Reviewer #1 (Public review):

This work regards the role of Aurora Kinase A (AurA) in trained immunity. The authors claim that AurA is essential to the induction of trained immunity. The paper starts with a series of experiments showing the effects of suppressing AurA on beta-glucan-trained immunity. This is followed by an account of how AurA inhibition changes the epigenetic and metabolic reprogramming that are characteristic of trained immunity. The authors then zoom in on specific metabolic and epigenetic processes (regulation of S-adenocylmethionine metabolism & histone methylation). Finally, an inhibitor of AurA is used to reduce beta-glucan's anti-tumour effects in a subcutaneous MC-38 model.

Strengths:

With the exception of my confusion around the methods used for relative gene expression measurements, the experimental methods are generally well-described. I appreciate the authors' broad approach to studying different key aspects of trained immunity (from comprehensive transcriptome/chromatin accessibility measurements to detailed mechanistic experiments). Approaching the hypothesis from many different angles inspires confidence in the results (although not completely - see weaknesses section). Furthermore, the large drug-screening panel is a valuable tool as these drugs are readily available for translational drug-repurposing research.

Weaknesses

(1) The manuscript contains factual inaccuracies such as:<br /> (a) Intro: the claim that trained cells display a shift from OXPHOS to glycolysis based on the paper by Cheng et al. in 2014; this was later shown to be dependent on the dose of stimulation and actually both glycolysis and OXPHOS are generally upregulated in trained cells (pmid 32320649)<br /> (b) Discussion: Trained immunity was first described as such in 2011, not decades ago.

(2) The authors approach their hypothesis from different angles, which inspires a degree of confidence in the results. However, the statistical methods and reporting are underwhelming.<br /> (a) Graphs depict mean +/- SEM, whereas mean +/- SD is almost always more informative.<br /> (b) The use of 1-tailed tests is dubious in this scenario. Furthermore, in many experiments/figures the case could be made that the comparisons should be considered paired (the responses of cells from the same animal are inherently not independent due to their shared genetic background and, up until cell isolation, the same host factors like serum composition/microbiome/systemic inflammation etc).<br /> (c) It could be explained a little more clearly how multiple testing correction was done and why specific tests were chosen in each instance.<br /> (d) Most experiments are done with n = 3, some experiments are done with n = 5. This is not a lot. While I don't think power analyses should be required for simple in vitro experiments, I would be wary of drawing conclusions based on n = 3. It is also not indicated if the data points were acquired in independent experiments. ATAC-seq/RNA-seq was, judging by the figures, done on only 2 mice per group. No power calculations were done for the in vivo tumor model.<br /> (e) Furthermore, the data spread in many experiments (particularly BMDM experiments) is extremely small. I wonder if these are true biological replicates, meaning each point represents BMDMs from a different animal? (disclaimer: I work with human materials where the spread is of course always much larger than in animal experiments, so I might be misjudging this.).

(3) Maybe the authors are reserving this for a separate paper, but it would be fantastic if the authors would report the outcomes of the entire drug screening instead of only a selected few. The field would benefit from this as it would save needless repeat experiments. The list of drugs contains several known inhibitors of training (e.g. mTOR inhibitors) so there must have been more 'hits' than the reported 8 Aurora inhibitors.

(4) Relating to the drug screen and subsequent experiments: it is unclear to me in supplementary figure 1B which concentrations belong to secondary screens #1/#2 - the methods mention 5 µM for the primary screen and "0.2 and 1 µM" for secondary screens, is it in this order or in order of descending concentration?<br /> (a) It is unclear if the drug screen was performed with technical replicates or not - the supplementary figure 1B suggests no replicates and quite a large spread (in some cases lower concentration works better?)

(5) The methods for (presumably) qPCR for measuring gene expression in Figure 1C are missing. Which reference gene was used and is this a suitably stable gene?

(6) From the complete unedited blot image of Figure 1D it appears that the p-Aurora and total Aurora are not from the same gel (discordant number of lanes and positioning). This could be alright if there are no/only slight technical errors, but I find it misleading as it is presented as if the actin (loading control to account for aforementioned technical errors!) counts for the entire figure.

(7) Figure 2: This figure highlights results that are by far not the strongest ones - I think the 'top hits' deserve some more glory. A small explanation on why the highlighted results were selected would have been fitting.

(8) Figure 3 incl supplement: the carbon tracing experiments show more glucose-carbon going into TCA cycle (suggesting upregulated oxidative metabolism), but no mito stress test was performed on the seahorse.

(9) Inconsistent use of an 'alisertib-alone' control in addition to 'medium', 'b-glucan', 'b-glucan + alisertib'. This control would be of great added value in many cases, in my opinion.

(10) Figure 4A: looking at the unedited blot images, the blot for H3K36me3 appears in its original orientation, whereas other images appear horizontally mirrored. Please note, I don't think there is any malicious intent but this is quite sloppy and the authors should explain why/how this happened (are they different gels and the loading sequence was reversed?)

(11) For many figures, for example prominently figure 5, the text describes 'beta-glucan training' whereas the figures actually depict acute stimulation with beta-glucan. While this is partially a semantic issue (technically, the stimulation is 'the training-phase' of the experiment), this could confuse the reader.

(12) Figure 6: Cytokines, especially IL-6 and IL-1β, can be excreted by tumour cells and have pro-tumoral functions. This is not likely in the context of the other results in this case, but since there is flow cytometry data from the tumour material it would have been nice to see also intracellular cytokine staining to pinpoint the source of these cytokines.

Reviewer #1 (Public Review):

HLA genes have long been known to harbor trans-species polymorphism (TSP). This manuscript aimed to use state-of-the-art analyses and updated genotyping data to rigorously test for the presence of TSP in HLA genes, quantify the timescales associated with HLA TSP, and relate HLA disease associations to evolutionary rates. To do this, the authors chose HLA alleles across great apes, old world monkeys, and new world monkeys on which to perform phylogenetic analyses, alongside non-parametric tests that compare patterns of synonymous diversity. Finally, HLA genetic associations with the disease were correlated with evolutionary rate.

Strengths:

The manuscript is well written and neatly organized, the figures are clear, and there are many supplementary analyses that will make this paper a great resource for MHC phylogenetics at allelic resolution.

Deployment of modern methodology such as BEAST2 can also test if the hypothesis of TSP is supported while accounting for uncertainties in tree topology and evolutionary rates, necessary additions to analyses of the MHC.

Weaknesses:

Because TSP has already been convincingly demonstrated to occur in the MHC, the primary benefit of the current study is to ensure these previous observations are still supported by the wealth of genetic data that is now available and modern phylogenetic approaches. However, the benefit of using the robust BEAST2 method comes with the weakness of not using all available data. Focusing on single gene trees with only a small subset of alleles may bias results, and inclusion/exclusion criteria should be better defined.

One major point that is somewhat overlooked is the presence of multiple copy numbers for the MHC genes through classic birth and death evolution. For example, MHC-B in new world monkeys is duplicated many times (up to 10; PMID: 23715823). This duplication is naturally accompanied by gene loss and pseudogene formation. All of these things muddy the waters considerably yet are not addressed here. A good example is MHC-A, where it has been very difficult to apportion orthologs, even amongst closely related species, due to alternative or incomplete duplication/loss across the species, or region configuration polymorphism (e.g. PMID: 26371256). An example is chimpanzee Patr-AL which shares similarities with human HLA-A*02 lineage, but is a separate locus, could this show up as TSP under the current analysis?

Similarly, an alternative hypothesis for TSP is convergent gene conversion mutations: intergenic gene conversion has been repeatedly observed in HLA genes and the possibility of it occurring with the same two genes becomes more realistic over 45 million years. If the same two MHC genes recombined in humans and in an NWM, each on their own lineages, this would appear as TSP and would cause an overlap of pairwise synonymous divergence between human-human and human-NWM allele comparisons. This might be especially possible in MHC-DR and MHC-DQ genes presented in Figure 2 since both humans and NWM have multiple MHC-DRB and DQB genes (unless e.g. were genes besides HLA/MHC-DRB1 such as DRB3,4,5 included in the DRB phylogenies?). While BEAST2 may be a good way of robustly modeling and identifying TSP, and I understand these analyses cannot support many more sequences, the authors should consider adding an analysis that rules out gene conversion as an explanation for their results (especially the often repeated claim of 45 million year TSP). For example, can the authors use BLAST to ensure that the alleles that underlie 45 million years of TSP do not share close similarities to other HLA genes present in their respective human and NWM genomes? This seems like it could be fairly quickly performed for all genes, and even if it argued against TSP, it would be an interesting finding.

Finally, the authors have limited themselves to a small subset of HLA/MHC alleles and do not provide sufficient information in the methods to understand how these were chosen nor sufficient discussion surrounding how inclusion/exclusion criteria could bias results. For example, the authors say the alleles were chosen at 2-digit (i.e. 1 field) resolution, but in the phylogenies of Fig. 2, I see variable numbers of alleles chosen for each 2-digit allele family - what metric was used to decide on these alleles?

"We also collected associations between amino acids and TCR phenotypes". It is not clear either what was analyzed, or the results for this part of the analysis. This is a topic of much debate and none of the previous work has been discussed (PMID: 18304006, PMID: 29636542 as primers for this contentious subject)

MHC class I also interact with NK cell receptors, including polymorphic KIR. Through their interactions during infection control and reproduction, the two complexes co-evolve across primates, contributing to the maintenance of MHC diversity. Interaction with KIR likely has a greater impact on HLA polymorphism than interactions with TCR, yet this is not factored into any of the models, or indeed mentioned in the text.

One additional reason inclusion of the KIR binding is important relates to the point above about gene conversion, where it is established that gene conversion reproducibly swaps KIR-binding motifs among MHC class I alleles and genes. HLA-A*23, *24, and *25, *32, for example, are characterized by the acquisition of the 'Bw4' motif from HLA-B (PMID: 26284483), likely followed by positive natural selection. For exon 2 (which encodes the motif), these alleles turn up in a clade distinct from other human HLA-A (Fig 2-S1). What is the impact of the Bw4 motif on this phylogeny? Could this shuffling of motifs be interpreted as indirect TSP?

The analysis that shows the most rapidly evolving sites occur in the peptide binding domain brings little new to the field. This has been established by the Hughes and Nei (cited) and Parham, Lawlor, etc of 1988 (e.g. PMID: 3375250), and replicated multiple times across human populations and many other species.<br /> Likewise, the disease association part. It is nice to have a summary of the known associations, but there are others out there and this one is far from thorough. Here, 50% of the information about infectious diseases appears to be taken from one reference, leaving out some major bodies of work; for example identifying specific peptide binding residues or peptides that associate with HIV (PMID: 22896606) or malaria control (PMID: 1280333). It is also missing some major concepts -such as the DRB1 'shared epitope' of peptide binding residues that predispose to Rheumatoid Arthritis and protects from Parkinson's disease (35 years of work from PMID: 2446635 through PMID: 30910980). The nasopharyngeal carcinoma and EBV story (e.g. PMID: 23209447). Another huge gap here is the pregnancy syndromes -associations of specific HLA C and NK cell receptor allotypes with preeclampsia for example. There are thousands of HLA associations not considered in this section, and to do them justice would likely require an enormous amount of work.<br /> Thus - neither the idea that HLA/MHC polymorphism is focused on peptide binding nor that this binding drives resistance to infection and associations with the disease are new concepts. The previous work in these areas is inadequately acknowledged.

The paper is written in a very approachable language, which is nice to read and friendly to non-experts, but perhaps a little too much so in places. I find that the paper follows a very non-traditional format with respect to for example the results section, which seems a mixture of Introduction/methods/figure legends/discussion with no real solid result description.

Reviewer #1 (Public review):

Summary:

The manuscript discusses the role of phosphorylated ubiquitin (pUb) by PINK1 kinase in neurodegenerative diseases. It reveals that elevated levels of pUb are observed in aged human brains and those affected by Parkinson's disease (PD), as well as in Alzheimer's disease (AD), aging, and ischemic injury. The study shows that increased pUb impairs proteasomal degradation, leading to protein aggregation and neurodegeneration. The authors also demonstrate that PINK1 knockout can mitigate protein aggregation in aging and ischemic mouse brains, as well as in cells treated with a proteasome inhibitor. While this study provided some interesting data, several important points should be addressed before being further considered.

Strengths:

(1) Reveals a novel pathological mechanism of neurodegeneration mediated by pUb, providing a new perspective on understanding neurodegenerative diseases.<br /> (2) The study covers not only a single disease model but also various neurodegenerative diseases such as Alzheimer's disease, aging, and ischemic injury, enhancing the breadth and applicability of the research findings.

Weaknesses:

(1) PINK1 has been reported as a kinase capable of phosphorylating Ubiquitin, hence the expected outcome of increased p-Ub levels upon PINK1 overexpression. Figures 5E-F do not demonstrate a significant increase in Ub levels upon overexpression of PINK1 alone, whereas the evident increase in Ub expression upon overexpression of S65A is apparent. Therefore, the notion that increased Ub phosphorylation leads to protein aggregation in mouse hippocampal neurons is not yet convincingly supported.<br /> (2) The specificity of PINK1 and p-Ub antibodies requires further validation, as a series of literature indicate that the expression of the PINK1 protein is relatively low and difficult to detect under physiological conditions.<br /> (3) In Figure 6, relying solely on Western blot staining and golgi staining under high magnification is insufficient to prove the impact of PINK1 overexpression on neuronal integrity and cognitive function. The authors should supplement their findings with immunostaining results for MAP2 or NeuN to demonstrate whether neuronal cells are affected.<br /> (4) The authors should provide more detailed figure captions to facilitate the understanding of the results depicted in the figures.<br /> (5) While the study proposes that pUb promotes neurodegeneration by affecting proteasomal function, the specific molecular mechanisms and signaling pathways remain to be elucidated.

Reviewer #1 (Public review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with two lipid compositions similar to native viral membranes. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region. The revised manuscript demonstrates that these lipid interactions are robust to alterations in membrane composition and rigidity. However, it does not address the reverse-that phospholipids known experimentally not to associate with these antibodies (if any such lipids exist) also fail to interact in MD simulations.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. These simulations recapitulate lipid binding interactions solved in published crystallographic studies but also lead to the discovery of a novel lipid binding site the authors term the "Loading Site", which could guide future experiments with this antibody.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. These CG simulations , which cannot resolve atomistic interactions, are nonetheless compelling because negative controls (ab 13h11, BSA) that should not associate with membrane indeed sample significantly less membrane.

Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive, creative, and biologically inspired. Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #1 (Public review):

In this work, the authors examine the mechanism of action of MOTS-c and its impact on monocyte-derived macrophages. In the first part of the study, they show that MOTS-c acts as a host defense peptide with direct antibacterial activity. In the second part of the study, the authors aim to demonstrate that MOTS-c influences monocyte differentiation into macrophages via transcriptional regulation.

Major strengths. Methods used to study the bactericidal activity of MOTS-c are appropriate and the results convincing.

Major weaknesses. Methods used to study the impact on monocyte differentiation are inappropriate and the conclusions not fully supported by the data shown. A major issue is the use of the THP-1 cell line, a transformed monocytic line which does not mimic physiological monocyte biology. In particular, THP-1 differentiation is induced by PMA, which is a completely artificial system and conclusions from this approach cannot be generalized to monocyte differentiation. The authors would need to perform this series of experiments using freshly isolated monocytes, either from mouse or human. The read-out used for macrophage differentiation (adherence to plastic) is also not very robust, and the authors would need to analyze other parameters such as cell surface markers. It is also not clear whether MOTS-c could act in a cell-intrinsic fashion, as the authors have exposed cells to exogenous MOTS-c in all their experiments. The authors have also analyzed the transcriptomic changes induced by MOTS-c exposure in macrophages derived from young or old mice. While the results are potentially interesting, the differences observed seem independent from MOTS-c and mainly related to age, therefore the conclusions from this figure are not clear. The physiological relevance of this study is also unclear.

Reviewer #1 (Public review):

Summary:

This paper examines patterns of diversity and divergence in two closely related sub-species of Zea mays. While the data are interesting and the authors have tried to exclude multiple confounding factors, many patterns cannot clearly be ascribed to one cause or another.

Strengths:

The paper presents interesting data from sets of sympatric populations of the two sub-species, maize and teosinte. This sampling offers unique insights into the diversity and divergence between the two, as well as the geographic structure of each. Many analyses and simulations to check analyses have been carried out.

Weaknesses:

The strength of conclusions that can be drawn from the analyses was low, partly because there are many strange patterns. The authors have done a good job of adding caveats, but clearly, these species do not meet many assumptions of our methods

Reviewer #1 (Public review):

Summary:

In this manuscript, Shao et al. investigate the contribution of different cortical areas to working memory maintenance and control processes, an important topic involving different ideas about how the human brain represents and uses information when no longer available to sensory systems. In two fMRI experiments, they demonstrate that human frontal cortex (area sPCS) represents stimulus (orientation) information both during typical maintenance, but even more so when a categorical response demand is present. That is, when participants have to apply an added level of decision control to the WM stimulus, sPCS areas encode stimulus information more than conditions without this added demand. These effects are then expanded upon using multi-area neural network models, recapitulating the empirical gradient of memory vs control effects from visual to parietal and frontal cortices. Multiple experiments and analysis frameworks provide support for the authors' conclusions, and control experiments and analysis are provided to help interpret and isolate the frontal cortex effect of interest. While some alternative explanations/theories may explain the roles of frontal cortex in this study and experiments, important additional analyses have been added that help ensure a strong level of support for these results and interpretations.

Strengths:

- The authors use an interesting and clever task design across two fMRI experiments that is able to parse out contributions of WM maintenance alone along with categorical, rule-based decisions. Importantly, the second experiment only uses one fixed rule, providing both an internal replication of Experiment 1's effects and extending them to a different situation when rule switching effects are not involved across mini-blocks.

- The reported analyses using both inverted encoding models (IEM) and decoders (SVM) demonstrate the stimulus reconstruction effects across different methods, which may be sensitive to different aspects of the relationship between patterns of brain activity and the experimental stimuli.

- Linking the multivariate activity patterns to memory behavior is critical in thinking about the potential differential roles of cortical areas in sub-serving successful working memory. Figure 3's nicely shows a similar interaction to that of Figure 2 in the role of sPCS in the categorization vs. maintenance tasks. This is an important contribution to the field when we consider how a distributed set of interacting cortical areas supports successful working memory behavior.

- The cross-decoding analysis in Figure 4 is a clever and interesting way to parse out how stimulus and rule/category information may be intertwined, which would have been one of the foremost potential questions or analyses requested by careful readers.

- Additional ROI analyses in more anterior regions of the PFC help to contextualize the main effects of interest in the sPCS (and no effect in the inferior frontal areas, which are also retinotopic, adds specificity). And, more explanation for how motor areas or preparation are likely not involved strengthens the takeaways of the study (M1 control analysis).

Weaknesses:

- An explicit, quantitative link between the RNN and fMRI data is perhaps a last point that would integrate the RNN conclusion and analyses in line with the human imaging data.

- As Rev 2 mentions, multiple types of information codes may be present, and the response letter Figure 5 using representational similarity (RSA) gets at this question. It would strengthen the work to, at minimum, include this analysis as an extended or supplemental figure.

To sum up the results, a possible, brief schematic of each cortical area analyzed and its contribution to information coding in WM and successful subsequent behavior may help readers take away important conclusions of the cortical circuitry involved.

Reviewer #1 (Public review):

Summary:

This paper proposes a new model of perceptual habituation and tests it over two experiments with both infants and adults. The model combines a neural network for visual processing with a Bayesian rational model for attention (i.e., looking time) allocation. This Bayesian framework allows the authors to measure elegantly diverse factors that might drive attention, such as expected information gain, current information gain, and surprise. The model is then fitted to infant and adult participants' data over two experiments, which systematically vary the amount of habituation trials (Experiment 1) and the type of dishabituation stimulus (familiarity, pose, number, identity, and animacy). Results show that a model based on (expected) information gain performs better than a model based on surprise. Additionally, while novelty preference is observed when exposure to familiar stimuli is elevated, no familiarity preference is observed when exposure to familiar stimuli is low or intermediate, which is in contrast with past work.

Strengths:

There are three key strengths of this work:

(1) It integrates a neural network model with a Bayesian rational learner, thus bridging the gap between two fields that have often been disconnected. This is rarely seen in the cognitive science field, but the advantages are very clear from this paper: It is possible to have computational models that not only process visual information, but also actively explore the environment based on overarching attentional processes.

(2) By varying parametrically the amount of stimulus exposure and by testing the effects of multiple novel stimulus types, this work allowed the authors to put classical theories of habituation to the test on much finer scales than previous research has done.

(3) The Bayesian model allows the authors to test what specific aspects are different in infants and adults, showing that infants display greater values for the noise parameter.

Weaknesses:

Although a familiarity preference is not found, it is possible that this is related to the nature of the stimuli and the amount of learning that they offer. While infants here are exposed to the same perceptual stimulus repeatedly, infants can also be familiarised to more complex stimuli or scenarios. Classical statistical learning studies for example expose infants to specific pseudo-words during habituation/familiarisation, and then test their preference for familiar vs novel streams of pseudo-words. The amount of learning progress in these probabilistic learning studies is greater than in perceptual studies, and familiarity preferences may thus be more likely to emerge there. For these reasons, I think it is important to frame this as a model of perceptual habituation. This would also fit well with the neural net that was used, which is processing visual stimuli rather than probabilistic structures. If statements in the discussion are limited to perceptual paradigms, they would make the arguments more compelling.

Reviewer #1 (Public review):

In the manuscript "Identification of neurodevelopmental organization of the cell populations of Juvenile Huntington's disease using dorso-ventral HD organoids and HD mouse embryos," the authors establish a fused dorso-ventral system that mimics cortex-striatum interactions within a single organoid and use this system to investigate neurodevelopmental impairments caused by HD. Specifically, they describe certain phenotypes in 60-day HD organoids and the brains of humanized mouse embryos, utilizing both wet-lab and single-cell sequencing techniques. The authors also develop dorsal/ventral and ventral/dorsal mosaic control/HD organoids, showing a capacity to rescue some HD phenotypes.

The manuscript could be a valuable contribution to the field, however it has relevant drawbacks, the most significant being a lack of clarity regarding the replicates used for each genotype in the sequencing analyses. The lack of information on replicates raises the possibility that only a single replicate was analyzed for each organoid and brain sample. This approach may lead to concerns regarding the reproducibility of the findings, and it may be necessary for the authors to generate additional data to strengthen their conclusions. In addition, the analysis of the HD samples was conducted by pooling distinct cell populations from different brain regions (CTX, HIP, ChP for the dorsal brain, and STR, HYP, TH for the ventral brain). It is unclear why scRNA seq was used on pooled brain regions, which could obscure region-specific insights.

Another issue pertains to their proposed outcome: "Finally, we found that TTR protein, a choroid plexus marker, is elevated in the adult HD mouse serum, indicating that TTR may be a promising marker for detecting HD". This statement appears to lack statistical support, which makes this set of data potentially misleading and inconclusive.

The authors are encouraged to provide evidence of biological replicates, remove outcomes that lack statistical support, and address a series of points as detailed elsewhere.

Reviewer #1 (Public review):

Shin et al. conduct extensive electrophysiological and behavioral experiments to study the mechanisms of short-term synaptic plasticity at excitatory synapses in layer 2/3 of the rat medial prefrontal cortex. The authors interestingly find that short-term facilitation is driven by progressive overfilling of the readily releasable pool, and that this process is mediated by phospholipase C/diacylglycerol signaling and synaptotagmin-7 (Syt7). Specifically, knockdown of Syt7 not only abolishes the refilling rate of vesicles with high fusion probability, but it also impairs the acquisition of trace fear memory.

Overall, the authors offer novel insight to the field of synaptic plasticity through well-designed experiments that incorporate a range of techniques.

Reviewer #1 (Public review):

Summary:

In this study, the authors explore a novel mechanism linking aging to chromosome mis-segregation and aneuploidy in yeast cells. They reveal that, in old yeast mother cells, chromosome loss occurs through asymmetric partitioning of chromosomes to daughter cells, a process coupled with the inheritance of an old Spindle Pole Body. Remarkably, the authors identify that remodeling of the nuclear pore complex (NPC), specifically the displacement of its nuclear basket, triggers these asymmetric segregation events. This disruption also leads to the leakage of unspliced pre-mRNAs into the cytoplasm, highlighting a breakdown in RNA quality control. Through genetic manipulation, the study demonstrates that removing introns from key chromosome segregation genes is sufficient to prevent chromosome loss in aged cells. Moreover, promoting pre-mRNA leakage in young cells mimics the chromosome mis-segregation observed in old cells, providing further evidence for the critical role of nuclear envelope integrity and RNA processing in aging-related genome instability.

Strengths:

The findings presented are not only intriguing but also well-supported by robust experimental data, highlighting a previously unrecognized connection between nuclear envelope integrity, RNA processing, and genome stability in aging cells, deepening our understanding of the molecular basis of chromosome loss in aging.

Weaknesses:

Further analysis of yeast aging data from microfluidic experiments will provide important information about the dynamic features and prevalence of the key aging phenotypes, e.g. pre-mRNA leakage and chromosome loss, reported in this work. In addition, a discussion would be needed to clarify the relationship between "chromosome loss" in this study and "genomic missegregation" reported previously in yeast aging.

Reviewer #1 (Public review):

Summary:

The manuscript uses state-of-the-art analysis technology to document the spatio-temporal dynamics of brain activity during the processing of threats. The authors offer convincing evidence that complex spatio-temporal aspects of brain dynamics are essential to describe brain operations during threat processing.

Strengths:

Rigorous complex analyses well suited to the data.

Weaknesses:

Lack of a simple take-home message about discovery of a new brain operation.

Reviewer #1 (Public review):

Summary:

The topic of nanobody-based PET imaging is important and holds great potential for real-world applications since nanobodies have many advantages over full sized immunoglobulins and small molecules.

Strengths:

The submitted manuscript contains quite a bit of interesting data from a collaborative team of well-respected researchers. The authors are to be congratulated for presenting results that may not have turned out the way they had hoped, and doing so in a transparent fashion.

Weaknesses:

However, the manuscript could be considered to be a collection of exploratory findings rather than a complete and mature scientific exposition. Most of the sample sizes were 3 per group, which is fine for exploratory work, but insufficient to draw strong statistically robust conclusions for definitive results.

Reviewer #1 (Public review):

Summary:

The paper is well-organized, with clearly defined sections. The systematic review methodology is thorough, with clear eligibility criteria, search strategy, and data collection methods. The risk of bias assessment is also detailed and useful for evaluating the strength of evidence. The involvement of a patient panel is noticeable and positive, ensuring the research addresses real-world concerns and aligning scientific inquiry with patient perspectives. The statistical approach used for analyzing seems appropriate.

The authors are encouraged to take into account the following points:

As the authors have acknowledged, there is a high risk of bias across all included studies, particularly in randomization, selective outcome reporting, and incomplete data, which could be highlighted more explicitly in the paper's discussion section, particularly the potential implications for the generalizability of the results. The authors can also suggest mitigation strategies for future studies (e.g., better randomization, blinding, reporting standards, etc.). None of the studies include female animals, and the use of young adult animals (instead of aged models) limits the applicability of the findings to the human stroke population, where stroke incidence is higher in older adults and perhaps the gender issue must be included to reflect the translational aspects. The authors can add to the paper's discussion section that perhaps future preclinical studies should include both sexes and aged animals to align better with the clinical population and improve the translation of findings. Another point is the comorbidity. Comorbidities such as diabetes and hypertension are prevalent in stroke patients. How can these be considered in preclinical designs? The authors should emphasize the importance of future research incorporating such comorbid models to enhance clinical relevance.

None of the studies had independent replication of their findings, which is a key limitation, especially for a field with high translational expectations. This should be highlighted as a critical next step for validating the efficacy of CCR5 antagonists.

The studies accessed limited cognitive outcomes (only one reported a cognitive outcome). Given the importance of cognitive recovery post-stroke, this is a gap to highlight in the discussion. Future studies should include more diverse and comprehensive behavioral assessments, including cognitive and emotional domains, to fully evaluate the therapeutic potential.

