Reviewer #1 (Public Review):

Summary:

In their manuscript, Schmidlin, Apodaca, et al try to answer fundamental questions about the evolution of new phenotypes and the trade-offs associated with this process. As a model, they use yeast resistance to two drugs, fluconazole and radicicol. They use barcoded libraries of isogenic yeasts to evolve thousands of strains in 12 different environments. They then measure the fitness of evolved strains in all environments and use these measurements to examine patterns in fitness trade-offs. They identify only six major clusters corresponding to different trade-off profiles, suggesting the vast genotypic landscape of evolved mutants translates to a highly constrained phenotypic space. They sequence over a hundred evolved strains and find that mutations in the same gene can result in different phenotypic profiles.

Overall, the authors deploy innovative methods to scale up experimental evolution experiments, and in many aspects of their approach tried to minimize experimental variation.

Weaknesses:

(1) One of the objectives of the authors is to characterize the extent of phenotypic diversity in terms of resistance trade-offs between fluconazole and radicicol. To minimize noise in the measurement of relative fitness, the authors only included strains with at least 500 barcode counts across all time points in all 12 experimental conditions, resulting in a set of 774 lineages passing this threshold. This corresponds to a very small fraction of the starting set of ~21 000 lineages that were combined after experimental evolution for fitness measurements. As the authors briefly remark, this will bias their datasets for lineages with high fitness in all 12 environments, as all these strains must be fit enough to maintain a high abundance. One of the main observations of the authors is phenotypic space is constrained to a few clusters of roughly similar relative fitness patterns, giving hope that such clusters could be enumerated and considered to design antimicrobial treatment strategies. However, by excluding all lineages that fit in only one or a few environments, they conceal much of the diversity that might exist in terms of trade-offs and set up an inclusion threshold that might present only a small fraction of phenotypic space with characteristics consistent with generalist resistance mechanisms or broadly increased fitness. This has important implications regarding the general conclusions of the authors regarding the evolution of trade-offs.

(2) Most large-scale pooled competition assays using barcodes are usually stopped after ~25 to avoid noise due to the emergence of secondary mutations. The authors measure fitness across ~40 generations, which is almost the same number of generations as in the evolution experiment. This raises the possibility of secondary mutations biasing abundance values, which would not have been detected by the whole genome sequencing as it was performed before the competition assay.

(3) The approach used by the authors to identify and visualize clusters of phenotypes among lineages does not seem to consider the uncertainty in the measurement of their relative fitness. As can be seen from Figure S4, the inter-replicate difference in measured fitness can often be quite large. From these graphs, it is also possible to see that some of the fitness measurements do not correlate linearly (ex.: Med Flu, Hi Rad Low Flu), meaning that taking the average of both replicates might not be the best approach. Because the clustering approach used does not seem to take this variability into account, it becomes difficult to evaluate the strength of the clustering, especially because the UMAP projection does not include any representation of uncertainty around the position of lineages. This might paint a misleading picture where clusters appear well separate and well defined but are in fact much fuzzier, which would impact the conclusion that the phenotypic space is constricted.

(4) The authors make the decision to use UMAP and a gaussian mixed model to cluster and represent the different fitness landscapes of their lineages of interest. Their approach has many caveats. First, compared to PCA, the axis does not provide any information about the actual dissimilarities between clusters. Using PCA would have allowed a better understanding of the amount of variance explained by components that separate clusters, as well as more interpretable components. Second, the advantages of dimensional reduction are not clear. In the competition experiment, 11/12 conditions (all but the no drug, no DMSO conditions) can be mapped to only three dimensions: concentration of fluconazole, concentration of radicicol, and relative fitness. Each lineage would have its own fitness landscape as defined by the plane formed by relative fitness values in this space, which can then be examined and compared between lineages. Third, the choice of 7 clusters as the cutoff for the multiple Gaussian model is not well explained. Based on Figure S6A, BIC starts leveling off at 6 clusters, not 7, and going to 8 clusters would provide the same reduction as going from 6 to 7. This choice also appears arbitrary in Figure S6B, where BIC levels off at 9 clusters when only highly abundant lineages are considered. This directly contradicts the statement in the main text that clusters are robust to noise, as more a stringent inclusion threshold appears to increase and not decrease the optimal number of clusters. Additional criteria to BIC could have been used to help choose the optimal number of clusters or even if mixed Gaussian modeling is appropriate for this dataset.

(5) Large-scale barcode sequencing assays can often be noisy and are generally validated using growth curves or competition assays. Having these types of results would help support the accuracy of the main assay in the manuscript and thus better support the claims of the authors.

Reviewer #1 (Public Review):

Mehrdad Kashefi et al. investigated the availability of planning future reaches while simultaneously controlling the execution of the current reach. Through a series of experiments employing a novel sequential arm reaching paradigm they developed, the authors made several findings: 1) participants demonstrate the capability to plan future reaches in advance, thereby accelerating the execution of the reaching sequence, 2) planning processes for future movements are not independent one another, however, it's not a single chunk neither, 3) Interaction among these planning processes optimizes the current movement for the movement that comes after for it.

The question of this paper is very interesting, and the conclusions of this paper are well supported by data.

Reviewer #1 (Public Review):

Summary:

HIV associated nephropathy (HIVAN) is a rapidly progressing form of kidney disease that manifests secondary to untreated HIV infection and is predominantly seen in individuals of African descent. Tg26 mice carrying an HIV transgene lacking gag and pol exhibit high levels of albuminuria and rapid decline in renal function that recapitulates many features of HIVAN in humans. HIVAN is seen predominantly in individuals carrying two copies of missense variants in the APOL1 gene, and the authors have previously shown that APOL1 risk variant mRNA induces activity of the double strand RNA sensor kinase PKR. Because of the tight association between the APOL1 risk genotype and HIVAN, the authors hypothesized that PKR activation may mediate the renal injury in Tg26 mice, and tested this hypothesis by treating mice with a commonly used PKR inhibitory compound called C16. Treatment with C16 substantially attenuated renal damage in the Tg26 model as measured by urinary albumin/creatinine ratio, urinary NGAL/creatinine ratio and improvement in histology. The authors then performed bulk and single-nucleus RNAseq on kidneys from mice from different treatment groups to identify pathways and patterns of cell injury associated with HIV transgene expression as well as to determine the mechanistic basis for the effect of C16 treatment. They show that proximal tubule nuclei from Tg26 mice appear to have more mitochondrial transcripts which was reversed by C16 treatment and suggest that this may provide evidence of mitochondrial dysfunction in this model. They explore this hypothesis by showing there is a decrease in the expression of nuclear encoded genes and proteins involved in oxidative phosphorylation as well as a decrease in respiratory capacity via functional assessment of respiration in tubule and glomerular preparations from these mouse kidneys. All of these changes were reversed by C16 treatment. The authors propose the existence of a novel injured proximal tubule cell-type characterized by the leak of mitochondrial transcripts into the nucleus (PT-Mito). Analysis of HIV transgene expression showed high level expression in podocytes, consistent with the pronounced albuminuria that characterizes this model and HIVAN, but transcripts were also detected in tubular and endothelial cells. Because of the absence of mitochondrial transcripts in the podocytes, the authors speculate that glomerular mitochondrial dysfunction in this model is driven by damage to glomerular endothelial cells.

Strengths:

The strengths of this study include the comprehensive transcriptional analysis of the Tg26 model, including an evaluation of HIV transgene expression, which has not been previously reported. This data highlights that HIV transcripts are expressed in a subset of podocytes, consistent with the highly proteinuric disease seen in mouse and humans. However, transcripts were also seen in other tubular cells, notably intercalated cells, principal cells and injured proximal tubule cells. Though the podocyte expression makes sense, the relevance of the tubular expression to human disease is still an open question.

The data in support of mitochondrial dysfunction are also robust and rely on combined evidence from downregulation of transcripts involved in oxidative phosphorylation, decreases in complex I and II as determined by immunoblot, and assessments of respiratory capacity in tubular and glomerular preparations. These data are largely consistent with other preclinical renal injury model reported in the literature as well as previous, less thorough assessments in the Tg26 model.

Comments on latest version:

The authors have revised the manuscript to acknowledge the potential limitations of the C16 tool compound used and have performed some additional analyses that suggest the PT-Mito population can be identified in samples from KPMP. The authors added some control images for the in situ hybridizations, which are helpful, though they don't get to the core issue of limited resolution to determine whether mitochondrial RNA is present in the nuclei of injured PT cells. Some additional work has been done to show that C16 treatment results in a decrease in phospho-PKR, a readout of PKR inhibition. These changes strengthen the manuscript by providing some evidence for the translatability of the PT-mito cluster to humans and some evidence for on-target activity for C16. It would be helpful if the authors could quantify the numbers of cells in IHC with nuclear transcripts as well as pointing out some specific examples in the images provided, as comparator data for the snRNAseq studies in which 3-6% of cortex cells had evidence of nuclear mitochondrial transcripts.

Reviewer #1 (Public Review):

This study is convincing because they performed time-resolved X-ray crystallography under different pH conditions using active/inactive metal ions and PpoI mutants, as with the activity measurements in solution in conventional enzymatic studies. Although the reaction mechanism is simple and maybe a little predictable, the strength of this study is that they were able to validate that PpoI catalyzes DNA hydrolysis through "a single divalent cation" because time-resolved X-ray study often observes transient metal ions which are important for catalysis but are not predictable in previous studies with static structures such as enzyme-substrate analog-metal ion complexes. The discussion of this study is well supported by their data. This study visualized the catalytic process and mutational effects on catalysis, providing a new insight into the catalytic mechanism of I-PpoI through a single divalent cation. The authors found that His98, a candidate of proton acceptor in the previous experiments, also affects the Mg2+ binding for catalysis without the direct interaction between His98 and the Mg2+ ion, suggesting that "Without a proper proton acceptor, the metal ion may be prone for dissociation without the reaction proceeding, and thus stable Mg2+ binding was not observed in crystallo without His98". In the future, this interesting feature observed in I-PpoI should be investigated by biochemical, structural and computational analyses using other one metal-ion dependent nucleases.

Reviewer #1 (Public Review):

Zheng et al. study the 'glass' transitions that occurs in proteins at ca. 200K using neutron diffraction and differential isotopic labeling (hydrogen/deuterium) of the protein and solvent. To overcome limitations in previous studies, this work is conducted in parallel with 4 proteins (myoglobin, cytochrome P450, lysozyme and green fluorescent protein) and experiments were performed at a range of instrument time resolutions (1ns - 10ps). The author's data looks compelling, and suggests that transitions in the protein and solvent behavior are not coupled and contrary to some previous reports, the apparent water transition temperature is a 'resolution effect'; i.e. instrument response is limited. This is likely to be important in the field, as a reassessment of solvent 'slaving' and the role of the hydration shell on protein dynamics should be reassessed in light of these findings.

Reviewer #1 (Public Review):<br /> Summary:

Zhu et al., investigate the cellular defects in glia as a result of loss in DEGS1/ifc encoding the dihydroceramide desaturase. Using the strength of Drosophila and its vast genetic toolkit, they find that DEGS1/ifc is mainly expressed in glia and its loss leads to profound neurodegeneration. This supports a role for DEGS1 in the developing larval brain as it safeguards proper CNS development. Loss of DEGS1/ifc leads to dihydroceramide accumulation in the CNS and induces alteration in the morphology of glial subtypes and a reduction in glial number. Cortex and ensheathing glia appeared swollen and accumulated internal membranes. Astrocyte-glia on the other hand displayed small cell bodies, reduced membrane extension and disrupted organization in the dorsal ventral nerve cord. They also found that DEGS1/ifc localizes primarily to the ER. Interestingly, the authors observed that loss of DEGS1/ifc drives ER expansion and reduced TGs and lipid droplet numbers. No effect on PC and PE and a slight increase in PS.

The conclusions of this paper are well supported by the data. The study could be further strengthened by a few additional controls and/or analyses.

Strengths:

This is an interesting study that provides new insight into the role of ceramide metabolism in neurodegeneration.

The strength of the paper is the generation of LOF lines, the insertion of transgenes and the use of the UAS-GAL4/GAL80 system to assess the cell-autonomous effect of DEGS1/ifc loss in neurons and different glial subtypes during CNS development.

The imaging, immunofluorescence staining and EM of the larval brain and the use of the optical lobe and the nerve cord as a readout are very robust and nicely done.

Drosophila is a difficult model to perform core biochemistry and lipidomics but the authors used the whole larvae and CNS to uncover global changes in mRNA levels related to lipogenesis and the unfolded protein responses as well as specific lipid alterations upon DEGS1/ifc loss.

Weaknesses:

The authors performed lipidomics and RTqPCR on whole larvae and larval CNS from which it is impossible to define the cell type-specific effects. Ideally, this could be further supported by performing single cell RNAseq on larval brains to tease apart the cell-type specific effect of DEGS1/ifc loss.

It's clear from the data that the accumulation of dihydroceramide in the ER triggers ER expansion but it remains unclear how or why this happens. Additionally, the authors assume that, because of the reduction in LD numbers, that the source of fatty acids comes from the LDs. But there is no data testing this directly.

The authors performed a beautiful EMS screen identifying several LOF alleles in ifc. However, the authors decided to only use KO/ifcJS3. The paper could be strengthened if the authors could replicate some of the key findings in additional fly lines.

The authors use M{3xP3-RFP.attP}ZH-51D transgene as a general glial marker. However, it would be advised to show the % overlap between the glial marker and the RFP since a lot of cells are green positive but not perse RFP positive and vice versa.

The authors indicate that other 3xP3 RFP and GFP transgenes at other genomic locations also label most glia in the CNE. Do they have a preferential overlap with the different glial subtypes?

Reviewer #1 (Public Review):

Summary:

The authors present a theoretical treatment of what they term the "Wright-Fisher-Haldane" model, a claimed modification of the standard model of genetic drift that accounts for variability in offspring number, and argue that it resolves a number of paradoxes in molecular evolution. Ultimately, I found this manuscript quite strange. The notion of effective population size as inversely related to the variance in offspring number is well known in the literature, and not exclusive to Haldane's branching process treatment. However, I found the authors' point about variance in offspring changing over the course of, e.g. exponential growth fairly interesting, and I'm not sure I'd seen that pointed out before. Nonetheless, I don't think the authors' modeling, simulations, or empirical data analysis are sufficient to justify their claims.

Weaknesses:

I have several outstanding issues. First of all, the authors really do not engage with the literature regarding different notions of an effective population. Most strikingly, the authors don't talk about Cannings models at all, which are a broad class of models with non-Poisson offspring distributions that nonetheless converge to the standard Wright-Fisher diffusion under many circumstances, and to "jumpy" diffusions/coalescents otherwise (see e.g. Mohle 1998, Sagitov (2003), Der et al (2011), etc.). Moreover, there is extensive literature on effective population sizes in populations whose sizes vary with time, such as Sano et al (2004) and Sjodin et al (2005). Of course in many cases here the discussion is under neutrality, but it seems like the authors really need to engage with this literature more.

The most interesting part of the manuscript, I think, is the discussion of the Density Dependent Haldane model (DDH). However, I feel like I did not fully understand some of the derivation presented in this section, which might be my own fault. For instance, I can't tell if Equation 5 is a result or an assumption - when I attempted a naive derivation of Equation 5, I obtained E(K_t) = 1 + r/c*(c-n)*dt. It's unclear where the parameter z comes from, for example. Similarly, is equation 6 a derivation or an assumption? Finally, I'm not 100% sure how to interpret equation 7. I that a variance effective size at time t? Is it possible to obtain something like a coalescent Ne or an expected number of segregating sites or something from this?

Similarly, I don't understand their simulations. I expected that the authors would do individual-based simulations under a stochastic model of logistic growth, and show that you naturally get variance in offspring number that changes over time. But it seems that they simply used their equations 5 and 6 to fix those values. Moreover, I don't understand how they enforce population regulation in their simulations---is N_t random and determined by the (independent) draws from K_t for each individual? In that case, there's no "interaction" between individuals (except abstractly, since logistic growth arises from a model that assumes interactions between individuals). This seems problematic for their model, which is essentially motivated by the fact that early during logistic growth, there are basically no interactions, and later there are, which increases variance in reproduction. But their simulations assume no interactions throughout!

The authors also attempt to show that changing variance in reproductive success occurs naturally during exponential growth using a yeast experiment. However, the authors are not counting the offspring of individual yeast during growth (which I'm sure is quite hard). Instead, they use an equation that estimates the variance in offspring number based on the observed population size, as shown in the section "Estimation of V(K) and E(K) in yeast cells". This is fairly clever, however, I am not sure it is right, because the authors neglect covariance in offspring between individuals. My attempt at this derivation assumes that I_t | I_{t-1} = \sum_{I=1}^{I_{t-1}} K_{i,t-1} where K_{i,t-1} is the number of offspring of individual i at time t-1. Then, for example, E(V(I_t | I_{t-1})) = E(V(\sum_{i=1}^{I_{t-1}} K_{i,t-1})) = E(I_{t-1})V(K_{t-1}) + E(I_{k-1}(I_{k-1}-1))*Cov(K_{i,t-1},K_{j,t-1}). The authors have the first term, but not the second, and I'm not sure the second can be neglected (in fact, I believe it's the second term that's actually important, as early on during growth there is very little covariance because resources aren't constrained, but at carrying capacity, an individual having offspring means that another individuals has to have fewer offspring - this is the whole notion of exchangeability, also neglected in this manuscript). As such, I don't believe that their analysis of the empirical data supports their claim.

Thus, while I think there are some interesting ideas in this manuscript, I believe it has some fundamental issues: first, it fails to engage thoroughly with the literature on a very important topic that has been studied extensively. Second, I do not believe their simulations are appropriate to show what they want to show. And finally, I don't think their empirical analysis shows what they want to show.

References:

Möhle M. Robustness results for the coalescent. Journal of Applied Probability. 1998;35(2):438-447. doi:10.1239/jap/1032192859

Sagitov S. Convergence to the coalescent with simultaneous multiple mergers. Journal of Applied Probability. 2003;40(4):839-854. doi:10.1239/jap/1067436085

Der, Ricky, Charles L. Epstein, and Joshua B. Plotkin. "Generalized population models and the nature of genetic drift." Theoretical population biology 80.2 (2011): 80-99

Sano, Akinori, Akinobu Shimizu, and Masaru Iizuka. "Coalescent process with fluctuating population size and its effective size." Theoretical population biology 65.1 (2004): 39-48

Sjodin, P., et al. "On the meaning and existence of an effective population size." Genetics 169.2 (2005): 1061-1070

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes a series of experiments using human intracranial neural recordings designed to evaluate processing of self-generated speech in the setting of feedback delays. Specifically, the authors aim to address the question about the relationship between speech-induced suppression and feedback sensitivity in the auditory cortex, which, relationship has been conflicting in the literature. They found a correlation between speech suppression and feedback delay sensitivity, suggesting a common process. Additional controls were done for possible forward suppression/adaptation, as well as controlling for other confounds due to amplification, etc.

Strengths:<br /> The primary strength of the manuscript is the use of human intracranial recording, which is a valuable resource and gives better spatial and temporal resolution than many other approaches. The use of delayed auditory feedback is also novel and has seen less attention than other forms of shifted feedback during vocalization. Analyses are robust and include demonstrating a scaling of neural activity with the degree of feedback delay, more robust evidence for error encoding than simply using a single feedback perturbation.

Weaknesses:<br /> Some of the analyses performed differ from those used in past work, which limits the ability to directly compare the results. Notably, past work has compared feedback effects between production and listening, which was not done here. There were also some unusual effects in the data, such as increased activity with no feedback delay when wearing headphones, that the authors attempted to control for with additional experiments, but remain unclear. Confounds by behavioral results of delayed feedback are also unclear.

Overall the work is well done and clearly explained. The manuscript addresses an area of some controversy and does so in a rigorous fashion, namely the correlation between speech-induced suppression and feedback sensitivity (or lack thereof). While the data presented overlap that collected and used for a previous paper, this is expected given the rare commodity these neural recordings represent. Contrasting these results to previous ones using pitch-shifted feedback should spawn additional discussion and research, including verification of the previous finding, looking at how the brain encodes feedback during speech over multiple acoustic dimensions, and how this information can be used in speech motor control.

Reviewer #1 (Public Review):

Summary:

The authors want to elucidate which are the mechanisms that regulate the immune response in physiological conditions in cortical development. To achieve this goal, authors used a wide range of mutant mice to analyse the consequences of immune activation in the formation of cortical ectopia in mice.

Strengths:

The authors demonstrated that Abeta monomers are anti-inflammatory and inhibit microglial activation. This is a novel result that demonstrates the physiological role of APP in cortical development.

Weaknesses:

-On the other hand, cortical ectopia has been already described in mouse models in which the amyloid signalling has been disrupted (Herms et al., 2004; Guenette et al., 2006), making the current study less novel.

One of the molecules analysed is Ric8a, a GTPase activator involved in neuronal development. Authors used the conditional mutant mice Emx1-Ric8a to delete Ric8a from early progenitors and glutamatergic neurons in the pallium. Emx1-Ric8a mutant mice present cortical ectopias and authors attributed this malformation to the increase in inflammatory response due to Ric8a deletion in microglia. Several discordances do not fit this interpretation:

-The role of Ric8a in cortical development and function has been already described in several papers, but none of them has been cited in the current manuscript (Kask et al., 2015, 2018; Ruisu et al., 2013; Tonissoo et al., 2006).

-Ectopia formation in the cortex has been already described in Nestin-Ric8a cKO mice (Kask et al., 2015). In the current manuscript, authors analyzed the same mutant mice (Nestin-Ric8a), but they did not detect any ectopia. Authors should discuss this discordance.

-Authors claim that microglia express Emx1, and therefore, Ric8a is deleted in microglia cells. However, the arguments for this assumption are very weak and the evidence suggests that this is not the case. This is an important point considering that authors want to emphasise the role of Ric8a in microglia activation, and therefore, additional experiments should demonstrate that Ric8a is deleted in microglia in Emx1-Ric8a mutant mice.

Reviewer #1 (Public Review):

Summary:<br /> Meissner et al describe an update on the collection of split-GAL4 lines generated by a consortium led by Janelia Research Campus. This follows the same experimental pipeline described before and presents as a significant increment to the present collection. This will strengthen the usefulness and relevance of "splits" as a standard tool for labs that already use this tool and attract more labs and researchers to use it.

Strengths:<br /> This manuscript presents a solid step to establish Split-GAL4 lines as a relevant tool in the powerful Drosophila toolkit. Not only does the raw number of available lines contribute to the relevance of this tool in the "technical landscape" of genetic tools, but additional features of this effort contribute to the successful adoption. These include:<br /> (1) A description of expression patterns in the adult and larvae, expanding the "audience" for these tools<br /> (2) A classification of line combination according to quality levels, which provides a relevant criterion while deciding to use a particular set of "splits".<br /> (3) Discrimination between male and female expression patterns, providing hints regarding the potential role of these gender-specific circuits.<br /> (4) The search engine seems to be user-friendly, facilitating the retrieval of useful information.<br /> Overall, the authors employed a pipeline that maximizes the potential of the Split-GAL4 collection to the scientific community.

Weaknesses:<br /> The following aspects apply:<br /> The use of split-GAL4 lines has improved tremendously the genetic toolkit of Drosophila and this manuscript is another step forward in establishing this tool in the genetic repertoire that laboratories use. Thus, this would be a perfect opportunity for the authors to review the current status of this tool, addressing its caveats and how to effectively implement it into the experimental pipeline.

(1) While the authors do bring up a series of relevant caveats that the community should be aware of while using split-GAL4 lines, the authors should take the opportunity to address some of the genetic issues that frequently arise while using the described genetic tools. This is particularly important for laboratories that lack the experience using split-GAL4 lines and wish to use them. Some of these issues are covertly brought up, but not entirely clarified.<br /> First, why do the authors (wisely) rescreen the lines using UAS-CsChrimson-mVenus? One reason is that using another transgene (such as UAS-GFP) and/or another genomic locus can drive a different expression pattern or intensities. Although this is discussed, this should be made more explicit and the readers should be aware of this.<br /> Second, it would be important to include a discussion regarding the potential of hemidriver lines to suffer from transvection effects whenever there is a genetic element in the same locus. These are serious issues that prevent a more reliable use of split-GAL4 lines that, once again, should be discussed.

(2) The authors simply mention that the goal of the manuscript is to "summarize the results obtained over the past decade.". A better explanation would be welcomed in order to understand the need of a dedicated manuscript to announce the availability of a new batch of lines when previous publications already described the Split-GAL4 lines. At the extreme, one might question why we need a manuscript for this when a simple footnote on Janelia's website would suffice.

Reviewer #1 (Public Review):

Their findings elucidate the mechanisms underlying 2-AA-mediated reduction of pyruvate transport into mitochondria, which impairs the interaction between ERRα and PGC1α, consequently suppressing MPC1 expression and reducing ATP production in tolerized macrophages.

This paper presents a novel discovery regarding the mechanisms through which PA regulates the bioenergetics of tolerized macrophages. This paper will provide valuable insights for the journal's broad readership of scientists.

Reviewer #1 (Public Review):

Summary:

Sumarac et al investigate differences in globus pallidus internus (GPi) spike activity and short- and long-term plasticity of direct pathway projections in patients with Parkinson's disease (PD) and dystonia. Their main claims are that GPi neurons exhibit distinct characteristics in these two disorders, with PD associated with specific power-frequency oscillations and dystonia showing lower firing rates, increased burstiness, and less regular activity. Additionally, long-term plasticity and synaptic depression appear to differ between the two conditions. The authors suggest that these findings support the concept of hyperfunctional GPi output in PD and hypofunctional output in dystonia, possibly driven by variations in plasticity of striato-pallidal synapses. Overall enthusiasm is relatively high, but I think the discussion omits discussing findings that don't align well with standard models.

Strengths:

- These types of studies are valuable as the data arise from patients who have dystonia or PD. This could provide unique insights into disease pathophysiology that might not be recapitulated in animal systems work.

Comments on latest version:

The authors addressed my concerns in their revision.

Reviewer #1 (Public Review):

Summary:<br /> The authors aim to consider the effects of phonotactics on the effectiveness of memory reactivation during sleep. They have created artificial words that are either typical or atypical and showed that reactivation improves memory for the latter but not the former.

Strengths:<br /> This is an interesting design and a creative way of manipulating memory strength and typicality. In addition, the spectral analysis on both the wakefulness data and the sleep data is well done. The article is clearly written and provides a relevant and comprehensive of the literature and of how the results contribute to it.

Weaknesses:<br /> (1) Unlike most research involving artificial language or language in general, the task engaged in this manuscript did not require (or test) learning of meaning or translation. Instead, the artificial words were arbitrarily categorised and memory was tested for that categorisation. This somewhat limits the interpretation of the results as they pertain to language science, and qualifies comparisons with other language-related sleep studies that the manuscript builds on.

(2) Participants had to determine whether words are linked with reward or omission of punishment (if correctly categorised). Therefore, the task isn't a mere item categorisation task (group A/B), but also involves the complicated effects of reward (e.g., reward/loss asymmetries as predicted by prospect theory). This is not, in itself, a flaw, but there isn't a clear hypothesis as to the effects of reward on categorisation, and therefore no real justification for this design. This aspect of the task may add unneeded complexity (at best) or some reward-related contamination of the results (at worst).

