall, the study appears to be a bit premature, as several parts need to be worked out more to support the claims of the paper and to increase its impact.

First, the functional implication of modulatory feedback is not really clear. The "only feedforward" model (is a drive-only model meant?) attains the same performance as the composite model (with modulatory feedback) on virtually all tasks tested, it just takes a bit longer to learn for some tasks, but then is also faster at others. It even reproduces the visual bias on the audiovisual switching task. Therefore, the claims "Altogether, our results demonstrate that the distinction between feedforward and feedback inputs has clear computational implications, and that ANN models of the brain should therefore consider top-down feedback as an important biological feature." and "More broadly, our work supports the conclusion that both the cellular neurophysiology and structure of feed-back inputs have critical functional implications that need to be considered by computational models of brain function" are not sufficiently supported by the results of the study. Moreover, the latter points would require showing that this model describes neural data better, e.g., by comparing representations in the model with and without top-down feedback to recorded neural activity.

Second, the analyses are not supported by supplementary material, hence it is difficult to evaluate parts of the claims. For example, it would be helpful to investigate the impact of the process time after which the output is taken for evaluation of the model. This is especially important because in recurrent and feedback models the convergence should be checked, and if the network does not converge, then it should be discussed why at which point in time the network is evaluated.

Third, the descriptions of the models in the methods are hard to understand, i.e., parameters are not described and equations are explained by referring to multiple other studies. Since the implications of the results heavily rely on the model, a more detailed description of the model seems necessary.

Lastly, the discussion and testable predictions are not very well worked out and need more details. For example, the point "This represents another testable prediction flowing from our study, which could be studied in humans by examining the optical flow (Pines et al., 2023) between auditory and visual regions during an audiovisual task" needs to be made more precise to be useful as a prediction. What did the model predict in terms of "optic flow", how can modulatory from simple driving effect be distinguished, etc.

Reviewer #2 (Public review):

In this manuscript, the authors present an approach to correct GRIN lens aberrations, which primarily cause a decrease in signal-to-noise ratio (SNR), particularly in the lateral regions of the field-of-view (FOV), thereby limiting the usable FOV. The authors propose to mitigate these aberrations by designing and fabricating aspherical corrective lenses using ray trace simulations and two-photon lithography, respectively; the corrective lenses are then mounted on the back aperture of the GRIN lens.

This approach was previously demonstrated by the same lab for GRIN lenses shorter than 4.1 mm (Antonini et al., eLife, 2020). In the current work, the authors extend their method to a new class of GRIN lenses with lengths exceeding 6 mm, enabling access to deeper brain regions as most ventral region of the mouse brain. Specifically, they designed and characterized corrective lenses for GRIN lenses measuring 6.4 mm and 8.8 mm in length. Finally, they applied these corrected long micro-endoscopes to perform high-precision calcium signal recordings in the olfactory cortex.

Compared with alternative approaches using adaptive optics, the main strength of this method is that it does not require hardware or software modifications, nor does it limit the system's temporal resolution. The manuscript is well-written, the data are clearly presented, and the experiments convincingly demonstrate the advantages of the corrective lenses.

The implementation of these long corrected micro-endoscopes, demonstrated here for deep imaging in the mouse olfactory bulb, will also enable deep imaging in larger mammals such as rats or marmosets.

Comments on revisions:

The authors have clearly addressed all my comments.

Reviewer #2 (Public Review):

Summary:

The authors aimed to explore the role of climbing fibers (CFs) in cerebellar learning, with a focus on optokinetic reflex (OKR) adaptation. Their goal was to understand how CF activity influences memory acquisition, memory consolidation, and memory retrieval by optogenetically suppressing CF inputs at various stages of the learning process.

Strengths:

The study addresses a significant question in the cerebellar field by focusing on the specific role of CFs in adaptive learning. The authors use optogenetic tools to manipulate CF activity. This provides a direct method to test the causal relationship between CF activity and learning outcomes.