The timing of CCR5 administration across studies varies widely (from pre-stroke to several days post-stroke) complicating the interpretation and comparison of results. The authors are encouraged to add that future preclinical studies could focus on narrowing the therapeutic window to more clinically relevant time points.<br /> The paper identifies some alignment with clinical trials, but there are several gaps, too, particularly in the types of behavioral tests used in preclinical studies versus those in clinical trials. If this systematic review and meta-analysis aim to formulate a set of recommendations for future studies, it is important that the authors also propose specific preclinical behavioral tasks that could better align with clinical measures used in trials, like functional assessments related to human stroke outcomes.

The discussion needs some revisions. It could benefit from an expanded explanation of CCR5's mechanistic role in neuroplasticity and stroke recovery. For instance, linking CCR5 antagonism more closely with molecular pathways related to synaptic repair and remyelination would enhance the quality of the discussion and understanding of the drugs' potential.

While the tool is used to assess the risk of bias, it might be helpful to integrate a broader framework for evaluating the quality of included studies. This could include sample size justifications, statistical power analysis, or the use of pre-registration in animal studies. These elements can also introduce bias or minimize those if in place.

Please also highlight confounding factors that might have influenced the results in the included studies, such as variation in stroke models, dosing regimens, or behavioral assessment methods.

There is some discussion of the meta-analysis' limitations due to the few studies, but this point could be more thoroughly addressed. Please consider including a more critical discussion of the limitations of pooling data from heterogeneous study designs, stroke models, and outcome measures. What can this lead to? Is it reliable to do so, or does it lack scientific rigor? The authors are encouraged to formulate a balanced discussion adding, positive and negative aspects.<br /> The conclusion should more explicitly acknowledge that while CCR5 antagonists show potential, the findings are still preliminary due to the limitations in the preclinical studies (high bias risk, lack of diverse animal models). Overall, the conclusion can end with a call for rigorous, well-controlled, and replicated studies with improved alignment to clinical populations and trials to show that the conclusion remains inconclusive, considering what has been analyzed here.

Reviewer #1 (Public review):

Summary:

This work made a lot of efforts to explore the multifaceted roles of the inferior colliculus (IC) in auditory processing, extending beyond traditional sensory encoding. The authors recorded neuronal activity from the IC at single unit level when monkeys were passively exposed or actively engaged in behavioral task. They concluded that 1）IC neurons showed sustained firing patterns related to sound duration, indicating their roles in temporal perception, 2) IC neuronal firing rates increased as sound sequences progress, reflecting modulation by behavioral context rather than reward anticipation, 3) IC neurons encode reward prediction error and their capability of adjusting responses based on reward predictability, 4) IC neural activity correlates with decision-making. In summary, this study tried to provide a new perspective on IC functions by exploring its roles in sensory prediction and reward processing, what are not traditionally associated with this structure.

Strengths:

The major strength of this work is that the authors performed electrophysiological recordings from the IC of behaving monkeys. Compared with the auditory cortex and thalamus, the IC in monkeys has not been adequately explored.

Comments on revised version:

The authors have adequately addressed all my concerns.

Reviewer #1 (Public review):

In this manuscript, the authors recorded cerebellar unipolar brush cells (UBCs) in acute brain slices. They confirmed that mossy fiber (MF) inputs generate a continuum of UBC responses. Using systematic and physiological trains of MF electrical stimulation, they demonstrated that MF inputs either increased or decreased UBC firing rates (UBC ON vs. OFF) or induced complex, long-lasting modulation of their discharges. The MF influence on UBC firing was directly associated with a specific combination of metabotropic glutamate receptors, mGluR2/3 (inhibitory) and mGluR1 (excitatory). Ultimately, the amount and ratio of these two receptors controlled the time course of the effect, yielding specific temporal transformations such as phase shifts. The experiments are well-executed and properly analyzed.

Strengths:

(1) A wide range of MF stimulation based on activity patterns observed in vivo was explored, including burst duration and frequency dependency, which could serve as a valuable foundation for explicit modeling of temporal transformations in the granule cell layer.<br /> (2) The pharmacological blockade of mGluR2/3, mGluR1, AMPA, and NMDA receptors helped identify the specific roles of these glutamate receptors.<br /> (3) The experiments convincingly demonstrate the key role of mGluR1 receptors in temporal information processing by UBCs.

Weaknesses:

(1) This study is a follow up of previous work (Guo et al., Nat. Commun., 2021).<br /> (2) The MF activity used to mimic natural stimulation was previously collected from primates, whereas the recordings were conducted in mice.

Comments on revisions:

The authors included a discussion about inhibition, but I still disagree with their claim that it was not possible to study the MF-UBC connection with inhibition unblocked. This group has already conducted experiments on Golgi cell inhibition in slices.

Reviewer #1 (Public review):

Summary:

In the submitted manuscript, Solomon et al carefully detail shifts in tissue-specific myeloid populations associated with trained immunity using intraperitoneal BCG injection as a model for induction. They define the kinetics of shifts in myeloid populations within the spleen and the transcriptional response associated with IP BCG exposure. In lineage tracing experiments, they demonstrate that tissue-resident macrophages, red-pulp macrophages (RPM) that are rapidly depleted after BCG exposure, are replenished from recruited monocytes and expansion of tissue-resident cells; they use transcriptional profiling to characterize those cells. In contrast to previous descriptions of BCG-driven immune training, they do not find BCG in the bone marrow in their model, suggesting that there is not direct training of myeloid precursor populations in the bone marrow. They then link the observed trained immunity phenotype (restriction of heterologous infection with ST) with early activation of STAT1 through IFN-γ.

Strengths:

The work includes careful detaining of shifts and origins of myeloid populations within tissue associated with trained immunity and is a meaningful advance for the field.

Caveats:<br /> Given that the authors demonstrate that BCG persists in the spleen, it is possible that some level of BCG persistence in the spleen is a necessary contributor (together with signaling through STAT1) to the observed tissue-specific T1 phenotype.

Whether ongoing signaling through the axes are required for ongoing protection is not specifically addressed in this work. There is recent work by other groups that partially addresses these caveats, and it would be helpful context to reference those papers.

Reviewer #1 (Public review):

This manuscript by Kleinman & Foster investigates the dependence of hippocampal replay on VTA activity. They recorded neural activity from the dorsal CA1 region of the hippocampus while chemogenetically silencing VTA dopamine neurons as rats completed laps on a linear track with reward delivery at each end. Reward amount changed across task epochs within a session on one end of the track. The authors report that VTA activity is necessary for an increase in sharp-wave rate to remain localized to the feeder that undergoes a change in reward magnitude, an effect that was especially pronounced in a novel environment. They follow up on this result with a second experiment in which reward magnitude varies unpredictably at one end of the linear track and report that changes in sharp-wave rate at the variable location reflect both the amount of reward rats just received there, in addition to a smaller modulation that is reminiscent of reward prediction error coding, in which the previous reward rats received at the variable location affects the magnitude of the subsequent change in sharp-wave rate that occurs on the present visit.

This work is technically innovative, combining neural recordings with chemogenetic inactivation. The question of how VTA activity affects replay in the hippocampus is interesting and important given that much of the work implicating hippocampal replay in memory consolidation and planning comes from reward-motivated behavioral tasks.

Comments on revisions:

Overall, I think the authors have done everything they could to address reviewer concerns, short of collecting more data. The more consistent statistical approach makes the paper easier to read and follow. It's helpful to have more details/rationale for the variability in CNO dose and timing. I think some of the results are still not fully convincing, especially the reward volatility experiment (which the authors also note requires additional validation). Given the small number of rats, the small effect sizes, and the complexity of the experimental manipulations, I still have concerns about whether these effects would hold with larger groups sizes.

Reviewer #1 (Public review):

The paper proposes an interesting perspective on the spatio-temporal relationship between FC in fMRI and electrophysiology. The study found that while similar networks configurations are found in both modalities, there is a tendency for the networks to spatially converge more commonly at synchronous than asynchronous timepoints. However, my confidence in the findings and their interpretation is undermined by an incomplete justification for the expected outcomes for each of the proposed scenarios.

Main Concern

Fig 1 makes sense to me conceptually, including the schematics of the trajectories, i.e.:

- Scenario1. Temporally convergent, same trajectories through connectome state space<br /> - Scenario2. Temporally divergent, different trajectories through connectome state space

However, based on my understanding (and apologies if I am mistaken), I am concerned that these scenarios do not necessarily translate into the schematic CRP plots shown in fig 2C, or the statements in the main text, i.e.:

- For scenario1, "epochs of cross-modal spatial similarity should occur more frequently at on-diagonal (synchronous) than off-diagonal (asynchronous) entries, resulting in an on-/off-diagonal ratio larger than unity"<br /> - For scenario2, "epochs of spatial similarity could occur equally likely at on-diagonal and off-diagonal entries (ratio≈1)"

Where do the authors get these statements and the schematics in fig2C from? They do not seem to be fully justified via previous literature, theory, or simulations?

In particular, I am not convinced based on the evidence currently in the paper, that the ratio of off- to on-diagonal entries (and under what assumptions) is a definitive way to discriminate between scenarios 1 and 2.

For example, what about the case where the same network configuration reoccurs in both modalities at multiple time points. It seems to me that you would get a CRP with entries occurring equally on the on-diagonal as on the off-diagonal, regardless of whether the dynamics are matched between the two modalities or not (i.e. regardless of scenario 1 or 2 being true).

This thought experiment example might have a flaw in it, and the authors might ultimately be correct, but nonetheless a systematic justification needs to be provided for using the ratio of off- to on-diagonal entries to discriminate between scenario 1 and 2 (and under what assumptions it is valid).

In the absence of theory, the authors could use surrogate data for scenario 1 and 2. For example:

a. For scenario 1, run the CRP using a single modality. E.g. feed in the EEG into the analysis as both modality 1 AND modality 2. This should provide at least one example of CRP under scenario 1 (although it does not ensure that all CRPs under this scenario will look like this, it is at least a useful sanity check).<br /> b. For scenario 2, run the CRP using a single modality plus a shuffled version. E.g. feed in the EEG into the analysis as both modality 1 AND a temporally shuffled version of the EEG as modality 2. The temporal shuffling of the EEG could be done by simple splitting the data into blocks of say ~10s and then shuffling them into a new order. This should provide a version of the CRP under scenario 2 (although it does not ensure that all CRPs under this scenario will look like this, it is at least a useful sanity check)

The authors have provided CRP plots for option a. It shows a CRP, as expected, consistent with scenario 1. This is a useful sanity check. However, as mentioned above, it does not ensure that all CRPs under this scenario will look like this.

However, the authors have not shown a CRP as per option b. As such, there is an incomplete justification for the expected outcomes of the scenarios.

Note that another option, which has not been carried out, is to use full simulations, with clearly specified assumptions, for scenario1 and 2. One way of doing this is to use a simplified (state-space) setup where you randomly simulate N spatially fixed networks that are independently switching on and off over time (i.e. "activation" is 0 or 1). Note that this would result in a N-dimensional connectome state space.

Using this, you can simulate and compute the CRPs for the two scenarios:

a. Scenario 1: where the simulated activation timecourses are set to be the same between both modalities<br /> b. Scenario 2: where the simulated activation timecourses are simulated separately for each of the modalities

Minor Concern

Leakage correction. The paper states: "To mitigate this issue, we provide results from source-localized data both with and without leakage correction (supplementary and main text, respectively)." It is great that the authors provide both. However, given that FC in EEG is almost totally dominated by spatial leakage (see Hipp paper), the main results/figures for the scalp EEG should be done using spatial leakage corrected EEG data.

Reviewer #1 (Public review):

Summary:

The anatomical connectivity of the claustrum and the role of its output projections has, thus far, not been studied in detail. The aim of this study was to map the outputs of the endopiriform (EN) region of the claustrum complex, and understand their functional role. Here the authors have combined sophisticated intersectional viral tracing techniques, and ex vivo electrophysiology to map the neural circuitry of EN outputs to vCA1, and shown that optogenetic inhibition of the EN→vCA1 projection impairs both social and object recognition memory. Interestingly the authors find that the EN neurons target inhibitory interneurons providing a mechanism for feedforward inhibition of vCA1.

Strengths:

The strength of this study was the application of a multilevel analysis approach combining a number of state-of-the-art techniques to dissect the contribution of the EN→vCA1 to memory function.

In addition the authors conducted behavioural analysis of locomotor activity, anxiety and fear memory, and complemented the analysis of discrimination with more detailed description of the patterns of exploratory behaviour.

Reviewer #1 (Public review):

Summary:

Liu and colleagues applied the hidden Markov model on fMRI to show three brain states underlying speech comprehension. Many interesting findings were presented: brain state dynamics were related to various speech and semantic properties, timely expression of brain states (rather than their occurrence probabilities) was correlated with better comprehension, and the estimated brain states were specific to speech comprehension but not at rest or when listening to non-comprehensible speech.

Strengths:

Recently, the HMM has been applied to many fMRI studies, including movie watching and rest. The authors cleverly used the HMM to test the external/linguistic/internal processing theory that was suggested in comprehension literature. I appreciated the way the authors theoretically grounded their hypotheses and reviewed relevant papers that used the HMM on other naturalistic datasets. The manuscript was well written, the analyses were sound, and the results had clear implications.

Reviewer #1 (Public review):

The authors in this paper investigate the nature of the activity in the rodent EPN during a simple freely moving cue-reward association task. Given that primate literature suggest movement coding whereas other primate and rodent studies suggest mainly reward outcome coding in the EPNs, it is important try to tease apart the two views. Through careful analysis of behavior kinematics, position, and the neural activity in the EPNs, the authors reveal an interesting and complex relationship between the EPN and mouse behavior.

Strengths:

(1) The authors use a novel freely moving task to study EPN activity, which displays rich movement trajectories and kinematics. Given that previous studies have mostly looked at reward coding during head fixed behavior, this study adds a valuable dataset to the literature.

(2) The neural analysis is rich and thorough. Both single neuron level and population level (i.e. PCA) analysis are employed to reveal what EPN encodes.

Discussion:<br /> EPN is one of the major output nuclei of the basal ganglia. What information is present within EPN is still unclear, and under investigation. The authors have used electrophysiology to determine the nature of information present within EPN that is likely to be valuable to the field. Future studies should try to address whether this information is specific to certain cell types within EPN or whether there is topography within EPN that reflects the kinematic information present within EPN. This will require more careful dissection of EPN activity based on anatomy. Future experiments should also consider tasks that isolate a single limb (i.e. joystick tasks) in order to better understand the kinematic encoding of forelimb movement. This, combined with recording in forelimb encoding region of EPN, should give us insights into the nature of kinematic control of EPN. Overall, this study will be useful to inspire future investigations in the function of EPN.

Reviewer #1 (Public review):

Kreeger and colleagues have explored the balance of excitation and inhibition in the cochlear nucleus octopus cells of mice using morphological, electrophysiological and computational methods. On the surface, the conclusion, that synaptic inhibition is present, does not seem like a leap. However, the octopus cells have been in the past portrayed as lacking synaptic inhibition. This view was supported by the paucity of glycinergic fibers in the octopus cell area and the lack of apparent IPSPs. Here, Kreeger et al., used beautiful immunohistochemical and mouse genetic methods to quantify the inhibitory and excitatory boutons over the complete surface of individual octopus cells and further analyzed the proportions of the different subtypes of spiral ganglion cell inputs. I think the analysis of synaptic distribution and the origin of the excitatory inputs stands as one of the most complete descriptions of any neuron, leaving little doubt about the presence of glycinergic boutons.

Kreeger et al then examined inhibition physiologically. Recordings from these neurons are notoriously difficult to make because of the enormous leak currents that shunt membrane stimuli and currents, and complicate voltage clamp. The authors have tried to overcome these limitations using drugs to block leak conductances, and computational approaches based on realistic parameters. They conclude that dendritic inhibition can modify the size and kinetics of excitatory signals, and may play out in computations made on temporally dispersed stimuli as might be experienced during a ramp in sound frequency or complex natural sounds like vocalizations.

Reviewer #1 (Public review):

Summary:

The authors use an innovative behavior assay (chamber preference test) and standard calcium imaging experiments on cultured dorsal root ganglion (DRG) neurons to evaluate the consequences of global knockout of TRPV1 and TRPM2, and overexpression of TRPV1, on warmth detection. They find a profound effect of TRPM2 elimination in the behavioral assay, whereas the elimination of TRPV1 has the largest effect on the neuronal responses. These findings are very important, as there is substantial ongoing discussion in the field regarding the contribution of TRP channels to different aspects of thermosensation.

Strengths:

The chamber preference test is an important innovation compared to the standard two-plate test, as it depends on thermal information sampled from the entire skin, as opposed to only the plantar side of the paws. With this assay, and the detailed analysis, the authors provide strong supporting evidence for a role of TRPM2 in warmth avoidance. The conceptual framework using the Drift Diffusion Model provides a first glimpse of how this decision of a mouse to change between temperatures can be interpreted and may form the basis for further analysis of thermosensory behavior.

Weaknesses:

The authors juxtapose these behavioral data with calcium imaging data using isolated DRG neurons. As the authors acknowledge, it remains unclear whether the clear behavioral effect seen in the TRPM2 knockout animals is directly related to TRPM2 functioning as a warmth sensor in sensory neurons. The effects of the TRPM2 KO on the proportion of warmth sensing neurons are very subtle, and TRPM2 may also play a role in the behavioral assay through its expression in thermoregulatory processes in the brain. Future behavioral experiments on sensory-neuron specific TRPM2 knockout animals will be required to clarify this important point.

Reviewer #2 (Public review):

Summary:

This manuscript reports analyses of fMRI data from infants and toddlers watching naturalistic movies. Visual areas in the infant brain show distinct functions, consistent with previous studies using resting state and awake task-based infant fMRI. The pattern of activity in visual regions contains some features predicted by the regions' retinotopic responses. The revised version of the manuscript provides additional validation of the methodology, and clarifies the claims. As a result, the data provide clear support for the claims.

Strengths:

The authors have collected a unique dataset: the same individual infants both watched naturalistic animations and a specific retinotopy task. Using these data position the authors show that activity evoked by movies, in infants' visual areas, is correlated with the regions' retinopic response. The revised manuscript validates this methodology, using adult data. The revised manuscript also shows that an infant's movie watching data is not sufficient or optimal to predict their visual areas' retinotopic responses; anatomical alignment with a group of previous participants provides more accurate prediction of a new participant's retinotopic response.

Weaknesses:

A key step in the analysis of the movie-watching data is the selection of independent components of the movie evoked response that resemble retinotopic spatial patterns. While the trained researcher was unlikely to be biased by this infant's own retinotopy, he/she was actively looking for ICs that resemble average patterns of retinotopic response. To show that these ICs didn't arise by chance (i.e. in noise), the authors proposed an additional analysis in the revised manuscript, by misaligning the functional and anatomical data for a subset of participants. This only partially confirms the reliability of the original components, since when the (new) coder tried to be conservative to avoid false components, he/she identified just over half of the 'true' components (13 vs 22 estimated over the group of 6 infants).

Reviewer #2 (Public review):

Summary:

In this paper, the authors investigated the admixture history of domestic cattle since they were introduced into Iberia, by studying genomic data from 24 ancient samples dated to ~2000-8000 years ago and comparing them to modern breeds. They aimed to (1) characterize genomic variation of skeletal remains and concordance (or discordance) with morphological features; (2) test for hybridization between wild aurochs and domestic cattle; (3) test for correlation between genetic ancestry and stable isotope levels (which are indicative of ecological niche); and (4) test for previously hypothesized higher aurochs ancestry in a modern breed of fighting bulls.

Strengths:

Overall, this study collects valuable new data and tests several important hypotheses regarding the evolutionary history and genomic variation of domestic cattle in Iberia, such as admixture between domestic and wild populations, and correlation between genome-wide aurochs ancestry and aggressiveness.

Weaknesses:

Most conclusions are well supported by the data presented, with the strengths and caveats of each analysis clearly explained. The presence of admixed individuals in prehistorical periods strongly support hybridization between wild and domestic populations, although the evidence for sex-biased introgression and ecological niche sharing is relatively weak. Lastly, the authors presented convincing evidence for relatively constant aurochs ancestry across all modern breeds, including the Lidia breed that has been bred for aggressiveness for centuries.

Major comments:

As the authors pointed out, a major limitation of this study is uncertainty in the "population identity" for most sampled individuals (i.e., whether an individual belonged to the domesticated or wild herd when they were alive). Based on chronology, morphology and genetic data, it is clear the Mesolithic samples from the Artusia and Mendandia sites are bona fide aurochs, but the "population identities" of individuals from the other two sites are much less certain. Indeed, archeological and morphological evidence from El Portalon supports the presence of both domestic animals and wild aurochs, which is echoed by the inter-individual heterogeneity in genetic ancestry. Despite the strong evidence of hybridization, it is unclear whether these admixed individuals were raised in the domestic population or lived in the wild population and hunted, limiting the authors' ability to draw conclusions regarding the direction of gene flow.

In general, detecting sex-bias admixture is an inherently challenging problem, especially given limited data. The differential ancestry proportions (estimated by f4 ratios) on autosomes and X chromosome are indicative of sex-biased hybridization and consistent with previous mtDNA results and other non-genetic data. However, as shown in Fig 3, the confidence intervals of X and autosomal estimates overlap for all but a couple of individuals, despite the overall trend of the point estimates. Moreover, even if there is significant difference, it only suggests existence of sex-bias but does not speak to the extent (unless further quantitative argument is made). Statements such as "it was mostly aurochs males who contributed wild ancestry to domestic herds" is too strong and may be interpreted as extreme bias. The authors did a good job noting the caveats of this analysis and down-toned the statement in the main text, but claims regarding sex-bias hybridization that use the phrase "mostly" in the abstract and discussion need to be further weakened.

The stable isotope analysis is very under-powered, due to issues of categorization of wild vs domestic Bos, as discussed by the authors. Although the considerable overlap in stable isotope values between domestic and wild groups is consistent with shared ecological niche, but the absence of evidence (ie significant difference between groups) is not evidence of absence. Two alternative, non-mutually exclusive scenarios are (1) prevalent errors in classification of wild vs domestic individuals; (2) different ecological niches share similar isotope profiles. Thus, the claim "suggesting that wild and domesticated groups often did not occupy different niches in Iberia" is still too strong.

Reviewer #1 (Public review):

Summary:<br /> The authors set out to explore the role of upstream open reading frames (uORFs) in stabilizing protein levels during Drosophila development and evolution. By utilizing a modified ICIER model for ribosome translation simulations and conducting experimental validations in Drosophila species, the study investigates how uORFs buffer translational variability of downstream coding sequences. The findings reveal that uORFs significantly reduce translational variability, which contributes to gene expression stability across different biological contexts and evolutionary timeframes.

Strengths:<br /> (1) The study introduces a sophisticated adaptation of the ICIER model, enabling detailed simulation of ribosomal traffic and its implications for translation efficiency.<br /> (2) The integration of computational predictions with empirical data through knockout experiments and translatome analysis in Drosophila provides a compelling validation of the model's predictions.<br /> (3) By demonstrating the evolutionary conservation of uORFs' buffering effects, the study provides insights that are likely applicable to a wide range of eukaryotes.

Weaknesses:<br /> (1) Although the study is technically sound, it does not clearly articulate the mechanisms through which uORFs buffer translational variability. A clearer hypothesis detailing the potential molecular interactions or regulatory pathways by which uORFs influence translational stability would enhance the comprehension and impact of the findings.<br /> (2) The study could be further improved by a discussion regarding the evolutionary selection of uORFs. Specifically, it would be beneficial to explore whether uORFs are favored evolutionarily primarily for their role in reducing translation efficiency or for their capability to stabilize translation variability. Such a discussion would provide deeper insights into the evolutionary dynamics and functional significance of uORFs in genetic regulation.

Reviewer #1 (Public review):

Summary:

Cook et al. have presented an important study on the transcriptomic and epigenomic signature underlying craniofacial development in marsupials. Given the lack of a dunnart genome, the authors also prepared long and short-read sequence datasets to assemble and annotate a novel genome to allow for the mapping of RNAseq and ChIPseq data against H3K4me3 and H3K27ac, which allowed for the identification of putative promoter and enhancer sites in dunnart. They found that genes proximal to these regulatory loci were enriched for functions related to bone, skin, muscle and embryonic development, highlighting the precocious state of newborn dunnart facial tissue. When compared with mouse, the authors found a much higher proportion of promoter regions aligned between species than for enhancer regions, and subsequent profiling identified regulatory elements conserved across species and are important for mammalian craniofacial development. In contrast, the identification of dunnart-specific enhancers and patterns of RNA expression further confirm the precocious state of muscle development, as well as for sensory system development, in dunnart suggesting that early formation of these features are critical for neonate marsupials likely to assist with detecting and responding to cues that direct the joeys to the mother's teat after birth. This is one of the few epigenomic studies performed in marsupials (of any organ) and the first performed in fat-tailed dunnart (also of any organ). Marsupials are emerging as an important model for studying mammalian development and evolution and the authors have performed a novel and thorough analysis, impressively including the assembly of a new marsupial reference genome that will benefit many future studies.

Strengths:

The study provides multiple pieces of evidence supporting the important role enhancer elements play in mammalian phenotypic evolution, namely the finding of a lower proportion of peaks present in both dunnart and mouse for enhancers than for promoters, and dunnart showing more genes uniquely associated with it's active enhancers than any other combination of mouse and dunnart samples, whereas this pattern was less pronounced than for promoter-associated genes. In addition, rigorous parameters were used for the cross-species analyses to identify the conserved regulatory elements and the dunnart-specific enhancers. For example, for the results presented in Figure 1, I agree that it is a little surprising that the average promoter-TSS distance is greater than that for enhancers, but that this could be related to the possible presence of unannotated transcripts between genes. The authors addressed this well by examining the distribution of promoter-TSS distances and using proximal promoters (cluster #1) as high confidence promoters for downstream analyses.

The genome assembly method was thorough, using two different long read methods (Pacbio and ONT) to generate the long reads for contig and scaffold construction, increasing the quality of the final assembled genome.

Weaknesses:

Biological replicates of facial tissue were collected at a single developmental time point of the fat-tailed dunnart within the first postnatal day (P0), and analysed this in the context of similar mouse facial samples from the ENCODE consortium at six developmental time points, where previous work from the authors have shown that the younger mouse samples (E11.5-12.5) approximately corresponds to the dunnart developmental stage (Cook et al. 2021). However, it would be useful to have samples from at least one older dunnart time point, for example, at a developmental stage equivalent to mouse E15.5. This would provide additional insight into the extent of accelerated face development in dunnart relative to mouse, i.e. how long do the regulatory elements that activated early in dunnart remain active for and does their function later influence other aspects of craniofacial development?

The authors refer to the development of the CNS being delayed in marsupials relative to placental mammals, however, evidence shows how development of the dunnart brain (whole brain or cortex) is protracted compared to mouse, by a factor of at least 2 times, rather than delayed per se (Workman et al. 2013; Paolino et al. 2023). In addition, there is evidence that cortical formation and cell birth may begin at approximately the same stage across species equivalent to the neonate period in dunnart (E10.5 in mouse), and that shortly after this at the stage equivalent to mouse E12.5, the dunnart cortex shows signs of advanced neurogenesis followed by a protracted phase of neuronal maturation (Paolino et al. 2023). Therefore, it is possible that marsupial CNS development appears delayed relative to mouse but instead begins at the same stage and then proceeds to develop on a different timing scale.

Reviewer #1 (Public review):

Summary:

This study by Howe and colleagues investigates the role of the posterolateral cortical amygdala (plCoA) in mediating innate responses to odors, specifically attraction and aversion. By combining optogenetic stimulation, single-cell RNA sequencing, and spatial analysis, the authors identify a topographically organized circuit within plCoA that governs these behaviors. They show that specific glutamatergic neurons in the anterior and posterior regions of plCoA are responsible for driving attraction and avoidance, respectively, and that these neurons project to distinct downstream regions, including the medial amygdala and nucleus accumbens, to control these responses.

Strengths:

The major strength of the study is the thoroughness of the experimental approach, which combines advanced techniques in neural manipulation and mapping with high-resolution molecular profiling. The identification of a topographically organized circuit in plCoA and the connection between molecularly defined populations and distinct behaviors is a notable contribution to understanding the neural basis of innate motivational responses. Additionally, the use of functional manipulations adds depth to the findings, offering valuable insights into the functionality of specific neuronal populations.

