Reviewer #1 (Public review):

Summary:

The authors performed experimental evolution of MreB mutants that have a slow growing round phenotype and studied the subsequent evolutionary trajectory using analysis tool from molecular biology. It was remarkable and interesting that they found that the original phenotype was not restored (most common in these studies) but that the round phenotype was maintained.

Strengths:

The finding that the round phenotype was maintained during evolution rather than that the original phenotype, rod shape cells, was recovered is interesting. The paper extensively investigates what happens during adaptation with various different techniques. Also the extensive discussion of the findings at the end of the paper is well thought through and insightful.

Weaknesses:

I find there are three general weaknesses<br /> (1) Although the paper states in the abstract that it emphasizes "new knowledge to be gained" it remains unclear what this concretely is. At page 4 they state 3 three research questions, these could be more extensively discussed in the abstract. Also these questions read more like genetics questions while the paper is a lot about cell biological findings.<br /> (2) It is not clear to me from the text what we already know about restoration of MreB loss from suppressors studies (in the literature). Are there supressor screens in the literature and which part of the findings is consistent with suppressor screens and which parts are new knowledge?<br /> (3) The clarity of the figures, captions and data quantification need to be improved.

Reviewer #1 (Public review):

Summary:

In this study, Avila et al. tested the hypothesis that chronic pain states are associated with changes in excitability of the medial prefrontal cortex (mPFC). The authors used the slope of the aperiodic component of the EEG power spectrum (= the aperiodic exponent) as a novel, non-invasive proxy for the cortical excitation-inhibition ratio. They performed source localization to estimate the EEG signals generated specifically by the mPFC. By pooling resting-state EEG recordings from three existing datasets, the authors were able to compare the aperiodic exponent in the mPFC and across the whole brain (at all modeled cortical sources) between 149 chronic pain patients and 115 healthy controls. Additionally, they assessed the relationship between the aperiodic exponent and pain intensity reported by the patients. To account for heterogeneity in pain etiology, the analysis was also performed separately for two patient subgroups with different chronic pain conditions (chronic back pain and chronic widespread pain). The study found robust evidence against differences in the aperiodic exponent in the mPFC between people with chronic pain and healthy participants, and no correlation was observed between the aperiodic exponent and pain intensity. These findings were consistent across different patient subgroups and were corroborated by the whole-brain analysis.

Strengths:

The study is based on sound scientific reasoning and rigorously employs suitable methods to test the hypothesis. It follows a pre-registered protocol, which greatly increases the transparency and, consequently, the credibility of the reported results. In addition to the planned steps, the authors used a multiverse analysis to ensure the robustness of the results across different methodological choices. I find this particularly interesting, as the EEG aperiodic exponent has only recently been linked to network excitability, and the most appropriate methods for its extraction and analysis are still being determined. The methods are clearly and comprehensively described, making this paper very useful for researchers planning similar studies. The results are convincing, supported by informative figures, and the lack of the expected difference in mPFC excitability between the tested groups is thoroughly and constructively discussed.

Weaknesses:

Firstly, to augment the sample size, the authors pooled data recorded by different researchers using different experimental protocols. This inevitably increases sample variability and may limit the availability of certain measures, as was the case here with the reports of pain intensity in the patient group. Secondly, the analysis heavily relies on the estimation of cortical sources, an approach that may yield imprecise results, especially when default conduction models, source models, and electrode coordinates are used (as was the case here).

Comments on revisions:

The authors satisfactorily revised the manuscript and responded to previous questions and suggestions. I have no further comments.

Reviewer #1 (Public review):

This study tests whether Little Swifts exhibit optimal foraging, which the data seem to indicate is the case. This is unsurprising as most animals would be expected to optimize the energy income : expenditure ratio, however it hasn't been explicitly quantified before the way it was in this manuscript.

The major strength of this work is the sheer volume of tracking data and the accuracy of those data. The ATLAS tracking system really enhanced this study and allowed for pinpoint monitoring of the tracked birds. These data could be used to ask and answer many questions beyond just the one tested here.

The major weakness of this work lies in the sampling of insect prey abundance at a single point on the landscape, 6.5 km from the colony. This sampling then requires the authors to work under the assumption that prey abundance is simultaneously even across the study region. It may be fair to say that prey populations might be correlated over space but are not equal. It is uncertain whether other aspects of the prey data are problematic. For example, the radar only samples insects at 50m or higher from the ground - how often do Little Swifts forage under 50m high?

The finding that Little Swifts forage optimally is indeed supported by the data, notwithstanding some of the shortcomings in the prey abundance data. The authors achieved their aims and the results support their conclusions.

At its centre, this work adds to our understanding of Little Swift foraging and extends to a greater understanding of aerial insectivores in general. While unsurprising that Little Swifts act as optimal foragers, it is good to have quantified this and show that the population declines observed in so many aerial insectivores are not necessarily a function of inflexible foraging habits. Further, the methods used in this research have great potential for other work. For example, the ATLAS system poses some real advantages and an exciting challenge to existing systems, like MOTUS. The radar that was used to quantify prey abundance also presents exciting possibilities if multiple units could be deployed to get a more spatially-explicit view.

To improve the context of this work, it is worth noting that this research goes into much further depth than any previous studies on a similar topic in several flycatcher and swallow species. A further justification is posited that this research is needed due to dramatic insect population declines, however, the magnitude and extent of such declines are fiercely debated in the literature.

Reviewer #1 (Public review):

Summary:

In the first half of this study, Pham et al. investigate the regulation of TEAD via ubiquitination and PARylation, identifying an E3 ubiquitin ligase, RNF146, as a negative regulator of TEAD activity through an siRNA screen of ubiquitin-related genes in MCF7 cells. The study also finds that depletion of PARP1 reduced TEAD4 ubiquitination levels, suggesting a certain relationship between TEAD4 PARylation and ubiquitination which was also explored through an interesting D70A mutation. Pham et al. subsequently tested this regulation in D. melanogaster by introducing Hpo loss-of-function mutations and rescuing the overgrowth phenotype through RNF146 overexpression.

In the second half of this study, Pham et al. designed and assayed several potential TEAD degraders with a heterobifunctional design, which they term TEAD-CIDE. Compounds D and E were found to effectively degrade pan-TEAD, an effect which could be disrupted by treatment with TEAD lipid pocket binders, proteasome inhibitors, or E1 inhibitors, demonstrating that the TEAD-CIDEs operate in a proteasome-dependent manner. These TEAD-CIDEs could reduce cell proliferation in OVCAR-8, a YAP deficient cell line, but not SK-N-FI, a Hippo pathway independent cell line. Finally, this study also utilizes ATAC-seq on Compound D to identify reductions in chromatin accessibility at the regions enriched for TEAD DNA binding motifs.

Strengths:

The study provides compelling evidence that the E3 ubiquitin ligase RNF146 is a novel negative regulator of TEAD activity. The authors convincingly delineate the mechanism through multiple techniques and approaches. The authors also describe the development of heterobifunctional pan-degraders of TEAD, that could serve as valuable reagents to more deeply study TEAD biology.

Weaknesses:

The scope of this study is extremely broad. The first half of the paper highlights the mechanisms underlying TEAD degradation; however, the connection to the second half of the paper on small molecule degraders of TEAD is jarring, and it seems as though two separate stories were combined into this single massive study. In my opinion, the study would be stronger if it chose to focus on only one of these topics and instead went deeper.

Additionally, the figure clarity needs to be substantially improved, as readability and interpretation was difficult in many panels. Lastly, there are numerous typos and poor grammar throughout the text that need to be addressed.

Comments on revisions:

The authors have addressed most of our critiques. The manuscript has improved significantly, particularly in the clarity of the figures and the flow of the text. The findings of this study contribute valuable insights into TEAD biology in cancer and provide useful resources for further research into TEAD.

However, as noted by other reviewers, the manuscript still feels somewhat disjointed, despite the attempt to connect the two parts on RNF146-mediated TEAD degradation and the development of TEAD degraders. Certain data inconsistencies and technical limitations may have made some aspects of the data hard to interpret accurately and could benefit from further clarification.

Reviewer #1 (Public review):

The revision by Ruan et al clarifies several aspects of the original manuscript that were difficult to understand, and I think it presents some useful and interesting ideas. I understand that the authors are distinguishing their model from the standard Wright-Fisher model in that the population size is not imposed externally, but is instead a consequence of the stochastic reproduction scheme. Here, the authors chose a branching process but in principle any Markov chain can probably be used. Within this framework, the authors are particularly interested in cases where the variance in reproductive success changes through time, as explored by the DDH model, for example. They argue with some experimental results that there is a reason to believe that the variance in reproductive success does change over time.

One of the key aspects of the original manuscript that I want to engage with is the DDH model. As the authors point out, their equations 5 and 6 are assumptions, and not derived from any principles. In essence, the authors are positing that that the variance in reproductive success, given by 6, changes as a function of the current population size. There is nothing "inherent" to a negative binomial branching mechanism that results in this: in fact, the the variance in offspring number could in principle be the same for all time. As relates to models that exist in the literature, I believe that this is the key difference: unlike Cannings models, the authors allow for a changing variance in reproduction through time.

This is, of course, an interesting thing to consider, and I think that the situation the authors point out, in which drift is lower at small population sizes and larger at large population sizes, is not appreciated in the literature. However, I am not so sure that there is anything that needs to be resolved in Paradox 1. A very strong prediction of that model is that Ne and N could be inversely related, as shown by the blue line in Fig 3b. This suggests that you could see something very strange if you, for example, infer a population size history using a Wright-Fisher framework, because you would infer a population *decline* when there is in fact a population *expansion*. However, as far as I know there are very few "surprising population declines" found in empirical data. An obvious case where we know there is very rapid population growth is human populations; I don't think I've ever seen an inference of recent human demographic history from genetic data that suggests anything other than a massive population expansion. While I appreciate the authors empirical data supporting their claim of Paradox 1 (more on the empirical data later), it's not clear to me that there's a "paradox" in the literature that needs explaining so much as this is a "words of caution about interpreting inferred effective population sizes". To be clear, I think those words of caution are important, and I had never considered that you might be so fundamentally misled as to infer decline when there is growth, but calling it a "paradox" seems to suggest that this is an outstanding problem in the literature, when in fact I think the authors are raising a *new* and important problem. Perhaps an interesting thing for the authors to do to raise the salience of this point would be to perform simulations under this model and then infer effective population sizes using e.g. dadi or psmc and show that you could identify a situation in which the true history is one of growth, but the best fit would be one of decline

The authors also highlight that their approach reflects a case where the population size is determined by the population dynamics themselves, as opposed to being imposed externally as is typical in Cannings models. I agree with the authors that this aspect of population regulation is understudied. Nonetheless, several manuscripts have dealt with the case of population genetic dynamics in populations of stochastically fluctuating size. For example, Kaj and Krone (2003) show that under pretty general conditions you get something very much like a standard coalescent; for example, combining their theorem 1 with their arguments on page 36 and 37, they find that exchangeable populations with stochastic population dynamics where the variance does not change with time still converge to exactly the coalescent you would expect from Cannings models. This is strongly suggestive that the authors key result isn't about stochastic population dynamics per se, but instead related to arguing that variance in reproductive success could change through time. In fact, I believe that the result of Kaj and Krone (2003) is substantially more general than the models considered in this manuscript. That being said, I believe that the authors of this manuscript do a much better job of making the implications for evolutionary processes clear than Kaj and Krone, which is important---it's very difficult to understand from Kaj and Krone the conditions under which effective population sizes will be substantially impacted by stochastic population dynamics.

I also find the authors exposition on Paradox 3 to be somewhat strange. First of all, I'm not sure there's a paradox there at all? The authors claim that the lack of dependence of the fixation probability on Ne is a paradox, but this is ultimately not surprising---fixation of a positively selected allele depends mostly on escaping the boundary layer, which doesn't really depend on the population size (see Gillespie's book "The Causes of Molecular Evolution" for great exposition on boundary layer effects). Moreover, the authors *use a Cannings-style argument* to get gain a good approximation of how the fixation probability changes when there is non-Poisson reproduction. So it's not clear that the WFH model is really doing a lot of work here. I suppose they raise the interesting point that the particularly simple form of p(fix) = 2s is due to the assumption that variance in offspring is equal to 1.

In addition, I raised some concerns about the analysis of empirical results on reproductive variance in my original review, and I don't believe that the authors responded to it at all. I'm not super worried about that analysis, but I think that the authors should probably respond to me.

Overall, I feel like I now have a better understanding of this manuscript. However, I think it still presents its results too strongly: Paradox 1 contains important words of caution that reflect what I am confident is an under appreciated possibility, and Paradox 3 is, as far as I'm concerned, not a paradox at all. I have not addressed Paradox 2 very much because I think that another reviewer had solid and interesting comments on that front and I am leaving it to them. That being said, I do think Paradox 2 actually presents a deep problem in the literature and that the authors' argument may actually represent a path toward a solution.

This manuscript can be a useful contribution to the literature, but as it's presented at the moment, I think most of it is worded too strongly and it continues to not engage appropriately with the literature. Theoretical advances are undoubtedly important, and I think the manuscript presents some interesting things to think about but ultimately needs to be better situated and several of the claims strongly toned down.

References:<br /> Kaj, I., & Krone, S. M. (2003). The coalescent process in a population with stochastically varying size. Journal of Applied Probability, 40(1), 33-48.

Reviewer #1 (Public review):

Summary:

The authors used multiple approaches to study salt effects in liquid-liquid phase separation (LLPS). Results on both wild-type Caprin1 and mutants and on different types of salts contribute to a comprehensive understanding.

Strengths:

The main strength of this work is the thoroughness of investigation. This aspect is highlighted by the multiple approaches used in the study, and reinforced by the multiple protein variants and different salts studied.

Weaknesses:

(1) The multiple computational approaches are a strength, but they're cruder than explicit-solvent all-atom molecular dynamics (MD) simulations and may miss subtle effects of salts. In particular, all-atom MD simulations demonstrate that high salt strengthens pi-types of interactions (ref. 42 and MacAinsh et al, https://www.biorxiv.org/content/10.1101/2024.05.26.596000v3).<br /> (2) The paper can be improved by distilling the various results into a simple set of conclusions. By example, based on salt effects revealed by all-atom MD simulations, MacAinsh et al. presented a sequence-based predictor for classes of salt dependence. Wild-type Caprin1 fits right into the "high net charge" class, with a high net charge and a high aromatic content, showing no LLPS at 0 NaCl and an increasing tendency of LLPS with increasing NaCl. In contrast, pY-Caprin1 belongs to the "screening" class, with a high level of charged residues and showing a decreasing tendency of LLLPS.<br /> (3) Mechanistic interpretations can be further simplified or clarified. (i) Reentrant salt effects (e.g., Fig. 4a) are reported but no simple explanation seems to have been provided. Fig. 4a,b look very similar to what has been reported as strong-attraction promotor and weak-attraction suppressor, respectively (ref. 50; see also PMC5928213 Fig. 2d,b). According to the latter two studies, the "reentrant" behavior of a strong-attraction promotor, CL- in the present case, is due to Cl-mediated attraction at low to medium [NaCl] and repulsion between Cl- ions at high salt. Do the authors agree with this explanation? If not, could they provide another simple physical explanation? (ii) The authors attributed the promotional effect of Cl- to counterion-bridged interchain contacts, based on a single instance. There is another simple explanation, i.e., neutralization of the net charge on Caprin1. The authors should analyze their simulation results to distinguish net charge neutralization and interchain bridging; see MacAinsh et al.<br /> (4) The authors presented ATP-Mg both as a single ion and as two separate ions; there is no explanation of which of the two versions reflects reality. When presenting ATP-Mg as a single ion, it's as though it forms a salt with Na+. I assume NaCl, ATP, and MgCl2 were used in the experiment. Why is Cl- not considered? Related to this point, it looks ATP is just another salt ion studied and much of the Results section is on NaCl, so the emphasis of ATP ("Diverse Roles of ATP" in the title is somewhat misleading.

Comments on revisions:

This revision addressed all my previous comments.

Reviewer #1 (Public review):

Summary:

This manuscript explores the multiple cell types present in the wall of murine collecting lymphatic vessels with the goal of identifying cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the use of genetic models to delete individual genes or detect cytosolic calcium in specific cell types, the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction.

Strengths:

The experiments are rigorously performed, the data justify the conclusions and the limitations of the study are appropriately discussed.

There is a need to identify therapeutic targets to improve lymphatic contraction and this work helps identify lymphatic muscle cells as potential cellular targets for intervention.

Reviewer #1 (Public Review):

This study presents valuable observations of white matter organisation from diffusion MRI and two types of synchrotron imaging in both monkeys and mice. Cross-modality comparisons are interesting as the different methods are able to probe anatomical structures at different length scales, from single axons in high-resolution synchrotron (ESRF) imaging, to clusters of axons in lower-resolution synchrotron (DEXY) data, to axon populations at the mm-scale in diffusion MRI. By acquiring all modalities in monkey and mouse ex vivo samples, the authors can observe principles of fibre organisation, and characterise how fibre characteristics, such as tortuosity and micro-dispersion, vary across select brain regions and in healthy tissue versus a demyelination model.

One very interesting result is the observation of apparent laminar organisation of fibres in ex vivo monkey white matter samples. DESY data from the corpus callosum shows fibres with two dominant orientations (one L-R, one slightly inclined), clustered in laminar structures within this major fibre bundle. Thanks to the authors providing open data, I was able to look through the raw DESY volume and observe regions with different "textures" (different orientations) in the described laminar arrangement. That this organisation can be observed by eye, as well as by structure tensor, is fairly convincing.

Reviewer #1 (Public review):

This study extends the previous interesting work of this group to address the potentially differential control of movement and posture. Their earlier work explored a broad range of data to make the case for a downstream neural integrator hypothesized to convert descending velocity movement commands into postural holding commands. Included in that data were observations from people with hemiparesis due to stroke. The current study uses similar data, but pushes into a different, but closely related direction, suggesting that these data may address the independence of these two fundamental components of motor control. I find the logic laid out in the second sentence of the abstract ("The paretic arm after stroke is notable for abnormalities both at rest and during movement, thus it provides an opportunity to address the relationships between control of reaching, stopping, and stabilizing") less then compelling, but the study does make some interesting observations. Foremost among them, is the relation between the resting force postural bias and the effect of force perturbations during the target hold periods, but not during movement. While this interesting observation is consistent with the central mechanism the authors suggest, it seems hard to me to rule out other mechanisms, including peripheral ones. These limitations should should be discussed.

Reviewer #1 (Public review):

Summary:

Govindan and Conrad use a genome-wide CRISPR screen to identify genes regulating retention of intron 4 in OGT, leveraging an intron retention reporter system previously described (PMID: 35895270). Their OGT intron 4 reporter reliably responds to O-GlcNAc levels, mirroring the endogenous splicing event. Through a genome-wide CRISPR knockout library, they uncover a range of splicing-related genes, including multiple core spliceosome components, acting as negative regulators of OGT intron 4 retention. They choose to follow up on SFSWAP, a largely understudied splicing regulator shown to undergo rapid phosphorylation in response to O-GlcNAc level changes (PMID: 32329777). RNA-sequencing reveals that SFSWAP depletion not only promotes OGT intron 4 splicing but also broadly induces exon inclusion and intron splicing, affecting decoy exon usage. While this study offers interesting insights into intron retention and O-GlcNAc signaling regulation, the RNA sequencing experiments lack the essential controls needed to provide full confidence to the authors' conclusions.

Strengths:

(1) This study presents an elegant genetic screening approach to identify regulators of intron retention, uncovering core spliceosome genes as unexpected positive regulators of intron retention.

(2) The work proposes a novel functional role for SFSWAP in splicing regulation, suggesting that it acts as a negative regulator of splicing and cassette exon inclusion, which contrasts with expected SR-related protein functions.

(3) The authors suggest an intriguing model where SFSWAP, along with other spliceosome proteins, promotes intron retention by associating with decoy exons.

Weaknesses:

(1) The conclusions on SFSWAP impact on alternative splicing are based on cells treated with two pooled siRNAs for five days. This extended incubation time without independent siRNA treatments raises concerns about off-target effects and indirect effects from secondary gene expression changes, potentially limiting confidence in direct SFSWAP-dependent splicing regulation. Rescue experiments and shorter siRNA-treatment incubation times could address these issues.

(2) The mechanistic role of SFSWAP in splicing would benefit from further exploration. Key questions remain, such as whether SFSWAP directly binds RNA, specifically the introns and exons (including the decoy exons) it appears to regulate. Furthermore, given that SFSWAP phosphorylation is influenced by changes in O-GlcNAc signaling, it would be interesting to investigate this relationship further. While generating specific phosphomutants may not yield definitive insights due to redundancy and also beyond the scope of the study, the authors could examine whether distinct SFSWAP domains, such as the SR and SURP domains, which likely overlap with phosphorylation sites, are necessary for regulating OGT intron 4 splicing.

(3) Data presentation could be improved (specific suggestions are included in the recommendations section). Furthermore, Excel tables with gene expression and splicing analysis results should be provided as supplementary datasheets. Finally, a more detailed explanation of statistical analyses is necessary in certain sections.

Reviewer #1 (Public review):

Summary:

This study explores how heterozygosity for specific neurodevelopmental disorder-associated Trio variants affects mouse behavior, brain structure, and synaptic function, revealing distinct impacts on motor, social, and cognitive behaviors linked to clinical phenotypes. Findings demonstrate that Trio variants yield unique changes in synaptic plasticity and glutamate release, highlighting Trio's critical role in presynaptic function and the importance of examining variant heterozygosity in vivo.

Strengths:

This study generated multiple mouse lines to model each Trio variant, reflecting point mutations observed in human patients with developmental disorders. The authors employed various approaches to evaluate the resulting behavioral, neuronal morphology, synaptic function, and proteomic phenotypes.

Weaknesses:

While the authors present extensive results, the flow of experiments is challenging to follow, raising concerns about the strength of the experimental conclusions. Additionally, the connection between sex, age, behavioral data, brain regions, synaptic transmission, and plasticity lacks clarity, making it difficult to understand the rationale behind each experiment. Clearer explanations of the purpose and connections between experiments are recommended. Furthermore, the methodology requires more detail, particularly regarding mouse breeding strategies, timelines for behavioral tests, electrophysiology conditions, and data analysis procedures.

Reviewer #1 (Public review):

Summary:<br /> This study addresses the roles of polyunsaturated fatty acids (PUFAs) in animal physiology and membrane function. A C. elegans strain carrying the fat-2(wa17) mutation possess a very limited ability to synthesize PUFAs and there is no dietary input because the E. coli diet consumed by lab grown C. elegans does not contain any PUFAs. The fat-2 mutant strain was characterized to confirm that the worms grow slowly, have rigid membranes, and have a constitutive mitochondrial stress response. The authors showed that chemical treatments or mutations known to increase membrane fluidity did not rescue growth defects. A thorough genetic screen was performed to identify genetic changes to compensate for the lack of PUFAs. The newly isolated suppressor mutations that compensated for FAT-2 growth defects included intergenic suppressors in the fat-2 gene, as well as constitutive mutations in the hypoxia sensing pathway components EGL-9 and HIF-1, and loss of function mutations in ftn-2, a gene encoding the iron storage protein ferritin. Taken together, these mutations lead to the model that increased intracellular iron, an essential cofactor for fatty acid desaturases, allows the minimally functional FAT-2(wa17) enzyme to be more active, resulting in increased desaturation and increased PUFA synthesis.

Strengths:<br /> (1) This study provides new information further characterizing fat-2 mutants. The authors measured increased rigidity of membranes compared to wild type worms, however this rigidity is not able to be rescued with other fluidity treatments such as detergent or mutants. Rescue was only achieved with polyunsaturated fatty acid supplementation.<br /> (2) A very thorough genetic suppressor screen was performed. In addition to some internal fat-2 compensatory mutations, the only changes in pathways identified that are capable of compensating for deficient PUFA synthesis was the hypoxia pathway and the iron storage protein ferritin. Suppressor mutations included an egl-9 mutation that constitutively activates HIF-1, and Gain of function mutations in hif-1 that are dominant. This increased activity of HIF conferred by specific egl-9 and hif-1 mutations lead to decreased expression of ftn-2. Indeed, loss of ftn-2 leads to higher intracellular iron. The increased iron apparently makes the FAT-2 fatty acid desaturase enzyme more active, allowing for the production of more PUFAs.<br /> (3) The mutations isolated in the suppressor screen show that the only mutations able to compensate for lack of PUFAs were ones that increased PUFA synthesis by the defective FAT-2 desaturase, thus demonstrating the essential need for PUFAs that cannot be overcome by changes in other pathways. This is a very novel study, taking advantage of genetic analysis of C. elegans, and it confirms the observations in humans that certain essential PUFAs are required for growth and development.<br /> (4) Overall, the paper is well written, and the experiments were carried out carefully and thoroughly. The conclusions are well supported by the results.

Weaknesses:<br /> Overall, there are not many weaknesses. The main one I noticed is that the lipidomic analysis shown in Figs 3C, 7C, S1 and S3. Whie these data are an essential part of the analysis and provide strong evidence for the conclusions of the study, it is unfortunate that the methods used did not enable the distinction between two 18:1 isomers. These two isomers of 18:1 are important in C. elegans biology, because one is a substrate for FAT-2 (18:1n-9, oleic acid) and the other is not (18:1n-7, cis vaccenic acid). Although rarer in mammals, cis-vaccenic acid is the most abundant fatty acid in C. elegans and is likely the most important structural MUFA. The measurement of these two isomers is not essential for the conclusions of the study, but the manuscript should include a comment about the abundance of oleic vs vaccenic acid in C. elegans (authors can find this information, even in the fat-2 mutant, in other publications of C. elegans fatty acid composition). Otherwise, readers who are not familiar with C. elegans might assume the 18:1 that is reported is likely to be mainly oleic acid, as is common in mammals.

Other suggestions to authors to improve the paper:<br /> (1) The title could be less specific; it might be confusing to readers to include the allele name in the title.<br /> (2) There are two errors in the pathway depicted in Figure 1A. The16:0-16:1 desaturation can be performed by FAT-5, FAT-6, and FAT-7. The 18:0-18:1 desaturation can only be performed by FAT-6 and FAT-7

Reviewer #1 (Public review):

Summary:

In this article, Nedbalova et al. investigate the biochemical pathway that acts in circulating immune cells to generate adenosine, a systemic signal that directs nutrients toward the immune response, and S-adenosylmethionine (SAM), a methyl donor for lipid, DNA, RNA, and protein synthetic reactions. They find that SAM is largely generated through the uptake of extracellular methionine, but that recycling of adenosine to form ATP contributes a small but important quantity of SAM in immune cells during the immune response. The authors propose that adenosine serves as a sensor of cell activity and nutrient supply, with adenosine secretion dominating in response to increased cellular activity. Their findings of impaired immune action but rescued larval developmental delay when the enzyme Ahcy is knocked down in hemocytes are interpreted as due to effects on methylation processes in hemocytes and reduced production of adenosine to regulate systemic metabolism and development, respectively. Overall this is a strong paper that uses sophisticated metabolic techniques to map the biochemical regulation of an important systemic mediator, highlighting the importance of maintaining appropriate metabolite levels in driving immune cell biology.

Strengths:

The authors deploy metabolic tracing - no easy feat in Drosophila hemocytes - to assess flux into pools of the SAM cycle. This is complemented by mass spectrometry analysis of total levels of SAM cycle metabolites to provide a clear picture of this metabolic pathway in resting and activated immune cells.

The experiments show that the recycling of adenosine to ATP, and ultimately SAM, contributes meaningfully to the ability of immune cells to control infection with wasp eggs.

This is a well-written paper, with very nice figures showing metabolic pathways under investigation. In particular, the italicized annotations, for example, "must be kept low", in Figure 1 illustrate a key point in metabolism - that cells must control levels of various intermediates to keep metabolic pathways moving in a beneficial direction.

Experiments are conducted and controlled well, reagents are tested, and findings are robust and support most of the authors' claims.

Weaknesses:

The authors posit that adenosine acts as a sensor of cellular activity, with increased release indicating active cellular metabolism and insufficient nutrient supply. It is unclear how generalizable they think this may be across different cell types or organs.