Weaknesses:

Despite shedding light on the potential role of CFs in cerebellar learning, the study is hampered by significant methodological issues that question the validity of its conclusions. The absence of detailed evidence on the effectiveness of CF suppression and concerns over tissue damage from optogenetic stimulation weakens the argument that CFs are not essential for memory consolidation. These challenges make it difficult to confirm whether the study's objectives were fully met or if the findings conclusively support the authors' claims. The research commendably attempts to unravel the temporal involvement of CFs in learning but also underscores the difficulties in pinpointing specific neural mechanisms that underlie the phases of learning. Addressing these methodological issues, investigating other signals that might instruct consolidation, and understanding CFs' broader impact on various learning behaviors are crucial steps for future studies.

[Editors' note: we have included the original concerns, which the Reviewing Editor agrees with. Methodological concerns remain after revisions.]

Reviewer #2 (Public review):

Summary:

Nagarajan et al. investigate the role of the anterior cingulate cortex (ACC) in vocal development of infant marmoset monkeys using lesions in this brain area. Many previous studies show that ACC plays an important role in volitional and emotion-driven vocal behavior in mammals. The experiments Nagarajan et al. performed strengthen the long-standing hypothesis that ACC influences the development of social-vocal behavior in non-human primates. Furthermore, their anatomical studies support the idea of cortical structures exerting cognitive control over subcortical networks for innate vocalization, and thus, enabling mammals to perform flexible social-vocal communication.

Strengths:

Many invasive behavioral studies in monkeys often use 2-3 animals. The authors used a sufficiently high number of animals for their experiments. This increases the power of their conclusions.

The study also investigates the impact of ACC lesions on downstream areas important for innate vocal production. This adds further evidence to the role of ACC on influencing these subcortical regions during vocal development and vocal behavior in general.

Weaknesses:

The study only provides data up to the 6th week after birth. Given the plasticity of the cortex, it would be interesting to see if these impairments in vocal behavior persist throughout adulthood or if the lesioned marmosets will recover their social-vocal behavior compared to the control animals. The authors give a reasonable explanation for why they did not provide this data.

Even though this study focuses entirely on the development of social vocalizations, providing data about altered social non-vocal behaviors that accompany ACC lesions is missing. This data can provide further insights and generate new hypothesis about the exact role of ACC in social-vocal development. For example, do these marmosets behave differently towards their conspecifics or family members and vice versa, and is this an alternate cause for the observed changes in social-vocal development? Unfortunately, the authors are unable to provide that data. Hopefully, this will be the goal of future studies.

Reviewer #2 (Public review):

Summary:

It is generally believed that higher-order areas in the prefrontal cortex guide selection during working memory and attention through signals that selectively recruiting neuronal populations in sensory areas that encode the relevant feature. In this work, Parto-Dezfouli and colleagues tested how these prefrontal signals influence activity in visual area V4 using a spatial working memory task. They recorded neuronal activity from visual area V4 and found that information about visual features at the behaviorally relevant part of space during the memory period is carried in a spatially selective manner in the timing of spikes relative to a beta oscillation (phase coding) rather than in the average firing rate (rate code). The authors further tested whether there is a causal link between prefrontal input and the phase encoding of visual information during the memory period. They found that indeed inactivation of the frontal eye fields, a prefrontal area known to send spatial signal to V4, decreased beta oscillatory activity in V4 and information about the visual features. The authors went one step further to develop a neural model that replicated the experimental findings and suggested that changes in the average firing rate of individual neurons might be a result of small changes in the exact beta oscillation frequency within V4. These data provide important new insights on the possible mechanisms through which top-down signals can influence activity in hierarchically lower sensory areas and can therefore have a significant impact on the Systems, Cognitive and Computational Neuroscience fields.

Strengths:

This is a well-written paper with a well-thought-out experimental design. The authors used a smart variation of the memory-guided saccade task to assess how information about the visual features of stimuli is encoded during the memory period. By using a grating of various contrasts and orientations as the background the authors ensured that bottom-up visual input would drive responses in visual area V4 in the delay period, something that is not commonly done in experimental settings in the same task. Moreover, one of the major strengths of the study is the use of different approaches including analysis of electrophysiological data using advanced computational methods of analysis, manipulation of activity through inactivation of prefrontal cortex to establish causality of top-down signals on local activity signatures (beta oscillations, spike locking and information carried) as well as computational neuronal modeling. This has helped extend an observation into a possible mechanism well supported by the results.