Weaknesses:

There are some weaknesses in the study's methods and interpretation. The lack of clarity regarding the behavior of the mice during head-fixed imaging experiments raises the possibility that restricted behavior could explain the absence of valence encoding at the population level. Furthermore, while the authors employ chemogenetic inhibition of specific pathways, the rationale for this choice over optogenetic inhibition is not fully addressed, and this could potentially affect the interpretation of the results. Additionally, the choice of the mplCoA for manipulation, rather than the more directly implicated anterior and posterior subregions, is not well-explained, which could undermine the conclusions drawn about the topographic organization of plCoA.

Despite these concerns, the work provides significant insights into the neural circuits underlying innate behaviors and opens new avenues for further research. The findings are particularly relevant for understanding the neural basis of motivational behaviors in response to sensory stimuli, and the methods used could be valuable for researchers studying similar circuits in other brain regions. If the authors address the methodological issues raised, this work could have a substantial impact on the field, contributing to both basic neuroscience and translational research on the neural control of behavior.

Reviewer #1 (Public review):

Summary:

MHC (Major Histocompatibility Complex) genes have long been mentioned as cases of trans-species polymorphism (TSP), where alleles might have their most recent common ancestor with alleles in a different species, rather than other alleles in the same species (e.g., a human MHC allele might coalesce with a chimp MHC allele, more recently than the two coalesce with other alleles in either species). This paper provides a more complete estimate of the extent and ages of TSP in primate MHC loci. The data clearly support deep TSP linking alleles in humans to (in some cases) old world monkeys, but the amount of TSP varies between loci.

Strengths:

The authors use publicly available datasets to build phylogenetic trees of MHC alleles and loci. From these trees they are able to estimate whether there is compelling support for Trans-species polymorphisms (TSPs) using Bayes Factor tests comparing different alternative hypotheses for tree shape. The phylogenetic methods are state-of-the-art and appropriate to the task.

The authors supplement their analyses of TSP with estimates of selection (e.g., dN/dS ratios) on motifs within the MHC protein. They confirm what one would suspect: classical MHC genes exhibit stronger selection at amino acid residues that are part of the peptide binding region, and non-classical MHC exhibit less evidence of selection. The selected sites are associated with various diseases in GWAS studies.

Weaknesses:

An implication drawn from this paper (and previous literature) is that MHC has atypically high rates of TSP. However, rates of TSP are not estimated for other genes or gene families, so readers have no basis of comparison. No framework to know whether the depth and frequency of TSP is unusual for MHC family genes, relative to other random genes in the genome, or immune genes in particular. I expect (from previous work on the topic), that MHC is indeed exceptional in this regard, but some direct comparison would provide greater confidence in this conclusion.

Given the companion paper's evidence of genic gain/loss, it seems like there is a real risk that the present study under-estimates TSP, if cases of TSP have been obscured by the loss of the TSP-carrying gene paralog from some lineages needed to detect the TSP. Are the present analyses simply calculating rates of TSP of observed alleles, or are you able to infer TSP rates conditional on rates of gene gain/loss?

Figure 5 (and 6) provide regression model fits (red lines in panel C) relating evolutionary rates (y axis not labeled) to site distance from the peptide binding groove, on the protein product. This is a nice result. I wonder, however, whether a linear model (as opposed to non-linear) is the most biologically reasonable choice, and whether non-linear functions have been evaluated. The authors might consider generalized additive models (GAMs) as an alternative that relaxes linearity assumptions.

The connection between rapidly evolving sites, and disease associations (lines 382-3) is very interesting. However, this is not being presented as a statistical test of association. The authors note that fast-evolving amino acids all have at least one association: but is this really more disease-association than a random amino acid in the MHC? Or, a randomly chosen polymorphic amino acid in MHC? A statistical test confirming an excess of disease associations would strengthen this claim.

Reviewer #1 (Public review):

Summary:

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:

This manuscript presents a solid step to establish Split-GAL4 lines as a relevant tool in the powerful Drosophila toolkit. Not only 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:

(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 /> (5) An acknowledgement of the caveats and challenges that splits (like any other genetic tool) can carry.<br /> Overall, the authors employed a pipeline that maximizes the potential of the Split-GAL4 collection to the scientific community.

Weaknesses:

My concerns were resolved regarding the existence of caveats while using these tools that researchers should be aware of, particularly those using them for the first time.

Reviewer #1 (Public review):

Summary:

This study provides convincing data showing that expression of the PIK3R1(deltaExon11) dominant negative mutation in Activated PI3K Delta Syndrome 1/2 (APDS1/2) patient-derived cells reduces AKT activation and p110δ protein levels. Using a 3T3-L1 model cell system, the authors show that overexpressed p85α(deltaExon 11) displays reduced association with the p110α catalytic subunit but strongly interacts with Irs1/2. Overexpression of PIK3R1 dominant negative mutants inhibit AKT phosphorylation and reduce cellular differentiation of preadipocytes. The experimental design, interpretation, and quantification broadly support the authors' conclusions, which establishes a new paradigm that warrants future studies.

Strengths:

The strength of this study is the clear results derived from Western blots analysis of cell signaling markers (e.g. pAKT1), and co-immunoprecipitation of PI3K holoenzyme complexes and associated regulatory factors (e.g. Irs1/2). The authors analyze a variety of PIK3R1 mutants (i.e. deltaExon11, E489K, R649W, and Y657X), which reveals a range of phenotypes that support the proposed model for dominant negative activity. The use of clonal cell lines with doxycycline induced expression of the PIK3R1 mutants (deltaExon 11, R649W, and Y657X) provides convincing experimental data concerning the relationship between p85α mutant expression and AKT phosphorylation in vivo. This approach for overexpression is excellent and should be utilized more broadly by cell biologists. The authors convincingly show that p85α(deltaExon11, R649W, or Y657X) is unable to associate with p110α but instead more strongly associates with Irs1/2 compared to wild type p85α. Overall, this article does a great job of motivating future studies of SHORT and APDS2 PIK3R1 mutants expressed from their endogenous loci (e.g. knock-in mice).

Weaknesses:

The limitations for this study lie in the complexity of the cell signaling pathway under investigation, rather than a lack of rigor by the authors. Future experimentation will help reconcile the cell type specific differences (e.g. APDS2 patient derived cells vs. the 3T3-L1 cell model system) in PIK3R1 mutant behavior reported by the authors. This is also intimately linked to variable expression of PIK3R1 mutants and cell-type specific regulatory factors. Although beyond the scope of this work, an unbiased proteomic study that broadly evaluates the cell signaling landscape could provide a more holistic understanding of the APDS2 and SHORT mutants compared to a candidate-based approach. Additional structural biochemistry of the p110α/p85α(deltaExon 11) complex is needed to explain why PIK3R1 mutant regulatory subunits do not strongly associate with the p110 catalytic subunit. A more comprehensive biochemical analysis of p110α/p85α, p110β/p85α, and p110δ/p85α mutant protein complexes will also be necessary to explain various cell signaling phenotypes. A minor limitation of this study is the use of single end point assays to measure PI3K lipid kinase activity in the presence of one regulatory input (i.e. RTK-derived pY peptide). An expanded biochemical analysis of purified mutant PI3K complexes across the canonical membrane signaling landscape will be important for deciphering how competition between wild-type and mutant regulatory subunits are regulated in different cell signaling contexts.

Reviewer #1 (Public review):

This study presents an investigation into the physiological functions of RIPK1 within the context of liver physiology, particularly during short-term fasting. Through the use of hepatocyte-specific Ripk1-deficient mice (Ripk1Δhep), the authors embarked on an examination of the consequences of Ripk1 deficiency in hepatocytes under fasting conditions. They discovered that the absence of RIPK1 sensitized the liver to acute injury and hepatocyte apoptosis during fasting, a finding of significant interest given the crucial role of the liver in metabolic adaptation. Employing a combination of transcriptomic profiling and single-cell RNA sequencing techniques, the authors uncovered intricate molecular mechanisms underlying the exacerbated proinflammatory response observed in Ripk1Δhep mice during fasting. While the investigation offers valuable insights into the consequences of Ripk1 deficiency in hepatocytes during fasting conditions, there appears to be a primarily descriptive nature to the study with a lack of clear connection between the experiments. Thus, a stronger focus is warranted, particularly on understanding the dialogue between hepatocytes and macrophages. Moreover, the data would benefit from reinforcement through additional experiments such as Western blotting, flow cytometry, and rescue experiments, which would offer a more quantitative aspect to the findings. By incorporating these enhancements, the study could achieve a more comprehensive understanding of the underlying mechanisms and ultimately strengthen the overall impact of the research.

Comments on revision:

The authors have addressed my comments accordingly.

Reviewer #1 (Public Review):

Summary:

The emergence of Drosophila EM connectomes has revealed numerous neurons within the associative learning circuit. However, these neurons are inaccessible for functional assessment or genetic manipulation in the absence of cell-type-specific drivers. Addressing this knowledge gap, Shuai et al. have screened over 4000 split-GAL4 drivers and correlated them with identified neuron types from the "Hemibrain" EM connectome by matching light microscopy images to neuronal shapes defined by EM. They successfully generated over 800 split-GAL4 drivers and 22 split-LexA drivers covering a substantial number of neuron types across layers of the mushroom body associative learning circuit. They provide new labeling tools for olfactory and non-olfactory sensory inputs to the mushroom body; interneurons connected with dopaminergic neurons and/or mushroom body output neurons; potential reinforcement sensory neurons; and expanded coverage of intrinsic mushroom body neurons. Furthermore, the authors have optimized the GR64f-GAL4 driver into a sugar sensory neuron-specific split-GAL4 driver and functionally validated it as providing a robust optogenetic substitute for sugar reward. Additionally, a driver for putative nociceptive ascending neurons, potentially serving as optogenetic negative reinforcement, is characterized by optogenetic avoidance behavior. The authors also use their very large dataset of neuronal anatomies, covering many example neurons from many brains, to identify neuron instances with atypical morphology. They find many examples of mushroom body neurons with altered neuronal numbers or mistargeting of dendrites or axons and estimate that 1-3% of neurons in each brain may have anatomic peculiarities or malformations. Significantly, the study systematically assesses the individualized existence of MBON08 for the first time. This neuron is a variant shape that sometimes occurs instead of one of two copies of MBON09, and this variation is more common than that in other neuronal classes: 75% of hemispheres have two MBON09's, and 25% have one MBON09 and one MBON08. These newly developed drivers not only expand the repertoire for genetic manipulation of mushroom body-related neurons but also empower researchers to investigate the functions of circuit motifs identified from the connectomes. The authors generously make these flies available to the public. In the foreseeable future, the tools generated in this study will allow important advances in the understanding of learning and memory in Drosophila.

Strengths:

(1) After decades of dedicated research on the mushroom body, a consensus has been established that the release of dopamine from DANs modulates the weights of connections between KCs and MBONs. This process updates the association between sensory information and behavioral responses. However, understanding how the unconditioned stimulus is conveyed from sensory neurons to DANs, and the interactions of MBON outputs with innate responses to sensory context remains less clear due to the developmental and anatomic diversity of MBONs and DANs. Additionally, the recurrent connections between MBONs and DANs are reported to be critical for learning. The characterization of split-GAL4 drivers for 30 major interneurons connected with DANs and/or MBONs in this study will significantly contribute to our understanding of recurrent connections in mushroom body function.

(2) Optogenetic substitutes for real unconditioned stimuli (such as sugar taste or electric shock) are sometimes easier to implement in behavioral assays due to the spatial and temporal specificity with which optogenetic activation can be induced. GR64f-GAL4 has been widely used in the field to activate sugar sensory neurons and mimic sugar reward. However, the authors demonstrate that GR64f-GAL4 drives expression in other neurons not necessary for sugar reward, and the potential activation of these neurons could introduce confounds into training, impairing training efficiency. To address this issue, the authors have elaborated on a series of intersectional drivers with GR64f-GAL4 to dissect subsets of labeled neurons. This approach successfully identified a more specific sugar sensory neuron driver, SS87269, which consistently exhibited optimal training performance and triggered ethologically relevant local searching behaviors. This newly characterized line could serve as an optimized optogenetic tool for sugar reward in future studies.

(3) MBON08 was first reported by Aso et al. 2014, exhibiting dendritic arborization into both ipsilateral and contralateral γ3 compartments. However, this neuron could not be identified in the previously published Drosophila brain connectomes. In the present study, the existence of MBON08 is confirmed, occurring in one hemisphere of 35% of imaged flies. In brains where MBON08 is present, its dendrite arborization disjointly shares contralateral γ3 compartments with MBON09. This remarkable phenotype potentially serves as a valuable resource for understanding the stochasticity of neurodevelopment and the molecular mechanisms underlying mushroom body lobe compartment formation.

Reviewer #1 (Public review):

Summary:

Knudstrup et al. use two-photon calcium imaging to measure neural responses in the mouse primary visual cortex (V1) in response to image sequences. The authors presented mice with many repetitions of the same four-image sequence (ABCD) for four days. Then on the fifth day, they presented unexpected stimulus orderings where one stimulus was either omitted (ABBD) or substituted (ACBD). After analyzing trial-averaged responses of neurons pooled across multiple mice, they observed that stimulus omission (ABBD) caused a small, but significant, strengthening of neural responses but observed no significant change in the response to stimulus substitution (ACBD). Next, they performed population analyses of this dataset. They showed that there were changes in the correlation structure of activity and that many features about sequence ordering could be reliably decoded. This second set of analyses is interesting and exhibited larger effect sizes than the first results about predictive coding. However, concerns about the design of the experiment temper my enthusiasm.

The most recent version of this manuscript makes a few helpful changes (entirely in supplemental figures--the main text figures are unchanged). It does not resolve any of the larger weaknesses of the experimental design, or even perform single-neuron tracking in the one case where it was possible (between similar FOVs shown in Supplemental Figure 1).

Strengths:

(1) The topic of predictive coding in the visual cortex is exciting, and this task builds on previous important work by the senior author (Gavornik and Bear 2014) where unexpectedly shuffling sequence order caused changes in LFPs recorded from visual cortex.

(2) Deconvolved calcium responses were used appropriately here to look at the timing of the neural responses.

(3) Neural decoding results showing that the context of the stimuli could be reliably decoded from trial-averaged responses were interesting. But I have concerns about how the data was formatted for performing these analyses.

Weaknesses:

(1) All analyses were performed on trial-averaged neural responses that were pooled across mice (except for Supplementary Figure 6, see below). Owing to differences between subjects in behavior, experimental preparation quality, and biological variability, it seems important to perform most analyses on individual datasets to assess how behavioral training might differently affect each animal.

In the most recent draft, a single-mouse analysis was added for Figure 4C (Supplementary Figure 6). This effect of "representational drift" was not statistically quantified in either the single-mouse results or in the main text figure panel. Moreover, the apparent correlational drift could be accounted for by a reduction in SNR as a consequence of photobleaching.

(2) The correlation analyses presented in Figure 3 (labeled the second Figure 2 in the text) should be conducted on a single-animal basis. Studying population codes constructed by pooling across mice, particularly when there is no behavioral readout to assess whether learning has had similar effects on all animals, appears inappropriate to me. If the results in Figure 3 hold up on single animals, I think that is definitely an interesting result.

In the most recent draft, this analysis was still not performed on single mice. I was referring to the "decorrelation of responses" analysis in Figure 3, not the "representational drift" analysis in Figure 4. See my comments on Supplementary Figure 6 above.

(3) On Day 0 and Day 5, the reordered stimuli are presented in trial blocks where each image sequence is shown 100 times. Why wasn't the trial ordering randomized as was done in previous studies (e.g. Gavornik and Bear 2014)? Given this lack of reordering, did neurons show reduced predictive responses because the unexpected sequence was shown so many times in quick succession? This might change the results seen in Figure 2, as well as the decoder results where there is a neural encoding of sequence order (Figure 4). It would be interesting if the Figure 4 decoder stopped working when the higher order block structure of the task were disrupted.

In the rebuttal letter for the most recent draft, the authors refer to recent work in press (Hosmane et al. 2024) suggesting that because sleep may be important for plastic changes between sessions, they do not expect much change to be apparent within a session. However, they admit that this current study is too underpowered to know for sure--and do not cite or mention this yet unpublished work in the manuscript itself.

As a control, I would be interested to at least know how much variance in neural responses is observed between intermediate "training" sessions with identical stimuli, e.g. between Day 1 and Day 4, but this is not possible as imaging was not performed on these days.

Despite being referred to as "similar" I do not think early and late responses are clearly shown--aside from the histograms comparing "early traces" to "all traces" which include early traces in Figure 5B and Figure 6A. Showing variance in single-cell responses would be helpful to add in Supplementary Figure 3 and Supplementary Figure 4.

(4) A primary advantage of using two-photon calcium imaging over other techniques like extracellular electrophysiology is that the same neurons can be tracked over many days. This is a standard approach that can be accomplished by using many software packages-including Suite2P (Pachitariu et al. 2017), which is what the authors already used for the rest of their data preprocessing. The authors of this paper did not appear to do this. Instead, it appears that different neurons were imaged on Day 0 (baseline) and Day 5 (test). This is a significant weakness of the current dataset.

In the most recent draft, this concern has not been mitigated. Despite Supplementary Figure 1 showing similar FOVs, mostly different neurons were still extracted. In all other sessions, it is not reported how far apart the other recorded FOVs were from each other.

The rebuttal comment that the PE statistic is computed on an individual cell within-session basis is reasonable. Moreover, the bootstrapped version of the PE analysis in Supplementary Figure 8 is an improvement of the main analysis in the paper. As a control, it would have been helpful to compute the stability of the PE ratio statistics between training days (e.g. between day 1 and day 4). How much change would have been observed when none is expected? Unfortunately, imaging was not performed on these training days so this analysis will not be readily possible to perform. Moreover, the PE statistic requires averaging across cells and trials and is therefore very likely to wash out many interesting effects. Even if it is the population response that is changing, why would it be the arithmetic mean that changes in particular vs. some other projection of the population activity? The experimental and analysis design of the paper here remains weak in my mind.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors provide a method aiming to accurately reflect the individual deviation of longitudinal/temporal change compared to the normal temporal change characterized based on pre-trained population normative model (i.e., a Bayesian linear regression normative model), which was built based on cross-sectional data. This manuscript aims at solving a recently identified problem of using normative models based on cross-sectional data to make inferences about longitudinal change.

Strengths:

The efforts of this work make a good contribution to addressing an important question of normative modeling. With the greater availability of cross-sectional studies for normative modeling than longitudinal studies, and the inappropriateness of making inferences about longitudinal subject-specific changes using these cross-sectional data-based normative models, it's meaningful to try to address this gap from the aspect of methodological development.

Reviewer #1 (Public review):

Summary:

This work aims at understanding the role of thalamus POm in dorsal lateral striatum (DLS) projection in learning a sensorimotor associative task. The authors first confirm that POm forms "en passant" synapses with some of the DLS neuronal subtypes. They then perform a go/no-go associative task that consists of the mouse learning to discriminate between two different textures and to associate one of them with an action. During this task they either record the activity of the POm to DLS axons using endoscopy or silence their activity. They report that POm axons in the DLS are activated around the sensory stimulus but that the activity is not modulated by the reward. Last, they showed that silencing the POm axons at the level of DLS slows down learning the task.

The authors show convincing evidence of projections from POm to DLS and that POm inputs to DLS code for whisking whatever the outcome of the task is. However, their results do not allow to conclude if more neurones are recruited during the learning process or if the already activated fibres get activated more strongly. Last, because POm fibres in the DLS are also projecting to S1, silencing the POm fibres in the DLS could have affected inputs in S1 as well and therefore, the slowdown in acquiring the task is not necessarily specific to the POm to DLS pathway.

Strengths:

One of the main strengths of the paper is to go from slice electrophysiology to behaviour to get an in-depth characterization of one pathway. The authors did a comprehensive description of the POm projections to the DLS using transgenic mice to unambiguously identify the DLS neuronal population. They also used a carefully designed sensorimotor association task, and they exploited the results in depth.

It is a very nice effort to have measured the activity of the axons in the DLS not only after the mice have learned the task but throughout the learning process. It shows the progressive increase of activity of POm axons in the DLS, which could imply that there is a progressive strengthening of the pathway. The results show convincingly that POm axons in the DLS are not activated by the outcome of the task but by the whisker activity, and that this activity in average increases with learning.

Weaknesses:

One of the main targets of the striatum from thalamic input are the cholinergic neurons that weren't investigated here, is there information that could be provided?

It is interesting to know that the POm projects to all neuronal types in the DLS, but this information is not used further down the manuscript so the only take-home message of Figure 1 is that the axons that they image or silence in the DLS are indeed connected to DLS neurons and not just passing fibres. In this line, are these axons the same as the ones projecting to S1? If this is the case, why would we expect a different behaviour of the axon activity at the DLS level compared to S1?

The authors used endoscopy to measure the POm axons in the DLS activity, which makes it impossible to know if the progressive increase of POm response is due to an increase of activity from each individual neurons or if new neurons are progressively recruited in the process.

The picture presented in Figure 4 of the stimulation site is slightly concerning as there are hardly any fibres in neocortical layer 1 while there seems to be quite a lot of them in layer 4, suggesting that the animal here was injected in the VB. This is especially striking as the implantation and projection sites presented in Figure 1 and 2 are very clean and consistent with POm injection.

Comment after review: The weaknesses remain as concerns have not been addressed. The dataset is interesting but the interpretation, due partly to the lack of control (especially relative to VPM contamination), is difficult.

Reviewer #1 (Public review):

Summary:

In this series of studies, Locantore et al. investigated the role of SST-expressing neurons in the entopeduncular nucleus (EPNSst+) in probabilistic switching tasks, a paradigm that requires continued learning to guide future actions. In prior work, this group had demonstrated EPNSst+ neurons co-release both glutamate and GABA and project to the lateral habenula (LHb), and LHb activity is also necessary for outcome evaluation necessary for performance in probabilistic decision-making tasks. Previous slice physiology works have shown that the balance of glutamate/GABA co-release is plastic, altering the net effect of EPN on downstream brain areas and neural circuit function. The authors used a combination of in vivo calcium monitoring with fiber photometry and computational modelling to demonstrate that EPNSst+ neural activity represents movement, choice direction and reward outcomes in their behavioral task. However, viral-genetic manipulations to synaptically silence these neurons or selectively eliminate glutamate release had no effect on behavioral performance in well-trained animals. The authors conclude that despite their representation of task variables, EPN Sst+ neuron synaptic output is dispensable for task performance.

Strengths and Weaknesses:

Overall, the manuscript is exceptionally scholarly, with a clear articulation of the scientific question and a discussion of the findings and their limitations. The analyses and interpretations are careful and rigorous. This review appreciates the thorough explanation of the behavioral modelling and GLM for deconvolving the photometry signal around behavioral events, and the transparency and thoroughness of the analyses in the supplemental figures. This extra care has the result of increasing the accessibility for non-experts, and bolsters confidence in the results. To bolster a reader's understanding of results, we suggest it would be interesting to see the same mouse represented across panels (i.e. Fig 1 F-J, Supp 1 F,K etc i.e via inclusion of faint hash lines connecting individual data points across variables. Additionally, Fig 3E demonstrates that eliminating the 'reward' and 'choice and reward' terms from the GLM significantly worsens model performance; to demonstrate the magnitude of this effect, it would be interesting to include a reconstruction of the photometry signal after holding out of both or one of these terms, alongside the 'original' and 'reconstructed' photometry traces in panel D. This would help give context for how the model performance degrades by exclusion of those key terms. Finally, the authors claimed calcium activity increased following ipsilateral movements. However, figure 3C clearly shows that both SXcontra and SXisi increase beta coefficients. Instead, the choice direction may be represented in these neurons, given that beta coefficients increase following CXipsi and before SEipsi, presumably when animals make executive decisions. Could the authors clarify their interpretation on this point? Also, it is not clear if there is a photometry response related to motor parameters (i.e. head direction or locomotion, licking), which could change the interpretation of the reward outcome if it is related to a motor response; could the authors show photometry signal from representative 'high licking' or 'low licking' reward trials, or from spontaneous periods of high. Vs low locomotor speeds (if the sessions are recorded) to otherwise clarify this point?

There are a few limitations with the design and timing of the synaptic manipulations that would improve the manuscript if discussed or clarified. The authors take care to validate the intersectional genetic strategies: Tetanus Toxin virus (which eliminates synaptic vesicle fusion) or CRISPR editing of Slc17a6, which prevents glutamate loading into synaptic vesicles. The magnitude of effect in the slice physiology results are striking. However, this relies on co-infection of a second AAV to express channelrhodopsin for the purposes of validation, and it is surely the case that there will not be 100% overlap between the proportion of cells infected. Alternative means of glutamate packaging (other VGluT isoforms, other transporters etc) could also compensate for the partial absence of VGluT2, which should be discussed. The authors do not perform a complimentary experiment to delete GABA release (i.e. via VGAT editing), which is understandable, given the absence of an effect with the pan-synaptic manipulation. A more significant concern is the timing of these manipulations as the authors acknowledge. The manipulations are all done in well-trained animals, who continue to perform during the length of viral expression. Moreover, after carefully showing that mice use different strategies on the 70/30 version vs the 90/10 version of the task, only performance on the 90/10 version is assessed after the manipulation. Together, the observation that EPNsst activity does not alter performance on a well learned, 90/10 switching task decreases the impact of the findings, as this population may play a larger role during task acquisition or under more dynamic task conditions. Additional experiments could be done to strengthen the current evidence, although the limitations is transparently discussed by the authors.

Finally, intersectional strategies target LHb-projecting neurons, although in the original characterization it is not entirely clear that the LHb is the only projection target of EPNsst neurons. A projection map would help clarify this point.

Overall, the authors used a pertinent experimental paradigm and common cell-specific approaches to address a major gap in the field, which is the functional role of glutamate/GABA co-release from the major basal ganglia output nucleus in action selection and evaluation. The study is carefully conducted, their analyses are thorough, and the data are often convincing and thought-provoking. However, the limitations of their synaptic manipulations with respect to the behavioral assays reduces generalizability and to some extent the impact of their findings.

Comments on the latest version:

Specifically, they have included more thorough analyses to address several concerns related to interpreting activity patterns of EPSst+ neurons. The authors clearly point out that calcium activity increased during ipsilateral movements, and the increase was statistically larger during the choice phase (Figure 2 supplement 1F-G), indicating that these neurons may represent movement and additional factors (e.g. executive decision-making). Correspondingly, we appreciate the thorough explanation of using a GLM model to determine which behavioural variables contribute to observed physiological signals and adding the example reconstructed signal with direction and reward variables omitted in Figure 3 supplements 1 and 2.

Although no new manipulation experiment is added to the manuscript, the authors respond to common critiques related to testing the behavioural effect after the manipulations in well-trained mice. The discussion related to technical limitations, possible compensatory mechanisms and alternative interpretations is thorough and overall satisfying. Based on the behaviour modeling results, the authors speculate that animals need to integrate more evidence from the past to guide choice in a more uncertain environment (70/30 version), instead of adopting a 'win-stay, lose-shift' strategy in the more deterministic 90/10 version. The authors expand the discussion, but the possibility that EPNSst+ neurons contribute to task performance in well-trained animals under uncertainty is not directly tested. Along with other alternative explanations discussed in the manuscript, we think the paper is valuable literature for future studies to understand the basal ganglia circuits in learning and decision-making.

Reviewer #2 (Public review):

This study by Bell et al. focuses on understanding the roles of two alternatively spliced exons in the single Drosophila Cav2 gene cac. The authors generate a series of cac alleles in which one or the other mutually exclusive exons are deleted to determine the functional consequences at the neuromuscular junction. They find alternative splicing at one exon encoding part of the voltage sensor impacts the activation voltage as well as localization to the active zone. In contrast, splicing at the second exon pair does not impact Cav2 channel localization, but it appears to determine the abundance of the channel at active zones. Together, the authors propose that alternative splicing at the Cac locus enables diversity in Cav2 function generated through isoform diversity generated at the single Cav2 alpha subunit gene encoded in Drosophila.