The authors extrapolate the findings in Figure 3 of decreased extracellular adenosine in ex vivo cultures of hemocytes with knockdown of Ahcy (panel B) to the in vivo findings of a rescue of larval developmental delay in wasp egg-infected larvae with hemocyte-specific Ahcy RNAi (panel C). This conclusion (discussed in lines 545-547) should be somewhat tempered, as a number of additional metabolic abnormalities characterize Ahcy-knockdown hemocytes, and the in vivo situation may not mimic the ex vivo situation. If adenosine (or inosine) measurements were possible in hemolymph, this would help bolster this idea. However, adenosine at least has a very short half-life.

Reviewer #1 (Public review):

Summary:

In this study, the authors aim to understand the neural basis of implicit causal inference, specifically how people infer causes of illness. They use fMRI to explore whether these inferences rely on content-specific semantic networks or broader, domain-general neurocognitive mechanisms. The study explores two key hypotheses: first, that causal inferences about illness rely on semantic networks specific to living things, such as the 'animacy network,' given that illnesses affect only animate beings; and second, that there might be a common brain network supporting causal inferences across various domains, including illness, mental states, and mechanical failures. By examining these hypotheses, the authors aim to determine whether causal inferences are supported by specialized or generalized neural systems.

The authors observed that inferring illness causes selectively engaged a portion of the precuneus (PC) associated with the semantic representation of animate entities, such as people and animals. They found no cortical areas that responded to causal inferences across different domains, including illness and mechanical failures. Based on these findings, the authors concluded that implicit causal inferences are supported by content-specific semantic networks, rather than a domain-general neural system, indicating that the neural basis of causal inference is closely tied to the semantic representation of the specific content involved.

Strengths:

(1) The inclusion of the four conditions in the design is well thought out, allowing for the examination of the unique contribution of causal inference of illness compared to either a different type of causal inference (mechanical) or non-causal conditions. This design also has the potential to identify regions involved in a shared representation of inference across general domains.

(2) The presence of the three localizers for language, logic, and mentalizing, along with the selection of specific regions of interest (ROIs), such as the precuneus and anterior ventral occipitotemporal cortex (antVOTC), is a strong feature that supports a hypothesis-driven approach (although see below for a critical point related to the ROI selection).

(3) The univariate analysis pipeline is solid and well-developed.

(4) The statistical analyses are a particularly strong aspect of the paper.

Weaknesses:

Based on the current analyses, it is not yet possible to rule out the hypothesis that inferring illness causes relies on neurocognitive mechanisms that support causal inferences irrespective of their content, neither in the precuneus nor in other parts of the brain.

(1) The authors, particularly in the multivariate analyses, do not thoroughly examine the similarity between the two conditions (illness-causal and mechanical-causal), as they are more focused on highlighting the differences between them. For instance, in the searchlight MVPA analysis, an interesting decoding analysis is conducted to identify brain regions that represent illness-causal and mechanical-causal conditions differently, yielding results consistent with the univariate analyses. However, to test for the presence of a shared network, the authors only perform the Causal vs. Non-causal analysis. This analysis is not very informative because it includes all conditions mixed together and does not clarify whether both the illness-causal and mechanical-causal conditions contribute to these results.

(2) To address this limitation, a useful additional step would be to use as ROIs the different regions that emerged in the Causal vs. Non-causal decoding analysis and to conduct four separate decoding analyses within these specific clusters:<br /> (a) Illness-Causal vs. Non-causal - Illness First;<br /> (b) Illness-Causal vs. Non-causal - Mechanical First;<br /> (c) Mechanical-Causal vs. Non-causal - Illness First;<br /> (d) Mechanical-Causal vs. Non-causal - Mechanical First.<br /> This approach would allow the authors to determine whether any of these ROIs can decode both the illness-causal and mechanical-causal conditions against at least one non-causal condition.

(3) Another possible analysis to investigate the existence of a shared network would be to run the searchlight analysis for the mechanical-causal condition versus the two non-causal conditions, as was done for the illness-causal versus non-causal conditions, and then examine the conjunction between the two. Specifically, the goal would be to identify ROIs that show significant decoding accuracy in both analyses.

(4) Along the same lines, for the ROI MVPA analysis, it would be useful not only to include the illness-causal vs. mechanical-causal decoding but also to examine the illness-causal vs. non-causal conditions and the mechanical-causal vs. non-causal conditions. Additionally, it would be beneficial to report these data not just in a table (where only the mean accuracy is shown) but also using dot plots, allowing the readers to see not only the mean values but also the accuracy for each individual subject.

(5) The selection of Regions of Interest (ROIs) is not entirely straightforward:<br /> In the introduction, the authors mention that recent literature identifies the precuneus (PC) as a region that responds preferentially to images and words related to living things across various tasks. While this may be accurate, we can all agree that other regions within the ventral occipital-temporal cortex also exhibit such preferences, particularly areas like the fusiform face area, the occipital face area, and the extrastriate body area. I believe that at least some parts of this network (e.g., the fusiform gyrus) should be included as ROIs in this study. This inclusion would make sense, especially because a complementary portion of the ventral stream known to prefer non-living items (i.e., anterior medial VOTC) has been selected as a control ROI to process information about the mechanical-causal condition. Given the main hypothesis of the study - that causal inferences about illness might depend on content-specific semantic representations in the 'animacy network' - it would be worthwhile to investigate these ROIs alongside the precuneus, as they may also yield interesting results.

(6) Visual representation of results:<br /> In all the figures related to ROI analyses, only mean group values are reported (e.g., Figure 1A, Figure 3, Figure 4A, Supplementary Figure 6, Figure 7, Figure 8). To better capture the complexity of fMRI data and provide readers with a more comprehensive view of the results, it would be beneficial to include a dot plot for a specific time point in each graph. This could be a fixed time point (e.g., a certain number of seconds after stimulus presentation) or the time point showing the maximum difference between the conditions of interest. Adding this would allow for a clearer understanding of how the effect is distributed across the full sample, such as whether it is consistently present in every subject or if there is greater variability across individuals.

(7) Task selection:<br /> (a) To improve the clarity of the paper, it would be helpful to explain the rationale behind the choice of the selected task, specifically addressing: (i) why an implicit inference task was chosen instead of an explicit inference task, and (ii) why the "magic detection" task was used, as it might shift participants' attention more towards coherence, surprise, or unexpected elements rather than the inference process itself.<br /> (b) Additionally, the choice to include a large number of catch trials is unusual, especially since they are modeled as regressors of non-interest in the GLM. It would be beneficial to provide an explanation for this decision.

Reviewer #1 (Public review):

Summary:

Horizontal gene transfer is the transmission of genetic material between organisms through ways other than reproduction. Frequent in prokaryotes, this mode of genetic exchange is scarcer in eukaryotes, especially in multicellular eukaryotes. Furthermore, the mechanisms involved in eukaryotic HGT are unknown. This article by Banerjee et al. claims that HGT occurs massively between cells of multicellular organisms. According to this study, the cell free chromatin particles (cfChPs) that are massively released by dying cells are incorporated in the nucleus of neighboring cells. These cfChPs are frequently rearranged and amplified to form concatemers, they are made of open chromatin, expressed, and capable of producing proteins. Furthermore, the study also suggests that cfChPs transmit transposable elements (TEs) between cells on a regular basis, and that these TEs can transpose, multiply, and invade receiving cells. These conclusions are based on a series of experiments consisting in releasing cfChPs isolated from various human sera into the culture medium of mouse cells, and using FISH and immunofluorescence to monitor the state and fate of cfChPs after several passages of the mouse cell line.

Strengths:

The results presented in this study are interesting because they may reveal unsuspected properties of some cell types that may be able to internalize free-circulating chromatin, leading to its chromosomal incorporation, expression, and unleashing of TEs. The authors propose that this phenomenon may have profound impacts in terms of diseases and genome evolution. They even suggest that this could occur in germ cells, leading to within-organism HGT with long-term consequences.

Weaknesses:

The claims of massive HGT between cells through internalization of cfChPs are not well supported because they are only based on evidence from one type of methodological approach: immunofluorescence and fluorescent in situ hybridization (FISH) using protein antibodies and DNA probes. Yet, such strong claims require validation by at least one, but preferably multiple, additional orthogonal approaches. This includes, for example, whole genome sequencing (to validate concatemerization, integration in receiving cells, transposition in receiving cells), RNA-seq (to validate expression), ChiP-seq (to validate chromatin state).

Another weakness of this study is that it is performed only in one receiving cell type (NIH3T3 mouse cells). Thus, rather than a general phenomenon occurring on a massive scale in every multicellular organism, it could merely reflect aberrant properties of a cell line that for some reason became permeable to exogenous cfChPs. This begs the question of the relevance of this study for living organisms.

Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed in Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism. Yet, telomere-to-telomere genomes have been produced for many eukaryote species, calling into question the conclusions of this study.

Reviewer #1 (Public review):

Summary:

Formins are complex proteins with multiple effects on actin filament assembly, including nucleation, capping with processive elongation, and bundling. Determining which of these activities is important for a given biological process and normal cellular function is a major challenge.

Here, the authors study the formin FHOD3L, which is essential for normal sarcomere assembly in muscle cells. They identify point mutants of FHOD3L in which formin nucleation and elongation/bundling activities are functionally separated. Expression of these mutants in neonatal rat ventricular myocytes shows that the control of actin filament elongation by formin is the major activity required for the normal assembly of functional sarcomeres.

Strengths:

The strength of this work is to combine sensitive biochemical assays with excellent work in neonatal rat ventricular myocytes. This combination of approaches is highly effective for analyzing the function of proteins with multiple activities in vitro.

Weaknesses:

FHOD3L does not seem to be the easiest formin to study because of its relatively weak nucleation activity and the short duration of capping events. This difficulty imposes rigorous biochemical analysis and careful interpretation of the data, which should be improved in this work.

Reviewer #1 (Public review):

Summary:

The authors seek to establish whether triadic interaction can promote affiliative relationships in the context of strict dominance hierarchies, and whether the vasopressinergic system is involved in such affiliations. To address this, they experimentally examine how male same-sex affiliations form by testing triadic cohabitation in large-billed crows, a species where males are known to develop and maintain same-sex affiliative relationships within a strict linear social hierarchy. They show a reduction in aggressive behavior over time with cohabitation and the formation of affiliative relationships, as measured by reciprocal allopreening, between two members (dyad) of the triad. The authors then administer a V1aR antagonist to each member of the triad, finding that allopreening decreases and dominance/submissive behaviors reemerge only in the dyad that developed an affiliated relationship ("affiliated dyad") with blockade of V1aR, demonstrating that V1aR mediates maintenance of affiliative peer relationships. The questions of how peer affiliations form, particularly in the context of dominance hierarchies, and the role of V1aR in regulating these behaviors are impactful for the field of social behavior. While the experimental paradigm provides a new way of approaching these questions, we have outlined below our concerns regarding the collection and interpretation of the data that limit the impact of this particular study.

Strengths:

(1) The authors develop a behavioral paradigm and experimental sequence using large-billed crows that allows them to identify the formation of stable, affiliated dyads within triadic groups that are robust to subsequent testing and are sensitive to pharmacological manipulation.

(2) The effects of V1aR antagonism on allopreening and respective dominance or submissive behaviors appear significant and specific to the affiliated dyad, which supports the view that V1aR plays a role in context-dependent, flexible regulation of aggressive behaviors across species. However, these results are difficult to interpret with respect to the authors' main claims given the weaknesses outlined below.

Weaknesses:

(1) The authors claim that the data demonstrates that a triadic social group facilitates the formation of affiliative dyads and go further to claim that these relationships have relevance to understanding coalition formation. It is difficult to say whether the triadic structure actually facilitates or promotes the formation of these affiliative interactions as stated without direct comparisons to alternately sized groupings. Further, the relevance to coalitions is weak without expanded behavioral testing.

(2) Aspects of the experimental design introduce confounding factors that make it difficult to interpret the resulting data. In experiment 1, 6 of the 18 animals that are used for testing are part of multiple triads. This is not accounted for in either the experimental design (wash-out period prior to reuse of animals) or statistical analysis (including repeated testing as a factor in the model) or is not described. Further, while the authors do randomize and counterbalance the two dose trials for the antagonist, vehicle vs drug exposure is not randomized.

(3) The re-emergence of dominance-related agonistic behaviors with V1aR antagonism specifically in the affiliated dyads is interesting, but difficult to interpret without further description and analysis of the dyadic behavior, particularly given the absence of dominance-related behaviors in either affiliated or unaffiliated dyads during the cohabitation period. In addition, the current data does not support the hypothesis that V1aR is also required to form affiliative relationships, as stated in the discussion (Lines 464-5, 472, 494), since the authors did not administer V1aR antagonist during the initial period of triadic cohabitation.

(4) Sentences are often repetitive or duplicated (lines 424-426), and paragraphs should be condensed for easier reading, especially in the discussion. Further, some of the discussion might be better presented in an "Ideas and Speculation" subsection, which would help readers appropriately assess the validity of the conclusions based on the data vs the larger implications suggested by the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate the role of microtubule dynamics and its effects on neuronal aging. Using C. elegans as a model, the authors investigate the role of evolutionarily conserved Hippo pathway in microtubule dynamics of touch receptor neurons (TRNs) in an age-dependent manner. Using genetic, molecular, behavioral, and pharmacological approaches, the authors show that age-dependent loss of microtubule dynamics might underlie structural and functional aging of TRNs. Further, the authors show that the Hippo pathway specifically functions in these neurons to regulate microtubule dynamics. Specifically, authors show that hyperactivation of YAP-1, a downstream component of the Hippo pathway that is usually inhibited by the kinase activity of the upstream components of the pathway, results in microtubule stabilization and that might underlie the structural and functional decline of TRNs with age. However, how the Hippo pathway regulates microtubule dynamics and neuronal aging was not investigated by the authors.

Strengths:

This is a well-conducted and well-controlled study, and the authors have used multiple approaches to address different questions.

Weaknesses:

There are no major weaknesses identified, except that the effect of the Hippo pathway seems to be specific to only a subset of neurons. I would like the authors to address the specificity of the effect of the Hippo pathway in TRNs, in their resubmission.

for - stats - climate crisis - example - positive impacts of mutualisation / sharing - car sharing - 1 Shared car can replace 9 to 13 cars without loss of mobility - from Substack article - The Cosmo-Local Plan for our Next Civilization - Michel Bauwens - 2024, Dec 20

Reviewer #1 (Public Review):

Summary

The authors asked if parabrachial CGRP neurons were only necessary for a threat alarm to promote freezing or were necessary for a threat alarm to promote a wider range of defensive behaviors, most prominently flight.

Major Strengths of Methods and Results

The authors performed careful single-unit recording and applied rigorous methodologies to optogenetically tag CGRP neurons within the PBN. Careful analyses show that single-units and the wider CGRP neuron population increases firing to a range of unconditioned stimuli. The optogenetic stimulation of experiment 2 was comparatively simpler but achieved its aim of determining the consequence of activating CGRP neurons in the absence of other stimuli. Experiment 3 used a very clever behavioral approach to reveal a setting in which both cue-evoked freezing and flight could be observed. This was done by having the unconditioned stimulus be a "robot" traveling along a circular path at a given speed. Subsequent cue presentation elicited mild flight in controls and optogenetic activation of CGRP neurons significantly boosted this flight response. This demonstrated for the first time that CGRP neuron activation does more than promote freezing. The authors conclude by demonstrating that bidirectional modulation of CGRP neuron activity bidirectionally affects freezing in a traditional fear conditioning setting and affects both freezing and flight in a setting in which the robot served as the unconditioned stimulus. Altogether, this is a very strong set of experiments that greatly expand the role of parabrachial CGRP neurons in threat alarm.

Weaknesses

In all of their conditioning studies the authors did not include a control cue. For example, a sound presented the same number of times but unrelated to US (shock or robot) presentation. This does not detract from their behavioral findings. However, it means the authors do not know if the observed behavior is a consequence of pairing. Or is a behavior that would be observed to any cue played in the setting? This is particularly important for the experiments using the robot US.

The authors make claims about the contribution of CGRP neurons to freezing and fleeing behavior, however, all of the optogenetic manipulations are centered on the US presentation period. Presently, the experiments show a role for these neurons in processing aversive outcomes but show little role for these neurons in cue responding or behavior organizing. Claims of contributions to behavior should be substantiated by manipulations targeting the cue period.

Appraisal

The authors achieved their aims and have revealed a much greater role for parabrachial CGRP neurons in threat alarm.

Discussion

Understanding neural circuits for threat requires us (as a field) to examine diverse threat settings and behavioral outcomes. A commendable and rigorous aspect of this manuscript was the authors decision to use a new behavioral paradigm and measure multiple behavioral outcomes. Indeed, this manuscript would not have been nearly as impactful had they not done that. This novel behavior was combined with excellent recording and optogenetic manipulations - a standard the field should aspire to. Studies like this are the only way that we as a field will map complete neural circuits for threat.

Reviewer #1 (Public review):

Summary:

This study investigates the potential of targeting specific regions within the RNA genome of the Porcine Epidemic Diarrhea Virus (PEDV) for antiviral drug development. The authors used SHAPE-MaP to analyze the structure of the PEDV RNA genome in infected cells. They categorized different regions of the genome based on their structural characteristics, focusing on those that might be good targets for drugs or small interfering RNAs (siRNAs).

They found that dynamic single-stranded regions can be stabilized by compounds (e.g., to form G-quadruplexes), which inhibit viral proliferation. They demonstrated this by targeting a specific G4-forming sequence with a compound called Braco-19. The authors also describe stable (structured) single-stranded regions that they used to design siRNAs showing that they effectively inhibited viral replication.

Strengths:

There are a number of strengths to highlight in this manuscript.

(1) The study uses a sophisticated technique (SHAPE-MaP) to analyze the PEDV RNA genome in situ, providing valuable insights into its structural features.

(2) The authors provide a strong rationale for targeting specific RNA structures for antiviral development.

(3) The study includes a range of experiments, including structural analysis, compound screening, siRNA design, and viral proliferation assays, to support their conclusions.

(4) Finally, the findings have potential implications for the development of new antiviral therapies against PEDV and other RNA viruses.

Overall, this interesting study highlights the importance of considering RNA structure when designing antiviral therapies and provides a compelling strategy for identifying promising RNA targets in viral genomes.

Weaknesses:

I have some concerns about the utility of the 3D analyses, the effects of their synonymous mutants on expression/proliferation, a potentially missed control for studies of mutants, and the therapeutic utility of the compound they tested vs. G-quadruplexes.

Reviewer #1 (Public review):

Summary:<br /> In the manuscript "Intergenerational transport of double-stranded RNA limits heritable epigenetic changes," Shugarts and colleagues investigate intergenerational dsRNA transport in the nematode C. elegans. By inducing oxidative damage, they block dsRNA import into cells, which affects heritable gene regulation in the adult germline (Fig. 2). They identify a novel gene, sid-1-dependent gene-1 (sdg-1), upregulated upon SID-1 inhibition (Fig. 3). Both transient and genetic depletion of SID-1 lead to the upregulation of sdg-1 and a second gene, sdg-2 (Fig. 5). Interestingly, while sdg-1 expression suggests a potential role in dsRNA transport, neither its overexpression nor loss-of-function impacts dsRNA-mediated silencing in the germline (Fig. 7).

Strengths:<br /> • The authors employ a robust neuronal stress model to systematically explore SID-1 dependent intergenerational dsRNA transport in C. elegans.<br /> • They discover two novel SID-1-dependent genes, sdg-1 and sdg-2.<br /> • The manuscript is well-written and addresses the compelling topic of dsRNA signaling in C. elegans.

Weaknesses:<br /> • The molecular mechanism downstream of SDG-1 remains unclear. Testing whether sdg-2 functions redundantly with sdg-1could provide further insights.<br /> • SDG-1 dependent genes in other nematodes remain unknown.

Reviewer #1 (Public review):

Summary:

Recommendations for the authors In this study, Liu, Jiang, Diao et.al. investigated the role of GSDMD in psoriasis-like skin inflammation in mice. The authors have used full-body GSDMD knock-out mice and Gsdm floxed mice crossed with the S100A8- Cre. In both mice, the deficiency of GSDMD ameliorated the skin phenotype induced by the imiquimod. The authors also analyzed RNA sequencing data from the psoriatic patients to show an elevated expression of GSDMD in the psoriatic skin.

Strengths:

It has the potential to unravel the new role of neutrophils.

Comments on revisions:

The authors have addressed the majority of comments and concerns and highlighted the potential limitations wherever not possible.

Reviewer #1 (Public review):

Fuchs describes a novel method of enzymatic protein-protein conjugation using the enzyme Connectase. The author is able to make this process irreversible by screening different Connectase recognition sites to find an alternative sequence that is also accepted by the enzyme. They are then able to selectively render the byproduct of the reaction inactive, preventing the reverse reaction, and add the desired conjugate with the alternative recognition sequence to achieve near-complete conversion. I agree with the authors that this novel enzymatic protein fusion method has several applications in the field of bioconjugation, ranging from biophysical assay conduction to therapeutic development. Previously the author has published on the discovery of the Connectase enzymes and has shown its utility in tagging proteins and detecting them by in-gel fluorescence. They now extend their work to include the application of Connectase in creating protein-protein fusions, antibody-protein conjugates, and cyclic/polymerized proteins. As mentioned by the author, enzymatic protein conjugation methods can provide several benefits over other non-specific and click chemistry labeling methods. Connectase specifically can provide some benefits over the more widely used Sortase, depending on the nature of the species that is desired to be conjugated. However, due to a similar lengthy sequence between conjugation partners, the method described in this paper does not provide clear benefits over the existing SpyTag-SpyCatcher conjugation system. Additionally, specific disadvantages of the method described are not thoroughly investigated, such as difficulty in purifying and separating the desired product from the multiple proteins used. Overall, this method provides a novel, reproducible way to enzymatically create protein-protein conjugates.

The manuscript is well-written and will be of interest to those who are specifically working on chemical protein modifications and bioconjugation.

Reviewer #1 (Public review):

Summary:

This study by Lo et al. seeks to explain the cellular defects underlying the brain phenotypes of Lowe syndrome (LS). There have been limited studies on this topic and hence this is a timely study.

Strengths:

Studies such as these can contribute to an understanding of the cellular and developmental mechanisms of brain disorders.

Weaknesses:

This study by Lo et al. seeks to explain the cellular defects underlying the brain phenotypes of Lowe syndrome (LS). There have been limited studies on this topic and hence this is a timely study.

The study uses two models: (1) an LS IOB knockout mouse and (2) neurons derived from iPSC lines from LS patients. These two models are used to present three separate findings: (1) altered mitochondria function, (2) altered numbers of neurons and glia in both models, and (3) some evidence of altered Sonic Hedgehog signaling projected as a defect in cilia.

Conceptually, there are some problems of serious concern which must be carefully considered:<br /> (1) The IOB mouse was very extensively phenotyped when it was generated by Festa et.al HMM, 2019. It does not have any obvious phenotypes of brain deficits although the studies in this paper were very detailed indeed.<br /> (2) Reduced brain size is reported as a phenotype of the IOB mouse in this study. Yet over the many clinical studies of LS published over the years, altered brain size has not been noted, either in clinical examination or in the many MRI reports of LS patients.

While reading through these results it is striking that the link between the three reported phenotypes is at least tenuous, and in fact may not exist at all. The link between mitochondria and neurogenesis is based on a single paper that has been cited incorrectly and out of context. There is no evidence presented for a link between the Shh signaling defect reported and the mitochondrial phenotype.

General comments

(1) The preparation of the manuscript requires improvement. There are many errors in the presentation of data.<br /> (2) The use of references needs to be re-considered. Sometimes a reference is used when in fact the results included in that paper are the opposite of what the authors intend.<br /> (3) The authors conclude the paper by claiming that mitochondrial dysfunction and impairments of the ciliary SHH contribute to abnormal neuronal differentiation in LS, but the mechanism by which this sequence of events might happen hasn't been shown.

Final comments:

(1) Phenotype of increased astrocytes:<br /> The phenotype of increased astrocytes in both the IOB mouse brain or iPSC-derived cultures iN cells requires clarification as one of the markers used as an astrocyte marker, BRN2, is commonly used as a neuronal marker. As LS is a neurodevelopmental disorder, and the phenotype in question is related to differentiation, it is crucial to shed light on the developmental timeline in which this phenotype is seen in the mouse brain.

(2) Ciliary homeostasis:<br /> Mitochondrial dysfunction in astrocytes has been shown to induce a ciliogenic program. However, almost the opposite is shown in this paper, with regards to ciliation. Morphology of the cilia was not assessed either, which is an important feature of ciliary homeostasis. The improper ciliary homeostasis here appears to be the improper Shh signalling, which has not been shown to be related to mitochondrial dysfunction. This leaves one wondering how exactly the different phenotypes shown in this paper are connected.

(3) This paper lacks a clear mechanistic approach. While the data validates the 3 broad phenotypes mentioned, there is a lack of connection between these phenotypes or an answer to why these phenotypes appear. While the discussion attempts to shed light on this by referencing previous studies, some of the referenced studies show contradicting results. Hence, it would be beneficial to clarify these gaps with further experiments and address the larger question of the connection between the mitochondria, Shh signalling, and astrocyte formation.

(4) Most importantly, there is no mention of how the loss of OCRL, a 5-phosphatase enzyme, results in the appearance of the mentioned phenotypes. Since there are multiple studies in the field of Lowe Syndrome that shed light on the various functions of OCRL, both catalytic and non-catalytic, it is important to address the role of OCRL in resulting in these phenotypes.

(5) There are numerous errors in the qPCR experiments performed with regard to the genes that were assayed. The genes mentioned in the text section do not match those indicated in the graphs or legends. This takes away the confidence of the reader in this data.

Reviewer #1 (Public review):

Summary:

The authors develop a set of biophysical models to investigate whether a constant area hypothesis or a constant curvature hypothesis explains the mechanics of membrane vesiculation during clathrin-mediated endocytosis.

Strengths:

The models that the authors choose are fairly well-described in the field and the manuscript is well-written.

Weaknesses:

One thing that is unclear is what is new with this work. If the main finding is that the differences are in the early stages of endocytosis, then one wonders if that should be tested experimentally. Also, the role of clathrin assembly and adhesion are treated as mechanical equilibrium but perhaps the process should not be described as equilibria but rather a time-dependent process. Ultimately, there are so many models that address this question that without direct experimental comparison, it's hard to place value on the model prediction.<br /> While an attempt is made to do so with prior published EM images, there is excessive uncertainty in both the data itself as is usually the case but also in the methods that are used to symmetrize the data. This reviewer wonders about any goodness of fit when such uncertainty is taken into account.

Reviewer #1 (Public review):

Summary:

Al Asafen and colleagues apply a set of scanning fluorescence correlation spectroscopic approaches (Raster Image Correlation Spectroscopy (RICS), cross-correlation RICS, and pair-correlation function spectroscopy) to address the nuclear-cytoplasmic kinetics of the Dorsal (Dl) transcription factor in early Drosophila embryos. The Toll/Dl system has long been appreciated to establish dorsal-ventral polarity of the embryo through Toll-dependent control of Dl nuclear localization, and provides an example of a morphogen gradient produced with high enough precision to yield robust biophysical measurements of general transcription factor activity and function. By measuring GFP-tagged Dl protein, either in wild-type embryos or in mutant embryos with low/medium/high levels of Toll signaling, the authors report diffusivity of Dl in nuclear and cytoplasmic compartments of the embryo, as well as the fraction of mobile and immobile Dl, which can be correlated with DNA binding through cross-correlation RICS. A model is presented where Cactus/IkB is implicated in preventing Dl from binding to DNA.

Strengths:

The experiments on wild-type GFP-tagged Dorsal are performed well, are mostly reported well, and are interpreted fairly.

Weaknesses:

The discrepancy between experiment and theory as pertains to Michaelis-Menten kinetics is not fully motivated in the text, and could benefit from a more clear presentation. The experiments performed to distinguish between the contribution of Toll-dependent phosphorylation and Cactus interaction models for limiting Dorsal DNA binding are possibly confounded by the presence of wild-type, GFP-tagged Dorsal protein.

Reviewer #1 (Public review):

Summary:

This study presents useful insights into the in vivo dynamics of insulin-producing cells (IPCs), key cells regulating energy homeostasis across the animal kingdom. The authors further provide compelling evidence using adult Drosophila melanogaster that IPCs, unlike neighboring DH44 cells, do not respond to glucose directly, but that glucose can indirectly regulate IPC activity after ingestion supporting an incretin-like mechanism in flies similar to mammals. The authors link decreased activity of IPCs to hyperactivity observed in starved flies, a locomotive behavior aimed to increase food search. Furthermore, the authors provide evidence that IPCs receive inhibitory inputs from Dh44 neurons, which are linked to increased locomotor activity.