(3) The study starts off with a sample size of N=39 but excludes 17 participants for some crucial analyses. This is a high number, and exclusion criteria were not pre-registered. Having said that, some criteria seem very reasonable (e.g., excluding participants who were not fully exposed to words during sleep).

(4) Relatedly, the final N is low for a between-subjects study (N=11 per group). This is adequately mentioned as a limitation, but since it does qualify the results, it seemed important to mention it here.

(5) The linguistic statistics used for establishing the artificial words are all based on American English, and are therefore in misalignment with the spoken language of the participants (which was German). This is a limitation of the study. The experimenters did not check whether participants were fluent in English. In all fairness, the behavioural effects presented in Figure 2A are convincing, providing a valuable manipulation test.

Reviewer #1 (Public Review):

Summary:

The authors sought to understand the stage-dependent regulation of exophergenesis, a process thought to contribute to promoting neuronal proteostasis in C. elegans. Focusing on the ALMR neuron, they show that the frequency of exopher production correlates with the timing of reproduction. Using many genetic tools, they dissect the requirements of this pathway to eventually find that occupancy of the uterus acts as a signal to induce exophergenesis. Interestingly, the physical proximity of neurons to the egg zone correlates with exophergenesis frequency. The authors conclude that communication between the uterus and proximal neurons occurs through the sensing of mechanic forces of expansion normally provided by egg occupancy to coordinate exophergenesis with reproduction.

Strengths:

The genetic data presented is thorough and solid, and the observation is novel.

Weaknesses:

The authors have addressed the main weakness of the study in the revised manuscript, by providing data showing stimulation of exopher production in a single-copy transgenic line. Whether this process is related to the extrusion of cellular damage by the neurons in relatively young day 2 animals should be addressed in future studies.

Reviewer #1 (Public Review):

This study by Paoli et al. used a resonant scanning multiphoton microscope to examine olfactory representation in the projection neurons (PNs) of the honeybee with improved temporal resolution. PNs were classified into 9 groups based on their response patterns. Authors found that excitatory repose in the PNs precedes the inhibitory responses for ~40ms, and ~50% of PN responses contain inhibitory components. They built the neural circuit model of the mushroom body (MB) with evolutionally conserved features such as sparse representation, global inhibition, and a plasticity rule. This MB model fed with the experimental data could reproduce a number of phenomena observed in experiments using bees and other insects, including dynamical representations of odor onset and offset by different populations of Kenyon cells, prolonged representations of after-smell, different levels of odor-specificity for early/delay conditioning, and shift of behavioral timing in delay conditioning. The trace conditioning was also tested experimentally, although bees did not shift the timing of PER response to the post-odor period as the model predicted. The experimental data and the model provide a solid basis for future studies.

Reviewer #1 (Public Review):

Summary:

The manuscript by Tuberosa et al outlines the generation of a new set of transgenic mice that express different recombinases specifically in Smim32 positive cells. They show that Smim32 is a useful marker of the mouse claustrum. Therefore, these mice could be useful for functional studies focused on measuring claustrum activity or manipulating the claustrum using optogenetic and pharmacogenetic tools.

Strengths:

The manuscript provides a new genetic approach to target claustrum neurons, using Smim32. The work may help future studies where claustrum excitatory neurons are measured or manipulated.

Weaknesses:

A toolbox is only useful if others can use it. Therefore, these mice should be made available to the community through commercial vendors. Without this added step, this toolbox and method does not provide any utility to the research community.

The data presented and quantified in each figure subpanel are from N = 1 mouse. This is not acceptable or conventional. Replication is an important aspect of any paper, and currently, there are no replicates contained in the manuscript. Additional examples of female mice should also be included and separately quantified. Mice from different litters should be used for replicates.

Given the preliminary nature of these data from the minimum possible number of mice, a better characterization of all data should be undertaken.

The tone of the paper implies that this is the superior way to locate the claustrum. A more balanced discussion of the strengths and weaknesses of these mice should be included. Several sentences highlighting the shortfalls of other approaches are overstated and should be toned down.

Reviewer #1 (Public Review):

Summary

In this study, Takagi and colleagues demonstrate that changes in axonal arborization of the segmental wave motor command neurons are sufficient to change behavioral motor output.

The authors identify the Wnt receptors DFz2 and DFz4 and the ligand Wnt4 as modulators of stereotypic segmental arborization patterns of segmental wave neurons along the anterior-posterior body axis. Based on both embryonic expression pattern analysis and genetic manipulation of the signaling components in wave neurons (receptors) and the neuropil (Wnt4) the authors convincingly demonstrate that Wnt4 acts as a repulsive ligand for DFz2 that restricts posterior axon guidance of both anterior and posterior wave neurons. They also provide the first evidence that Wnt4 potentially acts as an attractive ligand for Df4 to promote the posterior extension of p-wave neurons. Interestingly, artificial optogenetic activation of all wave neurons that normally induces backward locomotion due to the activity of anterior wave neurons, fails to induce backward locomotion in a DFz2 knockdown condition with altered axonal extensions of all wave neurons towards posterior segments. In addition, the authors now observe enhanced fast-forward locomotion, a feature normally induced by posterior wave neurons. Consistent with these findings, they observe that the natural response to an anterior tactile stimulus is similarly altered in DFz2 knockdown animals. The animals respond with less backward movement and increased fast forward motion. These results suggest that alterations in the innervation pattern of wave motor command neurons are sufficient to switch behavioral response programs.

Strengths

The authors convincingly demonstrate the importance of Wnt signaling for anterior-posterior axon guidance of a single class of motor command neurons in the larval CNS. The demonstration that alteration of the expression level of a single axon guidance receptor is sufficient to not only alter the innervation pattern but to significantly modify the behavioral response program of the animal provides a potential entry point to understanding behavioral adaptations during evolution.

Weaknesses

While the authors demonstrate an alteration of the behavioral response to a natural tactile stimulus the observed effects, a reduction of backward motion and increased fast-foward locomotion, currently cannot be directly correlated to the morphological alterations observed in the single-neuron analyses. The authors do not report any loss of innervation in the "normal" target region but only a small additional innervation of more posterior regions. An analysis of synaptic connectivity and/or a more detailed morphological analysis that is supported by a larger number of analyzed neurons both in control and experimental animals would further strengthen the confidence of the study. As the authors suggest an alteration of the command circuitry, a direct observation of the downstream activation pattern in response to selective optogenetic stimulation of anterior wave neurons would further strengthen their claims (analogous to Takagi et al., 2017, Figure 4).

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors present a novel CRISPR/Cas9-based genetic tool for the dopamine receptor dop1R2. Based on the known function of the receptor in learning and memory, they tested the efficacy of the genetic tool by knocking out the receptor specifically in mushroom body neurons. The data suggest that dop1R2 is necessary for longer-lasting memories through its action on ⍺/ß and ⍺'/ß' neurons but is dispensable for short-term memory and thus in ɣ neurons. The experiments impressively demonstrate the value of such a genetic tool and illustrate the specific function of the receptor in subpopulations of KCs for longer-term memories. The data presented in this manuscript are significant.

Reviewer #1 (Public Review):

Summary:

This paper explores how diverse forms of inhibition impact firing rates in models for cortical circuits. In particular, the paper studies how the network operating point affects the balance of direct inhibition from SOM inhibitory neurons to pyramidal cells, and disinhibition from SOM inhibitory input to PV inhibitory neurons. This is an important issue as these two inhibitory pathways have largely been studies in isolation. Support for the main conclusions is generally solid, but could be strengthened by additional analyses.

Strengths:

A major strength of the paper is the systematic exploration of how circuit architecture effects the impact of inhibition. This includes scans across parameter space to determine how firing rates and stability depend on effective connectivity. This is done through linearization of the circuit about an effective operating point, and then the study of how perturbations in input effect this linear approximation.

Weaknesses:

The linearization approach means that the conclusions of the paper are valid only on the linear regime of network behavior. The paper would be substantially strengthened with a test of whether the conclusions from the linearized circuit hold over a large range of network activity. Is it possible to simulate the full network and do some targeted tests of the conclusions from linearization? Those tests could be guided by the linearization to focus on specific parameter ranges of interest.

The results illustrated in the figures are generally well described but there is very little intuition provided for them. Are there simplified examples or explanations that could be given to help the results make sense? Here are some places such intuition would be particularly helpful:<br /> page 6, paragraph starting "In sum ..."<br /> Page 8, last paragraph<br /> Page 10, paragraph starting "In summary ..."<br /> Page 11, sentence starting "In sum ..."

Reviewer #1 (Public Review):

Summary:

Dr. Santamaria's group previously utilized antigen-specific nanomedicines to induce immune tolerance in treating autoimmune diseases. The success of this therapeutic strategy has been linked to expanded regulatory mechanisms, particularly the role of T-regulatory type-1 (TR1) cells. However, the differentiation program of TR1 cells remained largely unclear. Previous work from the authors suggested that TR1 cells originate from T follicular helper (TFH) cells. In the current study, the authors aimed to investigate the epigenetic mechanisms underlying the transdifferentiation of TFH cells into IL-10-producing TR1 cells. Specifically, they sought to determine whether this process involves extensive chromatin remodeling or is driven by pre-existing epigenetic modifications. Their goal was to understand the transcriptional and epigenetic changes facilitating this transition and to explore the potential therapeutic implications of manipulating this pathway.

The authors successfully demonstrated that the TFH-to-TR1 transdifferentiation process is driven by pre-existing epigenetic modifications rather than extensive new chromatin remodeling. The comprehensive transcriptional and epigenetic analyses provide robust evidence supporting their conclusions.

Strengths:

(1) The study employs a broad range of bulk and single-cell transcriptional and epigenetic tools, including RNA-seq, ATAC-seq, ChIP-seq, and DNA methylation analysis. This comprehensive approach provides a detailed examination of the epigenetic landscape during the TFH-to-TR1 transition.

(2) The use of high-throughput sequencing technologies and sophisticated bioinformatics analyses strengthens the foundation for the conclusions drawn.

(3) The data generated can serve as a valuable resource for the scientific community, offering insights into the epigenetic regulation of T-cell plasticity.

(4) The findings have significant implications for developing new therapeutic strategies for autoimmune diseases, making the research highly relevant and impactful.

Weaknesses:

(1) While the scope of this study lies in transcriptional and epigenetic analyses, the conclusions need to be validated by future functional analyses.

(2) This study successfully identified key transcription factors and epigenetic marks. How these factors mechanistically drive chromatin closure and gene expression changes during the TFH-to-TR1 transition requires further investigation.

(3) The study provides a snapshot of the epigenetic landscape. Future dynamic analysis may offer more insights into the progression and stability of the observed changes.

Reviewer #1 (Public Review):

Summary:

The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.

Strengths:

(1) 7T high-resolution MRS is used.

(2) This study combines the behavioral tests, MRS, and fMRI.

Weaknesses:

Major:

(1) In Melnick (2013) IQ scores were measured by the full set of WAIS-III, including all subtests. However, this study only used visual spatial domain of gF. I wonder why only the visuo-spatial subtest was used not the full WAIS-III? I am wondering whether other subtests were conducted and, if so, please include the results as well to have comprehensive comparisons with Melnick (2013).

Minor:

(1) Table 1 and Table supplementary 1-3 contain many correlation results. But what are the main points of these values? Which values do the authors want to highlight? Why are only p-values shown with significance symbols in Table supplementary 2??

(2) Line 27, it is unclear to me what is "the canonical theory".

(3) Throughout the paper, the authors use "MT+", I would suggest using "hMT+" to indicate the human MT complex, and to be consistent with the human fMRI literature.

(4) At the beginning of the results section, I suggest including the total number of subjects. It is confusing what "31/36 in MT+, and 28/36 in V1" means.

(5) Line 138, "This finding supports the hypothesis that motion perception is associated with neural activity in MT+ area". This sentence is strange because it is a well established finding in numerous human fMRI papers. I think the authors should be more specific about what this finding implies.

(6) There are no unit labels for all x- and y-axies in Figure 1. I only see the unit for Conc is mmol per kg wet weight.

(7) Although the correlations are not significant in Figure supplement 2&3, please also include the correlation line, 95% confidence interval, and report the r values and p values (i.e., similar format as in Figure 1C).

(8) There is no need to separate different correlation figures into Figure supplementary 1-4. They can be combined into the same figure.

(9) Line 213, as far as I know, the study (Melnick et al., 2013) is a psychophysical study and did not provide evidence that the spatial suppression effect is associated with MT+.

(10) At the beginning of the results, I suggest providing more details about the motion discrimination tasks and the measurement of the BDT.

(11) Please include the absolute duration thresholds of the small and large sizes of all subjects in Figure 1.

(12) Figure 5 is too small. The items in plot a and b can be barely visible.

Reviewer #1 (Public Review):

In this revised manuscript, authors have conducted epigenetic and transcriptomic profiling to understand how environmental chemicals such as BPS can cause epimutations that can propagate to future generations. They used isolated somatic cells from mice (Sertoli, granulosa), pluripotent cells to model preimplantation embryos (iPSCs) and cells to model the germline (PGCLCs). This enabled them to model sequential steps in germline development, and when/how epimutations occur. The major findings were that BPS induced unique epimutations in each cell type, albeit with qualitative and quantitative cell-specific differences; that these epimutations are prevalent in regions associated with estrogen-response elements (EREs); and that epimutations induced in iPSCs are corrected as they differentiate into PGCLCs, concomitant with the emergence of de novo epimutations. This study will be useful in understanding the multigenerational effects of EDCs, and underlying mechanisms.

Strengths include:

(1) Using different cell types representing life stages of epigenetic programming and during which exposures to EDCs have different effects. This progression revealed information both about the correction of epimutations and the emergence of new ones in PGCLCs.

(2) Work conducted by exposing iPSCs to BPS or vehicle, then differentiating to PGCLCs, revealed that novel epimutations emerged.

(3) Relating epimutations to promoter and enhancer regions

During the review process, authors improved the manuscript through better organization, clarifying previous points from reviewers, and providing additional data.

Reviewer #1 (Public Review):

Summary:

Kan et al. report the serendipitous discovery of a Bacillus amyloliquefaciens strain that kills N. gonorrhoeae. They use TnSeq to identify that the anti-gonococcal agent is oxydifficidin and show that it acts at the ribosome and that one of the dedA gene products in N. gonorrhoeae MS11 is important for moving the oxydifficidin across the membrane.

Strengths:

This is an impressive amount of work, moving from a serendipitous observation through TnSeq to characterize the mechanism by which Oxydifficidin works.

Weaknesses:

(1) There are important gaps in the manuscript's methods.

(2) The work should evaluate antibiotics relevant to N. gonorrhoeae.

(3) The genetic diversity of dedA and rplL in N. gonorrhoeae is not clear, neither is it clear whether oxydifficidin is active against more relevant strains and species than tested so far.

Reviewer #1 (Public Review):

Summary:

This paper details a study of endothelial cell vessel formation during zebrafish development. The results focus on the role of aquaporins, which mediate the flow of water across the cell membrane, leading to cell movement. The authors show that actin and water flow together drive endothelial cell migration and vessel formation. If any of these two elements are perturbed, there are observed defects in vessels. Overall, the paper significantly improves our understanding of cell migration during morphogenesis in organisms.

Strengths:

The data are extensive and are of high quality. There is a good amount of quantification with convincing statistical significance. The overall conclusion is justified given the evidence.

Weaknesses:

There are two weaknesses, which if addressed, would improve the paper.

(1) The paper focuses on aquaporins, which while mediates water flow, cannot drive directional water flow. If the osmotic engine model is correct, then ion channels such as NHE1 are the driving force for water flow. Indeed this water is shown in previous studies. Moreover, NHE1 can drive water intake because the export of H+ leads to increased HCO3 due to the reaction between CO2+H2O, which increases the cytoplasmic osmolarity (see Li, Zhou and Sun, Frontiers in Cell Dev. Bio. 2021). If NHE cannot be easily perturbed in zebrafish, it might be of interest to perturb Cl channels such as SWELL1, which was recently shown to work together with NHE (see Zhang, et al, Nat. Comm. 2022).

(2) In some places the discussion seems a little confusing where the text goes from hydrostatic pressure to osmotic gradient. It might improve the paper if some background is given. For example, mention water flow follows osmotic gradients, which will build up hydrostatic pressure. The osmotic gradients across the membrane are generated by active ion exchangers. This point is often confused in literature and somewhere in the intro, this could be made clearer.

Reviewer #1 (Public Review):

Summary:

Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head restrained mice running down a virtual linear path. Mice were trained to collect water reward at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

The revised manuscript included additional evidence of increased (but transient) signal in LC axons after a transition to a novel environment during periods of immobility, and also that a change from dark to familiar environment induces a peak in LC axon activity, showing that LC input to dCA1 may not solely signal novelty.

Strengths:

The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis at the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

The authors have addressed my concerns in a thorough manner. The reviewer also appreciates the increased transparency of reporting in the revised manuscript.

Weaknesses:

Listed below are some remaining comments.<br /> The increase in LC activity with any change in environment (from familiar to novel or from dark to familiar) suggests that LC input acts not solely as a novelty signal, but as a general arousal or salience signal in response to environmental changes. Based on this, I have a couple of questions:

• Is the overall claim that LC input to the dHC signals novelty still valid based on observed findings - as claimed throughout the manuscript?<br /> • Would the omission of a reward be considered a salient change in the environment that activates LC signals, or is the LC not involved with processing reward-related information? Has the activity of LC and VTA axons been analysed in the seconds following reward presentation and/or omission?

Reviewer #1 (Public Review):

Summary:

Otero-Coronel et al. address an important question for neuroscience - how does a premotor neuron capable of directly controlling behavior integrate multiple sources of sensory inputs to inform action selection? For this, they focused on the teleost Mauthner cell, long known to be at the core of a fast escape circuit. What is particularly interesting in this work is the naturalistic approach they took. Classically, the M-cell was characterized, both behaviorally and physiologically, using an unimodal sensory space. Here the authors make the effort (substantial!) to study the physiology of the M-cell taking into account both the visual and auditory inputs. They performed well-informed electrophysiological approaches to decipher how the M-cell integrates the information of two sensory modalities depending on the strength and temporal relation between them.

Strengths:

The empirical results are convincing and well-supported. The manuscript is well-written and organized. The experimental approaches and the selection of stimulus parameters are clear and informed by the bibliography. The major finding is that multisensory integration increases the certainty of environmental information in an inherently noisy environment.

Weaknesses:

Even though the manuscript and figures are well organised, I found myself struggling to understand key points of the figures.

For example, in Figure 1 it is not clear what are actually the Tonic and Phasic components. The figure will benefit from more details on this matter. Then, in Figure 4 the label for the traces in panel A is needed since I was not able to pick up that they were coming from different sensory pathways.

In line 338 it should be optic tectum and not "optical tectum".

Reviewer #1 (Public Review):

Summary:

This work identified new NMD inhibitors and tested them for cancer treatment, based on the hypothesis that inhibiting NMD could lead to the production of cancer neoantigens from the stabilized mutant mRNAs, thereby enhancing the immune system's ability to recognize and kill cancer cells. Key points of the study include:

• Development of an RNA-seq based method for NMD analysis using mixed isogenic cells that express WT or mutant transcripts of STAG2 and TP53 with engineered truncation mutations.

• Application of this method for a drug screen and identified several potential NMD inhibitors.

• Demonstration that one of the identified compounds, LY3023414, inhibits NMD by targeting the SMG1 protein kinase in the NMD pathway in cultured cells and mouse xenografts.

• Due to the in vivo toxicity observed for LY3023414, the authors developed 11 new SMG1 inhibitors (KVS0001-KVS0011) based on the structures of the known SMG1 inhibitor SMG1i-11 and the SMG1 protein itself.

• Among these, KVS0001 stood out for its high potency, excellent bioavailability and low toxicity in mice. Treatment with KVS0001 caused NMD inhibition and increased presentation of neoantigens on MHC-I molecules, resulting in the clearance of cancer cells in vitro by co-cultured T cells and cancer xenografts in mice by the immune system.

These findings support the strategy of targeting the NMD pathway for cancer treatment and provide new research tools and potential lead compounds for further exploration.

Strengths:

The RNA-seq based NMD analysis, using isogenic cell lines with specific NMD-inducing mutations, represents a novel approach for the high-throughput identification of potential NMD modulators or genetic regulators. The effectiveness of this method is exemplified by the identification of a new activity of AKT1/mTOR inhibitor LY3023414 in inhibiting NMD.

The properties of KVS0001 described in the manuscript as a novel SMG1 inhibitor suggest its potential as a lead compound for further testing the NMD-targeting strategies in cancer treatment. Additionally, this compound may serve as a useful research tool.

The results of the in vitro cell killing assay and in vivo xenograft experiments in both immuno-proficient and immune-deficient mice indicate that inhibiting NMD could be a viable therapeutic strategy for certain cancers.

Weaknesses:

The authors did not address the potential effects of NMD/SMG1 inhibitors on RNA splicing. Given that the transcripts of many RNA-binding proteins are natural targets of NMD, inhibiting NMD could significantly alter splicing patterns. This, in turn, might influence the outcomes of the RNA-seq-based method for NMD analysis and result interpretation.

While the RNA-seq based approach offers several advantages for analyzing NMD, the effects of NMD/SMG1 inhibitors observed through this method should be confirmed using established NMD reporters. This step is crucial to rule out the possibility that mutations in STAG2 or TP53 affect NMD in cells, as well as to address potential clonal variations between different engineered cell lines.

The results from the SMG1/UPF1 knockdown and SMG1i-11 experiments presented in Figure 3 correlate with the effects seen for LY3023414, but they do not conclusively establish SMG1 as the direct target of LY3023414 in NMD inhibition. An epistatic analysis with LY3023414 and SMG1-knockdown is needed.

Comment on the revised version:

Although KVS0001 exhibits promising properties as an SMG1 inhibitor for cancer treatment, it remains unclear if it is superior to existing SMG1 inhibitors, as no direct comparisons have been made.

Reviewer #1 (Public Review):

Summary:

Trutti and colleagues used 7T fMRI to identify brain regions involved in subprocesses of updating the content of working memory. Contrary to past theoretical and empirical claims that the striatum serves a gating function when new information is to be entered into working memory, the relevant contrast during a reference-back task did not reveal significant subcortical activation. Instead, the experiment provided support for the role of subcortical (and cortical) regions in other subprocesses.

Strengths:

The use of high-field imaging optimized for subcortical regions in conjunction with the theory-driven experimental design mapped well to the focus on a hypothetical striatal gating mechanism.

Consideration of multiple subprocesses and the transparent way of identifying these, summarized in a table, will make it easy for future studies to replicate and extend the present experiment.

Weaknesses:

The reference-back paradigm seems to only require holding a single letter in working memory (X or O; Figure 1). It remains unclear how such low demand on working memory influences associated fMRI updating responses. It is also not clear whether reference-switch trials with 'same' response truly tax working-memory updating (and gate opening), as the working-memory content/representation does not need to be updated in this case. These potential design issues, together with the rather low number of experimental trials, raise concerns about the demonstrated absence of evidence for striatal gate opening.

The authors provide a motivation for their multi-step approach to fMRI analyses. Still, the three subsections of fMRI results (3.2.1; 3.2.2; 3.3.3) for 4 subprocesses each (gate opening, gate closing, substitution, updating mode) made the Results section complex and it was not always easy to understand why some but not other approaches revealed significant effects (as the midbrain in gate opening).

The many references to the role of dopamine are interesting, but the discussion of dopaminergic pathways and signals remains speculative and must be confirmed in future studies (e.g., with PET imaging).

Reviewer #1 (Public Review):

The authors investigate pleiotropy in the genetic loci previously associated to a range of neuropsychiatric disorders: Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Parkinson's disease, and schizophrenia. The local statistical fine-mapping and variant colocalisation approaches they use have the potential to uncover not only shared loci but also shared causal variants between these disorders. There is existing literature describing the pleiotropy between ALS and these other disorders but here the authors apply state-of-the-art, local genetic correlation approaches to further refine any relationships.

Complex disease and GWAS is not my area of expertise but the authors managed to present their methods and results in a clear, easy-to-follow manner. Their results statistically support several correlations between the disorders and, for ALS and AD, a shared variant in the vicinity of the lead SNP from the original ALS GWAS. Such findings could have important implications for our understanding of the mechanisms of such disorders and eventually the possibility of managing and treating them.

The authors have built a useful pipeline that plugs together all the gold-standard, existing software to perform this analysis and made it openly available which is commendable. However, there is little discussion of what software is available to perform global and local correlation analysis and, if there are multiple tools available, why they consider the ones they selected to be the gold-standard.

There is some mention of previous findings of genetic pleiotropy between ALS and these other disorders in the introduction, and discussion of their improved ALS-AD evidence relative to previous work. However, detailed comparisons of their other correlations to what was described before for the same pairs of disorders (if any) is missing. Adding this would strengthen the impact of this paper.

Finally, being new to this approach I found the abstract a little confusing. Initially, the shared causal variant between ALS and AD is mentioned but immediately in the following sentence they describe how their study "suggested that disease- implicated variants in these loci often differ between traits". After reading the whole paper I understood that the ALS-AD shared variant was the exception but it may be best to restructure this part of the abstract. Additionally, in the abstract the authors state that different variants "suggests the role of distinct mechanisms across diseases despite shared loci". Is it not possible that different variants in the same regulatory region or protein-coding parts of a gene could be having the same effect and mechanism? Or does the methodology to establish that different variants are involved automatically mean that the variants are too distant for this to be possible?

These concerns were addressed in the revised version of this manuscript.

Reviewer #1 (Public Review):

Summary:

In this paper, Steinemann et al. characterized the nature of stochastic signals underlying the trial-averaged responses observed in lateral intraparietal cortex (LIP) of non-human primates (NHPs), while these performed the widely used random dot direction discrimination task. Ramp-up dynamics in the trial averaged LIP responses were reported in numerous papers before. But the temporal dynamics of these signals at the single-trial level have been subject to debate. Using large scale neuronal recordings with Neuropixels in NHPs, allows the authors to settle this debate rather compellingly. They show that drift-diffusion like computations account well for the observed dynamics in LIP.

Strengths:

This work uses innovative technical approaches (Neuropixel recordings in behaving macaque monkeys). The authors tackle a vexing question that requires measurements of simultaneous neuronal population activity and hence leverage this advanced recording technique in a convincing way.

They use different population decoding strategies to help interpret the results.

They also compare how decoders relying on the data-driven approach using dimensionality reduction of the full neural population space compares to decoders relying on more traditional ways to categorize neurons that are based on hypotheses about their function. Intriguingly, although the functionally identified neurons are a modest fraction of the population, decoders that only rely on this fraction achieve comparable decoding performance to those relying on the full population. Moreover, decoding weights for the full population did not allow the authors to reliably identify the functionally identified subpopulation.

The revision addressed the minor weaknesses to our satisfaction.

Reviewer #1 (Public Review):

This is an interesting and thorough paper describing the modes of locomotion of the nematode C. elegans in the context of random exploration or response to an aversive stimulus. The authors collect extensive statistics on various locomotor states and compare findings to a minimal mathematical model inspired by the data. Their data reveal biases in two modes of turning- gradual and sharp- which define the path structure of the animal moving on an agar plate. The authors also find that animals tend to overcome inherent anatomical/physiological biases to locomotion when escaping aversive stimuli.