Overall this is an excellent, rigorously validated study that defines unanticipated functions for alternative splicing in Cav2 channels. The authors have generated an important toolkit of mutually exclusive Cac splice isoforms that will be of broad utility for the field, and show convincing evidence for distinct consequences of alternative splicing of this single Cav2 channel at synapses. Importantly, the authors use electrophysiology and quantitative live sptPALM imaging to determine the impacts of Cac alternative splicing on synaptic function. There remain some questions regarding the mechanisms underlying the changes in Cac localization to somatodendritic compartments. Nonetheless, this is a compelling investigation of alternative splicing in Cav2 channels that should be of interest to many researchers.

Reviewer #1 (Public review):

Summary:

Authors benchmarked 5 IBD detection methods (hmmIBD, isoRelate, hap-IBD, phasedIBD, and Refined IBD) in Plasmodium falciparum using simulated and empirical data. Plasmodium falciparum has a mutation rate similar to humans but a much higher recombination rate and lower SNP density. Thus, the authors evaluated how recombination rate and marker density affect IBD segment detection. Next, they performed parameter optimization for Plasmodium falciparum and benchmarked the robustness of downstream analyses (selection detection and NE inference) using IBD detected by each of the methods. They also tracked the computational efficiency of these methods. The authors work is valuable for the tested species and the analyses presented appear to support their claim that users should be cautious calling IBD when SNP density is low and recombination rate is high.

Strengths:

The study design was solid. The authors set up their reasoning for using P. falciparum very well. The high recombination rate and similar mutation rate to human is indeed an interesting case. Further, they chose methods that were developed explicitly for each species. This was a strength of the work, as well as incorporating both simulated and empirical data to support their goal that IBD detection should be benchmarked in P. falciparum.

Weaknesses:

The scope of the optimization and application of results from the work are narrow, in that everything is fine-tuned for Plasmodium. Some of the results were not entirely unexpected for users of any of the tested software that was developed for humans. For example, it is known that Refined IBD is not going to do well with the combination of short IBD segments and low SNP density. Lastly, it appears the authors only did one large-scale simulation (there are no reported SDs).

Reviewer #1 (Public review):

Summary:

This Tanzanian study focused on the relationship between human genetic ancestry, Mycobacterium tuberculosis complex (MTBC) diversity, and tuberculosis (TB) disease severity. The authors analyzed the genetic ancestry of 1,444 TB patients and genotyped the corresponding MTBC strains isolated from the same individuals. They found that the study participants predominantly possess Bantu-speaking genetic ancestry, with minimal European and Asian ancestry. The MTBC strains identified were diverse and largely resulted from introductions from South or Central Asia. Unfortunately, no associations were identified between human genetic ancestry, the MTBC strains, or TB severity. The authors suggest that social and environmental factors are more likely to contribute to TB severity in this setting.

Strengths:

In comparison to other studies investigating the role of human genetics in TB phenotypes, this study is relatively large, with more than 1,400 participants.

The matched human-MTBC strain collection is valuable and offers the opportunity to address questions about human-bacterium co-evolution.

Weaknesses:

Although the authors had genome-wide genotyping and whole genome sequencing data, they only compared the associations between human ancestry and MTBC strains. Given the large sample size, they had the opportunity to conduct a genome-wide association study similar to that of Muller et al. (https://doi.org/10.1016/j.ygeno.2021.04.024).

The authors tested whether human genetic ancestry is associated with TB severity. However, the basis for this hypothesis is unclear. The studies cited as examples all focused on progression to active TB (from a latent infection state), which should not be conflated with disease severity. It is difficult to ascertain whether the role of genetic ancestry in disease severity would be detectable through this study design, as some participants might simply have been sicker for longer before being diagnosed (despite the inquiry about cough duration). This delay in diagnosis would not be influenced solely by human genetics, which is the conclusion of the study.

Additionally, the study only included participants who attended the TB clinic.

Including healthy controls from the general population would have provided an interesting comparison to see if ancestry proportions differ.

Although the authors suggest that social and environmental factors contribute to TB severity, only age, smoking, and HIV status were characterised in the study.

Reviewer #1 (Public review):

Summary:

This work introduces the differentiable Gillespie algorithm, DGA, which is a differentiable variant of the celebrated (and exact) Gillespie algorithm commonly used to perform stochastic simulations across numerous fields, notably in the life sciences. The proposed DGA approximates the exact Gillespie algorithm using smooth functions yielding a suitable approximate differentiable stochastic system as a proxy for the underlying discrete stochastic system, where DGA stochastic reactions have continuous reaction index and the species abundances. To illustrate their methodology, the authors specifically consider in detail the case of a well-studied two-state promoter gene regulation system that they analyze using a machine learning approach, and by combining simulation data with analytical results. For the two-state promoter gene system, the DGA is benchmarked by accurately reproducing the results of the exact Gillespie algorithm. For this same simple system, the authors also show that how the DGA can be used for estimating kinetic parameters of both simulated and real noisy experimental data. This let them argue convincingly that the DGA can become a powerful computation tool for applications in quantitative and synthetic biology. In order to argue that the DGA can be employed to design circuits with ad-hoc input-output relations, these considerations are then extended to a more complex four-state promoter model of gene regulation.

Strengths:

The main strength of the paper is its clarity and its pedagogical presentation of the simulation methods.

Weaknesses:

It would have been useful to have a brief discussion, based on a concrete example, of what can be achieved with the DGA and is totally beyond the reach of the Gillespie algorithm and the numerous existing stochastic simulation methods. A more comprehensive and quantitative analysis of the limitations of the DGA, e.g. for rare events, would have also been helpful.

Reviewer #1 (Public review):

Summary:

The authors provide a resource to the systems neuroscience community, by offering their Python-based CLoPy platform for closed-loop feedback training. In addition to using neural feedback, as is common in these experiments, they include a capability to use real-time movement extracted from DeepLabCut as the control signal. The methods and repository are detailed for those who wish to use this resource. Furthermore, they demonstrate the efficacy of their system through a series of mesoscale calcium imaging experiments. These experiments use a large number of cortical regions for the control signal in the neural feedback setup, while the movement feedback experiments are analyzed more extensively.

Strengths:

The primary strength of the paper is the availability of their CLoPy platform. Currently, most closed-loop operant conditioning experiments are custom built by each lab and carry a relatively large startup cost to get running. This platform lowers the barrier to entry for closed-loop operant conditioning experiments, in addition to making the experiments more accessible to those with less technical expertise.

Another strength of the paper is the use of many different cortical regions as control signals for the neurofeedback experiments. Rodent operant conditioning experiments typically record from the motor cortex and maybe one other region. Here, the authors demonstrate that mice can volitionally control many different cortical regions not limited to those previously studied, recording across many regions in the same experiment. This demonstrates the relative flexibility of modulating neural dynamics, including in non-motor regions.

Finally, adapting the closed-loop platform to use real-time movement as a control signal is a nice addition. Incorporating movement kinematics into operant conditioning experiments has been a challenge due to the increased technical difficulties of extracting real-time kinematic data from video data at a latency where it can be used as a control signal for operant conditioning. In this paper they demonstrate that the mice can learn the task using their forelimb position, at a rate that is quicker than the neurofeedback experiments.

Weaknesses:

There are several weaknesses in the paper that diminish the impact of its strengths. First, the value of the CLoPy platform is not clearly articulated to the systems neuroscience community. Similarly, the resource could be better positioned within the context of the broader open-source neuroscience community. For an example of how to better frame this resource in these contexts, I recommend consulting the pyControl paper. Improving this framing will likely increase the accessibility and interest of this paper to a less technical neuroscience audience, for instance by highlighting the types of experimental questions CLoPy can enable.

While the dataset contains an impressive amount of animals and cortical regions for the neurofeedback experiment, and an analysis of the movement-feedback experiments, my excitement for these experiments is tempered by the relative incompleteness of the dataset, as well as its description and analysis in the text. For instance, in the neurofeedback experiment, many of these regions only have data from a single mouse, limiting the conclusions that can be drawn. Additionally, there is a lack of reporting of the quantitative results in the text of the document, which is needed to better understand the degree of the results. Finally, the writing of the results section could use some work, as it currently reads more like a methods section.

Suggestions for improved or additional experiments, data or analyses:

Not necessary for this paper, but it would be interesting to see if the CLNF group could learn without auditory feedback.

There are no quantitative results in the results section. I would add important results to help the reader better interpret the data. For example, in: "Our results indicated that both training paradigms were able to lead mice to obtain a significantly larger number of rewards over time," You could show a number, with an appropriate comparison or statistical test, to demonstrate that learning was observed.

For: "Performing this analysis indicated that the Raspberry Pi system could provide reliable graded feedback within ~63 {plus minus} 15 ms for CLNF experiments." The LED test shows the sending of the signal, but the actual delay for the audio generation might be longer. This is also longer than the 50 ms mentioned in the abstract.

It could be helpful to visualize an individual trial for each experiment type, for instance how the audio frequency changes as movement speed / calcium activity changes.

The sample sizes are small (n=1) for a few groups. I am excited by the variety of regions recorded, so it could be beneficial for the authors to collect a few more animals to beef up the sample sizes.

I am curious as to why 60 trials sessions were used. Was it mostly for the convenience of a 30 min session, or were the animals getting satiated? If the former, would learning have occurred more rapidly with longer sessions?

Figure 4 E is interesting, it seems like the changes in the distribution of deltaF was in both positive and negative directions, instead of just positive. I'd be curious as to the author's thoughts as to why this is the case. Relatedly, I don't see Figure 4E, and a few other subplots, mentioned in the text. As a general comment, I would address each subplot in the text.

For: "In general, all ROIs assessed that encompassed sensory, pre-motor, and motor areas were capable of supporting increased reward rates over time," I would provide a visual summary showing the learning curves for the different types of regions.

Relatedly, I would further explain the fast vs slow learners, and if they mapped onto certain regions.

Also I would make the labels for these plots (e.g. Supp Fig3) more intuitive, versus the acronyms currently used.

The CLMF animals showed a decrease in latency across learning, what about the CLNF animals? There is currently no mention in the text or figures.

Reviewer #1 (Public review):

Summary:

Fluorescence imaging has become an increasingly popular technique for monitoring neuronal activity and neurotransmitter concentrations in the living brain. However, factors such as brain motion and changes in blood flow and oxygenation can introduce significant artifacts, particularly when activity-dependent signals are small. Yogesh et al. quantified these effects using GFP, an activity-independent marker, under two-photon and wide-field imaging conditions in awake behaving mice. They report significant GFP responses across various brain regions, layers, and behavioral contexts, with magnitudes comparable to those of commonly used activity sensors. These data highlight the need for robust control strategies and careful interpretation of fluorescence functional imaging data.

Strengths:

The effect of hemodynamic occlusion in two-photon imaging has been previously demonstrated in sparsely labeled neurons in V1 of anesthetized animals (see Shen and Kara et al., Nature Methods, 2012). The present study builds on these findings by imaging a substantially larger population of neurons in awake, behaving mice across multiple cortical regions, layers, and stimulus conditions. The experiments are extensive, the statistical analyses are rigorous, and the results convincingly demonstrate significant GFP responses that must be accounted for in functional imaging experiments. However, whether these GFP responses are driven by hemodynamic occlusion remains less clear, given the complexities associated with awake imaging and GFP's properties (see below).

Weaknesses:

(1) The authors primarily attribute the observed GFP responses to hemodynamic occlusion. While this explanation is plausible, other factors may also contribute to the observed signals. These include uncompensated brain movement (e.g., axial-direction movements), leakage of visual stimulation light into the microscope, and GFP's sensitivity to changes in intracellular pH (see e.g., Kneen and Verkman, 1998, Biophysical Journal). Although the correlation between GFP signals and blood vessel diameters supports a hemodynamic contribution, it does not rule out significant contributions from these (or other) factors. Consequently, whether GFP fluorescence can reliably quantify hemodynamic occlusion in two-photon microscopy remains uncertain.

(2) Regardless of the underlying mechanisms driving the GFP responses, these activity-independent signals must be accounted for in functional imaging experiments. However, the present manuscript does not explore potential strategies to mitigate these effects. Exploring and demonstrating even partial mitigation strategies could have significant implications for the field.

(3) Several methodology details are missing from the Methods section. These include: (a) signal extraction methods for two-photon imaging data (b) neuropil subtraction methods (whether they are performed and, if so, how) (c) methods used to prevent visual stimulation light from being detected by the two-photon imaging system (d) methods to measure blood vessel diameter/area in each frame. The authors should provide more details in their revision.

Reviewer #1 (Public review):

Summary:

This study aims to investigate the links between social behaviors observed in free-moving situations and behavioral performances measured in well-controlled, laboratory settings. The authors assessed general social tendencies and dyadic relationships among four monkeys in a group by scoring agonistic (aggression) and affiliative (grooming and proximity) behaviors in each pair. By measuring the saccadic reaction time in a classic social interference task, the authors reported that the monkeys with higher SEIs (i.e., more social individuals) were less distracted by the faces of other monkeys. These effects were enhanced when the distractors were out-group monkey faces rather than in-group ones. Lastly, oxytocin administration increased the impact of the out-group monkey faces in the social interference task, while reducing the magnitude of general social tendencies measured with SEI.

Strengths:

(1) The combination of behavioral data obtained in a colony room and in a laboratory environment is rare and important.<br /> (2) The evaluation of social interactions were successfully performed based on an automated target detection algorithm. The resulting multi-dimensional, complicated social interactions were summarized into simple indices (SEI and IEI). These indices provide a good measure for the social tendencies of each monkey.<br /> (3) Well-designed and robust experiments in the laboratory environment that are linked nicely with the general social tendencies observed in spontaneous behaviors.

Weaknesses:

(1) While the overall results are interesting, I am somewhat left confused about how to interpret the difference in the scores derived from different conditions. For example, the authors stated "Comparing the weights for in-group and out-group distractors, the effect of proximity was larger than that of aggression and grooming" in p.8. Does this mean that the proximity is indeed the type of behavior most affected in the out-group condition compared to the in-group condition? The out-group effects are difficult to examine with actual behavioral data, but some in-group effects such as those involving OT can be tested, which possibly provides good insights into interpreting the differences of the weights observed across the experimental conditions.

(2) I think it is important to provide how variable spontaneous social interactions were across sessions and how impactful the variability of the interactions is on the SEI and IEI, as it helps to understand how meaningful the differences of weights are across the conditions, but such data are missing. In line with this point, although the conclusions still hold as those data were obtained during the same experimental periods, shouldn't the weights in Fig. 3f and Figs. 4g and 4h (saline) be expected to be similar, if not the same?

Reviewer #1 (Public review):

Summary:

In this manuscript, Kondo et al. developed a method to suppress somatic action potentials while recording spine calcium signals using two-photon imaging in the L2/3 visual cortex in response to visual stimuli. The authors identified different patterns of dendritic spine activation by visual stimuli and analyzed how the different patterns of spine responses may contribute to somatic visual responses. Their analysis results suggest that spines on dendrites with a clustered arrangement can potentially generate sharply tuned output.

Strengths:

This is an interesting study addressing a standing question of how previously reported pepper-and-salt-like distributed sensory inputs on individual spines may give rise to somatic sensory selectivity. The method of somatic inhibition to prevent bAPs appears new and effective. The measurements of spine activity are carefully done. The finding that a small number of spines located in the same branch with similar tuning properties would predict the somatic tuning is consistent with local dendritic nonlinear integration mechanisms.

Weaknesses:

(1) The demonstration of the effectiveness of soma-specific inhibition is inadequate. Figure 1 only provides a single example trace showing the inhibition of somatic visual responses. The authors should provide statistical analysis over grouped data. For the effect of soma-specific inhibition on spine activity, the authors provided mostly negative results, lacking effects on spine responses for both soma inhibition and bAP subtraction. This is confusing. One possible explanation is that bAPs normally have little influence on spine activity. However, this would conflict with the known fact that somatic APs can easily invade spines in L2/3 neurons (e.g., Chen et al., Nature 2011). Another possibility is that under the current experimental conditions, somatic APs were rarely evoked by the visual stimulus. The authors should also rule out the possibility that the spines they imaged are from different neurons than the ones with somatic inhibition. The authors may consider identifying those cases where somatic APs have a significant impact on spine activity or spine tuning and show how bAP inhibition influences the dendritic and spine responses.

(2) Figure 4 shows that the proportion of spines with a preferred orientation similar to the soma (ΔOri {less than or equal to} 30{degree sign}) was 60%, which is surprisingly high. It is intriguing that without somatic AP invasion, there could be such a high degree of similarity between spine activity and somatic tuning. What is the ratio without soma inhibition? One could reason that with bAP invasion, there should be even more spines showing visual responses similar to those of the soma. Moreover, with such a high proportion of spines showing similar sensory tuning to the soma, it is inevitable that many branches contain more spines with similar tuning as the soma, exhibiting an apparent branch-specific clustering. While such apparent clustering may well predict somatic tuning, it primarily reflects a correlational relationship rather than a causal synaptic integration mechanism.

(3) There has been extensive work studying how the integration of spine activity or sub-branch activity gives rise to somatic output. The proposed main contribution of this study is to use an improved method to inhibit somatic activity in order to more confidently measure spine-specific activity and examine the integration mechanisms. However, the results showed that the measured spine-specific activity under soma inhibition was not significantly different from that measured under normal conditions (see point 1). It becomes unclear how this new method contributes to obtaining new insights into the synaptic integration mechanism.

(4) Figure 6 shows how the tuning similarity between spines depends on the distance between them. It is unclear what new information was acquired regarding the functional clustering of spines. This result can be largely explained by the overall higher proportion of similarly tuned spines (60%) compared to the soma's preferred orientations. Moreover, the authors did not demonstrate how such clustering may contribute to nonlinear synaptic integration.

(5) The results shown in Figure 7 can again be largely explained by the static property of a higher proportion of spines tuned similarly to the soma. These results do not reveal any active dendritic integration mechanisms.

Reviewer #1 (Public review):

In this manuscript, Chang et al. investigated the cell type-specific role of the integrin activator Shv in activity-dependent synaptic remodeling. Using the Drosophila larval neuromuscular junction as a model, they show that glial-secreted Shv modulates synaptic plasticity by maintaining the extracellular balance of neuronal Shv proteins and regulating ambient extracellular glutamate concentrations, which in turn affects postsynaptic glutamate receptor abundance. Furthermore, they report that genetic perturbation of glial morphogenesis phenocopies the defects observed with the loss of glial Shv. Altogether, their findings propose a role for glia in activity-induced synaptic remodeling through Shv secretion. While the conclusions are intriguing, several issues related to experimental design and data interpretation merit further discussion.

Reviewer #1 (Public review):

Summary:

As TDP-43 mislocalization is a hallmark of multiple neurodegenerative diseases, the authors seek to identify pathways that modulate TDP-43 levels. To do this, they use a FACS based genome wide CRISPR KD screen in a Halo tagged TDP-43 KI iPSC line. Their screen identifies a number of genetic modulators of TDP-43 expression including BORC which plays a role in lysosome transport.

Strengths:

Genome wide CRISPR based screen identifies a number of modulators of TDP-43 expression to generate hypotheses regarding RNA BP regulation and perhaps insights into disease.

Weaknesses:

It is unclear how altering TDP-43 levels may relate to disease where TDP-43 is not altered in expression but mislocalized. This is a solid cell biology study, but the relation to disease is not clear without providing evidence of BORC alterations in disease or manipulation of BORC reversing TDP-43 pathology in disease.

The mechanisms by which BORC and lysosome transport modulate TDP-43 expression are unclear. Presumably, this may be through altered degradation of TDP protein but this is not addressed.

Previous studies have demonstrated that TDP-43 levels can be modulated by altering lysosomal degradation so the identification of lysosomal pathways is not particularly novel.

It is unclear whether this finding is specific to TDP-43 levels or whether lysosome localization may more broadly impact proteostasis in particular of other RNA BPs linked to disease.

Unclear whether BORC depletion alters lysosome function or simply localization.

Reviewer #1 (Public review):

Summary of what is achieved: This manuscript validates and extends upon the sigh generating circuit between the NMB/GRP+ RTN/parafacial neurons and the NMBR/GRPR+ preBötC neurons established in Li et al., 2016. The authors generate multiple transgenic lines that enable selective targeting of these various sub-populations of cells and demonstrate the sufficiency of each type in generating a sigh breath. Additionally, they show that NMBR and GPRP preBötC neurons are glutamatergic, have overlapping and distinct expression, and do not express SST. Beyond this validation, the authors show that ectopic stimulation of SST neurons is sufficient to evoke sighs and that they are necessary for NMB/GRP induced sighing. This data is the first time that preBötC neurons downstream of NMBR/GRPR neurons have been identified that transform a eupneic breath into a sign breath. The five conclusions stated at the end of the introduction are supported by the data.

Summary of a primary weakness: A strong emphasis throughout the manuscript is the identification of an unsubstantiated slow sigh rhythm that is produced by NMBR/GRPR neurons. It is even suggested that this is an intrinsic property of these neurons. However, to make such a novel (and quite surprising) claim requires many more studies and the conclusion is dependent on how the authors have defined a sigh. Moreover, some data within the paper conflicts with this idea. The resubmitted manuscript does not contain any revisions and the rebuttal does not sufficiently address the critiques.

In summary, the optogenetic and chemogenetic characterization of the neuropeptide pathway transgenic lines nicely aligns with and provides important validation of the previous study by Li et. al., 2016 and the SST neuron studies provide a new mechanism for the transformation of NMBR/GRPR neuropeptide activation into a sigh. These are important findings, and they should be the points emphasized. The proposal of a slow sigh rhythm should be more rigorously established with new experiments and analysis or should be more carefully described and discussed.

Reviewer #1 (Public review):

Summary:

Dong et al here have studied the impact of the small Ras-like GTPase Rab10 on the exocytosis of dense core vesicles (DVC), which are important mediators of neuropeptide signaling in brain. They use optical imaging to show that lentiviral depletion of Rab10 in mouse hippocampal neurons in culture independent of the established defects in neurite outgrowth hamper DCV exocytosis. They further demonstrate that such defects are paralleled by changes in ER morphology and defective ER-based calcium buffering as well as reduced ribosomal protein expression in Rab10-depleted neurons. Re-expression of Rab10 or supplementation of exogenous L-leucine to restore defective neuronal protein synthesis rescues impaired DCV secretion. Based on these results they propose that Rab10 regulates DCV release by maintaining ER calcium homeostasis and neuronal protein synthesis.

Strengths:

This work provides interesting and potentially important new insights into the connection between ER function and the regulated secretion of neuropeptides via DCVs. The authors combine advanced optical imaging with light and electron microscopy, biochemistry and proteomics approaches to thoroughly assess the effects of Rab10 knockdown at the cellular level in primary neurons. The proteomic dataset provided may be valuable in facilitating future studies regarding Rab10 function. This work will thus be of interest to neuroscientists and cell biologists.

Weaknesses:

Whether and how the phenotypes of Rab10 reported in this study are linked remains an open question. Likewise, a possible role of Rab10 in exocytosis cannot be excluded at this stage.

Comments on revisions:

My previous questions and concerns have been satisfactorily addressed by the authors.

Reviewer #2 (Public review):

This work aggregates data across 5 openly available stopping studies (3 at 7 tesla and 2 at 3 tesla) to evaluate activity patterns across the common contrasts of Failed Stop (FS) > Go, FS > stop success (SS), and SS > Go. Previous work has implicated a set of regions that tend to be positively active in one or more of these contrasts, including the bilateral inferior frontal gyrus, preSMA, and multiple basal ganglia structures. However, the authors argue that upon closer examination, many previous papers have not found subcortical structures to be more active on SS than FS trials, bringing into question whether they play an essential role in (successful) inhibition. In order to evaluate this with more data and power, the authors aggregate across five datasets and find many areas that are *more* active for FS than SS, including bilateral preSMA, GPE, thalamus, and VTA. They argue that this brings into question the role of these areas in inhibition, based upon the assumption that areas involved in inhibition should be more active on successful stop than failed stop trials, not the opposite as they observed.

Comments on revisions:

The authors have been responsive to the feedback of both reviewers and they have significantly improved the manuscript. I now judge the work as valuable and solid. The authors have achieved their aims to characterize subcortical BOLD activation in the stop-signal paradigm.

Reviewer #1 (Public review):

The authors set out to define the molecular basis for LP as the origin of BRCA1-deficient breast cancers. They showed that LPs have the highest level of replicative stress, and hypothesise that this may account for their tendency to transform. They went on to identify ELF3 as a candidate driver of LP transformation and showed that ELF3 expression is up-regulated in response to replicative stress as well as BRCA1 deficiency. They went on to show that ELF3 inactivation led to a higher level of DNA damage, which may result from compromised replicative stress responses.

While the manuscript supports the interesting idea wherein ELF3 may fuel LP cell transformation, it remains obscure how ELF3 promotes cell tolerance to DNA damage. Interestingly the authors proposed that ELF3 suppresses excessive genomic instability, but in my opinion, I do not see any evidence that supports this claim. In fact, one might think that genomic instability is key to cell transformation.

Comments on revisions:

The authors have addressed most of my concerns.

This being said, the one major criticism raised by both Reviewers is the lack of evidence to support ELF3 as a driver of transformation of and in LP cells. The authors appear to have invested much resource and time but were not successful in isolating LP cells for experimentations. I would therefore suggest that the authors tone down their claims throughout the manuscript.

Reviewer #1 (Public review):

Summary:

This study analyzed biomarker data from 28 subjects with geographic atrophy (GA) in a Phase I/II clinical trial of PPY988, a subretinal AAV2 complement factor I (CFI) gene therapy, to evaluate pharmacokinetics and pharmacodynamics. Post-treatment, a 2-fold increase in vitreous humor (VH) FI was observed, correlating with a reduction in FB breakdown product Ba but minimal changes in other complement factors. The aqueous humor (AH) was found to be an unreliable proxy for VH in assessing complement activation. In vitro assays showed that the increase in FI had a minor effect on the complement amplification loop compared to the more potent C3 inhibitor pegcetacoplan. These findings suggest that PPY988 may not provide enough FI protein to effectively modulate complement activation and slow GA progression, highlighting the need for thorough biomarker review to determine optimal dosing in future studies.

Strengths:

This manuscript provides critical data on the efficacy of gene therapy for the eye, specifically introducing complement FI expression. It presents the results from a halted clinical trial, making the publication of this data essential for understanding the outcomes of this gene therapy approach. The findings offer valuable insights and lessons for future gene therapy attempts in similar contexts.

Weaknesses:

No particular weaknesses. The study was carefully performed and limitations are discussed.

I have just some concerns about the methodology used. The authors use the MILLIPLEX assays, which allow for multiplexed detection of complement proteins and they mention extensive validation. How are the measurements with this assay correlating with gold standard methods? Is the specificity and the expected normal ranges preserved with this assay? This also stands for the Olink assay. Some of the proteins are measured by both assay and/or by standard ELISA. How do these measurements correlate?

Comments on revisions:

The authors answered part of my comments. Only one remained - please provide a comparison between ELISA/Multiplex and Olink data to judge the robustness of the Olinkl assay for complement.

Reviewer #1 (Public review):

Summary:

Chlamydia spp. has a biphasic developmental cycle consisting of an extracellular, infectious form called an elementary body (EB) and an intracellular, replicative form known as a reticular body (RB). The structural stability of EBs is maintained by extensive cross linking of outer membrane proteins while the outer membrane proteins of RBs are in a reduced state. The overall redox state of EBs is more oxidized than RBs. The authors propose that redox state may be a controlling factor in the developmental cycle. To test this, alkyl hydroperoxide reductase subunit C (ahpC) was overexpressed or knocked down to examine effects on developmental gene expression. KD of ahpC induced increased expression of EB-specific genes and accelerated EB production. Conversely, overexpression of phpC delayed differentiation to EBs. The results suggest that chlamydial redox state may play a role in differentiation.

Strengths:

Uses modern genetic tools to explore the difficult area of temporal gene expression throughout the chlamydial developmental cycle.

Weaknesses:

The environmental signals triggering ahpC expression/activity are not determined.

Comments on revisions:

I am satisfied with the modifications made to the manuscript.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors evaluate the attenuation, immunogenicity, and protection efficacy of a live-attenuated SARS-CoV-2 vaccine candidate (BK2102) against SARS-CoV-2.

Strengths:

The authors demonstrate that intranasal inoculation of BK2102 is safe and able to induce humoral and cellular immune responses in hamsters, without apparent signs of damage in the lungs, that protects against homologous SARS-CoV-2 and Omicron BA.5 challenge. Safety of BK2102 was further confirmed in a new hACE2 transgenic mouse model generated by the authors.