This paper is of outstanding interest to scientists aiming to understand metabolic control of circuit dynamics, in particular for internal state-linked behaviors competing with the feeding state.

Strengths:

(1) By using whole cell patch clamp recording, the authors convincingly showed the activity pattern and regulation of IPCs and neighboring DH44 neurons under different feeding states and in various refeeding paradigms.<br /> (2) The paper provides compelling evidence that IPCs are not directly and acutely activated by glucose, but rather through a post-ingestive incretin-like mechanism. In addition, the authors show that Dh44 neurons located adjacent to the IPCs respond to bath application of nutritive sugars contrary to the IPCs.<br /> (3) The paper also provides useful data on the regulation of IPC activity by Dh44 neurons, which is useful to understand their regulation in vivo.

No major weaknesses remain in the revised version of this work.

Reviewer #1 (Public review):

Summary:

The authors test the "OHC-fluid-pump" hypothesis by assaying the rates of kainic acid dispersal both in quiet and in cochleae stimulated by sounds of different levels and spectral content. The main result is that sound (and thus, presumably, OHC contractions and expansions) result in faster transport along the duct. OHC involvement is corroborated using salicylate, which yielded results similar to silence. Especially interesting is the fact that some stimuli (e.g., tones) seem to provide better/faster pumping than others (e.g., noise), ostensibly due to the phase profile of the resulting cochlear traveling-wave response.

Strengths:

The experiments appear well controlled and the results are novel and interesting. Some elegant cochlear modeling that includes coupling between the organ of Corti and the surrounding fluid as well as advective flow supports the proposed mechanism.

The current limitations and future directions of the study, including possible experimental tests, extensions of the modeling work, and practical applications to drug delivery, are thoughtfully discussed.

Reviewer #1 (Public review):

I have reviewed the manuscript "Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast" with interest. The described findings are relevant and useful for daily practice in periodontology. The paper is concise, professionally written, and easy to read. In this study, Jiayao et al. revealed the role of miR-335-3p in psychological stress-induced osteoporosis. CUMS mice were constructed to observe the femur phenotype, osteoclasts were identified as the main research object, and miRNA-seq was used to find the key miRNAs linking the brain and peripheral tissues. This study showed that miR-335-3p expression was simultaneously reduced in murine NAC, serum, and bone under psychological stress. The miR-335-3p/Fos/NFATC1 signaling pathway was validated in osteoclasts to reveal the potential mechanism of enhanced osteoclast activity under psychological stress. This study, from a new perspective of miRNAs, indicates a possible cause of disturbed bone metabolism due to psychological stress and may suggest a new approach to treating osteoporosis.

Reviewer #1 (Public review):

Batra, Cabrera and Spence et al. present a model which integrates histone posttranslational modification (PTM) data across cell models to predict gene expression with the goal of using this model to better understand epigenetic editing. This gene expression prediction model approach is useful if a) it predicts gene expression in specific cell lines b) it predicts expression values rather than a rank or bin, c) if it helps us to better understand the biology of gene expression or d) it helps us to understand epigenome editing activity. Problematically for point a) and b) it is easier to directly measure gene expression than to measure multiple PTMs and so the real usefulness of this approach mostly relates to c) and d).

Other approaches have been published that use histone PTM to predict expression (e.g. PMID 27587684, 36588793). Is this model better in some way? No comparisons are made although a claim is made that direct comparisons are difficult. I appreciate that the authors have not used the histone PTM data to predict gene expression levels of an "average cell" but rather that they are predicting expression within specific cell types or for unseen cell types. Approaches that predict expression levels are much more useful whereas some previous approaches have only predicted expressed or not expressed or a rank order or bin-based ranking. The paper does not seem to have substantial novel insights into understanding the biology of gene expression.

The approach of using this model to predict epigenetic editor activity on transcription is interesting and to my knowledge novel although only examined in the context of a p300 editor. As the author point out the interpretation of the epigenetic editing data is convoluted by things like sgRNA activity scoring and to fully understand the results likely would require histone PTM profiling and maybe dCas9 ChIP-seq for each sgRNA which would be a substantial amount of work.

Furthermore from the model evaluation of H3K9me3 is seems the model is performing modestly for other forms of epigenetic or transcriptional editing- e.g. we know for the best studied transcriptional editor which is CRISPRi (dCas9-KRAB) that recruitment to a locus is associated with robust gene repression across the genome and is associated with H3K9me3 deposition by recruitment of KAP1/HP1/SETDB1 (PMID: 35688146, 31980609, 27980086, 26501517).

One concern overall with this approach is that dCas9-p300 has been observed to induce sgRNA independent off target H3K27Ac (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349887/ see Figure S5D) which could convolute interpretation of this type of experiment for the model.

Reviewer #1 (Public review):

Summary:

In this article, the authors set out to understand how people's food decisions change when they are hungry vs. sated. To do so, they used an eye-tracking experiment where participants chose between two food options, each presented as a picture of the food plus its "Nutri-Score". In both conditions, participants fasted overnight, but in the sated condition, participants received a protein shake before making their decisions. The authors find that participants in the hungry condition were more likely to choose the tastier option. Using variants of the attentional drift-diffusion model, they further find that the best-fitting model has different attentional discounts on the taste and health attributes and that the attentional discount on the health information was larger for the hungry participants.

Strengths:

The article has many strengths. It uses a food-choice paradigm that is established in neuroeconomics. The experiment uses real foods, with accurate nutrition information, and incentivized choices. The experimental manipulation is elegant in its simplicity - administering a high-calorie protein shake. It is also commendable that the study was within-participant. The experiment also includes hunger and mood ratings to confirm the effectiveness of the manipulation. The modeling work is impressive in its rigor - the authors test 9 different variants of the DDM, including recent models like the mtDDM and maaDDM, as well as some completely new variants (maaDDM2phi and 2phisp). The model fits decisively favor the maaDDM2phi.

Weaknesses:

First, in examining some of the model fits in the supplements, e.g. Figures S9, S10, S12, S13, it looks like the "taste weight" parameter is being constrained below 1. Theoretically, I understand why the authors imposed this constraint, but it might be unfairly penalizing these models. In theory, the taste weight could go above 1 if participants had a negative weight on health. This might occur if there is a negative correlation between attractiveness and health and the taste ratings do not completely account for attractiveness. I would recommend eliminating this constraint on the taste weight.

Second, I'm not sure about the mediation model. Why should hunger change the dwell time on the chosen item? Shouldn't this model instead focus on the dwell time on the tasty option?

Third, while I do appreciate the within-participant design, it does raise a small concern about potential demand effects. I think the authors' results would be more compelling if they replicated when only analyzing the first session from each participant. Along similar lines, it would be useful to know whether there was any effect of order.

Fourth, the authors report that tasty choices are faster. Is this a systematic effect, or simply due to the fact that tasty options were generally more attractive? To put this in the context of the DDM, was there a constant in the drift rate, and did this constant favor the tasty option?

Fifth, I wonder about the mtDDM. What are the units on the "starting time" parameters? Seconds? These seem like minuscule effects. Do they align with the eye-tracking data? In other words, which attributes did participants look at first? Was there a correlation between the first fixations and the relative starting times? If not, does that cast doubt on the mtDDM fits? Did the authors do any parameter recovery exercises on the mtDDM?

Reviewer #1 (Public review):

In this meta-analysis, Ng and colleagues review the association between slow-oscillation spindle coupling during sleep and overnight memory consolidation. The coupling of these oscillations (and also hippocampal sharp-wave ripples) have been central to theories and mechanistic models of active systems consolidation, that posit that the coupling between ripples, spindles, and slow oscillations (SOs) coordinate and drive the coordinated reactivation of memories in hippocampus and cortex, facilitating cross-regional information and ultimately memory strengthening and stabilisation.

Given the importance that these coupling mechanisms have been given in theory, this is a timely and important contribution to the literature in terms of determining whether these theoretical assumptions hold true in human data. The results show that the timing of sleep spindles relative to the SO phase, and the consistency of that timing, predicted overnight memory consolidation in meta-analytic models. The overall amount of coupling events did not show as strong a relationship. The coupling phase in particular was moderated by a number of variables including spindle type (fast, slow), channel location (frontal, central, posterior), age, and memory type. The main takeaway is that fast spindles that consistently couple close to the peak of the SO in frontal channel locations are optimal for memory consolidation, in line with theoretical predictions.

I did not follow the logic behind including spindle amplitude in the meta-analysis. This is not a measure of SO-spindle coupling (which is the focus of the review), unless the authors were restricting their analysis of the amplitude of coupled spindles only. It doesn't sound like this is the case though. The effect of spindle amplitude on memory consolidation has been reviewed in another recent meta-analysis (Kumral et al, 2023, Neuropsychologia). As this isn't a measure of coupling, it wasn't clear why this measure was included in the present meta-analysis. You could easily make the argument that other spindle measures (e.g., density, oscillatory frequency) could also have been included, but that seems to take away from the overall goal of the paper which was to assess coupling.

At the end of the first paragraph of section 3.1 (page 13), the authors suggest their results "... further emphasise the role of coupling compared to isolated oscillation events in memory consolidation". This had me wondering how many studies actually test this. For example, in a hierarchical regression model, would coupled spindles explain significantly more variance than uncoupled spindles? We already know that spindle activity, independent of whether they are coupled or not, predicts memory consolidation (e.g., Kumral meta-analysis). Is the variance in overnight memory consolidation fully explained by just the coupled events? If both overall spindle density and coupling measures show an equal association with consolidation, then we couldn't conclude that coupling compared to isolated events is more important.

It was very interesting to see that the relationship between the fast spindle coupling phase and overnight consolidation was strongest in the frontal electrodes. Given this, I wonder why memory promoting fast spindles shows a centro-parietal topography? Surely it would be more adaptive for fast spindles to be maximally expressed in frontal sites. Would a participant who shows a more frontal topography of fast spindles have better overnight consolidation than someone with a more canonical centro-parietal topography? Similarly, slow spindles would then be perfectly suited for memory consolidation given their frontal distribution, yet they seem less important for memory.

The authors rightly note the issues with multiple comparisons in sleep physiology and memory studies. Multiple comparison issues arise in two ways in this literature. First are comparisons across multiple electrodes (many studies now use high-density systems with 64+ channels). Second are multiple comparisons across different outcome variables (at least 3 ways to quantify coupling (phase, consistency, occurrence) x 2 spindle types (fast, slow). Can the authors make some recommendations here in terms of how to move the field forward, as this issue has been raised numerous times before (e.g., Mantua 2018, Sleep; Cox & Fell 2020, Sleep Medicine Reviews for just a couple of examples). Should researchers just be focusing on the coupling phase? Or should researchers always report all three metrics of coupling, and correct for multiple comparisons? I think the use of pre-registration would be beneficial here, and perhaps could be noted by the authors in the final paragraph of section 3.5, where they discuss open research practices.

Joint Public Review:

Summary:

Hossain and coworkers investigate the mechanisms of recognition of xCas9, a variant of Cas9 with expanded targeting capability for DNA. They do so by using molecular simulations and combining different flavors of simulation techniques, ranging from long classical MD simulations, to enhanced sampling, to free energy calculations of affinity differences. Through this, the authors are able to develop a consistent model of expanded recognition based on the enhanced flexibility of the protein receptor.

Strengths:

The paper is solidly based on the ability of the authors to master molecular simulations of highly complex systems. In my opinion, this paper shows no major weaknesses. The simulations are carried out in a technically sound way. Comparative analyses of different systems provide valuable insights, even within the well-known limitations of MD. Plus, the authors further investigate why xCas9 exhibits improved recognition of the TGG PAM sequence compared to SpCas9 via well-tempered metadynamics simulations focusing on the binding of R1335 to the G3 nucleobase and the DNA backbone in both SpCas9 and xCas9. In this context, the authors provide a free-energy profiling that helps support their final model.

The implementation of FEP calculations to mimic directed evolution improvement of DNA binding is also interesting, original and well-conducted.

Overall, my assessment of this paper is that it represents a strong manuscript, competently designed and conducted, and highly valuable from a technical point of view.

Weaknesses:

To make their impact even more general, the authors may consider expanding their discussion on entropic binding to other recent cases that have been presented in the literature recently (such as e.g. the identification of small molecules for Abeta peptides, or the identification of "fuzzy" mechanisms of binding to protein HMGB1). The point on flexibility helping adaptability and expansion of functional properties is important, and should probably be given more evidence and more direct links with a wider picture.

Reviewer #1 (Public review):

Summary:

The work provides more evidence of the importance of data quality and representation for ligand-based virtual screening approaches. The authors have applied different machine learning (ML) algorithms and data representation using a new dataset of BRAF ligands. First, the authors evaluate the ML algorithms, and demonstrate that independently of the ML algorithm, predictive and robust models can be obtained in this BRAF dataset. Second, the authors investigate how the molecular representations can modify the prediction of the ML algorithm. They found that in this highly curated dataset the different molecule representations are adequate for the ML algorithms since almost all of them obtain high accuracy values, with Estate fingerprints obtaining the worst performing predictive models and ECFP6 fingerprints producing the best classificatory models. Third, the authors evaluate the performance of the models on subsets of different composition and size of the BRAF dataset. They found that given a finite number of active compounds, increasing the number of inactive compounds worsens the recall and accuracy. Finally, the authors analyze if the use of "less active" molecules affect the model's predictive performance using "less active" molecules taken from ChEMBl Database or using decoys from DUD-E. As results, they found that the accuracy of the model falls as the number of "less active" examples in the training dataset increases while the implementation of decoys in the training set generates results as good as the original models or even better in some cases. However, the use of decoys in the training set worsens the predictive power in the test sets that contain active and inactive molecules.

Strengths:

This is a highly relevant topic in medicinal chemistry and drug discovery. The manuscript is well-written, with a clear structure that facilitates easy reading, and it includes up-to-date references. The hypotheses are clearly presented and appropriately explored. The study provides valuable insights into the importance of deriving models from high-quality data, demonstrating that, when this condition is met, complex computational methods are not always necessary to achieve predictive models. Furthermore, the generated BRAF dataset offers a valuable resource for medicinal chemists working in ligand-based virtual screening.

Weaknesses:

While the work highlights the importance of using high-quality datasets to achieve better and more generalizable results, it does not present significant novelty, as the analysis of training data has been extensively studied in chemoinformatics and medicinal chemistry. Additionally, the inclusion of "AI" in the context of data-centric AI is somewhat unclear, given that the dataset curation is conducted manually, selecting active compounds based on IC50 values from ChEMBL and inactive compounds according to the authors' criteria.

Moreover, the conclusions are based on the analysis of only two high-quality datasets. To generalize these findings, it would be beneficial to extend the analysis to additional high-quality datasets (at least 10 datasets for a robust benchmarking exercise).

A key aspect that could be improved is the definition of an "inactive" compound, which remains unclear. In the manuscript, it is stated:

• "The inactives were carefully selected based on the fact that they have no known pharmacological activity against BRAF."<br /> Does the lack of BRAF activity data necessarily imply that these compounds are inactive?<br /> • "We define a compound as 'inactive' if there are no known pharmacological assays for the said compound on our target, BRAF."<br /> However, in the authors' response, they mention:<br /> • "We selected certain compounds that we felt could not possibly be active against BRAF, such as ligands for neurotransmitter receptors, as inactives."

Given that the definition of "inactive" is one of the most critical concepts in the study, I believe it should be clearly and consistently explained.

Lastly, while statistical comparison is not always common in machine learning, it would greatly enhance the value of this work, especially when comparing models with small differences in accuracy.

Joint Public Review:

Riva et al uncovered the neural substrate underlying the oviposition rhythm in Drosophila melanogaster using a novel device that automates egg collection from individual mated females over the course of multiple days. By systematically knocking down the clock gene period in specific clock neurons the authors show that three cryptochrome (cry) positive dorso-lateral neurons (LNds) present in each hemisphere of the fly brain are critical to generating a female, sex-specific rhythm in oviposition. Interestingly, these neurons are not essential for freerunning locomotor activity. By contrast, the LNvs (lateral ventral neurons), which are essential for freerunning locomotor activity rhythmicity, were not involved in controlling the circadian rhythmicity of oviposition. Thus, this work has identified the first truly sex-specific circadian circuit in Drosophila. Using available Drosophila hemibrain connectome data they identify bidirectional connections between cry-expressing LNd and oviposition-related neurons.

Strengths:

This paper established a new semi-automatic device to register egg-laying activity, in Drosophila and found a specific role for a subset of clock neurons in the control of a female-specific circadian behavior. They also lay the groundwork for understanding how these neurons are connected to the neurons that control egg laying.

Weaknesses:

(1) Controls for the genetic background are incomplete, leaving open the possibility that the observed oviposition timing defects may be due to targeted knockdown of the period (per) gene but from the GAL4, Gal80, and UAS transgenes themselves. To resolve this issue the authors should determine the egg-laying rhythms of the relevant controls (GAL4/+, UAS-RNAi/+, etc); this only needs to be done for those genotypes that produced an arrhythmic egg-laying rhythm.

(2) Reliance on a single genetic tool to generate targeted disruption of clock function leaves the study vulnerable to associated false positive and false negative effects: a) The per RNAi transgene used may only cause partial knockdown of gene function, as suggested by the persistent rhythmicity observed when per RNAi was targeted to all clock neurons. This could indicate that the results in Fig 2C-H underestimate the phenotypes of targeted disruption of clock function. b) Use of a single per RNAi transgene makes it difficult to rule out that off-target effects contributed significantly to the observed phenotypes. We suggest that the authors repeat the critical experiments using a separate UAS-RNAi line (for period or for a different clock gene), or, better yet, use the dominant negative UAS-cycle transgene produced by the Hardin lab (https://doi.org/10.1038/22566).

(3) The egg-laying profiles obtained show clear damping/decaying trends which necessitates careful trend removal from the data to make any sense of the rhythm. Further, the detrending approach used by the authors is not tested for artefacts introduced by the 24h moving average used.

(4) According to the authors the oviposition device cannot sample at a resolution finer than 4 hours, which will compel any experimenter to record egg laying for longer durations to have a suitably long time series which could be useful for circadian analyses.

(5) Despite reducing the interference caused by manually measuring egg-laying, the rhythm does not improve the signal quality such that enough individual rhythmic flies could be included in the analysis methods used. The authors devise a workaround by combining both strongly and weakly rhythmic (LSpower > 0.2 but less than LSpower at p < 0.05) data series into an averaged time series, which is then tested for the presence of a 16-32h "circadian" rhythm. This approach loses valuable information about the phase and period present in the individual mated females, and instead assumes that all flies have a similar period and phase in their "signal" component while the distribution of the "noise" component varies amongst them. This assumption has not yet been tested rigorously and the evidence suggests a lot more variability in the inter-fly period for the egg-laying rhythm.

(6) This variability could also depend on the genotype being tested, as the authors themselves observe between their Canton-S and YW wild-type controls for which their egg-laying profiles show clearly different dynamics. Interestingly, the averaged records for these genotypes are not distinguishable but are reflected in the different proportions of rhythmic flies observed. Unfortunately, the authors also do not provide further data on these averaged profiles, as they did for the wild-type controls in Figure 1, when they discuss their clock circuit manipulations using perRNAi. These profiles could have been included in Supplementary figures, where they would have helped the reader decide for themselves what might have been the reason for the loss of power in the LS periodogram for some of these experimental lines.

(7) By selecting 'the best egg layers' for inclusion in the oviposition analyses an inadvertent bias may be introduced and the results of the assays may not be representative of the whole population.

(8) An approach that measures rhythmicity for groups of individual records rather than separate individual records is vulnerable to outliers in the data, such as the inclusion of a single anomalous individual record. Additionally, the number of individual records that are included in a group may become a somewhat arbitrary determinant for the observed level of rhythmicity. Therefore, the experimental data used to map the clock neurons responsible for oviposition rhythms would be more convincing if presented alongside individual fly statistics, in the same format as used for Figure 1.

(9) The features in the experimental periodogram data in Figures 3B and D are consistent with weakened complex rhythmicity rather than arrhythmicity. The inclusion of more individual records in the groups might have provided the added statistical power to demonstrate this. Graphs similar to those in 1G and 1I, might have better illustrated qualitative and quantitative aspects of the oviposition rhythms upon per knockdown via MB122B and Mai179; Pdf-Gal80.

Wider context:

The study of the neural basis of oviposition rhythms in Drosophila melanogaster can serve as a model for the analogous mechanisms in other animals. In particular, research in this area can have wider implications for the management of insects with societal impact such as pests, disease vectors, and pollinators. One key aspect of D. melanogaster oviposition that is not addressed here is its strong social modulation (see Bailly et al.. Curr Biol 33:2865-2877.e4. doi:10.1016/j.cub.2023.05.074). It is plausible that most natural oviposition events do not involve isolated individuals, but rather groups of flies. As oviposition is encouraged by aggregation pheromones (e.g., Dumenil et al., J Chem Ecol 2016 https://link.springer.com/article/10.1007/s10886-016-0681-3) its propensity changes upon the pre-conditioning of the oviposition substrates, which is a complication in assays of oviposition rhythms that periodically move the flies to fresh substrate.

Reviewer #1 (Public review):

Summary:

The authors demonstrate a fully unsupervised, high throughput (meaning very low human interaction required) approach to quantifying marmoset behavior in unconstrained environments.

Strengths:

The authors provide an approach that is scalable, easy to implement at face value, and highly robust. Currently, most behavioral quantification approaches do not work well on marmosets, or the published examples that do look promising do not scale towards high throughput as demonstrated by the authors.

While marmosets can certainly be a useful translational research model devoid of free behavior quantification, the authors make a compelling point about how this approach can be useful in the study of treatments of emerging marmoset disease models.

Overall this is a very exhaustive manuscript that overcomes significant shortcomings in previous work and speaks highly to the use of marmosets for unconstrained behavioral and neural assessment.

Weaknesses:

Recording marmoset behavior with a 60Hz frame rate is a significant limitation to the approach which is hopefully easily alleviated in the future through better cameras/reconstruction pipelines. Marmosets (in the reviewers' experience) have a lot of motion energy above the 30Hz nyquist limit imposed by this system and are agile to a degree requiring higher frame rates.

The manuscript neglects recent approaches to non-human primate behavioral quantification from other groups that should be included. Simians are simians after all.

As a minor weakness, this reviewer would have liked to see code shared for the reviewers to evaluate, especially pertaining to the high throughput and robustness of the approach.

Reviewer #1 (Public review):

Summary:

Ren et al developed a novel computational method to investigate cell evolutionary trajectory for scRNA-seq samples. This method, MGPfact, estimates pseudotime and potential branches in the evolutionary path through explicitly modeling the bifurcations in a Gaussian process. They benchmarked this method using synthetic as well as real world samples and showed superior performance for some of the tasks in cell trajectory analysis. They further demonstrated the utilities of MGPfact using single cell RNA-seq samples derived from microglia or T cells and showed that it can accurately identify the differentiation timepoint and uncover biologically relevant gene signatures.

Strengths:

Overall I think this is a useful new tool that could deliver novel insights for the large body of scRNA-seq data generated in the public domain. The manuscript is written is a logical way and most parts of the method are well described.

Comments on revisions:

In this revision, the authors have sufficiently addressed all of my concerns. I don't have any follow-up comments.

Reviewer #1 (Public review):

This study is focused on a population of neurons in the mouse parasubthalamic nucleus (pSTN) that express Tackhykinin1 (Tac1). This gene has been used before to target pSTN for functional circuit studies because it is fairly selective for pSTN in this region, though it targets only a subset of pSTN neurons. Prior work has shown that activity in these neurons can impact motivated behaviors, including feeding and drinking behaviors, and that their activity is associated with aversion or avoidance behaviors. While not breaking much new ground, this study adds to that work by making use of a 2-way active avoidance assay, where a CS predicts a US (footshock), that the mice can escape. Using fiber photometry the authors show convincing evidence that Tac1 neurons in pSTN increase their activity in response to a US footshock, and that after some pairings the neurons will start responding to the CS too, though to a lesser extent than the US. Their most important data shows that either ablation or optogenetic inhibition of these cells can hugely block the active avoidance (escape) behavior, suggesting these neurons are key for the performance of this task, which they interpret as key for learning the task (but see more below). They show that optogenetic stimulation is aversive in a real-time place assay, and when paired with footshock can enhance active avoidance behavior. Finally, they show that Tac1 pSTN axons in PVT recapitulate these effects while showing that axons in CEA or PBN may only recapitulate some of these effects (more below). Overall I think the data is solid and shows that the activity of Tac1 pSTN neurons in the 2 way active avoidance task is causally related to avoidance behavior in the direction that would be predicted by recent literature. However, I think the authors overstate the conclusions in the title, abstract, and text. I do not think the data make a strong case for a role for these cells in learning, at least in any classical sense, as used in the title and abstract and elsewhere. Also the statement in the abstract that the pSTN mediates its effects 'differentially' through its downstream targets is not convincingly supported by data.

Major concerns:

(1) The authors infer that the activity in the Tac1 pSTN neurons is necessary for aversive or avoidance 'learning'. But this is not well defined, what exactly does that mean and what types of evidence would support or falsify such a hypothesis? Moreover, the authors show convincingly, and in line with prior reports, that these cells are activated by aversive stimuli (here footshock), and that activation of these cells is sufficient to induce avoidance behavior. Because manipulation of these cells can serve as a primary negative reinforcer, it becomes even more challenging and important to explain how experiments that manipulate these cells while measuring behavior/performance can discriminate between changes in: (1) primary aversion, (2) motivation to avoid, (3) associative learning, or (4) memory/retrieval. The authors seem to favor #3, but they don't make a clear case for this point of view or else what they mean by 'avoidance learning'. In my opinion, the data do not well discriminate between possibilities 1 through 3. The authors should clarify their logic and temper their conclusions throughout.

(2) Abstract line 37 is not well supported. The authors focus mostly on pSTN projections to PVT and show that the measurements or manipulation of these axons recapitulates the effects seen with pSTN cell bodies. The authors do fewer studies of axons in CeA and PBN, but do find that they can recapitulate the effects with opsin inhibition, but detect no effects with opsin stimulation. However, the lack of effect with opsin stimulation in Figure S7a-e proves very little on its own. It could be technical, due to inadequate expression or functional efficacy. It is not supported by histological and functional evidence that the manipulation was effective. Overall I can only conclude that the projections to these regions might be very similar (based on the inhibition data), or might be a little different. The data are thus inadequate to support the authors' claim that the pSTN mediates learning differentially through its downstream targets.

Other concerns:

(3) Line 93 is not adequately supported by data in Figure 1b. Additional data is needed that shows expression across cases, including any spread that may be visible when zooming out from pSTN. Additional methods are needed to indicate what exclusion criteria were applied and how many mice were excluded. These data could help support the statement on line 93 that expression was largely restricted within pSTN.

(4) From the results and methods it is not clear where the GFP signal would come from in the mice expressing Casp3 for the ablation studies. It is therefore not clear if the absence of GFP should be taken as evidence of cell loss. For example, it is not clear if multiple vectors were used, if volumes and titers were carefully matched between control groups, or if competition/occlusion between AAVs could be ruled out. It is also not clear how this was quantified, that is how many sections/subjects and how counting was done. It is not clear how long was waited between the AAV infusion, behavior, and euthanasia, perhaps especially important for the ablation done after avoidance learning occurred.

(5) The authors should consider showing individual measurements and not just mean/sem wherever feasible, for example, to support the statement on line 141 that 'all ablated mice showed...'.

(6) S3 is an important control for interpreting data in Figure 2d-i. Something similar is needed to support the inferences made in 2j-u. The very strong effect showing a lack of active avoidance in response to CS or the US when pSTN Tac1 neurons are inhibited during CS or during US suggests that something gross may be going on, such as a gross motor or sensory response that supersedes the effect of footshock. The authors do not comment on whether there are any gross behavioral responses to the inhibition, but an experiment as in S3 is needed, for example, to show that behavior is intact during pSTN inhibition if delivered after the mice already learned to associate CS with US.

(7) The authors use 100 shocks of 0.8 mA for 7 days. I think this is quite strong and in the pSTN inhibition experiments it seems to be functionally 'inescapable' and could thus produce behaviors similar to 'learned helplessness'. Can the authors consider whether this might contribute to the striking findings they observed in their opsin inhibition assays?