Understanding animal navigation is a window for revealing efficient algorithms for exploration of space, and also allows testing of the extent to which we understand how the nervous system produces specific behaviors. This paper adds important analysis towards these goals. I have a couple of comments that may be worth considering:

(1) The authors place a circular barrier of SDS near the edges of their plates and assume that this aversive stimulus is only sensed when the animal is near the barrier. However, it is possible that the SDS diffuses enough into the interior of the plate to affect the navigation statistics. In this case, the data they have accumulated may in fact be some sort of combination of exploratory locomotion and a general background SDS aversive stimulus. Can the authors control for this? Perhaps test the plates at different distances and times for SDS diffusion? Or replace the barrier with a physical one and not a chemical one?

(2) The authors do not look at mutants or perturb the physiology in defined ways relevant to the locomotion being studied to test their model. Specifically, it would be of interest to identify neural circuits that govern some of the parameters in the model. Although the authors bring this up in their Discussion section, it seems appropriate for this paper, as it would considerably bolster the impact of the work.

Public Review:

The study addresses the role of enkephalins, which are specifically expressed by regulatory T cells (Treg), in sensory perception in mice. The authors used a combination of transcriptomic databases available online to characterize the molecular signature of Treg. The proenkephalin gene Penk is among the most enriched transcripts, suggesting that Treg plays an analgesic role through the release of endogenous opioids. In addition, in silico analysis suggests that Penk is regulated by the TNFR superfamily; this being experimentally confirmed. Using flow cytometry analysis, the authors then show that Penk is mostly expressed in Treg of the skin and colon, compared to other immune cells. Finally, genetic conditional excision of Penk, selectively in Treg, results in heat hypersensitivity, as assessed by behavior analysis.

Editors' note: The authors accepted most if not all the suggestions given by the reviewers and the revised version of the manuscript is substantially improved.

Reviewer #2 (Public Review):

Main comment from 1st review:

Weaknesses:<br /> The modeling is interesting, with the integration of tension through tension triangulation around vertices and thus integrating force inference directly in the vertex model. However, the authors are not using it to test their hypothesis and support their analysis at the tissue level. Thus, although interesting, the analysis at the tissue level stays mainly descriptive.

Comments on the revised version:

My main concern was that the author did not use the analysis of mutant contexts such as Snail and Twist to confirm their predictions. They made a series of modifications, clarifying their conclusions. In particular, they now included an analysis of Snail mutant and show that isogonal deformations in the ventro-lateral regions are absent when the external pulling force of the VF is abolished, supporting the idea that isogonal strain could be used as an indicator of external forces (Fig7 and S6).

They further discuss their results in the context of what was published regarding the mutant backgrounds (fog, torso-like, scab, corkscrew, ksr) where midgut invagination is disrupted, and where germ band buckles, and propose that this supports the importance of internal versus external forces driving GBE.<br /> Overall, these modifications, in addition to clarifications in the text, clearly strengthen the manuscript.

Reviewer #1 (Public Review):

Summary and strength:<br /> The authors undertook to assemble and annotate the genome sequence of the Malabar grouper fish, with the aim to provide molecular resources for fundamental and applied research. Even though this is more mainstream, the task is still daunting and labor intensive. Currently, high quality and fully annotated genome sequences are of strategic importance in modern biology. The authors make use of the resource to address the endocrine control of an ecologically and developmentally relevant life cycle transition, metamorphosis. As opposed to amphibian and flat fish where body plan changes, fish metamorphosis is anatomically more subtle and much less known, although it is clear that thyroid hormone (TH) signaling is a key player. The authors thus provide a repertoire of TH-relevant gene expression changes during development and across post-embryonic transitions and correlate developmental stages with changes of gene expression. Overall, this work represents a significant advance in the field.

Fish 'metamorphosis' is well known because it is not as spectacular as amphibians. This work clearly provides technical and theoretical resources to address in a more systematic manner the molecular changes occurring during development and post-embryonic transitions. Heterochrony is a major source of functional and life cycle diversity in fish, which blurs our anatomy-based understanding of fish biology, and has a direct impact on the protocols and rearing procedures used to produce live stocks. This work illustrates how, by using genomics coupled to simple experimental endocrinology, one directly addresses these challenges.

Reviewer #1 (Public Review):

The authors present data on outer membrane vesicle (OMV) production in different mutants, but they state that this is beyond the scope of the current manuscript, which I disagree with. This data could provide valuable physiological context that is otherwise lacking. The preliminary blots suggest that YafK does not alter OMV biogenesis. I recommend repeating these blots with appropriate controls, such as blotting for proteins in the culture media, an IM protein, periplasmic protein and an OM protein to strengthen the reliability of these findings. Including this data in the manuscript, even if it does not directly support the initial hypothesis, would enhance the physiological relevance of the study. Currently, the manuscript relies completely on the experimental setup (labeling-mass spec) previously developed by the authors, which limits the broader scope and interpretability of this study.

Additionally susceptibility of strains to detergents like SDS can be tested to provide a much needed physisological context to the study.

In summary, the authors should consider revising the manuscript to improve clarity, substantiate their claims with more detailed evidence, and include additional experimental results that provide necessary physiological context to their study.

Reviewer #1 (Public Review):

Koren et al. derive and analyse a spiking network model optimised to represent external signals using the minimum number of spikes. Unlike most prior work using a similar setup, the network includes separate populations of excitatory and inhibitory neurons. The authors show that the optimised connectivity has a like-to-like structure, leading to the experimentally observed phenomenon of feature competition. They also characterise the impact of various (hyper)parameters, such as adaptation timescale, ratio of excitatory to inhibitory cells, regularisation strength, and background current. These results add useful biological realism to a particular model of efficient coding. However, not all claims seem fully supported by the evidence. Specifically, several biological features, such as the ratio of excitatory to inhibitory neurons, which the authors claim to explain through efficient coding, might be contingent on arbitrary modelling choices. In addition, earlier work has already established the importance of structured connectivity for feature competition. A clearer presentation of modelling choices, limitations, and prior work could improve the manuscript.

Major comments:

(1) Much is made of the 4:1 ratio between excitatory and inhibitory neurons, which the authors claim to explain through efficient coding. I see two issues with this conclusion: (i) The 4:1 ratio is specific to rodents; humans have an approximate 2:1 ratio (see Fang & Xia et al., Science 2022 and references therein); (ii) the optimal ratio in the model depends on a seemingly arbitrary choice of hyperparameters, particularly the weighting of encoding error versus metabolic cost. This second concern applies to several other results, including the strength of inhibitory versus excitatory synapses. While the model can, therefore, be made consistent with biological data, this requires auxiliary assumptions.

(2) A growing body of evidence supports the importance of structured E-I and I-E connectivity for feature selectivity and response to perturbations. For example, this is a major conclusion from the Oldenburg paper (reference 62 in the manuscript), which includes extensive modelling work. Similar conclusions can be found in work from Znamenskiy and colleagues (experiments and spiking network model; bioRxiv 2018, Neuron 2023 (ref. 82)), Sadeh & Clopath (rate network; eLife, 2020), and Mackwood et al. (rate network with plasticity; eLife, 2021). The current manuscript adds to this evidence by showing that (a particular implementation of) efficient coding in spiking networks leads to structured connectivity. The fact that this structured connectivity then explains perturbation responses is, in the light of earlier findings, not new.

(3) The model's limitations are hard to discern, being relegated to the manuscript's last and rather equivocal paragraph. For instance, the lack of recurrent excitation, crucial in neural dynamics and computation, likely influences the results: neuronal time constants must be as large as the target readout (Figure 4), presumably because the network cannot integrate the signal without recurrent excitation. However, this and other results are not presented in tandem with relevant caveats.

(4) On repeated occasions, results from the model are referred to as predictions claimed to match the data. A prediction is a statement about what will happen in the future - but most of the "predictions" from the model are actually findings that broadly match earlier experimental results, making them "postdictions". This distinction is important: compared to postdictions, predictions are a much stronger test because they are falsifiable. This is especially relevant given (my impression) that key parameters of the model were tweaked to match the data.

Reviewer #1 (Public Review):

Summary:

The question of whether eyespots mimic eyes has certainly been around for a very long time and led to a good deal of debate and contention. This isn't purely an issue of how eyespots work either, but more widely an example of the potential pitfalls of adopting 'just-so-stories' in biology before conducting the appropriate experiments. Recent years have seen a range of studies testing eye mimicry, often purporting to find evidence for or against it, and not always entirely objectively. Thus, the current study is very welcome, rigorously analysing the findings across a suite of papers based on evidence/effect sizes in a meta-analysis.

Strengths:

The work is very well conducted, robust, objective, and makes a range of valuable contributions and conclusions, with an extensive use of literature for the research. I have no issues with the analysis undertaken, just some minor comments on the manuscript. The results and conclusions are compelling. It's probably fair to say that the topic needs more experiments to really reach firm conclusions but the authors do a good job of acknowledging this and highlighting where that future work would be best placed.

Weaknesses:

There are few weaknesses in this work, just some minor amendments to the text for clarity and information.

Reviewer #1 (Public Review):

Summary:

Weiss and co-authors presented a versatile probabilistic tool. aTrack helps in classifying tracking behaviors and understanding important parameters for different types of single particle motion types: Brwonian, Confined, or Directed motion. The tool can be used further to analyze populations of tracks and the number of motion states. This is a stand-alone software package, making it user-friendly for a broad group of researchers.

Strengths:

This manuscript presents a novel method for trajectory analysis.

Weaknesses:

(1) In the results section, is there any reason to choose the specific range of track length for determining the type of motion? The starting value is fine, and would be short enough, but do the authors have anything to report about how much is too long for the model?

(2) Robustness to model mismatches is a very important section that the authors have uplifted diligently. Understanding where and how the model is limited is important. For example, the authors mentioned the limitation of trajectory length, do the authors have any information on the trajectory length range at which this method works accurately? This would be of interest to readers who would like to apply this method to their own data.

(3) aTrack extracts certain parameters from the trajectories to determine the motion types. However, it is not very clear how certain parameters are calculated. For example, is the diffusion coefficient D calculated from fitting, and how is the confinement factor defined and estimated, with equations? This information will help the readers to understand the principles of this algorithm.

(4) The authors mentioned the scenario where a particle may experience several types of motion simultaneously. How do these motions simulated and what do they mean in terms of motion types? Are they mixed motion (a particle switches motion types in the same trajectory) or do they simply present features of several motion types? It is not intuitive to the readers that a particle can be diffusive (Brownian) and direct at the same time.

Reviewer #1 (Public Review):

Summary:

As our understanding of the immune system increases it becomes clear that murine models of immunity cannot always prove an accurate model system for human immunity. However, mechanistic studies in humans are necessarily limited. To bridge this gap many groups have worked on developing humanised mouse models in which human immune cells are introduced into mice allowing their fine manipulation. However, since human immune cells will attack murine tissues, it has proven complex to establish a human-like immune system in mice. To help address this, Vecchione et al have previously developed several models using human cell transfer into mice with or without human thymic fragments that allow negative selection of autoreactive cells. In this report they focus on the examination of the function of the B-helper CD4 T-cell subsets T-follicular helper (Tfh) and T-peripheral helper (Tph) cells. They demonstrate that these cells are able to drive both autoantibody production and can also induce B-cell independent autoimmunity.

Strengths:

A strength of this paper is that currently there is no well-established model for Tfh or Tph in HIS mice and that currently there is no clear murine Tph equivalent making new models for the study of this cell type of value. Equally, since many HIS mice struggle to maintain effective follicular structures Tfh models in HIS mice are not well established giving additional value to this model.

Weaknesses:

A weakness of the paper is that the models seem to lack a clear ability to generate germinal centres. For Tfh it is unclear how we can interpret their function without the structure where they have the greatest influence. In some cases, the definition of Tph does not seem to differentiate well between Tph and highly activated CD4 T-cells in general.

Reviewer #1 (Public Review):

Summary:

Cording et al. investigated how deletion of CNTNAP2, a gene associated with autism spectrum disorder, alters corticostriatal engagement and behavior. Specifically, the authors present slice electrophysiology data showing that striatal projection neurons (SPNs) are more readily driven to fire action potentials in response to stimulation of corticostriatal afferents, and this is due to increases in SPN intrinsic excitability rather than changes in excitatory or inhibitory synaptic inputs. The authors show that CNTNAP2 mice display repetitive behaviors, enhanced motor learning, and cognitive inflexibility. Overall the authors' conclusions are supported by their data, but a few claims could use some more evidence to be convincing.

Strengths:

The use of multiple behavioral techniques, both traditional and cutting-edge machine learning-based analyses, provides a powerful means of assessing repetitive behaviors and behavioral transitions/rigidity. Characterization of both excitatory and inhibitory synaptic responses in slice electrophysiology experiments offers a broad survey of the synaptic alterations that may lead to increased corticostriatal engagement of SPNs.

Weaknesses:

(1) The authors conclude that increased cortical engagement of SPNs is due to changes in SPN intrinsic excitability rather than synaptic strength (either excitatory or inhibitory). One weakness is that only AMPA receptor-mediated responses were measured. Though the holding potential used for experiments in Figure 1F-I wasn't clear, recordings were presumably performed at a hyperpolarized potential that limits NMDA receptor-mediated responses. Because the input-output experiments used to conclude that corticostriatal engagement of SPNs is elevated (Figure 1B-E) were conducted in the current clamp, it is possible that enhanced NMDA receptor engagement contributed to increased SPN responses to cortical stimulation. Confirming that NMDA receptor-mediated EPSC components are not altered would strengthen the main conclusion.

(2) Data clearly show that SPN intrinsic excitability is increased in knockout mice. Given that CNTNAP2 has been linked to potassium channel regulation, it would be helpful to show and quantify additional related electrophysiology data such as negative IV curve responses and action potential hyperpolarization.

(3) As it stands, the reported changes in dorsolateral striatum SPN excitability are only correlative with reported changes in repetitive behaviors, motor learning, and cognitive flexibility.

Reviewer #1 (Public Review):

Summary:

Boldt et al test several possible relationships between trandiagnostically-defined compulsivity and cognitive offloading in a large online sample. To do so, they develop a new and useful cognitive task to jointly estimate biases in confidence and reminder-setting. In doing so, they find that over-confidence is related to less utilization of reminder-setting, which partially mediates the negative relationship between compulsivity and lower reminder-setting. The paper thus establishes that, contrary to the over-use of checking behaviors in patients with OCD, greater levels of transdiagnostically-defined compulsivity predict less deployment of cognitive offloading. The authors offer speculative reasons as to why (perhaps it's perfectionism in less clinically-severe presentations that lowers the cost of expending memory resources), and set an agenda to understand the divergence in cognition between clinical and nonclinical samples. Because only a partial mediation had robust evidence, multiple effects may be at play, whereby compulsivity impacts cognitive offloading via overconfidence and also by other causal pathways.

Strengths:

The study develops an easy-to-implement task to jointly measure confidence and replicates several major findings on confidence and cognitive-offloading. The study uses a useful measure of cognitive offloading - the tendency to set reminders to augment accuracy in the presence of experimentally manipulated costs. Moreover, the utilizes multiple measures of presumed biases - overall tendency to set reminders, the empirically estimated indifference point at which people engage reminders, and a bias measure that compares optimal indifference points to engage reminders relative to the empirically-observed indifference points. That the study observes convergenence along all these measures strengthens the inferences made relating compulsivity to the under-use of reminder-setting. Lastly, the study does find evidence for one of several a priori hypotheses and sets a compelling agenda to try to explain why such a finding diverges from an ostensible opposing finding in clinical OCD samples and the over-use of cognitive offloading.

Weaknesses:

Although I think this design and study are very helpful for the field, I felt that a feature of the design might reduce the tasks's sensitivity to measuring dispositional tendencies to engage cognitive offloading. In particular, the design introduces prediction errors, that could induce learning and interfere with natural tendencies to deploy reminder-setting behavior. These PEs comprise whether a given selected strategy will be or not be allowed to be engaged. We know individuals with compulsivity can learn even when instructed not to learn (e.g., Sharp, Dolan, and Eldar, 2021, Psychological Medicine), and that more generally, they have trouble with structure knowledge (eg Seow et al; Fradkin et al), and thus might be sensitive to these PEs. Thus, a dispositional tendency to set reminders might be differentially impacted for those with compulsivity after an NPE, where they want to set a reminder, but aren't allowed to. After such an NPE, they may avoid more so the tendency to set reminders. Those with compulsivity likely have superstitious beliefs about how checking behaviors leads to a resolution of catastrophes, which might in part originate from inferring structure in the presence of noise or from purely irrelevant sources of information for a given decision problem.

It would be good to know if such learning effects exist if they're modulated by PE (you can imagine PEs are higher if you are more incentivized - e.g., 9 points as opposed to only 3 points - to use reminders, and you are told you cannot use them), and if this learning effect confounds the relationship between compulsivity and reminder-setting.

A more subtle point, I think this study can be more said to be an exploration than a deductive test of a particular model -> hypothesis -> experiment. Typically, when we test a hypothesis, we contrast it with competing models. Here, the tests were two-sided because multiple models, with mutually exclusive predictions (over-use or under-use of reminders) were tested. Moreover, it's unclear exactly how to make sense of what is called the direct mechanism, which is supported by partial (as opposed to complete) mediation.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors investigate the impact of rare and extreme events on rodents' decision-making under risk, in gain and loss contexts. They describe the behavior of 20 rats performing a four-armed bandit task, where probabilistic gains (sugar pellets) and losses (time-out punishments) can - in some arms - incorporate extremely large - but rare - outcomes. They report that most rats are sensitive to rare and extreme outcomes despite their infrequent occurrence, and that this sensitivity is primarily driven by extreme loss events which they try to avoid, rather than extreme gains that they seek to obtain.

They finally propose a modification of standard reinforcement-learning, which features a specific sensitivity to rare and extreme outcomes and can account for the observed behavior.

Strengths:

The manuscript really taps into a surprisingly neglected but very relevant aspect of decision-making: the effect of rare and extreme events (REE). The authors have developed an experimental setup that seemingly allows investigation of this aspect, which is not trivial given the idiosyncratic properties of rare and extreme events.

The parameters of the experimental setup seem also to be well thought off: basically, in the absence of REE, some options are objectively better than others (because, in expectation, they overall deliver more food, or minimize time-out punishments), but this ordering reverses if REE are taken into account. This allows for a clean test of the integration of REE in the rodent's decision-making model.

The data is presented and analyzed in a very descriptive but exhaustive and transparent way, down to the description of individual rodent's behavior.

Weaknesses:

While the description and analyses of the behavioral patterns are rigorously done under the economic lens of risky decision-making, the authors' interpretation heavily relies on the assumption that rodents have built the correct model of the task during the training. Extensive details are provided about the training procedure, and the observed behavior at the end of the training, but it remains virtually impossible to disambiguate choices due to imperfect learning to choices made due to intrinsic preferences for risk or REE.

By nature, gains (food pellets) and losses (time-out punishments) are somewhat incommensurable so the interpretation of the asymmetry due to outcome valence is also subject to interpretation. There might be some additional subtleties due e.g. satiety that could come from gaining REE (i.e. the delivery of 80 pellets from the Jackpot).

In its current form, the paper is quite hard to digest. This is naturally the case with interdisciplinary work (here mixing economists and neurobiologists). But I am afraid that with the current frame, the paper is going to miss its target, in terms of audience.

The proposed model seems somewhat disconnected from the behavioral patterns: while the model suggests an effect of REE at the decision stage (i.e. with specific decision weights for those rare events), this formalism seems at odds with the observation that REE (notably in the loss domain) has an impact of subsequent behavior - (Black Swans tend to reinforce Total Sensitivity to REE) which rather suggests an effect at the learning stage.

Discussion:

This study convincingly demonstrates that REEs are processed rather uniquely, which makes sense given their evolutionary relevance. REE has indeed been somewhat neglected in previous research, and this study therefore opens an interesting new front on the fundamental aspects of decision under risk. The authors have devised an original theoretical and empirical framework that will be useful for the community, and the combination of economics analysis and rodent behavior constitutes a thought-provoking ground to think about the nature of risk preferences. The interpretation and mechanistic account of these aspects, as well as their generalizability outside the specific context of this study, remain to be strengthened.

Reviewer #1 (Public Review):

Summary:

Lloyd et al employ an evolutionary comparative approach to study how sleep deprivation affects DNA damage repair in Astyanax mexicanus, using the cave vs surface species evolution as a playground. The work shows, convincingly, that the cavefish population has evolved an impaired DNA damage response both following sleep deprivation or a classical paradigm of DNA damage (UV).

Strengths:

The study employs a thorough multidisciplinary approach. The experiments are well conducted and generally well presented.

Weaknesses:

Having a second experimental mean to induce DNA damage would strengthen and generalise the findings.

Overall, the study represents a very important addition to the field. The model employed underlines once more the importance of using an evolutionary approach to study sleep and provides context and caveats to statements that perhaps were taken a bit too much for granted before. At the same time, the paper manages to have an extremely constructive approach, presenting the platform as a clear useful tool to explore the molecular aspects behind sleep and cellular damage in general. The discussion is fair, highlighting the strengths and weaknesses of the work and its implications.

Reviewer #1 (Public Review):

Summary:

In this article, the authors investigated how the brain anticipates sequences of potential sensory events, using temporal predictability to enhance perception. To do so, they combined a tone detection task, electrophysiological recordings, and recurrent neural network models. The stimuli consisted of continuous white noise embedded with either a single tone presented at one of 3 equidistant (500ms) temporal locations, or no tone. The main analyses were carried out on no-tone trials, in which subjects only anticipated future events. First, a modulation power spectrum analysis revealed 4 frequency clusters, and a coupling analysis allowed the authors to group 3 of them together into cluster 234. The time course of the latter aligned with the temporal locations, reaching a local maximum following each of them. The power of cluster 234 during no-tone trials was positively correlated with behavioral performance (d') during tone trials, but not with false alarm rate. Then, the authors trained several continuous-time recurrent neural networks to model the experimental paradigm. After the networks were tuned to reflect the average d' of human subjects, a neural network analogue of EEG was extracted from the activity of neurons. The latter displayed a peak at 2Hz, its time course aligned with the temporal locations, reaching a local maximum both before and after each of them, and its d' score was higher for tones located at one of the temporal locations. A network trained with randomly occurring tones displayed no 2Hz activity and d' independent from tone location. Finally, the authors perturbed the excitatory/inhibitory ratio of neurons within the network, finding that more inhibition resulted in earlier peaks in the neural network activity.

Strengths:

(1) The experimental paradigm introduced in this study is original and well-built, allowing for the study of the targeted phenomenon. The fact that relevant neural signals were found despite the absence of sensory cues proves the setup is promising, opening the way for future works, playing with different parameters: number of tones, time between tones, sequence of temporal locations complexity, sequence of events...etc.

(2) The statistical analysis was exhaustive, the authors consistently introduced controls for different conditions and alternative hypotheses, thoroughly explaining each step of the analysis as well as the choices behind them. The supplementary figures further helped understand the data and answer interrogation one might have. This comprehensive approach was well-appreciated.

(3) The use of more biologically plausible networks, compared to traditional RNNs, to model the response of subjects is a promising approach, which can give clues as to the mechanism at play, but also make predictions that can then be proven (or disproven) by future experiments.

The authors provided a work of good technical quality and reported their methods and findings transparently, making for good reproducibility and evaluation.

Weaknesses:

(1) The most glaring weakness of the paper lies in its interpretation of the different results. Conclusions are scattered around the paper, mostly unclear, and do not always make much sense with regard to the data. For example, the authors never address the absence of a peak before the first temporal location: why would subjects not "suppress" noise before the first temporal location given its (strong) predictability? Moreover, they immediately assume a functional role for the neural signature they found, as well as a direct link between the mechanisms at play in their RNN and the human brain, thus jumping to hasty and unreliable conclusions. The authors seemed to have a strong bias towards a hypothesis (predictive gain control) and tried to fit their data into it.

- The authors cited very few relevant papers on related fields, notably on omission, and therefore did not build efficiently on previous works (e.g., Yabe, Raij, Schröger, Bekinschtein, Chait, Auksztulewicz...). Moreover, at several points in the paper, they make choices about their analysis or model without proper justification or cited sources, even when explicitly pointing to the existence of research supporting said choices.

- Only a single electrode (out of 64) was used (Cz) to carry out every analysis. Without proper justification, this choice could be misinterpreted. Moreover, adopting instead a multivariate approach (incorporating all channels) would give more strength to the paper.

- Overall, the observed electrophysiological results could be more simply explained by a mechanism akin to a go/no-go (a tone/no-tone) or omission response happening after each temporal location, as subjects have learned when to make that inference. The delay of the response with regards to temporal location would change due to error accumulation in time perception, rather than "the anticipation of the first temporal location facilitating the anticipation of the second", which makes little sense. Moreover, a response in Cz could be expected.

- As for the results of RNN, not only is the analogy with actual neurophysiological activity limited, both in principle (simple E/I dynamics) and in implementation (inference is only done at the end of each trial), but the authors do not address the activity before the first temporal location, which is a major difference with human data. Their assumption that both RNN and cluster 234 are functionally related to gain control is thus further flawed. Moreover, the analysis of the RNN is lacking, for example, the authors did not compare false positive/negative of different delays, or analyzed Wout.

- The phrasing and introduction of the paper are misleading, as confusion can arise between predicting a sequence of events (several events in a row) and predicting a single event appearing at different potential locations. It should be clarified that the paper does not address sequences of events at any point.

It seems the authors already drew their conclusion beforehand and fit the data to match this bias. As such, the interpretation of the data is messy, flawed, and often hasty, drawing erroneous conclusions and parallels.

Overall, the manuscript is of good technical quality and communicated results very transparently, but the authors seem to have a strong confirmation bias towards temporal anticipation and gain control, thus leading to flawed interpretations.

Reviewer #1 (Public Review):

Summary:

In this study, Gu et al. employed novel viral strategies, combined with in vivo two-photon imaging, to map the tone response properties of two groups of cortical neurons in A1. The thalamocortical recipient (TR neurons) and the corticothalamic (CT neurons). They observed a clear tonotopic gradient among TR neurons but not in CT neurons. Moreover, CT neurons exhibited high heterogeneity of their frequency tuning and broader bandwidth, suggesting increased synaptic integration in these neurons. By parsing out different projecting-specific neurons within A1, this study provides insight into how neurons with different connectivity can exhibit different frequency response-related topographic organization.

Strengths:

This study reveals the importance of studying neurons with projection specificity rather than layer specificity since neurons within the same layer have very diverse molecular, morphological, physiological, and connectional features. By utilizing a newly developed rabies virus CSN-N2c GCaMP-expressing vector, the authors can label and image specifically the neurons (CT neurons) in A1 that project to the MGB. To compare, they used an anterograde trans-synaptic tracing strategy to label and image neurons in A1 that receive input from MGB (TR neurons).

Weaknesses:

- Perhaps as cited in the introduction, it is well known that tonotopic gradient is well preserved across all layers within A1, but I feel if the authors want to highlight the specificity of their virus tracing strategy and the populations that they imaged in L2/3 (TR neurons) and L6 (CT neurons), they should perform control groups where they image general excitatory neurons in the two depths and compare to TR and CT neurons, respectively. This will show that it's not their imaging/analysis or behavioral paradigms that are different from other labs.  

- Figures 1D and G, the y-axis is Distance from pia (%). I'm not exactly sure what this means. How does % translate to real cortical thickness? 

