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 #2 (Public review):

Summary:

This manuscript describes the creation and curation of a collection of genetic driver lines that specifically label small numbers of neurons, often just a single to handful of cell types, in the central nervous system of the fruit fly, Drosophila melanogaster. The authors screened over 77,000 split hemidriver combinations to yield a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These genetic driver lines have already contributed to several important publications and will no doubt continue to do so. It is a truly valuable resource that represents the cooperation of several labs throughout the Drosophila community.

Strengths:

The authors have thoughtfully curated and documented the lines that they have created, so that they may be maximally useful to the greater community. This documentation includes confocal images of neurons labeled by each driver line and when possible, a list of cell types labeled by the genetic driver line and their identity in an EM connectome dataset. The authors have also made available some information from the other lines they created and tested but deemed not specific or strong enough to be included as part of the collection. This additional resource will be a valuable aid for those seeking to label cell types that may not be included in the main collection.

The added revisions help to clarify important points relating to the creation of the lines, which lines were included as part of this specific collection, and caveats to be mindful of when using any of the described lines. These revisions will increase the manuscript's utility to users who may be less familiar with this resource.

Weaknesses:

The major weakness, which is also in some ways a strength, is the stringent requirement that lines that be included be highly specific across the CNS. As a result, the lines that are part of this specific collection are sparse and specific but also limited in which cell types they cover. Doubtless there are many missing cell types.

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 #2 (Public review):

Patsy R. Tomlinson et al; investigated the impact of different p85 alpha variants associated with SHORT syndrome or APDS2 on insulin mediated signaling in dermal fibroblasts and preadipocytes. They perform this study as APDS2 patients oftern present with features of SHORT syndrome. They found no evidence of hyperactive PI3K signalling monitored by pAKT in a APDS2 patient-derived dermal fibroblast cells. In these cells p110 alpha protein levels were comparable to levels in control cells, however, p110 delta protein levels were strongly reduced. Remarkably, the truncated APDS2-causal p85 alpha variant was less abundant in these cells than p85 alpha wildtype. Afterwards they studied the impact of ectopically expressed p85 alpha variants on insulin mediated PI3K signaling in 3T3-L1 preadipocytes. Interestingly they found that the truncated APDS2-causal p85 alpha variant impaired insulin induced signaling. Using immunoprecipitation of p110 alpha they did not find truncated APDS2-causal p85 alpha variant in p110 alpha precipitates. Furthermore, by immunoprecipitating IRS1 and IRS2 they observed that the truncated APDS2-causal p85 alpha variant was very abundant in IRS1 and IRS2 precipitates, even in the absence of insulin stimulation. These important findings add in an interesting way possible mechanistic explanation for the growing number of APDS2 patients described with features of SHORT syndrome.

Strengths:

Based on state-of-the-art functional studies, the authors show that the p85 alpha variant responsible for APDS2, known to be associated with increased PI3K-delta signaling, can attenuate PI3K-alpha signalling in preadipocytes, providing a possible mechanistic explanation for the growing number of APDS2 patients with features of SHORT syndrome.

Weaknesses:

The proposed paradigm is based on one cell line derived from an APDS2 patient and an overexpressing system. The investigation of a larger number of cell lines derived from APDS2 patients would further substantiate the conclusion.

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 #2 (Public review):

Summary:

Zhang et al. analyzed the functional role of hepatocyte RIPK1 during metabolic stress, particularly its scaffold function rather than kinase function. They show that Ripk1 knockout sensitizes the liver to cell death and inflammation in response to short-term fasting, a condition that would not induce obvious abnormality in wild-type mice.

Strengths:

The findings are based on a knockout mouse model and supported by bulk RNA-seq and scRNA-seq. The work consolidates the complex role of RIPK1 in metabolic stress.

Comments on revision:

The authors have addressed my concerns. The added experiments consolidated the findings. I do not have further comments.

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 #2 (Public Review):

Summary:

The article by Shuai et al. describes a comprehensive collection of over 800 split-GAL4 and split-LexA drivers, covering approximately 300 cell types in Drosophila, aimed at advancing the understanding of associative learning. The mushroom body (MB) in the insect brain is central to associative learning, with Kenyon cells (KCs) as primary intrinsic neurons and dopaminergic neurons (DANs) and MB output neurons (MBONs) forming compartmental zones for memory storage and behavior modulation. This study focuses on characterizing sensory input as well as direct upstream connections to the MB both anatomically and, to some extent, behaviorally. Genetic access to specific, sparsely expressed cell types is crucial for investigating the impact of single cells on computational and functional aspects within the circuitry. As such, this new and extensive collection significantly extends the range of targeted cell types related to the MB and will be an outstanding resource to elucidate MB-related processes in the future.

Strengths:

The work by Shuai et al. provides novel and essential resources to study MB-related processes and beyond. The resulting tools are publicly available and, together with the linked information, will be foundational for many future studies. The importance and impact of this tool development approach, along with previous ones, for the field cannot be overstated. One of many interesting aspects arises from the anatomical analysis of cell types that are less stereotypical across flies. These discoveries might open new avenues for future investigations into how such asymmetry and individuality arise from development and other factors, and how it impacts the computations performed by the circuitry that contains these elements.

Reviewer #3 (Public Review):

Summary:

Previous research on the Drosophila mushroom body (MB) has made this structure the best-understood example of an associative memory center in the animal kingdom. This is in no small part due to the generation of cell-type specific driver lines that have allowed consistent and reproducible genetic access to many of the MB's component neurons. The manuscript by Shuai et al. now vastly extends the number of driver lines available to researchers interested in studying learning and memory circuits in the fly. It is an 800-plus collection of new cell-type specific drivers target neurons that either provide input (direct or indirect) to MB neurons or that receive output from them. Many of the new drivers target neurons in sensory pathways that convey conditioned and unconditioned stimuli to the MB. Most drivers are exquisitely selective, and researchers will benefit from the fact that whenever possible, the authors have identified the targeted cell types within the Drosophila connectome. Driver expression patterns are beautifully documented and are publicly available through the Janelia Research Campus's Flylight database where full imaging results can be accessed. Overall, the manuscript significantly augments the number of cell type-specific driver lines available to the Drosophila research community for investigating the cellular mechanisms underlying learning and memory in the fly. Many of the lines will also be useful in dissecting the function of the neural circuits that mediate sensorimotor circuits.

Strengths:

The manuscript represents a huge amount of careful work and leverages numerous important developments from the last several years. These include the thousands of recently generated split-Gal4 lines at Janelia and the computational tools for pairing them to make exquisitely specific targeting reagents. In addition, the manuscript takes full advantage of the recently released Drosophila connectomes. Driver expression patterns are beautifully illustrated side-by-side with corresponding skeletonized neurons reconstructed by EM. A comprehensive table of the new lines, their split-Gal4 components, their neuronal targets, and other valuable information will make this collection eminently useful to end-users. In addition to the anatomical characterization, the manuscript also illustrates the functional utility of the new lines in optogenetic experiments. In one example, the authors identify a specific subset of sugar reward neurons that robustly promotes associative learning.

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 #2 (Public review):

Liu et al. applied hidden Markov models (HMM) to fMRI data from 64 participants listening to audio stories. The authors identified three brain states, characterized by specific patterns of activity and connectivity, that the brain transitions between during story listening. Drawing on a theoretical framework proposed by Berwick et al. (TICS 2023), the authors interpret these states as corresponding to external sensory-motor processing (State 1), lexical processing (State 2), and internal mental representations (State 3). States 1 and 3 were more likely to transition to State 2 than between one another, suggesting that State 2 acts as a transition hub between states. Participants whose brain state trajectories closely matched those of an individual with high comprehension scores tended to have higher comprehension scores themselves, suggesting that optimal transitions between brain states facilitated narrative comprehension.

Overall, the conclusions of the paper are well-supported by the data. Several recent studies (e.g., Song, Shim, and Rosenberg, eLife, 2023) have found that the brain transitions between a small number of states; however, the functional role of these states remains under-explored. An important contribution of this paper is that it relates the expression of brain states to specific features of the stimulus in a manner that is consistent with theoretical predictions.

The correlation between narrative features and brain state expression was reliable, but relatively low (~0.03). As discussed in the manuscript, this could be due to measurement noise, as well as narrative features accounting for a small proportion of cognitive processes underlying the brain states.

A strength of the paper is that the authors repeated the HMM analyses across different tasks (Figure 5) and an independent dataset (Figure S3) and found that the data was consistently best fit by 3 brain states. Across tasks, however, the spatial regions associated with each state varied. For example, state 2 during narrative comprehension was similar to both states 2 and 3 during rest (Fig. 5A), suggesting that the organization of the three states was task dependent.