(8) The description of the experiment in S5 is inadequate. What are the adjacent areas? Where do the authors see spread? The use of the word 'case' in figure S5 implies an individual case, but the legend says 5 mice were used for 'case 1' and 3 mice were used for 'case 2'. The use of the word 'off-target in the figure implies that the expression was of the intended target. But the text of results and methods implies it was intentional targeting of unnamed and unshown adjacent regions. This should be clarified.

(9) The authors suggest the CPA study is divergent from Serra et al 2023. Though I think this could be due to how the conditioning was done, it would be helpful for the authors to include less processed data. This would aid in possible interpretations for any divergences across studies. Can the authors include raw data (in seconds of time spent) in each compartment for each group across baseline and test days?

Reviewer #1 (Public review):

Summary:

The investigators in this study analyzed the dataset assembly from 540 Salmonella isolates, and those from 45 recent isolates from Zhejiang University of China. The analysis and comparison of the resistome and mobilome of these isolates identified a significantly higher rate of cross-region dissemination compared to localized propagation. This study highlights the key role of the resistome in driving the transition and evolutionary history of S. Gallinarum.

Strengths:

The isolates included in this study were from 16 countries in the past century (1920 to 2023). While the study uses S. Gallinarun as the prototype, the conclusion from this work will likely apply to other Salmonella serotypes and other pathogens.

Weaknesses:

While the isolates came from 16 countries, most strains in this study were originally from China.

Comments on revisions:

This reviewer is happy with the detailed responses from the authors regarding revising this manuscript. I do not have further comments.

Reviewer #1 (Public review):

Summary:

Cell metabolism exhibits a well-known behavior in fast-growing cells, which employ seemingly wasteful fermentation to generate energy even in the presence of sufficient environmental oxygen. This phenomenon is known as Overflow Metabolism or the Warburg effect in cancer. It is present in a wide range of organisms, from bacteria and fungi to mammalian cells.

In this work, starting with a metabolic network for Escherichia coli based on sets of carbon sources, and using a corresponding coarse-grained model, the author applies some well-based approximations from the literature and algebraic manipulations. These are used to successfully explain the origins of Overflow Metabolism, both qualitatively and quantitatively, by comparing the results with E. coli experimental data.

By modeling the proteome energy efficiencies for respiration and fermentation, the study shows that these parameters are dependent on the carbon source quality constants K_i (p.115 and 116). It is demonstrated that as the environment becomes richer, the optimal solution for proteome energy efficiency shifts from respiration to fermentation. This shift occurs at a critical parameter value K_A(C).<br /> This counter intuitive results qualitativelly explains Overflow Metabolism.

Quantitative agreement is achieved through the analysis of the heterogeneity of the metabolic status within a cell population. By introducing heterogeneity, the critical growth rate is assumed to follow a Gaussian distribution over the cell population, resulting in accordance with experimental data for E. coli. Overflow metabolism is explained by considering optimal protein allocation and cell heterogeneity.

The obtained model is extensively tested through perturbations: 1) Introduction of overexpression of useless proteins; 2) Studying energy dissipation; 3) Analysis of the impact of translation inhibition with different sub-lethal doses of chloramphenicol on Escherichia coli; 4) Alteration of nutrient categories of carbon sources using pyruvate. All model perturbations results are corroborated by E. coli experimental results.

Strengths:

In this work, the author effectively uses modeling techniques typical of Physics to address complex problems in Biology, demonstrating the potential of interdisciplinary approaches to yield novel insights. The use of Escherichia coli as a model organism ensures that the assumptions and approximations are well-supported in existing literature. The model is convincingly constructed and aligns well with experimental data, lending credibility to the findings. In this version, the extension of results from bacteria to yeast and cancer is substantiated by a literature base, suggesting that these findings may have broad implications for understanding diverse biological systems.

Weaknesses:

The author explores the generalization of their results from bacteria to cancer cells and yeast, adapting the metabolic network and coarse-grained model accordingly. In previous version this generalization was not completedly supported by references and data from the literature. This drawback, however, has been treated in this current version, where the authors discuss in much more detail and give references supporting this generalization.

Reviewer #1 (Public review):

The authors sought to examine the associations between child age, reports of parent-child relationship quality, and neural activity patterns while children (and also their parents) watched a movie clip. Major methodological strengths include the sample of 3-8 year-old children in China (rare in fMRI research for both age range and non-Western samples), use of a movie clip previously demonstrated to capture theory of mind constructs at the neural level, measurement of caregiver-child neural synchrony, and assessment of neural maturity. Results provide important new information about parent-child neural synchronization during this movie and associations with reports of parent-child relationship quality. The work is a notable advance in understanding the link between the caregiving context and the neural construction of theory of mind networks in the developing brain.

There are several theoretical and methodological limitations of the manuscript in its current form:

(1) We appreciate that the authors wanted to show support for a mediational mechanism. However, we suggest that the authors drop the structural equation modeling because the data are cross-sectional so mediation is not appropriate. Other issues include the weak justification of including the parent-child neural synchronization as part of parenting.... it could just as easily be a mechanism of change or driven by the child rather than a component of parenting behavior. The paper would be strengthened by looking at associations between selected variables of interest that are MOST relevant to the imaging task in a regression type of model. Furthermore, the authors need to be more explicit about corrections for multiple comparisons throughout the manuscript; some of the associations are fairly weak so claims may need to be tempered if they don't survive correction.

(2) Reverse correlation analysis is sensible given what prior developmental fMRI studies have done. But reverse correlation analysis may be more prone to overfitting and noise, and lacks sensitivity to multivariate patterns. Might inter-subject correlation be useful for *within* the child group? This would minimize noise and allow for non-linear patterns to emerge.

(3) No learning effects or temporal lagged effects are tested in the current study, so the results do not support the authors' conclusions that the data speak to Bandura's social learning theory. The authors do mention theories of biobehavioral synchrony in the introduction but do not discuss this framework in the discussion (which is most directly relevant to the data). The data can also speak to other neurodevelopmental theories of development (e.g.,neuroconstructivist approaches), but the authors do not discuss them. The manuscript would benefit from significantly revising the framework to focus more on biobehavioral synchrony data and other neurodevelopmental approaches given the prior work done in this area rather than a social psychology framework that is not directly evaluated.

(4) The significance and impact of the findings would be clearer if the authors more clearly situated the findings in the context of (a) other movie and theory of mind fMRI task data during development; and (b) existing data on parent-child neural synchrony (often uses fNIRS or EEG). What principles of brain and social cognition development do these data speak to? What is new?

(5) There is little discussion about the study limitations, considerations about the generalizability of the findings, and important next steps and future directions. What can the data tell us, and what can it NOT tell us?

Reviewer #1 (Public review):

Summary:

The authors present NeuroSCAN, an accessible and interactive tool for visualizing and summarizing data from multiple previously annotated C. elegans connectomes. NeuroSCAN provides a useful entry point for streamlined observation of neuronal morphology, and the membrane contacts and synaptic connectivity between neurons across developmental stages and individual connectomes readily extracted from existing data.

Strengths:

Koonce et al. have generated a web-based visualization tool for exploring C. elegans neuronal morphology, contact area between neurons, and synaptic connectivity data. Here, the authors integrate volumetric segmentation of neurons and visualization of contact area patterns of individual neurons generated from Diffusion Condensation and C-PHATE embedding based on previous work from adult volumetric electron microscopy (vEM) data, extended to available vEM data for earlier developmental stages, which effectively summarizes modularity within the collated C. elegans contactomes to date. Overall, NeuroSCAN's relative ease of use for generating visualizations, its ability to quickly toggle between developmental stages, and its integration of a concise visualization of individual neurons' contact patterns strengthen its utility.

Weaknesses:

NeuroSCAN provides an accessible and convenient platform. However, many of the characteristics of NeuroSCAN overlap with that of an existing tool for visualizing connectomics data, Neuroglancer, which is a widely-used and shared platform with data from other organisms. The authors do not make clear their motivation for generating this new tool rather than building on a system that has already collated previous connectomics data. Although the field will benefit from any tool that collates connectomics data and makes it more accessible and user-friendly, such a tool is only useful if it is kept up-to-date, and if data formatting for submitting electron microscopy data to be added to the tool is made clear. It is unclear from this manuscript whether NeuroSCAN will be updated with recently published and future C. elegans connectomes, or how additional datasets can be submitted to be added in the future.

The interface for visualizing contacts and synapses would be improved with better user access to the quantitative underlying data. When contact areas or synapses are added to the viewer, adding statistics on the magnitude of the contact area, the number of synapses, and the rank of these values among the neuron's top connections, would make the viewer more useful for hypothesis generation. Furthermore, synapses are currently listed individually, with names that are not very legible to the web user. Grouping them by pre- and postsynaptic neurons and linking these groups across developmental stages would also be an improvement.

While the DC/C-PHATE visualizations are a useful tool for the user, it is difficult to understand when grouping or splitting of cell contact patterns is biologically significant. DC is a deterministic algorithm applied to a contactome from a single organism, and the authors do not provide quantitative metrics of distances between individual neurons or a number of DC iterations on the C-PHATE plot, nor is the selection process for the threshold for DC described in this manuscript. In the application of DC/C-PHATE to larval stage nerve ring strata organization shown by the authors, qualitative observations of C-PHATE plots colored based on adult data seem to be the only evidence shown for persistent strata during development (Figure 3) or changing architectural motifs across stages (Figure 4). Quantitation of differences in neuron position within the DC hierarchy, or differences in modularity across stages, is needed to support these conclusions. Furthermore, illustrating the quantitative differences in C-PHATE plots used to make these conclusions will provide a more instructive guide for users of NeuroSCAN in generating future hypotheses.

While the case studies presented by the authors help to highlight the utility of the different visualizations offered by the NeuroSCAN platform, the authors need to be more careful with the claims they make from these correlative observations. For example, in Figure 4, the authors use C-PHATE clustering patterns to make conclusions about changes in clustering patterns of individual neurons across development based on single animal datasets. In this and many other cases presented in this study with the limited existing datasets, it is difficult to differentiate between developmental changes and individual variability between the neurite positions, contacts, and synapse differences within these data. This caveat needs to be clearly addressed.

Reviewer #1 (Public review):

Summary:

In this lovely paper, McDermott and colleagues tackle an enduring puzzle in the cognitive neuroscience of perceptual prediction. Though many scientists agree that top-down predictions shape perception, previous studies have yielded incompatible results - with studies showing 'sharpened' representations of expected signals, and others showing a 'dampening' of predictable signals to relatively enhance surprising prediction errors. To deepen the paradox further, it seems like there are good reasons that we would want to see both influences on perception in different contexts.

Here, the authors aim to test one possible resolution to this 'paradox' - the opposing process theory (OPT). This theory makes distinct predictions about how the time course of 'sharpening' and 'dampening' effects should unfold. The researchers present a clever twist on a leading-trailing perceptual prediction paradigm, using AI to generate a large dataset of test and training stimuli so that it is possible to form expectations about certain categories without repeating any particular stimuli. This provides a powerful way of distinguishing expectation effects from repetition effects - a perennial problem in this line of work.

Using EEG decoding, the researchers find evidence to support the OPT. Namely, they find that neural encoding of expected events is superior in earlier time ranges (sharpening-like) followed by a relative advantage for unexpected events in later time ranges (dampening-like). On top of this, the authors also show that these two separate influences may emerge differently in different phases of learning - with superior decoding of surprising prediction errors being found more in early phases of the task, and enhanced decoding of predicted events being found in the later phases of the experiment.

Strengths:

As noted above, a major strength of this work lies in important experimental design choices. Alongside removing any possible influence of repetition suppression mechanisms in this task, the experiment also allows us to see how effects emerge in 'real-time' as agents learn to make predictions. This contrasts with many other studies in this area - where researchers 'over-train' expectations into observers to create the strongest possible effects or rely on prior knowledge that was likely to be crystallised outside the lab.

Weaknesses:

This study reveals a great deal about how certain neural representations are altered by expectation and learning on shorter and longer timescales, so I am loath to describe certain limitations as 'weaknesses'. But one limitation inherent in this experimental design is that, by focusing on implicit, task-irrelevant predictions, there is not much opportunity to connect the predictive influences seen at the neural level to the perceptual performance itself (e.g., how participants make perceptual decisions about expected or unexpected events, or how these events are detected or appear).

The behavioural data that is displayed (from a post-recording behavioural session) shows that these predictions do influence perceptual choice - leading to faster reaction times when expectations are valid. In broad strokes, we may think that such a result is broadly consistent with a 'sharpening' view of perceptual prediction, and the fact that sharpening effects are found in the study to be larger at the end of the task than at the beginning. But it strikes me that the strongest test of the relevance of these (very interesting) EEG findings would be some evidence that the neural effects relate to behavioural influences (e.g., are participants actually more behaviourally sensitive to invalid signals in earlier phases of the experiment, given that this is where the neural effects show the most 'dampening' a.k.a., prediction error advantage?)

Reviewer #1 (Public review):

Summary:

The authors test the hypotheses, using an effort-exertion and an effort-based decision-making task, while recording brain dynamics with EEG, that the brain processes reward outcomes for effort differentially when they earned for themselves versus others.

Strengths:

The strengths of this experiment include what appears to be a novel finding of opposite signed effects of effort on the processing of reward outcomes when the recipient is self versus others. Also, the experiment is well-designed, the study seems sufficiently powered, and the data and code are publicly available.

Weaknesses:

Inferences rely heavily on the results of mixed effects models which may or may not be properly specified and are not supported by complementary analyses. Also, not all results hang together in a sensible way. For example, participants report feeling less subjective effort, but also more disliking of tasks when they were earning rewards for others versus self. Given that participants took longer to complete tasks when earning effort for others, it is conceivable that participants might have been working less hard for others versus themselves, and this may complicate the interpretation of results.

Reviewer #1 (Public review):

The manuscript by Coquel et al. investigates the effects of BKC and IBC, two compounds found in Psoralea corylifolia in DNA replication and the response to DNA damage, and explores their potential use in cancer treatment. These compounds have been previously shown to affect different cellular pathways and the authors use transformed cancer cells of different origins and a non-transformed cell line to question if their combination is toxic in cancer versus non-cancer cells. They propose that BKC inhibits DNA polymerases while IBC targets CHK2. Their results show that both compounds do affect DNA replication, inducing replication stress and affecting double strand break repair. They also show that their combined use increases their toxicity in a synergistic manner.

Comments on current version:

The authors have addressed the main questions raised in the original manuscript. The new data provide stronger evidence supporting the inhibition of DNA polymerases by BKC and the effect of IBC on CHK2. In addition, the new data provides information about the potential mechanism of action of IBC in cells and xenograft models. Together, the revised manuscript has notably increased the relevance and impact of the results with stronger conclusions and better controlled experiments.

Reviewer #1 (Public review):

Summary:

Ngo et. al use several computational methods to determine and characterize structures defining the three major states sampled by the human voltage-gated potassium channel hERG: the open, closed, and inactivated state. Specifically, they use AlphaFold and Rosetta to generate conformations that likely represent key features of the open, closed, and inactivated states of this channel. Molecular dynamics simulations confirm that ion conduction for structure models of the open but not the inactivated state. Moreover, drug docking in silico experiments show differential binding of drugs to the conformation of the three states; the inactivated one being preferentially bound by many of them. Docking results are then combined with a Markov model to get state-weighted binding free energies that are compared with experimentally measured ones.

Strengths:

The study uses state-of-the art modeling methods to provide detailed insights into the structure-function relationship of an important human potassium channel. AlphaFold modeling, MD simulations, and Markov modeling are nicely combined to investigate the impact of structural changes in the hERG channel on potassium conduction and drug binding.

Weaknesses:

(1) The selection of inactivated conformations based on AlphaFold modeling seems a bit biased. The authors base their selection of the "most likely" inactivated conformation on the expected flipping of V625 and the constriction at G626 carbonyls. This follows a bit of the "Streetlight effect". It would be better to have selection criteria that are independent of what they expect to find for the inactivated state conformations. Using cues that favour sampling/modeling of the inactivated conformation, such as the deactivated conformation of the VSD used in the modeling of the closed state, would be more convincing. There may be other conformations that are more accurately representing the inactivated state. I see no objective criteria that justify the non-consideration of conformations from cluster 3 of the inactivated state modeling. I am not sure whether pLDDT is a good selection criterion. It reports on structural confidence, but that may not relate to functional relevance.

(2) The comparison of predicted and experimentally measured binding affinities lacks an appropriate control. Using binding data from open-state conformations only is not the best control. A much better control is the use of alternative structures predicted by AlphaFold for each state (e.g. from the outlier clusters or not considered clusters) in the docking and energy calculations. Using these docking results in the calculations would reveal whether the initially selected conformations (e.g. from cluster 2 for the inactivated state) are truly doing a better job in predicting binding affinities. Such a control would strengthen the overall findings significantly.

(3) Figures where multiple datapoints are compared across states generally lack assessment of the statistical significance of observed trends (e,g. Figure 3d).

(4) Figure 3 and Figures S1-S4 compare structural differences between states. However, these differences are inferred from the initial models. The collection of conformations generated via the MD runs allow for much more robust comparisons of structural differences.

Reviewer #1 (Public review):

Summary:

The study by Fang et al. reports a 3D MERFISH method that enable spatial transcriptomics for tissues up to 200um in thickness. MERFISH as well other spatial transcriptomics technologies have been mainly used for thin (e.g, 10um) tissue slices, which limits the dimension of spaital transcriptomics technique. Therefore, expanding the capacity of MERFISH to thick tissues represents a major technical advance to enable 3D spatial transcriptomics. Here the authors provide detailed technical descriptions of the new method, troubleshooting, optimization, and application examples to demonstrate its technical capacity, accuracy, sensitivity, and utility. The method will likely have major impact on future spatial transcriptomics studies to benefit diverse biomedical fields.

Strengths:

The study was well-designed, executed, and presented. Extensive protocol optimization and quality assessments were carried out and conclusions are well supported by the data. The methods were sufficiently detailed and the results are solid and compelling.

Weaknesses:

Thorough performance comparison with other existing technologies can be done in the future.

Comments on revisions:

The authors have sufficiently addressed the previous comments.

Reviewer #1 (Public review):

Summary:

In this study, Kume et al examined the role of the protein Semaphorin 4a in steady state skin homeostasis and how this relates to skin changes seen in human psoriasis and imiquimod-induced psoriasis-like disease in mice. The authors found that human psoriatic skin has reduced expression of Sema4a in the epidermis. While Sema4a has been shown to drive inflammatory activation in different immune populations, this finding suggested Sema4a might be important for negatively regulating Th17 inflammation in the skin. The authors go on to show that Sema4a knockout mice have skin changes in key keratinocyte genes, increased gdT cells, and increased IL-17 similar to differences seen in non-lesional psoriatic skin, and that bone marrow chimera mice with WT immune cells and Sema4a KO stromal cells develop worse IMQ-induced psoriasis-like disease, further linking expression of Sema4a in the skin to maintaining skin homeostasis. The authors next studied downstream pathways that might mediate the homeostatic effects of Sema4a, focusing on mTOR given its known role in keratinocyte function. Like for the immune phenotypes, Sema4a KO mice had increased mTOR activation in the epidermis in a similar pattern to mTOR activation noted in non-lesional psoriatic skin. The authors next targeted the mTOR pathway and showed rapamycin could reverse some of the psoriasis-like skin changes in Sema4a KO mice, confirming the role of increased mTOR in contributing to the observed skin phenotype.

In the revised manuscript, the authors expand on the potential relevance to psoriasis by demonstrating similar findings in an IL-23-diriven model of skin inflammation, which is an orthogonal model of psoriasis to their original IMQ model. They also show that in addition to reversing steady state differences in skin thickness between Sema4a KO mice and WT mice, rapamycin improves metrics of disease in the IMQ model of psoriasis. These additional studies further bolster their conclusions that Sema4a may play a protective role in by preventing over-activation of mTOR in the skin in psoriasis.

Strengths:

The most interesting finding is the tissue-specific role for Sema4a, where it has previously been considered to play a mostly pro-inflammatory role in immune cells, this study shows that when expressed by keratinocytes, Sema4a plays a homeostatic role that when missing leads to development of psoriasis-like skin changes. This has important implications in terms of targeting Sema4a pharmacologically. It also may yield a novel mouse model to study mechanisms of psoriasis development in mice separate from the commonly used IMQ model. The included experiments are well-controlled and executed rigorously.

The new experiments provide additional data to support the conclusions through an orthogonal model of psoriasis and demonstrating rapamycin-induced reversal of changes in the IMQ disease model.

Weaknesses:

While the main weakness of these studies, lack of tissue-specific Sema4a knockout mice (e.g. in keratinocytes only), remains, generating these mice and performing the necessary experiments is beyond the scope of completing these particular studies. Similarly, it is understandable that additional bone marrow chimeras would be costly and labor intensive without adding much more in the absence of tissue-specific knockouts.

Reviewer #1 (Public review):

The authors focus on the molecular mechanisms by which EMT cells confer resistance to cancer cells. The authors use a wide range of methods to reveal that overexpression of Snail in EMT cells induces cholesterol/sphingomyelin imbalance via transcriptional repression of biosynthetic enzymes involved in sphingomyelin synthesis. The study also revealed that ABCA1 is important for cholesterol efflux and thus for counterbalancing the excess of intracellular free cholesterol in these snail-EMT cells. Inhibition of ACAT, an enzyme catalyzing cholesterol esterification, also seems essential to inhibit the growth of snail-expressing cancer cells.

However, It seems important to analyze the localization of ABCA1, as it is possible that in the event of cholesterol/sphingomyelin imbalance, for example, the intracellular trafficking of the pump may be altered.<br /> The authors should also analyze ACAT levels and/or activity in snail-EMT cells that should be increased. Overall, the provided data are important to better understand cancer biology.

Reviewer #1 (Public review):

Summary:

Howard et al. performed deep mutational scanning on the MC4R gene, using a reporter assay to investigate two distinct downstream pathways across multiple experimental conditions. They validated their findings with ClinVar data and previous studies. Additionally, they provided insights into the application of DMS results for personalized drug therapy and differential ligand responses across variant types.

Strengths:

They captured over 99% of variants with robust signals and investigated subtle functionalities, such as pathway-specific activities and interactions with different ligands, by refining both the experimental design and analytical methods.

Weaknesses:

While the study generated informative results, it lacks a detailed explanation regarding the input library, replicate correlation, and sequencing depth for a given number of cells. Additionally, there are several questions that it would be helpful for authors to clarify.

(1) It would be helpful to clarify the information regarding the quality of the input library and experimental replicates. Are variants evenly represented in the library? Additionally, have the authors considered using long-read sequencing to confirm the presence of a single intended variant per construct? Finally, could the authors provide details on the correlation between experimental replicates under each condition?

(2) Since the functional readout of variants is conducted through RNA sequencing, it seems crucial to sequence a sufficient number of cells with adequate sequencing saturation. Could the authors clarify the coverage depth used for each RNA-seq experiment and how this depth was determined? Additionally, how many cells were sequenced in each experiment?

(3) It appears that the frequencies of individual RNA-seq barcode variants were used as a proxy for MR4C activity. Would it be important to also normalize for heterogeneity in RNA-seq coverage across different cells in the experiment? Variability in cell representation (i.e., the distribution of variants across cells) could lead to misinterpretation of variant effects. For example, suppose barcode_a1 represents variant A and barcode_b1 represents variant B. If the RNA-seq results show 6 reads for barcode_a1 and 7 reads for barcode_b1, it might initially appear that both variants have similar effect sizes. However, if these reads correspond to 6 separate cells each containing 1 copy of barcode_a1, and only 1 cell containing 7 copies of barcode_b1, the interpretation changes significantly. Additionally, if certain variants occupy a larger proportion of the cell population, they are more likely to be overrepresented in RNA sequencing.

(4) Although the assay system appears to effectively represent MC4R functionality at the molecular level, we are curious about the potential disparity between the DMS score system and physiological relevance. How do variants reported in gnomAD distribute within the DMS scoring system?

(5) To measure Gq signaling, the authors used the GAL4-VPR relay system. Is there additional experimental data to support that this relay system accurately represents Gq signaling?

(6) Identifying the variants responsive to the corrector was impressive. However, we are curious about how the authors confirmed that the restoration of MC4R activity was due to the correction of the MC4R protein itself. Is there a possibility that the observed effect could be influenced by other factors affected by the corrector? When the corrector was applied to the cells, were any expected or unexpected differential gene expression changes observed?

(7) As mentioned in the introduction, gain-of-function (GoF) variants are known to be protective against obesity. It would be interesting to see further studies on the observed GoF variants. Do the authors have any plans for additional research on these variants?

Reviewer #1 (Public review):

The authors, Zhang et al., demonstrate the beneficial effects of treating degenerate human primary intervertebral disc (IVD) cells with recombinant human PDGF-AB/BB on the senescence transcriptomic signatures. Utilizing a combination of degenerate cells from elderly humans and experimentally induced senescence in young, healthy IVD cells, the authors show the therapeutic effects on mRNA transcription as well as cellular processes through informatics approaches.

One notable strength of this study is the use of human primary cells and recombinant forms of human PDGF-AB/BB proteins, which increases the translational potential of these in vitro studies. The manuscript is well-written, and the informatics analyses are thorough and clearly presented.

However, in its current form, the study does not provide sufficient experimental details, and clarifications are needed. These are as follows:

(1) The source of PDGF-AB/BB proteins is not detailed.<br /> (2) The irradiation parameters are not adequately reported - the authors should consider (PMCID: PMC5495460) for the parameters that should be reported.<br /> (3) The criteria for young and old patient donors are not explicitly described - though from the table, one presumes the cut-off for young is 27 years old.<br /> (4) What is the rationale for using different concentrations of PDGF-AB/BB in the degenerate cell and irradiation experiments?

There are also a number of other issues the authors could consider. First, in the title and throughout the manuscript, the effects of PDGF-AB/BB are described as protective, yet in all the experiments, PDGF-AB/BB appears to be administered following either in vivo degeneration or in vitro irradiation, where protective effects (e.g., administration prior to insult) were not tested. Therefore, the effects of PDGF-AB/BB may be more accurately described as mitigating or therapeutic rather than protective.

The authors state that the focus on NP (nucleus pulposus) cell studies is due to NP being the first site impacted during degeneration. However, this reviewer believes that this is because changes in the NP are more clinically evident (by imaging methods), despite degeneration often initiating from the AF (annulus fibrosus), e,g. through tears/microtears.

A prior study has examined the effects of X-ray irradiation on NF-kB signaling in young and aged IVDs (PMCID: PMC5495460), and the authors may wish to consider this work.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors discovered MYL3 of marine medaka (Oryzias melastigma) as a novel NNV entry receptor, elucidating its facilitation of RGNNV entry into host cells through macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 pathway.

Strengths:

In this manuscript, the authors have performed in vitro and in vivo experiments to prove that MnMYL3 may serve as a receptor for NNV via macropinocytosis pathway. These experiments with different methods include Co-IP, RNAi, pulldown, SPR, flow cytometry, immunofluorescence assays, and so on. In general, the results are clearly presented in the manuscript.

Weaknesses:

For the writing in the introduction and discussion sections, the author Yao et al mainly focus on the viral pathogens and fish in Aquaculture, the meaning and novelty of results provided in this manuscript are limited, and not broad in biology. The authors should improve the likely impact of their work on the viral infection field, maybe also in the evolutionary field with the fish model.

(1) Myosin is a big family, why did authors choose MYL3 as a candidate receptor for NNV?

(2) What is the relationship between MmMYL3 and MmHSP90ab1 and other known NNV receptors? Why does NNV have so many receptors? Which one is supposed to serve as the key entry receptor?

(3) In vivo knockout of MYL3 using CRISPR-Cas9 should be conducted to verify whether the absence of MYL3 really inhibits NNV infection. Although it might be difficult to do it in marine medaka as stated by the authors, the introduction of zebrafish is highly recommended, since it has already been reported that zebrafish could serve as a vertebrate model to study NNV (doi: 10.3389/fimmu.2022.863096).

(4) The results shown in Figure 6 are not enough to support the conclusion that "RGNNV triggers macropinocytosis mediated by MmMYL3". Additional electron microscopy of macropinosomes (sizes, morphological characteristics, etc.) will be more direct evidence.

(5) MYL3 is "predominantly found in muscle tissues, particularly the heart and skeletal muscles". However, NNV is a virus that mainly causes necrosis of nervous tissues (brain and retina). If MYL3 really acts as a receptor for NNV, how does it balance this difference so that nervous tissues, rather than muscle tissues, have the highest viral titers?