- For Figure 2G and H, is each circle a neuron or an animal? Why are they staggered on top of each other on the x-axis? If the x-axis is the distance from caudal to rostral, each neuron should have a different distance? Also, it seems like it's because Figure 2H has more circles, which is why it has more variation, thus not significant (for example, at 600 or 900um, 2G seems to have fewer circles than 2H).  

- Similarly, in Figures 2J and L, why are the circles staggered on the y-axis now? And is each circle now a neuron or a trial? It seems they have many more circles than Figure 2G and 2H. Also, I don't think doing a correlation is the proper stats for this type of plot (this point applies to Figures 3H and 3J).

- What does the inter-quartile range of BF (IQRBF, in octaves) imply? What's the interpretation of this analysis? I am confused as to why TR neurons show high IQR in HF areas compared to LF areas, which means homogeneity among TR neurons (lines 213 - 216). On the same note, how is this different from the BF variability?  Isn't higher IQR equal to higher variability?

- Figure 4A-B, there are no clear criteria on how the authors categorize V, I, and O shapes. The descriptions in the Methods (lines 721 - 725) are also very vague.

Reviewer #1 (Public Review):

Summary:

This study resolves a cryo-EM structure of the GPCR, GPR30, which was recently identified as a bicarbonate receptor by the authors' lab. Understanding the ligand and the mechanism of activation is of fundamental importance to the field of receptor signaling. However, the main claim of the paper, the identification of the bicarbonate binding site, is only partly supported by the structural and functional data, leaving the study incomplete.

Strengths:

The overall structure, and proposed mechanism of G-protein coupling seem solid. The authors perform fairly extensive unbiased mutagenesis to identify a host of positions that are important to G-protein signaling. To my knowledge, bicarbonate is the only physiological ligand that has been identified for GPR30, making this study a particularly important contribution to the field.

Weaknesses:

Without higher resolution structures and/or additional experimental assessment of the binding pocket, the assignment of the bicarbonate remains highly speculative. The local resolution is especially poor in the ECL loop region where the ligand is proposed to bind (4.3 - 4 .8 Å range). Of course, sometimes it is difficult to achieve high structural resolution, but in these cases, the assignment of ligands should be backed up by even more rigorous experimental validation.

The functional assay monitors activation of GPR30, and thus reports on not only bicarbonate binding, but also the integrity of the allosteric network that transduces the binding signal across the membrane. Thus, disruption of bicarbonate signaling by mutagenesis of the putative coordinating residues does not necessarily mean that bicarbonate binding has been disrupted. Moreover, the mutagenesis was apparently done prior to structure determination, meaning that residues proposed to directly surround bicarbonate binding, such as E218, were not experimentally validated. Targeted mutagenesis based on the structure would strengthen the story.

Moreover, the proposed bicarbonate binding site is surprising in a chemical sense, as it is located within an acidic pocket. The authors cite several other structural studies to support the surprising observation of anionic bicarbonate surrounded by glutamate residues in an acidic pocket (references 31-34). However, it should be noted that in general, these other structures also possess a metal ion (sodium or calcium) and/or a basic sidechain (arginine or lysine) in the coordination sphere, forming a tight ion pair. Thus, the assigned bicarbonate binding site in GPR30 remains an anomaly in terms of the chemical properties of the proposed binding site.

Reviewer #1 (Public Review):

Summary:

The study "Impact of Maximal Overexpression of a Non-toxic Protein on Yeast Cell Physiology" by Fujita et al. aims to elucidate the physiological impacts of overexpressing non-toxic proteins in yeast cells. By identifying model proteins with minimal cytotoxicity, the authors claim to provide insights into cellular stress responses and metabolic shifts induced by protein overexpression.

Strengths:

The study introduces a neutrality index to quantify cytotoxicity and investigates the effects of protein burden on yeast cell physiology. The study identifies mox-YG (a non-fluorescent fluorescent protein) and Gpm1-CCmut (an inactive glycolytic enzyme) as proteins with the lowest cytotoxicity, capable of being overexpressed to more than 40% of total cellular protein while maintaining yeast growth. Overexpression of mox-YG leads to a state resembling nitrogen starvation probably due to TORC1 inactivation, increased mitochondrial function, and decreased ribosomal abundance, indicating a metabolic shift towards more energy-efficient respiration and defects in nucleolar formation.

Weaknesses:

While the introduction of the neutrality index seems useful to differentiate between cytotoxicity and protein burden, the biological relevance of the effects of overexpression of the model proteins is unclear.

Reviewer #1 (Public Review):

In this manuscript, Satouh et al. report giant organelle complexes in oocytes and early embryos. Although these structures have often been observed in oocytes and early embryos, their exact nature has not been characterized. The authors named these structures "endosomal-lysosomal organelles form assembly structures (ELYSAs)". ELYSAs contain organelles such as endosomes, lysosomes, and probably autophagic structures. ELYSAs are initially formed in the perinuclear region and then migrate to the periphery in an actin-dependent manner. When ELYSAs are disassembled after the 2-cell stage, the V-ATPase V1 subunit is recruited to make lysosomes more acidic and active. The ELYSAs are most likely the same as the "endolysosomal vesicular assemblies (ELVAs)", reported by Elvan Böke's group earlier this year (Zaffagnini et al. doi.org/10.1016/j.cell.2024.01.031). However, it is clear that Satouh et al. identified and characterized these structures independently. These two studies could be complementary. Although the nature of the present study is generally descriptive, this paper provides valuable information about these giant structures. The data are mostly convincing, and only some minor modifications are needed for clarification and further explanation to fully understand the results.

Reviewer #1 (Public Review):

Summary:

PROTACs are heterobifunctional molecules that utilize the Ubiquitin Proteasome System to selectively degrade target proteins within cells. Upon introduction to the cells, PROTACs capture the activity of the E3 ubiquitin ligases for ubiquitination of the targeted protein, leading to its subsequent degradation by the proteasome. The main benefit of PROTAC technology is that it expands the "druggable proteome" and provides numerous possibilities for therapeutic use. However, there are also some difficulties, including the one addressed in this manuscript: identifying suitable target-E3 ligase pairs for successful degradation. Currently, only a few out of about 600 E3 ligases are used to develop PROTAC compounds, which creates the need to identify other E3 ligases that could be used in PROTAC synthesis. Testing the efficacy of PROTAC compounds has been limited to empirical tests, leading to lengthy and often failure-prone processes. This manuscript addressed the need for faster and more reliable assays to identify the compatible pairs of E3 ligases-target proteins. The authors propose using the RiPA assay, which depends on rapamycin-induced dimerization of FKBP12 protein with FRB domain. The PROTAC technology is advancing rapidly, making this manuscript both timely and essential. The RiPA assay might be useful in identifying novel E3 ligases that could be utilized in PROTAC technology. Additionally, it could be used at the initial stages of PROTAC development, looking for the best E3 ligase for the specific target.

The authors described an elegant assay that is scalable, easy-to-use, and applicable to a wide range of cellular models. This method allows for the quantitative validation of the degradation efficacy of a given pair of E3 ligase-target proteins, using luciferase activity as a measure. Importantly, the assay also enables the measurement of kinetics in living cells, enhancing its practicality.

Strengths:

(1) The authors have addressed the crucial needs that arise during PROTAC development. In the introduction, they nicely describe the advantages and disadvantages of the PROTAC technology and explain why such an assay is needed.

(2) The study includes essential controls in experiments (important for generating new assay), such as using the FRB vector without E3 ligase as a negative control, testing different linkers (which may influence the efficacy of the degradation), and creating and testing K-less vectors to exclude the possibility of luciferase or FKBP12 ubiquitination instead of WDR5 (the target protein). Additionally, the position of the luc in the FKBP12 vector and the position of VHL in the FRB vector are tested. Different E3 ligases are tested using previously identified target proteins, confirming the assay's utility and accuracy.

(3) The study identified a "new" E3 ligase that is suitable for PROTAC technology (FBXL).

Weaknesses:

It is not clear how feasible it would be to adapt the assay for high-throughput screens. In some experiments, the efficacy of WDR5 degradation tested by immunoblotting appears to be lower than luciferase activity (e.g., Figure 2G and H).

Reviewer #1 (Public Review):

This manuscript presents insights into biased signaling in GPCRs, namely cannabinoid receptors. Biased signaling is of broad interest in general, and cannabinoid signaling is particularly relevant for understanding the impact of new drugs that target this receptor. Mechanistic insight from work like this could enable new approaches to mitigate the public health impact of new psychoactive drugs. Towards that end, this manuscript seeks to understand how new psychoactive substances (NPS, e.g. MDMB-FUBINACA) elicit more signaling through β-arrestin than classical cannabinoids (e.g. HU-210). The authors use an interesting combination of simulations and machine learning.

The caption for Figure 3 doesn't explain the color scheme, so it's not obvious what the start and end states of the ligand are.

For the metadynamics simulations were multiple Gaussian heights/widths tried to see what, if any, impact that has on the unbinding pathway? That would be useful to help ensure all the relevant pathways were explored.

It would be nice to acknowledge previous applications of metadynamics+MSMs and (separately) TRAM, such as the Simulation of spontaneous G protein activation... (Sun et al. eLife 2018) and Estimation of binding rates and affinities... (Ge and Voelz JCP 2022).

What is KL divergence analysis between macrostates? I know KL divergence compares probability distributions, but it is not clear what distributions are being compared.

I suggest being more careful with the language of universality. It can be "supported" but "showing" or "proving" its universal would require looking at all possible chemicals in the class.

Reviewer #1 (Public Review):

Summary:

In this study, the authors identified and described the transcriptional trajectories leading to CMs during early mouse development, and characterized the epigenetic landscapes that underlie early mesodermal lineage specification.

The authors identified two transcriptomic trajectories from a mesodermal population to cardiomyocytes, the MJH and PSH trajectories. These trajectories are relevant to the current model for the First Heart Field (FHF) and the Second Heart Field (SHF) differentiation. Then, the authors characterized both gene expression and enhancer activity of the MJH and PSH trajectories, using a multiomics analysis. They highlighted the role of Gata4, Hand1, Foxf1, and Tead4 in the specification of the MJH trajectory. Finally, they performed a focused analysis of the role of Hand1 and Foxf1 in the MJH trajectory, showing their mutual regulation and their requirement for cardiac lineage specification.

Strengths:

The authors performed an extensive transcriptional and epigenetic analysis of early cardiac lineage specification and differentiation which will be of interest to investigators in the field of cardiac development and congenital heart disease. The authors considered the impact of the loss of Hand1 and Foxf1 in-vitro and Hand1 in-vivo.

Weaknesses:

The authors used previously published scRNA-seq data to generate two described transcriptomic trajectories.

(1) Details of the re-analysis step should be added, including a careful characterization of the different clusters and maker genes, more details on the WOT analysis, and details on the time stamp distribution along the different pseudotimes. These details would be important to allow readers to gain confidence that the two major trajectories identified are realistic interpretations of the input data.

The authors have also renamed the cardiac trajectories/lineages, departing from the convention applied in hundreds of papers, making the interpretation of their results challenging.

(2) The concept of "reverse reasoning" applied to the Waddington-OT package for directional mass transfer is not adequately explained. While the authors correctly acknowledged Waddington-OT's ability to model cell transitions from ancestors to descendants (using optimal transport theory), the justification for using a "reverse reasoning" approach is missing. Clarifying the rationale behind this strategy would be beneficial.

(3) As the authors used the EEM cell cluster as a starting point to build the MJH trajectory, it's unclear whether this trajectory truly represents the cardiac differentiation trajectory of the FHF progenitors:<br /> - This strategy infers that the FHF progenitors are mixed in the same cluster as the extra-embryonic mesoderm, but no specific characterization of potential different cell populations included in this cluster was performed to confirm this.

- The authors identified the EEM cluster as a Juxta-cardiac field, without showing the expression of the principal marker Mab21l2 per cluster and/or on UMAPs.

- As the FHF progenitors arise earlier than the Juxta-cardiac field cells, it must be possible to identify an early FHF progenitor population (Nkx2-5+; Mab21l2-) using the time stamp. It would be more accurate to use this FHF cluster as a starting point than the EEM cluster to infer the FHF cardiac differentiation trajectory.

These concerns call into question the overall veracity of the trajectory analysis, and in fact, the discrepancies with prior published heart field trajectories are noted but the authors fail to validate their new interpretation. Because their trajectories are followed for the remainder of the paper, many of the interpretations and claims in the paper may be misleading. For example, these trajectories are used subsequently for annotation of the multiomic data, but any errors in the initial trajectories could result in errors in multiomic annotation, etc, etc.

(4) As mentioned in the discussion, the authors identified the MJH and PSH trajectories as non-overlapping. But, the authors did not discuss major previously published data showing that both FHF and SHF arise from a common transcriptomic progenitor state in the primitive streak (DOI: 10.1126/science.aao4174; DOI: 10.1007/s11886-022-01681-w). The authors should consider and discuss the specifics of why they obtained two completely separate trajectories from the beginning, how these observations conflict with prior published work, and what efforts they have made at validation.

(5) Figures 1D and E are confusing, as it's unclear why the authors selected only cells at E7.0. Also, panels 1D 'Trajectory' and 'Pseudotime' suggest that the CM trajectory moves from the PSH cells to the MJH. This result is confusing, and the authors should explain this observation.

(6) Regarding the PSH trajectory, it's unclear how the authors can obtain a full cardiac differentiation trajectory from the SHF progenitors as the SHF-derived cardiomyocytes are just starting to invade the heart tube at E8.5 (DOI: 10.7554/eLife.30668).

The above notes some of the discrepancies between the author's trajectory analysis and the historical cardiac development literature. Overall, the discrepancies between the author's trajectory analysis and the historical cardiac development literature are glossed over and not adequately validated.

(7) The authors mention analyzing "activated/inhibited genes" from Peng et al. 2019 but didn't specify when Peng's data was collected. Is it temporally relevant to the current study? How can "later stage" pathway enrichment be interpreted in the context of early-stage gene expression?

(8) Motif enrichment: cluster-specific DAEs were analyzed for motifs, but the authors list specific TFs rather than TF families, which is all that motif enrichment can provide. The authors should either list TF families or state clearly that the specific TFs they list were not validated beyond motifs.

(9) The core regulatory network is purely predictive. The authors again should refrain from language implying that the TFs in the CRN have any validated role.

Regarding the in vivo analysis of Hand1 CKO embryos, Figures 6 and 7:

(10) How can the authors explain the presence of a heart tube in the E9.5 Hand1 CKO embryos (Figure 6B) if, following the authors' model, the FHF/Juxta-cardiac field trajectory is disrupted by Hand1 CKO? A more detailed analysis of the cardiac phenotype of Hand1 CKO embryos would help to assess this question.

(11) The cell proportion differences observed between Ctrl and Hand1 CKO in Figure 6D need to be replicated and an appropriate statistical analysis must be performed to definitely conclude the impact of Hand1 CKO on cell proportions.

(12) The in-vitro cell differentiations are unlikely to recapitulate the complexity of the heart fields in-vivo, but they are analyzed and interpreted as if they do.

(13) The schematic summary of Figure 7F is confusing and should be adjusted based on the following considerations:<br /> (a) the 'Wild-type' side presents 3 main trajectories (SHF, Early HT and JCF), but uses a 2-color code and the authors described only two trajectories everywhere else in the article (aka MJH and PSH). It's unclear how the SHF trajectory (blue line) can contribute to the Early HT, when the Early HT is supposed to be FHF-associated only (DOI: 10.7554/eLife.30668). As mentioned previously in Major comment 3., this model suggests a distinction between FHF and JCF trajectories, which is not investigated in the article.<br /> (b) the color code suggests that the MJH (FHF-related) trajectory will give rise to the right ventricle and outflow tract (green line), which is contrary to current knowledge.

Minor comments:

(1) How genes were selected to generate Figure 1F? Is this a list of top differentially expressed genes over each pseudotime and/or between pseudotimes?

(2) Regarding Figure 1G, it's unclear how inhibited signaling can have an increased expression of underlying genes over pseudotimes. Can the authors give more details about this analysis and results?

(3) How do the authors explain the visible Hand1 expression in Hand1 CKO in Figure S7C 'EEM markers'? Is this an expected expression in terms of RNA which is not converted into proteins?

(4) The authors do not address the potential presence of doublets (merged cells) within their newly generated dataset. While they mention using "SCTransform" for normalization and artifact removal, it's unclear if doublet removal was explicitly performed.

Reviewer #1 (Public Review):

Summary:

This paper presents a class of small molecule inhibitors of tau aggregation which was discovered through a computational screen. Analogs were generated and tested for their ability to inhibit fibril formation.

Strengths:

A few of the analogs were found to have sub-stoichiometric activity. A comparison of unseeded and seeded aggregation kinetics suggests that these compounds preferentially target early-stage aggregation.

Weaknesses:

The authors state their interest is in finding compounds that target monomeric states of tau, but their only detection method is late-stage fibril formation. In this respect, they have not really defined a mechanism of action. They state their plan to use hydrogen-exchange mass spectrometry, but there are other techniques, such as single-molecule FRET and measurement of intramolecular reconfiguration. Additionally, there is information that can be gleaned from detailed kinetic modeling of the ThT kinetics to include monomer dynamics, formation of oligomers, and secondary nucleation of fibrils.

Reviewer #1 (Public Review):

Summary:

The study conducted by the Shouldiner's group advances the understanding of mitochondrial biology through the utilization of their bi-genomic (BiG) split-GFP assay, which they had previously developed and reported. This research endeavors to consolidate the catalog of matrix and inner membrane mitochondrial proteins. In their approach, a genetic framework was employed wherein a GFP fragment (GFP1-10) is encoded within the mitochondrial genome. Subsequently, a collection of strains was created, with each strain expressing a distinct protein tagged with the GFP11 fragment. The reconstitution of GFP fluorescence occurs upon the import of the protein under examination into the mitochondria.

Strengths:

Notably, this assay was executed under six distinct conditions, facilitating the visualization of approximately 400 mitochondrial proteins. Remarkably, 50 proteins were conclusively assigned to mitochondria for the first time through this methodology. The strains developed and the extensive dataset generated in this study serve as a valuable resource for the comprehensive study of mitochondrial biology. Specifically, it provides a list of 50 "eclipsed" proteins whose role in mitochondria remains to be characterized.

Weaknesses:

The work could include some functional studies of at least one of the newly identified 50 proteins.

Reviewer #1 (Public Review):

Summary:<br /> Overall, this study provides a meticulous comparison of developmental transcriptomes between two sub-species of the annelid Streblospio benedicti. Different lineages of S. benedicti maintain one of two genetically programmed alternative life histories, the ancestral planktotrophic or derived lecithotrophic forms of development. This contrast is also seen at the inter-species level in many marine invertebrate taxa, such as echinoderms and molluscs. The authors report relatively (surprisingly?) modest differences in transcriptomes overall, but also find some genes whose expression is essentially morph-specific (which they term "exclusive").

Strengths:<br /> The study is based on dense and appropriately replicated sampling of early development. The tight clustering of each stage/morph combination in PCA space suggests the specimens were accurately categorized. The similar overall trajectories of the two morphs was surprising to me for two stage: 1) the earliest stage (16-cell), at which we might expect maternal differences due to the several-fold difference in zygote size, and 2) the latest stage (1-week), where there appears to be the most obvious morphological difference. This is why we need to do experiments!

The examination of F1 hybrids was another major strength of the study. It also produced one of the most surprising results: though intermediate in phenotype, F1 embryos have the most distinct transcriptomes, and reveal a range of fixed, compensatory differences in the parental lines. Further, the F1 lack expression of nearly all transcripts identified as morph-specific in the pure parental lines. Since the F1 larvae present intermediate traits combining the features of both morphs, this implies that morph-specific transcripts are not actually necessary for morph-specific traits. This is interesting and somewhat counter to what one might naively expect.

Weaknesses:<br /> Overall I really enjoyed this paper, and in its revised form it addresses some concerns I had in the first version. I still see a few places where it can be tightened and made more insightful.

Reviewer #1 (Public Review):

Summary:

The endocannabinoid system (ECS) components are dysregulated within the lesion microenvironment and systemic circulation of endometriosis patients. Using endometriosis mouse models and genetic loss of function approaches, Lingegowda et al. report that canonical ECS receptors, CNR1 and CNR2, are required for disease initiation, progression, and T-cell dysfunction.

Strengths:

The approach uses genetic approaches to establish in vivo causal relationships between dysregulated ECS and endometriosis pathogenesis. The experimental design incorporates both bulk and single-cell RNAseq approaches, as well as imaging mass spectrometry to characterize the mouse lesions. The identification of immune-related and T-cell-specific changes in the lesion microenvironment of CNR1 and CNR2 knockout (KO) mice represents a significant advance

Weaknesses:

Although the mouse phenotypic analyses involves a detailed molecular characterization of the lesion microenvironment using genomic approaches, detailed measurements of lesion size/burden and histopathology would provide a better understanding of how CNR1 or CNR2 loss contributes to endometriosis initiation and progression. The cell or tissue-specific effects of the CNR1 and CNR2 are not incorporated into the experimental design of the studies. Although this aspect of the approach is recognized as a major limitation, global CNR1 and CNR2 KO may affect normal female reproductive tract function, ovarian steroid hormone levels, decidualization response, or lead to preexisting alterations in host or donor tissues, which could affect lesion establishment and development in the surgically induced, syngeneic mouse model of endometriosis.

Reviewer #1 (Public Review):

The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.

The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.

The authors have addressed most of my previous concerns.

Reviewer #1 (Public Review):

Summary:

In this well-designed study, the authors of the manuscript have analyzed the impact of individually silencing 90 lipid transfer proteins on the overall lipid composition of a specific cell type. They confirmed some of the evidence obtained by their own and other research groups in the past, and additionally, they identified an unreported role for ORP9-ORP11 in sphingomyelin production at the trans-Golgi. As they delved into the nature of this effect, the authors discovered that ORP9 and ORP11 form a dimer through a helical region positioned between their PH and ORD domains.

Strengths:

This well-designed study presents compelling new evidence regarding the role of lipid transfer proteins in controlling lipid metabolism. The discovery of ORP9 and ORP11's involvement in sphingolipid metabolism invites further investigation into the impact of the membrane environment on sphingomyelin synthase activity.

Weaknesses:

There are a couple of weaknesses evident in this manuscript. Firstly, there's a lack of mechanistic understanding regarding the regulatory role of ORP9-11 in sphingomyelin synthase activity. Secondly, the broader role of hetero-dimerization of LTPs at ER-Golgi membrane contact sites is not thoroughly addressed. The emerging theme of LTP dimerization through coiled domains has been reported for proteins such as CERT, OSBP, ORP9, and ORP10. However, the specific ways in which these LTPs hetero and/or homo-dimerize and how this impacts lipid fluxes at ER-Golgi membrane contact sites remain to be fully understood.

Regardless of the unresolved points mentioned above, this manuscript presents a valuable conceptual advancement in the study of the impact of lipid transfer on overall lipid metabolism. Moreover, it encourages further exploration of the interplay among LTP actions across various cellular organelles.

Reviewer #2 (Public Review):

Summary:

In this work, Mohamed Y. El-Naggar and co-workers present a detailed electronic characterization of cable bacteria from Southern California freshwater sediments. The cable bacteria could be reliably enriched in laboratory incubations, and subsequent TEM characterization and 16S rRNA gene phylogeny demonstrated their belonging to the genus Candidatus Electronema. Atomic force microscopy and two-point probe resistance measurements were then used to map out the characteristics of the conductive nature, followed by microelectrode four-probe measurements to quantify the conductivity.

Interestingly, the authors observe that some freshwater cable bacteria filaments displayed a higher degree of robustness upon oxygen exposure than what was previously reported for marine cable bacteria. Finally, a single nanofiber conductivity on the order of 0.1 S/cm is calculated, which matches the expected electron current densities linking electrogenic sulphur oxidation to oxygen reduction in sediment and is consistent with hopping transport.

Strengths and weaknesses:

A comprehensive study is applied to characterise the conductive properties of the sampled freshwater cable bacteria. Electrostatic force microscopy and conductive atomic force microscopy provide direct evidence of the location of conductive structures. Four-probe microelectrode devices are used to quantify the filament resistance, which presents a significant advantage over commonly used two-probe measurements that include contributions from contact resistances. While the methodology is convincing, I find that some of the conclusions seem to be drawn on very limited sample sizes, which display widely different behaviour. In particular:

The authors observe that the conductivity of freshwater filaments may be less sensitive to oxygen exposure than previously observed for marine filaments. This is indeed the case for an interdigitated array microelectrode experiment (presented in Figure 5) and for a conductive atomic force microscopy experiment (described in line 391), but the opposite is observed in another experiment (Figure S1). It is therefore difficult to assess the validity of the conclusion until sufficient experimental replications are presented.

The calculation of a single nanofiber conductivity is based on experiment and calculation with significant uncertainty. E.g. for the number of nanofibres in a single filament that varies depending on the filament size (Frontiers in microbiology, 2018, 9: 3044.), and the measured CB resistance, which does not scale well with inner probe separation (Figure 5). A more rigorous consideration of these uncertainties is required.

Comments on revised version:

The authors address all of the comments carefully.

Reviewer #1 (Public Review):

Little is known about the local circuit mechanisms in the preoptic area (POA) that regulate body temperature. This carefully executed study investigates the role of GABAergic interneurons in the POA that express neurotensin (NTS). The principal finding is that GABA-release from these cells inhibits neighboring neurons, including warm-activated PACAP neurons, thereby promoting hyperthermia, whereas NTS released from these cells has the opposite effect, causing a delayed activation and hypothermia. This is shown through an elegant series of experiments that include slice recordings alongside matched in vivo functional manipulations. The roles of the two neurotransmitters are distinguished using a cell-type-specific knockout of Vgat as well as pharmacology to block GABA and NTS receptors. Overall, this is an excellent study that is noteworthy for revealing local circuit mechanisms in the POA that control body temperature and also for highlighting how amino acid neurotransmitters and neuropeptides released from the same cell can have opposing physiologic effects.

Reviewer #1 (Public Review):

Summary:

The authors aim to assess the effect of salt stress on root:shoot ratio, identify the underlying genetic mechanisms, and evaluate their contribution to salt tolerance. To this end, the authors systematically quantified natural variations in salt-induced changes in root:shoot ratio. This innovative approach considers the coordination of root and shoot growth rather than exploring biomass and the development of each organ separately. Using this approach, the authors identified a gene cluster encoding eight paralog genes with a domain-of-unknown-function 247 (DUF247), with the majority of SNPs clustering into SR3G (At3g50160). In the manuscript, the authors utilized an integrative approach that includes genomic, genetic, evolutionary, histological, and physiological assays to functionally assess the contribution of their genes of interest to salt tolerance and root development.

Strengths:

The holistic approach and integrative methodologies presented in the manuscript are essential for gaining a mechanistic understanding of a complex trait such as salt tolerance. The authors focused on At3g50160 but included in their analyses additional DUF247 paralogs, which further contributes to the strength of their approach. In addition, the authors considered the developmental stage (young seedlings, early or late vegetative stages) and growth conditions of the plants (agar plates or soil) when investigating the role of SR3G in salt tolerance and root or shoot development.