The three states identified in the manuscript correspond rather well to areas with short, medium, and long temporal timescales (see Hasson, Chen & Honey, TiCs, 2015). Given the relationship with behavior, where State 1 responds to acoustic properties, State 2 responds to word-level properties, and State 3 responds to clause-level properties, a "single-process" account where the states differ in terms of the temporal window for which one needs to integrate information over may offer a more parsimonious account than a multi-process account where the states correspond to distinct processes. This possibility is mentioned briefly in the introduction, but not developed further.

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):

Knudstrup and colleagues investigate response to short and rapid sequences of stimuli in layer 2/3 of mouse visual cortex. To quote the authors themselves: "the work continues the recent tradition of providing ambiguous support for the idea that cortical dynamics are best described by predictive coding models". Unfortunately, the ambiguity here is largely a result of the choice of experimental design and analysis, and the data provide only incomplete support for the authors' conclusions.

The authors have addressed some of the concerns of the first revision. However, many still remain.

(1) From the first review: "There appears to be some confusion regarding the conceptual framing of predictive coding. Assuming the mouse learns to expect the sequence ABCD, then ABBD does not probe just for negative prediction errors, and ACBD not just positive prediction errors. With ABBD, there is a combination of a negative prediction error for the missing C in the 3rd position, and a positive prediction error for B in 3rd. Likewise, with ACBD, there is negative prediction error for the missing B at 2nd and missing C at 3rd, and a positive prediction error for the C in 2nd and B in 3rd. Thus, the authors' experimental design does not have the power to isolate either negative or positive prediction errors. Moreover, looking at the raw data in Figure 2C, this does not look like an "omission" response to C, more like a stronger response to a longer B. The pitch of the paper as investigating prediction error responses is probably not warranted - we see no way to align the authors' results with this interpretation."

The authors acknowledge in their response that this is a problem, but do not appear to discuss this in the manuscript. This should be fixed.

(2) From the first review: "Recording from the same neurons over the course of this paradigm is well within the technical standards of the field, and there is no reason not to do this. Given that the authors chose to record from different neurons, it is difficult to distinguish representational drift from drift in the population of neurons recorded. "

The authors respond by pointing out that what they mean by "drift" is within day changes. This has been clarified. However, the analyses in Figures 3 and 5 still are done across days. Figure 3: "Experience modifies activity in PCA space ..." and figure 5: "Stimulus responses shift with training". Both rely on comparisons of population activity across days. This concern remains unchanged here. It would probably be best to remove any analysis done across days - or use data where the same neurons were tracked. Performing chronic two-photon imaging experiments without tracking the same neurons is simply bad practice (assuming one intends to do any analysis across recording sessions).

(3) From the first revision: "The block paradigm to test for prediction errors appears ill chosen. Why not interleave oddball stimuli randomly in a sequence of normal stimuli? The concern is related to the question of how many repetitions it takes to learn a sequence. Can the mice not learn ACBD over 100x repetitions? The authors should definitely look at early vs. late responses in the oddball block. Also the first few presentations after block transition might be potentially interesting. The authors' analysis in the paper already strongly suggests that the mice learn rather rapidly. The authors conclude: "we expected ABCD would be more-or-less indistinguishable from ABBD and ACBD since A occurs first in each sequence and always preceded by a long (800 ms) gray period. This was not the case. Most often, the decoder correctly identified which sequence stimulus A came from." This would suggest that whatever learning/drift could happen within one block did indeed happen and responses to different sequences are harder to interpret."

Again, the authors acknowledge the problem and state that "there is no indication that this is a learned effect". However, they provide no evidence for this and perform no analysis to mitigate the concern.

(4) Some of the minor comments also appear unaddressed and uncommented. E.g. the response amplitudes are still shown in "a.u." instead of dF/F or z-score or spikes.

Reviewer #3 (Public review):

Summary:

This work provides insights into predictive coding models of visual cortex processing. These models predict that visual cortex neurons will show elevated responses when there are unexpected changes to learned sequential stimulus patterns. This model is currently controversial, with recent publications providing conflicting evidence. In this work, the authors test two types of unexpected pattern variations in layer 2/3 of the mouse visual cortex. They show that pattern omission evokes elevated responses, in favor of a predictive coding model, but find no evidence for prediction errors with substituted patterns, which conflicts with both prior results in L4, and with the expectations of a predictive coding model. They also report that with sequence training, responses sparsify and decorrelate, but surprisingly find no changes in the ability of an ideal observer to decode stimulus identity or timing.

These results are an important contribution to the understanding of how temporal sequences and expectations are encoded in the primary visual cortex

Comments on revisions:

In this revision, the authors address several of the concerns in the original manuscript. However, the primary issue, raised by all three reviewers, was the block design of the experiments. This design makes disentangling the effects of any rapid (within block) plasticity from any longer term (across days) plasticity-which nominally is the subject of the paper-extremely difficult.

Although it may be the case that re-running the experiments with an interleaved design is beyond the scope of this paper, unfortunately, the revised manuscript still does not adequately discuss this potential confound. The authors note that stimulus A in ABCD, ABBD, and ACBD could be distinguished on day 0, indicating that within block changes do occur. In both the original and revised manuscript this finding is discussed in terms of representational drift, but the authors fail to discuss how such within block plasticity may impact their primary findings of prediction error effects.

This remains a significant concern with the revised manuscript.

Many of the other issues in the original manuscript have been addressed, and in these areas the revised manuscript is both clearer and more accurately reflects the presented data. The additional analyses and controls shown in the supplemental figures aid in the interpretation of the findings.

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 #2 (Public review):

Summary:

Shin et al aim to identify in a very extensive piece of work a mechanism that contributes to dynamic regulation of synaptic output in the rat cortex at the second time scale. This mechanism is related to a new powerful model is well versed to test if the pool of SV ready for fusion is dynamically scaled to adjust supply demand aspects. The methods applied are state-of-the-art and both address quantitative aspects with high signal to noise. In addition, the authors examine both excitatory output onto glutamatergic and GABAergic neurons, which provides important information on how general the observed signals are in neural networks, The results are compellingly clear and show that pool regulation may be predominantly responsible. Their results suggests that a regulation of release probability, the alternative contender for regulation, is unlikely to be involved in the observed short term plasticity behavior (but see below). Besides providing a clear analysis pof the underlying physiology, they test two molecular contenders for the observed mechanism by showing that loss of Synaptotagmin7 function and the role of the Ca dependent phospholipase activity seems critical for the short term plasticity behavior. The authors go on to test the in vivo role of the mechanism by modulating Syt7 function and examining working memory tasks as well as overall changes in network activity using immediate early gene activity. Finally, they model their data, providing strong support for their interpretation of TS pool occupancy regulation.

Strengths:

This is a very thorough study, addressing the research question from many different angles and the experimental execution is superb. The impact of the work is high, as it applies recent models of short term plasticity behavior to in vivo circuits further providing insights how synapses provide dynamic control to enable working memory related behavior through nonpermanent changes in synaptic output.

Weaknesses:

While this work is carefully examined and the results are presented and discussed in a detailed manner, the reviewer is still not fully convinced that regulation of release provability is not a putative contributor to the observed behavior. No additional work is needed but in the moment I am not convinced that changes in release probability are not in play. One solution may be to extend the discussion of changes in rules probability as an alternative.

Fig 3 I am confused about the interpretation of the Mean Variance analysis outcome. Since the data points follow the curve during induction of short term plasticity, aren't these suggesting that release probability and not the pool size increases? Related, to measure the absolute release probability and failure rate using the optogenetic stimulation technique is not trivial as the experimental paradigm bias the experiment to a given output strength, and therefore a change in release probability cannot be excluded.

Fig4B interprets the phorbol ester stimulation to be the result of pool overfilling, however, phorbol ester stimulation has also been shown to increase release probability without changing the size of the readily releasable pool. The high frequency of stimulation may occlude an increased paired pulse depression in presence of OAG, which others have interpreted in mammalian synapses as an increase in release probability.

The literature on Syt7 function is still quite controversial. An observation in the literature that loss of Syt7 function in the fly synapse leads to an increase of release probability. Thus the observed changes in short term plasticity characteristics in the Syt7 KD experiments may contain a release probability component. Can the authors really exclude this possibility? Figure 5 shows for the Syt7 KD group a very prominent depression of the EPSC/IPSC with the second stimulus, particularly for the short interpulse intervals, usually a strong sign of increased release probability, as lack of pool refilling can unlikely explain the strong drop in synaptic output.