Reviewer #1 (Public review):

In this manuscript, Domingo et al. present a novel perturbation-based approach to experimentally modulate the dosage of genes in cell lines. Their approach is capable of gradually increasing and decreasing gene expression. The authors then use their approach to perturb three key transcription factors and measure the downstream effects on gene expression. Their analysis of the dosage response curve of downstream genes reveals marked non-linearity.

One of the strengths of this study is that many of the perturbations fall within the physiological range for each cis gene. This range is presumably between a single-copy state of heterozygous loss-of-function (log fold change of -1) and a three-copy state (log fold change of ~0.6). This is in contrast with CRISPRi or CRISPRa studies that attempt to maximize the effect of the perturbation, which may result in downstream effects that are not representative of physiological responses.

Another strength of the study is that various points along the dosage-response curve were assayed for each perturbed gene. This allowed the authors to effectively characterize the degree of linearity and monotonicity of each dosage-response relationship. Ultimately, the study revealed that many of these relationships are non-linear, and that the response to activation can be dramatically different than the response to inhibition.

To test their ability to gradually modulate dosage, the authors chose to measure three transcription factors and around 80 known downstream targets. As the authors themselves point out in their discussion about MYB, this biased sample of genes makes it unclear how this approach would generalize genome-wide. In addition, the data generated from this small sample of genes may not represent genome-wide patterns of dosage response. Nevertheless, this unique data set and approach represents a first step in understanding dosage-response relationships between genes.

Another point of general concern in such screens is the use of the immortalized K562 cell line. It is unclear how the biology of these cell lines translates to the in vivo biology of primary cells. However, the authors do follow up with cell-type-specific analyses (Figures 4B, 4C, and 5A) to draw a correspondence between their perturbation results and the relevant biology in primary cells and complex diseases.

The conclusions of the study are generally well supported with statistical analysis throughout the manuscript. As an example, the authors utilize well-known model selection methods to identify when there was evidence for non-linear dosage response relationships.

Gradual modulation of gene dosage is a useful approach to model physiological variation in dosage. Experimental perturbation screens that use CRISPR inhibition or activation often use guide RNAs targeting the transcription start site to maximize their effect on gene expression. Generating a physiological range of variation will allow others to better model physiological conditions.

There is broad interest in the field to identify gene regulatory networks using experimental perturbation approaches. The data from this study provides a good resource for such analytical approaches, especially since both inhibition and activation were tested. In addition, these data provide a nuanced, continuous representation of the relationship between effectors and downstream targets, which may play a role in the development of more rigorous regulatory networks.

Human geneticists often focus on loss-of-function variants, which represent natural knock-down experiments, to determine the role of a gene in the biology of a trait. This study demonstrates that dosage response relationships are often non-linear, meaning that the effect of a loss-of-function variant may not necessarily carry information about increases in gene dosage. For the field, this implies that others should continue to focus on both inhibition and activation to fully characterize the relationship between gene and trait.

Reviewer #1 (Public review):

Summary:

In this paper, Blancke Soares and Stäcker et al serendipitously identify a domain of the Plasmodium falciparum protein MSRP6 that mediates both export from the parasite into the infected red blood cell and association with the Maurer's cleft organelles found in the infected cell. The authors use this domain to identify a putative complex of proteins at the Maurer's cleft via proximity biotinylation. Six members of the complex are confirmed to interact with MSRP6 by co-immunoprecipitation.

The functions of select proteins of this complex are further investigated with regard to the formation of Maurer's clefts. Disruption of PeMP2, PIESP2, and Pf332 resulted in morphological changes to the Maurer's clefts and prevented the anchoring of the Maurer's clefts to the infected red blood cell plasma membrane that normally occurs in the trophozoite stage. Curiously, disruption of MSRP6, the central member of the complex, did not affect Maurer's cleft anchoring. Mechanistically, how this complex affects Maurer's cleft structure and anchoring remains unclear.

Finally, the authors show that the loss of Maurer's cleft anchoring observed upon disruption of PIESP2 or Pf332 does not affect cytoadherence of infected red blood cells via PfEMP1, arguing against a prior assumption that cleft tethering is required for the presentation of parasite-exported proteins on the infected red blood cell surface.

Strengths:

Maurer's clefts are enigmatic organelles found in red blood cells infected by Plasmodium falciparum that are presumed to play a role in trafficking exported parasite proteins to the surface of the red blood cells, though little is known about their biogenesis and function. The authors here convincingly identify a protein complex present at the Maurer's clefts using multiple orthogonal tools, and carry out assays that indicate this protein complex has a role in shaping and anchoring the Maurer's clefts at their final location at the red blood cell membrane. The data indicating that Maurer's cleft anchoring is dispensable for trafficking of P. falciparum exported proteins to the infected red blood cell membrane has implications for understanding the function of this organelle.

Weaknesses:

In many instances, the data lack appropriate controls that would be desirable for the highest level of rigor. Many, if not most, fluorescence microscopy assays lack untagged/parental controls (prepared in parallel and captured with the same settings) that are necessary to determine the validity of the data - that the observed signal is specific to the protein of interest and not due to autofluorescence or bleed-through from other channels. In other cases, wild-type controls are missing where data from disruption mutants are presented. Additionally, while some phenotypes are quantified, others are only qualitatively described where a more thorough quantitative investigation would be valuable. Finally, where phenotypes have been quantified, in many instances it is not clear that the analyses have included biological replicates as would be expected.

Joint Public Review:

Previously, this group showed that Tgfbr1 regulates the reorganization of the epiblast and primitive streak into the chordo-neural hinge and tailbud during the trunk-to-tail transition. Gdf11 signaling plays a crucial role in orchestrating the transition from trunk to tail tissues in vertebrate embryos, including the reallocation of axial progenitors into the tailbud and Tgfbr1 plays a key role in mediating its signaling activity. Progenitors that contribute to the extension of the neural tube and paraxial mesoderm into the tail are located in this region. In this work, the authors show that Tgfbr1 also regulates the reorganization of the posterior primitive streak/base of allantois and the endoderm as well.

By analyzing the morphological phenotypes and marker gene expression in Tgfbr1 mutant mouse embryos, they show that it regulates the merger of somatic and splanchnic layers of the lateral plate mesoderm, the posterior streak derivative. They also present evidence suggesting that Tgfbr1 acts upstream of Isl1 (key effector of Gdf11 signaling for controlling differentiation of lateral mesoderm progenitors) and regulates the remodelling of the major blood vessels, the lateral plate mesoderm and endoderm associated with the trunk-to-tail transition. Through a detailed phenotypic analysis, the authors observed that, similarly to Isl1 mutants, the lack of Tgfbr1 in mouse embryos hinders the activation of hindlimb and external genitalia maker genes and results in a failure of lateral plate mesoderm layers to converge during tail development. As a result, they interpret that ventral lateral mesoderm, which generates the peri cloacal mesenchyme and genital tuberculum, fails to specify.

They also show defects in the morphogenesis of the dorsal aorta at the trunk/tail juncture, resulting in an aberrant embryonic/extraembryonic vascular connection. Endoderm reorganization defects following abnormal morphogenesis of the gut tube in the Tgfbr1 mutants cause failure of tailgut formation and cloacal enlargement. Thus, Tgfbr1 activity regulates the morphogenesis of the trunk/tail junction and the morphogenetic switch in all germ layers required for continuing post-anal tail development. Taken together with the previous studies, this work places Gdf11/8 - Tgfbr1 signaling at the pivot of trunk-to-tail transition and the authors speculate that critical signaling through Tgfbr1 occurs in the posterior-most part of the caudal epiblast, close to the allantois.

The data shown is solid with excellent embryology/developmental biology. This work demonstrates meticulous execution and is presented in a comprehensive and coherent manner. Although not completely novel, the results/conclusions add to the known function of Gdf11 signaling during the trunk-to-tail transition.

Reviewer #1 (Public review):

Summary:

The authors investigate ligand and protein-binding processes in GPCRs (including dimerization) by the multiple walker supervised molecular dynamics method. The paper is interesting and it is very well written.

Strengths:

The authors' method is a powerful tool to gain insight on the structural basis for the pharmacology of G protein-coupled receptors.

Reviewer #1 (Public review):

Summary:

The authors describe that the endocytic pathway is crucial for ColI fibrillogenesis. ColI is endocytosed by fibroblasts, prior to exocytosis and formation of fibrils, which can include a mixture of endogenous/nascent ColI chains and exogenous ColI. ColI uptake and fibrillogenesis are regulated by circadian rhythm as described by the authors in 2020, thanks to the dependence of this pathway on circadian-clock-regulated protein VPS33B. Cells are capable of forming fibrils with recently endocytosed ColI along when nascent chains are not available. Previously identified VPS33B is demonstrated not to have a role in endocytosis of ColI, but to play a role in fibril formation, which the authors demonstrate by showing the loss of fibril formation in VPS33B KO, and an excess of insoluble fibrils - along-side a decrease in soluble ColI secretion - in VPS33B overexpression conditions. A VPS33B binding protein VIPAS39 is also shown to be required for fibrillogenesis and to colocalise with ColI. The authors thus conclude that ColI is internalised into endosomal structures within the cell, and that ColI, VPS33B and VIPA39 are co-trafficked to the site of fibrillogenesis, where along with ITGA11, which by mass spectrometric analysis is shown to be regulated by VPS33B levels, ColI fibrils are formed. Interestingly, in involved human skin sections from idiopathic pulmonary fibrosis (IPF) patients, ITGA11 and VPS33B expression is increased compared to healthy tissue, while in patient-derived fibroblasts, uptake of fluorescently-labelled ColI is also increased. This suggests that there may be a significant contribution of endocytosis-dependent fibrillogenesis in the formation of fibrotic and chronic wound-healing diseases in humans.

Strengths:

This is an interesting paper that contributes an exciting novel understanding of the formation of fibrotic disease, which despite its high occurrence, still has no robust therapeutic options. The precise mechanisms of fibrillogenesis are also not well understood, so a study devoted to this complex and key mechanism is well appreciated. The dependence of fibrillogenesis on VPS33B and VIPA39 is convincing and robust, while the distinction between soluble ColI secretion and insoluble fibrillar ColI is interesting and informative.

Weaknesses:

There are a number of limitations to this study in its current state. Inhibition of ColI uptake is performed using Dyngo4a, which although proposed as an inhibitor of Clathrin-dependent endocytosis is known to be quite un-specific. This may not be a problem however, as the endocytic mechanism for ColI also does not seem to be well defined in the literature, in fact, the principle mechanism described in the papers referred to by the authors is that of phagocytosis. It would be interesting to explore this important part of the mechanism further, especially in relation to the intracellular destination of ColI. The circadian regulation does not appear as robust as the authors last paper, however, there could be a larger lag between endocytosis of ColI and realisation of fibrils. The authors state that the endocytic pathway is the mechanism of trafficking and that they show ColI, VPS33B and VIPA39 are co-trafficked. However, the only link that is put forward to the endosomes is rather tenuously through VPS33B/VIPA39. There is no direct demonstration of ColI localisation to endosomes (ie. immunofluorescence), and this is overstated throughout the text. Demonstrating the intracellular trafficking and localisation of ColI, and its actual relationship to VPS33B and VIPA39, followed by ITGA11, would broaden the relevance of this paper significantly to incorporate the field of protein trafficking. Finally, the "self-formation" of ColI fibrils is discussed in relation to the literature and the concentration of fluorescently-tagged ColI, however as the key message of the paper is the fibrillogenesis from exocytosed colI, I do not feel like it is demonstrated to leave no doubt. Specific inhibition of intracellular trafficking steps, or following the progressive formation of ColI fibrils over time by immunofluorescence would demonstrate without any further doubt that ColI must be endocytosed first, to form fibrils as a secondary step, rather than externally-added ColI being incorporated directly to fibrils, independent of cellular uptake.

Joint Public Review:

Summary:

This work provides a new general tool for predicting post-ERCP pancreatitis before the procedure depending on pancreatic calcification, female sex, intraductal papillary mucinous neoplasm, a native papilla of Vater, or the use of pancreatic duct procedures. Even though it is difficult for the endoscopist to predict before the procedure which case might have post-ERCP pancreatitis, this new model score can help with the maneuver and when the patient is at high risk of pancreatitis, sometimes can be deadly), so experienced endoscopists can do the procedure from the start. This paper provides a model for stratifying patients before the ERCP procedure into low, moderate, and high risk for pancreatitis. To be validated, this score should be done in many countries and on large numbers of patients. Risk factors can also be identified and added to the score to increase rank.

Strengths:

(1) One of the severe complications of endoscopic retrograde cholangiopancreatography procedure is pancreatitis, so investigators try all the time to find a score that can predict which patients will probably have pancreatitis after the procedure. Most scores depend on the intraprocedural maneuver. Some studies discuss the preprocedural score that can predict pancreatitis before the procure. This study discusses a new preprocedural score for post-ERCP pancreatitis.

(2) Depending on this score that identifies low, moderate, and high-risk patients for post-pancreatitis, so from the start, experienced and well-trained endoscopists can do the procedure or can refer patients to tertiary hospitals or use interventional radiology or endoscopic retrograde cholangiopancreatography.

(3) The number of patients in this study is sufficient to analyze data correctly.

Weaknesses:

(1) It is a single-country, retrospective study.

(2) Many cases were excluded, so the score cannot be applied to those patients.

Comments on revised version:

Depending on old references cannot help us know the current situation. What if there are better more recent predictive tools? It would be better to test the validity of that score against, if present, a proven score to check its validity.

for - Great Separations, Three Great Separations, alienation, alienation - industrial revolution, John Ikerd, 3 separation - from - Post Capital Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - Title - The Three “Great Separations” that Unravelled Our Connection to Earth and Each Other - Author - John Kkerd

from - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - https://hyp.is/kSvpDre2Ee-CsF-EO4pwTg/www.youtube.com/watch?v=dk6F4IlEbAk

Reviewer #1 (Public review):

Summary

The authors determine the phylogenetic relation of the roughly two dozen wtf elements of 21 S. pombe isolates and show that none of them in the original S. pombe are essential for robust mitotic growth. It would be interesting to test their meiotic function by simply crossing each deletion mutant with the parent and analyzing spores for non-Mendelian inheritance. If this has been reported already, that information should be added to the manuscript. If not, I suggest the authors do these simple experiments and add this information.

Strengths:

The most interesting data (Figure 4) show that one recombinant (wtfC4) between wtf18 and wtf23 produces in mitotic growth a poison counteracted by its own antidote but not by the parental antidotes. Again, it would be interesting to test this recombinant in a more natural setting - meiosis between it and each of the parents.

Weaknesses:

In the opinion of this reviewer, some minor rewriting is needed.

Reviewer #1 (Public review):

In the manuscript Cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and gates rapid phase shifts of the circadian clock Brenna et al., study the role of Cdk5 on circadian rhythms, the authors aim to elucidate the role of Cyclin-Dependent Kinase 5 (Cdk5) in modulating circadian rhythms, particularly in response to light cues. They hypothesized that Cdk5 acts as a gatekeeper, regulating the sensitivity of the circadian clock to light-induced phase shifts.

Strengths:

• Novelty: The study presents a novel mechanism by which Cdk5 influences circadian rhythms, particularly its role in modulating the light-induced phase-shifting response.<br /> • Experiments: The authors have employed a combination of molecular, cellular, and behavioural techniques, including genetic manipulations, biochemical assays, and electrophysiology, to investigate the role of Cdk5. The set of experiments performed in this work is non-trivial, done to a high standard and the additional experiments, data and textual alterations presented following the 1st round of review needs to be lauded.<br /> • Data: The data is well-presented in clear figures and appropriately described in the text.

Weaknesses:

• Although I found the data on Cdk5 gating light responses highly convincing there could be additional mechanisms which the authors have duly acknowledged and discussed in their text.<br /> In my assessment, the authors have convincingly demonstrated that Cdk5 plays a critical role in gating the light-induced phase-shifting response of the circadian clock. Their results strongly support their conclusions, as evidenced by their findings:<br /> This study provides valuable insights into the molecular mechanisms underlying circadian rhythm regulation and the impact of light on the circadian clock. The findings have the potential to influence future research in the field of chronobiology and may have implications for understanding and treating circadian rhythm disorders.<br /> The methods and data presented in this study are valuable to the field and can be used to further investigate the role of Cdk5 and other signalling pathways in circadian rhythm regulation.<br /> Broader context<br /> The circadian clock is a fundamental biological process that regulates various physiological functions, including sleep-wake cycles, hormone secretion, and metabolism. Disruptions to the circadian clock have been linked to a variety of health problems, such as sleep disorders, metabolic disorders, and cancer. Understanding the molecular mechanisms that underlie circadian rhythm regulation is essential for developing effective treatments for these disorders.

All in all, I have no reservations regarding the manuscript titled "Cyclin-dependent kinase 5 (Cdk5) activity is modulated by light and gates rapid phase shifts of the circadian clock by Brenna et al. After consideration of the authors' revisions, I believe the manuscript has been significantly improved. I commend the authors for their diligence in addressing the reviewers' comments and for the quality of their research.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors train mice on a two-armed bandit task, in which the reward value associated with the arms suddenly switches in a pseudorandom fashion. Their first finding is that the mice are able to anticipate the reward value switch points after long blocks, evident both prior to the switch point with higher rates of switching to the less-rewarded arm, and after the switch point with faster transition to the more-rewarded arm. They next find that unilateral ACAd/MO lesion / optogenetic silencing (surprisingly) causes greater anticipation of reward switch points, both prior to and after the switch point. They use behavioral modeling to argue that the unilateral ACAd/MO lesion effects are due to an increase in the contralateral hazard rate. Finally, they found that bilateral lesions did not have any effect on the hazard rate, suggesting that the unilateral lesion effect is due to balancing between hemispheres. This manuscript employed a clever behavioral design and analysis approach, though the effects were somewhat difficult to interpret and the author's interpretation relies heavily on the accuracy of their underlying behavioral model.

Strengths:

This paper employs a well-designed task that allows the researchers to detect whether mice have noticed a change in reward value both before and after the change takes place. The use of unilateral and bilateral inactivation experiments allowed the authors to test the role of the ACAd/MO region in the change point estimation. They found that unilateral inactivation, but not bilateral inactivation, had a significant effect on behavior. They performed sophisticated behavioral analysis to determine how ACAd/MO perturbations affect decision-making variables. This topic is of interest to the field, and the results are presented clearly and generally convincing.

Weaknesses:

The observed effects of the lesions are somewhat counterintuitive, with lesions appearing to affect persistence within a block more than change point detection itself-the mice actually adjusted more quickly to changes in reward values. Moreover, they had no issue detecting change points after bilateral inactivation. As a result, I'm not sure if the main framing of the article (including the title) is supported by their findings. Finally, I was unsure how the differences between unilateral and bilateral inactivation could be explained by their behavioral model.

Reviewer #1 (Public review):

Of course, there is always another layer of the onion, VAMP-seq measures contributions from isolated thermodynamic stability, stability conferred by binding partners (small molecule and protein), synthesis/degradation balance (especially important in "degron" motifs), etc. Here the authors' goal is to create simple models that can act as a baseline for two main reasons:<br /> (1) how to tell when adding more information would be helpful for a global model;<br /> (2) how to detect when a residue/mutation has an unusual profile indicative of an unbalanced contribution from one of the factors listed above.

As such, the authors state that this manuscript is not intended to be a state-of-the-art method in variant effect prediction, but rather a direction towards considering static structural information for the VAMP-seq effects. At its core, the method is a fairly traditional asymmetric substitution matrix (I was surprised not to see a comparison to BLOSUM in the manuscript) - and shows that a subdivision by burial makes the model much more predictive. Despite only having 6 datasets, they show predictive power even when the matrices are based on a smaller number. Another success is rationalizing the VAMPseq results on relevant oligomeric states.

Specific Feedback:

Major points:

The authors spend a good amount of space discussing how the six datasets have different distributions in abundance scores. After the development of their model is there more to say about why? Is there something that can be leveraged here to design maximally informative experiments?

They compare to one more "sophisticated model" - RosettaddG - which should be more correlated with thermodynamic stability than other factors measured by VAMP-seq. However, the direct head-to-head comparison between their matrices and ddG is underdeveloped. How can this be used to dissect cases where thermodynamics are not contributing to specific substitution patterns OR in specific residues/regions that are predicted by one method better than the other? This would naturally dovetail into whether there is orthogonal information between these two that could be leveraged to create better predictions.

Perhaps beyond the scope of this baseline method, there is also ThermoMPNN and the work from Gabe Rocklin to consider as other approaches that should be more correlated only with thermodynamics.

I find myself drawn to the hints of a larger idea that outliers to this model can be helpful in identifying specific aspects of proteostasis. The discussion of S109 is great in this respect, but I can't help but feel there is more to be mined from Figure S9 or other analyses of outlier higher than predicted abundance along linear or tertiary motifs.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate the cellular mechanism underlying suppression of adrenergic effects on excitatory transmission onto hypothalamic CRH neurons by stress. Experiments in ex-vivo slices show that this is a long-lasting effect that occurs through endocytosis of receptors. The authors then move into an immortalized hypothalamic cell line to enable investigation of the mechanism of changes in receptor trafficking. They use a series of immunohistochemistry, FRET, and biochemical experiments to show that application of corticosterone increases targeting of alpha1 adrenergic receptors to the late endosome and lysosome rather than the recycling endosome. Perhaps most interesting, they find that alpha1 receptors and glucocortioid receptors form a complex that is ultimately transferred to the nucleus.

Strengths:

Overall, the studies in this manuscript are rigorous and well-conducted. The data supports their conclusions, and they've shown convincingly that glucocorticoid signaling affects trafficking of alpha1 receptors in the culture system they are using. These findings are important for the field of stress research, both in understanding how two components of the stress system (norepinephrine and HPA axis) interact with each other and in neuromodulatory modulation of hypothalamic CRH neurons. Their finding that alpha1 receptors and glucocorticoid receptors form a complex is particularly interesting and maybe impactful outside of the immediate application in the hypothalamus.

Weaknesses:

The study has two primary weaknesses. First, the majority of the experiments were conducted in an immortalized hypothalamic cell line. This was necessary to conduct the type of experiments needed to test the author's hypothesis, but it remains unclear how closely these cells resemble CRH neurons, or how the same mechanism may be preserved or altered in an intact circuit. Further discussion of these points would strengthen the manuscript.<br /> Second, while experiments are generally well-designed, the authors do not show that the effects of corticosterone can be blocked with a glucocorticoid receptor antagonist. This is fairly standard pharmacology and would strengthen confidence in the findings presented in the study.

Reviewer #1 (Public Review):

The authors were curious about the formation of the electrosensory lateral line, which is found in non-traditional model organisms. This issue has traditionally hampered studies because those organisms are not amenable to controlled experimental work.

The authors skillfully use CRIPR-based technologies to overcome this limitation. Together with exceptionally good whole-mount in situ hybridisation, they produced a well-supported conclusion that Bmp signalling has different roles in the development of electrosensory ampullary organs.

I would not entirely agree that Bmp signalling has "opposing" roles because the authors do not show evidence of opposition via gain-of-function experiments at different developmental times. Instead, they are simply different at different periods of organogenesis.

The study is important for understanding the development of a still-mysterious sensory system, and for its implications in evolutionary biology more generally.

Reviewer #1 (Public review):

Summary:

"Neural noise", here operationalized as an imbalance between excitatory and inhibitory neural activity, has been posited as a core cause of developmental dyslexia, a prevalent learning disability that impacts reading accuracy and fluency. This is study is the first to systematically evaluate the neural noise hypothesis of dyslexia. Neural noise was measured using neurophysiological (electroencephalography [EEG]) and neurochemical (magnetic resonance spectroscopy [MRS]) in adolescents and young adults with and without dyslexia. The authors did not find evidence of elevated neural noise in the dyslexia group from EEG or MRS measures, and Bayes factors generally informed against including the grouping factor in the models. Although the comparisons between groups with and without dyslexia did not support the neural noise hypothesis, a mediation model that quantified phonological processing and reading abilities continuously revealed that EEG beta power in the left superior temporal sulcus was positively associated with reading ability via phonological awareness. This finding lends support for analysis of associations between neural excitatory/inhibitory factors and reading ability along a continuum, rather than as with a case/control approach, and indicates the relevance of phonological awareness as an intermediate trait that may provide a more proximal link between neurobiology and reading ability. Further research is needed across developmental stages and over a broader set of brain regions to more comprehensively assess the neural noise hypothesis of dyslexia, and alternative neurobiological mechanisms of this disorder should be explored.

Strengths:

The inclusion of multiple methods of assessing neural noise (neurophysiological and neurochemical) is a major advantage of this paper. MRS at 7T confers an advantage of more accurately distinguishing and quantifying glutamate, which is a primary target of this study. In addition, the subject-specific functional localization of the MRS acquisition is an innovative approach. MRS acquisition and processing details are noted in the supplementary materials using according to the experts' consensus recommended checklist (https://doi.org/10.1002/nbm.4484). Commenting on rigor the EEG methods is beyond my expertise as a reviewer.<br /> Participants recruited for this study included those with a clinical diagnosis of dyslexia, which strengthens confidence in the accuracy of the diagnosis. The assessment of reading and language abilities during the study further confirms the persistently poorer performance of the dyslexia group compared to the control group.<br /> The correlational analysis and mediation analysis provide complementary information to the main case-control analyses, and the examination of associations between EEG and MRS measures of neural noise is novel and interesting.<br /> The authors follow good practice for open science, including data and code sharing. They also apply statistical rigor, using Bayes Factors to support conclusions of null evidence rather than relying only on non-significant findings. In the discussion, they acknowledge the limitations and generalizability of the evidence and provide directions for future research on this topic.

Weaknesses:

Though the methods employed in the paper are generally strong, the MRS acquisition was not optimized to quantify GABA, so the findings (or lack thereof) should be interpreted with caution. Specifically, while 7T MRS affords the benefit of quantifying metabolites, such as GABA, without spectral editing, this quantification is best achieved with echo times (TE) of 68 or 80 ms in order to minimize the spectral overlap between glutamate and GABA and reduce contamination from the macromolecular signal (Finkelman et al., 2022, https://doi.org/10.1016/j.neuroimage.2021.118810). The data in the present study were acquired at TE=28 ms, and are therefore likely affected by overlapping Glu and GABA peaks at 2.3 ppm that are much more difficult to resolve at this short TE, which could directly affect the measures that are meant to characterize the Glu/GABA+ ratio/imbalance. In future research, MRS acquisition schemes should be optimized for the acquisition of Glutamate, GABA, and their relative balance.

As the authors note in the discussion, additional factors such as MRS voxel location, participant age, and participant sex could influence associations between neural noise and reading abilities and should be considered in future studies.

Appraisal:

The authors present a thorough evaluation of the neural noise hypothesis of developmental dyslexia in a sample of adolescents and young adults using multiple methods of measuring excitatory/inhibitory imbalances as an indicator of neural noise. The authors concluded that there was not support for the neural noise hypothesis of dyslexia in their study based on null significance and Bayes factors. This conclusion is justified, and further research is called for to more broadly evaluate the neural noise hypothesis in developmental dyslexia.

Impact:

This study provides an exemplar foundation for the evaluation of the neural noise hypothesis of dyslexia. Other researcher may adopt the model applied in this paper to examine neural noise in various populations with/without dyslexia, or across a continuum of reading abilities, to more thoroughly examine evidence (or lack thereof) for this hypothesis. Notably, the lack of evidence here does not rule out the possibility for a role of neural noise in dyslexia, and the authors point out that presentation with co-occurring conditions, such as ADHD, may contribute to neural noise in dyslexia. Dyslexia remains a multi-faceted and heterogenous neurodevelopmental condition, and many genetic, neurobiological and environmental factors play a role. This study demonstrates one step toward evaluating neurobiological mechanisms that may contribute to reading difficulties.

Reviewer #1 (Public review):

Summary:

This study focuses on metabolic changes in the paraventricular hypothalamic (PVH) region of the brain during acute periods of cold exposure. The authors point out that in comparison to the extensive literature on the effects of cold exposure in peripheral tissues, we know relatively little about its effects on the brain. They specifically focus on the hypothalamus, and identify the PVH as having changes in Atgl and Hsl gene expression changes during cold exposure. They then go on to show accumulation of lipid droplets, increased Fos expression, and increased lipid peroxidation during cold exposure. Further, they show that neuronal activation is required for the formation of lipid droplets and lipid peroxidation.