Weaknesses:

The authors' claims and interpretation of the results are not fully supported by the data and analyses. In several cases, the authors report differences that are not statistically significant (e.g., Figures 4A, 7C, 8B, S14, S16B, S17C), use inappropriate statistical tests (e.g., t-test instead of Dunnett Test/ANOVA as in Figures 10B-C, S19-23), present standard errors that do not seem to be consistent with the post-hoc Tukey HSD Test (e.g., Figures 4, 9B-C, S16B), or lack controls (e.g., Figure 5C-E, staining of the truncated versions with FM4-64 is missing).

In other cases, traits of root system architecture and expression patterns are inconsistent between different assays despite similar growth conditions (e.g., Figures S17A-B vs. 10A-C vs. 6A, and Figures S16B vs. 4A/9B), or T-DNA insertion alleles of WRKY75 that are claimed to be loss-of-function show comparable expression of WRKY75 as WT plants. Additionally, several supplemental figures are mislabeled (Figures S6-9), and some figure panels are missing (e.g., Figures S16C and S17E).

Consequently, the authors' decisions regarding subsequent functional assays, as well as major conclusions about gene function, including SR3G function in root system architecture, involvement in root suberization, and regulation of cellular damage are incomplete.

Reviewer #1 (Public Review):

Summary:

Assessment of cardiac LEC transcriptomes post-MI may yield new targets to improve lymphatic function. scRNAseq is a valid approach as cardiac LECs are rare compared to blood vessel endothelial cells.

Strengths:

Extensive bioinformatics approaches employed by the group.

Weaknesses:

Too few cells are included in scRNAseq data set and the spatial transcriptomics data that was exploited has little relevance, or rather specificity, for cardiac lymphatics. This study seems more like a collection of preliminary transcriptomic data than a conclusive scientific report to help advance the field.

Reviewer #1 (Public Review):

Summary:

In this study, the authors developed three case studies: (1) transcriptome profiling of two human cell cultures (HEK293 and HeLa), (2) identification of experimentally enriched transcripts in cell culture (RiboMinus and RiboPlus treatments), and (3) identification of experimentally manipulated genes in yeast strains (gene knockouts or strains transformed with plasmids containing the deleted gene for overexpression). Sequencing was performed using the Oxford Nanopore Technologies (ONT), the only technology that allows for real-time analysis. The real-time transcriptomic analysis was performed using NanopoReaTA, a recent toolbox for comparative transcriptional analyses of Nanopore-seq data, developed by the group (Wierczeiko and Pastore et al. 2023). The authors aimed to show the use of the tool developed by them in data generated by ONT, evidencing the versatility of the tool and the possibility of cost reduction since the sequencing by ONT can be stopped at any time since enough data were collected.

Strengths:

Given that Oxford Nanopore Technologies offers real-time sequencing, it is extremely useful to develop tools that allow real-time data analysis in parallel with data generation. The authors demonstrated that this strategy is possible for both human cell lines and yeasts in the case studies presented. It is a useful strategy for the scientific community and it has the potential to be integrated into clinical applications for rapid and cost-effective quality checks in specific experiments such as overexpression of genes.

Weaknesses:

In relation to the RNA-Seq analyses, for a proper statistical analysis, a greater number of replicates should have been performed. The experiments were conducted with a minimal number of replicates (2 replicates for case study 1 and 2 and 3 replicates for case study 3).

Regarding the experimental part, some problems were observed in the conversion to double-stranded and loading for Nanopore-Seq, which were detailed in Supplementary Material 2. This fact is probably reflected in the results where a reduction in the overall sequencing throughput and detected gene number for HEK293 compared to HeLa were observed (data presented in Supplementary Figure 2). It is necessary to use similar quantities of RNA/cDNA since the sequencing occurs in real-time. The authors should have standardized the experimental conditions to proceed with the sequencing and perform the analyses.

Reviewer #1 (Public Review):

In this work, the authors provide a valuable transcriptomic resource for the intermediate free-living transmission stage (miracidium larva) of the blood fluke. The single-cell transcriptome inventory is beautifully supplemented with in situ hybridization, providing spatial information and absolute cell numbers for many of the recovered transcriptomic states. The identification of sex-specific transcriptomic states within the populations of stem cells was particularly unexpected. The work comprises a rich resource to complement the biology of this complex system.

Comments on revised version:

I have read through the responses and the revised manuscript. I think together this results in an improved version.

Reviewer #1 (Public Review):

This study of mixed glutamate/GABA transmission from axons of the supramammillary nucleus to dentate gyrus seeks to sort out whether the two transmitters are released from the same or different synaptic vesicles. This conundrum has been examined in other dual-transmission cases and even in this particular pathway, there are different views. The authors use a variety of electrophysiological and immunohistochemical methods to reach the surprising (to me) conclusion that glutamate and GABA-filled vesicles are distinct yet released from the same nerve terminals. The strength of the conclusion rests on the abundance of data (approaches) rather than the decisiveness of any one approach, and I came away believing that the boutons may indeed produce and release distinct types of vesicles, but have reservations. Accepting the conclusion, one is now left with another conundrum, not addressed even in the discussion: how can a single bouton sort out VGLUTs and VIAATs to different vesicles, position them in distinct locations with nm precision, and recycle them without mixing? And why do it this way instead of with single vesicles having mixed chemical content? For example, could a quantitative argument be made that separate vesicles allow for higher transmitter concentrations? I feel the paper needs to address these problems with some coherent discussion, at minimum.

Major concerns:

(1) Throughout the paper, the authors use repetitive optogenetic stimulation to activate SuM fibers and co-release glutamate and GABA. There are several issues here: first, can the authors definitively assure the reader that all the short-term plasticity is presynaptic and not due to ChR2 desensitization? This has not been addressed. Second, can the authors also say that all the activated fibers release both transmitters? If for example 20% of the fibers retained a one-transmitter identity and had distinct physiological properties, could that account for some of the physiological findings?

(2) PPR differences in Figures 1F-I are statistically significant but still quite small. You could say they are more similar than different in fact, and residual differences are accounted for by secondary factors like differential receptor saturation.

(3) The logic of the GPCR experiments needs a better setup. I could imagine different fibers released different transmitters and had different numbers of mGluRs, so that one would get different modulations. On the assumption that all the release is from a single population of boutons, then either the mGluRs are differentially segregated within the bouton, or the vesicles have differential responsiveness to the same modulatory signal (presumably a reduced Ca current). This is not developed in the paper.

(4) The biphasic events of Figures 3 and S3: I find these (unaveraged) events a bit ambiguous. Another way to look at them is that they are not biphasic per se but rather are not categorizable. Moreover, these events are really tiny, perhaps generated by only a few receptors whose open probability is variable, thus introducing noise into the small currents.

(5) Figure 4 indicates that the immunohistochemical analysis is done on SuM terminals, but I do not see how the authors know that these terminals come from SuM vs other inputs that converge in DG.

(6) Figure 4E also shows many GluN1 terminals not associated with anything, not even Vglut, and the apparent numbers do not mesh with the statistics. Why?

(7) Do the conclusions based on the fluorescence immuno mesh with the apparent dimensions of the EM active zones and the apparent intermixing of labeled vesicles in immuno EM?

(8) Figure 6 is not so interesting to me and could be removed. It seems to test the obvious: EPSPs promote firing and IPSPs oppose it.

Reviewer #2 (Public Review):

Summary:

Previous research shows that humans tend to adjust learning in environments where stimulus-outcome contingencies become more volatile. This learning rate adaptation is impaired in some psychiatric disorders, such as depression and anxiety. In this tudy the authors reanalyze previously published data on a reversal learning task with two volatility levels. Through a new model they provide some evidence for an alternative explanation whereby the learning rate adaptation is driven by different decision-making strategies and not learning deficits. In particular, they propose that adjusting of learning can be explained by deviations from the optimal decision-making strategy (based on maximizing expected utility) due to response stickiness or focus on reward magnitude. Furthermore, a factor related to general psychopathology of individuals with anxiety and depression negatively correlated with the weight on the optimal strategy and response stickiness, while it correlated positively with the magnitude strategy (a strategy that ignores the probability of outcome).

The main strength of the study is a novel and interesting explanation of an otherwise well-established finding in human reinforcement learning. This proposal is supported by rigorously conducted parameter retrieval and the comparison of the novel model to a wide range of previously published models. The authors explore from many angles, if and why the predictions from the new proposed model are superior to previously applied models.

My previous concerns were addressed in the revised version of the manuscript. I believe that the article now provides a new perspective on a well-established learning effect and offer a novel set of interesting response models that can be applied to a wide array of decision-making problems.

I see two limitations of the study not mentioned in the discussion of the manuscript. First, the task features binary inputs and responses, therefore unexpected uncertainty (volatility) is impossible to differentiate from the uncertainty about outcomes, and exploration is inseparable from random choices. Future work could validate these findings in task designs that allow to distinguish these processes. Second, clinical results are based on a small sample of patients and should be interpreted with this in mind.

Reviewer #1 (Public Review):

Summary:

Das and Menon describe an analysis of a large open-source iEEG dataset (UPENN-RAM). From encoding and recall phases of memory tasks, they analyzed power and phase-transfer entropy as a measure of directed information flow in regions across a hypothesized tripartite network system. The anterior insula (AI) was found to have heightened high gamma power during encoding and retrieval, which corresponded to suppression of high gamma power in the posterior cingulate cortex (PCC) during encoding but not recall. In contrast, directed information flow from (but not to) AI to mPFC/PCC and dorsal posterior parietal/middle frontal cortex is high during both time periods when PTE is analyzed with broadband but not narrowband activity. They claim that these findings significantly advance an understanding of how network communication facilitates cognitive operations such as control over memory and that the AI of the salience network (SN) is responsible for governing the switch between the frontoparietal network (FPN) and default-mode network (DMN) when shifting between externally- and internally-driven processing.

I find this question interesting and important and agree with the authors that iEEG presents a unique opportunity to investigate the temporal dynamics within network nodes. However, I am not convinced that their claims are supported by the results currently presented. In particular, the fact that network-level communication is not modulated significantly compared to rest and does not relate to behavior suggests that PTE analyses may not be tapping into task-relevant communication. Moreover, dissociation of network effects - present during both encoding and recall - from local power suppression effects - present only during encoding - suggests that these sets of results may index separate and not unitary task processes.

Strengths:

- The authors present results from an impressively sized iEEG sample. For reader context, this type of invasive human data is difficult and time-consuming to collect and many similar studies in high-level journals include 5-20 participants, typically not all of whom have electrodes in all regions of interest. It is excellent that they have been able to leverage open-source data in this way.

- Preprocessing of iEEG data also seems sensible and appropriate based on field standards.

- The authors tackle the replication issues inherent in much of the literature by replicating findings across task contexts, demonstrating that the principles of network communication evidenced by their results generalize in multiple task memory contexts. Again, the number of iEEG patients who have multiple tasks' worth of data is impressive.

Weaknesses:

• The motivation for investigating the tripartite network during memory tasks is not currently well-elaborated. Though the authors mention, for example, that "the formation of episodic memories relies on the intricate interplay between large-scale brain networks (p. 4)", there are no citations provided for this statement, and the reader is unable to evaluate whether the nodes and networks evidenced to support these processes are the same as networks measured here.

• In addition, though the tripartite network has been proposed to support cognitive control processes, and the neural basis of cognitive control is the framed focus of this work, the authors do not demonstrate that they have measured cognitive control in addition to simple memory encoding and retrieval processes. Tasks that have investigated cognitive control over memory (such as those cited on p. 13 - Badre et al., 2005; Badre & Wagner, 2007; Wagner et al., 2001; Wagner et al., 2005) generally do not simply include encoding, delay, and recall (as the tasks used here), but tend to include some manipulation that requires participants to engage control processes over memory retrieval, such as task rules governing what choice should be made at recall (e.g., from Badre et al., 2005 Fig. 1: congruency of match, associative strength, number of choices, semantic similarity). Moreover, though there are task-responsive signatures in the nodes of the tripartite networks, concluding that cognitive control is present because cognitive control networks are active would be a reverse inference.

• It is currently unclear if the directed information flow from AI to DMN and FPN nodes truly arises from task-related processes such as cognitive control or if it is a function of static brain network characteristics constrained by anatomy (such as white matter connection patterns, etc.). This is a concern because the authors did not find that influences of AI on DMN or FPN are increased relative to a resting baseline (collected during the task) or that directed information flow differs in successful compared to unsuccessful retrieval. I doubt that this AI influence is 1) supporting a switch between the DMN and FPN via the SN or 2) relevant for behavior if it doesn't differ from baseline-active task or across accuracy conditions. An additional comparison that may help investigate whether this is reflective of static connectivity characteristics would be a baseline comparison during non-task rest or sleep periods.

• Related to the above concern, it is also questionable how directed information flow from AI facilitates switching between FPN and DMN during both encoding and recall if high gamma activity does not significantly differ in AI versus PCC or mPFC during recall as it does during encoding. It seems erroneous to conclude that the network-level communication is happening or happening with the same effect during both task time points when these effects are decoupled in such a way from the power findings.

• Missing information about the methods used for time-frequency conversion for power calculation and the power normalization/baseline-correction procedure bars a thorough evaluation of power calculation methods and results.

If revisions to the manuscript can address concerns about directed information flow possibly being due to anatomical constraints - such as by indicating that directed information flow is not present during non-task rest or sleep - this work may convey important information about the structure and order of communication between these networks during attention to tasks in general. However, the ability of the findings to address cognitive control-specific communication and the nature of neurophysiological mechanisms of this communication - as opposed to the temporal order and structure of recruited networks - may be limited.

Because phase-transfer entropy is presented as a "causal" analysis in this investigation (PTE), I also believe it is important to highlight for readers recent discussions surrounding the description of "causal mechanisms" in neuroscience (see "Confusion about causation" section from Ross and Bassett, 2024, Nature Neuroscience). A large proportion of neuroscientists (admittedly, myself included) use "causal" only to refer to a mechanism whose modulation or removal (with direct manipulation, such as by lesion or stimulation) is known to change or control a given outcome (such as a successful behavior). As Ross and Bassett highlight, it is debatable whether such mechanistic causality is captured by Granger "causality" (a.k.a. Granger prediction) or the parametric PTE, and the imprecise use of "causation" may be confusing. The authors could consider amending language regarding this analysis if they are concerned about bridging these definitions of causality across a wide audience.

Reviewer #1 (Public Review):

Summary:

The investigation delves into allosteric modulation within the glycosylated SARS-CoV-2 spike protein, focusing on the fatty acid binding site. This study uncovers intricate networks connecting the fatty acid site to crucial functional regions, potentially paving the way for developing innovative therapeutic strategies.

Strengths:

This article's key strength lies in its rigorous use of dynamic nonequilibrium molecular dynamics (D-NEMD) simulations. This approach provides a dynamic perspective on how the fatty acid binding site influences various functional regions of the spike. A comprehensive understanding of these interactions is crucial in deciphering the virus's behavior and identifying potential targets for therapeutic intervention.

Weaknesses:

The presented evidence is compelling but could be better if this study is supported with sequence analysis to facilitate a complete view of the allosteric networks. The thorough analysis of the simulation results is partially aligned with the discussion because observed in the replicates and the monomers an asymmetry in the perturbations generated by D-NEMD, even when we're using 210 nonequilibrium MD of 10 ns. While the authors claim that the strategy used in this article has been previously validated, the complexity of the spike and the interactions analyzed have required a robust statistical analysis, which is not shown quantitatively. The investigation examines the allosteric modulation within the glycosylated SARS-CoV-2 spike protein, emphasizing the significance of the fatty acid binding site in influencing the structural dynamics and communication pathways essential for viral function, potentially facilitating the development of novel therapeutic strategies. The presented evidence is compelling but needs to be supported by sequence analysis, which will facilitate understanding of the scientific community.

Minor considerations:

Figure S3 shows a discrepancy in the presentation of residue values S325 in the plots of Chains A, B, and C. While chain A shows a value near 0.1, chains B and C plots do not have any value.

Please explain why the plots of figures S6, S7, and S8 show significant changes in several regions, such as RBM and Furin Site. Can these changes be explained?

The flow of the allosteric interaction is complex to visualize just by looking at structures. Could you please include a diagram showing the flow of allosteric interactions (in a sequence diagram or using the structure of the protein)? Or could you include a vector showing how the perturbation done in the FA Active site takes contact with other relevant regions of the Spike protein?

Reviewer #1 (Public Review):

The authors effectively delineate the differential distribution and behaviour of MNPs within the heart, noting that these cells can be characterised by their expression levels of csf1ra and mpeg1.1. Key findings include the identification of distinct origins for larval macrophage populations and the sustained presence of csf1ra-expressing cells on the surface of the adult heart. The study examines the embryonic development of these MNPs, revealing that csf1ra+ cells begin populating the heart from embryonic day 3, while mpeg1.1+ cells colonise the heart around day 10, with a significant increase by day 17. Given that the emergence of mpeg1.1+ cells coincides with the reported timing for the onset of haematopoietic stem cell-derived haematopoiesis, the authors combined kaede-lineage tracing experiments and mutant backgrounds to conclude that the earliest tissue-resident macrophages in the heart are derived from primitive haematopoiesis.

The authors also note that the spatial distribution of MNPs varies, with csf1ra+ cells found on the atrium and ventricle surfaces, while mpeg1.1+ cells are initially located on the surface but later distributed throughout the cardiac tissue. Notably, the study demonstrates that tissue-resident macrophages proliferate rapidly following cardiac injury. The authors observe an increased number of proliferating csf1ra+ cells, especially in csf1ra mutant zebrafish, which likely correspond to primitive-derived tissue-resident macrophages that rapidly respond to injury and contribute to the reduced scarring observed in these mutants.

This manuscript makes an important contribution to the field by enhancing our understanding of the ontogeny of tissue-resident macrophages in the heart and their cellular behaviour in a vertebrate model capable of heart regeneration.

Strengths:

This work presents a landmark study on the ontogeny and cellular behaviour of macrophages in the zebrafish heart as it comprehensively examines their development and distribution in both embryonic and adult stages.

One of the key strengths of this study is its thorough cellular description using a range of available genetic tools. By employing transgenic lines to differentiate between a few MNP subtypes, the authors provide a detailed and nuanced understanding of these cells' origins, distribution, and behaviour. This approach allows for high-resolution characterisation of MNP populations, revealing significant insights into their potential role in cardiac homeostasis and regeneration.

Furthermore, the study's findings are significant as they parallel those observed in mouse models, thereby reinforcing the validity and relevance of the zebrafish as a model organism for studying macrophage function in the context of cardiac injury. This comparative aspect underscores the evolutionary conservation of these cellular processes and enhances the study's impact.

Another notable strength is the use of ex vivo imaging techniques, which enable the authors to observe and study the dynamic behaviour of MNPs in heart tissue in real-time. This live imaging capability is crucial for understanding how these cells interact with their environment, particularly in response to cardiac injury. The ability to visualise MNP proliferation and movement provides valuable insights into the mechanisms underlying tissue repair and regeneration.

Weaknesses:

While the manuscript offers significant insights into the ontogeny and behaviour of MNPs in the zebrafish heart, a few limitations described below should be considered:

One potential issue lies in the lineage tracing experiments using the photoconversion Tg(csf1ra:Gal4); Tg(UAS:kaede) line. The authors photoconverted all cardiac tissue macrophages present at 2 days post-fertilisation (dpf) and examined the hearts of these fish at 21 dpf. Although photoconverted macrophages were still observed at 21 dpf, the majority of cells present in the heart at that time were non-photoconverted (cyan) csf1ra+ cells. While this suggests that early-seeded embryonic csf1ra+ macrophages are retained during late larval stages, the contribution of macrophages derived from haematopoietic stem cells (HSCs) might be overestimated. An important concern is that the kaede-converted cells could have proliferated during the embryonic timeframe analysed, thereby diluting and extinguishing the converted kaede protein. This dilution effect could lead to an underestimation of the contribution of primitive embryonic macrophages relative to the HSC-derived cells, resulting in an inaccurate assessment of the proportion of embryonic-derived tissue-resident macrophages over time.

Moreover, the study reports no significant difference in immune cell numbers in the hearts of cmyb-/- mutants, which have normal primitive haematopoiesis but lack HSCs, at 5 dpf. Given the authors' suggestion that mpeg+ cells originate from the HSC wave, it is essential to assess the number of mpeg+ cells in these mutants at later stages. This assessment would clarify whether mpeg+ cells are indeed HSC-derived or if csf1ra+ cells later switch on mpeg expression. Without this additional data, conclusions about the origins of mpeg+ cells remain speculative.

The study's reliance on available genetic tools, while a strength, also introduces limitations. The use of only a few transgenic lines will not fully capture the complexity and diversity of MNP populations, leading to an incomplete understanding of their roles and dynamics.

Furthermore, while the use of ex vivo imaging provides dynamic insights into cell behaviour, it may not fully capture the complexity of in vivo conditions, possibly overlooking interactions and influences present in the living organism.

The manuscript would benefit from increasing the sample sizes to ensure the robustness of the findings. The use of Phalloidin staining to delineate single cells more accurately would also enhance the precision of cell counting and improve the overall data quality.

The study could also benefit from a more in-depth exploration of the functional consequences of MNP heterogeneity in the heart. While the cellular characterisations are thorough, the molecular and regulatory insights provided by the study are limited to a couple of RT-PCRs for some known genes.

Overall, the manuscript by Moyse and colleagues significantly advances our understanding of the ontogeny and behaviour of macrophages in the zebrafish heart, revealing important parallels with mammalian models. However, the points above should be carefully considered when interpreting the results presented in this study.

Reviewer #1 (Public Review):

Summary:

The authors aimed to investigate the contribution of antigenic drift in the HA and NA genes of seasonal influenza A(H3N2) virus to their epidemic dynamics. Analyzing 22 influenza seasons before the COVID-19 pandemic, the study explored various antigenic and genetic markers, comparing them against indicators characterizing the epidemiology of annual outbreaks. The central findings highlight the significant influence of genetic distance on A(H3N2) virus epidemiology and emphasize the role of A(H1N1) virus incidence in shaping A(H3N2) epidemics, suggesting subtype interference as a key factor.

Major Strengths:

The paper is well-organized, written with clarity, and presents a comprehensive analysis. The study design, incorporating a span of 22 seasons, provides a robust foundation for understanding influenza dynamics. The inclusion of diverse antigenic and genetic markers enhances the depth of the investigation, and the exploration of subtype interference adds valuable insights.

Major Weaknesses:

While the analysis is thorough, some aspects require deeper interpretation, particularly in the discussion of certain results. Clarity and depth could be improved in the presentation of findings, and minor adjustments are suggested. Furthermore, the evolving dynamics of H3N2 predominance post-2009 need better elucidation.

Comments on revised version:

The authors have addressed each of the comments well. I have no further comments.

Reviewer #1 (Public Review):

Summary:

In this study, Maestri et al. use an integrative framework to study the evolutionary history of coronaviruses. They find that coronaviruses arose recently rather than having undergone ancient codivergences with their mammalian hosts. Furthermore, recent host switching has occurred extensively, but typically between closely related species. Humans have acted as an intermediate host, especially between bats and other mammal species.

Strengths:

The study draws on a range of data sources to reconstruct the history of virus-host codivergence and host switching. The analyses include various tests of robustness and evaluations through simulation.

Weaknesses:

The analyses are limited to a single genetic marker (RdRp) from coronaviruses, but using other sections of the genome might lead to different conclusions. The genetic marker also lacks resolution for recent divergences, which precludes the detailed examination of recent host switches. Careful and detailed reconstruction of the timescale would be helpful for clarifying the evolutionary history of coronaviruses alongside their hosts.

Reviewer #1 (Public Review):

Summary:

This study, titled "Enhancing Bone Regeneration and Osseointegration using rhPTH(1-34) and Dimeric R25CPTH(1-34) in an Osteoporotic Beagle Model," provides valuable insights into the therapeutic effects of two parathyroid hormone (PTH) analogs on bone regeneration and osseointegration. The research is methodologically sound, employing a robust animal model and a comprehensive array of analytical techniques, including micro-CT, histological/histomorphometric analyses, and serum biochemical analysis.

Strengths:

The use of a large animal model, which closely mimics postmenopausal osteoporosis in humans, enhances the study's relevance to clinical applications. The study is well-structured, with clear objectives, detailed methods, and a logical flow from introduction to conclusion. The findings are significant, demonstrating the potential of rhPTH(1-34) and dimeric R25CPTH(1-34) in enhancing bone regeneration, particularly in the context of osteoporosis.

Weaknesses: There are no major weaknesses.

Reviewer #1 (Public Review):

This paper by Ionescu et al. applies novel brain connectivity measures based on fMRI and serotonin PET both at baseline and following ecstasy use in rats. There are multiple strengths to this manuscript. First, the use of connectivity measures using temporal correlations of 11C-DASB PET, especially when combined with resting state fMRI, is highly novel and powerful. The effects of ecstasy on molecular connectivity of the serotonin network and salience network are also quite intriguing.

I would like the authors to discuss and justify their use of high-dose (1.3%) isolfurane. A recent consensus paper on rat fMRI (Grandjean et al., "A Consensus Protocol for Functional Connectivity Analysis in the Rat Brain.") found that medetomidine combined with low dose isoflurane provided optimal control of physiology and fMRI signal. To overcome any doubts about the effects of the high-dose anaesthetic I'd encourage the authors to show the results of their functional connectivity specificity using the same or similar image processing protocol as described in that consensus paper. This is especially true since the fMRI ICs in Figure 2A appear fairly restricted.

I'd also be interested to read more about why the cerebellum was chosen as a reference region, given that serotonin is highly expressed in the cerebellum, and what effects the choice of reference region has on their quantification.

The PET ICs appear less bilateral than the fMRI ICs. Is that simply a thresholding artefact or is it a real signal?

"The data will be made available upon reasonable request" is not sufficient - please deposit the data in an open repository and link to its location.

Reviewer #1 (Public Review):

Summary:

The experiment is interesting and well executed and describes in high detail fish behaviour in thermally stratified waters. The evidence is strong but the experimental design cannot distinguish between temperature and vertical position of the treatments.

Strengths:

High statistical power, solid quantification of behaviour.

Weaknesses:

A major issue with the experimental design is the vertical component of the experiment. Many thermal preference and avoidance experiments are run using horizontal division in shuttlebox systems or in annular choice flumes. These remove the vertical stratification component so that hot and cold can be compared equally, without the vertical layering as a confounding factor. The method chosen, with its vertical stratification, is inherently unable to control for this effect because warm water is always above, and cold water is always below. This complicates the interpretations and makes firm conclusions about thermal behaviour difficult.

Reviewer #1 (Public Review):

Allodynia is commonly measured in the pain field using von Frey filaments, which are applied to a body region (usually hindpaw if studying rodents) by a human. While humans perceive themselves as being objective, as the authors noted, humans are far from consistent when applying these filaments. Not to mention, odors from humans, including those of different sexes, can influence animal behavior. There is thus a major unmet need for a way to automate this tedious von Frey testing process and to remove humans from the experiment. I have no major scientific concerns with the study, as the authors did an outstanding job of comparing this automated system to human experimenters in a rigorous and quantitative manner. They even demonstrated that their automated system can be used in conjunction with in vivo imaging techniques.