Reviewer #3 (Public review):

Summary:

The report by Shin, Lee, Kim, and Lee entitled "Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex" describes electrophysiological experiments of short-term synaptic plasticity during repetitive presynaptic stimulation at synapses between layer 2/3 pyramidal neurons and nearby target neurons. Manipulations include pharmacological inhibition of PLC and actin polymerization, activation of DAG receptors, and shRNA knockdown of Syt7. The results are interpreted as support for the hypothesis that synaptic vesicle release sites are vacant most of the time at resting synapses (i.e., p_occ is low) and that facilitation (and augmentation) components of short-term enhancement are caused by an increase in occupancy, presumably because of acceleration of the transition from not-occupied to occupied. The report additionally describes behavioural experiments where trace fear conditioning is degraded by knocking down syt7 in the same synapses.

Strengths:

The strength of the study is in the new information about short-term plasticity at local synapses in layer 2/3, and the major disruption of a memory task after eliminating short-term enhancement at only 15% of excitatory synapses in a single layer of a small brain region. The local synapses in layer 2/3 were previously difficult to study, but the authors have overcome a number of challenges by combining channel rhodopsins with in vitro electroporation, which is an impressive technical advance.

Weaknesses:

The question of whether or not short-term enhancement causes an increase in p_occ (i.e., "readily releasable pool overfilling") is important because it cuts to the heart of the ongoing debate about how to model short term synaptic plasticity in general. However, my opinion is that, in their current form, the results do not constitute strong support for an increase in p_occ, even though this is presented as the main conclusion. Instead, there are at least two alternative explanations for the results that both seem more likely. Neither alternative is acknowledged in the present version of the report.

The evidence presented to support overfilling is essentially two-fold. The first is strong paired pulse depression of synaptic strength when the interval between action potentials is 20 or 25 ms, but not when the interval is 50 ms. Subsequent stimuli at frequencies between 5 and 40 Hz then drive enhancement. The second is the observation that a slow component of recovery from depression after trains of action potentials is unveiled after eliminating enhancement by knocking down syt7. Of the two, the second is predicted by essentially all models where enhancement mechanisms operate independently of release site depletion - i.e., transient increases in p_occ, p_v, or even N - so isn't the sort of support that would distinguish the hypothesis from alternatives (Garcia-Perez and Wesseling, 2008, https://doi.org/10.1152/jn.01348.2007).

Regarding the paired pulse depression: The authors ascribe this to depletion of a homogeneous population of release sites, all with similar p_v. However, the details fit better with the alternative hypothesis that the depression is instead caused by quickly reversing inactivation of Ca2+ channels near release sites, as proposed by Dobrunz and Stevens to explain a similar phenomenon at a different type of synapse (1997, PNAS,<br /> https://doi.org/10.1073/pnas.94.26.14843). The details that fit better with Ca2+ channel inactivation include the combination of the sigmoid time course of the recovery from depression (plotted backwards in Fig1G,I) and observations that EGTA (Fig2B) increases the paired-pulse depression seen after 25 ms intervals. That is, the authors ascribe the sigmoid recovery to a delay in the activation of the facilitation mechanism, but the increased paired pulse depression after loading EGTA indicates, instead, that the facilitation mechanism has already caused p_r to double within the first 25 ms (relative to the value if the facilitation mechanism was not active). Meanwhile, Ca2+ channel inactivation would be expected to cause a sigmoidal recovery of synaptic strength because of the sigmoidal relationship between Ca2+-influx and exocytosis (Dodge and Rahamimoff, 1967, https://doi.org/10.1113/jphysiol.1967.sp008367).

The Ca2+-channel inactivation hypothesis could probably be ruled in or out with experiments analogous to the 1997 Dobrunz study, except after lowering extracellular Ca2+ to the point where synaptic transmission failures are frequent. However, a possible complication might be a large increase in facilitation in low Ca2+ (Fig2B of Stevens and Wesseling, 1999, https://doi.org/10.1016/s0896-6273(00)80685-6).

On the other hand, even if the paired pulse depression is caused by depletion of release sites rather than Ca2+-channel inactivation, there does not seem to be any support for the critical assumption that all of the release sites have similar p_v. And indeed, there seems to be substantial emerging evidence from other studies for multiple types of release sites with 5 to 20-fold differences in p_v at a wide variety of synapse types (Maschi and Klyachko, eLife, 2020, https://doi.org/10.7554/elife.55210; Rodriguez Gotor et al, eLife, 2024, https://doi.org/10.7554/elife.88212 and refs. therein). If so, the paired pulse depression could be caused by depletion of release sites with high p_v, whereas the facilitation could occur at sites with much lower p_v that are still occupied. It might be possible to address this by eliminating assumptions about the distribution of p_v across release sites from the variance-mean analysis, but this seems difficult; simply showing how a few selected distributions wouldn't work - such as in standard multiple probability fluctuation analyses - wouldn't add much.

In any case, the large increase - often 10-fold or more - in enhancement seen after lowering Ca2+ below 0.25 mM at a broad range of synapses and neuro-muscular junctions noted above is a potent reason to be cautious about the LS/TS model. There is morphological evidence that the transitions from a loose to tight docking state (LS to TS) occur, and even that the timing is accelerated by activity. However, 10-fold enhancement would imply that at least 90 % of vesicles start off in the LS state, and this has not been reported. In addition, my understanding is that the reverse transition (TS to LS) is thought to occur within 10s of ms of the action potential, which is 10-fold too fast to account for the reversal of facilitation seen at the same synapses (Kusick et al, 2020, https://doi.org/10.1038/s41593-020-00716-1).

Individual points:

(1) An additional problem with the overfilling hypothesis is that syt7 knockdown increases the estimate of p_occ extracted from the variance-mean analysis, which would imply a faster transition from unoccupied to occupied, and would consequently predict faster recovery from depression. However, recovery from depression seen in experiments was slower, not faster. Meanwhile, the apparent decrease in the estimate of N extracted from the mean-variance analysis is not anticipated by the authors' model, but fits well with alternatives where p_v varies extensively among release sites because release sites with low p_v would essentially be silent in the absence of facilitation.

(2) Figure S4A: I like the TTX part of this control, but the 4-AP part needs a positive control to be meaningful (e.g., absence of TTX).

(3) Line 251: At least some of the previous studies that concluded these drugs affect vesicle dynamics used logic that was based on some of the same assumptions that are problematic for the present study, so the reasoning is a bit circular.

(4) Line 329 and Line 461: A similar problem with circularity for interpreting earlier syt7 studies.

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 #2 (Public review):

Summary:

The study investigates the brain's functional connectivity (FC) dynamics across different timescales using simultaneous recordings of intracranial EEG/source-localized EEG and fMRI. The primary research goal was to determine which of three convergence/divergence scenarios is the most likely to occur.

The results indicate that despite similar FC patterns found in different data modalities, the timepoints were not aligned, indicating spatial convergence but temporal divergence.

The researchers also found that FC patterns in different frequencies do not overlap significantly, emphasizing the multi-frequency nature of brain connectivity. Such asynchronous activity across frequency bands supports the idea of multiple connectivity states that operate independently and are organized into a multiplex system.

Strengths:

The data supporting the authors' claims are convincing and come from simultaneous recordings of fMRI and iEEG/EEG, which has been recently developed and adapted.

The analysis methods are solid and involved a novel approach to analyzing the co-occurrence of FC patterns across modalities (cross-modal recurrence plot, CRP) and robust statistics, including replication of the main results using multiple operationalizations of the functional connectome (e.g., amplitude, orthogonalized, and phase-based coupling).

In addition, the authors provided a detailed interpretation of the results, placing them in the context of recent advances and understanding of the relationships between functional connectivity and cognitive states.

The authors also did a control analysis and verified the effect of temporal window size or different functional connecvitity operationalizations. I also applaud their effort to make the analysis code open-sourced.

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 #2 (Public review):

Summary:

Yonk and colleagues show that the posterior medial thalamus (POm), which is interconnected with sensory and motor systems, projects directly to major categories of neurons in the striatum, including direct and indirect pathway MSNs, and PV interneurons. Activity in POm-striatal neurons during a sensory-based learning task indicates a relationship between reward expectation and arousal. Inhibition of these neurons slows reaction to stimuli and overall learning. This circuit is positioned to feed salient event activation to the striatum to set the stage for effective learning and action selection.