Weaknesses:

Although the authors provide support for their conclusions from different approaches, a few conceptual gaps make it harder for the reader to appreciate the mechanisms that lead to the observed results and evaluate whether and how these may apply to other cases of top-down control. The fact that the visual features under study were behaviorally irrelevant make it difficult to appreciate the relevance of the finding and its relation to top-down spatial attention mechanisms that involve similar/overlapping circuits. In the same vein, the use of the memory-guided saccade task has certain disadvantages in the context of this study. Although delay activity is interpreted as memory activity by the authors, it is in principle possible that it reflects preparation for the upcoming saccade, spatial attention (particularly since there is a stimulus in the RF) etc. This could potentially change the conclusion and perspective.

Moreover, encoding of the two visual features that are manipulated in the context of the study (contrast and orientation) seems to be affected differently in certain cases, which leaves a reader wondering about the source of this variability.

Finally, although the study provides evidence in favor of a role of FEF in influencing phase coding of visual features in V4 in beta frequencies, important analysis that could have revealed the long-range mechanisms of such an effect including the analysis of intra-FEF and interareal (FEF-V4) neuronal interactions is missing from this paper

Reviewer #2 (Public review):

Summary:

While selective attention is a crucial ability of human beings, previous studies on selective attention are primarily conducted in a strictly controlled context, leaving a notable gap in underlying the complexity and dynamic nature of selective attention in a naturalistic context. This issue is particularly important for classroom learning in individuals with ADHD, as selecting the target and ignoring the distractions are pretty difficult for them but are the pre-requirement of effective learning. The authors of this study have addressed this challenge using a well-motivated study. I believe the findings of this study will be a nice addition to the fields both cognitive neuroscience and educational neuroscience.

Strengths:

To achieve the purpose of setting up a naturalistic context, the authors have based their study on a novel Virtual Reality platform. This is clever as it is usually difficult to perform such a study in the real classroom. Moreover, various techniques such as brain imaging, eye-tracking and physiological measurement are combined to collect multi-level data. They found that, different from the controls, individuals with ADHD had higher neural responses to the irrelevant rather than the target sounds, reduced speech tracking of the teacher. Additionally, the power of alpha-oscillations and frequency of gaze-shifts away from the teacher are found to be associated with the ADHD symptoms. These results provide new insights into the mechanism of selective attention among ADHD populations.

Weaknesses:

It is worth noting that nowadays there has been some studies trying to do so in the real classroom, and thus the authors should acknowledge the difference between the virtual and real classroom context and foresee the potential future changes.<br /> The approach of combining multi-level data owns advantage to obtain reliable results, but also raises significant difficult for the readers to understand the main results.

- An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

As expected, individuals with ADHD showed anomalous pattern of neural responses, and eye-tracking pattern, compared to the controls. But there are also some similarities between groups such as amount of time paying attention to teachers, etc. In general, their conclusions are supported.

- A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.

The findings are an extension of previous efforts in understanding selective attention in the naturalistic context. The findings of this study are particularly helpful in inspiring teacher's practice and advancing the research of educational neuroscience. This study demonstrates, again, that it is important to understand the complexity of cognitive process in the naturalistic context.

Comments on revisions:

The authors have appropriately responded to my concerns. I do not have other comments. I do hope to see more data and results from the authors in future.

Reviewer #2 (Public review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

Strengths:

Technically this is an impressive study, using an emerging approach that allows single cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population level activity in CA1.

Comments on revisions:

I have no further major requests and thank the authors for the additional data and analyses.

Reviewer #2 (Public review):

Summary:

The basal ganglia is often understood within a reinforcement learning (RL) framework, where dopamine neurons convey a reward prediction error which modulates cortico-striatal connections onto spiny projection neurons (SPNS) in the striatum. However, current models of plasticity rules are inconsistent with learning in a reinforcement learning framework.

This paper proposes a new model that describes how distinct learning rules in direct and indirect pathway striatal neurons allows them to implement reinforcement learning models. It proposes that two distinct component of striatal activity affect action selection and learning. They show that the proposed implementation allows learning in simple tasks and is consistent with experimental data from calcium imaging data in direct and indirect SPNs in freely moving mouse.