While it is somewhat unclear how easy and inexpensive this device will be, I anticipate everyone in the pain field will be clamoring to get their hands on a system like this. And given the mechanical nature of the device and the propensity for mice to urinate on things, I also wonder how frequently the device breaks/needs to be repaired. Perhaps some details regarding the cost and reliability of the device would be helpful to include, as these are the two things that could make researchers hesitant to adopt immediately.

The only major technical concern, which is easy to address, is whether the device generates ultrasonic sounds that rodents can hear when idle or operational, across the ultrasonic frequencies that are of biological relevance (20-110 kHz). These sounds are generally alarm vocalizations and can create stress in animals, and/or serve as cues of an impending stimulus (if indeed they are produced by the device).

Reviewer #1 (Public Review):

Summary:

The authors set out to evaluate the regulation of interferon (IFN) gene expression in fish, using mainly zebrafish as a model system. Similar to more widely characterized mammalian systems, fish IFN is induced during viral infection through the action of the transcription factor IRF3 which is activated by phosphorylation by the kinase TBK1. It has been previously shown in many systems that TBK1 is subjected to both positive and negative regulation to control IFN production. In this work, the authors find that the cell cycle kinase CDK2 functions as a TBK1 inhibitor by decreasing its abundance through the recruitment of the ubiquitinylation ligase, Dtx4, which has been similarly implicated in the regulation of mammalian TBK1. Experimental data are presented showing that CDK2 interacts with both TBK1 and Dtx4, leading to TBK1 K48 ubiquitinylation on K567 and its subsequent degradation by the proteasome.

Strengths:

The strengths of this manuscript are its novel demonstration of the involvement of CDK2 in a process in fish that is controlled by different factors in other vertebrates and its clear and supportive experimental data.

Weaknesses:

The weaknesses of the study include the following. 1) It remains unclear whether the function described for CDK2 is regulatory, that is, it affects TBK1 levels during physiological responses such as viral infection or cell cycle progression, or if it is homeostatic, governing the basal abundance of TBK1 but not responding to signaling. 2) The authors have not explored whether the catalytic activity of CDK2 is required for TBK1 ubiquitinylation and, if so, what its target specificity is. 3) Given the multitude of CDK isoforms in fish, it remains unexplored whether the identified fish CDK2 homolog is a requisite cell cycle regulator or if its action in the cell cycle is redundant with other CDKs.

Reviewer #1 (Public Review):

Faiz et al. investigate small molecule-driven direct lineage reprogramming of mouse postnatal mouse astrocytes to oligodendrocyte lineage cells (OLCs). They use a combination of in vitro, in vivo, and computational approaches to confirm lineage conversion and to examine the key underlying transcription factors and signaling pathways. Lentiviral delivery of transcription factors previously reported to be essential in OLC fate determination-Sox10, Olig2, and Nkx2.2-to astrocytes allows for lineage tracing. They found that these transcription factors are sufficient in reprogramming astrocytes to iOLCs, but that the OLCs range in maturity level depending on which factor they are transfected with. They followed up with scRNA-seq analysis of transfected and control cultures 14DPT, confirming that TF-induced astrocytes take on canonical OLC gene signatures. By performing astrocyte lineage fate mapping, they further confirmed that TF-induced astrocytes give rise to iOLCs. Finally, they examined the distinct genetic drivers of this fate conversion using scRNA-seq and deep learning models of Sox10- astrocytes at multiple time points throughout the reprogramming. These findings are certainly relevant to diseases characterized by the perturbation of OLC maturation and/or myelination, such as Multiple Sclerosis and Alzheimer's Disease. Their application of such a wide array of experimental approaches gives more weight to their findings and allows for the identification of additional genetic drivers of astrocyte to iOLC conversion that could be explored in future studies. Overall, I find this manuscript thoughtfully constructed and only have a few questions to be addressed.

(1) The authors suggest that Sox10- and Olig2- transduced astrocytes result in distinct subpopulations iOLCs. Considering it was discussed in the introduction that these TFs cyclically regulate one another throughout differentiation, could they speculate as to why such varying iOLCs resulted from the induction of these two TFs?

(2) In Figure 1B it appears that the Sox10- MBP+ tdTomato+ cells decreases from D12 to D14. Does this make sense considering MBP is a marker of more mature OLCs?

(3) Previous studies have shown that MBP expression and myelination in vitro occurs at the earliest around 4-6 weeks of culturing. When assessing whether further maturation would increase MBP positivity, authors only cultured cells up to 22 DPT and saw no significant increase. Has a lengthier culture timeline been attempted?

(4) Figure S4D is described as "examples of tdTomatonegzsGreen+OLCmarker+ cells that arose from a tdTomatoneg cell with an astrocyte morphology." The zsGreen+ tdTomato- cell is not convincingly of "astrocyte morphology"; it could be a bipolar OLC. To strengthen the conclusions and remove this subjectivity, more extensive characterizations of astrocyte versus OLC morphology in the introduction or results are warranted. This would make this observation more convincing since there is clearly an overlap in the characteristics of these cell types.

Joint Public Review:

The manuscript "Engineering of PAClight1P78A: A High-Performance Class-B1 GPCR-Based Sensor for PACAP1-38" by Cola et al. presents the development of a novel genetically encoded sensor, PAClight1P78A, based on the human PAC1 receptor. The authors provide a thorough in vitro and in vivo characterization of this sensor, demonstrating its potential utility across various applications in life sciences, including drug development and basic research.

The main criticism of this manuscript after initial review is that the PACLight1 sensor has not been shown to detect the release of endogenous PACAP, whether in culture, in vivo, or ex vivo. The authors appear to be cognizant of this significant limitation (for a PACAP sensor) but no significant changes to address this limitation are provided in the revision.

While the sensor that is described here is new and the experimental results support the conclusions, the sensor reported here is not suited for the detection of endogenous PACAP release in vivo. In some respects, this manuscript could be seen as a stepping stone for further development either by the authors or other groups. Indeed, in many cases initial versions of genetically encoded sensors undergo substantial development post-publication, as exemplified by the evolution of GCaMP. However, the situation with the PAClight sensor reported here requires a different approach. Unlike GCaMP, which was one of the first genetically encoded calcium indicators, PAClight is another variant in a series of GPCR-fluorophore conjugates, following methodologies similar to those developed in the Lin Tian lab and the multiple GRAB-based sensors from Yulong Li's lab. These sensors have already demonstrated in vivo applicability, setting a standard that PAClight must meet or exceed to confirm its value and novelty.

Given that the title of the manuscript, "Probing PAC1 receptor activation across species with an engineered sensor," implies broader applicability, it potentially misleads readers about the sensor's utility in vivo, where "in vivo" should be understood as referring to the detection of endogenous PACAP release.

To align the manuscript with the expectations set by its title, it is crucial that the authors either provide substantial in vivo validation (ability to detect endogenous release of PACAP) or revise the title and the text to clarify that the sensor is primarily intended to detect exogenously applied PACAP. This clarification will ensure that the manuscript accurately reflects the sensor's current capabilities and scope of use.

Reviewer #1 (Public Review):

Summary:

Non-B DNA structures such as G4s and R-loops have the potential to impact genome stability, gene transcription, and cell differentiation. This study investigates the distribution of G4s and R-loops in human and mouse cells using some interesting technical modifications of existing Tn5-based approaches. This work confirms that the helicase DHX9 could regulate the formation and/or stability of both structures in mouse embryonic stem cells (mESCs). It also provides evidence that the lack of DHX9 in mESCs interferes with their ability to differentiate.

Strengths:

HepG4-seq, the new antibody-free strategy to map G4s based on the ability of Hemin to act as a peroxidase when complexed to G4s, is interesting. This study also provides more evidence that the distribution pattern of G4s and R-loops might vary substantially from one cell type to another.

Weaknesses:

This study is essentially descriptive and does not provide conclusive evidence that lack of DHX9 does interfere with the ability of mESCs to differentiate by regulating directly the stability of either G4 or R-loops. In the end, it does not substantially improve our understanding of DHX9's mode of action.

There is no in-depth comparison of the newly generated data with existing datasets and no rigorous control was presented to test the specificity of the hemin-G4 interaction (a lot of the hemin-dependent signal seems to occur in the cytoplasm, which is unexpected).

The authors talk about co-occurrence between G4 and R-loops but their data does not actually demonstrate co-occurrence in time. If the same loci could form alternatively either R-loops or G4 and if DHX9 was somehow involved in determining the balance between G4s and R-loops, the authors would probably obtain the same distribution pattern. To manipulate R-loop levels in vivo and test how this affects HEPG4-seq signals would have been helpful.

This study relies exclusively on Tn5-based mapping strategies. This is a problem as global changes in DNA accessibility might strongly skew the results. It is unclear at this stage whether the lack of DHX9, BLM, or WRN has an impact on DNA accessibility, which might underlie the differences that were observed. Moreover, Tn5 cleaves DNA at a nearby accessible site, which might be at an unknown distance away from the site of interest. The spatial accuracy of Tn5-based methods is therefore debatable, which is a problem when trying to demonstrate spatial co-occurrence. Alternative mapping methods would have been helpful.

Reviewer #1 (Public Review):

Summary:

Medina et al, 2023 investigated the peripheral blood transcriptional responses in patients with diversifying disease outcomes. The authors characterized the blood transcriptome of four non-hospitalized individuals presenting mild disease and four patients hospitalized with severe disease. These individuals were observed longitudinally at three timepoints (0-, 7-, and 28-days post recruitment), and distinct transcriptional responses were observed between severe hospitalized patients and mild non-hospitalized individuals, especially during 0- and 7-day collection timepoints. Particularly, the authors found that increased expression of genes associated with NK cell cytotoxicity is associated with mild outcomes. Additional co-regulated gene network analyses positively correlates T cell activity with mild disease and neutrophil degranulation with severe disease.

Strengths:

The longitudinal measurements in individual participants at consistent collection intervals can offer an added dimension to the dataset that involves temporal trajectories of genes associated with disease outcomes and is a key strength of the study. The use of co-expressed gene networks specific to the cohort to complement enrichment results obtained from pre-determined gene sets can offer valuable insights into new associations/networks associated with disease progression and warrants further analyses on the biological functions enriched within these co-expressed network modules.

Weaknesses:

There is a large difference in the infection timeline (onset of symptom to recruitment) between mild and severe patient cohort. As immune responses during early infection can be highly dynamic, the differences in infection timeline may bias transcriptional signatures observed between the groups. The study is also limited by a small cohort size.

Comments on revised version:

The authors have addressed the specific concerns brought forth by the reviewers.

Reviewer #3 (Public Review):

Summary:

The authors discovered that the RdnE effector possesses DNase activity, and in competition, P. mirabilis having RdnE outcompetes the null strain. Additionally, they presented evidence that the RdnI immunity protein binds to RdnE, suppressing its toxicity. Interestingly, the authors demonstrated that the RdnI homolog from a different phylum (i.e., Actinomycetota) provides cross-species protection against RdnE injected from P. mirabilis, despite the limited identity between the immunity sequences. Finally, using metagenomic data from human-associated microbiomes, the authors provided bioinformatic evidence that the rdnE/rdnI gene pair is widespread and present in individual microbiomes. Overall, the discovery of broad protection by non-cognate immunity is intriguing, although not necessarily surprising in retrospect, considering the prolonged period during which Earth was a microbial battlefield/paradise.

Strengths:

The authors presented a strong rationale in the manuscript and characterized the molecular mechanism of the RdnE effector both in vitro and in the heterologous expression model. The utilization of the bacterial two-hybrid system, along with the competition assays, to study the protective action of RdnI immunity is informative. Furthermore, the authors conducted bioinformatic analyses throughout the manuscript, examining the primary sequence, predicted structural, and metagenomic levels, which significantly underscore the significance and importance of the EI pair.

Weaknesses:

(1) The interaction between RdnI and RdnE appears to be complex and requires further investigation. The manuscript's data does not conclusively explain how RdnI provides a "promiscuous" immunity function, particularly regarding the RdnI mutant/chimera derivatives. The lack of protection observed in these cases might be attributed to other factors, such as a decrease in protein expression levels or misfolding of the proteins. Additionally, the transient nature of the binding interaction could be insufficient to offer effective defenses.<br /> (2) The results from the mixed population competition would benefit from quantitative analysis. The swarm competition assays only yield binary outcomes (Yes or No), limiting the ability to obtain more detailed insights from the data.<br /> (3) The discovery of cross-species protection is solely evident in the heterologous expression-competition model. It remains uncertain whether this is an isolated occurrence or a common characteristic of RdnI immunity proteins across various scenarios. Further investigations are necessary to determine the generality of this behavior.

Public Review:

The authors used an innovative technic to study the visual vigilance based on high-acuity vision, the fovea. Combining motion-capture features and visual space around the head, the authors were able to estimate the visual fixation of free-feeding pigeon at any moment. Simulating predator attacks on screens, they showed that 1) pigeons used their fovea to inspect predators cues, 2) the behavioural state (feeding or head-up) influenced the latency to use the fovea and 3) the use of the fovea decrease the latency to escape of both the individual that foveate the predators cues but also the other flock members.

The paper is very interesting, and combines innovative technic well adapted to study the importance of high-acuity vision for spotting a predator, but also of improving the behavioural response (escaping). The results are strong and the models used are well-adapted. This paper is a major contribution to our understanding of the use of visual adaptation in a foraging context when at risk. This is also a major contribution to the understanding of individual interaction in a flock.

Reviewer #1 (Public Review):

Summary:

In this preprint, the authors systematically and rigorously investigate how specific classes of residue mutations alter the critical temperature as a proxy for the driving forces for phase separation. The work is well executed, the manuscript well-written, and the results reasonable and insightful.

Strengths:

The introductory material does an excellent job of being precise in language and ideas while summarizing the state of the art. The simulation design, execution, and analysis are exceptional and set the standard for these types of large-scale simulation studies. The results, interpretations, and Discussion are largely nuanced, clear, and well-motivated.

Weaknesses:

This is not exactly a weakness, but I think it would future-proof the authors' conclusions to clarify a few key caveats associated with this work. Most notably, given the underlying implementation of the Mpipi model, temperature dependencies for intermolecular interactions driven by solvent effects (e.g., hydrophobic effect and charge-mediated interactions facilitated by desolvation penalties) are not captured. This itself is not a "weakness" per se, but it means I would imagine CERTAIN types of features would not be well-captured; notably, my expectation is that at higher temperatures, proline-rich sequences drive intermolecular interactions, but at lower temperatures, they do not. This is likely also true for the aliphatic residues, although these are found less frequently in IDRs. As such, it may be worth the authors explicitly discussing.

Similarly, prior work has established the importance of an alpha-helical region in TDP-43, as well as the role of aliphatic residues in driving TDP-43's assembly (see Schmidt et al 2019). I recognize the authors have focussed here on a specific set of mutations, so it may be worth (in the Discussion) mentioning [1] what impact, if any, they expect transient or persistent secondary structure to have on their conclusions and [2] how they expect aliphatic residues to contribute. These can and probably should be speculative as opposed to definitive.

Again - these are not raised as weaknesses in terms of this work, but the fact they are not discussed is a minor weakness, and the preprint's use and impact would be improved on such a discussion.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors had 2 aims:

(1) Measure macaques' aversion to sand and see if its' removal is intentional, as it is likely in an unpleasurable sensation that causes tooth damage.

(2) Show that or see if monkeys engage in suboptimal behavior by cleaning foods beyond the point of diminishing returns, and see if this was related to individual traits such as sex and rank, and behavioral technique.

They attempted to achieve these aims through a combination of geochemical analysis of sand, field experiments, and comparing predictions to an analytical model.

The authors' conclusions were that they verified a long-standing assumption that monkeys have an aversion to sand as it contains many potentially damaging fine-grained silicates and that removing it via brushing or washing is intentional.

They also concluded that monkeys will clean food for longer than is necessary, i.e. beyond the point of diminishing returns, and that this is rank-dependent.

High and low-ranking monkeys tended not to wash their food, but instead over-brushed it, potentially to minimize handling time and maximize caloric intake, despite the long-term cumulative costs of sand.

This was interpreted through the *disposable soma hypothesis*, where dominants maximize immediate needs to maintain rank and increase reproductive success at the potential expense of long-term health and survival.

Strengths:

The field experiment seemed well-designed, and their quantification of physical and mineral properties of quartz particles (relative to human detection thresholds) seemed good relative to their feret diameter and particle circularity (to a reviewer who is not an expert in sand). The *Rank Determination* and *Measuring Sand* sections were clear.

In achieving Aim 1, the authors validated a commonly interpreted, but unmeasured function, of macaque and primate behavior-- a key study/finding in primate food processing and cultural transmission research.

I commend their approach in developing a quantitative model to generate predictions to compare to empirical data for their second aim.

This is something others should strive for.

I really appreciated the historical context of this paper in the introduction, and found it very enjoyable and easy to read.

I do think that interpreting these results in the context of the *disposable soma hypothesis* and the potential implications in the *paleolithic matters* section about interpreting dental wear in the fossil record are worthwhile.

Weaknesses:

Most of the weaknesses in this paper lie in statistical methods, visualization, and a missing connection to the marginal value theorem and optimal foraging theory.

I think all of these weaknesses are solvable.

The data and code were not submitted. Therefore I was unable to better understand the simulation or to provide useful feedback on the stats, the connection between the two, and its relevance to the broader community.

(1) Statistics:

(a) AIC and outcome distributions

The use of AIC for hierarchical models, and models with different outcome distributions brought up several concerns.

The authors appear to use AIC to help inform which model to use for their primary analyses in Tables S1 and S2. It is unclear which of these models are analyzed in Tables S3 and S4.

AIC should not be used on hierarchical models, and something like WAIC (or DIC which has other caveats) would be more appropriate.

Also, using information criteria on Mixture Models like Negative Binomials (aka Gamma-Poisson) should be done with extreme caution, or not at all, as the values are highly sensitive to the data structure.

Some researchers also say that information criteria should not be used to compare models with different outcome distributions - although this might be slightly less of a concern as all of your models are essentially variations on a Poisson GLM.

Discussion on this can be found in McElreath Statistical Rethinking (Section 12.1.3) and Gelman et al. BDA3 (Chapter 7).

Choosing an outcome distribution, based on your understanding of the data generating process is a better approach than relying on AIC, especially in this context where it can be misleading.

(b) Zeros

I also had some concerns about how zeros were treated in the models.

In lines 217-218, they mentioned that "if a monkey consumed a cucumber slice without brushing or washing it, the zero-second duration was included in both GLMMs."

This zero implies no processing and should not be treated as a length 0 duration of processing.

This suggests to me that a zero-inflated poisson or zero-inflated negative binomial, would be the best choice for modelling the data as it is essentially a 2-step process:<br /> (i) Do they process the cucumber at all?<br /> (ii) If so do they wash or brush, and how is this predicted by rank and treatment?

(2) Absence of Links to Foraging Theory

Optimal cleaning time model: the optimality model was not well described including how it was programmed. Better description and documentation of this model, along with code (Mathematica judging from the plot?) is needed.

There seems to be much conceptual and theoretical overlap with foraging theory models that were not well described - namely the *marginal value theorem (Charnov (1976), Krebs et al. (1974),) and its subsequent advances* (see https://doi.org/10.1016/j.jaa.2016.03.002 and https://doi.org/10.1086/283929 for examples).

In the suggestions, I attached the R code where I replicated their model to show that it is *mathematically identical to the marginal value theorem*. This was not mentioned at all in the text or citations.

This is a well-studied literature since the 1970's and there is a history of studies that compare behavior to an optimality model and fail (or do find) instances where animals conform or diverge with its predictions (https://doi.org/10.1146/annurev.es.15.110184.002515). This link should be highlighted, and interpreting it in that theoretical context will make it more broadly applicable to behavioral ecologists.

The data was subsetted to include instances where there were < 3 monkeys present to avoid confounds of rank, but it is important to know that optimal behavior might vary by individual, and can change in a social context depending on rank (see https://doi.org/10.1016/j.tree.2022.06.010). Discussion of this, and further exploration of it in the data would strengthen the overall contribution of this manuscript to the field, but I understand that the researchers wish to avoid that in this paper for it is a complex topic, which this dataset is uniquely suited to address.

(3) Interpretation and validity of model relative to data

In lines 92-102, they present summary statistics (I think) showing that time spent brushing and washing is consistent with washing or brushing to remove sand.

In the **mitigating tooth wear** section (line 73) and corresponding Figure S1 showing surface sand removed, more detail about how these numbers were acquired, and statistical modelling, is needed.

This is important as uncertainty and measurement error around these metrics are key to the central finding and interpretation of Aim 2 in this paper.

It appears that the researchers simulated the monkey's brushing and washing behaviors (similar to https://doi.org/10.1007/s10071-009-0230-3).

How many researchers simulated monkey behavior and how many times?

What are the repeat points in Figure S1?

What is the number of trials or number of people?

This effect appears stronger for washing than brushing as well - if so, why?

More info about this data, and the uncertainty in this is important, as it is key to the second central claim of this paper.

The estimates of removing between 76% +/- 7 and 93% +/- 4 of sand (visualized in Figure S1), are statistical estimates.

I would find the argument more convincing if after propagating for the uncertainty in handling in sand removal rates, and the corresponding half-saturation constants, if this processing for food is too long, after accounting for diminishing returns held true.<br /> It is very possible that after accounting for uncertainty and variation in handling time and removal rates, the second result may not hold true.

I was not able to convince myself of this via reanalysis as the description of the data in the text was not enough to simulate it myself.

Essentially, this would imply that in Figure 3 the predicted value would have some variation around it (informed by boundary conditions of time being positive, and percents having floors and ceilings) and that a range of predicting cleaning times (optimal give-up times) would be plotted in Figure 3.

This could be accomplished in a Bayesian approach, Or by simply plotting multiple predictions given some confidence interval around, c and h.

Reviewer #1 (Public Review):

Summary:

The authors attempt to understand how cells forage for spatially heterogeneous complex polysaccharides. They aimed to quantify the foraging behavior and interrogate its genetic basis. The results show that cells aggregate near complex polysaccharides and disperse when simpler byproducts are added. Dispersing cells tend to move towards the polysaccharide. The authors also use transcriptomics to attempt to understand which genes support each of these behaviors - with motility and transporter related genes being highly expressed during dispersal, as expected.

Strengths:

The paper is well written and builds on previous studies by some of the authors showing similar behavior by a different species of bacteria (Caulobacter) on another polysaccharide (xylan). The conceptual model presented at the end encapsulates the findings and provides an interesting hypothesis. I also find the observation of chemotaxis towards the polysaccharide in the experimental conditions interesting.

Weaknesses:

Much of the genetic analysis, as it stands, is quite speculative and descriptive. I found myself confused about many of the genes (e.g., quorum sensing) that pop up enriched during dispersal quite in contrast to my expectations. While the authors do discuss this in the text as worth following up on, I think the analysis as it stands is speculative about the behaviors observed. In the authors' defense, I acknowledge that it might have the potential to generate hypotheses and thus aid future studies.

Reviewer #1 (Public Review):

The present paper presents a new, simple, and cost-effective technique for multimodal EM imaging that combines the strengths of volume scanning electron microscopy (SEM) and electron microscopic tomography. The novel ATUM-Tomo approach enables the consecutive inspection of selected areas of interest by correlated serial SEM and TEM, optionally in combination with CLEM, as demonstrated. The most important finding of ATUM-Tomo and particularly correlative ATUM-Tomo is that it can bridge several scales from the cellular to the high-resolution subcellular scale, from the micrometer to low nanometer resolution, which is particularly important for the ultrastructural analysis of biological regions of interest as demonstrated here by focal pathologies or rare cellular and subcellular structures. Both imaging modalities are non-destructive, thus allowing re-imaging and hierarchical imaging at the SEM and TEM levels, which is particularly important for precious samples, such as human biopsies or specimens from complex CLEM experiments. The paper demonstrates that the new approach is very helpful in analyses of pathologically altered brains, including humans brain tissue samples, that require high-resolution SEM and TEM in combination with immunohistochemistry for analysis. Even the combination with tracers would be possible. In sum, ATUM-Tomo opens up new possibilities in multimodal volume EM imaging for diverse biological areas of research.

Strengths

This paper is a very nice piece of work. It combines modern, high-end, state-of-the-art technology that allows to investigate diverse biological questions in different fields and at multiple scales. The paper is clear and well-written. It is accompanied by excellent figures, supplementals, and colored 3D-reconstructions that make it easy for the reader to follow the experimental procedure and the scientific context alike.

Weaknesses

There is a bit of an imbalance between the description of the state-of-the-art methodology and the scientific context. The discussion of the latter could be expanded.

Reviewer #1 (Public Review):

Summary:

In this study, Faniyan and colleagues build on their recent finding that renal Glut2 knockout mice display normal fasting blood glucose levels despite massive glucosuria. Renal Glut2 knockout mice were found to exhibit increased endogenous glucose production along with decreased hepatic metabolites associated with glucose metabolism. Crh mRNA levels were higher in the hypothalamus while circulating ACTH and corticosterone was elevated in this model. While these mice were able to maintain normal fasting glucose levels, ablating afferent renal signals to the brain caused low fasting blood glucose levels. In addition, the higher CRH and higher corticosterone levels of the knockout mice were lost following this denervation. Finally, acute phase proteins were altered, plasma Gpx3 was lower, and major urinary protein MUP18 and its gene expression were higher in renal Glut2 knockout mice. Overall, the main conclusion that afferent signaling from the kidney is required for renal glut2 dependent increases in endogenous glucose production is well supported by these findings.

Strengths:

An important strength of the paper is the novelty of the identification of kidney to brain communication as being important for glucose homeostasis. Previous studies had focused on other functions of the kidney modulated by or modulating brain function. This work is likely to promote interest in CNS pathways that respond to afferent renal signals and the response of the HPA axis to glucosuria. Additional strengths of this paper stem from the use of incisive techniques. Specifically, the authors use isotope enabled measurement of endogenous glucose production by GC-MS/MS, capsaicin ablation of afferent renal nerves, and multifiber recording from the renal nerve. The authors also paid excellent attention to rigor in the design and performance of these studies. For example, they used appropriate surgical controls, confirmed denervation through renal pelvic CGRP measurement, and avoided the confounding effects of nerve regrowth over time. These factors strengthen confidence in their results. Finally, humans with glucose transporter mutations and those being treated with SGLT2 inhibitors show a compensatory increase in endogenous glucose production. Therefore, this study strengthens the case for using renal Glut2 knockout mice as a model for understanding the physiology of these patients.

Comments on latest version:

My concerns have been addressed.

Reviewer #1 (Public Review):

Summary:

Authors explore how sex-peptide (SP) affects post-mating behaviours in adult females, such as receptivity and egg laying. This study identifies different neurons in the adult brain and the VNC that become activated by SP, largely by using an intersectional gene expression approach (split-GAL4) to narrow down the specific neurons involved. They confirm that SP binds to the well-known Sex Peptide Receptor (SPR), initiating a cascade of physiological and behavioural changes related to receptivity and egg laying.

Areas of improvement and suggestions:

(1) "These results suggest the SP targets interneurons in the brain that feed into higher processing centers from different entry points likely representing different sensory input" and "All together, these data suggest that the abdominal ganglion harbors several distinct type of neurons involved in directing PMRs"<br /> The characterization of the post-mating circuitry has been largely described by the group of Barry Dickson and other labs. I suggest ruling out a potential effect of mSP in any of the well-known post-mating neuronal circuitry, i.e: SPSN, SAG, pC1, vpoDN or OviDNs neurons. A combination of available split-Gal4 should be sufficient to prove this.