Strengths:

The results are well presented and offer interesting insight into an understudied thalamostriatal circuit. In general, this work is important as part of a general need for an increased understanding of thalamostriatal circuits in complex learning and action selection processes, which have generally received less attention than corticostriatal systems.

Weaknesses:

There could be a stronger connection between the connectivity part of the data - showing that POm neurons context D1, D2, and PV neurons in striatum but with some different properties - and the functional side of the project. One wonders whether the POm neurons projecting to these subtypes or striatal neurons have unique signaling properties related to learning, or if there is a uniform, bulk signal sent to striatum. This is not a weakness per se, as it's reasonable for these questions to be answered in future papers.

All the in vivo activity-related conclusions stem from data from just 5 mice, which is a relatively small sample set. Optogenetic groups are also on the small side.

Comments on revisions:

The revision has a lot of thoughtful discussion added. I think overall the paper is more thorough and will also be a nice set up for a number of future research questions.

Reviewer #3 (Public review):

Yonk and colleagues investigate the role of the thalamostriatal pathway. Specifically, they studied the interaction of the posterior thalamic nucleus (PO) and the dorsolateral striatum in the mouse. First, they characterize connectivity by recording DLS neurons in in vitro slices and optogenetically activating PO terminals. PO is observed to establish depressing synapses onto D1 and D2 spiny neurons as well as PV neurons. Second, the image PO axons are imaged by fiber photometry in mice trained to discriminate textures. Initially, no trial-locked activity is observed, but as the mice learn PO develops responses timed to the audio cue that marks the start of the trial and precedes touch. PO does appear to encode the tactile stimulus type or outcome. Optogenetic suppression of PO terminals in striatum slow task acquisition. The authors conclude that PO provides a "behaviorally relevant arousal-related signal" and that this signal "primes" striatal circuitry for sensory processing.

A great strength of this paper is its timeliness. Thalamostriatal processing has received almost no attention in the past, and the field has become very interested in the possible functions of PO. Additionally, the experiments exploit multiple cutting-edge techniques.

There seem to be some technical/analytical weaknesses. The in vitro experiments appear to have some contamination of nearby thalamic nuclei by the virus delivering the opsin, which could change the interpretation. Some of the statistical analysis of these data also appear inappropriate. The correlative analysis of Pom activity in vivo, licking, and pupil could be more convincingly done.

The bigger weakness is conceptual - why should striatal circuitry need "priming" by thalamus in order to process sensory stimuli? Why would such circuitry even be necessary? Why is a sensory signal from cortex insufficient? Why should the animal more slowly learn the task? How does this fit with existing ideas of striatal plasticity? It is unclear from the experiments that the thalamostriatal pathway exists for priming sensory processing. In fact the optogenetic suppression of the thalamostriatal pathway seems to speak against that idea.

Comments on revisions:

The authors have only tweaked the Discussion and not necessarily in ways that addressed our previous comments. They could have fairly easily analyzed the effect of distance of recording from injection site and compared subsets of data depending on contamination beyond PO (my comments 1 and 2) or effects of movements (3 and 4). Minimally, they could have given caveats in the Results and Discussion about these, and I would strongly encourage them to be explicit about the caveats. The analyses would probably be better.

The suggestion that the effects have something to do with priming (5), seems a grasp for function of the circuit.

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):

Summary:

This paper aimed to determine the role EP sst+ neurons play in a probabilistic switching task.

Strengths:

- The in vivo recording of the EP sst+ neurons activity in the task is one of the strongest parts of this paper. Previous work had recorded from the EP-LHb population in rodents and primates in head fixed configurations, the recordings of this population in a freely moving context is a valuable addition to these studies and has highlighted more clearly that these neurons respond both at the time of choice and outcome.

- The use of a refined intersectional technique to record specifically the EP sst+ neurons is also an important strength of the paper. This is because previous work has shown that there are two genetically different types of glutamatergic EP neurons that project to the LHb. Previous work had not distinguished between these types in their recordings so the current results showing that the bidirectional value signaling is present in the EP sst+ population is valuable.

Weaknesses:

- One of the main weaknesses of the paper is to do with how the effect of the EP sst+ neurons on the behavior was assessed.

o All the manipulations (blocking synaptic release and blocking glutamatergic transmission) are chronic and more importantly the mice are given weeks of training after the manipulation before the behavioral effect is assessed. This means that as the authors point out in their discussion the mice will have time to adjust to the behavioral manipulation and compensate for the manipulations. The results do show that mice can adapt to these chronic manipulations and that the EP sst+ are not required to perform the task. What is unclear is whether the mice have compensated for the loss of EP sst+ neurons and whether they play a role in the task under normal conditions. Acute manipulations or chronic manipulations without additional training would be needed to assess this.

o Another weakness is that the effect of the manipulations was assessed in the 90/10 contingency version of the task. Under these contingencies, mice integrate past outcomes over fewer trials to determine their choice and animals act closer to a simple win-stay-lose switch strategy. Due to this it is unclear if the EP sst+ neurons would play a role in the task when they must integrate over a larger number of conditions in the less deterministic 70/30 version of the task. Indeed it is not clear that lesioning any other regions involved in evaluation of action outcomes such as VTA dopamine neurons, that encode reward prediction errors, would have any deficit when assessed in this way. Due to this, it's not clear if the mice have adapted to solve the task without evaluating action outcomes at all and are just acting in a more deterministic lose switch manner that would not presumably involve any of the circuitry in evaluating action outcomes.

- The authors conclude that they do not see any evidence for bidirectional prediction errors. It is not possible to conclude this. First, they see a large response in the EP sst+ neurons to the omission of an expected reward. This is what would be expected of a negative reward prediction error. There are much more specific well controlled tests for this that are commonplace in head-fixed and freely moving paradigms that could be tested to probe this. The authors do look at the effect of previous trials on the response and do not see strong consistent results, but this is not a strong formal test of what would be expected of a prediction error, either a positive or negative. The other way they assess this is by looking at the size of the responses in different recording sessions with different reward contingencies. They claim that the size of the reward expectation and prediction error should scale with the different reward probabilities. If all the reward probabilities were present in the same session this should be true as lots of others have shown for RPE. Because however this data was taken from different sessions it is not expected that the responses should scale, this is because reward prediction errors have been shown to adaptively scale to cover the range of values on offer (Tobler et al., Science 2005). A better test of positive prediction error would be to give a larger than expected reward on a subset of trials. Either way there is already evidence that responses reflect a negative prediction error in their data and more specific tests would be needed to formally rule in or out prediction error coding especially as previous recordings have shown it is present in previous primate and rodent recordings.

- There are a lot of variables in the GLM that occur extremely close in time such as the entry and exit of a port. If two variables occur closely in time and are always correlated it will be difficult if not impossible for a regression model to assign weights accurately to each event. This is not a large issue, but it is misleading to have regression kernels for port entry and exits unless the authors can show these are separable due to behavioral jitter or a lack of correlation under specific conditions, which does not seem to be the case.

Reviewer #3 (Public review):

Summary:

The authors find that Sst-EPN neurons, which project to the lateral habenula, encode information about response directionality (left vs right) and outcome (rewarded vs unrewarded). Surprisingly, chronic impairment of vesicular signaling in these neurons onto their LHb targets did not impair probabilistic choice behavior.

Strengths:

Strengths of the current work include extremely detailed and thorough analysis of data at all levels, not only of the physiological data, but also an uncommonly thorough analysis of behavioral response patterns.

Weaknesses:

In this revised manuscript, the authors have addressed my earlier critiques.

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 #3 (Public review):

Summary:

Bell and colleagues studied how different splice isoforms of voltage-gated CaV2 calcium channels affect channel expression, localization, function, synaptic transmission, and locomotor behavior at the larval Drosophila neuromuscular junction. They reveal that one mutually exclusive exon located in the fourth transmembrane domain encoding the voltage sensor is essential for calcium channel expression, function, active zone localization, and synaptic transmission. Furthermore, a second mutually exclusive exon residing in an intracellular loop containing the binding sites for Caβ and G-protein βγ subunits promotes the expression and synaptic localization of around ~50% of CaV2 channels, thereby contributing to ~50% of synaptic transmission. This isoform enhances release probability, as evident from increased short-term depression, is vital for homeostatic potentiation of neurotransmitter release induced by glutamate receptor impairment, and promotes locomotion. The roles of the two other tested isoforms remain less clear.

Strengths:

The study is based on solid data that was obtained with a diverse set of approaches. Moreover, it generated valuable transgenic flies that will facilitate future research on the role of calcium channel splice isoforms in neural function.