Strengths:

Despite the success of reward prediction errors at characterizing the responses of dopamine neurons as the temporal difference error within an RL framework, the implementation of RL algorithms in the rest of the basal ganglia has been unclear. A key missing aspect has been the lack of a RL implementation that is consistent with the distinction of direct- and indirect SPNs. This paper proposes a new model that is able to learn successfully in simple RL tasks and explains recent experimental results.

The author shows that their proposed model, unlike previous implementations, this model can perform well in RL tasks. The new model allows them to make experimental predictions. They test some of these predictions and show that the dynamics of dSPNs and iSPNs correspond to model predictions.

More generally, this new model can be used to understand striatal dynamics across direct and indirect SPNs in future experiments.

Weaknesses:

The authors could characterize better the reliability of their experimental predictions and the description of the parameters of some of the simulations

The authors propose some ideas about how the specificity of the striatal efferent inputs but should highlight better that this is a key feature of the model whose anatomical implementation has yet to be resolved.

Comments on revisions:

I thank the authors for their response to public and private reviews and for the clarifications and changes to the manuscript which have strengthened it. I understand the inability to implement some of the proposed additional simulation due to authors having left academia and the request for a version of record.

Reviewer #2 (Public review):

Summary:

The study characterized the dependence of spike-timing-dependent long-term depression (tLTD) on presynaptic NMDA receptors and the intracellular cascade after NMDAR activation possibly involved in the observed decrease in glutamate probability release at L5-L5 synapses of the visual cortex in mouse brain slices.

Strengths:

The genetic and electrophysiological experiments are thorough. The experiments are well-reported and mainly support the conclusions. This study confirms and extends current knowledge by elucidating additional plasticity mechanisms at cortical synapses, complementing existing literature.

Weaknesses:

While one of the main conclusions (preNMDARs mediating presynaptic LTD) is resolved in a very convincing genetic approach, the second main conclusion of the manuscript (non-ionotropic preNMDARs) relies on the use of a high concentration of extracellular blockers (MK801, 2 mM; 7-clorokinurenic acid: 100 microM), but no controls for the specific actions of these compounds are shown. In addition, no direct testing for ions passing through preNMDAR has been performed.

It is not known if the results can be extrapolated to adult brain as the data were obtained from 11-18 days-old mice slices, a period during which synapses are still maturing and the cortex is highly plastic.

Reviewer #2 (Public review):

Summary:

In this study, Goyal et al demonstrate that the assembly of proteins with polyphosphate into either condensates or aggregates can reveal information on the initial protein ensemble. They show that, unlike DNA, polyphosphate is able to effectively discriminate against initial protein ensembles with different conformational heterogeneity, structure, and compactness. The authors further show that the protein native ensemble is vital on whether polyphosphate induces phase separation or aggregation, whereas DNA induces a similar outcome regardless of the initial protein ensemble. This work provides a way to improve our mechanistic understanding of how conformational transitions of proteins may regulate or drive LLPS condensate and aggregate assemblies within biological systems.

Strengths:

This is a thoroughly conducted study that provides an alternative route for inducing phase separation that is more informative on the initial protein ensemble involved. This is particularly useful and a complementary means to investigate the role played by protein dynamics and plasticity in phase transitions. The authors use an appropriate set of techniques to investigate unique phase transitions within proteins induced by polyphosphates. An alternative protein system is used to corroborate their findings that the unique assemblies induced by polyphosphates when compared to DNA are not restricted to a single system. The work here is well-documented, easy to interpret, and of relevance for the condensate community.