(2) Authors must show how specific is their "head" (elav/otd-flp) and "trunk" (elav/tsh) expression of mSP by showing images of the same constructs driving GFP.

(3) VT3280 is termed as a SAG driver. However, VT3280 is a SPSN specific driver (Feng et al., 2014; Jang et al., 2017; Scheunemann et al., 2019; Laturney et al., 2023). The authors should clarify this.

(4) Intersectional approaches must rule out the influence of SP on sex-peptide sensing neurons (SPSN) in the ovary by combining their constructs with SPSN-Gal80 construct. In line with this, most of their lines targets the SAG circuit (4I, J and K). Again, here they need to rule out the involvement of SPSN in their receptivity/egg laying phenotypes. Especially because "In the female genital tract, these split-Gal4 combinations show expression in genital tract neurons with innervations running along oviduct and uterine walls (Figures S3A-S3E)".

(5) The authors separate head (brain) from trunk (VNC) responses, but they don't narrow down the neural circuits involved on each response. A detailed characterization of the involved circuits especially in the case of the VNC is needed to (a) show that the intersectional approach is indeed labelling distinct subtypes and (b) how these distinct neurons influence oviposition.

Reviewer #1 (Public Review):

Previous Review:

The authors have identified the predicted EBE of PthA4 in the promoter of Cs9g12620, which is induced by Xcc. The authors identified a homolog of Cs9g12620, which has variations in the promoter region. The authors show PthA4 suppresses Cs9g12620 promoter activity independent of the binding action. The authors also show that CsLOB1 binds to the promoter of Cs9g12620. Interestingly, the authors show that PthA4 interacts with CsLOB1 at protein level. Finally, it shows that Cs9g12620 is important for canker symptoms. Overall, this study has reported some interesting discoveries and the writing is generally well done. However, the discoveries are affected by the reliability of the data and some flaws of the experimental designs.

Here are some major issues:

The authors have demonstrated that Cs9g12620 contains the EBE of PthA4 in the promoter region, to show that PthA4 controls Cs9g12620, the authors need to compare to the wild type Xcc and pthA4 mutant for Cs9g12620 expression. The data in Figure 1 is not enough.

The authors confirmed the interaction between PthA4 and the EBE in the promoter of Cs9g12620 using DNA electrophoretic mobility shift assay (EMSA). However, Fig. 2B is not convincing. The lane without GST-PthA4 also clearly showed mobility shift. For EMSA assay, the authors need also to include non-labeled probe as competitor to verify the specificity. The description of the EMSA in this paper suggests that it was not done properly. It is suggested the authors to redo this EMSA assay following the protocol: Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions PMID: 17703195.

The authors also claimed that PthA4 suppresses the promote activity of Cs9g12620. The data is not convincing and also contradicts with their own data that overexpression of Cs9g12620 causes canker and silencing of it reduces canker considering PthA4 is required for canker development. The authors conducted the assays using transient expression of PthA4. It should be done with Xcc wild type, pthA4 mutant and negative control to inoculate citrus plants to check the expression of Cs9g12620.

Fig. 6 AB is not convincing. There are no apparent differences. The variations shown in B is common in different wild type samples. It is suggested that the authors to conduct transgenic instead of transient overexpression.

Gene silencing data needs more appropriate controls. Fig. D. seems to suggest canker symptoms actually happen for the RNAi treated. The authors need to make sure same amount of Xcc was used for both CTV empty vector and the RNAi. It is suggested a blink test is needed here.

Comments on revised version:

Point 1: Addressed well.

Point 2: The EMSA was reconducted with adding unlabeled DNA, however, the results are still not convincing. Firstly, in fig.3D lane 5, with the absence of unlabeled DNA, the shifted bound signal wasn't reduced significantly. Secondly, still in fig.3D lane 5, the free labeled DNA probe at the bottom of the gel didn't increase. Which together mean that the unlabeled DNA was unable to compete with the labeled DNA and the "bound" shifted bands might not be true positive.

Point 3: The authors didn't address the question clearly regarding the connection between the inhibition of Cs9g12620 promoter by PthA4 and the positive function of Cs9g12620 on citrus canker.

Point 4: The comment was not addressed. Fig.7A and B are not convincing. Firstly, no evidence shows the expression of transiently expressed genes. Secondly, hard to tell the difference in 7A. Thirdly, since CsLOB1 positively regulates Cs9g12620, why expressing of CsLOB1 is unable to cause phenotype, while expression of PthA4 does?

Point 5: addressed.

Reviewer #1 (Public Review):

Summary:

Orlovski and his colleagues revealed an interesting phenomenon that SAP54-overexpressing leaf exposure to leafhopper males is required for the attraction of followed females. By transcriptomic analysis, they demonstrated that SAP54 effectively suppresses biotic stress response pathways in leaves exposed to the males. Furthermore, they clarified how SAP54, by targeting SVP, heightens leaf vulnerability to leafhopper males, thus facilitating female attraction and subsequent plant colonization by the insects.

Strengths:

The phenomenon of this study is interesting and exciting.

Weaknesses:

The underlying mechanisms of this phenomenon are not convincing.

Reviewer #1 (Public Review):

Summary:

The authors used video tracking of 4 larval cichlid species and medaka to quantify prey-capture behaviors.

Strengths:

Comparing these behaviors is in principle an interesting question, and helps to address the typicality of the much better-understood zebrafish model. The authors make a good effort to analyze their data quantitatively.

Weaknesses:

(1) The overall conclusion, as summarized in the abstract as "Together, our study documents the diversification of locomotor and oculomotor adaptations among hunting teleost larvae" is not that compelling. What would be much more interesting would be to directly relate these differences to different ecological niches (e.g. different types of natural prey, visual scene conditions, height in water column etc), and/or differences in neural circuit mechanisms. While I appreciate that this paper provides a first step on this path, by itself it seems on the verge of stamp collecting, i.e. collecting and cataloging observations without a clear, overarching hypothesis or theoretical framework.

(2) The data to support some of the claims is either weak or lacking entirely.

Reviewer #1 (Public Review):

Summary and Strengths:

The study focuses on PIM1 and 2 in CD8 T cell activation and differentiation. These two serine/threonine kinases belong to a large network of Serine/Threonine kinases that acts following engagement of the TCR and of cytokine receptors and phosphorylates proteins that control transcriptional, translational and metabolic programs that result in effector and memory T cell differentiation. The expression of PIM1 and PIM2 is induced by the T-cell receptor and several cytokine receptors. The present study capitalized on high-resolution quantitative analysis of the proteomes and transcriptomes of Pim1/Pim2-deficient CD8 T cells to decipher how the PIM1/2 kinases control TCR-driven activation and IL-2/IL-15-driven proliferation, and differentiation into effector T cells.<br /> Quantitative mass spectrometry-based proteomics analysis of naïve OT1 CD8 T cell stimulated with their cognate peptide showed that the PIM1 protein was induced within 3 hours of TCR engagement and its expression was sustained at least up to 24 hours. The kinetics of PIM2 expression was protracted as compared to that of PIM1. Such TCR-dependent expression of PIM1/2 correlated with the analysis of both Pim1 and Pim2 mRNA. In contrast, Pim3 mRNA was only expressed at very low levels and the PIM3 protein was not detected by mass spectrometry. Therefore, PIM1 and 2 are the major PIM kinases in recently activated T cells. Pim1/Pim2 double knockout (Pim dKO) mice were generated on a B6 background and found to express a lower number of splenocytes. No difference in TCR/CD28-driven proliferation was observed between WT and Pim dKO T cells over 3 days in culture. Quantitative proteomics of >7000 proteins further revealed no substantial quantitative or qualitative differences in protein content or proteome composition. Therefore, other signaling pathways can compensate for the lack of PIM kinases downstream of TCR activation.

Considering that PIM1 and PIM2 kinase expression is regulated by IL-2 and IL-15, antigen-primed CD8 T cells were expanded in IL-15 to generate memory phenotype CD8 T cells or expanded in IL-2 to generate effector cytotoxic T lymphocytes (CTL). Analysis of the survival, proliferation, proteome, and transcriptome of Pim dKO CD8 T cells kept for 6 days in IL-15 showed that PIM1 and PIM2 are dispensable to drive the IL-15-mediated metabolic or differentiation programs of antigen-primed CD8 T cells. Moreover, Pim1/Pim2-deficiency had no impact on the ability of IL-2 to maintain CD8 T cell viability and proliferation. However, WT CTL downregulated the expression of CD62L whereas the Pim dKO CTL sustained higher CD62L expression. Pim dKO CTL was also smaller and less granular than WT CTL. Comparison of the proteome of day 6 IL-2 cultured WT and Pim dKO CTL showed that the latter expressed lower levels of the glucose transporters, SLC2A1 and SLC2A3, of a number of proteins involved in fatty acid and cholesterol biosynthesis, and CTL effector proteins such as granzymes, perforin, IFNg, and TNFa. Parallel transcriptomics analysis showed that the reduced expression of perforin and some granzymes correlated with a decrease in their mRNA whereas the decreased protein levels of granzymes B and A, and the glucose transporters SLC2A1 and SLC2A3 did not correspond with decreased mRNA expression. Therefore, PIM kinases are likely required for IL-2 to maximally control protein synthesis in CD8 CTL. Along that line, the translational repressor PDCD4 was increased in Pim dKO CTL and pan-PIM kinase inhibitors caused a reduction in protein synthesis rates in IL-2-expanded CTL. Finally, the differences between Pim dKO and WT CTL in terms of CD62L expression resulted in Pim dKO CTL but not WT CTL retained the capacity to home to secondary lymphoid organs. In conclusion, this thorough and solid study showed that the PIM1/2 kinases shape the effector CD8 T cell proteomes rather than transcriptomes and are important mediators of IL2-signalling and CD8 T cell trafficking.

Weaknesses:

None identified by this reviewer.

Joint Public Review:

Summary:

This paper by Beath et. al. identifies a potential regulatory role for proteins involved in cytoplasmic streaming and maintaining the grouping of paternal organelles: holding sperm contents in the fertilized embryos away from the oocyte meiotic spindle so that they don't get ejected into the polar body during meiotic chromosome segregation. The authors show that by time-lapse video, paternal mitochondria (used as a readout for sperm and its genome) is excluded from yolk granules and maternal mitochondria, even when moving long distances by cytoplasmic streaming. To understand how this exclusion is accomplished, they first show that it is independent of both internal packing and the engulfment of the paternal chromosomes by the maternal endoplasmic reticulum creating an impermeable barrier. They then test whether the control of cytoplasmic steaming affects this exclusion by knocking down two microtubule motors, Katanin and kinesis I. They find that the ER ring, which is used as a proxy for paternal chromosomes, undergoes extensive displacement with these treatments during anaphase I and interacts with the meiotic spindle, supporting their hypothesis that the exclusion of paternal chromosomes is regulated by cytoplasmic streaming. Next, they test whether a regulator of maternal ER organization, ATX-2, disrupts sperm organization so that they can combine the double depletion of ATX-2 and KLP-7, presumably because klp-7 RNAi (unlike mei-1 RNAi) does not affect polar body extrusion and they can report on what happens to paternal chromosomes. They find that the knockdown of both ATX-2 and KLP-7 produces a higher incidence of what appears to be the capture of paternal chromosomes by the meiotic spindle (5/24 vs 1/25). However, this capture event appears to halt the cell cycle, preventing the authors from directly observing whether this would result in the paternal chromosomes being ejected into the polar body.

The authors addressed the vast majority of the Reviewer's comments including the addition of new figures, re-wording of data interpretation and discussion points to better reflect the claims of the paper. There remain a few outstanding points which were not addressed.

In many cases the number of embryos analyzed or events capture remains low and the authors conclude that these sample sizes prevented statistical significance. It's not clear if more embryos were analyzed or if more capture would lead to statistical significance. Language capturing this caveat should also be included in the manuscript. A specific example of this is given below:

In the double knockdown of ATX-2 and KLP-7, there was no significant difference between single and double knockdowns and the ER ring displacement was not analyzed in this double mutant. Further, there was no difference in the frequency of sperm capture between single and double ATX-2 and KLP-7 due to low sample size, the the strength of the conclusion of this manuscript would be greatly improved if both of these results were further explored.

Reviewer #2 (Public Review):

Summary:<br /> The authors used four datasets spanning 30 countries to examine funding success and research quality score for various disciplines. They examined whether funding or research quality score were influenced by majority gender of the discipline and whether these affected men, women, or both within each discipline. They found that disciplines dominated by women have lower funding success and research quality score than disciplines dominated by men. These findings are surprising because even the men in women-dominated fields experienced lower funding success and research quality score.

Strengths:<br /> - The authors utilized a comprehensive dataset covering 30 countries to explore the influence of the majority gender in academic disciplines on funding success and research quality scores.<br /> - Findings suggest a systemic issue where disciplines with a higher proportion of women have lower evaluations and funding success for all researchers, regardless of gender.<br /> - The manuscript is notable for its large sample size and the diverse international scope, enhancing the generalizability of the results.<br /> - The work accounts for various factors including age, number of research outputs, and bibliometric measures, strengthening the validity of the findings.<br /> - The manuscript raises important questions about unconscious bias in research evaluation and funding decisions, as evidenced by lower scores in women-dominated fields even for researchers that are men.<br /> - The study provides a nuanced view of gender bias, showing that it is not limited to individuals but extends to entire disciplines, impacting the perception and funding and quality or worth of research.<br /> - This work underscores the need to explore motivations behind gender distribution across fields, hinting at deep-rooted societal and institutional barriers.<br /> - The authors have opened a discussion on potential solutions to counter bias, like adjusting funding paylines or anonymizing applications, or other practical solutions.<br /> - While pointing out limitations such as the absence of data from major research-producing countries, the manuscript paves the way for future studies to examine whether its findings are universally applicable.<br /> - The study carefully uses the existing data (including PBRF funding panel gender diversity) to examine gender bias. These types of datasets are often not readily accessible for analysis. Here, the authors have used the available data to the fullest extent possible.

The authors have addressed the concerns I had about the original version.

Reviewer #1 (Public Review):

Summary: The authors present a close to complete annotation of the male Drosophila ventral nerve cord, a critical part of the fly's central nervous system.

Strengths: The manuscript describes an enormous amount of work that takes the first steps towards presenting and comprehending the complexity and organization of the ventral nerve cord. The analysis is thorough and complete. It also makes the effort to connect this EM-centric view of the nervous system to more classical analyses, such as the previously defined hemilineages, that also describe the organization of the fly nervous system. There are many, many insights that come from this work that will be valuable to the field for the foreseeable future.

Weaknesses: With more than 60 primary figures, the paper is overwhelming and cannot be read and digested in a single sitting. The result is more like a detailed resource rather than a typical research paper.

Reviewer #1 (Public Review):

This study generated 3D cell constructs from endometrial cell mixtures that were seeded in the Matrigel scaffold. The cell assemblies were treated with hormones to induce a "window of implantation" (WOI) state.

The authors did their best to revise their study according to the reviewers' comments. However, the study remains unconvincing and at the same time too dense and not focused enough.

(1) The use of the term organoids is still confusing and should be avoided. Organoids are epithelial tissue-resembling structures. Hence, the multiple-cell aggregates developed here are rather "co-culture models" (or "assembloids"). It is still unexpected (unlikely) that these structures containing epithelial, stromal and immune cells can be robustly passaged in the epithelial growth conditions used. All other research groups developing real organoids from endometrium have shown that only the epithelial compartment remains in culture at passaging (while the stromal compartment is lost). If authors keep to their idea, they should perform scRNA-seq on both early and late (passage 6-10) "organoids". And they should provide details of culturing/passaging/plating etc that are different with other groups and might explain why they keep stromal and immune cells in their culture for such a long time. In other words, they should then in detail compare their method to the standard method of all other researchers in the field, and show the differences in survival and growth of the stromal and immune cells.<br /> (2) The paper is still much too dense, touching upon all kind of conclusions from the manifold bioinformatic analyses. The latter should be much clearer and better described, and then some interesting findings (pathways/genes) should be highlighted without mentioning every single aspect that is observed. The paper needs a lot of editing to better focus and extract take-home messages, not bombing the reader with a mass of pathways, genes etc which makes the manuscript just not readable or 'digest-able'. There is no explanation whatever and no clear rationale why certain genes are included in a list while others are not. There is the impression that mass bioinformatics is applied without enough focus.<br /> (3) The study is much too descriptive and does not show functional validation or exploration (except glycogen production). Some interesting findings extracted from the bioinformatics must be functionally tested.<br /> (4) In contrast to what was found in vivo (Wang et al. 2020), no abrupt change in gene expression pattern is mentioned here from the (early-)secretory to the WoI phase. Should be discussed. Although the bioinformatic analyses point into this direction, there are major concerns which must be solved before the study can provide the needed reliability and credibility for revision.<br /> (5) All data should be benchmarked to the Wang et al 2020 and Garcia-Alonso et al. 2021 papers reporting very detailed scRNA-seq data, and not only the Stephen R. Quake 2020 paper.<br /> (6) Fig. 2B: Vimentin staining is not at all clear. F-actin could be used to show the typical morphology of the stromal cells?<br /> (7) Where does the term "EMT-derived stromal cells" come from? On what basis has this term been coined?<br /> (8) CD44 is shown in Fig. 2D but the text mentions CD45 (line 159)?<br /> (9) All quantification experiments (of stainings etc) should be in detail described how this was done. It looks very difficult (almost not feasible) when looking at the provided pictures to count the stained cells.<br /> (10) Fig. 3C: it is unclear how quantification can be reliably done. Moreover, OLFM4 looks positive in all cells of Ctrl, but authors still see an increase?<br /> (11) Fig. 3F: Met is downregulated which is not in accordance with the mentioned activation of the PI3K-AKT pathway.<br /> (12) Lines 222-226: transcriptome and proteome differences are not significant; so, how meaningful are the results then? Then, it is very hard to conclude an evolution from secretory phase to WoI.<br /> (13) WoI organoids show an increased number of cilia. However, some literature shows the opposite, i.e. less ciliated cells in the endometrial lining at WoI (to keep the embryo in place). How to reconcile?<br /> (14) How are pinopodes distinguished from microvilli? Moreover, Fig. 3 does not show the typical EM structure of cilia.<br /> (15) There is a recently published paper demonstrating another model for implantation. This paper should be referenced as well (Shibata et al. Science Advances, 2024).<br /> (16) Line 78: two groups were the first here (Turco and Borreto) and should both be mentioned.<br /> (17) Line 554: "as an alternative platform" - alternative to what? Authors answer reviewers' comments by just changing one word, but this makes the text odd.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors conducted an important study that explored an innovative regenerative treatment for pediatric craniofacial bone loss, with a particular focus on investigating the impacts of JAGGED1 (JAG1) signaling.

Strengths:

Building on their prior research involving the effect of JAG1 on murine cranial neural crest cells, the authors demonstrated successful bone regeneration in an in vivo murine bone loss model with a critically-sized cranial defect, where they delivered JAG1 with pediatric human bone-derived osteoblast-like cells in the hydrogel. Additionally, their findings unveiled a crucial mechanism wherein JAG1 induces pediatric osteoblast commitment and bone regeneration through the phosphorylation of p70 S6K. This discovery offers a promising avenue for potential treatment, involving targeted delivery of JAG1 and activation of downstream p70 s6K, for pediatric craniofacial bone loss. Overall, the experimental design is appropriate, and the results are clearly presented.

Reviewer #1 (Public Review):

Summary:

In this paper, proteomics analysis of the plasma of human subjects that underwent an exercise training regime consisting of a combination of endurance and resistance exercise led to the identification of several proteins that were responsive to exercise training. Confirming previous studies, many exercise-responsive secreted proteins were found to be involved in the extra-cellular matrix. The protein CD300LG was singled out as a potential novel exercise biomarker and the subject of numerous follow-up analyses. The levels of CD300LG were correlated with insulin sensitivity. The analysis of various open-source datasets led to the tentative suggestion that CD300LG might be connected with angiogenesis, liver fat, and insulin sensitivity. CD300LG was found to be most highly expressed in subcutaneous adipose tissue and specifically in venular endothelial cells. In a subset of subjects from the UK Biobank, serum CD300LG levels were positively associated with several measures of physical activity - particularly vigorous activity. In addition, serum CD300LG levels were negatively associated with glucose levels and type 2 diabetes. Genetic studies hinted at these associations possibly being causal. Mice carrying alterations in the CD300LG gene displayed impaired glucose tolerance, but no change in fasting glucose and insulin. Whether the production of CD300LG is changed in the mutant mice is unclear.

Strengths:

The specific proteomics approach conducted to identify novel proteins impacted by exercise training is new. The authors are resourceful in the exploitation of existing datasets to gain additional information on CD300LG.

Weaknesses:

While the analyses of multiple open-source datasets are necessary and useful, they lead to relatively unspecific correlative data that collectively insufficiently advance our knowledge of CD300LG and merely represent the starting point for more detailed investigations. Additional more targeted experiments of CD300LG are necessary to gain a better understanding of the role of CD300LG and the mechanism by which exercise training may influence CD300LG levels. One should also be careful to rely on external data for such delicate experiments as mouse phenotyping. Can the authors vouch for the quality of the data collected?

Reviewer #3 (Public Review):

In multiple cancers, the key roles of B cells are emerging in the tumor microenvironment (TME). The authors of this study appropriately introduce that B cells are relatively under-characterised in the TME and argue correctly that it is not known how the B cell receptor (BCR) repertoires across tumor, lymph node and peripheral blood relate. The authors therefore supply a potentially useful study evaluating the tumor, lymph node and peripheral blood BCR repertoires and site-to-site as well as intra-site relationships. The authors employ sophisticated analysis techniques, although the description of the methods is incomplete.

Major strengths:

(1) The authors provide a unique analysis of BCR repertoires across tumor, dLN, and peripheral blood. The work provides useful insights into inter- and intra-site BCR repertoire heterogeneity. While patient-to-patient variation is expected, the findings with regard to intra-tumor and intra-dLN heterogeneity with the use of fragments from the same tissue are of importance, contribute to the understanding of the TME, and will inform future study design.

(2) A particular strength of the study is the detailed CDR3 physicochemical properties analysis which leads the authors to observations that suggest a less-specific BCR repertoire of TIL-B compared to circulating B cells.

Concerns and comments on current version:

The revision has improved the manuscript but, in my opinion, remains inadequate. While most of my requested changes have been made, I do not see an expansion of Fig1A legend to incorporate more details about the analysis. Lacking details of methodology was a concern from all reviewers. Similarly, the 'fragmented' narrative was a concern of all reviewers. These matters have not been dealt with adequately enough - there are parts of the manuscript which remain fragmented and confusing. The narrative and analysis does not explain how the plasma cell bias has been dealt with adequately and in fact is simply just confusing. There is a paragraph at the beginning of the discussion re the plasma cell bias, which should be re-written to be clearer and moved to have a prominent place early in the results. Why are these results not properly presented? They are key for interpretation of the manuscript. Furthermore, the sorted plasma cell sequencing analysis also has only been performed on two patients. Another issue is that some disease cohorts are entirely composed of patients with metastasis, some without but metastasis is not mentioned. Metastasis has been shown to impact the immune landscape.

A reviewer brought up a concern about the overlap analysis and I also asked for an explanation on why this F2 metric chosen. Part of the rebuttal argues that another metric was explored showing similar results, thus conclusion reached is reasonable. Remarkably, these data are not only omitted from the manuscript, but is not even provided for the reviewers.

This manuscript certainly includes some interesting and useful work. Unfortunately, a comprehensive re-write was required to make the work much clearer and easier to understand and this has not been realised.

Reviewer #1 (Public Review):

Summary:

Kroeg et al. describe a novel method for 2D culture human induced pluripotent stem cells (hiPSCs) to form cortical tissue in a multiwell format. The method claims to offer a significant advancement over existing developmental models. Their approach allows them to generate cultures with precise, reproducible dimensions and structure with a single rosette; consistent geometry; incorporating multiple neuronal and glial cell types (cellular diversity); avoiding the necrotic core (often seen in free-floating models due to limited nutrient and oxygen diffusion). The researchers demonstrate the method's capacity for long-term culture, exceeding ten months, and show the formation of mature dendritic spines and considerable neuronal activity. The method aims to tackle multiple key problems of in vitro neural cultures: reproducibility, diversity, topological consistency, and electrophysiological activity. The authors suggest their potential in high-throughput screening and neurotoxicological studies.

Strengths:

The main advances in the paper seem to be: The culture developed by the authors appears to have optimal conditions for neural differentiation, lineage diversification, and long-term culture beyond 300 days. These seem to me as a major strength of the paper and an important contribution to the field. The authors present solid evidence about the high cell type diversity present in their cultures. It is a major point and therefore it could be better compared to the state of the art. I commend the authors for using three different IPS lines, this is a very important part of their proof. The staining and imaging quality of the manuscript is of excellent quality.

Weaknesses:

(1) The title is misleading: The presented cultures appear not to be organoids, but 2D neural cultures, with an insufficiently described intermediate EB stage. For nomenclature, see: doi: 10.1038/s41586-022-05219-6. Should the tissue develop considerable 3D depth, it would suffer from the same limited nutrient supply as 3D models - as the authors point out in their introduction.

(2) The method therefore should be compared to state-of-the-art (well-based or not) 2D cultures, which seems to be somewhat overlooked in the paper, therefore making it hard to assess what the advance is that is presented by this work.

(3) Reproducibility is prominently claimed throughout the manuscript. However, it is challenging to assess this claim based on the data presented, which mostly contain single frames of unquantified, high-resolution images. There are almost no systematic quantifications presented. The ones present (Figure S1D, Figure 4) show very large variability. However, the authors show sets of images across wells (Figure S1B, Figure S3) which hint that in some important aspects, the culture seems reproducible and robust.

(4) What is in the middle? All images show markers in cells present around the center. The center however seems to be a dense lump of cells based on DAPI staining. What is the identity of these cells? Do these cells persist throughout the protocol? Do they divide? Until when? Addressing this prominent cell population is currently lacking.

(5) This manuscript proposes a new method of 2D neural culture. However, the description and representation of the method are currently insufficient.<br /> (a) The results section would benefit from a clear and concise, but step-by-step overview of the protocol. The current description refers to an earlier paper and appears to skip over some key steps. This section would benefit from being completely rewritten. This is not a replacement for a clear methods section, but a section that allows readers to clearly interpret results presented later.<br /> (b) Along the same lines, the graphical abstract should be much more detailed. It should contain the time frames and the media used at the different stages of the protocol, seeding numbers, etc.

Reviewer #2 (Public Review):

Summary:

The manuscript by Kelbert et al. presents results on the involvement of the yeast transcription factor Sfp1 in the stabilisation of transcripts whose synthesis it stimulates. Sfp1 is known to affect the synthesis of a number of important cellular transcripts, such as many of those that code for ribosomal proteins. The hypothesis that a transcription factor can remain bound to the nascent transcript and affect its cytoplasmic half-life is attractive. However, the association of Sfp1 with cytoplasmic transcripts remains to be validated, as explained in the following comments:

A two-hybrid based assay for protein-protein interactions identified Sfp1, a transcription factor known for its effects on ribosomal protein gene expression, as interacting with Rpb4, a subunit of RNA polymerase II. Classical two-hybrid experiments depend on the presence of the tested proteins in the nucleus of yeast cells, suggesting that the observed interaction occurs in the nucleus. Unfortunately, the two-hybrid method cannot determine whether the interaction is direct or mediated by nucleic acids. The revised version of the manuscript now states that the observed interaction could be indirect.