Weaknesses:

Comments on revisions:

The authors addressed most points. However, from my point of view, the new data (somatodendritic cac currents in adult motoneurons of IS4B mutants without the pre-pulse, and localization of IS4A channels in the larval brain) do not strongly support that the IS4B exon is required for cacophony localization. According to their definition of localization, IS4B is required for cacophony channels to enter motoneuron boutons and to localize to active zones. In case of a true localization defect (without degradation, as they claim), IS4A channels should mislocalize to the soma, axon, or dendrite. However, they do not find them in motoneurons of IS4B mutants. Furthermore, I find the interpretation of the voltage clamp data in flight motoneurons rather difficult. On the one hand, sustained HVA cac currents are strongly attenuated/absent in IS4B mutants. On the other hand, total cac currents (without the -50 mV pre-pulse, not shown in the original submission) are less affected in IS4B mutants. Based on these data, they conclude that IS4B is required for sustained HVA cac currents and that IS4A channel isoforms are expressed and functional. How does this relate to a localization defect at the NMJ? Finally, detecting IS4A channels in other cell types and regions is not a strong argument for a localization defect at the NMJ. I, therefore, suggest toning down the conclusions regarding a localization defect in IS4B mutants/a role for the IS4B exon in cac localization. It should be also discussed how a splice isoform in S4 may result in no detectable cac channels at the NMJ or regulate subcellular channel localization.

I have a few additional points, mainly related to the responses to my previous points:

(1) The authors state "active zones at the NMJ contain only cac isoforms with the IS4B exon. Nevertheless, the small representative EPSC remaining in IS4B mutants suggests that there is synchronous release in the absence of IS4B (Fig. 3B). Are the small EPSCs in dIS4B (Fig. 3B) indeed different from noise/indicative of evoked release? If yes, which cac channels may drive these EPSCs? IS4A channels?<br /> (2) (Related to previous point 4) The authors argue that EPSC amplitudes are not statistically different between Canton S and IS4A mutants (Fig. 2F). However, the Canton S group appears undersampled, thus precluding conclusions based on statistics. Moreover, the effect size Canton S vs. dIS4A looks similar to the one of Canton S vs. dIS4A/dIS4B.<br /> (3) (Related to previous point 11): Can they cite a paper relating calcium channel inactivation to EPSC half width/decay kinetics to support their speculation that "decreased EPSC half width could be caused by significantly faster channel inactivation kinetics" (p. 42, l.42). In addition, many papers have demonstrated that mini decay kinetics provide valuable insights into GluR subunit composition at the Drosophila NMJ (e.g., Schmid et al., 2008 https://doi.org/10.1038/nn.2122). Thus, the statement "Mini decay kinetic analysis because this depends strongly on the distance of the recording electrode to the actual site of transmission in these large muscle cells" is not valid and should be revised.

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 #2 (Public review):

Summary:

Guo et al. benchmarked and optimized methods for detecting Identity-By-Descent (IBD) segments in Plasmodium falciparum (Pf) genomes, which are characterized by high recombination rates and low marker density. Their goal was to address the limitations of existing IBD detection tools, which were primarily developed for human genomes and do not perform well in the genomic context of highly recombinant genomes. They first analysed various existing IBD callers, such as hmmIBD, isoRelate, hap-IBD, phased-IBD, refinedIBD. They focused on the impact of recombination on the accuracy, which was calculated based on two metrics, the false negative rate and the false positive rate. The results suggest that high recombination rates significantly reduce marker density, leading to higher false negative rates for short IBD segments. This effect compromises the reliability of IBD-based downstream analyses, such as effective population size (Ne) estimation.<br /> They showed that the best tool for IBD detection in Pf is hmmIBD, because it has relatively low FN/FP error rates and is less biased for relatedness estimates. However, this method is the less computationally efficient.<br /> Their suggestion is to optimize human-oriented IBD methods and use hmmIBD only for the estimation of Ne.

Strengths:

Although I am not an expert on Plasmodium falciparum genetics, I believe the authors have developed a valuable benchmarking framework tailored to the unique genomic characteristics of this species. Their framework enables a thorough evaluation of various IBD detection tools for non-human data, such as high recombination rates and low marker density, addressing a key gap in the field.<br /> This study provides a comparison of multiple IBD detection methods, including probabilistic approaches (hmmIBD, isoRelate) and IBS-based methods (hap-IBD, Refined IBD, phased IBD). This comprehensive analysis offers researchers valuable guidance on the strengths and limitations of each tool, allowing them to make informed choices based on specific use cases. I think this is important beyond the study of Pf.<br /> The authors highlight how optimized IBD detection can help identify signals of positive selection, infer effective population size (Ne), and uncover population structure.<br /> They demonstrate the critical importance of tailoring analytical tools to suit the unique characteristics of a species. Moreover, the authors provide practical recommendations, such as employing hmmIBD for quality-sensitive analyses and fine-tuning parameters for tools originally designed for non-P. falciparum datasets before applying them to malaria research.

Overall, this study represents a meaningful contribution to both computational biology and malaria genomics, with its findings and recommendations likely to have an impact on the field.

Weaknesses:

One weakness of the study is the lack of emphasis on the broader importance of studying Plasmodium falciparum as a critical malaria-causing organism. Malaria remains a significant global health challenge, causing hundreds of thousands of deaths annually. The authors could have introduced better the topic, even though I understand this is a methodological paper. While the study provides a thorough technical evaluation of IBD detection methods and their application to Pf, it does not adequately connect these findings to the broader implications for malaria research and control efforts. Additionally, the discussion on malaria and its global impact could have framed the study in a more accessible and compelling way, making the importance of these technical advances clearer to a broader audience, including researchers and policymakers in the fight against malaria.

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 #2 (Public review):

Summary:

This manuscript reports the results of an observational study conducted in Dar es Salaam, Tanzania, investigating potential associations between genetic variation in M. tuberculosis and human host vs. disease severity. The headline finding is that no such associations were found, either for host / bacillary genetics as main effects or for interactions between them.

Strengths:

Strengths of the study include its large size and rigorous approaches to classification of genetic diversity for host and bacillus.

Weaknesses:

(1) There are some limitations of the disease severity read-outs employed: X-ray scores and Xpert cycle thresholds from sputum analysis can only take account of pulmonary disease. CXR is an insensitive approach to assessing 'lung damage', especially when converted to a binary measure. What was the basis for selection of Ralph score of 71 to dichotomise patients? If outcome measures were analysed as continuous variables, would this have been more sensitive in capturing associations of interest?

(2) There is quite a lot of missing data, especially for TB scores - could this have introduced bias? This issue should be mentioned in the discussion.

(3) The analysis adjusted for age, sex, HIV status, age, smoking and cough duration - but not for socio-economic status. This will likely be a major determinant of disease severity. Was adjustment made for previous TB (i.e. new vs repeat episode) and drug-sensitivity of the isolate? Cough duration will effectively be a correlate/consequence of more severe disease - thus likely highly collinear with disease severity read-outs - not a true confounder. How does removal of this variable from the model affect results? Data on socioeconomic status should be added to models, or if not possible then lack of such data should be noted as a limitation.

(4) Recruitment at hospitals may have led to selection bias due to exclusion of less severe, community cases. The authors already acknowledge this limitation in the Discussion however.

(5) Introduction: References refer to disease susceptibility, but the authors should also consider the influences of host/pathogen genetics on host response - both in vitro (PMIDs 11237411, 15322056) and in vivo (PMID 23853590). The last of these studies encompassed a broader range of ethnic variation than the current study, and showed associations between host ancestry and immune response - null results from the current study may reflect the relative genetic homogeneity of the population studied.

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 #2 (Public review):

Summary:

In this work, the authors present a differentiable version of the widely-used Gillespie Algorithm. The Gillespie Algorithm has been used for decades to simulate the behavior of stochastic biochemical reaction networks. But while the Gillespie Algorithm is a powerful tool for the forward simulation of biochemical systems given some set of known reaction parameters, it cannot be used for reverse process, i.e. inferring reaction parameters given a set of measured system characteristics. The Differentiable Gillespie Algorithm ("DGA") overcomes this limitation by approximating two discontinuous steps in the Gillespie Algorithm with continuous functions. This makes it possible to calculate of gradients for each step in the simulation process which, in turn, allows the reaction parameters to be optimized via powerful backpropagation techniques. In addition to describing the theoretical underpinnings of DGA, the authors demonstrate different potential use-cases for the algorithm in the context of simple models of stochastic gene expression.