Weaknesses:

The authors have addressed my previous comments on the first submission of the document.

Reviewer #1 (Public Review):

Summary:

The present study's main aim is to investigate the mechanism of how VirR controls the magnitude of MEV release in Mtb. The authors used various techniques, including genetics, transcriptomics, proteomics, and ultrastructural and biochemical methods. Several observations were made to link VirR-mediated vesiculogenesis with PG metabolism, lipid metabolism, and cell wall permeability. Finally, the authors presented evidence of a direct physical interaction of VirR with the LCP proteins involved in linking PG with AG, providing clues that VirR might act as a scaffold for LCP proteins and remodel the cell wall of Mtb. Since the Mtb cell wall provides a formidable anatomical barrier for the entry of antibiotics, targeting VirR might weaken the permeability of the pathogen along with the stimulation of the immune system due to enhanced vesiculogenesis. Therefore, VirR could be an excellent drug target. Overall, the study is an essential area of TB biology.

Strengths:

The authors have done a commendable job of comprehensively examining the phenotypes associated with the VirR mutant using various techniques. Application of Cryo-EM technology confirmed increased thickness and altered arrangement of CM-L1 layer. The authors also confirmed that increased vesicle release in the mutant was not due to cell lysis, which contrasts with studies in other bacterial species.

Another strength of the manuscript is that biochemical experiments show altered permeability and PG turnover in the mutant, which fits with later experiments where authors provide evidence of a direct physical interaction of VirR with LCP proteins.

Transcriptomics and proteomics data were helpful in making connections with lipid metabolism, which the authors confirmed by analyzing the lipids and metabolites of the mutant.

Lastly, using three approaches, the authors confirm that VirR interacts with LCP proteins in Mtb via the LytR_C terminal domain.

Altogether, the work is comprehensive, experiments are designed well, and conclusions were made based on the data generated after verification using multiple complementary approaches.

Weaknesses:

The major weakness is that the mechanism of VirR-mediated EV release remains enigmatic. Most of the findings are observational and only associate enhanced vesiculogenesis observed in the VirR mutant with cell wall permeability and PG metabolism. Authors suggest that EV release occurs during cell division when PG is most fragile. However, this has yet to be tested in the manuscript-the AFM of the VirR mutant, which produces thicker PG with more pore density, displays enhanced vesiculogenesis. No evidence was presented to show that the PG of the mutant is fragile, and there are differences in cell division to explain increased vesiculogenesis. These observations, counterintuitive to the authors' hypothesis, need detailed experimental verification.

Transcriptomic data only adds a little substantial. Transcriptomic data do not correlate with the proteomics data. It remains unclear how VirR deregulates transcription. TLCs of lipids are not quantitative. For example, the TLC image of PDIM is poor; quantitative estimation needs metabolic labeling of lipids with radioactive precursors. Further, change in PDIMs is likely to affect other lipids (SL-1, PAT/DAT) that share a common precursor (propionyl- CoA).

The connection of cholesterol with cell wall permeability is tenuous. Cholesterol will serve as a carbon source and contribute to the biosynthesis of methyl-branched lipids such as PDIM, SL-1, and PAD/DAT. Carbon sources also affect other aspects of physiology (redox, respiration, ATP), which can directly affect permeability and import/export of drugs. Authors should investigate whether restoration of the normal level of permeability and EV release is not due to the maintenance of cell wall lipid balance upon cholesterol exposure of the VirR mutant.

Finally, protein interaction data is based on experiments done once without statistical analysis. If the interaction between VirR and LCP protein is expected on the mycobacterial membrane, how SPLIT_GFP system expressed in the cytoplasm is physiologically relevant. No explanation was provided as to why VirR interacts with the truncated version of LCP proteins and not with the full-length proteins.

Comments on revisions:

The authors have addressed my comments. I have no further issues.

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 large-scale simulation studies. The results, interpretations, and Discussion are largely nuanced, clear, and well-motivated, and the pedagogical nature with which sampling convergence is discussed is greatly appreciated. Finally, the underlying data are shared in a clear and accessible manner. Overall, the manuscript is a model

Weaknesses:

The simplicity of a one-bead-per-residue model parameterized to capture UCST-type phase behavior does perhaps impact some aspects of the generality of this work. That said, the authors carefully acknowledge these limitations, and overall, this is not seen as a major weakness of the conclusions drawn or the manuscript, given those conclusions are appropriately couched.

Joint Public Review:

This manuscript by Tao et al. reports on an effort to better specify the underlying interactions driving the effects of biodiversity on productivity in biodiversity experiments. The authors are especially concerned with the potential for competitive interactions to drive positive biodiversity-ecosystem functioning relationships by driving down the biomass of subdominant species. The authors suggest a new partitioning schema that utilizes a suite of partial density treatments to capture so-called competitive ability.

Readers are encouraged to consider the original reviews in full, which outline the strengths and weaknesses of the work:

First version: https://elifesciences.org/reviewed-preprints/98073v1/reviews

Second version: https://elifesciences.org/reviewed-preprints/98073v2/reviews

There are no further reviews for this version because the authors declined to make further improvements to their manuscript.

Reviewer #1 (Public review):

Summary

In this human neuroimaging and electrophysiology study, the authors aimed to characterise effects of a period of visual deprivation in the sensitive period on excitatory and inhibitory balance in the visual cortex. They attempted to do so by comparing neurochemistry conditions ('eyes open', 'eyes closed') and resting state, and visually evoked EEG activity between ten congenital cataract patients with recovered sight (CC), and ten age-matched control participants (SC) with normal sight.

First, they used magnetic resonance spectroscopy to measure in vivo neurochemistry from two locations, the primary location of interest in the visual cortex, and a control location in the frontal cortex. Such voxels are used to provide a control for the spatial specificity of any effects, because the single-voxel MRS method provides a single sampling location. Using MR-visible proxies of excitatory and inhibitory neurotransmission, Glx and GABA+ respectively, the authors report no group effects in GABA+ or Glx, no difference in the functional conditions 'eyes closed' and 'eyes open'. They found an effect of group in the ratio of Glx/GABA+ and no similar effect in the control voxel location. They then perform multiple exploratory correlations between MRS measures and visual acuity, and report a weak positive correlation between the 'eyes open' condition and visual acuity in CC participants.

The same participants then took part in an EEG experiment. The authors selected two electrodes placed in the visual cortex for analysis and report a group difference in an EEG index of neural activity, the aperiodic intercept, as well as the aperiodic slope, considered a proxy for cortical inhibition. Control electrodes in the frontal region did not present with the same pattern. They report an exploratory correlation between the aperiodic intercept and Glx in one out of three EEG conditions.

The authors report the difference in E/I ratio, and interpret the lower E/I ratio as representing an adaptation to visual deprivation, which would have initially caused a higher E/I ratio. Although intriguing, the strength of evidence in support of this view is not strong. Amongst the limitations are the low sample size, a critical control cohort that could provide evidence for higher E/I ratio in CC patients without recovered sight for example, and lower data quality in the control voxel. Nevertheless, the study provides a rare and valuable insight into experience-dependent plasticity in the human brain.

Strengths of study

How sensitive period experience shapes the developing brain is an enduring and important question in neuroscience. This question has been particularly difficult to investigate in humans. The authors recruited a small number of sight-recovered participants with bilateral congenital cataracts to investigate the effect of sensitive period deprivation on the balance of excitation and inhibition in the visual brain using measures of brain chemistry and brain electrophysiology. The research is novel, and the paper was interesting and well written.

Limitations

Low sample size. Ten for CC and ten for SC, and further two SC participants were rejected due to lack of frontal control voxel data. The sample size limits the statistical power of the dataset and increases the likelihood of effect inflation.

In the updated manuscript, the authors have provided justification for their sample size by pointing to prior studies and the inherent difficulties in recruiting individuals with bilateral congenital cataracts. Importantly, this highlights the value the study brings to the field while also acknowledging the need to replicate the effects in a larger cohort.

Lack of specific control cohort. The control cohort has normal vision. The control cohort is not specific enough to distinguish between people with sight loss due to different causes and patients with congenital cataracts with co-morbidities. Further data from a more specific populations, such as patients whose cataracts have not been removed, with developmental cataracts, or congenitally blind participants, would greatly improve the interpretability of the main finding. The lack of a more specific control cohort is a major caveat that limits a conclusive interpretation of the results.

In the updated version, the authors have indicated that future studies can pursue comparisons between congenital cataract participants and cohorts with later sight loss.

MRS data quality differences. Data quality in the control voxel appears worse than in the visual cortex voxel. The frontal cortex MRS spectrum shows far broader linewidth than the visual cortex (Supplementary Figures). Compared to the visual voxel, the frontal cortex voxel has less defined Glx and GABA+ peaks; lower GABA+ and Glx concentrations, lower NAA SNR values; lower NAA concentrations. If the data quality is a lot worse in the FC, then small effects may not be detectable.

In the updated version, the authors have added more information that informs the reader of the MRS quality differences between voxel locations. This increases the transparency of their reporting and enhances the assessment of the results.

Because of the direction of the difference in E/I, the authors interpret their findings as representing signatures of sight improvement after surgery without further evidence, either within the study or from the literature. However, the literature suggests that plasticity and visual deprivation drives the E/I index up rather than down. Decreasing GABA+ is thought to facilitate experience dependent remodelling. What evidence is there that cortical inhibition increases in response to a visual cortex that is over-sensitised to due congenital cataracts? Without further experimental or literature support this interpretation remains very speculative.

The updated manuscript contains key reference from non-human work to justify their interpretation.

Heterogeneity in patient group. Congenital cataract (CC) patients experienced a variety of duration of visual impairment and were of different ages. They presented with co-morbidities (absorbed lens, strabismus, nystagmus). Strabismus has been associated with abnormalities in GABAergic inhibition in the visual cortex. The possible interactions with residual vision and confounds of co-morbidities are not experimentally controlled for in the correlations, and not discussed.

The updated document has addressed this caveat.

Multiple exploratory correlations were performed to relate MRS measures to visual acuity (shown in Supplementary Materials), and only specific ones shown in the main document. The authors describe the analysis as exploratory in the 'Methods' section. Furthermore, the correlation between visual acuity and E/I metric is weak, not corrected for multiple comparisons. The results should be presented as preliminary, as no strong conclusions can be made from them. They can provide a hypothesis to test in a future study.

This has now been done throughout the document and increases the transparency of the reporting.

P.16 Given the correlation of the aperiodic intercept with age ("Age negatively correlated with the aperiodic intercept across CC and SC individuals, that is, a flattening of the intercept was observed with age"), age needs to be controlled for in the correlation between neurochemistry and the aperiodic intercept. Glx has also been shown to negatively correlates with age.

This caveat has been addressed in the revised manuscript.

Multiple exploratory correlations were performed to relate MRS to EEG measures (shown in Supplementary Materials), and only specific ones shown in the main document. Given the multiple measures from the MRS, the correlations with the EEG measures were exploratory, as stated in the text, p.16, and in Fig.4. yet the introduction said that there was a prior hypothesis "We further hypothesized that neurotransmitter changes would relate to changes in the slope and intercept of the EEG aperiodic activity in the same subjects." It would be great if the text could be revised for consistency and the analysis described as exploratory.

This has been done throughout the document and increases the transparency of the reporting.

The analysis for the EEG needs to take more advantage of the available data. As far as I understand, only two electrodes were used, yet far more were available as seen in their previous study (Ossandon et al., 2023). The spatial specificity is not established. The authors could use the frontal cortex electrode (FP1, FP2) signals as a control for spatial specificity in the group effects, or even better, all available electrodes and correct for multiple comparisons. Furthermore, they could use the aperiodic intercept vs Glx in SC to evaluate the specificity of the correlation to CC.

This caveat has been addressed. The authors have added frontal electrodes to their analysis, providing an essential regional control for the visual cortex location.

Comments on the latest version:

The authors have made reasonable adjustments to their manuscript that addressed most of my comments by adding further justification for their methodology, essential literature support, pointing out exploratory analyses, limitations and adding key control analyses. Their revised manuscript has overall improved, providing valuable information, though the evidence that supports their claims is still incomplete.

Reviewer #1 (Public review):

Contractile Injection Systems (CIS) are versatile machines that can form pores in membranes or deliver effectors. They can act extra or intracellularly. When intracellular they are positioned to face the exterior of the cell and hence should be anchored to the cell envelope. The authors previously reported the characterization of a CIS in Streptomyces coelicolor, including significant information on the architecture of the apparatus. However, how the tubular structure is attached to the envelope was not investigated. Here they provide a wealth of evidence to demonstrate that a specific gene within the CIS gene cluster, cisA, encodes a membrane protein that anchors the CIS to the envelope. More specifically, they show that:

- CisA is not required for assembly of the structure but is important for proper contraction and CIS-mediated cell death<br /> - CisA is associated to the membrane (fluorescence microscopy, cell fractionation) through a transmembrane segment (lacZ-phoA topology fusions in E. coli)<br /> - Structural prediction of interaction between CisA and a CIS baseplate component<br /> - In addition they provide a high-resolution model structure of the >750-polypeptide Streptomyces CIS in its extended conformation, revealing new details of this fascinating machine, notably in the baseplate and cap complexes.

All the experiments are well controlled including trans-complemented of all tested phenotypes.

One important information we miss is the oligomeric state of CisA.

While it would have been great to test the interaction between CisA and Cis11, to perform cryo-electron microscopy assays of detergent-extracted CIS structures to maintain the interaction with CisA, I believe that the toxicity of CisA upon overexpression or upon expression in E. coli render these studies difficult and will require a significant amount of time and optimization to be performed. It is worth mentioning that this study is of significant novelty in the CIS field because, except for Type VI secretion systems, very few membrane proteins or complexes responsible for CIS attachment have been identified and studied.

Reviewer #1 (Public review):

Summary:

Carter et al. present the eduWOSM imaging platform, a promising development in open-source microscopy for educational purposes. The paper outlines the construction and setup of this versatile microscope, demonstrating its capabilities through three key examples: single fluorophore tracking of tubulin heterodimers in gliding microtubules, 4D deconvolution imaging and tracking of chromosome movements in dividing human cells, and automated single-particle tracking in vitro and in live cells, with motion classified into sub-diffusive, diffusive, and super-diffusive behaviors.

The paper is well-written and could be strengthened by providing more empirical data on its performance, addressing potential limitations, and offering detailed insights into its educational impact. The project holds great potential and more discussion on long-term support and broader applications would provide a more comprehensive view of its relevance in different contexts.

Strengths:

(1) The eduWOSM addresses a crucial need in education, providing research-quality imaging at a lower cost (<$10k). The fact that it is open-source adds significant value, enabling broad accessibility even in under resourced areas.<br /> (2) There is availability of extensive resources, including a dedicated website, YouTube channel, and comprehensive tutorial guides to help users replicate the microscope.<br /> (3) The compact, portable, and stable design makes it easy to build multiple systems for use in diverse environments, including crowded labs and classrooms. This is further enhanced by the fact multiple kind of imaging experiments can be run on the system, from live imaging to super-resolution imaging.<br /> (4) The paper highlights the user-friendly nature of the platform, with the imaging examples in the paper being acquired by undergrad students.

Weaknesses:

(1) The paper mentions the microscope is suitable not just for education but even for research purposes. This claim needs validation through quantitative comparison to existing research-grade microscopes in terms of resolution, signal-to-noise ratio, and other key metrics. Adding more rigorous comparisons would solidify its credibility for research use, which would immensely increase the potential of the microscope.<br /> (2) The open-source microscope field is crowded with various options catering to hobby, educational, and research purposes (e.g., openFLexure, Flamingo, Octopi, etc.). The paper would benefit from discussing whether any aspects set the eduWOSM platform apart or fulfill specific roles that other microscopes do not.<br /> (3) While the eduWOSM platform is designed to be user-friendly, the paper would benefit from discussing whether the microscope can be successfully built and operated by users without direct help from the authors. It's important to know if someone with basic technical knowledge, relying solely on the provided resources (website, YouTube tutorials, and documentation), can independently assemble, calibrate, and operate the eduWOSM.<br /> (4) Ensuring long-term support and maintenance of the platform is crucial. The paper would benefit from addressing how the eduWOSM developers plan to support updates, improvements, or troubleshooting.

Reviewer #1 (Public review):

Summary:

The authors show that early life experience of juvenile bats shape their outdoor foraging behaviors. They achieve this by raising juvenile bats either in an impoverished or enriched environment. They subsequently test the behavior of bats indoors and outdoors. The authors show that behavioral measures outdoors were more reliable in delineating the effect of early life experiences as the bats raised in enriched environments were more bold, active and exhibit higher exploratory tendencies.

Strengths:

The major strength of the study is providing a quantitative study of animal "personality" and how it is likely shaped by innate and environmental conditions. The other major strength is the ability to do reliable long term recording of bats in the outdoors giving researchers the opportunity to study bats in their natural habitat. To this point, the study also shows that the behavioral variables measured indoors do not correlate to that measured outdoors, thus providing a key insight into the importance of testing animal behaviors in their natural habitat.

Weaknesses:

It is not clear from the analysis presented in the paper how persistent those environmentally induced changes, do they remain with the bats till the end of their lives.

Reviewer #1 (Public review):

Summary:

The manuscript aims to elucidate the impact of a prophage within the genome of Shewanella fidelis on its interaction with the marine tunicate Ciona robusta. The authors made a deletion mutant of S. fidelis that lacks one of its two prophages. This mutant exhibited an enhanced biofilm phenotype, as assessed through crystal violet staining, and showed reduced motility. The authors examined the effect of prophage deletion on several genes that could modulate cyclic-diGMP levels. While no significant changes were observed under in vitro conditions, the gene for one protein potentially involved in cyclic-diGMP hydrolysis was overexpressed during microbe-host interactions. The mutant was retained more effectively within a one-hour timeframe, whereas the wild-type (WT) strain became more abundant after 24 hours. Fluorescence microscopy was used to visualize the localization patterns of the two strains, which appeared to differ. Additionally, a significant difference in the expression of one immune protein was noted after one hour, but this difference was not evident after 23 hours. An effect of VCBC-C addition on the expression of one prophage gene was also observed.

Strengths:

I appreciate how the authors integrate diverse expertise and methods to address questions regarding the impact of prophages on gut microbiome-host interactions. The chosen model system is appropriate, as it allows for high-throughput experimentation and the application of simple imaging techniques.

Weaknesses:

My primary concern is that the manuscript primarily describes observations without providing insight into the molecular mechanisms underlying the observed differences. It is particularly unclear how the presence of the prophage leads to the phenotypic changes related to bacterial physiology and host-microbe interactions. Which specific prophage genes are critical, or is the insertion at a specific site in the bacterial genome the key factor? While significant effects on bacterial physiology are reported under in vitro conditions, there is no clear attribution to particular enzymes or proteins. In contrast, when the system is expanded to include the tunicate, differences in the expression of a cyclic-diGMP hydrolase become apparent. Why do we not observe such differences under in vitro conditions, despite noting variations in biofilm formation and motility? Furthermore, given that the bacterial strain possesses two prophages, I am curious as to why the authors chose to target only one and not both.

Regarding the microbe-host interaction, it is not clear why the increased retention ability of the prophage deletion strain did not lead to greater cell retention after 24 hours, especially since no differences in the immune response were observed at that time point.

Concerning the methodological approach, I am puzzled as to why the authors opted for qPCR instead of transcriptomics or proteomics. The latter approaches could have provided a broader understanding of the prophage's impact on both the microbe and the host.

Reviewer #1 (Public review):

Summary:

The authors showed the presence of Mtb in human liver biopsy samples of TB patients and reported that chronic infection of Mtb causes immune-metabolic dysregulation. Authors showed that Mtb replicates in hepatocytes in a lipid rich environment created by up regulating transcription factor PPARγ. Authors also reported that Mtb protects itself from anti-TB drugs by inducing drug metabolising enzymes.

Strengths:

It has been shown that Mtb induces storage of triacylglycerol in macrophages by induction of WNT6/ACC2 which helps in its replication and intracellular survival, however, creation of favorable replicative niche in hepatocytes by Mtb is not reported. It is known that Mtb infects macrophages and induces formation of lipid-laden foamy macrophages which eventually causes tissue destruction in TB patients. In a recent article it has been reported that "A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages" that shows how Mtb manipulates host defense mechanisms for its survival. In this manuscript, authors reported the enhancement of lipid droplets in Mtb infected hepatocytes and convincingly showed that fatty acid synthesis and triacylglycerol formation is important for growth of Mtb in hepatocytes. The authors also showed the molecular mechanism for accumulation of lipid and showed that the transcription factor associated with lipid biogenesis, PPARγ and adipogenic genes were upregulated in Mtb infected cells.

The comparison of gene expression data between macrophages and hepatocytes by authors is important which indicates that Mtb modulates different pathways in different cell type as in macrophages it is related to immune response whereas, in hepatocytes it is related to metabolic pathways.

Authors also reported that Mtb residing in hepatocytes showed drug tolerance phenotype due to up regulation of enzymes involved in drug metabolism and showed that cytochrome P450 monooxygenase that metabolize rifampicin and NAT2 gene responsible for N-acetylation of isoniazid were up regulated in Mtb infected cells.

Weaknesses:

There are reports of hepatic tuberculosis in pulmonary TB patients especially in immune-compromised patients, therefore finding granuloma in human liver biopsy samples is not surprising.<br /> Mtb infected hepatic cells showed induced DME and NAT and this could lead to enhanced metabolism of drug by hepatic cells as a result Mtb in side HepG2 cells get exposed to reduced drug concentration and show higher tolerance to drug. The authors mentioned that " hepatocyte resident Mtb may display higher tolerance to rifampicin". In my opinion higher tolerance to drugs is possible only when DME of Mtb inside is up regulated or the target is modified. Although, in the end authors mentioned that drug tolerance phenotype can be better attributed to host intrinsic factors rather than Mtb efflux pumps. It may be better if the Drug tolerant phenotype section can be rewritten to clarify the facts.

Reviewer #1 (Public review):

Summary:

This is an interesting theoretical study examining the viability of Virtual Circular Genome (VCG) model, a recently proposed scenario of prebiotic replication in which a relatively long sequence is stored as a collection of its shorter subsequences (and their compliments). It was previously pointed out that VCG model is prone to so-called sequence scrambling which limits the overall length of such a genome. In the present paper, additional limitations are identified. Specifically, it is shown that VCG is well replicated when the oligomers are elongated by sufficiently short chains from "feedstock" pool. However, ligation of oligomers from VCG itself results in a high error rate. I believe the research is of high quality and well written. However, the presentation could be improved and the key messages could be clarified.

(1) It is not clear from the paper whether the observed error has the same nature as sequence scrambling<br /> (2) The authors introduce two important lengths LS1 and LS2 only in the conclusions and do not explain enough which each of them is important. It would make sense to discuss this early in the manuscript.<br /> (3) It is not entirely clear why specific length distribution for VCG oligomers has to be assumed rather than emerged from simulations.<br /> (4) Furthermore, the problem has another important length, L0 that is never introduced or discussed: a minimal hybridization length with a lifetime longer than the ligation time. From the parameters given, it appears that L0 is sufficiently long (~10 bases). In other words, it appears that the study is done is a somewhat suboptimal regime: most hybridization events do not lead to a ligation. Am I right in this assessment? If that is the case, the authors might want to explore another regime, L0<br /> Strengths:

High-quality theoretical modeling of an important problem is implemented.

Weaknesses:

The conclusions are somewhat convoluted and could be presented better.

Reviewer #1 (Public review):

Summary:

Persistence is a phenomenon by which genetically susceptible cells are able to survive exposure to high concentrations of antibiotics. This is especially a major problem when treating infections caused by slow growing mycobacteria such as M. tuberculosis and M. abscessus. Studies on the mechanisms adopted by the persisting bacteria to survive and evade antibiotic killing can potentially lead to faster and more effective treatment strategies.

To address this, in this study, the authors have used a transposon mutagenesis based sequencing approach to identify the genetic determinants of antibiotic persistence in M. abscessus. To enrich for persisters they employed conditions, that have been reported previously to increase persister frequency - nutrient starvation, to facilitate genetic screening for this phenotype. M.abs transposon library was grown in nutrient rich or nutrient depleted conditions and exposed to TIG/LZD for 6 days, following which Tn-seq was carried out to identify genes involved in spontaneous (nutrient rich) or starvation-induced conditions. About 60% of the persistence hits were required in both the conditions. Pathway analysis revealed enrichment for genes involved in detoxification of nitrosative, oxidative, DNA damage and proteostasis stress. The authors then decided to validate the findings by constructing deletions of 5 different targets (pafA, katG, recR, blaR, Mab_1456c) and tested the persistence phenotype of these strains. Rather surprisingly only 2 of the 5 hits (katG and pafA) exhibited a persistence defect when compared to wild type upon exposure to TIG/LZD and this was complemented using an integrative construct. The authors then investigated the specificity of delta-katG susceptibility against different antibiotic classes and demonstrated increased killing by rifabutin. The katG phenotype was shown to be mediated through the production of oxidative stress which was reverted when the bacterial cells were cultured under hypoxic conditions. Interestingly, when testing the role of katG in other clinical strains of Mab, the phenotype was observed only in one of the clinical strains demonstrating that there might be alternative anti-oxidative stress defense mechanisms operating in some clinical strains.

Strengths:

While the role of ROS in antibiotic mediated killing of mycobacterial cells have been studied to some extent, this paper presents some new findings with regards to genetic analysis of M. abscessus susceptibility, especially against clinically used antibiotics, which makes it useful. Also, the attempts to validate their observations in clinical isolates is appreciated.

Weaknesses:

- Fig. 3 - 5 of the hits from the transposon screen were reconstructed as clean deletion strains and tested for persistence. However, only 1 (katG) gave a strong and 1 (Mab_1456c) exhibited a minor defect. Two of the clones did not show any persistence phenotype (blaR and recR) and one (pafA) showed a minor phenotype, however it was not clear if this difference was really relevant as the mutant exhibited differences at Day 0, prior to the addition of antibiotics. Considering these results from the validation, the conclusion would be that the Tn-seq approach to screen persistence defects is not reliable and is more likely to result in misses than hits.

- Fig 3 - Why is there such a huge difference in the extent of killing of the control strain in media, when exposed to TIG/LZD, when compared to Fig. 1C and Fig. 4. In Fig. 1C, M. abs grown in media decreases by >1 log by Day 3 and >4 log by Day 6, whereas in Fig. 3, the bacterial load decreases by <1 log by Day 3 and <2 log by Day 6. This needs to be clarified, if the experimental conditions were different, because if comparing to Fig. 1C data then the katG mutant strain phenotype is not very different.

Reviewer #1 (Public review):

Summary:

The imaging pipeline presented in this paper is a useful tool for visualizing and dynamically tracking bacterial colony formation at the individual worm level, enabling the study of microbiome colonization's association with host physiology, including lifespan, infection severity, and genetic mutations in real-time. This technique allows for certain biological information to be obtained that was previously missed such as pmk-1 mutants exhibiting a higher rate of colonization by E. coli OP50 than wild-type animals. Overall, this platform could be of interest to many labs studying C. elegans interactions with their microbiome and with bacterial pathogens.

Strengths:

This platform allows for unbiased quantifications of microbe colonization of bacteria at scale. This is particularly important in a field studying dynamic responses or potentially more subtle or variable phenotypes.

Platform could be adapted for multiple uses or potentially other species of nematodes for evolutionary comparisons.

The platform allows researchers to correlate bacterial colonization with predicted lifespan.

Weaknesses:

Platform will require optimization for any given bacteria species which restricts its ease of use for researchers that won't regularly be studying the same bacteria.

Requires the bacteria to be genetically tractable so cannot be easily adapted to microbes that do not have established ways of expressing GFP or other reporters.

This platform requires the use of relatively older adult animals that are more prone to larger gut colonies of bacteria. Thus, studies using this platform are restricted to studying older populations.

The relationship between bacterial colonization and host lifespan requires further investigation. The current SICKO platform and experimentation cannot fully address whether animals in poorer health are more susceptible to colonization, or whether colonization casually contributes to a decline in health. Furthermore, while such effects are statistically significant their effect size in some cases is modest.

for - colonialism - China faces backlash in Mozambique and DRC as violence erupts against corruption 2025, Jan 1

// - comment Widescale violence breaker out in Mozambique over fraudulent election results - Chinese businesses and infrastructure have been targeted as they are seen as corrupt supporters of the current government. - China's corrupt business practices that have kept ordinary people in poverty while destroying the environment. - Years if such exploitation reached a boiling point when the corrupt government won they election again.