Strengths:

A strength of the study is trying to better understand how metabolism in the brain is a dynamic process, much like how it has been viewed in other organs. The authors also use a creative approach to measuring in vivo lipid peroxidation via delivery of BD-C11 sensor through a cannula to the region in conjunction with fiber photometry to measure fluorescence changes deep in the brain.

Comments on revised version:

The authors have attempted to address concerns brought to their attention in the initial review. They have performed one or two additional experiments to address concerns (e.g. adding fiber photometry of PVH neurons and trying to manipulate lipid peroxidation) though many of the concerns from the original review stand. The authors have also revised the text to limit the extent of their claims and to improve clarity, which is appreciated.

Reviewer #1 (Public review):

Summary:

This manuscript addresses the question of whether spontaneous activity contributes to the clustering of retinogeniculate synapses before eye opening. The authors re-analyze a previously published dataset to answer the question. The authors conclude that synaptic clustering is eye-specific and activity dependent during the first postnatal week. While there is useful information in this manuscript, I don't see how the data meaningfully supports the claims made about clustering.<br /> In adult retinogeniculate connections, functionally specificity is supported by select pairings of retinal ganglion cells and thalamocortical cells forming dozens of synaptic connections in subcellular microcircuits called glomeruli. In this manuscript, the authors measure whether the frequency of nearby synapses is higher in the observed data than in a model where synapses are randomly distributed throughout the volume. Any real anatomical data will deviate from such a model. The interesting biological question is not whether a developmental state deviates from random. The interesting question is how much of the adult clustering occurs before eye opening. In trying to decode the analysis in this manuscript, I can't tell if the answer is 99% or 0.001%.

Strengths:

The source dataset is high resolution data showing the colocalization of multiple synaptic proteins across development. Added to this data is labeling that distinguishes axons from the right eye from axons from the left eye. The first order analysis of this data showing changes in synapse density and in the occurrence of multi-active zone synapses is useful information about the development of an important model system.

Weaknesses:

I don't think the analysis of clustering within this dataset improves our understanding of how the system works. It is possible that the result is clear to the authors based on looking at the images. As a reader trying to interpret the analysis, I ran into the following problems:

• It is not possible to estimate biologically meaningful effect sizes from the data provided. Spontaneous activity in the post natal week could be responsible for 99% or 0.001% of RGC synapse clustering.<br /> • There is no clear biological interpretation of the core measure of the publication, the normalized clustering index. The normalized clustering index starts with counting the fraction of single active zone synapses within various distances to the edge of synapses. This frequency is compared to a randomization model in which the positions of synapses are randomized throughout a volume. The authors found that the biggest deviation between the observed and randomized proximity frequency using a distance threshold of 1.5 um. They consider the deviation from the random model to be a sign of clustering. However, two RGC synapses 1.5 um apart have a good chance of coming from the same RGC axon. At this scale, real observations will, therefore, always look more clustered than a model where synapses are randomly placed in a volume. If you randomly place synapses on an axon, they will be much closer together than if you randomly place synapses within a volume. The authors normalize their clustering measure by dividing by the frequency of clustering in the normalized model. That makes the measure of clustering an ambiguous mix of synapse clustering, axon morphology, and synaptic density.<br /> • Other measures are also very derived. For instance, one argument is based on determining that the cumulative distribution of the distance of dominant-eye multi-active zone synapses with nearby single-active zone synapses from dominant-eye multi-active zone synapses is statistically different from the cumulative distribution of the distance of dominant-eye multi-active zones without nearby single-active zone synapses from dominant-eye multi-active zones. Multiple permutations of this measure are compared.<br /> • The sample size is too small for the kinds of comparisons being made. The authors point out that many STORM studies use an n of 1 while the authors have n = 3 for each of their six experimental groups. However, the critical bit is what kinds of questions you are trying to answer with a given sample size. This study depends on determining whether the differences between groups are due to age, genotype, or individual variation. This study also makes multiple comparisons of many different noisy parameters that test the same or similar hypothesis. In this context, it is unlikely that n = 3 sufficiently controls for individual variation.<br /> • There are major biological differences between groups that are difficult to control for. Between P2, P4, and P8, there are changes in cell morphology and synaptic density. There are also large differences in synapse density between wild type and KO mice. It is difficult to be confident that these differences are not responsible for the relatively subtle changes in clustering indices.<br /> • Many claims are based on complicated comparisons between groups rather than the predominating effects within the data. It is noted that: "In KO mice, dominant eye projections showed increased clustering around mAZ synapses compared to sAC synapses suggesting partial maintenance of synaptic clustering despite retinal wave defects". In contrast, I did not notice any discussion of the fact that the most striking trend in those measures is that the clustering index decreases from P2 to P8.<br /> • Statistics are improperly applied. In my first review I tried to push the authors to calculate confidence intervals for two reasons. First, I believed the reader should be able to answer questions such as whether 99% or 0.01% of RGC synaptic clustering occurred in the first postnatal week. Second, I wanted the authors to deal with the fact that n=3 is underpowered for many of the questions they were asking. While many confidence intervals can now be found leading up to a claim, it is difficult to find claims that are directly supported by the correct confidence interval. Many claims are still incorrectly based on which combinations of comparisons produced statistically significant differences and which combinations did not.

Joint Public Review:

Though the Norrin protein is structurally unrelated to the Wnt ligands, it can activate the Wnt/β-catenin pathway by binding to the canonical Wnt receptors Fzd4 and Lrp5/6, as well as the tetraspanin Tspan12 co-receptor. Understanding the biochemical mechanisms by which Norrin engages Tspan12 to initiate signaling is important, as this pathway plays an important role in regulating retinal angiogenesis and maintaining the blood-retina-barrier. Numerous mutations in this signaling pathway have also been found in human patients with ocular diseases. The overarching goal of the study is to define the biochemical mechanisms by which Tspan12 mediates Norrin signaling. Using purified Tspan12 reconstituted in lipid nanodiscs, the authors conducted detailed binding experiments to document the direct, high-affinity interactions between purified Tspan12 and Norrin. To further model this binding event, they used AlphaFold to dock Norrin and Tspan12 and identified four putative binding sites. They went on to validate these sites through mutagenesis experiments. Using the information obtained from the AlphaFold modeling and through additional binding competition experiments, it was further demonstrated that Tspan12 and Fzd4 can bind Norrin simultaneously, but Tspan12 binding to Norrin is competitive with other known co-receptors, such as HSPGs and Lrp5/6. Collectively, the authors proposed that the main function of Tspan12 is to capture low concentrations of Norrin at the early stage of signaling, and then "hand over" Norrin to Fzd4 and Lrp5/6 for further signal propagation. Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

Numerous pathways have been proposed to elucidate the nongenomic actions of progesterone within both male and female reproductive tissues. The authors employed the Xenopus oocyte system to investigate the PLA2 activity of ABHD2 and the downstream lipid mediators in conjunction with mPRb and P4, on their significance in meiosis. The research has been conducted extensively and is presented clearly.

Strengths:

While the interaction between membranous PR and ABHD2 is not a novel concept, this present study exhibits several strengths:

(1) mPRbeta, a member of the PAQR family, has been elusive in terms of detailed signal transduction. Through mutation studies involving the Zn binding domain, the authors discovered that the hydrolase activity of mPRbeta is not essential for meiosis and oocyte maturation. Instead, they suggest that ABHD2, acting as a coreceptor of mPRbeta, demonstrates phospholipase activity, indicating that downstream lipid mediators may play a dominant role when stimulated by progesterone.<br /> (2) Extensive exploration of downstream signaling pathways and the identification of several potential meiotic activity-related lipid mediators make this aspect of the study novel and potentially significant.

Weaknesses:

However, there are some weaknesses and areas that need further clarification:

(1) The mechanism governing the molecular assembly of mPRbeta and ABHD2 remains unclear. Are they constitutively associated or is their association ligand-dependent? Does P4 bind not only to mPRbeta but also to ABHD2, as indicated in Figure 6J? In the latter case, the reviewer suggests that the authors conduct a binding experiment using labeled P4 with ABHD2 to confirm this interaction and assess any potential positive or negative cooperativity with a partner receptor.

(2) The authors have diligently determined the metabolite profile using numerous egg cells. However, the interpretation of the results appears incomplete, and inconsistencies were noted between Figure 2F and Supplementary Figure 2C. Furthermore, PGE2 and D2 serve distinct roles and have different elution patterns by LC-MS/MS, thus requiring separate measurements. In addition, the extremely short half-life of PGI2 necessitates the measurement of its stable metabolite, 6-keto-PGF1a, instead. The authors also need to clarify why they measured PGF1a but not PGF2a. Unfortunately, even in the revision, authors did not adequately address the last issue (differential measurements of PGD2 and E2, 6-keto-PG!alpha be determined instead of PGI2).

(3) Although they propose PGs, LPA and S1P are important downstream mediators, the exact roles of the identified lipid mediators have not been clearly demonstrated, as receptor expression and activation were not demonstrated. While the authors showed S1PR3 expression and its importance by genetic manipulation, there was no observed change in S1P levels following P4 treatment (Supplementary Figure 2D). It is essential to identify which receptors (subtypes) are expressed and how downstream signaling pathways (PKA, Ca, MAPK, etc.) relate to oocyte phenotypes.

These clarifications and further experiments would enhance the overall impact and comprehensiveness of the study.

Comments on revisions:

Need correction and addition for differential analyses of PGD2 and PGE2, and measurement of 6-keto-PGF1alpha instead of PGI2 (Figure 2F). PGI2 is extremely unstable (T1/2, 1 min in neutral buffer) and rapidly converted nonenzymically to 6-keto-PGF1a.

Reviewer #1 (Public review):

Summary:

This study is focused an important aspect of axon guidance at the central nervous system (CNS) midline: how neurons extend axons that either do or do not cross the CNS midline. The authors here address contradictory work in the field relating to how cell surface expression of the slit receptor Robo1 is regulated so as to generate crossed and non-crossed axon trajectories during Drosophila neural development. They use fly genetics, cell lines, and biochemical assessments to define a complex consisting of the commissureless, Nedd4 and Robo1 proteins necessary for regulating Robo1 protein expression. This work resolves certain remaining questions in the field regarding midline axon guidance, with strengths out weighing weaknesses; however, addressing some of these weaknesses would strengthen this study.

Strengths:

Strengths include:<br /> - The use of well controlled genetic gain-of-function (over expression) approaches in vivo in Drosophila to show that phosphorylation sites (there are 2, and this study allows for assessment of the contributions made by each) in the commissureless (Comm) protein are indeed required for Comm function with respect to regulating axon midline guidance via their role in directing Comm-mediated Robo1 ubiquitination and degradation in the lysosome.<br /> - The demonstration that in vitro, and in a sensitized genetic background in vivo, the Nedd4 ubiquitin ligase regulates Robo1 protein cell surface distribution and also midline axon crossing in vivo.<br /> - Important evidence here that serves to resolve many questions raised by previous studies (not from these authors) regarding how Robo1 is regulated by Comm and Nedd4 family ubiquitin ligases. Further, these results are likely to have implications for thinking about the regulation of midline guidance in more complex nervous systems.

Weaknesses:

- A weakness beyond the purview of revision but important to mention is that the authors chose not to complement their GOF experiments with gene editing approaches to generate endogenous PY mutant alleles of Comm that might have been useful in genetic interaction experiments directed toward revealing roles for endogenous Comm in the regulation of Robo1.

Comments on revised version:

In this revised manuscript the authors provide new experiments and also reasonable explanations to address concerns raised in the initial review. I am satisfied that these efforts address satisfactorily the points raised in the initial review and that this study has been strengthened. This is an interesting body of work that adds to our understanding of CNS midline guidance molecular mechanisms.

Reviewer #1 (Public review):

This work addresses an important question in the field of Drosophila aggression and mating- prior social isolation is known to increase aggression in males by increased lunging, which is suppressed by group housing (GH). However, it is also known that single-housed (SH) males, despite their higher attempts to court females, are less successful. Here, Gao et al., developed a modified aggression assay, to address this issue by recording aggression in Drosophila males for 2 hours, over a virgin female which is immobilized by burying its head in the food. They found that while SH males frequently lunge in this assay, GH males switch to higher intensity but very low-frequency tussling. Constitutive neuronal silencing and activation experiments implicate cVA sensing Or67d neurons promoting high-frequency lunging, similar to earlier studies, whereas Or47b neurons promote low-frequency but higher intensity tussling. Using optogenetic activation they found that three pairs of pC1 neurons- pC1SS2 increase tussling. While P1a neurons, previously implicated in promoting aggression and courtship, did not increase tussling in optogenetic activation (in the dark), they could promote aggressive tussling in thermogenetic activation carried out in the presence of visible light. It was further suggested, using a further modified aggression assay that GH males use increased tussling and are able to maintain territorial control, providing them mating advantage over SI males and this may partially overcome the effect of aging in GH males.

Strengths:

Using a series of clever neurogenetic and behavioral approaches, subsets of ORNs and pC1 neurons were implicated in promoting tussling behaviors. The authors devised a new paradigm to assay for territory control which appears better than earlier paradigms that used a food cup (Chen et al, 2002), as this new assay is relatively clutter-free, and can be eventually automated using computer vision approaches. The manuscript is generally well-written, and the claims made are largely supported by the data.

Weaknesses:

I have a few concerns regarding some of the evidence presented and claims made as well as a description of the methodology, which needs to be clarified and extended further.

(1) Typical paradigms for assaying aggression in Drosophila males last for 20-30 minutes in the presence of nutritious food/yeast paste/females or all of these (Chen et al. 2002, Nilsen et al., 2004, Dierick et al. 2007, Dankert et al., 2009, Certel & Kravitz 2012). The paradigm described in Figure 1 A, while important and more amenable for video recording and computational analysis, seems a modification of the assay from Kravitz lab (Chen et al., 2002), which involved using a female over which males fight on a food cup. The modifications include a flat surface with a central food patch and a female with its head buried in the food, (fixed female) and much longer adaptation and recording times respectively (30 minutes, 2 hours), so in that sense, this is not a 'new' paradigm but a modification of an existing paradigm and its description as new should be appropriately toned down. It would also be important to cite these earlier studies appropriately while describing the assay.

(2) Lunging is described as a 'low intensity' aggression (line 111 and associated text), however, it is considered a mid to high-intensity aggressive behavior, as compared to other lower-intensity behaviors such as wing flicks, chase, and fencing. Lunging therefore is lower in intensity 'relative' to higher intensity tussling but not in absolute terms and it should be mentioned clearly.

(3) It is often difficult to distinguish faithfully between boxing and tussling and therefore, these behaviors are often clubbed together as box, tussle by Nielsen et al., 2004 in their Markov chain analysis as well as a more detailed recent study of male aggression (Simon & Heberlein, 2020). Therefore, authors can either reconsider the description of behavior as 'box, tussle' or consider providing a video representation/computational classifier to distinguish between box and tussle behaviors.

(4) Simon & Heberlein, 2020 showed that increased boxing & tussling precede the formation of a dominance hierarchy in males, and lunges are used subsequently to maintain this dominant status. This study should be cited and discussed appropriately while introducing the paradigm.

(5) It would be helpful to provide more methodological details about the assay, for instance, a video can be helpful showing how the males are introduced in the assay chamber, are they simply dropped to the floor when the film is removed after 30 minutes (Figures 1-2)?

(6) The strain of Canton-S (CS) flies used should be mentioned as different strains of CS can have varying levels of aggression, for instance, CS from Martin Heisenberg lab shows very high levels of aggressive lunges. Are the CS lines used in this study isogenized? Are various genetic lines outcrossed into this CS background? In the methods, it is not clear how the white gene levels were controlled for various aggression experiments as it is known to affect aggression (Hoyer et al. 2008).

(7) How important it is to use a fixed female for the assay to induce tussling? Do these females remain active throughout the assay period of 2.5 hours? Is it possible to use decapitated virgin females for the assay? How will that affect male behaviors?

(8) Raster plots in Figure 2 suggest a complete lack of tussling in SH males in the first 60 minutes of the encounter, which is surprising given the longer duration of the assay as compared to earlier studies (Nielsen et al. 2004, Simon & Heberlein, 2020 and others), which are able to pick up tussling in a shorter duration of recording time. Also, the duration for tussling is much longer in this study as compared to shorter tussles shown by earlier studies. Is this due to differences in the paradigm used, strain of flies, or some other factor? While the bar plots in Figure 2D show some tussling in SH males, maybe an analysis of raster plots of various videos can be provided in the main text and included as a supplementary figure to address this.

(9) Neuronal activation experiments suggesting the involvement of pC1SS2 neurons are quite interesting. Further, the role of P1a neurons was demonstrated to be involved in increasing tussling in thermogenetic activation in the presence of light (Figure 4, Supplement 1), which is quite important as the role of vision in optogenetic activation experiments, which required to be carried out in dark, is often not mentioned. However, in the discussion (lines 309-310) it is mentioned that PC1SS2 neurons are 'necessary and sufficient' for inducing tussling. Given that P1a neurons were shown to be involved in promoting tussling, this statement should be toned down.

(10) Are Or47b neurons connected to pC1SS2 or P1a neurons?

(11) The paradigm for territory control is quite interesting and subsequent mating advantage experiments are an important addition to the eventual outcome of the aggressive strategy deployed by the males as per their prior housing conditions. It would be important to comment on the 'fitness outcome' of these encounters. For instance, is there any fitness advantage of using tussling by GH males as compared to lunging by SH males? The authors may consider analyzing the number of eggs laid and eclosed progenies from these encounters to address this.

Reviewer #1 (Public Review):

The paper proposes a new source reconstruction method for electroencephalography (EEG) data and claims that it can provide far superior spatial resolution than existing approaches and also superior spatial resolution to fMRI. This primarily stems from abandoning the established quasi-static approximation to Maxwell's equations.

The proposed method brings together some very interesting ideas, and the potential impact is high. However, the work does not provide the evaluations expected when validating a new source reconstruction approach. I cannot judge the success or impact of the approach based on the current set of results. This is very important to rectify, especially given that the work is challenging some long-standing and fundamental assumptions made in the field.

I also find that the clarity of the description of the methods, and how they link to what is shown in the main results hard to follow.

I am insufficiently familiar with the intricacies of Maxwell's equations to assess the validity of the assumptions and the equations being used by WETCOW. The work therefore needs assessing by someone more versed in that area. That said, how do we know that the new terms in Maxwell's equations, i.e. the time-dependent terms that are normally missing from established quasi-static-based approaches, are large enough to need to be considered? Where is the evidence for this?

I have not come across EFD, and I am not sure many in the EEG field will have. To require the reader to appreciate the contributions of WETCOW only through the lens of the unfamiliar (and far from trivial) approach of EFD is frustrating. In particular, what impact do the assumptions of WETCOW make compared to the assumptions of EFD on the overall performance of SPECTRE?

The paper needs to provide results showing the improvements obtained when WETCOW or EFD are combined with more established and familiar approaches. For example, EFD can be replaced by a first-order vector autoregressive (VAR) model, i.e. y_t = A y_{t-1} + e_t (where y_t is [num_gridpoints x 1] and A is [num_gridpoints x num_gridpoints] of autoregressive parameters).

The authors' decision not to include any comparisons with established source reconstruction approaches does not make sense to me. They attempt to justify this by saying that the spatial resolution of LORETA would need to be very low compared to the resolution being used in SPECTRE, to avoid compute problems. But how does this stop them from using a spatial resolution typically used by the field that has no compute problems, and comparing with that? This would be very informative. There are also more computationally efficient methods than LORETA that are very popular, such as beamforming or minimum norm.

In short, something like the following methods needs to be compared:

(1) Full SPECTRE (EFD plus WETCOW)<br /> (2) WETCOW + VAR or standard ("simple regression") techniques<br /> (3) Beamformer/min norm plus EFD<br /> (4) Beamformer/min norm plus VAR or standard ("simple regression") techniques

This would also allow for more illuminating and quantitative comparisons of the real data. For example, a metric of similarity between EEG maps and fMRI can be computed to compare the performance of these methods. At the moment, the fMRI-EEG analysis amounts to just showing fairly similar maps.

There are no results provided on simulated data. Simulations are needed to provide quantitative comparisons of the different methods, to show face validity, and to demonstrate unequivocally the new information that SPECTRE can _potentially_ provide on real data compared to established methods. The paper ideally needs at least 3 types of simulations, where one thing is changed at a time, e.g.:

(1) Data simulated using WETCOW plus EFD assumptions<br /> (2) Data simulated using WETCOW plus e.g. VAR assumptions<br /> (3) Data simulated using standard lead fields (based on the quasi-static Maxwell solutions) plus e.g. VAR assumptions

These should be assessed with the multiple methods specified earlier. Crucially the assessment should be quantitative showing the ability to recover the ground truth over multiple realisations of realistic noise. This type of assessment of a new source reconstruction method is the expected standard.

Reviewer #1 (Public review):

Summary:

This study investigates the role of macrophage lipid metabolism in the intracellular growth of Mycobacterium tuberculosis. By using a CRISPR-Cas9 gene-editing approach, the authors knocked out key genes involved in fatty acid import, lipid droplet formation, and fatty acid oxidation in macrophages. Their results show that disrupting various stages of fatty acid metabolism significantly impairs the ability of Mtb to replicate inside macrophages. The mechanisms of growth restriction included increased glycolysis, oxidative stress, pro-inflammatory cytokine production, enhanced autophagy, and nutrient limitation. The study demonstrates that targeting fatty acid homeostasis at different stages of the lipid metabolic process could offer new strategies for host-directed therapies against tuberculosis.

The work is convincing and methodologically strong, combining genetic, metabolic, and transcriptomic analyses to provide deep insights into how host lipid metabolism affects bacterial survival.

Strengths:

The study uses a multifaceted approach, including CRISPR-Cas9 gene knockouts, metabolic assays, and dual RNA sequencing, to assess how various stages of macrophage lipid metabolism affect Mtb growth. The use of CRISPR-Cas9 to selectively knock out key genes involved in fatty acid metabolism enables precise investigation of how each step-lipid import, lipid droplet formation, and fatty acid oxidation affect Mtb survival. The study offers mechanistic insights into how different impairments in lipid metabolism lead to diverse antimicrobial responses, including glycolysis, oxidative stress, and autophagy. This deepens the understanding of macrophage function in immune defense.

The use of functional assays to validate findings (e.g., metabolic flux analyses, lipid droplet formation assays, and rescue experiments with fatty acid supplementation) strengthens the reliability and applicability of the results.

By highlighting potential targets for HDT that exploit macrophage lipid metabolism to restrict Mtb growth, the work has significant implications for developing new tuberculosis treatments.

Weaknesses:

The experiments were primarily conducted in vitro using CRISPR-modified macrophages. While these provide valuable insights, they may not fully replicate the complexity of the in vivo environment where multiple cell types and factors influence Mtb infection and immune responses.

Reviewer #1 (Public review):

Summary:

This study investigates what happens to the stimulus-driven responses of V4 neurons when an item is held in working memory. Monkeys are trained to perform memory-guided saccades: they must remember the location of a visual cue and then, after a delay, make an eye movement to the remembered location. In addition, a background stimulus (a grating) is presented that varies in contrast and orientation across trials. This stimulus serves to probe the V4 responses, is present throughout the trial, and is task-irrelevant. Using this design, the authors report memory-driven changes in the LFP power spectrum, changes in synchronization between the V4 spikes and the ongoing LFP, and no significant changes in firing rate.

Strengths:

(1) The logic of the experiment is nicely laid out.

(2) The presentation is clear and concise.

(3) The analyses are thorough, careful, and yield unambiguous results.

(4) Together, the recording and inactivation data demonstrate quite convincingly that the signal stored in FEF is communicated to V4 and that, under the current experimental conditions, the impact from FEF manifests as variations in the timing of the stimulus-evoked V4 spikes and not in the intensity of the evoked activity (i.e., firing rate).

Weaknesses:

I think there are two limitations of the study that are important for evaluating the potential functional implications of the data. If these were acknowledged and discussed, it would be easier to situate these results in the broader context of the topic, and their importance would be conveyed more fairly and transparently.

(1) While it may be true that no firing rate modulations were observed in this case, this may have been because the probe stimuli in the task were behaviorally irrelevant; if anything, they might have served as distracters to the monkey's actual task (the MGS). From this perspective, the lack of rate modulation could simply mean that the monkeys were successful in attending the relevant cue and shielding their performance from the potentially distracting effect of the background gratings. Had the visual probes been in some way behaviorally relevant and/or spatially localized (instead of full field), the data might have looked very different. With this in mind, it would be prudent to dial down the tone of the conclusions, which stretch well beyond the current experimental conditions (see recommendations).

(2) Another point worth discussing is that although the FEF delay-period activity corresponds to a remembered location, it can also be interpreted as an attended location, or as a motor plan for the upcoming eye movement. These are overlapping constructs that are difficult to disentangle, but it would be important to mention them given prior studies of attentional or saccade-related modulation in V4. The firing rate modulations reported in some of those cases provide a stark contrast with the findings here, and I again suspect that the differences may be due at least in part to the differing experimental conditions, rather than a drastically different encoding mode or functional linkage between FEF and V4.

Reviewer #1 (Public review):

Summary:

In this study, Masroor Ahmad Paddar and his/her colleagues explore the noncanonical roles of ATG5 and membrane atg8ylation in regulating retromer assembly and function. They begin by examining the interactomes of ATG5 and expand the scope of these effects to include homeostatic responses to membrane stress and damage.

Strengths:

This study provides novel insights into the noncanonical function of ATG8ylation in endosomal cargo sorting process.

Weaknesses:

The direct mechanism by which ATG8ylation regulates the retromer remains unsolved.

Comments on revisions:

After revision, though the major weakness remains unsolved, other questions have been addressed experimentally or further interpreted.

Reviewer #1 (Public review):

Overall I found the approach taken by the authors to be clear and convincing. It is striking that the conclusions are similar to those obtained in a recent study using a different computational approach (finite state controllers), and lend confidence to the conclusions about the existence of an optimal memory duration. There are a few points or questions that could be addressed in greater detail in a revision:

(1) Discussion of spatial encoding

The manuscript contrasts the approach taken here (reinforcement learning in a grid world) with strategies that involve a "spatial map" such as infotaxis. The authors note that their algorithm contains "no spatial information." However, I wonder if further degrees of spatial encoding might be delineated to better facilitate comparisons with biological navigation algorithms. For example, the gridworld navigation algorithm seems to have an implicit allocentric representation, since movement can be in one of four allocentric directions (up, down, left, right). I assume this is how the agent learns to move upwind in the absence of an explicit wind direction signal. However, not all biological organisms likely have this allocentric representation. Can the agent learn the strategy without wind direction if it can only go left/right/forward/back/turn (in egocentric coordinates)? In discussing possible algorithms, and the features of this one, it might be helpful to distinguish<br /> (1) those that rely only on egocentric computations (run and tumble),<br /> (2) those that rely on a single direction cue such as wind direction,<br /> (3) those that rely on allocentric representations of direction, and<br /> (4) those that rely on a full spatial map of the environment.

(2) Recovery strategy on losing the plume

While the approach to encoding odor dynamics seems highly principled and reaches appealingly intuitive conclusions, the approach to modeling the recovery strategy seems to be more ad hoc. Early in the paper, the recovery strategy is defined to be path integration back to the point at which odor was lost, while later in the paper, the authors explore Brownian motion and a learned recovery based on multiple "void" states. Since the learned strategy works best, why not first consider learned strategies, and explore how lack of odor must be encoded or whether there is an optimal division of void states that leads to the best recovery strategies? Also, although the authors state that the learned recovery strategies resemble casting, only minimal data are shown to support this. A deeper statistical analysis of the learned recovery strategies would facilitate comparison to those observed in biology.

(3) Is there a minimal representation of odor for efficient navigation?

The authors suggest (line 280) that the number of olfactory states could potentially be reduced to reduce computational cost. This raises the question of whether there is a maximally efficient representation of odors and blanks sufficient for effective navigation. The authors choose to represent odor by 15 states that allow the agent to discriminate different spatial regimes of the stimulus, and later introduce additional void states that allow the agent to learn a recovery strategy. Can the number of states be reduced or does this lead to loss of performance? Does the optimal number of odor and void states depend on the spatial structure of the turbulence as explored in Figure 5?

Reviewer #1 (Public review):

Summary:

The study by Nelson et al. is focused on formation of the Drosophila Posterior Signaling Center (PSC) which ultimately acts as a niche to support hematopoietic stem cells of the lymph gland (LG). Using a combination of genetics and live imaging, the authors show that PSC cells migrate as a tight collective and associate with multiple tissues during a trajectory that positions them at the posterior of the LG.