Overall, the DGA represents an important conceptual step forward for the field, and should lay the groundwork for exciting innovations in the analysis and design of stochastic reaction networks. At the same time, significantly more work is needed to establish when the approximations made by DGA are valid, and to demonstrate the viability of the algorithm in the context of complicated reaction networks.

Strengths:

This work makes an important conceptual leap by introducing a version of the Gillespie Algorithm that is end-to-end differentiable. This idea alone has the potential to drive a number of exciting innovations in the analysis, inference, and design of biochemical reaction networks. Beyond the theoretical adjustments, the authors also implement their algorithm in a Python-based codebase that combines DGA powerful optimization libraries like PyTorch. This codebase has the potential to be of interest to a wide range of researchers, even if the true scope of the method's applicability remains to be fully determined.

The authors also demonstrate how DGA can be used in practice both to infer reaction parameters from real experimental data (Figure 7) and to design networks with user-specified input-output characteristics (Figure 8). These illustrations should provide a nice roadmap for researchers interested in applying DGA to their own projects/systems.

Finally, although it does not stem directly from DGA, the exploration of pairwise parameter dependencies in different network architectures provides an interesting window into the design constraints (or lack thereof) that shape the architecture of biochemical reaction networks.

Weaknesses:

While it is clear that the DGA represents an important conceptual advancement, the authors do not do enough in the present manuscript to (i) validate the robustness of DGA inference and (ii) demonstrate that DGA inference works in the kinds of complex biochemical networks where it would actually be of legitimate use.

It is to the authors' credit that they are open and explicit about the potential limitations of DGA due to breakdowns in its continuous approximations. However they do not provide the reader with nearly enough empirical (i.e. simulation-based) or theoretical context to assess when, why, and to what extent DGA will fail in different situations. In Figure 2, they compare DGA to GA (i.e. ground-truth) in the context of a simple two state model of a stochastic transcription. Even in this minimal system, we see that DGA deviates notably from ground-truth both in the simulated mRNA distributions (Figure 2A) and in the ON/OFF state occupancy (Figure 2C). This begs the question of how DGA will scale to more complicated systems, or systems with non-steady state dynamics. Will the deviations become more severe? This is important because, in practice, there is really not much need for using DGA with a simple 2 state system-we have analytic solutions for this case. It is the more complex systems where DGA has the potential to move the needle.

A second concern is that the authors' present approach for parameter inference and error calculation does not seem to be reliable. For example, in Figure 5A, they show DGA inference results for the ON rate of a two-state system. We see substantial inference errors in this case, even though the inference problem should be non-degenerate in this case. One reason for this seems to be that the inference algorithm does not reliably find the global minimum of the loss function (Figure 2B). To turn DGA into a viable approach, it is paramount that the authors find some way to improve this behavior, perhaps by using multiple random initializations to better search the loss space.

Finally, the authors do a good job of illustrating how DGA might be used to infer biological parameters (Figure 7) and design reaction networks with desired input-output characteristics (Figure 8). However, analytic solutions exist for both of the systems they select for examples. This means that, in practice, there would be no need for DGA in these contexts, since one could directly optimize, e.g., the expressions for the mean and Fano Factor of the system in Figure 7A. I still believe that it is useful to have these examples, but it seems critical to add a use-case where DGA is the only option.

Reviewer #3 (Public review):

Summary:

This manuscript introduces a differentiable variant of the Gillespie algorithm (DGA) that allows gradient calculation using backpropagation. The most significant contribution of this work is the development of the DGA itself, a novel approach to making stochastic simulations differentiable. This is achieved by replacing discontinuous operations in the traditional Gillespie algorithm with smooth, differentiable approximations using sigmoid and Gaussian functions. This conceptual advance opens up new avenues for applying powerful gradient-based optimization techniques, prevalent in machine learning, to studying stochastic biological systems.

The method was tested on a simple two-state promoter model of gene expression. The authors found that the DGA accurately captured the moments of the steady-state distribution and other major qualitative features. However, it was less accurate at capturing information about the distribution's tails, potentially because rare events result from frequent low-probability reaction events where the approximations made by the DGA have a greater impact. The authors could further use the DGA to design a four-state promoter model of gene regulation that exhibited a desired input-output relationship. The DGA could learn parameters that produced a sharper response curve, which was achieved by consuming more energy.

The authors conclude that the DGA is a powerful tool for analyzing and designing stochastic systems.

Strengths:

The DGA allows gradient-based optimization techniques to estimate parameters and design networks with desired properties.

The DGA efficacy in estimating kinetic parameters from both synthetic and experimental data. This capability highlights the DGA's potential to extract meaningful biophysical parameters from noisy biological data.

The DGA's ability to design a four-state promoter architecture exhibiting a desired input-output relationship. This success indicates the potential of the DGA as a valuable tool for synthetic biology, enabling researchers to engineer biological circuits with predefined behaviours.

Weaknesses:

The study primarily focuses on analysing the steady-state properties of stochastic systems. It is unclear how and if this framework can be used beyond the steady-state data presented in the case studies, where it is already quite computationally heavy.<br /> A more in-depth exploration of the DGA's performance in analysing dynamic trajectories, which capture the system's evolution over time, would provide a more comprehensive view of the algorithm's capabilities.<br /> Gradient computations in the DGA can be susceptible to numerical instability, particularly when the sharpness parameters of the sigmoid and Gaussian approximations are set to high values. This issue could lead to challenges in convergence during the optimization process.

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 #2 (Public review):

Summary:

This is an interesting, timely, and high-quality study on the potential neuroprotective capabilities of C-C chemokine receptor type 5 (CCR5) antagonists in ischemic stroke. The focus is on preclinical investigations.

Strengths:

The results are timely and interesting. An outstanding feature is that stroke patient representatives have directly participated in the work. Although this is often called for, it is hardly realized in research practice, so the work goes beyond established standards.

The included studies were assessed regarding the therapeutic impact and their adherence to current quality assurance guidelines such as STAIR and SRRR, another important feature of this work. While overall results were promising, there were some shortcomings regarding guideline adherence.

The paper is very well written and concise yet provides much highly useful information. It also has very good illustrations and extremely detailed and transparent supplements.

Weaknesses:

Although the paper is of very high quality, a couple of items that may require the authors' attention to increase the impact of this exciting work further. Specifically:

Major aspects:

(1) I hope I did not miss that (apologies if I did), but when exactly was the search conducted? Is it possible to screen the recent literature (maybe up to 12/2024) to see whether any additional studies were published?

(2) Please clearly define the difference between "study" and "experiment," as this is not entirely clear. Is an "experiment" a distinct investigation within a particular publication (=study) that can describe more than one such "experiment"? Thanks for clarifying.

(3) Is there an opportunity to conduct a correlation analysis between the quality of a study (for instance, after transforming the ROB assessment into a kind of score) and reported effect sizes for particular experiments or studies? This might be highly interesting.

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 #2 (Public review):

Summary:

In this work, Gupta & Murphy present several parallel efforts. On one side, they present the hardware and software they use to build a head-fixed mouse experimental setup that they use to track in "real-time" the calcium activity in one or two spots at the surface of the cortex. On the other side, the present another setup that they use to take advantage of the "real-time" version of DeepLabCut with their mice. The hardware and software that they used/develop is described at length, both in the article and in a companion GitHub repository. Next, they present experimental work that they have done with these two setups, training mice to max out a virtual cursor to obtain a reward, by taking advantage of auditory tone feedback that is provided to the mice as they modulate either (1) their local cortical calcium activity, or (2) their limb position.

Strengths:

This work illustrates the fact that thanks to readily available experimental building blocks, body movement and calcium imaging can be carried using readily available components, including imaging the brain using an incredibly cheap consumer electronics RGB camera (RGB Raspberry Pi Camera). It is a useful source of information for researchers that may be interested in building a similar setup, given the highly detailed overview of the system. Finally, it further confirms previous findings regarding the operant conditioning of the calcium dynamics at the surface of the cortex (Clancy et al. 2020) and suggests an alternative based on deeplabcut to the motor tasks that aim to image the brain at the mesoscale during forelimb movements (Quarta et al. 2022).