To understand to which RNA Sfp1 might bind, the authors used an N-terminally tagged fusion protein in a cross-linking and purification experiment. This method identified 264 transcripts for which the CRAC signal was considered positive and which mostly correspond to abundant mRNAs, including 74 ribosomal protein mRNAs or metabolic enzyme-abundant mRNAs such as PGK1. The authors did not provide evidence for the specificity of the observed CRAC signal, in particular what would be the background of a similar experiment performed without UV cross-linking. This is crucial, as Figure S2G shows very localized and sharp peaks for the CRAC signal, often associated with over-amplification of weak signal during sequencing library preparation.

In a validation experiment, the presence of several mRNAs in a purified SFP1 fraction was measured at levels that reflect the relative levels of RNA in a total RNA extract. Negative controls showing that abundant mRNAs not found in the CRAC experiment were clearly depleted from the purified fraction with Sfp1 would be crucial to assess the specificity of the observed protein-RNA interactions (to complement Fig. 2D). The CRAC-selected mRNAs were enriched for genes whose expression was previously shown to be upregulated upon Sfp1 overexpression (Albert et al., 2019). The presence of unspliced RPL30 pre-mRNA in the Sfp1 purification was interpreted as a sign of co-transcriptional assembly of Sfp1 into mRNA, but in the absence of valid negative controls, this hypothesis would require further experimental validation. Also, whether the fraction of mRNA bound by Sfp1 is nuclear or cytoplasmic is unclear.

To address the important question of whether co-transcriptional assembly of Spf1 with transcripts could alter their stability, the authors first used a reporter system in which the RPL30 transcription unit is transferred to vectors under different transcriptional contexts, as previously described by the Choder laboratory (Bregman et al. 2011). While RPL30 expressed under an ACT1 promoter was barely detectable, the highest levels of RNA were observed in the context of the native upstream RPL30 sequence when Rap1 binding sites were also present. Sfp1 showed better association with reporter mRNAs containing Rap1 binding sites in the promoter region. Removal of the Rap1 binding sites from the reporter vector also led to a drastic decrease in reporter mRNA levels. Co-purification of reporter RNA with Sfp1 was only observed when Rap1 binding sites were included in the reporter. Negative controls for all the purification experiments might be useful.

To complement the biochemical data presented in the first part of the manuscript, the authors turned to the deletion or rapid depletion of SFP1 and used labelling experiments to assess changes in the rate of synthesis, abundance and decay of mRNAs under these conditions. An important observation was that in the absence of Sfp1, mRNAs encoding ribosomal protein genes not only had a reduced synthesis rate, but also an increased degradation rate. This important observation needs careful validation, as genomic run-on experiments were used to measure half-lives, and this particular method was found to give results that correlated poorly with other measures of half-life in yeast (e.g. Chappelboim et al., 2022 for a comparison). As an additional validation, a temperature shift to 42{degree sign}C was used to show that , for specific ribosomal protein mRNA, the degradation was faster, assuming that transcription stops at that temperature. It would be important to cite and discuss the work from the Tollervey laboratory showing that a temperature shift to 42{degree sign}C leads to a strong and specific decrease in ribosomal protein mRNA levels, probably through an accelerated RNA degradation (Bresson et al., Mol Cell 2020, e.g. Fig 5E). Finally, the conclusion that mRNA deadenylation rate is altered in the absence of Sfp1, is difficult to assess from the presented results (Fig. 3D).

The effects of SFP1 on transcription were investigated by chromatin purification with Rpb3, a subunit of RNA polymerase, and the results were compared with synthesis rates determined by genomic run-on experiments. The decrease in polII presence on transcripts in the absence of SFP1 was not accompanied by a marked decrease in transcript output, suggesting an effect of Sfp1 in ensuring robust transcription and avoiding RNA polymerase backtracking. To further investigate the phenotypes associated with the depletion or absence of Sfp1, the authors examined the presence of Rpb4 along transcription units compared to Rpb3. An effect of spf1 deficiency was that this ratio, which decreased from the start of transcription towards the end of transcripts, increased slightly. To what extent this result is important for the main message of the manuscript is unclear.

Suggestions: a) please clearly indicate in the figures when they correspond to reanalyses of published results. b) In table S2, it would be important to mention what the results represent and what statistics were used for the selection of "positive" hits.

Strengths:

- Diversity of experimental approaches used.<br /> - Validation of large-scale results with appropriate reporters.

Weaknesses:

- Lack of controls for the CRAC results and lack of negative controls for the co-purification experiments that were used to validate specific mRNA targets potentially bound by Sfp1.<br /> - Several conclusions are derived from complex correlative analyses that fully depend on the validity of the aforementioned Sfp1-mRNA interactions.

Reviewer #1 (Public Review):

This work seeks to understand how behaviour-related information is represented in the neural activity of the primate motor cortex. To this end, a statistical model of neural activity is presented that enables a non-linear separation of behaviour-related from unrelated activity. As a generative model, it enables the separate analysis of these two activity modes, here primarily done by assessing the decoding performance of hand movements the monkeys perform in the experiments. Several lines of analysis are presented to show that while the neurons with significant tuning to movements strongly contribute to the behaviourally-relevant activity subspace, less or un-tuned neurons also carry decodable information. It is further shown that the discovered subspaces enable linear decoding, leading the authors to conclude that motor cortex read-out can be linear.

Strengths:

In my opinion, using an expressive generative model to analyse neural state spaces is an interesting approach to understand neural population coding. While potentially sacrificing interpretability, this approach allows capturing both redundancies and synergies in the code as done in this paper. The model presented here is a natural non-linear extension of a previous linear model PSID) and uses weak supervision in a manner similar to a previous non-linear model (TNDM).

Weaknesses:

This revised version provides additional evidence to support the author's claims regarding model performance and interpretation of the structure of the resulting latent spaces, in particular the distributed neural code over the whole recorded population, not just the well-tuned neurons. The improved ability to linearly decode behaviour from the relevant subspace and the analysis of the linear subspace projections in my opinion convincingly demonstrates that the model picks up behaviour-relevant dynamics, and that these are distributed widely across the population. As reviewer 3 also points out, I would, however, caution to interpret this as evidence for linear read-out of the motor system - your model performs a non-linear transformation, and while this is indeed linearly decodable, the motor system would need to do something similar first to achieve the same. In fact to me it seems to show the opposite, that behaviour-related information may not be generally accessible to linear decoders (including to down-stream brain areas).

As in my initial review, I would also caution against making strong claims about identifiability although this work and TNDM seem to show that in practise such methods work quite well. CEBRA, in contrast, offers some theoretical guarantees, but it is not a generative model, so would not allow the type of analysis done in this paper. In your model there is a para,eter \alpha to balance between neural and behaviour reconstruction. This seems very similar to TNDM and has to be optimised - if this is correct, then there is manual intervention required to identify a good model.

Somewhat related, I also found that the now comprehensive comparison with related models shows that the using decoding performance (R2) as a metric for model comparison may be problematic: the R2 values reported in Figure 2 (e.g. the MC_RTT dataset) should be compared to the values reported in the neural latent benchmark, which represent well-tuned models (e.g. AutoLFADS). The numbers (difficult to see, a table with numbers in the appendix would be useful, see: https://eval.ai/web/challenges/challenge-page/1256/leaderboard) seem lower than what can be obtained with models without latent space disentanglement. While this does not necessarily invalidate the conclusions drawn here, it shows that decoding performance can depend on a variety of model choices, and may not be ideal to discriminate between models. I'm also surprised by the low neural R2 for LFADS I assume this is condition-averaged) - LFADS tends to perform very well on this metric.

One statement I still cannot follow is how the prior of the variational distribution is modelled. You say you depart from the usual Gaussian prior, but equation 7 seems to suggest there is a normal prior. Are the parameters of this distribution learned? As I pointed out earlier, I however suspect this may not matter much as you give the prior a very low weight. I also still am not sure how you generate a sample from the variational distribution, do you just draw one for each pass?

Summary:

This paper presents a very interesting analysis, but some concerns remain that mainly stem from the complexity of deep learning models. It would be good to acknowledge these as readers without relevant background need to understand where the possible caveats are.

Reviewer #1 (Public Review):

This study explored the relationship between sustained attention and substance use from ages 14 to 23 in a large longitudinal dataset. They found behaviour and brain connectivity associated with poorer sustained attention at age 14 predicted subsequent increase in cannabis and cigarette smoking from ages 14-23. They concluded that the brain network of sustained attention is a robust biomarker for vulnerability to substance use. The big strength of the study is a substantial sample size and validation of the generalization to an external dataset. In addition, various methods/models were used to prove the relationship between sustained attention and substance use over time.

Reviewer #1 (Public Review):

Summary:

This paper by Schommartz and colleagues investigates the neural basis of memory reinstatement as a function of both how recently the memory was formed (recent, remote) and its development (children, young adults). The core question is whether memory consolidation processes as well as the specificity of memory reinstatement differ with development. A number of brain regions showed a greater activation difference for recent vs. remote memories at the long versus shorter delay specifically in adults (cerebellum, parahippocampal gyrus, LOC). A different set showed decreases in the same comparison, but only in children (precuneus, RSC). The authors also used neural pattern similarity analysis to characterize reinstatement, though still in this revised paper I have substantive concerns about how the analyses were performed. While scene-specific reinstatement decreased for remote memories in both children and adults, claims about its presence cannot be made given the analyses. Gist-level reinstatement was observed in children but not adults, but I also have concerns about this analysis. Broadly, the behavioural and univariate findings are consistent with the idea memory consolidation differs between children and adults in important ways, and takes a step towards characterizing how.

Strengths:

The topic and goals of this paper are very interesting. As the authors note, there is little work on memory consolidation over development, and as such this will be an important data point in helping us begin to understand these important differences. The sample size is great, particularly given this is an onerous, multi-day experiment; the authors are to be commended for that. The task design is also generally well controlled, for example as the authors include new recently learned pairs during each session.

Weaknesses:

As noted above and in my review of the original submission, the pattern similarity analysis for both item and category-level reinstatement were performed in a way that is not interpretable given concerns about temporal autocorrelation within scanning run. Unfortunately these issues remain of concern in this revision because they were not rectified. Most of my review focuses on this analytic issue, though I also outline additional concerns.

(1) The pattern similarity analyses are largely uninterpretable due to how they were performed.

(a) First, the scene-specific reinstatement index: The authors have correlated a neural pattern during a fixation cross (delay period) with a neural pattern associated with viewing a scene as their measure of reinstatement. The main issue with this is that these events always occurred back-to-back in time. As such, the two patterns will be similar due simply to the temporal autocorrelation in the BOLD signal. Because of the issues with temporal autocorrelation within scanning run, it is always recommended to perform such correlations only across different runs. In this case, the authors always correlated patterns extracted from the same run, and which moreover have temporal lags that are perfectly confounded with their comparison of interest (i.e., from Fig 4A, the "scene-specific" comparisons will always be back-to-back, having a very short temporal lag; "set-based" comparisons will be dispersed across the run, and therefore have a much higher lag). The authors' within-run correlation approach also yields correlation values that are extremely high - much higher than would be expected if this analysis was done appropriately. The way to fix this would be to restrict the analysis to only cross-run comparisons, which is not possible given the design.

To remedy this, in the revision the authors have said they will refrain from making conclusions about the presence of scene-specific reinstatement (i.e., reinstatement above baseline). While this itself is an improvement from the original manuscript, I still have several concerns. First, this was not done thoroughly and at times conclusions/interpretations still seem to imply or assume the presence of scene reinstatement (e.g., line 979-985, "our research supports the presence of scene-specific reinstatement in 5-to-7-year-old children"; line 1138). Second, the authors' logic for the neural-behavioural correlations in the PLSC analysis involved restricting to regions that showed significant reinstatement for the gist analysis, which cannot be done for the analogous scene-specific reinstatement analysis. This makes it challenging to directly compare these two analyses since one was restricted to a small subset of regions and only children (gist), while scene reinstatement included both groups and all ROIs. Third, it is also unclear whether children and adults' values should be directly comparable given pattern similarity can be influenced by many factors like motion, among other things.

My fourth concern with this analysis relates to the lack of regional specificity of the effects. All ROIs tested showed a virtually identical pattern: "Scene-specific reinstatement" decreased across delays, and was greater in children than adults. I believe control analyses are needed to ensure artifacts are not driving these effects. This would greatly strengthen the authors' ability to draw conclusions from the "clean" comparison of day 1 vs. day 14. (A) The authors should present results from a control ROI that should absolutely not show memory reinstatement effects (e.g., white matter?). Results from the control ROI should look very different - should not differ between children and adults, and should not show decreases over time. (B) Do the recent items from day 1 vs. day 14 differ? If so, this could suggest something is different about the later scans (and if not, it would be reassuring). (C) If the same analysis was performed comparing the object cue and immediately following fixation (rather than the fixation and the immediately following scene), the results should look very different. I would argue that this should not be an index of reinstatement at all since it involves something presented visually rather than something reinstated (i.e., the scene picture is not included in this comparison). If this control analysis were to show the same effects as the primary analysis, this would be further evidence that this analysis is uninterpretable and hopelessly confounded.

(b) For the category-based neural reinstatement: (1) This suffers from the same issue of correlations being performed within run. Again, to correct this the authors would need to restrict comparisons to only across runs (i.e., patterns from run 1 correlated with patterns for run 2 and so on). The authors in their response letter have indicated that because the patterns being correlated are not derived from events in close temporal proximity, they should not suffer from the issue of temporal autocorrelation. This is simply not true. For example, see the paper by Prince et al. (eLife 2022; on GLMsingle). This is not the main point of Prince et al.'s paper, but it includes a nice figure that shows that, using standard modelling approaches, the correlation between (same-run) patterns can be artificially elevated for lags as long as ~120 seconds (and can even be artificially reduced after that; Figure 5 from that paper) between events. This would affect many of the comparisons in the present paper. The cleanest way to proceed is to simply drop the within-run comparisons, which I believe the authors can do and yet they have not. Relatedly, in the response letter the authors say they are focusing mainly on the change over time for reinstatement at both levels including the gist-type reinstatement; however, this is not how it is discussed in the paper. They in fact are mainly relying on differences from zero, as children show some "above baseline" reinstatement while adults do not, but I believe there were no significant differences over time (i.e., the findings the authors said they would lean on primarily, as they are arguably the most comparable). (2) This analysis uses a different approach of comparing fixations to one another, rather than fixations to scenes. In their response letter and the revised paper, the authors do provide a bit of reasoning as to why this is the most sensible. However, it is still not clear to me whether this is really "reinstatement" which (in my mind) entails the re-evoking of a neural pattern initially engaged during perception. Rather, could this be a shared neural state that is category specific? In any case, I think additional information should be added to the text to clarify that this definition differs from others in the literature. The authors might also consider using some term other than reinstatement. Again (as I noted in my prior review), the finding of no category-level reinstatement in adults is surprising and confusing given prior work and likely has to do with the operationalization of "reinstatement" here. I was not quite sure about the explanation provided in the response letter, as category-level reinstatement is quite widespread in the brain for adults and is robust to differences in analytic procedures etc. (3) Also from a theoretical standpoint-I'm still a bit confused as to why gist-based reinstatement would involve reinstatement of the scene gist, rather than the object's location (on the screen) gist. Were the locations on the screen similar across scene backgrounds from the same category? It seems like a different way to define memory retrieval here would be to compare the neural patterns when cued to retrieve the same vs. similar (at the "gist" level) vs. different locations across object-scene pairs. This is somewhat related to a point from my review of the initial version of this manuscript, about how scene reinstatement is not necessary. The authors state that participants were instructed to reinstate the scene, but that does not mean they were actually doing it. The point that what is being measured via the reinstatement analyses is actually not necessary to perform the task should be discussed in more detail in the paper.

(2) Inspired by another reviewer's comment, it is unclear to me the extent to which age group differences can be attributed to differences in age/development versus memory strength. I liked the other reviewer's suggestions about how to identify and control for differences in memory strength, which I don't think the authors actually did in the revision. They instead showed evidence that memory strength does seem to be lower in children, which indicates this is an interpretive confound. For example, I liked the reviewer's suggestion of performing analyses on subsets of participants who were actually matched in initial learning/memory performance would have been very informative. As it is, the authors didn't really control for memory strength adequately in my opinion, and as such their conclusions about children vs. adults could have been reframed as people with weak vs. strong memories. This is obviously a big drawback given what the authors want to conclude. Relatedly, I'm not sure the DDM was incorporated as the reviewer was suggesting; at minimum I think the authors need to do more work in the paper to explain what this means and why it is relevant. (I understand putting it in the supplement rather than the main paper, but I still wanted to know more about what it added from an interpretive perspective.)

(3) Some of the univariate results reporting is a bit strange, as they are relying upon differences between retrieval of 1- vs. 14-day memories in terms of the recent vs. report difference, and yet don't report whether the regions are differently active for recent and remote retrieval. For example in Figure 3A, neither anterior nor posterior hippocampus seem to be differentially active for recent vs. remote memories for either age group (i.e., all data is around 0). Precuneus also interestingly seems to show numerically recent>remote (values mostly negative), whereas most other regions show the opposite. This difference from zero (in either direction) or lack thereof seems important to the message. In response to this comment on the original manuscript, the authors seem to have confirmed that hippocampal activity was greater during retrieval than implicit baseline. But this was not really my question - I was asking whether hippocampus is (and other ROIs in this same figure are) differently engaged for recent vs. remote memories.

(4) Related to point 3, the claims about hippocampus with respect to multiple trace theory feel very unsupported by the data. I believe the authors want to conclude that children's memory retrieval shows reliance on hippocampus irrespective of delay, presumably because this is a detailed memory task. However the authors have not really shown this; all they have shown is that hippocampal involvement (whatever it is) does not vary by delay. But we do not have compelling evidence that the hippocampus is involved in this task at all. That hippocampus is more active during retrieval than implicit baseline is a very low bar and does not necessarily indicate a role in memory retrieval. If the authors want to make this claim, more data are needed (e.g., showing that hippocampal activity during retrieval is higher when the upcoming memory retrieval is successful vs. unsuccessful). In the absence of this, I think all the claims about multiple trace theory supporting retrieval similarly across delays and that this is operational in children are inappropriate and should be removed.

(5) There are still not enough methodological details in the main paper to make sense of the results. Some of these problems were addressed in the revision but others remain. For example, a couple of things that were unclear: that initially learned locations were split, where half were tested again at day 1 and the other half at day 14; what specific criterion was used to determine to pick the 'well-learned' associations that were used for comparisons at different delay periods (object-scene pairs that participants remembered accurately in the last repetition of learning? Or across all of learning?).

(6) In still find the revised Introduction a bit unclear. I appreciated the added descriptions of different theories of consolidation, though the order of presented points is still a bit hard to follow. Some of the predictions I also find a bit confusing as laid out in the introduction. (1) As noted in the paper multiple trace theory predicts that hippocampal involvement will remain high provided memories retained are sufficiently high detail. The authors however also predict that children will rely more on gist (than detailed) memories than adults, which would seem to imply (combined with the MTT idea) that they should show reduced hippocampal involvement over time (while in adults, it should remain high). However, the authors' actual prediction is that hippocampus will show stable involvement over time in both kids and adults. I'm having a hard time reconciling these points. (2) With respect to the extraction of gist in children, I was confused by the link to Fuzzy Trace Theory given the children in the present study are a bit young to be showing the kind of gist extraction shown in the Brainerd & Reyna data. Would 5-7 year olds not be more likely to show reliance on verbatim traces under that framework? Also from a phrasing perspective, I was confused about whether gist-like information was something different from just gist in this sentence: "children may be more inclined to extract gist information at the expense of detailed or gist-like information." (p. 8) - is this a typo?

(7) For the PLSC, if I understand this correctly, the profiles were defined for showing associations with behaviour across age groups. (1) As such, is it not "double dipping" to then show that there is an association between brain profile and behaviour-must this not be true by definition? If I am mistaken, it might be helpful to clarify this in the paper. (2) In addition, I believe for the univariate and scene-specific reinstatement analyses these profiles were defined across both age groups. I assume this doesn't allow for separate definition of profiles across the two group (i.e., a kind of "interaction"). If this is the case, it makes sense that there would not be big age differences... the profiles were defined for showing an association across all subjects. If the authors wanted to identify distinct profiles in children and adults they may need to run another analysis. (3) Also, as for differences between short delay brain profile and long delay brain profile for the scene-specific reinstatement - there are 2 regions that become significant at long delay that were not significant at a short delay (PC, and CE). However, given there are ceiling effects in behaviour at the long but not short delay, it's unclear if this is a meaningful difference or just a difference in sensitivity. Is there a way to test whether the profiles are statistically different from one another? (4) As I mentioned above, it also was not ideal in my opinion that all regions were included for the scene-specific reinstatement due to the authors' inability to have an appropriate baseline and therefore define above-chance reinstatement. It makes these findings really challenging to compare with the gist reinstatement ones.

(8) I would encourage the authors to be specific about whether they are measuring/talking about memory representations versus reinstatement, unless they think these are the same thing (in which case some explanation as to why would be helpful). For example, especially under the Fuzzy Trace framework, couldn't someone maintain both verbatim and gist traces of a memory yet rely more on one when making a memory decision?

(9) With respect to the learning criteria - it is misleading to say that "children needed between two to four learning-retrieval cycles to reach the criterion of 83% correct responses" (p. 9). Four was the maximum, and looking at the Figure 1C data it appears as though there were at least a few children who did not meet the 83% minimum. I believe they were included in the analysis anyway? Please clarify. Was there any minimum imposed for inclusion?

(10) For the gist-like reinstatement PLSC analysis, results are really similar a short and long delays and yet some of the text seems to implying specificity to the long delay. One is a trend and one is significant (p. 31), but surely these two associations would not be statistically different from one another?

(11) As a general comment, I had a hard time tying all of the (many) results together. For example adults show more mature neocortical consolidation-related engagement, which the authors say is going to create more durable detailed memories, but under multiple trace theory we would generally think of neocortical representations as providing more schematic information. If the authors could try to make more connections across the different neural analyses, as well as tie the neural findings in more closely with the behaviour & back to the theoretical frameworks, that would be really helpful.

Reviewer #1 (Public Review):

Summary:

This work by Passlick and colleagues set out to reveal the mechanism by which short bouts of ischemia perturb glutamate signalling. This manuscript builds upon previous work in the field that reported a paradoxical increase in synaptic transmission following acute, transient ischemia termed ischemic or anoxic long-term potentiation. Despite these observations how this occurs and the involvement of glutamate release and uptake mechanisms remained unanswered.

Here the authors employed two distinct chemical ischemia models, one lasting 2-minutes, the other 5-minutes. Recording evoked field excitatory postsynaptic potentials in acute brain slices, the authors revealed that shorter bouts of ischemia resulted in a transient decrease in postsynaptic responses followed by an overshoot and long-term potentiation. Longer bouts of chemical ischemia (5-minutes), however, resulted in synaptic failure that did not return to baseline levels over 50-minutes of recording (Figure 1).

Two-photon Imaging of fluorescent glutamate sensor iGluSnFR expressed in astrocytes matched postsynaptic responses with shorter ischemia resulting in a transient dip before increase in extracellular glutamate which was not the case with prolonged ischemia (Figure 2).

Mechanistically, the authors show that this increased glutamate levels and postsynaptic responses were not due to changes in glutamate clearance (Figure 3). Next using a competitive antagonist for postsynaptic AMPA receptors the authors show that synaptic glutamate release was enhanced by 2-minute chemical ischemia.

Taken together, these data reveal the underlying mechanism regarding ischemic long-term potentiation, highlighting presynaptic release as the primary culprit. Additionally, the authors show relative insensitivity of glutamate uptake mechanisms during ischemia, highlighting the resilience of astrocytes to this metabolic challenge.

Reviewer #1 (Public Review):

Summary:

This manuscript describes the crystallographic screening of a number of small molecules derived from the natural substrates S-adenosyl methionine (SAM) and adenine, against the SARS-CoV-2 2'-O-methyltransferase NSP16 in complex with its partner NSP10. High-quality structures for several of these are presented together with efforts to evaluate their potential biophysical binding and antiviral activities. The structures are of high quality and the data are well presented but do not yet show potency in biophysical binding. They only offer limited insights into the design of inhibitors of NSP16/10.

Strengths:

The main strengths of the study are the high quality of the structural data, and associated electron density maps making the structural data highly accurate and informative for future structure-based design. These results are clearly presented and communicated in the manuscript. Another strength is the authors' attempts to probe the binding of the identified fragments using biophysical assays. Although in general the outcome of these experiments shows negative data or very weak binding affinities the authors should be commended for attempting several techniques and showing the data clearly. This study is also useful as an example of the complexities associated with drug discovery on a bi-substrate target such as a methyltransferase, several of the observed binding poises were unexpected with compounds that are relatively similar to substrates binding in different parts of the active site or other unexpected orientations. This serves as an example of how experimental structural information is still of crucial importance to structure-based drug design. In general, the claims in the manuscript are well supported by the data.

Weaknesses:

The main limitations of the study are that the new structures generated in the study are fairly limited in terms of chemical space being similar to either SAM or RNA-CAP analogues. It feels a little bit of a lost opportunity to expand this to more diverse ligands which may reveal potential inhibitors that are distinct from current methyltransferase inhibitors based on SAM analogues and truly allow a selective targeting of this important target.

Another limitation is the potentially misleading nature of the antiviral assays. It is not possible to say if these compounds display on-target activity in these assays or even if the inhibition of NSP16/10 would have any effect in these assays. Whilst the authors do mention these points I think this should be emphasized more strongly.

Minor critical points:

The authors state that their crystals and protein preps have co-purified SAM occupying the active site of the crystals. Presumably, this complicates the interpretation of electron density maps as many of the ligands share overlap with the existing SAM density making traditional analysis of difference maps challenging. The authors did not utilize the PanDDA analysis for this step, perhaps this is related to the presence of SAM in the ground state datasets? Also, occupancies are reported in the manuscript in some cases to two significant figures, this seems to be an overestimation of the ability of refinement to determine occupancy based on density alone and the authors should clarify how these figures were reached.

The molecular docking approach to pre-selection of library compounds to soak did not appear to be successful. Could the authors make any observations about the compounds selected by docking or the docking approach used that may explain this?

Reviewer #1 (Public Review):

The comments below are from my review of the first submission of this article. I would now like to thank the authors for their hard work in responding to my comments. I am happy with the changes they have made, in particular the inclusion of further experimental evidence in Figures 2 and 4. I have no further comments to make.

In 'Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis', Patel et al. present a variety of analyses using different methodologies to investigate the importance of two miRNAs in regulating gene expression in a cellular model of cartilage development. They first re-analysed existing data to identify these miRNAs as one of the most dynamic across a chondrogenesis development timecourse. Next, they manipulated the expression of these miRNAs and showed that this affected the expression of various marker genes as expected. An RNA-seq experiment on these manipulations identified putative mRNA targets of the miRNAs which were also supported by bioinformatics predictions. These top hits were validated experimentally and, finally, a kinetic model was developed to demonstrate the relationship between the miRNAs and mRNAs studied throughout the paper.