This is an important study that identifies Slit-Robo signaling as a regulator of PSC morphogenesis, and highlights the complex relationship of interacting cell types - PSC, visceral mesoderm (VM) and cardioblasts (CBs) - in coordinated development of these three tissues during organ development. However, one point requiring clarification is the idea that PSC cells exhibit a collective cell migration; it is not clear that the cells are migrating rather than being pushed to a more dorsal position through dorsal closure and/or other similar large scale embryo movement. This does not detract from the very interesting analysis of PSC morphogenesis as presented.

Strengths:

• Using expression of Hid or Grim to ablate associated tissues, they find evidence that the VM and CB of the dorsal vessel affect PSC migration/morphology whereas the alary muscles do not. Slit is expressed by both VM and CBs, and therefore Slit-Robo signaling was investigated as PSCs express Robo.

• Using a combination of approaches, the authors convincingly demonstrate that Slit expression in the CBs and VM acts to support PSC positioning. A strength is the ability to knockdown slit levels in particular tissue types using the Gal4 system and RNAi.

• Although in the analysis of robo mutants, the PSC positioning phenotype is weaker in the individual mutants (robo1 and robo2) with only the double mutant (robo1,robo2) exhibiting a phenotype comparable to the slit RNAi. The authors make a reasonable argument that Slit-Robo signaling has an intrinsic effect, likely acting within PSCs, because PSCs show a phenotype even when CBs do not (Fig 4G).

• New insight into dorsal vessel formation by VM is presented in Fig 4A,B, as loss of the VM can affect dorsal vessel morphogenesis. This result additionally points to the VM as important.

Weaknesses:

• The authors are cautioned to temper the result that Slit-Robo signaling is intrinsic to PSC since loss of robo may affect other cell types (besides CBs and PSCs) to indirectly affect PSC migration/morphogenesis. In fact, in the robo2, robo1 mutant, the VM appears to be incorrectly positioned (Fig. 4G).

• If possible, the authors should use RNAi to knockdown Robo1 and Robo2 levels specifically in the PSCs if a Gal4 is available; might Antp.Gal4 (Fig 1K) be useful? Even if knockdown is achieved in PSCs+CBs, this would be a better/complementary experiment to support the approach outlined in Fig 4D.

• Movies are hard to interpret, as it seems unclear that the PSCs actively migrate rather than being pushed/moved indirectly due to association with VM and CBs/dorsal vessel.

Joint Public Review:

Summary

This manuscript explores the transcriptomic identities of olfactory ensheathing cells (OECs), glial cells that support life-long axonal growth in olfactory neurons, as they relate to spinal cord injury repair. The authors show that transplantation of cultured, immunopurified rodent OECs at a spinal cord injury site can promote injury-bridging axonal regrowth. They then characterize these OECs using single-cell RNA sequencing, identifying five subtypes and proposing functional roles that include regeneration, wound healing, and cell-cell communication. They identify one progenitor OEC subpopulation and also report several other functionally relevant findings, notably, that OEC marker genes contain mixtures of other glial cell type markers (such as for Schwann cells and astrocytes), and that these cultured OECs produce and secrete Reelin, a regrowth-promoting protein that has been disputed as a gene product of OECs.

Strengths

This manuscript offers an extensive, cell-level characterization of OECs, supporting their potential therapeutic value for spinal cord injury and suggesting potential underlying repair mechanisms. The authors use various approaches to validate their findings, providing interesting images that show the overlap between sprouting axons and transplanted OECs, and showing that OEC marker genes identified using single-cell RNA sequencing are present in vivo, in both olfactory bulb tissue and spinal cord after OEC transplantation.

Challenges

Despite the breadth of information presented, and although many of the suggestions in the initial review were addressed well, some points related to quantification and discussion of sex differences are not fully addressed in this revision.

(1) The request for quantification of OEC bridges is not fully addressed. We note that this revision includes the following statement (page 6): "We note, however, that such bridge formation is rare following a severe spinal cord injury in adult mammals." However, the title of the paper states that olfactory ensheathing cells promote neural repair and the abstract states that "OECs transplanted near the injury site modify the inhibitory glial scar and facilitate axon regeneration past the scar border and into the lesion." Statements such as these make it more crucial to include quantification of OEC bridges, because if single images are shown of remarkable, unusual bridges, but only one sentence acknowledges the low frequency of this occurrence, then this information taken together might present the wrong takeaway to readers.

Including some sort of quantification of bridging, whether it be the number of rats exhibiting bridges, the percentage area of OECs near a lesion site, or some other meaningful analysis, would add rigor and clarity to the manuscript.

(2) The additional discussion of sex differences in OEC bridging elaborates on the choice to study female rats, citing bladder challenges in male rats, but does not note salient clinical implications of this choice. Men account for ~80% of spinal cord injuries and likely also have worsened urinary tract issues, so it would be important to acknowledge this clinical fact and consider including males in future studies.

Reviewer #1 (Public review):

Huber proposes a theory where the role of the medial temporal lobe (MTL) is memory, where properties of spatial cells in the MTL can be explained through memory function rather than spatial processing or navigation. Instantiating the theory through a computational model, the author shows that many empirical phenomena of spatial cells can be captured, and may be better accounted through a memory theory. It is an impressive computational account of MTL cells with a lot of theoretical reasoning and aims to tightly relate to various spatial cell data.

In general, the paper is well written, and has been greatly improved after revision for clarity and situating the model in the context of the literature. Below are a few responses to the author's rebuttal.

(2 & 3) In response to my previous review point 2 and 3, the author has now added "According to this model, hexagonally arranged grid cells should be the exception rather than the rule when considering more naturalistic environments." It is good to know that it captures data that show non-grid like responses in more complex and realistic environments. However, the model still focuses on explaining the spatial firing aspect of grid cells even though they are not supposed to be spatial. I noted in my previous review, "If it's not encoding a spatial attribute, it doesn't have to have a spatial field. For example, it could fire in the whole arena". The author notes inhibitory drive and habituation. Habituation happens, but then spatial cell responses are supposed (or assumed) to be still strong after many visits to that environment. More generally, I am more convinced that grid-like and spatial coding are a special case - both in navigation and memory. In a way I believe the author agrees, though the work here focuses on capturing spatial properties (which is understandable given the literature). In conclusion, though there may be theoretical disagreements, I find the points the author raises fair.

(4) The difference between mEC and lEC or PRC for encoding non-spatial vs spatial attributes is still not clear to me - though not crucial for the point of this paper.

(5) Thank you for providing a video - this makes it extremely clear how learning occurs.

Reviewer #1 (Public review):

Summary:

The manuscript of He et al. compares the roles of Hox/Gbx genes between the well-established anthozoan model, the burrowing sea anemone Nematostella, and the new scleractinian model Montipora. The authors show staggered expression of Anthox6a.1, Anthox8 and Gbx of the Montipora larva and argue that their BMP-dependent expression is responsible for the segmentation of the endomesoderm, just like they have previously demonstrated in Nematostella (despite some differences in the timing, formation of extra mesenteries, etc). The authors posit that Hox/Gbx-dependent segmentation of the endomesoderm represents an ancestral anthozoan trait. The study addresses a remarkably interesting question, but it has several important shortcomings, which the authors should try to rectify.

Strengths:

The authors introduce a new scleractinian model Montipora and present interesting data on the composition of its compact Hox cluster, its embryonic and larval development, metamorphosis, and segmentation. They also show staggered expression of Gbx, Anthox6a.1, and Anthox8, which is suggestive of their involvement in the partitioning of the gastrodermis of the polyp.

Weaknesses:

He et al. claim that Gbx and Hox genes are responsible for the segmentation of the directive axis in Montipora based on expression patterns of these genes before the onset of segmentation. In the absence of functional analyses, this claim (although likely correct) is not supported. Moreover, the authors do not show that staggered Gbx and Hox gene expression correlates with the position of the segment boundaries.

The authors use two inhibitors of BMP signaling and show that segmentation is lost in the treated animals. However, they do not provide controls, which would show that the effect of the treatment is specific to the loss of BMP function. Moreover, their transcriptomic analyses suggest that the whole BMP signaling system in Montipora is wired completely differently than in Nematostella, but they do not acknowledge and discuss this striking difference. If true, this is a very interesting result, but it requires thorough validation.

Reviewer #1 (Public review):

Summary:

A theoretical model for microbial osmoresponse was proposed. The model assumes simple phenomenological rules: (i) the change of free water volume in the cell due to osmotic imbalance based on pressure balance, (ii) Osmoregulation that assumes change of the proteome partitioning depending on the osmotic pressure that affects the osmolyte-producing protein production, (iii) The cell-wall synthesis regulation where the change of the turgor pressure to the cell-wall synthesis efficiency to go back to the target turgor pressure, (iv) Effect of Intracellular crowding assuming that the biochemical reactions slow down for more crowding and stops when the protein density (protein mass divided by free water volume) reaches a critical value. The parameter values were found in the literature or obtained by fitting to the experimental data. The authors compare the model behavior with various microorganismcs (E. coli, B. subtils, S. Cerevisiae, S. pombe), and successfully reproduced the overall trend (steady state behavior for many of them, dynamics for S. pombe). In addition, the model predicts non-trivial behavior such as the fast cell growth just after the hypoosmotic shock, which is consistent with experimental observation. The authors further make experimentally testable predictions regarding mutant behavior and transient dynamics.

Strength:

The theory assumes simple mechanistic dependence between core variables without going into specific molecular mechanisms of regulations. The simplicity allows the theory to apply to different organisms by adjusting the time scales with parameters, and the model successfully explains broad classes of observed behaviours. Mathematically, the model provides analytical expressions of the parameter dependences and an understanding of the dynamics through the phase space without being buried in the detail. This theory can serve as a base to discuss the universality and diversity of microbial osmoresponse.

Weakness:

The core part of this model is that everything is coupled with growth physiology, and, as far as I understand, the assumption (iv) (eq. 8) that imposes the global reaction rate dependence on crowding plays a crucial role. I would think this is a strong and interesting assumption. However, the abstract or discussion does not discuss the importance of this assumption. In addition, the paper does not discuss gene regulation explicitly, and some comparison with a molecular mechanism-oriented model may be beneficial to highlight the pros and cons of the current approach.

Reviewer #1 (Public review):

In this important study, the authors characterized the transformation of neural representations of olfactory stimuli from the primary sensory cortex to multisensory regions in the medial temporal lobe and investigated how they were affected by non-associative learning. The authors used high-density silicon probe recordings from five different cortical regions while familiar vs. novel odors were presented to a head-restrained mouse. This is a timely study because unlike other sensory systems (e.g., vision), the progressive transformation of olfactory information is still poorly understood. The authors report that both odor identity and experience are encoded by all of these five cortical areas but nonetheless some themes emerge. Single neuron tuning of odor identity is broad in the sensory cortices but becomes narrowly tuned in hippocampal regions. Furthermore, while experience affects neuronal response magnitudes in early sensory cortices, it changes the proportion of active neurons in hippocampal regions. Thus, this study is an important step forward in the ongoing quest to understand how olfactory information is progressively transformed along the olfactory pathway.

The study is well-executed. The direct comparison of neuronal representations from five different brain regions is impressive. Conclusions are based on single neuronal level as well as population level decoding analyses. Among all the reported results, one stands out for being remarkably robust. The authors show that the anterior olfactory nucleus (AON), which receives direct input from the olfactory bulb output neurons, was far superior at decoding odor identity as well as novelty compared to all the other brain regions. This is perhaps surprising because the other primary sensory region - the piriform cortex - has been thought to be the canonical site for representing odor identity. A vast majority of studies have focused on aPCx, but direct comparisons between odor coding in the AON and aPCx are rare. The experimental design of this current study allowed the authors to do so and the AON was found to convincingly outperform aPCx. Although this result goes against the canonical model, it is consistent with a few recent studies including one that predicted this outcome based on anatomical and functional comparisons between the AON-projecting tufted cells vs. the aPCx-projecting mitral cells in the olfactory bulb (Chae, Banerjee et. al. 2022). Future experiments are needed to probe the circuit mechanisms that generate this important difference between the two primary olfactory cortices as well as their potential causal roles in odor identification.

The authors were also interested in how familiarity vs. novelty affects neuronal representation across all these brain regions. One weakness of this study is that neuronal responses were not measured during the process of habituation. Neuronal responses were measured after four days of daily exposure to a few odors (familiar) and then some other novel odors were introduced. This creates a confound because the novel vs. familiar stimuli are different odorants and that itself can lead to drastic differences in evoked neural responses. Although the authors try to rule out this confound by doing a clever decoding and Euclidian distance analysis, an alternate more straightforward strategy would have been to measure neuronal activity for each odorant during the process of habituation.

Reviewer #1 (Public review):

Summary:

The study by Gupta et al. investigates the role of mast cells (MCs) in tuberculosis (TB) by examining their accumulation in the lungs of M. tuberculosis-infected individuals, non-human primates, and mice. The authors suggest that MCs expressing chymase and tryptase contribute to the pathology of TB and influence bacterial burden, with MC-deficient mice showing reduced lung bacterial load and pathology.

Strengths:

(1) The study addresses an important and novel topic, exploring the potential role of mast cells in TB pathology.

(2) It incorporates data from multiple models, including human, non-human primates, and mice, providing a broad perspective on MC involvement in TB.

(3) The finding that MC-deficient mice exhibit reduced lung bacterial burden is an interesting and potentially significant observation.

Weaknesses:

(1) The evidence is inconsistent across models, leading to divergent conclusions that weaken the overall impact of the study.

(2) Key claims, such as MC-mediated cytokine responses and conversion of MC subtypes in granulomas, are not well-supported by the data presented.

(3) Several figures are either contradictory or lack clarity, and important discrepancies, such as the differences between mouse and human data, are not adequately discussed.

(4) Certain data and conclusions require further clarification or supporting evidence to be fully convincing.

Reviewer #1 (Public review):

Summary:

In this manuscript, BOUTRY et al examined a cnidarian Hydra model system where spontaneous tumors manifest in laboratory settings, and lineages featuring vertically transmitted neoplastic cells (via host budding) have been sustained for over 15 years. They observed that hydras harboring long-term transmissible tumors exhibit an unexpected augmentation in tentacle count. In addition, the presence of extra tentacles, enhancing the host's foraging efficiency, correlated with an elevated budding rate, thereby promoting tumor transmission vertically. This study provided the evidence that tumors, akin to parasitic entities, can also exert control over their hosts.

Strengths:

The manuscript is well-written, and the phenotype is intriguing.

Reviewer #3 (Public review):

In this study, O'Brien et al. address the need for scalable and cost-effective approaches to finding lead compounds for the treatment of the growing number of Mendelian diseases. They used state-of-the-art phenotypic screening based on an established high-dimensional phenotypic analysis pipeline in the nematode C. elegans.

First, a panel of 25 C. elegans models was created by generating CRISPR/Cas9 knock-out lines for conserved human disease genes. These mutant strains underwent behavioral analysis using the group's published methodology. Clustering analysis revealed common features for genes likely operating in similar genetic pathways or biological functions. The study also presents results from a more focused examination of ciliopathy disease models.

Subsequently, the study focuses on the NALCN channel gene family, comparing the phenotypes of mutants of nca-1, unc-77, and unc-80. This initial characterization identifies three behavioral parameters that exhibit significant differences from the wild type and could serve as indicators for pharmacological modulation.

As a proof-of-concept, O'Brien et al. present a drug repurposing screen using an FDA-approved compound library, identifying two compounds capable of rescuing the behavioral phenotype in a model with UNC80 deficiency. The relatively short time and low cost associated with creating and phenotyping these strains suggest that high-throughput worm tracking could serve as a scalable approach for drug repurposing, addressing the multitude of Mendelian diseases. Interestingly, by measuring a wide range of behavioural parameters, this strategy also simultaneously reveals deleterious side effects of tested drugs that may confound the analysis.

Considering the wealth of data generated in this study regarding important human disease genes, it is regrettable that the data is not made accessible to researchers less versed in data analysis methods. This diminishes the study's utility. It would have a far greater impact if an accessible and user-friendly online interface were established to facilitate data querying and feature extraction for specific mutants. This would empower researchers to compare their findings with the extensive dataset created here.

Another technical limitation of the study is the use of single alleles. Large deletion alleles were generated by CRISPR/Cas9 gene editing. At first glance, this seems like a good idea because it limits the risk that background mutations, present in chemically-generated alleles, will affect behavioral parameters. However, these large deletions can also remove non-coding RNAs or other regulatory genetic elements, as found, for example, in introns. Therefore, it would be prudent to validate the behavioral effects by testing additional loss-of-function alleles produced through early stop codons or targeted deletion of key functional domains.

Comments on revisions:

In this final round of revisions, the authors have improved their manuscript and provide useful information about analysis procedures and code and updated figures.

Reviewer #1 (Public review):

This paper examines the role of MLCK (myosin light chain kinase) and MLCP (myosin light chain phosphatase) in axon regeneration. Using loss-of-function approaches based on small molecule inhibitors and siRNA knockdown, the authors explore axon regeneration in cell culture and in animal models from central and peripheral nervous systems. Their evidence shows that MLCK activity facilitates axon extension/regeneration, while MLCP prevents it.

Major concerns:

(1) In the title, authors indicate that the observed effects from loss-of-function of MLCK/MLCP take place via F-actin redistribution in the growth cone. However, there are no experiments showing a causal effect between changes in axon growth mediated by MLCK/MLCP and F-actin redistribution.

(2) The author combines MLCK inhibitors with Bleb (Figure 6), trying to verify if both pairs of inhibitors act on the same target/pathway. MLCK may regulate axon growth independent of NMII activity. However, this has very important implications for the understanding not only on how NMII works and affects axon extension, but also in trying to understand what MLCP is doing. One wonders if MLCP actions, which are opposite of MLCK, also independent of NMII activity? The authors, in the discussion section, try to find an explanation for this finding, but I consider it fails since the whole rationale of the manuscript is still around how MLCK and MLCP affect NMII phosphorylation.

What follows is a discussion of the merits and limitations of different claims of the manuscript in light of the evidence presented.

(1) Using western blot and immunohistochemical analyses, authors first show that MLCK expression is increased in DRG sensory neurons following peripheral axotomy, concomitant to an increase in MLC phosphorylation, suggesting a causal effect (Figure 1). The authors claim that it is common that axon growth-promoting genes are upregulated. It would have been interesting at this point to study in this scenario the regulation of MLCP.

(2) Using DRG cultures and sciatic nerve crush in the context of MLCK inhibition (ML-7) and down-regulation, authors conclude that MLCK activity is required for mammalian peripheral axon regeneration both in vitro and in vivo (Figure 2). In parallel, the authors show that these treatments affect as expected the phosphorylation levels of MLC.

The in vitro evidence is of standard methods and convincing. However, here, as well as in all other experiments using siRNAs, no Control siRNAs were used. Authors do show that the target protein is downregulated, and they can follow transfected cells with GFP. Still, it should be noted that the standard control for these experiments has not been done.

(3) The authors then examined the role of the phosphatase MLCP in axon growth during regeneration. The authors first use a known MLCP blocker, phorbol 12,13-dibutyrate (PDBu), to show that is able to increase the levels of p-MLC, with a concomitant increase in the extent of axon regrowth of DRG neurons, both in permissive as well as non-permissive substrates. The authors repeat the experiments using the knockdown of MYPT1, a key component of the MLC-phosphatase, and again can observe a growth-promoting effect (Figure 3).

The authors further show evidence for the growth-enhancing effect in vivo, in nerve crush experiments. The evidence in vivo deserves more evidence and experimental details (see comment 2). A key weakness of the data was mentioned previously: no control siARN was used.

(4) In the next set of experiments (presented in Figure 4) authors extend the previous observations in primary cultures from the CNS. For that, they use cortical and hippocampal cultures, and pharmacological and genetic loss-of-function using the above-mentioned strategies. The expected results were obtained in both CNS neurons: inhibition or knockdown of the kinase decreases axon growth, whereas inhibition or knockdown of the phosphatase increases growth. A main weakness in this set is that drugs were used from the beginning of the experiment, and hence, they would also affect axon specification. As pointed in Materials and Method (lines 143-145) authors counted as "axons" neurites longer than twice the diameter of the cell soma, and hence would not affect the variable measured. In any case, to be sure one is only affecting axon extension in these cells, the drugs should have been used after axon specification and maturation, which occurs at least after 5 DIV.

(5) In Figure 7, the authors a local cytoskeletal action of the drug, but the evidence provided does not differentiate between a localized action of the drugs and a localized cell activity.

References:

(1) Eun-Mi Hur 1, In Hong Yang, Deok-Ho Kim, Justin Byun, Saijilafu, Wen-Lin Xu, Philip R Nicovich, Raymond Cheong, Andre Levchenko, Nitish Thakor, Feng-Quan Zhou. 2011. Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules. Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):5057-62. doi: 10.1073/pnas.1011258108.

(2) Garrido-Casado M, Asensio-Juárez G, Talayero VC, Vicente-Manzanares M. 2024. Engines of change: Nonmuscle myosin II in mechanobiology. Curr Opin Cell Biol. 2024 Apr;87:102344. doi: 10.1016/j.ceb.2024.102344.

(3) Karen A Newell-Litwa 1, Rick Horwitz 2, Marcelo L Lamers. 2015. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Dis Model Mech. 2015 Dec;8(12):1495-515. doi: 10.1242/dmm.022103.

Reviewer #1 (Public review):

Summary:

The work by Chuong et al. provides important new insights into the contribution of different molecular mechanisms in the dynamics of CNV formation. It will be of interest to anyone curious about genome architecture and evolution from yeast biologists to cancer researchers studying genome rearrangements.

Strengths:

Their results are especially striking in that the "simplest" mechanism of GAP1 amplification (non-allelic homologous recombination between the flanking Ty-LTR elements) is not the most common route taken by the cells, emphasizing the importance of experimentally testing what might seem on the surface to be obvious outcome. One of the important developments of their work is the use of their neural network simulation-based inference (nnSBI) model to derive rates of amplicon formation and their fitness effects.

Weaknesses:

The nnSBI model that derives rates of amplicon formation and fitness is still opaque to this reviewer. All of the other criticisms made in the first review have been clarified/corrected in this much-improved version of the manuscript.

Reviewer #1 (Public review):

Summary:

Motivated by the existence of different behavioral strategies (e.g. model-based vs. model-free), and potentially different neural circuits that underlie them, Venditto et al. introduce a new approach for inferring which strategies animals are using from data. In particular, they extend the mixture of agents (MoA) framework to accommodate the possibility that the weighting among different strategies might change over time. These temporal dynamics are introduced via a hidden Markov model (HMM), i.e. with discrete state transitions. These state transition probabilities and initial state probabilities are fit simultaneously along with the MoA parameters, which include decay/learning rate and mixture weightings, using the EM algorithm. The authors test their model on data from Miller et al., 2017, 2022, arguing that this formulation leads to (1) better fits and (2) improved interpretability over their original model, which did not include the HMM portion. Lastly, they claim that certain aspects of OFC firing are modulated by internal state as identified by the MoA-HMM.

Strengths:

The paper is very well written and easy to follow, especially for one with a significant modeling component. Furthermore, the authors do an excellent job explaining and then disentangling many threads that are often knotted together in discussions of animal behavior and RL: model-free vs. model-based choice, outcome vs. choice-focused, exploration vs. exploitation, bias, perserveration. Each of these concepts is quantified by particular parameters of their models. Model recovery (Fig. 3) is convincing post-revision and licenses their fits to animal behavior later. While the specific claims made about behavior and neural activity are not especially surprising (e.g. the animals begin a session, in which rare vs. common transitions are not yet known, in a more exploratory mode), the MoA-HMM framework seems broadly applicable to other tasks in the field and useful for the purpose of quantification here. Overall, I believe this paper is certainly worthy of publication in a journal like eLife.

Weaknesses:

I am pleased with the authors' responses to my initial comments, and I thank them for their efforts. My main note of caution to readers is just that when it comes to applying this method to neural data, the benefits may be subtle. On one extreme, it may be possible to capture many of these effects simply by explicitly modeling time, although the authors do a good job showing that they can beat this benchmark in their case. On the other extreme, there may be multiple switches that cannot simply be a monotonic time effect, but these might be at a faster timescale than can be easily captured in this model (in Fig. 7Aii, for example, there is still lots of variance unexplained by the latent state). Quantitative justification will be required for using this model over simpler alternatives, but again, I commend the authors for providing that justification in this paper.

Reviewer #1 (Public review):

Summary:

Wang et al. identify Hamlet, a PR-containing transcription factor, as a master regulator of reproductive development in Drosophila. Specifically, the fusion between the gonad and genital disc is necessary for the development of continuous testes and seminal vesicle tissue essential for fertility. To do this, the authors generate novel Hamlet null mutants by CRISPR/Cas9 gene editing and characterize the morphological, physiological, and gene expression changes of the mutants using immunofluorescence, RNA-seq, cut-tag, and in-situ analysis. Thus, Hamlet is discovered to regulate a unique expression program, which includes Wnt2 and Tl, that is necessary for testis development and fertility.

Strengths:

This is a rigorous and comprehensive study that identifies the Hamlet-dependent gene expression program mediating reproductive development in Drosophila. The Hamlet transcription targets are further characterized by Gal4/UAS-RNAi confirming their role in reproductive development. Finally, the study points to a role for Wnt2 and Tl as well as other Hamlet transcriptionally regulated genes in epithelial tissue fusion.

Weaknesses:

The image resolution and presentation of figures is a major issue in this study. As a non-expert, it is nearly impossible to see the morphological changes as described in the results. Quantification of all cell biological phenotypes is also lacking therefore reducing the impact of this study to those familiar with tissue fusion events in Drosophila development.

Reviewer #1 (Public review):

Summary:

The authors quantified information in gesture and speech, and investigated the neural processing of speech and gestures in pMTG and LIFG, depending on their informational content, in 8 different time-windows, and using three different methods (EEG, HD-tDCS and TMS). They found that there is a time-sensitive and staged progression of neural engagement that is correlated with the informational content of the signal (speech/gesture).

Strengths:

A strength of the paper is that the authors attempted to combine three different methods to investigate speech-gesture processing.

Weaknesses:

(1) One major issue is that there is a tight anatomical coupling between pMTG and LIFG. Stimulating one area could therefore also result in stimulation of the other area (see Silvanto and Pascual-Leone, 2008). I therefore think it is very difficult to tease apart the contribution of these areas to the speech-gesture integration process, especially considering that the authors stimulate these regions in time windows that are very close to each other in both time and space (and the disruption might last longer over time).

(2) Related to this point, it is unclear to me why the HD-TDCS/TMS is delivered in set time windows for each region. How did the authors determine this, and how do the results for TMS compare to their previous work from 2018 and 2023 (which describes a similar dataset+design)? How can they ensure they are only targeting their intended region since they are so anatomically close to each other?

(3) As the EEG signal is often not normally distributed, I was wondering whether the authors checked the assumptions for their Pearson correlations. The authors could perhaps better choose to model the different variables to see whether MI/entropy could predict the neural responses. How did they correct the many correlational analyses that they have performed?

(4) The authors use ROIs for their different analyses, but it is unclear why and on the basis of what these regions are defined. Why not consider all channels without making them part of an ROI, by using a method like the one described in my previous comment?

(5) The authors describe that they have divided their EEG data into a "lower half" and a "higher half" (lines 234-236), based on entropy scores. It is unclear why this is necessary, and I would suggest just using the entropy scores as a continuous measure.

Reviewer #1 (Public review):

Summary:

The drug Ivermectin is used to effectively treat a variety of worm parasites in the world, however resistance to Ivermectin poses a rising challenge for this treatment strategy. In this study, the authors found that loss of the E3 ubiquitin ligase UBR-1 in the worm C. elegans results in resistance to Ivermectin. In particular, the authors found that ubr-1 mutants are resistant to the effects of Ivermectin on worm viability, body size, pharyngeal pumping, and locomotion. The authors previously showed that loss of UBR-1 disrupts homeostasis of the amino acid and neurotransmitter glutamate resulting in increased levels of glutamate in C. elegans. Here, the authors found that the sensitivity of ubr-1 mutants to Ivermectin can be restored if glutamate levels are reduced using a variety of different methods. Conversely, treating worms with exogenous glutamate to increase glutamate levels also results in resistance to Ivermectin supporting the idea that increased glutamate promotes resistance to Ivermectin. The authors found that the primary known targets of Ivermectin, glutamate-gated chloride channels (GluCls), are downregulated in ubr-1 mutants providing a plausible mechanism for why ubr-1 mutants are resistant to Ivermectin. Although it is clear that loss of GluCls can lead to resistance to Ivermectin, this study suggests that one potential mechanism to decrease GluCl expression is via disruption of glutamate homeostasis that leads to increased glutamate. This study suggests that if parasitic worms become resistant to Ivermectin due to increased glutamate, their sensitivity to Ivermectin could be restored by reducing glutamate levels using drugs such as Ceftriaxone in a combination drug treatment strategy.