Weaknesses:

This work covers 3 separate research endeavors: (1) The development of two separate setups, their corresponding software. (2) A study that is highly inspired from the Clancy et al. 2020 paper on the modulation of the local cortical activity measured through a mesoscale calcium imaging setup. (3) A study of the mesoscale dynamics of the cortex during forelimb movements learning. Sadly, the analyses of the physiological data appears uncomplete, and more generally the paper tends to offer overstatements regarding several points:<br /> - In contrast to the introductory statements of the article, closed-loop physiology in rodents is a well-established research topic. Beyond auditory feedback, this includes optogenetic feedback (O'Connor et al. 2013, Abbasi et al. 2018, 2023), electrical feedback in hippocampus (Girardeau et al. 2009), and much more.<br /> - The behavioral setups that are presented are representative of the state of the art in the field of mesoscale imaging/head fixed behavior community, rather than a highly innovative design. In particular, the closed-loop latency that they achieve (>60 ms) may be perceived by the mice. This is in contrast with other available closed-loop setups.<br /> - Through the paper, there are several statements that point out how important it is to carry out this work in a closed-loop setting with an auditory feedback, but sadly there is no "no feedback" control in cortical conditioning experiments, while there is a no-feedback condition in the forelimb movement study, which shows that learning of the task can be achieved in the absence of feedback.<br /> - The analysis of the closed-loop neuronal data behavior lacks controls. Increased performance can be achieved by modulating actively only one of the two ROIs, this is not clearly analyzed (for instance looking at the timing of the calcium signal modulation across the two ROIs. It seems that overall ROIs1 and 2 covariate, in contrast to Clancy et al. 2020. How can this be explained?

Reviewer #3 (Public review):

Summary:

The study demonstrates the effectiveness of a cost-effective closed-loop feedback system for modulating brain activity and behavior in head-fixed mice. Authors have tested real-time closed-loop feedback system in head-fixed mice two types of graded feedback: 1) Closed-loop neurofeedback (CLNF), where feedback is derived from neuronal activity (calcium imaging), and 2) Closed-loop movement feedback (CLMF), where feedback is based on observed body movement. It is a python based opensource system, and authors call it CLoPy. The authors also claim to provide all software, hardware schematics, and protocols to adapt it to various experimental scenarios. This system is capable and can be adapted for a wide use case scenario.

Authors have shown that their system can control both positive (water drop) and negative reinforcement (buzzer-vibrator). This study also shows that using the close loop system mice have shown better performance, learnt arbitrary task and can adapt to change in the rule as well. By integrating real-time feedback based on cortical GCaMP imaging and behavior tracking authors have provided strong evidence that such closed-loop systems can be instrumental in exploring the dynamic interplay between brain activity and behavior.

Strengths:

Simplicity of feedback systems designed. Simplicity of implementation and potential adoption.

Weaknesses:

Long latencies, due to slow Ca2+ dynamics and slow imaging (15 FPS), may limit the application of the system.

Major comments:

(1) Page 5 paragraph 1: "We tested our CLNF system on Raspberry Pi for its compactness, general-purpose input/output (GPIO) programmability, and wide community support, while the CLMF system was tested on an Nvidia Jetson GPU device." Can these programs and hardware be integrated with windows-based system and a microcontroller (Arduino/ Tency). As for the broad adaptability that's what a lot of labs would already have (please comment/discuss)?

(2) Hardware Constraints: The reliance on Raspberry Pi and Nvidia Jetson (is expensive) for real-time processing could introduce latency issues (~63 ms for CLNF and ~67 ms for CLMF). This latency might limit precision for faster or more complex behaviors, which authors should discuss in the discussion section.

(3) Neurofeedback Specificity: The task focuses on mesoscale imaging and ignores finer spatiotemporal details. Sub-second events might be significant in more nuanced behaviors. Can this be discussed in the discussion section?

(4) The activity over 6s is being averaged to determine if the threshold is being crossed before the reward is delivered. This is a rather long duration of time during which the mice may be exhibiting stereotyped behaviors that may result in the changes in DFF that are being observed. It would be interesting for the authors to compare (if data is available) the behavior of the mice in trials where they successfully crossed the threshold for reward delivery and in those trials where the threshold was not breached. How is this different from spontaneous behavior and behaviors exhibited when they are performing the test with CLNF?

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 #2 (Public review):

Approach

In this study, Yogesh et al. aimed at characterizing hemodynamic occlusion in two photon imaging, where its effects on signal fluctuations are underappreciated compared to that in wide field imaging and fiber photometry. The authors used activity-independent GFP fluorescence, GCaMP and GRAB sensors for various neuromodulators in two-photon and widefield imaging during a visuomotor context to evaluate the extent of hemodynamic occlusion in V1 and ACC. They found that the GFP responses were comparable in amplitude to smaller GCaMP responses, though exhibiting context-, cortical region-, and depth-specific effects. After quantifying blood vessel diameter change and surrounding GFP responses, they argued that GFP responses were highly correlated with changes in local blood vessel size. Furthermore, when imaging with GRAB sensors for different neuromodulators, they found that sensors with lower dynamic ranges such as GRAB-DA1m, GRAB-5HT1.0, and GRAB-NE1m exhibited responses most likely masked by the hemodynamic occlusion, while a sensor with larger SNR, GRAB-ACh3.0, showed much more distinguishable responses from blood vessel change.

Strengths

This work is of broad interest to two photon imaging users and GRAB developers and users. It thoroughly quantifies the hemodynamic driven GFP response and compares it to previously published GCaMP data in a similar context, and illustrates the contribution of hemodynamic occlusion to GFP and GRAB responses by characterizing the local blood vessel diameter and fluorescence change. These findings provide important considerations for the imaging community and a sobering look at the utility of these sensors for cortical imaging.

Importantly, they draw clear distinctions between the temporal dynamics and amplitude of hemodynamic artifacts across cortical regions and layers. Moreover, they show context dependent (Dark versus during visual stimuli) effects on locomotion and optogenetic light-triggered hemodynamic signals.

Most of the first generation neuromodulator GRAB sensors showed relatively small responses, comparable to blood vessel changes in two photon imaging, which emphasizes a need for improved the dynamic range and response magnitude for future sensors and encourages the sensor users to consider removing hemodynamic artifacts when analyzing GRAB imaging data.

Weaknesses

The largest weakness of the paper is that, while they convincingly quantify hemodynamic artifacts across a range of conditions, they do not quantify any methods of correcting for them. The utility of the paper could have been greatly enhanced had they tested hemodynamic correction methods (e.g. from Ocana-Santero et al., 2024) and applied them to their datasets. This would serve both to verify their findings-proving that hemodynamic correction removes the hemodynamic signal-and to act as a guide to the field for how to address the problem they highlight.

The paper attributes the source of 'hemodynamic occlusion' primarily to blood vessel dilation, but leaves unanswered how much may be due to shifts in blood oxygenation. Figure 4 directly addresses the question of how much of the signal can be attributed to occlusion by measuring the blood vessel dilation, but notably fails to reproduce any of the positive transients associated with locomotion in Figure 2. Thus, an investigation into or at least a discussion of what other factors (movement? Hb oxygenation?) may drive these distinct signals would be helpful.

Along these lines, the authors carefully quantified the correlation between local blood vessel diameter and GFP response (or neuropil fluorescence vs blood vessel fluorescence with GRAB sensors). To what extent does this effect depend on proximity to the vessels? Do GFP/ GRAB responses decorrelate from blood vessel activity in neurons further from vessels (refer to Figure 5A and B in Neyhart et al., Cell Reports 2024)?

Raw traces are shown in Figure 2 but we are never presented with the unaveraged data for locomotion of stimulus presentation times, which limits the reader's ability to independently assess variability in the data. Inclusion of heatmaps comparing event aligned GFP to GCaMP6f may be of value to the reader.

More detailed analysis of differences between the kinds of dynamics observed in GFP vs GCaMP6f expressing neurons could aid in identifying artifacts in otherwise clean data. The example neurons in Figure 2A hint at this as each display unique waveforms and the question of whether certain properties of their dynamics can reveal the hemodynamic rather than indicator driven nature of the signal is left open. Eg. do the decay rate and rise times differ significantly from GCaMP6f signals?

The authors suggest that signal to noise ratio of an indicator likely affects the ability to separate hemodynamic response from the underlying fluorescence signal. Does the degree of background fluorescence affect the size of the artifact? If there was variation in background and overall expression level in the data this could potentially be used to answer this question. Could lower (or higher!) expression levels increase the effects of hemodynamic occlusion?<br /> The choice of the phrase 'hemodynamic occlusion' may cause some confusion as the authors address both positive and negative responses in the GFP expressing neurons, and there may be additional contributions from changes in blood oxygenation state.