Weaknesses:

The major weakness of this manuscript is that it is unclear if the information on the initial protein conformational ensemble can be determined solely from the assembly and maturation behavior and the discrimination abilities of polyphosphates. In both systems studied (CytR and FruR), polyphosphate discriminates and results in unique assemblies and maturation behaviors based on the initial protein ensemble. However, it seems the assembly and maturation behavior are not a direct result of the degree of conformational dynamics and plasticity in the initial protein. In the case of CytR, the fully-folded system forms condensates that resolubilize, while the highly disordered state immediately aggregates. Whereas, in the case of FruR, the folded state induces spontaneous aggregation, and the more dynamic, molten globular, system results in short-lived condensates. These results seem to suggest the polyphosphates' ability to discriminate between the initial protein ensemble may not be able to reveal what that initial protein ensemble is unless it is already known.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Appraisal and impact

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

Reviewer #2 (Public review):

I appreciate the author's responses to my original review. This is a comprehensive analysis of CAPE on C. difficile activity. It seems like this compound affects all aspects of C. difficile, which could make it effective during infection but also make it difficult to understand the mechanism. Even considering the authors responses, I think it is critical for the authors to work on the conclusions regarding the infection model. There is some protection from disease by CAPE but some parameters are not substantially changed. For instance, weight loss is not significantly different in the C. difficile only group versus the C. difficile + CAPE group. Histology analysis still shows a substantial amount of pathology in the C. difficile + CAPE group. This should be discussed more thoroughly using precise language.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase the likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

The sample size was relatively small and may have been underpowered to find differences in outcomes (i.e., food choice behaviors). Participants were all women with BN, which limits the generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. Moreover, it is unclear how long the negative affect persisted during the actual task. It is possible that any increases in negative affect would have dissipated by the time participants were engaged in the decision-making task.

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of vasopressin and vasopressin receptor 1A gene expression in the mouse brain using a high-resolution in situ hybridization method called RNAscope. Overall, the findings are useful in identifying brain regions expressing Avpr1a transcript. The impact of findings is decreased by incomplete or inadequate data analysis due to limited description of Avpr1a mRNA distribution within brain regions and limited statistical inference. A comprehensive overview of Avpr1a expression in the mouse brain has the potential to be highly informative and impactful. The current manuscript used RNAscope (a proprietary method of in situ hybridization) to assess the transcript abundance of Avp (arginine vasopressin, a neuropeptide) and its receptor (Avpr1a). The style of graphs, limited use of photomicrographs, and low number of subjects all combine to limit the impact of the dataset. The finding of Avp-expressing cells outside of the hypothalamus and extended amygdala is poorly documented but would be novel. The Avpr1a data suggest expression in numerous brain regions. However, the data presented are difficult to interpret, with every value being an extremely small density value for a large swath of the brain. How many cells are impacted? Are puncta spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield? For a descriptive study, there is minimal statistical inference and relatively little description. The authors make a case for the novel nature of the work but do not seem, at times, to recognize a robust literature developed over the last 50 years. In conclusion, the experimental data are important and informative; however, the low number of subjects, lack of statistical power, limited description of individual brain regions, and poor quality and design of data figures reduce the overall impact.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring the function of vasopressin in the mammalian brain and developing hypotheses about cell - and circuit-level function.

Weaknesses:

(1) The style and type of data presentation, focusing on the density of individual mRNA transcript across a whole brain region, seemed incomplete in so far as the data presentation did not provide a clear visualization of the distribution of Avpr1a-expressing cells or transcript itself. However, knowing which brain regions do express transcript is itself informative.

(2) The manuscript strongly emphases on the possibility of sex differences in Avp and Avpr1a expression. However, the low number of animals used does not provide adequate statistical power to make strong inferences regarding sex differences in the data.

(3) The manuscript's methods are minimal but adequate to understand data acquisition. The description of how quantitative analyses were conducted is inadequate and would be impossible to replicate beyond identifying the program used.

Reviewer #2 (Public review):

Summary:

The authors perform a series of studies to follow up on their previous work, which established a role for dorsal raphe dopamine neurons (DRN) in the regulation of social-isolation-induced rebound in mice. In the present study, Lee et. al, use a combination of modern circuit tools to investigate putatively distinct roles of DRN dopamine transporting containing (DAT) projections to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP). Notably, they reveal that optogenetic stimulation of distinct pathways confers specific behavioral states, with DRNDAT-BLP driving aversion, DRNDAT-BNST regulating non-social exploratory behavior, and DRNDAT-CeA promoting social ability. A combination of electrophysiological studies and in situ hybridization studies reveal heterogenous dopamine and neuropeptide expression and different firing properties, providing further evidence of pathway-specific neural properties. Lastly, the authors combine optogenetics and calcium imaging to resolve social encoding properties in the DRNDAT-CeA pathway, which correlates observed social behavior to socially engaged neural ensembles.