//

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.

Comments on revisions:

Your efforts in addressing concerns related to methodological details, narrative clarity, and data representation are commendable. The expanded descriptions of Fig. 1A and the experimental design, as well as the restructuring of the discussion, have greatly enhanced the manuscript's clarity and coherence.

Reviewer #1 (Public review):

Summary:

In this work, a screening platform is presented for rapid and cost-effective screening of candidate genes involved in Fragile Bone Disorders. The authors validate the approach of using crispants, generating FO mosaic mutants, to evaluate the function of specific target genes in this particular condition. The design of the guide RNAs is convincingly described, while the effectiveness of the method is evaluated to 60% to 92% of the respective target genes being presumably inactivated. Thus, injected F0 larvae can be directly used to investigate the consequences of this inactivation.

Skeletal formation is then evaluated at 7dpf and 14dpf, first using a transgenic reporter line revealing fluorescent osteoblasts, second using alizarin-red staining of mineralized structures. In general, it appears that the osteoblast-positive areas are more often affected in the crispants compared to the mineralized areas, an observation that appears to correlate with the observed reduced expression of bglap, a marker for mature osteoblasts, and the increased expression of col1a1a in more immature osteoblasts.

Finally, the injected fish (except two lines that revealed a high mortality) are also analyzed at 90dpf, using alizarin red staining and micro-CT analysis, revealing an increased incidence of skeletal deformities in the vertebral arches, fractures, as well as vertebral fusions and compressions for all crispants except those for daam2. Finally, the Tissue Mineral Density (TMD) as determined by micro-CT is proposed as an important marker for investigating genes involved in osteoporosis.<br /> Taken together, this manuscript is well presented, the data are clear and well analyzed, and the methods well described. It makes a compelling case for using the crispant technology to screen the function of candidate genes in a specific condition, as shown here for bone disorders.

Strengths:

Strengths are the clever combination of existing technologies from different fields to build a screening platform. All the required methods are comprehensively described.

Weaknesses:

One may have wished to bring one or two of the crispants to the stage of bona fide mutants, to confirm the results of the screening, however, this is done for some of the tested genes as laid out in the discussion.

Comments on latest version:

All my issues were resolved.

Reviewer #1 (Public review):

Bacterial effectors that interfere with the inner molecular workings of eukaryotic host cells are of great biological significance across disciplines. On the one hand they help us to understand the molecular strategies that bacteria use to manipulate host cells. On the other hand they can be used as research tools to reveal molecular details of the intricate workings of the host machinery that is relevant for the interaction/defence/symbiosis with bacteria. The authors investigate the function and biological impact of a rhizobial effector that interacts with and modifies, and curiously is modified by, legume receptors essential for symbiosis. The molecular analysis revealed a bacterial effector that cleaves a plant symbiosis signaling receptor to inhibit signaling and the host counterplay by phosphorylation via a receptor kinase. These findings have potential implications beyond bacterial interactions with plants.

Bao and colleagues investigated how rhizobial effector proteins can regulate the legume root nodule symbiosis. A rhizobial effector is described to directly modify symbiosis-related signaling proteins, altering the outcome of the symbiosis. Overall, the paper presents findings that will have a wide appeal beyond its primary field.

Out of 15 identified effectors from Sinorhizobium fredii, they focus on the effector NopT, which exhibits proteolytic activity and may therefore cleave specific target proteins of the host plant. They focus on two Nod factor receptors of the legume Lotus japonicus, NFR1 and NFR5, both of which were previously found to be essential for the perception of rhizobial nod factor, and the induction of symbiotic responses such as bacterial infection thread formation in root hairs and root nodule development (Madsen et al., 2003, Nature; Tirichine et al., 2003; Nature). The authors present evidence for an interaction of NopT with NFR1 and NFR5. The paper aims to characterize the biochemical and functional consequences of these interactions and the phenotype that arises when the effector is mutated.

Evidence is presented that in vitro NopT can cleave NFR5 at its juxtamembrane region. NFR5 appears also to be cleaved in vivo. and NFR1 appears to inhibit the proteolytic activity of NopT by phosphorylating NopT. When NFR5 and NFR1 are ectopically over-expressed in leaves of the non-legume Nicotiana benthamiana, they induce cell death (Madsen et al., 2011, Plant Journal). Bao et al., found that this cell death response is inhibited by the coexpression of nopT. Mutation of nopT alters the outcome of rhizobial infection in L. japonicus. These conclusions are well supported by the data.

The authors present evidence supporting the interaction of NopT with NFR1 and NFR5. In particular, there is solid support for cleavage of NFR5 by NopT (Figure 3) and the identification of NopT phosphorylation sites that inhibit its proteolytic activity (Figure 4C). Cleavage of NFR5 upon expression in N. benthamiana (Figure 3A) requires appropriate controls (inactive mutant versions) that have been provided, since Agrobacterium as a closely rhizobia-related bacterium might increase defense related proteolytic activity in the plant host cells.

Key results from N. benthamiana appear consistent with data from recombinant protein expression in bacteria. For the analysis in the host legume L. japonicus transgenic hairy roots were included. To demonstrate that the cleavage of NFR5 occurs during the interaction in plant cells the authors build largely on western blots. Regardless of whether Nicotiana leaf cells or Lotus root cells are used as the test platform, the Western blots indicate that only a small proportion of NFR5 is cleaved when co-expressed with nopT, and most of the NFR5 persists in its full-length form (Figures 3A-D). It is not quite clear how the authors explain the loss of NFR5 function (loss of cell death, impact on symbiosis), as a vast excess of the tested target remains intact. It is also not clear why a large proportion of NFR5 is unaffected by the proteolytic activity of NopT. This is particularly interesting in Nicotiana in the absence of Nod factor that could trigger NFR1 kinase activity.

Comments on latest version:

The presentation of the figures and the language has greatly improved and the specific mistakes pointed out in the last review have been corrected. I especially appreciate the new images used to illustrate the observed mutant phenotypes, which are much clearer and easier to understand. The pictures used to illustrate the mutant phenotypes seem to be of more comparable root regions than before. Overall, the requested changes have been implemented, with some exceptions described below.

• Figure 1: New representative images are shown for BAX1 and CERK1. These pictures are more consistent with the phenotype seen in other treatments, but since the data has not changed, I presume the data from leaf discs (where the leaf discs for these treatments looked very different) previously shown is still included. The criteria for what was considered cell death is in my opinion still not described in the legend. The cell death/total ratio has been added for all leaf discs, as requested.<br /> • Figure 2: the discussion of the figure now emphasizes direct protein interaction. There is still no size marker in 2D or a description of size in the figure legend, making it difficult to compare the result to Figure 3. If I understand the rebuttal comments correctly, there are other bands on the blot, including non-specific bands. This does not negate the need to include the full blot as a supplemental figure to show cleaved NFR5 as well as other bands. I do not see any other clarifications on this subject in the manuscript.<br /> • Figure 5: From the pictures, it is now easier to understand what is meant by "infection foci". Although there is no description in the methods of how these were distinguished from infection threads, I believe the images are clear enough.<br /> • Figure 6: The changes in the discussion are appreciated, but panel E still misrepresents the evidence in the paper, as from the drawing it still seems that the cleaved NFR5 is somehow directly responsible for suppressing infection when this was not shown

Reviewer #1 (Public review):

Summary:

The authors introduced their previous paper with the concise statement that "the relationships between lineage-specific attributes and genotypic differences of tumors are not understood" (Chen et al., JEM 2019, PMID: 30737256). For example, it is not clear why combined loss of RB1 and TP53 is required for tumorigenesis in SCLC or other aggressive neuroendocrine (NE) cancers, or why the oncogenic mutations in KRAS or EGFR that drive NSCLC tumorigenesis are found so infrequently in SCLC. This is the main question addressed by the previous and current papers.

One approach to this question is to identify a discrete set of genetic/biochemical manipulations that are sufficient to transform non-malignant human cells into SCLC-like tumors. One group reported transformation of primary human bronchial epithelial cells into NE tumors through a complex lentiviral cocktail involving inactivation of pRB and p53 and activation of AKT, cMYC and BCL2 (PARCB) (Park et al., Science 2018, PMID: 30287662). The cocktail previously reported by Chen and colleagues to transform human pluripotent stem-cell (hPSC)-derived lung progenitors (LPs) into NE xenografts was more concise: DAPT to inactivate NOTCH signaling combined with shRNAs against RB1 and TP53. However, the resulting RP xenografts lacked important characteristics of SCLC. Unlike SCLC, these tumors proliferated slowly and did not metastasize, and although small subpopulations expressed MYC or MYCL, none expressed NEUROD1.

MYC is frequently amplified or expressed at high levels in SCLC, and here, the authors have tested whether inducible expression of MYC could increase the resemblance of their hPSC-derived NE tumors to SCLC. These RPM cells (or RPM T58A with stabilized cMYC) engrafted more consistently and grew more rapidly than RP cells, and unlike RP cells, formed liver metastases when injected into the renal capsule. Gene expression analyses reveled that RPM tumor subpopulations expressed NEUROD1, ASCL1 and/or YAP1.

The hPSC-derived RPM model is a major advance over the previous RP model. This may become a powerful tool for understanding SCLC tumorigenesis and progression and for discovering gene dependencies and molecular targets for novel therapies. However, the specific role of cMYC in this model needs to be clarified.

Recommended Revision:

cMYC can drive proliferation, tumorigenesis or apoptosis in a variety of lineages depending on concurrent mutations. For example, in the Park et al., study, normal human prostate cells could be reprogrammed to form adenocarcinoma-like tumors by activation of cMYC and AKT alone, without manipulation of TP53 or RB1. In their previous manuscript, the authors carefully showed the role of each molecular manipulation in NE tumorigenesis. DAPT was required for NE differentiation of LPs to PNECs, shRB1 was required for expansion of the PNECs, and shTP53 was required for xenograft formation. cMYC expression could influence each of these steps, and importantly, could render some steps dispensable. For example, shRB1 was previously necessary to expand the DAPT-induced PNECs, as neither shTP53 nor activation of KRAS or EGFR had no effect on this population, but perhaps cMYC overexpression could expand PNECs even in the presence of pRB, or even induce LPs to become PNECs without DAPT. Similarly, both shRB1 and shTP53 were necessary for xenograft formation, but maybe not if cMYC is overexpressed. If a molecular hallmark of SCLC, such as loss of RB1 or TP53, has become dispensable with the addition of cMYC, this information is critically important in interpreting this as a model of SCLC tumorigenesis.

To interpret the role of cMYC expression in hPSC-derived RPM tumors, we need to know what this manipulation does without manipulation of pRB, p53 or NOTCH, alone or in combination. There are 7 relevant combinations that should be presented in this manuscript: (1) cMYC alone in LPs, (2) cMYC + DAPT, (3) cMYC + shRB1, (4) cMYC + DAPT + shRB1, (5) cMYC + shTP53, (6) cMYC + DAPT + shTP53, and (7) cMYC + shRB1 + shTP53. Wild-type cMYC is sufficient; further exploration with the T58A mutant would not be necessary.

Please present the effects of these combinations on LP differentiation to PNECs, expansion of PNECs as well as other lung cells, xenograft formation and histology, and xenograft growth rate and capacity for metastasis. If this could be clarified experimentally, and the results discussed in the context of other similar approaches such as the Park et al., paper, this study would be a major addition to the field.

Reviewer #1 (Public review):

Summary:

Here the authors convincingly identify and characterize the SERBP1 interactome and further define its role in the nucleus, where it is associated with complexes involved in splicing, cell division, chromosome structure, and ribosome biogenesis. Many of the SERBP1-associated proteins are RNA-binding proteins and SERBP1 exerts its impact, at least in part, through these players. SERBP1 is mostly disordered but along with its associated proteins displays a preference for G4 binding and can can bind to PAR and be PARylated. They present data that strongly suggest that complexes in which SERBP1 participates are assembled through G4 or PAR binding. The authors suggest that because SERBP1 lacks traditional functional domains yet is clearly involved in distinct regulatory complexes, SERBP1 likely acts in the early steps of assembly through the recognition of interacting sites present in RNA, DNA, and proteins.

Strengths:

The data is very convincing and demonstrated through multiple approaches.

Weaknesses:

None. The authors have adequately addressed earlier reviewer concerns.

Reviewer #1 (Public review):

Summary:

In cells undergoing Flavivirus infection, cellular translation is impaired but the viruses themselves escape this inhibition and are efficiently translated. In this study, the authors use very elegant and direct approaches to identify the regions in the 5' and 3' UTRs that are important for this phenomenon and then use them to retrieve two cellular proteins that associate with them and mediate translational shutoff evasion (DDX3 and PABP1). A number of experimental approaches are used with a series of well-controlled experiments that fully support the authors' conclusions.

Strengths:

The work identifies the regions in the 5' and 3' UTRs of the viral genome that mediate the escape of JEV from cellular transcriptional shutoff, they evaluate the infectivity of the mutant viruses bearing or not these structures and even explore their pathogenicity in mice. They then identify the cellular proteins that bind to these regions (DDX3 and PABP1) and determine their role in translation blockade escape, in addition to examining and assessing the conservation of the stem-loop identified in JEV in other Flaviviridae.

In almost all of their systematic analyses, translational effects are put in parallel with the replication kinetics of the different mutant viruses. The experimental thread followed in this study is rigorous and direct, and all experiments are truly well-controlled, fully supporting the authors' conclusions

Reviewer #1 (Public review):

Summary:

The authors revealed the cellular heterogeneity of companion cells (CCs) and demonstrated that the florigen gene FT is highly expressed in a specific subpopulation of these CCs in Arabidopsis. Through a thorough characterization of this subpopulation, they further identified NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1)-like transcription factors as potential new regulators of FT. Overall, these findings are intriguing and valuable, contributing significantly to our understanding of florigen and the photoperiodic flowering pathway. However, there is still room for improvement in the quality of the data and the depth of the analysis. I have several comments that may be beneficial for the authors.

Strengths:

The usage of snRNA-seq to characterize the FT-expressing companion cells (CCs) is very interesting and important. Two findings are novel: 1) Expression of FT in CCs is not uniform. Only a subcluster of CCs exhibits high expression level of FT. 2) Based on consensus binding motifs enriched in this subcluster, they further identify NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1)-like transcription factors as potential new regulators of FT.

Weaknesses:

(1) Title: "A florigen-expressing subpopulation of companion cells". It is a bit misleading. The conclusion here is that only a subset of companion cells exhibit high expression of FT, but this does not imply that other companion cells do not express it at all.<br /> (2) Data quality: Authors opted for fluorescence-activated nuclei sorting (FANS) instead of traditional cell sorting method. What is the rationale behind this decision? Readers may wonder, especially given that RNA abundance in single nuclei is generally lower than that in single cells. This concern also applies to snRNA-seq data. Specifically, the number of genes captured was quite low, with a median of only 149 genes per nucleus. Additionally, the total number of nuclei analyzed was limited (1,173 for the pFT:NTF and 3,650 for the pSUC2:NTF). These factors suggest that the quality of the snRNA-seq data presented in this study is quite low. In this context, it becomes challenging for the reviewer to accurately assess whether this will impact the subsequent conclusions of the paper. Would it be possible to repeat this experiment and get more nuclei?<br /> (3) Another disappointment is that the authors did not utilize reporter genes to identify the specific locations of the FT-high expressing cells (cluster 7 cells) within the CC population in vivo. Are there any discernible patterns that can be observed?<br /> (4) The final disappointment is that the authors only compared FT expression between the nigtQ mutants and the wild type. Does this imply that the mutant does not have a flowering time defect particularly under high nitrogen conditions?

Reviewer #1 (Public review):

Summary

Chabukswar et al analysed endogenous retrovirus (ERV) Env variation in a set of primate genomes using consensus Env sequences from ERVs known to be present in hominoids using a Blast homology search with the aim of characterising env gene changes over time. The retrieved sequences were analysed phylogenetically, and showed that some of the integrations are LTR-env recombinants.

Strengths

The strength of the manuscript is that such an analysis has not been performed yet for the subset of ERV Env genes selected and most of the publicly available primate genomes.

Weaknesses

Unfortunately, the weaknesses of the manuscript outnumber its strengths. Especially the methods section does not contain sufficient information to appreciate or interpret the results. The results section contains methodological information that should be moved, while the presentation of the data is often substandard. For instance, the long lists of genomes in which a certain Env was found could better be shown in tables. Furthermore, there is no overview of the primate genomes, or accession numbers, used. It is unclear whether the analyses, such as the phylogenetic trees, are based on nucleotide or amino acid sequences since this is not stated. tBLASTn was used in the homology searches, so one would suppose aa are retrieved. In the Discussion, both env (nt?) and Env (aa?) are used.

For the non-hominoids, genome assembly of publicly available sequences is not always optimal, and this may require Blasting a second genome from a species. Which should for instance be done for the HML2 sequences found in the Saimiri boliviensis genome, but not in the related Callithrix jacchus genome. Finally, the authors propose to analyse recombination in Env sequences but only retrieve env-LTR recombinant Envs, which should likely not have passed the quality check.

Since the Methods section does not contain sufficient information to understand or reproduce the results, while the Results are described in a messy way, it is unclear whether or not the aims have been achieved. I believe not, as characterisation of env gene changes over time is only shown for a few abberrant integrations containing part of the LTR in the env ORF.

Reviewer #1 (Public review):

Summary:

In this study, the authors sequenced emm89 serotype genomes of clinical isolates from patients in Japan, where the number of invasive Group A Streptococcus (GAS), especially those of the emm89 serotype, has drastically increased over the past 10-15 years. The sequences from this cohort were compared against a large collection of publicly available global isolates, yielding a total of almost 1000 genomes in the analysis. Because the researchers focused on the emm89 serotype, they could construct a common core genome, with subsequent ability to analyze genomic differences in accessory genes and intergenic regions that contributed to the invasive phenotype using multiple types of GWAS analysis (SNP, k-mer). Their analysis demonstrates some mutations responsible for invasiveness are specific to the Japanese strains, and that multiple independent virulence factors can contribute to invasiveness. None of the invasive phenotypes were correlated with new gene acquisition. Together, the data support that synergy between bacterial survival and upregulation of virulence factors contributes to the development of severe infection.

Strengths:

• The authors verify their analysis by confirming that covS is one of the more frequently mutated genes in invasive strains of GAS, as has been shown in other publications.

• A mutation in one of the SNPs attributed to invasiveness (SNP fhuB) was introduced into an invasive strain. The authors demonstrate that this mutant strain survives less well in human blood. Therefore, the authors have experimental data to support their claims that their analysis uncovered a new mutation/SNP that contributed to invasiveness.

Weaknesses:

• It would be helpful for the authors to highlight why their technique (large scale analysis of one emm type) can yield more information than a typical GWAS analysis of invasive vs. non-invasive strains. Are SNPs easier to identify using a large-scale core genome? Is it more likely evolutionarily to find mutations in non-coding regions as opposed to the core genome and accessory genes, and this is what this technique allows? Did the analysis yield unexpected genes or new genes that had not been previously identified in other GWAS analyses? These points may need to be made more apparent in the results and deserve some thought in the discussion section.

• The Alpha-fold data does not demonstrate why the mutations the authors identified could contribute to the invasive phenotype. It would be helpful to show an overlay of the predicted structures containing the different SNPs to demonstrate the potential structural differences that can occur due to the SNP. This would make the data more convincing that the SNP has a potential impact on the function of the protein. Similarly, the authors discuss modification of the hydrophobicity of the side chain in the ferrichrome transporter (lines 317-318) due to a SNP, but this is not immediately obvious in the figure (Fig. 5).

Reviewer #1 (Public review):

Overall, the manuscript reveals the role of actin polymerization to drive the fusion of myoblasts during adult muscle regeneration. This pathway regulates fusion in many contexts, but whether it was conserved in adult muscle regeneration remained unknown. Robust genetic tools and histological analyses were used to support the claims convincingly.

There are a few interpretations that could be adjusted.

The beginning of the results about macrophages traversing ghost fibers after regeneration was a surprise given the context in the abstract and introduction. These results also lead to new questions about this biology that would need to be answered to substantiate the claims in this section. Also, it is unclear the precise new information learned here because it seems obvious that macrophages would need to extravasate the basement membrane to enter ghost fibers and macrophages are known to have this ability. Moreover, the model in Figure 4D has macrophages and BM but there is not even mention of this in the legend. The authors may wish to consider removing this topic from the manuscript.

Which Pax7CreER line was used? In the methods, the Jax number provided is the Gaka line but in the results, Lepper et al 2009 are cited, which is not the citation for the Gaka line.

Did the authors assess regeneration in the floxed mice that do not contain Cre as a control? Or is it known these alleles do not perturb the function of the targeted gene?

The authors comment: 'Interestingly, expression of the fusogenic proteins, MymK and MymX, was up-regulated in the TA muscle of these mice (Fig. S4F), suggesting that fusogen overexpression is not able to rescue the SCM fusion defect resulted from defective branched actin polymerization.' It is unclear if fusogens are truly overexpressed because the analysis is performed at dpi 4 when the expression of fusogens may be decreased in control mice because they have already fused. Also, only two animals were analyzed and it is unclear if MymX is definitively increased. The authors should consider adjusting the interpretation to SCM fusion defect resulting from defective branched actin polymerization is unlikely to be caused by a lack of fusogen expression.

Reviewer #1 (Public review):

Summary:

The authors use FIB SEM methods to generate 3D volumes of almost all cells comprising a miniature wasp eye and describe the anatomy of each cell type in detail. The function of each cell type is determined through comparisons with descriptions using other methods from larger insect species.

Strengths:

The data show that, despite the small size, many elements of the eye are consistent with those found in larger insects. In addition, the powerful FIB-SEM technique revealed a hitherto unknown case of ectopic photoreceptors.

Weaknesses:

As this paper only uses anatomical analyses, no functional interpretations of cell function are tested.

The aim of this paper was to describe the ultrastructural organization of compound eyes in the extremely small wasp Megaphragma viggianii. The authors successfully achieved this aim and provided an incredibly detailed description of all cell types with respect to their location, volume, and dimensions. As this is the first of its kind, the results cannot easily be compared with previous work. The findings are likely to be an important reference for future work that uses similar techniques to reconstruct the eyes of other insect species. The FIB-SEM method used is being used increasingly often in structural studies of insect sensory organs and brains and this work demonstrates the utility of this method.

Reviewer #1 (Public review):

Summary:

Mallimadugula et al. combined Molecular Dynamics (MD) simulations, thiol-labeling experiments, and RNA-binding assays to study and compare the RNA-binding behavior of the Interferon Inhibitory Domain (IID) from Viral Protein 35 (VP35) of Zaire ebolavirus, Reston ebolavirus, and Marburg marburgvirus. Although the structures and sequences of these viruses are similar, the authors suggest that differences in RNA binding stem from variations in their intrinsic dynamics, particularly the opening of a cryptic pocket. More precisely, the dynamics of this pocket may influence whether the IID binds to RNA blunt ends or the RNA backbone.

Overall, the authors present important findings to reveal how the intrinsic dynamics of proteins can influence their binding to molecules and, hence, their functions. They have used extensive biased simulations to characterize the opening of a pocket which was not clearly seen in experimental results - at least when the proteins were in their unbound forms. Biochemical assays further validated theoretical results and linked them to RNA binding modes. Thus, with the combination of biochemical assays and state-of-the-art Molecular Dynamics simulations, these results are clearly compelling.

Strengths:

The use of extensive Adaptive Sampling combined with biochemical assays clearly points to the opening of the Interferon Inhibitory Domain (IID) as a factor for RNA binding. This type of approach is especially useful to assess how protein dynamics can affect its function.

Weaknesses :

Although a connection between the cryptic pocket dynamics and RNA binding mode is proposed, the precise molecular mechanism linking pocket opening to RNA binding still remains unclear.

Reviewer #1 (Public review):

Summary:

In this manuscript, Pakula et al. explore the impact of reactive oxygen species (ROS) on neonatal cerebellar regeneration, providing evidence that ROS activates regeneration through Nestin-expressing progenitors (NEPs). Using scRNA-seq analysis of FACS-isolated NEPs, the authors characterize injury-induced changes, including an enrichment in ROS metabolic processes within the cerebellar microenvironment. Biochemical analyses confirm a rapid increase in ROS levels following irradiation, and forced catalase expression, which reduces ROS levels, and impairs external granule layer (EGL) replenishment post-injury.

Strengths:

Overall, the study robustly supports its main conclusion and provides valuable insights into ROS as a regenerative signal in the neonatal cerebellum.

Weaknesses:

Below are specific comments and concerns:

(1) The diversity of cell types recovered from scRNA-seq libraries of sorted Nes-CFP cells is unexpected, especially the inclusion of minor types such as microglia, meninges, and ependymal cells. The authors should validate whether Nes and CFP mRNAs are enriched in the sorted cells; if not, they should discuss the potential pitfalls in sampling bias or artifacts that may have affected the dataset, impacting interpretation.<br /> (2) The authors should de-emphasize that ROS signaling and related gene upregulation exclusively in gliogenic NEPs. Genes such as Cdkn1a, Phlda3, Ass1, and Bax are identified as differentially expressed in neurogenic NEPs and granule cell progenitors (GCPs), with Ass1 absent in GCPs. According to Table S4, gene ontology (GO) terms related to ROS metabolic processes are also enriched in gliogenic NEPs, neurogenic NEPs, and GCPs.<br /> (3) The authors need to justify the selection of only the anterior lobe for EGL replenishment and microglia quantification.<br /> (4) Figure 1K: The figure presents linkages between genes and GO terms as a network but does not depict a gene network. The terminology should be corrected accordingly.<br /> (5) Figure 1H and S2: The x-axis appears to display raw p-values rather than log10(p.value) as indicated. The x-axis should ideally show -log10(p.adjust), beginning at zero. The current format may misleadingly suggest that the ROS GO term has the lowest p-values.<br /> (6) Genes such as Ppara, Egln3, Foxo3, Jun, and Nos1ap were identified by bulk ATAC-seq based on proximity to peaks, not by scRNA-seq. Without additional expression data, caution is needed when presenting these genes as direct evidence of ROS involvement in NEPs.<br /> (7) The authors should annotate cell identities for the different clusters in Table S2.<br /> (8) Reiterative clustering analysis reveals distinct subpopulations among gliogenic and neurogenic NEPs. Could the authors clarify the identities of these subclusters? Can we distinguish the gliogenic NEPs in the Bergmann glia layer from those in the white matter?<br /> (9) In the Methods section, the authors mention filtering out genes with fewer than 10 counts. They should specify if these genes were used as background for enrichment analysis. Background gene selection is critical, as it influences the functional enrichment of gene sets in the list.<br /> (10) Figure S1C: The authors could consider using bar plots to better illustrate cell composition differences across conditions and replicates.<br /> (11) Figures 4-6: It remains unclear how the white matter microglia contribute to the recruitment of BgL-NEPs to the EGL, as the mCAT-mediated microglia loss data are all confined to the white matter.

Reviewer #1 (Public review):

Summary:

This manuscript investigates genes that escape X-Chromosome Inactivation (XCI) across human tissues, using females that exhibit skewed or non-random XCI. The authors identified 2 female individuals with skewed XCI in the GTex database, in addition to the 1 female skewed sample in this database that has been described in a previous publication (Ref.16). The authors also determined the genes that escape XCI for 380 X-linked genes across 30 different tissues.

Strengths:

The novelty of this manuscript is that the authors have identified the XCI expression status for a total of 380 genes across 30 different human tissues, and also discovered the XCI status (escape, variable escape, or silenced) for 198 X-linked genes, whose status was previously not determined. This report is a good resource for the field of XCI, and would benefit from additional analyses and clarification of their comparisons of XCI status.

Weaknesses:

Specific comments:

(1) The authors state that they have reclassified the allelic expression status of 32 genes (shown in Table S5, Supplementary Figure 3). The concern is the source of the tissue or cell line which was originally used to make the classification of XCI status, and whether the comparisons are equivalent. For example, if cell lines (and not tissues) were used to define the XCI status for EGFL6, TSPAN6, and CXorf38, then how can the authors be sure that the escape status in whole tissues would be the same? Also along these lines, the authors should consider whether escape status in previous studies using immortalized/cancer cell lines (such as the meta analyses done in Balaton publication) would be different compared to healthy tissues (seems like it should be). Therefore making comparisons between healthy whole tissues and cancer cell lines doesn't make sense.