Strengths:

(1) The use of multiple independent assays (i.e., viability, body size, pharyngeal pumping, locomotion, and serotonin-stimulated pharyngeal muscle activity) to monitor the effects of Ivermectin

(2) The use of multiple independent approaches (got-1, eat-4, ceftriaxone drug, exogenous glutamate treatment) to alter glutamate levels to support the conclusion that increased glutamate in ubr-1 mutants contributes to Ivermectin resistance.

Weaknesses:

(1) The primary target of Ivermectin is GluCls so it is not surprising that alteration of GluCl expression or function would lead to Ivermectin resistance.

(2) It remains to be seen what percent of Ivermectin-resistant parasites in the wild have disrupted glutamate homeostasis as opposed to mutations that more directly decrease GluCl expression or function.

Reviewer #1 (Public review):

Summary:

IPF is a disease lacking regressive therapies which has a poor prognosis, and so new therapies are needed. This ambitious phase 1 study builds on the authors' 2024 experience in Sci Tran Med with positive results with autologous transplantation of P63 progenitor cells in patients with COPD. The current study suggests that P63+ progenitor cell therapy is safe in patients with ILD. The authors attribute this to the acquisition of cells from a healthy upper lobe site, removed from the lung fibrosis. There are currently no cell-based therapies for ILD and in this regard the study is novel with important potential for clinical impact if validated in Phase 2 and 3 clinical trials.

Strengths:

This study addresses the need for an effective therapy for interstitial lung disease. It offers good evidence that the cells used for therapy are safe. In so doing it addresses a concern that some P63+ progenitor cells may be proinflammatory and harmful, as has been raised in the literature (articles which suggested some P63+ cells can promote honeycombing fibrosis; references 26 &35). The authors attribute the safety they observed (without proof) to the high HOPX expression of administered cells (a marker found in normal Type 1 AECs. The totality of the RNASeq suggests the cloned cells are not fibrogenic. They also offer exploratory data suggesting a relationship between clone roundness and PFT parameters (and a negative association between patient age and clone roundness).

Weaknesses:

The authors can conclude they can isolate, clone, expand, and administer P63+ progenitor cells safely; but with the small sample size and lack of a placebo group, no efficacy should be implied.

Specific points:

(1) The authors acknowledge most study weaknesses including the lack of a placebo group and the concurrent COVID-19 in half the subjects (the high-dose subjects). They indicate a phase 2 trial is underway to address these issues.

(2) The authors suggest an efficacy signal on pages 18 (improvement in 2 subjects' CT scans) and 21 (improvement in DLCO) but with such a small phase 1 study and such small increases in DLCO (+5.4%) the authors should refrain from this temptation (understandable as it is).

(3) Likewise most CT scans were unchanged and those that improved were in the mid-dose group (albeit DLCO improved in the 2 patients whose CT scans improved).

(4) The authors note an impressive 58m increase in 6MWTD in the high-dose group but again there is no placebo group, and the low-dose group has no net change in 6MWTD at 24 weeks.

(5) I also raise the question of the enrollment criteria in which 5 patients had essentially normal DLCO/VA values. In addition there is no discussion as to whether the transplanted stem cells are retained or exert benefit by a paracrine mechanism (which is the norm for cell-based therapies).

Joint Public Review:

In this work, the authors develop a new computational tool, DeepTX, for studying transcriptional bursting through the analysis of single-cell RNA sequencing (scRNA-seq) data using deep learning techniques. This tool aims to describe and predict the transcriptional bursting mechanism, including key model parameters and the steady-state distribution associated with the predicted parameters. By leveraging scRNA-seq data, DeepTX provides high-resolution transcriptional information at the single-cell level, despite the presence of noise that can cause gene expression variation. The authors apply DeepTX to DNA damage experiments, revealing distinct cellular responses based on transcriptional burst kinetics. Specifically, IdU treatment in mouse stem cells increases burst size, promoting differentiation, while 5FU affects burst frequency in human cancer cells, leading to apoptosis or, depending on the dose, to survival and potential drug resistance. These findings underscore the fundamental role of transcriptional burst regulation in cellular responses to DNA damage, including cell differentiation, apoptosis, and survival. Although the insights provided by this tool are mostly well supported by the authors' methods, certain aspects would benefit from further clarification.

The strengths of this paper lie in its methodological advancements and potential broad applicability. By employing the DeepTXSolver neural network, the authors efficiently approximate stationary distributions of mRNA counts through a mixture of negative binomial distributions, establishing a simple yet accurate mapping between the kinetic parameters of the mechanistic model and the resulting steady-state distributions. This innovative use of neural networks allows for efficient inference of kinetic parameters with DeepTXInferrer, reducing computational costs significantly for complex, multi-gene models. The approach advances parameter estimation for high-dimensional datasets, leveraging the power of deep learning to overcome the computational expense typically associated with stochastic mechanistic models. Beyond its current application to DNA damage responses, the tool can be adapted to explore transcriptional changes due to various biological factors, making it valuable to the systems biology, bioinformatics, and mechanistic modelling communities. Additionally, this work contributes to the integration of mechanistic modelling and -omics data, a vital area in achieving deeper insights into biological systems at the cellular and molecular levels.

This work also presents some weaknesses, particularly concerning specific technical aspects. The tool was validated using synthetic data, and while it can predict parameters and steady-state distributions that explain gene expression behaviour across many genes, it requires substantial data for training. The authors account for measurement noise in the parameter inference process, which is commendable, yet they do not specify the exact number of samples required to achieve reliable predictions. Moreover, the tool has limitations arising from assumptions made in its design, such as assuming that gene expression counts for the same cell type follow a consistent distribution. This assumption may not hold in cases where RNA measurement timing introduces variability in expression profiles.

The authors present a deep learning pipeline to predict the steady-state distribution, model parameters, and statistical measures solely from scRNA-seq data. Results across three datasets appear robust, indicating that the tool successfully identifies genes associated with expression variability and generates consistent distributions based on its parameters. However, it remains unclear whether these results are sufficient to fully characterise the transcriptional bursting parameter space. The parameters identified by the tool pertain only to the steady-state distribution of the observed data, without ensuring that this distribution specifically originates from transcriptional bursting dynamics.

A primary concern with the TXmodel is its reliance on four independent parameters to describe gene state-switching dynamics. Although this general model can capture specific cases, such as the refractory and telegraph models, accurately estimating the parameters of the refractory model using only steady-state distributions and typical cell counts proves challenging in the absence of time-dependent data.

The claim that the GO analysis pertains specifically to DNA damage response signal transduction and cell cycle G2/M phase transition is not fully accurate. In reality, the GO analysis yielded stronger p-values for pathways related to the mitotic cell cycle checkpoint signalling. As presented, the GO analysis serves more as a preliminary starting point for further bioinformatics investigation that could substantiate these conclusions. Additionally, while GSEA analysis was performed following the GO analysis, the involvement of the cardiac muscle cell differentiation pathway remains unclear, as it was not among the GO terms identified in the initial GO analysis.

As the advancement is primarily methodological, it lacks a comprehensive comparison with traditional methods that serve similar functions. Consequently, the overall evaluation of the method, including aspects such as inference accuracy, computational efficiency, and memory cost, remains unclear. The paper would benefit from being contextualised alongside other computational tools aimed at integrating mechanistic modelling with single-cell RNA sequencing data. Additional context regarding the advantages of deep learning methods, the challenges of analysing large, high-dimensional datasets, and the complexities of parameter estimation for intricate models would strengthen the work.

Reviewer #1 (Public review):

Summary:

In this study, Hilda Tateossian et al. sought to identify the specific gene linked to hearing loss caused by otitis media effusion (OME) in individuals with Down syndrome (DS). They approached this by analyzing a series of mouse models of DS (referred to as the DpTyb lines), which include various duplications that encompass the regions of the mouse genome analogous to the human chromosome 21 (Hsa21). This allowed them to pinpoint genetic loci that may be associated with OME in DS. To control for external variables, such as genetic background and environmental influences, which could affect the development of chronic OME, all DpTyb mouse lines were maintained on a uniform C57BL/6J genetic background. The authors could show that chronic OME phenotypes were consistently reproducible across two research centers, the Francis Crick Institute and MRC Harwell Institute, supporting their conclusion while also reducing the likelihood that environmental factors could affect results.

The authors then focused on a significant locus on chromosome 16 in the Dp5Tyb mouse model that was strongly associated with OME. This locus contains only 12 genes, and it overlapped with the duplicated genomic regions in three additional mouse models (Dp1Tyb, Dp3Tyb, and Ts1Rhr), strengthening the link between this locus and OME. To identify the gene responsible within this critical interval, they conducted targeted crosses of Dp mouse lines (Dp1Tyb, Dp3Tyb, and Dp5Tyb) with gene knockout models. This strategy enabled them to normalize the copy number of specific genes within the progeny and assess the effect on OME. They found that reducing the gene dosage of Dyrk1a specifically restored a wild-type phenotype, implicating Dyrk1a as a key player in the development of OME in DS.

Given the broad biological roles of DYRK1A in various cellular pathways, the researchers also explored its effects on downstream proteins and pathways within the middle ear epithelium using immunohistochemistry and RT-qPCR. They uncovered several pathological mechanisms by which DYRK1A triplication could promote middle ear inflammation and increased vascular permeability. These mechanisms included the interaction between DYRK1A and TGFβ signaling, which affects proinflammatory cytokines IL-6 and IL-17, as well as elevated levels of VEGF in the middle ear that were accompanied by increased Hif1a expression.

At the morphological level, analyses by scanning electron microscopy further revealed a loss of cilia on the epithelial cells in the middle ears of 2-month-old Dp3Tyb and Dp5Tyb mutant mice, which likely contributes to the development of OME in DS.

Finally, to validate the relevance of their findings in humans, the researchers examined the expression of the 12 genes within the Dp5Tyb locus in samples from children with DS compared to unaffected parental controls, using qPCR. They found that among the 12 genes, DYRK1A showed the most significant fold increase in expression, further supporting its potential role in OME associated with DS.

Strengths:

(1) The manuscript is well-written and clearly presents both experimental design and results, together supporting the main conclusions.

(2) The experiments are carefully designed and executed, with data that convincingly support the identification of DYRK1A as a key gene involved in OME in DS. The use of gene knockouts to normalize Dyrk1a gene dosage within the Dp mouse lines was a thorough and successful strategy to strengthen and validate DYRK1A's causal inference in OME.

(3) The study goes beyond simple gene identification by exploring the downstream pathways and cellular effects of DYRK1A triplication. This mechanistic focus provides actionable insights into the potential molecular underpinnings of OME in DS.

(4) The study addresses a clinically important issue - OME in children with DS - and proposes DYRK1A as a practical therapeutic target. Based on data in mice and the high dose of DYRK1A in human clinical samples, the authors suggest that suppressing the activity of this gene by localized delivery of inhibitors to the middle ear cavity in DS patients can be a potential strategy for future treatment of OME.

Weaknesses:

No major weakness is identified.

The authors could discuss further the potential involvement of the other genes within the Dp5Tyb interval, and whether interactions among these genes could impact the disease or whether additional contributions to OME might be overlooked. Beyond DYRK1A expression, discussion of a more extensive analysis of the other genes within the locus in larger cohorts of individuals with DS and OME could add strength to the translational relevance of the findings.

Reviewer #1 (Public review):

Summary:

Ita Mehta and colleagues have investigated the role of putrescine in the pili-dependent surface motility of a laboratory strain of Escherichia coli. Enterobacteria, and particularly E. coli and Salmonella Typhimurium contain an enormous amount of putrescine and cadaverine compared to other bacteria. It has been estimated by Igarashi and colleagues that putrescine is present in E. coli at levels of at least 30 mM. Therefore, an investigation of the role of putrescine in E. coli is a welcome and important contribution to understanding polyamine function. The authors have used a comprehensive suite of E. coli gene deletion strains of putrescine biosynthetic, transport, and catabolic genes to understand the role of putrescine in pili-dependent surface motility.

Strengths:

Single gene deletions of arginine decarboxylase (speA) and agmatine ureohydrolase (speB), and a double gene deletion of the constitutive ornithine decarboxylase (speC) and the acid-inducible ornithine decarboxylase (speF), all of which are involved in putrescine biosynthesis, were found by the authors to be less efficient at pili-dependent surface motility. In addition, the putrescine transport genes plaP and potF are also required for efficient pili-dependent surface motility. Furthermore, the putrescine catabolic genes patA and puuA, when co-deleted, reduce pili-dependent surface motility. Transcriptomic analysis of the agmatine ureohydrolase (speB) gene deletion strain compared to the parental strain indicates a coordinated response to the speB gene deletion, including upregulation of ornithine biosynthetic genes and a downregulation of energy metabolic genes.

Weaknesses:

Because the cellular content of putrescine and other polyamines in the E. coli strains was not measured at any point in this study, and the gene deletions were not genetically complemented, it is not possible to definitively attribute physiological changes to the gene deletion strains specifically to changes in putrescine levels. Furthermore, the GT medium used for the mobility experiments contains trypsinated casein (tryptone), which may contain polyamines and most certainly contain arginine. There are two modes of putrescine biosynthesis in E. coli: one mode is the direct formation of putrescine from L-ornithine mediated by ornithine decarboxylase, and the other is the indirect pathway involving decarboxylation of arginine to form agmatine, followed by hydrolysis of agmatine to form putrescine and urea. In the absence of external arginine, putrescine is made by ornithine decarboxylase, however, in the presence of external arginine, ornithine biosynthesis is repressed and arginine decarboxylase becomes the primary biosynthetic route for putrescine biosynthesis. The GT medium used by the authors will tend to favor putrescine production from arginine. The speB gene deletion, which is used for the transcriptomic analyses, will even in the absence of external arginine, accumulate a very large amount of agmatine, greater than the level of putrescine. This will confound the interpretation of the effect of the speB gene deletion, because agmatine accumulation may be responsible for some of the effects, and the addition of external putrescine may repress agmatine accumulation. In the absence of polyamine level measurements, the relative levels of agmatine, the putrescine structural analog cadaverine, and the accumulation of decarboxylated S-adenosylmethionine, are not known. Changes to these metabolites could affect pili-dependent surface motility. Furthermore, it is not possible to conclude that the effects of gene deletions to biosynthetic, transport or catabolic genes on pili-dependent surface motility are due to changes in putrescine levels unless one takes it on faith that there must be changes to putrescine levels. Since E. coli contains such an enormous amount of putrescine, it is important to know how much putrescine must be depleted in order to exert a physiological effect.

The authors have tackled an important biomedical problem relevant to infections of the urogenital tract and also important for understanding the very unusual high level of putrescine in E. coli and related species. However, without confirmation of putrescine levels in their various strains, it would be difficult to unequivocally conclude that putrescine, or changes to its concentrations, are responsible for the physiological changes seen with the gene deletion strains.

Reviewer #1 (Public review):

This manuscript investigates homeostatic structural plasticity and its interplay with synaptic scaling. It uses an integrated approach with models and experiments.<br /> First, electrophysiology and chronic imaging are used to investigate the influence of different levels of AMPA-receptor antagonist NBQX, which allows for gradual activity reduction. Low levels of NBQX lead to a decrease of activity and a homeostatic increase of synapse density, whereas high levels block neural activity and lead to a reduced number of synapses after 3 days. The authors conclude that there must be a non-linear dependency between neuronal activities and rewiring. As a mathematical model for this, a biphasic structural plasticity rule is used, which, for increasing neural activities, switches from net synapse removal to growth and back, yielding two stable states at zero activity and the homeostatic target.<br /> This rule is tested in various situations in silico, yet without attempting to reproduce the experiment. First, in network development, the biphasic rule generates a lot of unconnected silent neurons and a reasonable network structure only emerges when the neurons are additionally supported by a facilitating input current. For comparison, a linear and a simpler nonlinear homeostatic plasticity model, which had been ruled out by the experimental data, need no external drive. Second, the consequences of lasting, altered stimulation in a subgroup of neurons is explored. As expected by the design of the rule, a small increase and decrease in stimulation leads to a decrease and increase of synaptic connectivity, respectively, and stimulation silencing led to a complete disconnection of the sub-population with restoration of activity. Unlike in previous studies, an asymmetry of pre- and postsynaptic plasticity mechanisms cannot rescue this. Third, silencing only for a short time period and then overstimulating the network led to overly strong activity, which may, however, also hold without silencing. For a transiently silenced stimulation, recovery is possible, but only when there is enough recurrent excitation from the rest of the network.<br /> Following this, the second part of the manuscript explores whether synaptic scaling may adapt and up-regulate the recurrent excitation, such that activity in a normally silenced subpopulation can be restored. Indeed, fast enough synaptic scaling leads to a recovery of neuronal activity in simulations, but leads to highly synchronous activity. A systematic model analysis shows at which scaling and rewiring speeds the activity and connectivity for a silenced sub-population can be restored. In between, however, the authors analyze spine sizes and changes in their whole population AMPAR-blocking experiments that demonstrate synaptic scaling and that structural plasticity and scaling effects may be jointly regulated. This experimental "break" between a simulation and its systematic analysis makes the paper harder to read and seems unnecessary as the analyses from the experiments are not repeated for the model.

Overall, the combination of experiments and simulations is a promising approach to investigate network self-organization. Especially the gradual blocking of activity is very valuable to inform mathematical models and distinguish them from alternatives. However, it remains unclear whether the model would actually reproduce the experiment. When switching from one to the other, this entails a detour to the conceptual level which makes the narrative sometimes hard to follow.

In summary, this manuscript makes a valuable contribution to discern the mathematical shape of a homeostatic structural plasticity model and understanding the necessity of synaptic scaling in the same network. Both experimental and computational methods are solid and well described. Yet, both parts could be linked better in order to obtain conclusions with more impact and generality.

Reviewer #1 (Public review):

Summary:

In this work, the authors investigate the functional difference between the most commonly expressed form of PTH, and a novel point mutation in PTH identified in a patient with chronic hypocalcemia and hyperphosphatemia. The value of this mutant form of PTH as a potential anabolic agent for bone is investigated alongside PTH(1-84), which is a current anabolic therapy. The authors have achieved the aims of the study. Their conclusion that this suggests a "new path of therapeutic PTH analog development" seems unfounded; the benefit of this PTH variant is not clear, but the work is still interesting.

The work does not identify why the patient with this mutation has hypocalcemia and hyperphosphatemia; this was not the goal of the study, but the data is useful for helping to understand it.

Strengths:

The work is novel, as it describes the function of a novel, naturally occurring, variant of PTH in terms of its ability to dimerise, to lead to cAMP activation, to increase serum calcium, and its pharmacological action compared to normal PTH.

Weaknesses:

(1) The use of very young, 10 week old, mice as a model of postmenopausal osteoporosis remains a limitation of this study, but this is now quite clearly described as a limitation,, including justifying the use of the primary spongiosa as a measurement site.

(2) Methods have been clarified. It is still necessary to properly define the micro-CT threshold in mm HA/cc^3. I think it might be at about 200mg HA/cc^3 in this study.

(3) The apparent contradiction between the cortical thickness data (where there is no difference between the two PTH formulations) and the mechanical testing data (where there is a difference) remains unresolved. It is still not clear whether there is a material defect in the bone, which can be partially assessed by reporting the 3 point bending test, corrected for the diameters of the bone (i.e. as stress / strain curves).

(4) It is also puzzling that both dimeric and monomeric PTH lead to a reduction in total bone area (cross sectional area?). This would suggest a reduction in bone growth. This should be discussed in the work.

Reviewer #1 (Public review):

Aging reduces tissue regeneration capacity, posing challenges for an aging population. In this study, the authors investigate impaired bone healing in aging, focusing on calvarial bones, and introduce a two-part rejuvenation strategy. Aging depletes osteoprogenitor cells and reduces their function, which hinders bone repair. Simply increasing the number of these cells does not restore their regenerative capacity in aged mice, highlighting intrinsic cellular deficits. The authors' strategy combines Wnt-mediated osteoprogenitor expansion with intermittent fasting, which remarkably restores bone healing. Intermittent fasting enhances osteoprogenitor function by targeting NAD+ pathways and gut microbiota, addressing mitochondrial dysfunction - an essential factor in aging. This approach shows promise for rejuvenating tissue repair, not only in bones but potentially across other tissues.

This study is exciting, impressive, and novel. The data presented is robust and supports the findings well.

Reviewer #1 (Public review):

Summary:

Nitric oxide (NO) has been implicated as a neuromodulator in the retina. Specific types of amacrine cells (ACs) produce and release NO in a light-dependent manner. NO diffuses freely through the retina and can modulate intracellular levels of cGMP, or directly modify and modulate proteins via S-nitrosylation, leading to changes in gap-junction coupling, synaptic gain, and adaptation. Although these system-wide effects have been documented, it is not well understood how the physiological function of specific neuronal types is affected by NO. This study aims to address this gap in our knowledge.

There are two major findings. 1) About a third of the retinal ganglion cells display cell-type specific adaptation to prolonged stimulus protocols. 2) Application of NO specifically affected Off-suppressed ganglion cells designated as G32 cells. The G32 cluster likely contains 3 ganglion cell types that are differentially affected.

This is the first comprehensive analysis of the functional effects of NO on ganglion cells in the retina. The cell-type specificity of the effects is surprising and provides the field with valuable new information.

Strengths:

NO was expected to produce small effects, and considerable effort was expended in validating the system to ensure that changes in the state of the preparation would not confound any effects of NO. The authors used a sequential stimulus protocol to control for changes in the sensitivity of the retina during the extended recording periods. The approach potentially increases the sensitivity of the measurements and allows more subtle effects to be observed.

Neural activity was measured by Ca-imaging. Responsive ganglion cells were grouped into 32 types using a clustering analysis. Initial control experiments demonstrated that the cell-types revealed by the analysis largely recapitulate those from their earlier landmark study using a similar approach.

Application of NO to the retina modulated responses of a single cluster of cells, labeled G32, while having little effect on the remaining 31 clusters. In separate experiments, ganglion cell spiking activity was recorded on a multi-electrode array (MEA). Together the Ca-imaging and MEA recordings provide complementary approaches and demonstrate that NO modulates the temporal but not spatial properties of affected cell-types.

Weaknesses:

The concentration of NO used in these experiments was ~0.25µM, which is 5- to 10-fold lower than the endogenous concentration previously measured in rodent retina. It is perhaps surprising that this relatively low NO concentration produced significant effects. However, the endogenous measurements were done in an eye-cup preparation, while the current experiments were performed in a bare (no choroid) preparation. Perhaps the resting NO level is lower in this preparation. It is also possible that the low concentration of NO promoted more selective effects.

for - definition - spiral of the - 5 Elements Mandala - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - need to move - from linear pyramid, neoliberal logic - to trends logic - multi-dimensional - reflexive - feedbacks - intertwingled - need to know what you stand for and - what you stand against ( the dominant neoliberal culture)

for - definition - ahistorical amnesia - plagues philanthropy - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - application - Deep Humanity BEing Journey - onto shift - ontological shift - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - key insight - challenging ourselves for authentic, transformative change - inner and outer work to dismantle oppressive, entrenched systems - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - ontological shift - from totalizing neoliberalism - to spiral logic - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - adjacency - ontological shift - Deep Humanity - asking these fundamental questions - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - definition - ontological shift - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - adjacency - Deep Humanity - can provide new vocabulary and ideas to support - the horizon 3 - ontological shift - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

adjacency - between - ontological shift to reach horizon 3 - Deep Humanity - adjacency relationship - Deep Humanity may offer a new language and vocabulary for this Horizon 3 shift ontology

for - quote - Flow of wealth to the lowest place - Colonial project stops flow to the lowest place - Todd James - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - quote / key insight - must interrogate the deeper thought patterns else - we risk repeating simplistic linear transition social movements that have failed over the past centuries - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - definition - Three Horizon Framework - developed by Bill Sharp - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - example - Three Horizon Framework

example - Three Horizon Framework - horizon 1 - carbon credits - carbon capture - green new deal - green growth - reforming democracy - more humane capitalism - horizon 2 - equity and justice - decolonization - transition pathways to disrupt ideologies - formative stage - ontological - still operating in frame of modernity - still operating in material realm - horizon 3 - new ways of being, living seeing, worldviews - dearth of imagination

for - definition - just transition movement - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - Mexican artist Patrick Cruz - redefining - anthropocene - to age of consequence - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy

for - key insight - Philanthropies have decided on the outside, which communities and which problems need to be solved - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

comment - So true! Who hasn't experienced the NGO coming into the community with a know-it-all attitude and already decided who will receive what funds for what project. It's all decided ahead of time then offered! - We don't want to fall into the same trap!

for - key insight - as individuals, we promote neoliberalism - via entrenched and unconscious colonialism - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - deep entrenchment and entrainment of neolieralism in our bodies - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

comment - The depth of entrainment of neoliberalism in our bodies is very pronounced - This is why it is so difficult to make adhoc change because it faces so much opposition emerging from the unconscious

for - key insight - severe limits of philanthropy - abiding by neoliberal logic severely constrains them - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

for - progress traps - science - technology - neoliberalism - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023

progress traps - science - technology - neoliberalism - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - It is with progress that the modernity shaped by centuries of scaled-out industrial,scientific and technological innovation has created all our crisis. - It is absurd to say that science, technology and industrialization did not play a major role in the creation of all the crisis of modernity - If science, technology and industry inherently embed separation, how do we use it in a meaningful way that doesn't lead to another progress trap? - What are the motives of those who fund technology?

for - key insight - neoliberalism and industrial capitalism were based on Descarte and our separation from the natural world - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - adjacency - materialism, science and neoliberalism - will technology save us? - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - to - The Three Great Separations

key insight / summary - neoliberalism and industrial capitalism were based on Descarte and our separation from the natural world - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - FIrst, Descarte separated the mind from the body. We have the paradox of: - godlike mind housed in - animalistic bodies - (incidentally, this sets us up for the exageration of the existential crisis of the denial of death in modernity - Ernest Becker) - Then we impose separation of external vs internal world - Then, we have separate categories of mind and nature, and we begin othering of: - women - other (indigenous) cultures - What Alnoor and Lynn forgot to mention was that there is another separation that preceded the industrial revolution, the separation of people into distinct classes of: - producer - consumer - Then with the advance of Newtonian physics and the wild success of materialist theory applied to create a plethora of industrial technologies, a wedding occurred between: - dualism and - materialism - Materialism decomposes everything into subatomic particles that a rational mind can understand - To those who think science and technology can save us from the crisis it helped create - the deeper understanding reveals that science and technology are themselves agents of separation.

to - See the three great separations - https://hyp.is/go?url=https%3A%2F%2Finthesetimes.com%2Farticle%2Findustrial-agricultural-revolution-planet-earth-david-korten&group=world

for - definition - neoliberalism - as the name of the dominant, totalizing, cultural operating system of modernity - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - summary - neoliberalism - as the name of the dominant, totalizing, cultural operating system of modernity - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 definition - neoliberalism - as the name of the dominant, totalizing, cultural operating system of modernity - Post Capitalist Philanthropy Webinar 1 - Alnoor Ladha - Lynn Murphy - 2023 - Neoliberalism is a totalizing, cultural operating system for modernity - It is all of these things: - a political philosophy - an economic practice - a cosmology - a wordview - an ontology - a theocracy - a religious worldview based on faith - Most of the dogmas of neoliberalism have been proven to be false, and yet it is still taught in most institutions of higher education summary - Some of the premises of neoliberalism are: - 1. humans are homo economicus - our chief concern is our selves and NOT others - Enlightenment theories - Scientism - Evolutionary theory - All our systems are designed on this false premise - 2. Hierarchy is inevitable and necessary for order. Without it, we would revert to beasts - The system embeds - Patriarchism - White Supremacy - Gender inequality - 3. The individual is the primary unit of power - together with 1) and 2), it creates inherent competition - 4. Material wealth and power is the measure of wellbeing - If you have money, you are considered a success, otherwise, you are considered a moral failure