Collectively, these studies provide an interesting way of dissecting out separable features of a complex multifaceted social-emotional state that accompanies social isolation and the perception of 'loneliness.' The main conclusions of the paper provide an important and interesting set of findings that increase our understanding of these distinct DRN projections and their role in a range of social (e.g., prosocial, dominance), non-social, and emotional behaviors. However, as noted below, the examination of these circuits within a homeostatic framework is limited given that a number of the datasets did not include an isolated condition. The DRNDAT-CeA pathway was investigated with respect to social homeostatic states in the present study for some of the datasets.

Strengths:

(1) The authors perform a comprehensive and elegant dissection of the anatomical, behavioral, molecular, and physiological properties of distinct DRN projections relevant to social, non-social, and emotional behavior, to address multifaceted and complex features of social state.

(2) This work builds on prior findings of isolation-induced changes in DRN neurons and provides a working framework for broader circuit elements that can be addressed across the social homeostatic state.

(3) This work characterizes a broader circuit implicated in social isolation and provides a number of downstream targets to explore, setting a nice foundation for future investigation.

(4) The studies account for social rank and anxiety-like behavior in several of the datasets, which are an important consideration to the interpretation of social motivation states, especially in male mice with respect to dominance behavior.

Weaknesses:

(1) The conceptual framework of the study is based on the premise of social isolation and perceived 'loneliness' under the framework of social homeostasis, analogous to hunger. In this framework, social isolation should provoke an aversive state and compensatory social contact behavior. In the authors' prior work, they demonstrate synaptic changes in DRN neurons and social rebound following acute social isolation. Thus, the prediction would be that downstream projections also would show state-dependent changes as a function of social housing conditions (e.g., grouped vs. isolated). In the current paper, a social isolation condition was not included for the majority of the studies conducted (e.g., Figures 1-6 do not include an isolated condition, Figures 7-8 do include an isolated condition). Thus, while Figure 1-6 adds a very interesting and compelling set of data that is of high value to the social behavior field with respect to social and emotional processing and general circuit characterization, these studies do not directly investigate the impacts of dynamic social homeostatic state. The main claim of the paper, including the title (e.g., separable DRN projections mediate facets of loneliness-like state), abstract, intro, and discussion presents the claim of this work under the framework of dynamic social homeostatic states, which should be interpreted with caution, as the majority of the work in the paper did not include a social isolation comparison.

(2) In Figure 1, the authors confirm co-laterals in the BNST and CeA via anatomical tracing studies. The goal of the optogenetic studies is to dissociate the functional/behavioral roles of distinct projections. However, one limitation of optogenetic projection targeting is the possibility of back-propagating action potentials (stimulation of terminals in one region may back-propagate to activate cell bodies, and then afferent projections to other regions), and/or stimulation of fibers of passage. Therefore, one limitation in the dataset for the optogenetic stimulation studies is the possibility of non-specific unintended activation of projections other than those intended (e.g., DRNDAT-CeA). This can be dealt with by administering lidocaine to prevent back-propagating action potentials.

(3) It is unclear from the test, but in the subjects' section of the methods, it appears that only male animals were included in the study, with no mention of female subjects. It should be clear to the reader that this was conducted in males only if that is the case, with consideration or discussion, about female subjects and sex as a biological variable.

(4) Averaged data are generally reported throughout the study in the form of bar graphs, across most figures. Individual data points would increase the transparency of the data.

Reviewer #2 (Public review):

The authors tested tactile acuity on the breast of females using several tasks and reported overall low acuity compared to the back, which is typically considered to have the worst acuity of all body parts. Moreover, there was evidence that acuity is worse the larger the breast; this finding mirrors similar findings for the hand and therefore suggests that the number of tactile sensors is fixed and must be distributed across a larger extent of skin when a body part is larger, thus resulting in comparably lower tactile acuity.

Strengths:

I find this an interesting paper with results that are relevant to the tactile community. The authors apply several tasks allowing them to link the paper with previous results. The methodology and psychophysical analysis are sound.