(2) The authors note that skewed XCI is prevalent in the human population, and cite some publications (references 8, 10-12). If RNAseq data is available from these female individuals with skewed XCI (such as ref 12), the authors should consider using their allelic expression pipeline to identify XCI status of more X-linked genes.

(3) It has been well established that the human inactive X has more XCI escape genes compared to the mouse inactive X. In light of the author's observations across human tissues, how does the XCI status compare with the same tissues in mice?

Reviewer #1 (Public review):

Summary<br /> In this beautiful paper the authors examined the role and function of NR2F2 in testis development and more specifically on fetal Leydig cells development. It is well known by now that FLC are developed from an interstitial steroidogenic progenitors at around E12.5 and are crucial for testosterone and INSL3 production during embryonic development, which in turn shapes the internal and external genitalia of the male. Indeed, lack of testosterone or INSL3 are known to cause DSD as well as undescended testis, also termed as cryptorchidism.<br /> The authors first characterized the expression pattern of the NR2R2 protein during testis development and then used two cKO systems of NR2F2, namely the Wt1-creERT2 and the Nr5a1-cre to explore the phenotype of loss of NR2F2. They found in both cases that mice are presenting with undescended testis and major reduction in FLC numbers. They show that NR2F2 has no effect on the amount and expression of the progenitor cells but in its absence, there are less FLC and they are immature.<br /> The effect of NR2F2 is cell autonomous and does not seem to affect other signalling pathways implemented in Leydig cell development as the DHH, PDGFRA and the NOTCH pathway.

Overall, this paper is excellent, very well written, fluent and clear. The data is well presented, and all the controls and statistics are in place. I think this paper will be of great interest to the field and paves the way for several interesting follow up studies as stated in the discussion

Reviewer #1 (Public review):

Summary:

This study examines the role of host blood meal source, temperature, and photoperiod on the reproductive traits of Cx. quinquefasciatus, an important vector of numerous pathogens of medical importance. The host use pattern of Cx. quinquefasciatus is interesting in that it feeds on birds during spring and shifts to feeding on mammals towards fall. Various hypotheses have been proposed to explain the seasonal shift in host use in this species but have provided limited evidence. This study examines whether the shifting of host classes from birds to mammals towards autumn offers any reproductive advantages to Cx. quinquefasciatus in terms of enhanced fecundity, fertility, and hatchability of the offspring. The authors found no evidence of this, suggesting that alternate mechanisms may drive the seasonal shift in host use in Cx. quinquefasciatus.

Strengths:

Host blood meal source, temperature, and photoperiod were all examined together.

Weaknesses:

The study was conducted in laboratory conditions with a local population of Cx. quinquefasciatus from Argentina. I'm not sure if there is any evidence for a seasonal shift in the host use pattern in Cx. quinquefasciatus populations from the southern latitudes.

Comments on the revision:

Overall, the manuscript is much improved. However, the introduction and parts of the discussion that talk about addressing the question of seasonal shift in host use pattern of Cx. quin are still way too strong and must be toned down. There is no strong evidence to show this host shift in Argentinian mosquito populations. Therefore, it is just misleading. I suggest removing all this and sticking to discussing only the effects of blood meal source and seasonality on the reproductive outcomes of Cx. quin.

Reviewer #1 (Public review):

The aim of this paper is to describe a novel method for genetic labelling of animals or cell populations, using a system of DNA/RNA barcodes.

Strengths:

• The author's attempt at providing a straightforward method for multiplexing Drosophila samples prior to scRNA-seq is commendable. The perspective of being able to load multiple samples on a 10X Chromium without antibody labelling is appealing.<br /> • The authors are generally honest about potential issues in their method, and areas that would benefit from future improvement.<br /> • The article reads well. Graphs and figures are clear and easy to understand.

Weaknesses:

• The usefulness of TaG-EM for phototaxis, egg laying or fecundity experiments is questionable. The behaviours presented here are all easily quantifiable, either manually or using automated image-based quantification, even when they include a relatively large number of groups and replicates. Despite their claims (e.g., L311-313), the authors do not present any real evidence about the cost- or time-effectiveness of their method in comparison to existing quantification methods.<br /> • Behavioural assays presented in this article have clear outcomes, with large effect sizes, and therefore do not really challenge the efficiency of TaG-EM. By showing a T-maze in Fig 1B, the authors suggest that their method could be used to quantify more complex behaviours. Not exploring this possibility in this manuscript seems like a missed opportunity.<br /> • Experiments in Figs S3 and S6 suggest that some tags have a detrimental effect on certain behaviours or on GFP expression. Whereas the authors rightly acknowledge these issues, they do not investigate their causes. Unfortunately, this question the overall suitability of TaG-EM, as other barcodes may also affect certain aspects of the animal's physiology or behaviour. Revising barcode design will be crucial to make sure that sequences with potential regulatory function are excluded.<br /> • For their single-cell experiments, the authors have used the 10X Genomics method, which relies on sequencing just a short segment of each transcript (usually 50-250bp - unknown for this study as read length information was not provided) to enable its identification, with the matching paired-end read providing cell barcode and UMI information (Macosko et al., 2015). With average fragment length after tagmentation usually ranging from 300-700bp, a large number of GFP reads will likely not include the 14bp TaG-EM barcode. When a given cell barcode is not associated with any TaG-EM barcode, then demultiplexing is impossible. This is a major problem, which is particularly visible in Figs 5 and S13. In 5F, BC4 is only detected in a couple of dozen cells, even though the Jon99Ciii marker of enterocytes is present in a much larger population (Fig 5C). Therefore, in this particular case, TaG-EM fails to detect most of the GFP-expressing cells. Similarly, in S13, most cells should express one of the four barcodes, however many of them (maybe up to half - this should be quantified) do not. Therefore, the claim (L277-278) that "the pan-midgut driver were broadly distributed across the cell clusters" is misleading. Moreover, the hypothesis that "low expressing driver lines may result in particularly sparse labelling" (L331-333) is at least partially wrong, as Fig S13 shows that the same Gal4 driver can lead to very different levels of barcode coverage.<br /> • Comparisons between TaG-EM and other, simpler methods for labelling individual cell populations are missing. For example, how would TaG-EM compare with expression of different fluorescent reporters, or a strategy based on the brainbow/flybow principle?<br /> • FACS data is missing throughout the paper. The authors should include data from their comparative flow cytometry experiment of TaG-EM cells with or without additional hexameric GFP, as well as FSC/SSC and fluorescence scatter plots for the FACS steps that they performed prior to scRNA-seq, at least in supplementary figures.<br /> • The authors should show the whole data described in L229, including the cluster that they chose to delete. At least, they should provide more information about how many cells were removed. In any case, the fact that their data still contains a large number of debris and dead cells despite sorting out PI negative cells with FACS and filtering low abundance barcodes with Cellranger is concerning.

Overall, although a method for genetic tagging cell populations prior to multiplexing in single-cell experiments would be extremely useful, the method presented here is inadequate. However, despite all the weaknesses listed above, the idea of barcodes expressed specifically in cells of interest deserves more consideration. If the authors manage to improve their design to resolve the major issues and demonstrate the benefits of their method more clearly, then TaG-EM could become an interesting option for certain applications.

Comments on revisions:

The authors have addressed many important points, providing reassurances about the initial weaknesses of their work. Although the TaG-EM is unlikely to have a significant influence on the field due to its limited benefits, the results are now sound and provide the reader with an unbiased view of the possibilities and limitations of the method.

Reviewer #1 (Public review):

Summary:

BMP signaling is, arguably, best known for its role in the dorsoventral patterning, but not in nematodes, where it regulates body size. In their paper, Vora et al. analyze ChIP-Seq and RNA-Seq data to identify direct transcriptional targets of SMA-3 (Smad) and SMA-9 (Schnurri) and understand the respective roles of SMA-3 and SMA-9 in the nematode model Caenorhabditis elegans. The Authors use SMA-3 and SMA-9 ChIP-Seq data and RNA-Seq data from SMA-3 and SMA-9 mutants, and bioinformatic analyses to identify the genes directly controlled by these two transcription factors (TFs) and find approximately 350 such targets for each. They show that all SMA-3-controlled targets are positively controlled by SMA-3 binding, while SMA-9-controlled targets can be either up- or downregulated by SMA-9. 129 direct targets were shared by SMA-3 and SMA-9, and, curiously, the expression of 15 of them was activated by SMA-3 but repressed by SMA-9. In case of such opposing effects, the SMA-9 appears to act epistatically to SMA-3. Since genes responsible for cuticle collagen production were eminent among the SMA-3 targets, the Authors focused on trying to understand the body size defect known to be elicited by the modulation of BMP signaling. Vora et al. provide compelling evidence that this defect is likely to be due to problems with the BMP signaling-dependent collagen secretion necessary for cuticle formation.

Strengths:

Vora et al. provide a valuable analysis of ChIP-Seq and RNA-Seq datasets, which will be very useful for the community. They also shed light on the mechanism of the BMP-dependent body size control by identifying SMA-3 target genes regulating cuticle collagen synthesis and by showing that downregulation of these genes affects body size in C. elegans.

Weaknesses:

(1) Although the analysis of the SMA-3 and SMA-9 ChIP-Seq and RNA-Seq data is extremely useful, the goal "to untangle the roles of Smad and Schnurri transcription factors in the developing C. elegans larva", has not been reached. While the role of SMA-3 as a transcriptional activator appears to be quite straightforward, the function of SMA-9 in the BMP signaling remains obscure.

(2) The Authors clearly show that both TFs can bind independently of each other, however, by using distances between SMA-3 and SMA-9 ChIP peaks, they claim that when the peaks are close these two TFs likely act as complexes. In the absence of proof that SMA-3 and SMA-9 physically interact (e.g. that they co-immunoprecipitate - as they do in Drosophila), this is an unfounded claim, which still has to be experimentally substantiated. In the revised version of the manuscript, the authors acknowledge this.

(3) The second part of the results (the collagen story) is loosely connected the first part. dpy-11 encodes an enzyme important for cuticle development, and it is a differentially expressed direct target of SMA-3. dpy-11 can be bound by SMA-9, but it is not affected by this binding according to RNA-Seq. Thus, technically, this part of the paper does not require any information about SMA-9. However, this can likely be improved by addressing the function of the 15 genes, with the opposing mode of regulation by SMA-3 and SMA-9.

Comments on revisions:

In comparison to the first version of the manuscript, the authors have significantly improved the "readability" of the paper, made the Discussion much better, and toned down some of the less supported arguments.

Reviewer #1 (Public review):

Summary:

The study aims to create a comprehensive repository about the changes in protein abundance and their modification during oocyte maturation in Xenopus laevis.

Strengths:

The results contribute meaningfully to the field.

Weaknesses:

The manuscript could have benefitted from more comprehensive analyses and clearer writing. Nonetheless, the key findings are robust and offer a valuable resource for the scientific community.

Reviewer #1 (Public review):

Summary:

In this article, Gupta and colleagues explore the parameters that could promote the elimination of active Ras cells when surrounded by WT cells. The elimination of active Ras cells by surrounding WT cells was previously described extensively and associated with a process named cell competition, a context dependant elimination of cells. Several mechanisms have been associated with competition, including more recently elimination processes based on mechanical stress. This was explored theoretically and experimentally and was either associated with differential growth and sensitivity to pressure and/or differences in homeostatic density/pressure. This was extensively validated for the case of Scribble mutant cells which are eliminated by WT MDCK cells due to their higher homeostatic density. However, there has been so far very little systematic characterisation of the mechanical parameters and properties of these different cell types and how this could contribute to mechanical competition.

Here, the authors used the context of active Ras cells in MDCK cells (with some observations in vivo in mice gut which are a bit more anecdotal) to explore the parameters causal to Ras cell elimination. Using for the first time traction force microscopy, stress microscopy combined with Bayesian inference, they first show that clusters of active Ras cells experience higher pressure compared to WT. Interestingly, this occurs in absence of differences in growth rate, and while Ras cells seems to have lower homeostatic density, in contractions with the previous models associated with mechanical cell competition. Using a self-propelled Voronoi model, they explored more systematically the conditions that will promote the compression of transformed cells, showing globally that higher Area compressibility and/or lower junctional tension are associated with higher compressibility. Using then an original and novel experimental method to measure bulk compressibility of cell populations, they confirmed that active Ras cells are globally twice more compressible than WT cells. This compressibility correlates with a disruption of adherens junctions. Accordingly, the higher pressure near transformed Ras cells can be completely rescued by increasing cell-cell adhesion through E-cad overexpression, which also reduces the compressibility of the transformed cells. Altogether, these results go along the lines of a previous theoretical work (Gradeci et al. eLife 2021) which was suggesting that reduced stiffness/higher compressibility was essential to promote loser cell elimination. Here, the authors provide for the first time a very convincing experimental measurement and validation of this prediction. Moreover, their modelling approach goes far beyond what was performed before in terms of exploration of conditions promoting compressibility, and their experimental data point at alternative mechanisms that may contribute to mechanical competition.

Strengths:

- Original methodologies to perform systematic characterisation of mechanical properties of Ras cells during cell competition, which include a novel method to measure bulk compressibility.<br /> - A very extensive theoretical exploration of the parameters promoting cell compaction in the context of competition.

Weaknesses:

- Most of the theoretical focus is centred on the bulk compressibility, but so far does not really explain the final fate of the transformed cells. Classic cell competition scenario (including the one involving active Ras cells) lead to the elimination of one cell population either by cell extrusion/cell death or global delamination. This aspect is absolutely not explored in this article, experimentally or theoretically, and as such it is difficult to connect all the observables with the final outcome of cell competition. For instance, higher compressibility may not lead to loser status if the cells can withstand high density without extruding compared to the WT cells (and could even completely invert the final outcome of the competition). Down the line, and as suggested in most of the previous models/experiments, the relationship between pressure/density and extrusion/death will be the key factor that determine the final outcome of competition. However, there is absolutely no characterisation of cell death/cell extrusion in the article so far.

- While the compressibility measurement are very original and interesting, this bulk measurement could be explained by very different cellular processes, from modulation of cell shape, to cell extrusion and tissue multilayering (which by the way was already observed for active Ras cells, see for instance https://pubmed.ncbi.nlm.nih.gov/34644109/). This could change a lot the interpretation of this measurement and to which extend it can explain the compression observed in mixed culture. This compressibility measurement could be much more informative if coupled with an estimation of the change of cell aspect ratio and the rough evaluation of the contribution of cell shape changes versus alternative mechanisms.

- So far, there is no clear explanation of why transformed Ras cells get more compacted in the context of mixed culture compared to pure Ras culture. Previously, the compaction of mutant Scribble cells could be explained by the higher homeostatic density of WT cells which impose their prefered higher density to Scribble mutant (see Wagstaff et al. 2016 or Gradeci et al 2021), however that is not the case of the Ras cells (which have even slightly higher density at confluency). If I understood properly, the Voronoid model assumes some directional movement of WT cell toward transformed which will actively compact the Ras cells through self-propelled forces (see supplementary methods), but this is never clearly discussed/described in the results section, while potentially being one essential ingredient for observing compaction of transformed cells. In fact, this was already described experimentally in the case of Scribble competition and associated with chemoattractant secretion from the mutant cells promoting directed migration of the WT (https://pubmed.ncbi.nlm.nih.gov/33357449/). It would be essential to show what happens in absence of directional propelled movement in the model and validate experimentally whether there is indeed directional movement of the WT toward the transformed cells. Without this, the current data does not really explain the competition process.

- Some of the data lack a bit of information on statistic, especially for all the stress microscopy and traction forces where we do no really know how representative at the stress patterns (how many experiment, are they average of several movies ? integrated on which temporal window ?)

Reviewer #1 (Public review):

Summary:

In this article, Gupta and colleagues explore the parameters that could promote the elimination of active Ras cells when surrounded by WT cells. The elimination of active Ras cells by surrounding WT cells was previously described extensively and associated with a process named cell competition, a context dependant elimination of cells. Several mechanisms have been associated with competition, including more recently elimination processes based on mechanical stress. This was explored theoretically and experimentally and was either associated with differential growth and sensitivity to pressure and/or differences in homeostatic density/pressure. This was extensively validated for the case of Scribble mutant cells which are eliminated by WT MDCK cells due to their higher homeostatic density. However, there has been so far very little systematic characterisation of the mechanical parameters and properties of these different cell types and how this could contribute to mechanical competition.

Here, the authors used the context of active Ras cells in MDCK cells (with some observations in vivo in mice gut which are a bit more anecdotal) to explore the parameters causal to Ras cell elimination. Using for the first time traction force microscopy, stress microscopy combined with Bayesian inference, they first show that clusters of active Ras cells experience higher pressure compared to WT. Interestingly, this occurs in absence of differences in growth rate, and while Ras cells seems to have lower homeostatic density, in contractions with the previous models associated with mechanical cell competition. Using a self-propelled Voronoi model, they explored more systematically the conditions that will promote the compression of transformed cells, showing globally that higher Area compressibility and/or lower junctional tension are associated with higher compressibility. Using then an original and novel experimental method to measure bulk compressibility of cell populations, they confirmed that active Ras cells are globally twice more compressible than WT cells. This compressibility correlates with a disruption of adherens junctions. Accordingly, the higher pressure near transformed Ras cells can be completely rescued by increasing cell-cell adhesion through E-cad overexpression, which also reduces the compressibility of the transformed cells. Altogether, these results go along the lines of a previous theoretical work (Gradeci et al. eLife 2021) which was suggesting that reduced stiffness/higher compressibility was essential to promote loser cell elimination. Here, the authors provide for the first time a very convincing experimental measurement and validation of this prediction. Moreover, their modelling approach goes far beyond what was performed before in terms of exploration of conditions promoting compressibility, and their experimental data point at alternative mechanisms that may contribute to mechanical competition.

Strengths:

- Original methodologies to perform systematic characterisation of mechanical properties of Ras cells during cell competition, which include a novel method to measure bulk compressibility.<br /> - A very extensive theoretical exploration of the parameters promoting cell compaction in the context of competition.

Weaknesses:

- Most of the theoretical focus is centred on the bulk compressibility, but so far does not really explain the final fate of the transformed cells. Classic cell competition scenario (including the one involving active Ras cells) lead to the elimination of one cell population either by cell extrusion/cell death or global delamination. This aspect is absolutely not explored in this article, experimentally or theoretically, and as such it is difficult to connect all the observables with the final outcome of cell competition. For instance, higher compressibility may not lead to loser status if the cells can withstand high density without extruding compared to the WT cells (and could even completely invert the final outcome of the competition). Down the line, and as suggested in most of the previous models/experiments, the relationship between pressure/density and extrusion/death will be the key factor that determine the final outcome of competition. However, there is absolutely no characterisation of cell death/cell extrusion in the article so far.

- While the compressibility measurement are very original and interesting, this bulk measurement could be explained by very different cellular processes, from modulation of cell shape, to cell extrusion and tissue multilayering (which by the way was already observed for active Ras cells, see for instance https://pubmed.ncbi.nlm.nih.gov/34644109/). This could change a lot the interpretation of this measurement and to which extend it can explain the compression observed in mixed culture. This compressibility measurement could be much more informative if coupled with an estimation of the change of cell aspect ratio and the rough evaluation of the contribution of cell shape changes versus alternative mechanisms.

- So far, there is no clear explanation of why transformed Ras cells get more compacted in the context of mixed culture compared to pure Ras culture. Previously, the compaction of mutant Scribble cells could be explained by the higher homeostatic density of WT cells which impose their prefered higher density to Scribble mutant (see Wagstaff et al. 2016 or Gradeci et al 2021), however that is not the case of the Ras cells (which have even slightly higher density at confluency). If I understood properly, the Voronoid model assumes some directional movement of WT cell toward transformed which will actively compact the Ras cells through self-propelled forces (see supplementary methods), but this is never clearly discussed/described in the results section, while potentially being one essential ingredient for observing compaction of transformed cells. In fact, this was already described experimentally in the case of Scribble competition and associated with chemoattractant secretion from the mutant cells promoting directed migration of the WT (https://pubmed.ncbi.nlm.nih.gov/33357449/). It would be essential to show what happens in absence of directional propelled movement in the model and validate experimentally whether there is indeed directional movement of the WT toward the transformed cells. Without this, the current data does not really explain the competition process.

- Some of the data lack a bit of information on statistic, especially for all the stress microscopy and traction forces where we do no really know how representative at the stress patterns (how many experiment, are they average of several movies ? integrated on which temporal window ?)

Reviewer #1 (Public review):

Summary:

Olfaction is fundamental to the survival and reproduction of animals, as they rely on olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) to detect volatile chemical cues in their environment. Most mature OSNs adhere to the 'one neuron one receptor' rule, wherein each neuron selects a single receptor for expression from a large repertoire of olfactory receptor genes. The precise regulation of olfactory receptor expression is critical for accurate odorant recognition. Since the seminal discovery of olfactory receptors by Linda Buck and Richard Axel in 1991, substantial efforts have been made to elucidate the mechanisms underlying OSN differentiation and receptor expression. However, these processes remain incompletely understood. The development of in vitro olfactory epithelium organoids offers a promising platform to address these fundamental questions. The in vivo OE is composed of a complex array of cell types, which has posed a significant challenge for recapitulating its structure and function in vitro. Previous attempts to generate olfactory organoids from adult human or mouse OE cells yielded tissue containing OSNs, but these constructs were structurally distinct from the in vivo OE and lacked the characteristic pseudostratified epithelium.

In this study, Kazuya et al. successfully established olfactory epithelium organoids from E13.5 mouse embryonic OE stem cells, which developed into a pseudostratified structure closely resembling the native OE. They further examined the influence of different cultural conditions on OE differentiation, confirming the pivotal role of niche factors in promoting OSN development. Through immunofluorescence staining and single-cell RNA sequencing, they demonstrated that the organoids encompass a diverse range of cell types analogous to those present in the in vivo OE. Notably, calcium imaging revealed that the organoids were functionally responsive to odorants, and single-cell transcriptomic analysis showed that the majority of mature OSNs conformed to the 'one neuron one receptor' rule. Using these organoids, the authors performed a preliminary investigation into the developmental trajectories of OSNs, developed a tool to predict subpopulations of mature OSNs, and identified novel markers associated with OSN maturation. Collectively, the data provide compelling evidence for the reliability and utility of this olfactory organoid model. Further in-depth analyses may enable readers to better assess and utilize this tool to advance the study of olfactory biology.

Strengths:

The authors developed and established olfactory epithelium organoids, with immunofluorescence imaging confirming the presence of a pseudostratified structure similar to that of the in vivo olfactory epithelium, representing a significant advancement. Single-cell sequencing and calcium imaging further demonstrated the utility of these organoids, as they contain multiple cell types analogous to the in vivo olfactory epithelium. Importantly, they are physiologically functional, capable of responding to odor stimuli.

Weakness:

Although the authors have made significant progress in the technique, there are some gaps in understanding its underlying principles. First, it remains unclear what specific characteristics of E13.5 embryonic olfactory stem cells enable them to generate organoids in vitro that more closely resemble the in vivo olfactory epithelium, compared to adult mouse olfactory stem cells. Second, it is not clearly defined which specific cell type(s) from the embryonic olfactory epithelium give rise to these organoids, and the efficiency of organoid formation from the isolated cells also warrants further clarification.

Reviewer #1 (Public review):

Summary:

van der Linden et al. report on the development of a new green-fluorescent sensor for calcium, following a novel rational design strategy based on the modification of the cyan-emissive sensor mTq2-CaFLITS. Through a mutational strategy similar to the one used to convert EGFP into EYFP, coupled with optimization of strategic amino acids located in proximity of the chromophore, they identify a novel sensor, G-CaFLITS. Through a careful characterization of the photophysical properties in vitro and the expression level in cell cultures, the authors demonstrate that G-CaFLITS combines a large lifetime response with a good brightness in both the bound and unbound states. This relative independence of the brightness on calcium binding, compared with existing sensors that often feature at least one very dim form, is an interesting feature of this new type of sensors, which allows for a more robust usage in fluorescence lifetime imaging. Furthermore, the authors evaluate the performance of G-CaFLITS in different subcellular compartments and under two-photon excitation in Drosophila. While the data appears robust and the characterization thorough, the interpretation of the results in some cases appears less solid, and alternative explanations cannot be excluded.

Strengths:

- The approach is innovative and extends the excellent photophysical properties of the mTq2-based to more red-shifted variants. While the spectral shift might appear relatively minor, as the authors correctly point out, it has interesting practical implications, such as the possibility to perform FLIM imaging of calcium using widely available laser wavelengths, or to reduce background autofluorescence, which can be a significant problem in FLIM.<br /> - The screening was simple and rationally guided, demonstrating that, at least for this class of sensors, a careful choice of screening positions is an excellent strategy to obtain variants with large FLIM responses without the need of high-throughput screening.<br /> - The description of the methodologies is very complete and accurate, greatly facilitating the reproduction of the results by others, or the adoption of similar methods. This is particularly true for the description of the experimental conditions for optimal screening of sensor variants in lysed bacterial cultures.<br /> - The photophysical characterization is very thorough and complete, and the vast amount of data reported in the supporting information is a valuable reference for other researchers willing to attempt a similar sensor development strategy. Particularly well done is the characterization of the brightness in cells, and the comparison on multiple parameters with existing sensors.<br /> - Overall, G-CaFLITS displays excellent properties for a FLIM sensor: very large lifetime change, bright emission in both forms and independence from pH in the physiological range.

Weaknesses:

- The paper demonstrates the application of G-CaFLITS in various cellular sub-compartments without providing direct evidence that the sensor's response is not affected by the targeting. Showing at least that the lifetime values in the saturated state are similar in all compartments would improve the robustness of the claims.<br /> - In some cases, the interpretation of the results is not fully convincing, leaving alternative hypotheses as a possibility. This is particularly the case for the claim of the origin of the strongly reduced brightness of G-CaFLITS in Drosophila. The explanation of the intensity changes of G-CaFLITS also shows some inconsistency with the basic photophysical characterization.<br /> - While the claims generally appear robust, in some cases they are conveyed with a lack of precision. Several sentences in the introduction and discussion could be improved in this regard. Furthermore, the use of the signal-to-noise ratio as a means of comparison between sensors appears to be imprecise, since it is dependent on experimental conditions.

Joint Public Review:

Summary of the work:

This manuscript defines the differential stress response signaling induced by nuclear and cytoplasmic protein misfolding. To accomplish this, the authors used superfolder GFP fused to a destabilized FKBP protein-bearing targeting signal for cytosolic or nuclear localization. When cells were grown in the presence of the ligand Shield-1, this protein was stable, allowing fluorescence of the GFP protein. Upon removal of Shield-1, the FKBP protein is unfolded targeting the entire fusion protein to proteasomal degradation. Using this approach, they performed RNAseq to probe similarities and differences in transcriptional responses to the accumulation of unfolded proteins in the cytosol or nucleus. As expected, many of the pathways upregulated in both datasets involved protein homeostasis pathways such as the proteasome and cytosolic chaperones. The increase in proteasome subunits correlated with the stabilization of Nrf1 under these conditions, suggesting that protein misfolding might induce proteasome subunits through an Nrf1-dependent mechanism, but this was not explicitly tested. In contrast, the authors report that the p53-dependent transcriptional response was selectively induced by protein misfolding stress in the nucleus, but not the cytosol. Deletion of p53 blocked this increase, indicating that this response is attributable to p53 stabilization. The increased p53 transcriptional activity corresponded with the stabilization of p53 and its target p21 in cells subjected to nuclear but not cytosolic protein misfolding stress. Using a reporter of nuclear proteasome activity, they show that nuclear proteasome activity is reduced in cells following protein misfolding stress in the nucleus, indicating that the stabilization of p53 (and other transcription factors such as NRF1) might be attributed to reduced proteasomal degradation. Additionally, the authors showed that nuclear misfolding stress also induces cell cycle arrest. However, this effect was not dependent on p53 deletion, indicating that this is mediated by other unknown mechanisms.

Major strengths and weaknesses of the methods and results:

The findings reported here define specific transcriptional outputs induced by targeted protein misfolding stress in the nucleus and cytosol, revealing new insights into the organelle-specific stress signaling. The approach is interesting and effective at revealing cellular responses induced by compartment-specific protein misfolding stress.

One major weakness of the study is the lack of mechanistic follow-up for the transcriptional study. For example, what is the mechanistic basis for p53 stabilization by nuclear-destabilized domain (Nuc DD)? Is this entirely caused by diminished nuclear degradation activity as shown in Figure 6 or are there additional factors to be considered? If limited proteasome degradation capacity is the main reason for p53 upregulation, wouldn't the authors also see stabilization of other short-lived transcription factors? The fact that Nrf1 and Nrf2 are also stabilized by Nuc DD is consistent with the authors' hypothesis. On the other hand, if Nuc DD also affects other short-lived transcription factors such as c-fos or c-myc via proteasome inhibition, why did the gene expression analysis only pick up the p53 pathway as the one differentially regulated by Nuc DD? Would this imply that only p53 is specifically targeted by the nuclear proteasome, whereas other short-lived transcription factors are degraded either by the cytosolic proteasome or by both nuclear and cytosolic proteasome like Nrf1? Is there any evidence in the literature that supports this speculation? Additionally, how does Nuc DD affect the UPS system in the nucleus? Does it clog the proteasome directly or affect other assisting factors like chaperones or ubiquitinating enzymes? Lastly, it isn't clear what the functional implications of p53 stabilization would be for cells subjected to nuclear protein misfolding stress, particularly as the small effect on cell cycle arrest is not dependent on p53. In the end, the lack of mechanistic and/or functional follow-up reduces the overall importance of this manuscript. While the reviewers do not expect the authors to answer all these questions by experiments, additional work/clarifications/discussions along these lines would significantly improve the paper (see the recommendations).

Another major weakness is the lack of statistical analysis (SA) to better support their conclusions. In fact, no SA was provided for many figures even though the authors tried to make many comparisons.

The failure of the DD reporter to mount a significant heat shock response was puzzling. The presence of non-native proteins is the primary trigger for the heat shock response, but the authors acknowledge that inducible chaperones such as Hspa1a/b and Hsp90aa1 were not significantly changed in their system (page 8). Could this suggest a problem with the approach? What exactly is the nature of the stress mounted by Nuc DD?

The cell cycle data presented in Figure 5 is less robust, particularly as the p53 data in panels C and D was collected only once.

The Western blot data shown in Figure 6 does not have quantification to show how representative the blot is and how robust the changes in protein levels are over time. Western blots are known to be variable with different replicates and therefore the authors need to mention the number of biological repeats represented by the blot.

Reviewer #1 (Public review):

This manuscript discusses from a theory point of view he mechanisms underlying the formation of specialized or mixed factories. To investigate this, a chromatin polymer model was developed to mimic the chromatin binding-unbinding dynamics of various complexes of transcription factors (TFs).

The model revealed that both specialized (i.e., demixed) and mixed clusters can emerge spontaneously, with the type of cluster formed primarily determined by cluster size. Non-specific interactions between chromatin and proteins were identified as the main factor promoting mixing, with these interactions becoming increasingly significant as clusters grow larger.

These findings, observed in both simple polymer models and more realistic representations of human chromosomes, reconcile previously conflicting experimental results. Additionally, the introduction of different types of TFs was shown to strongly influence the emergence of transcriptional networks, offering a framework to study transcriptional changes resulting from gene editing or naturally occurring mutations.

Overall I think this is an interesting paper discussing a valuable model of how chromosome 3D organisation is linked to transcription. I would only advise the authors to polish and shorten their text to better highlight their key findings and make it more accessible to the reader.

Reviewer #1 (Public review):

Summary:

This study has preliminarily revealed the role of ACVR2A in trophoblast cell function, including its effects on migration, invasion, proliferation, and clonal formation, as well as its downstream signaling pathways.

Strengths:

The use of multiple experimental techniques, such as CRISPR/Cas9-mediated gene knockout, RNA-seq, and functional assays (e.g., Transwell, colony formation, and scratch assays), is commendable and demonstrates the authors' effort to elucidate the molecular mechanisms underlying ACVR2A's regulation of trophoblast function. The RNA-seq analysis and subsequent GSEA findings offer valuable insights into the pathways affected by ACVR2A knockout, particularly the Wnt and TCF7/c-JUN signaling pathways.

Weaknesses:

The molecular mechanisms underlying this study require further exploration through additional experiments. While the current findings provide valuable insights into the role of ACVR2A in trophoblast cell function and its involvement in the regulation of migration, invasion, and proliferation, further validation in both in vitro and in vivo models is needed. Additionally, more experiments are required to establish the functional relevance of the TCF7/c-JUN pathway and its clinical significance, particularly in relation to pre-eclampsia. Additional techniques, such as animal models and more advanced clinical sample analyses, would help strengthen the conclusions and provide a more comprehensive understanding of the molecular pathways involved.

Reviewer #1 (Public review):

Summary:

Meissner-Bernard et al present a biologically constrained model of telencephalic area of adult zebrafish, a homologous area to the piriform cortex, and argue for the role of precisely balanced memory networks in olfactory processing.

This is interesting as it can add to recent evidence on the presence of functional subnetworks in multiple sensory cortices. It is also important in deviating from traditional accounts of memory systems as attractor networks. Evidence for attractor networks has been found in some systems, like in the head direction circuits in the flies. However, the presence of attractor dynamics in other modalities, like sensory systems, and their role in computation has been more contentious. This work contributes to this active line of research in experimental and computational neuroscience by suggesting that, rather than being represented in attractor networks and persistent activity, olfactory memories might be coded by balanced excitation-inhibitory subnetworks.

Strengths:

The main strength of the work is in: (1) direct link to biological parameters and measurements, (2) good controls and quantification of the results, and (3) comparison across multiple models.

(1) The authors have done a good job of gathering the current experimental information to inform a biological-constrained spiking model of the telencephalic area of adult zebrafish. The results are compared to previous experimental measurements to choose the right regimes of operation.<br /> (2) Multiple quantification metrics and controls are used to support the main conclusions, and to ensure that the key parameters are controlled for - e.g. when comparing across multiple models.<br /> (3) Four specific models (random, scaled I / attractor, and two variant of specific E-I networks - tuned I and tuned E+I) are compared with different metrics, helping to pinpoint which features emerge in which model.

In the revised manuscript, the authors have also:<br /> (a) made a good effort to provide a mechanistic explanation of their results (especially on the mechanism underlying medium amplification in specific E/I network models);<br /> (b) performed a systematic analysis of the parameter space by changing different parameters of E and I neurons (specifically showing that different time constants of E and I neurons do not change the results and therefore the main effects result from connectivity);<br /> (c) added further analysis and discussion on the potential functional and computational significance of balanced specific E-I subnetworks.

These additions substantially strengthen the study, presenting compelling evidence for how networks with specific E-I structure can underpin olfactory processing and memory representations. The findings have potential implications that extend beyond the olfactory system and may be applicable to other neural systems and species.

Reviewer #1 (Public review):

Summary:

It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost importance and a reproducible setup is essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.

Strengths:

It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.

Also, the comparison between manual and software analysis is appreciated.

Reviewer #1 (Public review):

Summary:

In this manuscript, Shibata describes a method to assess rapidly fluctuating CpG sites (fCpGs) from single-cell methylation sequencing (sc-MeSeq) data. Assuming that fCpGs are largely consistent over time with changes induced by inheritable events during replication, the author infers lineage relationships in available brain-derived sc-MeSeq. Supplementing current lineage tracing through genomic and mitochondrial mosaic variants is an interesting concept that could supplement current work or allow additional lineage analysis in existing data.

However, the author failed to convincingly show the power of fCpG analysis to determine lineages in the human brain. While the correlation with cellular division and distinction of cell types appears plausible and strong, the application to detect specific lineages is less convincing. Aspects of this might be due to a lack of clarity in presentation and erroneous use of developmental concepts. However, without addressing these problems it is challenging for a reader to come to the same conclusions as the author.

On the flip side, this novel application of fCpGs will allow the re-use of existing sc-MeSeq to infer additional features that were previously unavailable, once the biological relevance has been further elucidated.

Strengths:

• Novel re-analysis application of methylation data to infer the status of fCpGs and the use as a lineage marker<br /> • Application of this method to an innovative existing data set to benchmark this framework against existing developmental knowledge

Weaknesses:

• Inconsistent or erroneous use of neurodevelopmental concepts which hinders appropriate interpretation of the results.<br /> • Somewhat confusing presentation at times which makes it hard to judge the value of this novel approach.

Reviewer #1 (Public review):

Summary:

The authors intended to investigate the earliest mechanisms enabling self-prioritization, especially in the attention. Combining a temporal order judgement task with computational modelling based on the Theory of Visual Attention (TVA), the authors suggested that the shapes associated with the self can fundamentally alter the attentional selection of sensory information into awareness. This self-prioritization in attentional selection occurs automatically at early perceptual stages. Furthermore, the processing benefits obtained from attentional selection via self-relatedness and physical salience were separated from each other.

Strengths:

The manuscript is written in a way that is easy to follow. The methods of the paper are very clear and appropriate.

Comments on revisions:

The authors clearly showed the relationship between attention and self-prioritization.

Reviewer #1 (Public review):

Summary:

Aicardi-Goutières Syndrome (AGS) is a genetic disorder that primarily affects the brain and immune system through excessive interferon production. The authors sought to investigate the role of microglia in AGS by first developing bone-marrow-derived progenitors in vitro that carry the estrogen-regulated (ER) Hoxb8 cassette, allowing them to expand indefinitely in the presence of estrogen and differentiate into macrophages when estrogen is removed. When injected into the brains of Csf1r-/- mice, which lack microglia, these cells engraft and resemble wild-type (WT) microglia in transcriptional and morphological characteristics, although they lack Sall1 expression. The authors then generated CRISPR-Cas9 Adar1 knockout (KO) ER-Hoxb8 macrophages, which exhibited increased production of inflammatory cytokines and upregulation of interferon-related genes. This phenotype could be rescued using a Jak-Stat inhibitor or by concurrently mutating Ifih1 (Mda5). However, these Adar1-KO macrophages fail to successfully engraft in the brain of both Csf1r-/- and Cx3cr1-creERT2:Csf1rfl/fl mice. To overcome this, the authors used a mouse model with a patient-specific Adar1 mutation (Adar1 D1113H) to derive ER-Hoxb8 bone marrow progenitors and macrophages. They discovered that Adar1 D1113H ER-Hoxb8 macrophages successfully engraft the brain, although at lower levels than WT-derived ER-Hoxb8 macrophages, leading to increased production of Isg15 by neighboring cells. These findings shed new light on the role of microglia in AGS pathology.

Strengths:

The authors convincingly demonstrate that ER-Hoxb8 differentiated macrophages are transcriptionally and morphologically similar to bone marrow-derived macrophages. They also show evidence that when engrafted in vivo, ER-Hoxb8 microglia are transcriptomically similar to WT microglia. Furthermore, ER-Hoxb8 macrophages engraft the Csf1r-/- brain with high efficiency and rapidly (2 weeks), showing a homogenous distribution. The authors also effectively use CRISPR-Cas9 to knock out TLR4 in these cells with little to no effect on their engraftment in vivo, confirming their potential as a model for genetic manipulation and in vivo microglia replacement.

Weaknesses:

The robust data showing the quality of this model at the transcriptomic level can be strengthened with confirmation at protein and functional levels. The authors were unable to investigate the effects of Adar1-KO using ER-Hoxb8 cells and instead had to rely on a mouse model with a patient-specific Adar1 mutation (Adar1 D1113H). Additionally, ER-Hoxb8-derived microglia do not express Sall1, a key marker of microglia, which limits their fidelity as a full microglial replacement, as has been rightfully pointed out in the discussion.

Overall, this paper demonstrates an innovative approach to manipulating microglia using ER-Hoxb8 cells as surrogates. The authors present convincing evidence of the model's efficacy and potential for broader application in microglial research, given its ease of production and rapid brain engraftment potential in microglia-deficient mice. While Adar1-KO macrophages do not engraft well, the success of TLR4-KO line highlights the model's potential for investigating other genes. Using mouse-derived cells for transplantation reduces complications that can come with the use of human cell lines, highlighting the utility of this system for research in mouse models.

Joint Public Review:

Summary:

Microfossils from the Paleoarchean Eon represent the oldest evidence of life, but their nature has been strongly debated among scientists. To resolve this, the authors reconstructed the lifecycles of Archaean organisms by transforming a Gram-positive bacterium into a primitive lipid vesicle-like state and simulating early Earth conditions. They successfully replicated all morphologies and life cycles of Archaean microfossils and studied cell degradation processes over several years, finding that encrustation with minerals like salt preserved these cells as fossilized organic carbon. Their findings suggest that microfossils from 3.8 to 2.5 billion years ago were likely liposome-like protocells with energy conservation pathways but without regulated morphology.

Strengths:

The authors have crafted a compelling narrative about the morphological similarities between microfossils from various sites and proliferating wall-deficient bacterial cells, providing detailed comparisons that have never been demonstrated in this detail before. The extensive number of supporting figures is impressive, highlighting numerous similarities. While conclusively proving that these microfossils are proliferating protocells morphologically akin to those studied here is challenging, we applaud this effort as the first detailed comparison between microfossils and morphologically primitive cells.

Summary of reviewer comments on this revision:

Each of the original reviewers evaluated the revised manuscript and were complimentary about how the authors addressed their original concerns. One reviewer added: "It is a thought-provoking manuscript that will be well received." We encourage readers of this version of the paper to consider the original reviewer comments and the authors' responses: https://elifesciences.org/reviewed-preprints/98637/reviews

Reviewer #1 (Public review):

Summary:

In their manuscript, the authors provide compelling evidence that stimulus-frequency otoacoustic emission (SFOAE) phase-gradient delays predict the sharpness (quality factors) of auditory-nerve-fiber (ANF) frequency tuning curves in budgerigars. In contrast with mammals, neither SFOAE- nor ANF-based measures of cochlear tuning match the frequency dependence of behavioral tuning in this species of parakeet. Although the reason for the discrepant behavioral results (taken from previous studies) remains unexplained, the present data provide significant and important support for the utility of otoacoustic estimates of cochlear tuning, a methodology previously explored only in mammals.

Strengths:

* The OAE and ANF data appear solid and believable. (The behavioral data are taken from previous studies.)

* No other study in birds (and only a single previous study in mammals) has combined behavioral, auditory-nerve, and otoacoustic estimates of cochlear tuning in a single species.

* SFOAE-based estimates of cochlear tuning now avoid possible circularity and were are obtained by assuming that the tuning ratio estimated in chicken applies also to the budgerigar.

Weaknesses:

* In mammals, accurate prediction of neural Q_ERB from otoacoustic N_SFOAE involves the application of species-invariance of the tuning ratio combined with an attempt to compensate for possible species differences in the location of the so-called apical-basal transition (for a review, see Shera & Charaziak, Cochlear frequency tuning and otoacoustic emissions. Cold Spring Harb Perspect Med 2019; 9:pii a033498. doi: 10.1101/cshperspect.a033498; in particular, the text near Eq. 2 and the value of CFa|b).

Despite this history, the manuscript makes no mention of the apical-basal transition, its possible role in birds, or why it was ignored in the present analysis. As but one result, the comparative discussion of the tuning ratio (paragraph beginning on lines 383) is incomplete and potentially misleading. Although the paragraph highlights differences in the tuning ratio across groups, perhaps these differences simply reflect differences in the value of CFa|b. For example, if the cochlea of the budgerigar is assumed to be entirely "apical" in character (so that CFa|b is around 7-8 kHz), then the budgerigar tuning ratios appear to align remarkably well with those previously obtained in mammals (see Shera et al 2010, Fig 9).

* For the most part, the authors take previous behavioral results in budgerigar at face value, attributing the discrepant behavioral results to hypothesized "central specializations for the processing of masked signals". But before going down this easy road, the manuscript would be stronger if the authors discussed potential issues that might affect the reliability of the previous behavioral literature. For example, the ANF data show that thresholds rise rapidly above about 5 kHz. Might the apparent broadening of the behavioral filters arise as<br /> a consequence of off-frequency listening due to the need to increase signal levels at these frequencies? Or perhaps there are other issues. Inquiring readers would appreciate an informed discussion.

Reviewer #1 (Public review):

Tu, Wen, et al. investigated the activity of mPFC putative glutamatergic neurons during a probabilistic threat discrimination and avoidance learning task using miniaturized GRIN lens implantation and single-photon calcium imaging in freely moving mice. In conjunction with this cellular recording, they employed channelrhodopsin-mediated optogenetic excitation of terminals from basal forebrain cholinergic projection neurons coupled to the delivery of an air puff on either of two maze paths with differential threat probability. The authors found that the optogenetic manipulation altered mPFC encoding of outcomes and disrupted animals' behavioral adaptation. Over the course of multiple learning sessions, optogenetically stimulated mice lagged behind control animals in resolving the differential threat probabilities on the two paths and making adaptive choices. In particular, the animals with optogenetic stimulation of cholinergic terminals were significantly more likely to switch to the path with higher threat probability after having just gotten a rare air puff on the generally "safer" path. Combined with data from a deterministic version of the task showing that optogenetically stimulated mice could behaviorally discriminate between the paths appropriately under such circumstances, these results suggest an impairment in the experimental animals' ability to make use of threat history over multiple trials. This comparison of probabilistic and deterministic versions of the same task is a highlight of this paper, representing a thoughtfulness about what information can be gleaned from such variations in the design of behavioral experiments that is all too often lacking. These data are timely in contributing to an ongoing discussion in the field about the role of phasic cholinergic signaling to the cortex, about which relatively little is known.

While the ensemble recording of mPFC neurons during the task appears to be reliable and well-designed and the behavioral effects of the optogenetic stimulation are convincing, some major weaknesses of the paper limit its usefulness to others in the field:

(1) Optogenetic excitation of presynaptic terminals can lead to antidromic action potentials that alter the firing properties of the target cell (see the excellent review on challenges of and strategies for presynaptic optogenetic experiments Rost et al., Nat Neurosci 2022). To their credit, the authors explicitly acknowledge this fact, but they believe that the only alternative possibility is that their intervention could lead to increased acetylcholine release at collateral projections in other prefrontal subregions. In fact, we do not know that the mechanism mediating the behavioral changes observed involves acetylcholine at all, as many ChAT+ basal forebrain neurons co-transmit using GABA (Saunders et al., Nature, 2015; Saunders et al., eLife, 2015; Granger et al., Neuropharmacology, 2016). A very useful internal control, which is recommended by Rost et al. for such presynaptic excitation experiments, would be to locally infuse nicotinic or muscarinic cholinergic antagonists into the mPFC in an attempt to reverse the optogenetically induced deficit; this would resolve whether the effect is indeed mediated by cholinergic neurotransmission and if it is specific to the mPFC.

(2) In a similar vein, the fact that LED illumination in the no-opsin control group appears to increase activity in prefrontal neurons (Figure 2C) and, moreover, has a functional effect in disrupting location-selective cellular activity to a similar extent as in the ChrimsonR group (Figure S3) is inadequately explained and cause for concern. Although the authors argue that the degree or "robustness" of puff-evoked activity was significantly greater in the ChrimsonR group as compared to fluorophore-only controls, their statistical test for demonstrating this is the Kolmogorov-Smirnov test (Figure 2D), thus showing that the two samples likely are drawn from different distributions but little else.

(3) Throughout the paper, the authors rely heavily on the Kolmogorov-Smirnov and binomial tests (Figures 2D, 3, 4D, S3, S4) to compare distributions in this manner, but it is unclear to me why these would be the most appropriate statistical tests for what they seek to demonstrate. Given the holistic nature of these tests in comparing the shape and spread of distributions, I am concerned that they might be inflating the significance of the differences between groups. Even if the authors were seeking a nonparametric statistical test, which most likely would be quite appropriate, there are nonparametric versions of ANOVA that they could use (e.g. Kruskal-Wallis, Friedman). Indeed, in much of this data set a repeated measures statistical analysis would seem to be called for, whereas the Kolmogorov-Smirnov test assumes that the two samples must be independent of each other. The most notable example of this premise being violated is in Figure 3, where data from the same cell populations in the same animals are being compared between experimental days and across various trial types.

Reviewer #1 (Public review):

This manuscript presents an interesting exploration of the potential activation mechanisms of DLK following axonal injury. While the experiments are beautifully conducted and the data are solid, I feel that there is insufficient evidence to fully support the conclusions made by the authors.

In this manuscript, the authors exclusively use the puc-lacZ reporter to determine the activation of DLK. This reporter has been shown to be induced when DLK is activated. However, there is insufficient evidence to confirm that the absence of reporter activation necessarily indicates that DLK is inactive. As with many MAP kinase pathways, the DLK pathway can be locally or globally activated in neurons, and the level of DLK activation may depend on the strength of the stimulation. This reporter might only reflect strong DLK activation and may not be turned on if DLK is weakly activated. The results presented in this manuscript support this interpretation. Strong stimulation, such as axotomy of all synaptic branches, caused robust DLK activation, as indicated by puc-lacZ expression. In contrast, weak stimulation, such as axotomy of some synaptic branches, resulted in weaker DLK activation, which did not induce the puc-lacZ reporter. This suggests that the strength of DLK activation depends on the severity of the injury rather than the presence of intact synapses. Given that this is a central conclusion of the study, it may be worthwhile to confirm this further. Alternatively, the authors may consider refining their conclusion to better align with the evidence presented.

As noted by the authors, DLK has been implicated in both axon regeneration and degeneration. Following axotomy, DLK activation can lead to the degeneration of distal axons, where synapses are located. This raises an important question: how is DLK activated in distal axons? The authors might consider discussing the significance of this "synapse connection-dependent" DLK activation in the broader context of DLK function and activation mechanisms.

Reviewer #1 (Public review):

This study identifies two behavioral processes that underlie learned pathogen avoidance behavior in C. elegans: exiting and re-entry of pathogenic bacterial lawns. Long-term behavioral tracking indicates that animals increase the prevalence of both behaviors over long-term exposure to the pathogen Pseudomonas aeruginosa. Using an optogenetic silencing screen, the authors identify groups of neurons, whose activity regulates lawn occupancy. Surprisingly, they find that optogenetic inhibition of neurons during only the first two hours of pathogen exposure can establish subsequent long-term changes in pathogen aversion. By leveraging a compressed sensing approach, the authors define a set of neurons involved in either lawn exit or lawn re-entry behavior using a constrained set of transgenic lines that drive Arch-3 expression in overlapping groups of neurons. They then measure the calcium activity of the candidate neurons involved in lawn re-entry in freely moving animals using GCaMP, and observe a reduction in their neural activity after exposure to pathogen. Optogenetic inhibition of AIY and SIA neurons during acute pathogen exposure in naïve animals delays lawn entry whereas activating these neurons in animals previously exposed to pathogen enhances lawn entry, albeit transiently.

This work is missing experiments and analyses that are necessary to substantiate their claims. Although the authors convincingly show that neuronal inhibition experiments during pathogen exposure reveal separable groups of neurons controlling pathogenic lawn exiting and re-entry, their methods to validate these results at single neuron cell-type resolution lack rigor.

In Figure 4, the authors claim that the reduction in calcium activity in cells of interest following pathogen exposure encodes pathogen experience. However, they make no effort to correlate the observed decreased activity with concomitant shifts in increased immobility (decreased forward locomotion) or the increased age of the worms since pathogen exposure began (24 hours have elapsed), either of which could easily explain these results. A better comparison would be between age-matched naive animals and animals exposed to pathogen. More to the point, we are interested in the involvement of these neurons' activity patterns with the behavioral motifs associated with lawn exits and re-entries, so examining these activity patterns in the absence of any pathogen before or after long-term pathogen exposure yields little insight into their relevant signaling roles. To substantiate the authors' claims, a better experiment would measure these neurons' calcium activity during lawn exits and re-entries in naive and post-exposed age-matched worms.

In Figure 5, the authors attempt to show that manipulating AIY and SIA/SIB neuronal activity controls pathogenic lawn re-entry behavior. Although they show that inhibiting these neurons in naive animals increases latency to enter pathogenic lawns, they never test the effect of neuronal inhibition in post-exposed animals. Instead they activate these neurons using channelrhodopsin, whereby they observe an increase in lawn entry and exit behavior, indicative of high forward locomotion speed. Although suggestive, neither of these experiments prove these neurons' involvement in pathogenic lawn re-entry behavior following pathogen exposure. To rigorously test the hypothesis that AIY and SIA/SIB neurons are required to sustain higher latency to lawn re-entry following pathogen exposure, the authors should perform neuronal inhibition experiments in post-pathogen-exposed animals as well and compare the results. The interpretation of this figure is further complicated by the fact that Npr-4::ChR2 animals express ChR2 in AIY in addition to SIA/SIB neurons: experiments that calculated lawn re-entry rates in Npr-4::ChR2 activation in post-exposed animals may include the known effect of stimulating AIY alone (Fig. 5J) since no discernible attempt at structured illumination to limit excitation to SIA/SIB neurons was made in these animals (Fig. 5 K, L).

This work raises the interesting possibility that different sets of neurons control lawn exit and lawn re-entry behaviors following pathogen exposure. However, the authors never directly test this claim. To rigorously show this, the authors would need to show that lawn-exit promoting neurons (CEPs, HSNs, RIAs, RIDs, SIAs) are dispensable for lawn re-entry behavior and that lawn re-entry promoting neurons (AVK, SIA, AIY, MI) are dispensable for lawn exit behavior in pathogen-exposed animals. The authors identify AVK neurons as important for modulating lawn re-entry behavior by brief inhibition at the start of pathogen exposure but fail to find that these neurons are required for increased latency to re-entry in naïve animals (Fig. 5D). Recent work from Marquina-Solis et al (2024) shows that chronic silencing of these neurons delays pathogen lawn leaving, due to impaired release of flp-1 neuropeptide. Authors may wish to connect their work more closely with the existing literature by investigating the behavioral process by which AVK contributes to lawn evacuation.

Reviewer #1 (Public review):

Summary:

In this manuscript by Lopez-Blanch and colleagues, 21 microexons are selected for a deep analysis of their impacts on behavior, development, and gene expression. The authors begin with a systematic analysis of microexon inclusion and conservation in zebrafish and use these data to select 21 microexons for further study. The behavioral, transcriptomic, and morphological data presented are for the most part convincing. Furthermore, the discussion of the potential explanations for the subtle impacts of individual microexon deletions versus loss-of-function in srrm3 and/or srrm4 is quite comprehensive and thoughtful. One major weakness: data presentation, methods, and jargon at times affect readability / might lead to overstated conclusions. However, overall this manuscript is well-written, easy to follow, and the results are of broad interest.

Strengths:

(1) The study uses a wide variety of techniques to assess the impacts of microexon deletion, ranging from assays of protein function to regulation of behavior and development.

(2) The authors provide comprehensive analyses of the molecular impact of their microexon deletions, including examining how host-gene and paralog expression is affected.

Weaknesses / Major Points:

(1) According to the methods, it seems that srrm3 social behavior is tested by pairing a 3mpf srrm3 mutant with a 30dpf srrm3 het. Is this correct? The methods seem to indicate that this decision was made to account for a slower growth rate of homozygous srrm3 mutant fish. However, the difference in age is potentially a major confound that could impact the way that srrm3 mutants interact with hets and the way that srrm3 mutants interact with one another (lower spread for the ratio of neighbour in front value, higher distance to neighbour value). This reviewer suggests testing het-het behavior at 3 months to provide age-matched comparisons for del-del, testing age-matched rather than size-matched het-del behavior, and also suggests mentioning this in the main text / within the figure itself so that readers are aware of the potential confound.

(2) Referring to srrm3+/+; srrm4-/- controls for double mutant behavior as "WT for simplicity" is somewhat misleading. Why do the authors not refer to these as srrm4 single mutants?

(3) It's not completely clear how "neurally regulated" microexons are defined / how they are different from "neural microexons"? Are these terms interchangeable?

(4) Overexpression experiments driving srrm3 / srrm4 in HEK293 cells are not described in the methods.

(4) Suggest including more information on how neurite length was calculated. In representative images, it appears difficult to determine which neurites arise from which soma, as they cross extensively. How was this addressed in the quantification?