Weaknesses:

The analysis of localization error direction, with the result that the nipple area may be a landmark for tactile localization, is interesting and aligns the paper with some other recent papers that have suggested that such landmarks should exist. However, there are major issues with methodology and statistics, so that currently the conclusions are not supported.

In the following, line numbers refer to the re-formatted manuscript provided by the authors upon request and are mentioned for them to find the relevant passages faster.

(1) Comments on analysis of tactile acuity:

- I had a hard time understanding some parts of the report. What is meant by "broadly no relationship" in line 137?

- It is suggested that spatial expansion (which is correlated with body part size) is related between medial breast and hand - is this to say that women with large hands have large medial breast size? Nipple size was measured, but hand size was not measured, is this correct?

- It is furthermore unclear how the authors differentiate medial breast and NAC. The sentence in lines 140-141 seems to imply the two terms are considered the same, as a conclusion about NAC is drawn from a result about the medial breast. This requires clarification.

- Finally, given that the authors suspect that overall localization ability (or attention) may be overshadowed by a size effect, would not an analysis be adequate that integrates both, e.g. a regression with multiple predictors?

(2) Comments on analysis of "The nipple is a unit":

- Statistics in this section are not adequately described and may be partly false.

- In the paragraph about testing quadrants of the nipple, it is stated that only 3 of 10 participants barely outperformed chance with a p < 0.01. It is unclear how a significant t-test is an indication of "barely above chance".

- The final part of the paragraph on nipple quadrants (starting line 176) explains that there was a trend (4 of 10 participants) for lower tactile acuity being related to the inability to differentiate quadrants. It seems to me that such a result would not be expected: The stated hypothesis is that all participants have the same number of tactile sensors in their nipple and areola, independent of NAC size. In this section, participants determine the quadrant of a single touch. Theoretically, all participants should be equally able to perform this task, because they all have the same number of receptors in each quadrant of nipple and areola. Thus, the result in Figure 2C is curious.

(3) Comments on analysis of "Absolute localization on the breast is anchored to the nipple"

- Again, there are things that are unclear with the statistics and description of the analysis.

- This section reports an Anova (line 193/194) with a factor "participant". This doesn't appear sensible. Please clarify. The factor distance is also unclear; is this a categorical or a continuous variable? Line 400 implies a 6-level factor, but Anovas and their factors, respectively, are not described in methods (nor are any of the other statistical approaches).

- The analysis on imprecision using mean pairwise error (line 199) is unclear: does pairwise refer to x/y or to touch vs. center of the nipple?

- p8, upper text, what is meant by "relative over-representation of the depth axis"? Does this refer to the breast having depth but the equivalent area on the back not having depth? What are the horizontal planes (probably meant to be singular?) - do you simply mean that depth was ignored for the calculation of errors? This seems to be implied in Figure 3AB.

- Lines 232-241, I cannot follow the conclusions drawn here. First, it is not clear to a reader what the aim of the presented analyses is: what are you looking for when you analyze the vectors? Second, "vector strength" should be briefly explained in the main text. Third, it is not clear how the final conclusion is drawn. If there is a bias of all locations towards the nipple, then a point closer to the nipple cannot exhibit a large bias, because the nipple is close-by. Therefore, one would expect that points close to the nipple exhibit smaller errors, but this would not imply higher acuity - just less space for localizing anything. The higher acuity conclusion is at odds with the remaining results, isn't it: acuity is low on the outer breast, but even lower at the NAC, so why would it be high in between the two?

(4) Comments on the Discussion:

The discussion makes some concrete suggestions for sensors in implants (line 283). It is not clear how the stated numbers were computed. Also, why should 4 sensors nipple quadrants receive individual sensors if the result here was that participants cannot distinguish these quadrants?

Additional comments:

I would find it interesting to know whether participants with small breast measurement delta had breast acuity comparable to the back. Alternatively, it would be interesting to know whether breast and back acuity are comparable in men. Such a result would imply that the torso has uniform acuity overall, but any spatial extension of the breast is unaccounted for. The lowest single participant data points in Figure 1B appear similar, which might support this idea.