The choice of ACC as the frontal region provides a substantial contrast in location, brain movement, and vascular architecture as compared to V1. As the authors note, ACC is close to the superior sagittal sinus and thus is the region where the largest vascular effects are likely to occur. The reader is left to wonder how much of the ROI may or may not have included vasculature in the ACC vs V1 recordings as the only images of the recording sites provided are for V1. We are left unable to conclude whether the differences observed between these regions are due to the presence of visible vasculature, capillary blood flow or differences in neurovasculature coupling between regions. A less medial portion of M2 may have been a more appropriate comparison. At least, inclusion of more example imaging fields for ACC in the supplementary figures would be of value.

In Figure 3, How do the proportions of responsive GFP neurons compare to GCaMP6f neurons?

How is variance explained calculated in Figure 4? Is this from a linear model and R^2 value? Is this variance estimate for separate predictors by using single variable models? The methods should describe the construction of the model including the design matrix and how the model was fit and if and how cross validation was run.

Cortical depth is coarsely defined as L2/3 or L5, without numerical ranges in depth from pia.

Overall Assessment:

This paper is an important contribution to our understanding of how hemodynamic artifacts may corrupt GRAB and calcium imaging, even in two-photon imaging modes. Certain useful control experiments, such as intrinsic optical imaging in the same paradigms, were not reported, nor were any hemodynamic correction methods investigated. Thus, this limits both mechanistic conclusions and the overall utility with respect to immediate applications by end users. Nevertheless, the paper is of significant importance to anyone conducting two-photon or widefield imaging with calcium and GRAB sensors and deserves the attention of the broader neuroscience and in-vivo imaging community.

Reviewer #3 (Public review):

Summary:

In this study, the authors aimed to investigate if hemodynamic occlusion contributes to fluorescent signals measured with two-photon microscopy. For this, they image the activity-independent fluorophore GFP in 2 different cortical areas, at different cortical depths and in different behavioral conditions. They compare the evoked fluorescent signals with those obtained with calcium sensors and neuromodulator sensors and evaluate their relationship to vessel diameter as a readout of blood flow.<br /> They find that GFP fluorescence transients are comparable to GCaMP6f stimuli-evoked signals in amplitude, although they are generally smaller. Yet, they are significant even at the single neuronal level. They show that GFP fluorescence transients resemble those measured with the dopamine sensor GRAB-DA1m and the serotonin sensor GRAB-5HT1.0 in amplitude an nature, suggesting that signals with these sensors are dominated by hemodynamic occlusion. 
Moreover, the authors perform similar experiments with wide-field microscopy which reveals the similarity between the two methods in generating the hemodynamic signals. Together the evidence presented calls for the development and use of high dynamic range sensors to avoid measuring signals that have another origin from the one intended to measure. In the meantime, the evidence highlights the need to control for those artifacts such as with the parallel use of activity independent fluorophores.

Strengths:

- Comprehensive study comparing different cortical regions in diverse behavioral settings in controlled conditions.<br /> - Comparison to the state-of-the-art, i.e. what has been demonstrated with wide-field microscopy.<br /> - Comparison to diverse activity-dependent sensors, including the widely used GCaMP.

Weaknesses:

- The kinetics of GCaMP is stereotypic. An analysis/comment on if and how the kinetics of the signals could be used to distinguish the hemodynamic occlusion artefacts from calcium signals would be useful.<br /> - Is it possible that motion is affecting the signals in a certain degree? This issue is not made clear.<br /> - The causal relationship with blood flow remains open. Hemodynamic occlusion seems a good candidate causing changes in GFP fluorescence, but this remains to be well addressed in further research.

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 #2 (Public review):

Summary:

The study presents significant findings that elucidate the relationship between multi-dimensional social relationships and social attention in rhesus macaques. By integrating advanced computational methods, behavioral analyses, and neuroendocrine manipulation, the authors provide strong evidence for how oxytocin modulates attention within social networks. The results are robust and address critical gaps in understanding the dynamics of social attention in primates.

Strengths:

(1) The use of YOLOv5 for automatic behavioral detection is an exceptional methodological advance. The combination of automated analyses with manual validation enhances confidence in the data.<br /> (2) The study's focus on three distinct dimensions of social interaction (aggression, grooming, and proximity) is comprehensive and provides nuanced insights into the complexity of primate social networks.<br /> (3) The investigation of oxytocin's role adds a compelling neuroendocrine dimension to the findings, providing a bridge between behavioral and neural mechanisms.

Weaknesses:

(1) The study's conclusions are based on observations of only four monkeys, which limits the generalizability of the findings. Larger sample sizes could strengthen the validity of the results.<br /> (2) The limited set of stimulus images (in-group and out-group faces) may introduce unintended biases. This could be addressed by increasing the diversity of stimuli or incorporating a broader range of out-group members.

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 #2 (Public review):

Summary:

The paper from Kondo et al., addresses how the functional organization of synaptic inputs in 2/3 pyramidal neurons contributes to their output firing. Expressing GCamp6s to monitor calcium activity and the bi-stable inhibitory opsin SwiChR++ to inhibit the somatic activity of the imaged neurons, the authors were able to image up to ~5700 spines in basal dendrites from 6 neurons. Mapping the functional responses of such a large number of dendritic spines and relating it to the output firing of the parent neuron is a remarkable feat. The authors studied the clustering of similarly tuned spines within individual dendrites and found that while some dendrites are similarly tuned to the same orientation of the parent neuron, other dendrites exhibit tuning to other orientations and moreover a significant proportion of dendrites exhibit no tuning. Modelling work suggests that the clustering of spines in a small proportion of dendrites should suffice to give rise to the tuning of the parent cell.

Strengths:

(1) Removal of the potential confound of somatic firing via optogenetic inhibition is convincing and validates a useful tool for the neuroscientific community. As discussed by the authors the tool would be most valuable for the study of excitatory inputs in inhibitory neurons.

(2) The comparison of optogenetic inhibition of somatic responses and isolation of spine-specific signals using the removal of backpropagating action potential by robust regression is an important control and constitutes an important affirmation of previously published work.

(3) The large dataset size provides enough statistical power to test for clustering of similarly tuned spines in basal dendrites.

(4) The study provides a useful replication of previously published results.

(5) Modelling work in the study shows that as in the ferret visual cortex (Wilson et al., 2016), a combination of dendritic nonlinearity and spike thresholding contribute to the sharpness of orientation tuning in the mouse visual cortex.

Weaknesses:

(1) One of the main conclusions of the study, the classification of dendrites according to the presence or absence of visual responses, lacks quantification.

(2) Some of the statistics employed in combination with shuffling controls are not adequate.

(3) All the neurons imaged are very highly tuned (with a very high orientation selectivity index (OSI)). The performance of the models is evaluated by the correlation coefficient between the predicted and the measured somatic tuning curve. The high OSI of the neurons reduces the sensitivity of the evaluation of the models, as it results in extremely high or low correlation coefficients (Figure 8a). It would be important to recapitulate the results from the model for neurons with lower OSI, given that not all L2/3 neurons are so highly tuned.

(4) It is very hard to understand how the modelling results relate to the experimental data, as the definitions of what constitutes a clustered dendrite in the model or in the experimental data are unclear.

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 #2 (Public review):

In this paper Chang et al follow up on their lab's previous findings about the secreted protein Shv and its role in activity-induced synaptic remodeling at the fly NMJ. Previously they reported that shv mutants have impaired synaptic plasticity. Normally a high stimulation paradigm should increase bouton size and GluR expression at synapses but this does not happen in shv mutants. The phenotypes relating to activity dependent plasticity were completely recapitulated when Shv was knocked down only in neurons and could be completely rescued by incubation in exogenously applied Shv protein. The authors also showed that Shv activation of integrin signaling on both the pre- and post- synapse was the molecular mechanism underlying its function. Here they extend their study to consider the role of Shv derived from glia in modulating synaptic features at baseline and remodeling conditions. This study is important to understand if and how glia contribute to these processes. Using cell-type specific knockdown of Shv only in glia causes abnormally high baseline GluR expression and prevents activity-dependent increases in bouton size or GluR expression post-stimulation. This does not appear to be a developmental defect as the authors show that knocking down Shv in glia after basic development has the same effects as life long knockdown, so Shv is acting in real time. Restoring Shv in ONLY glia in mutant animals is sufficient to completely rescue the plasticity phenotypes and baseline GluR expression, but glial-Shv does not appear to activate integrin signaling which was shown to be the mechanism for neuronally derived Shv to control plasticity. This led the authors to hypothesize that glial Shv works by controlling the levels of neuronal Shv and extracellular glutamate. They provide evidence that in the absence of glial Shv, synaptic levels of Shv go up overall, presumably indicating that neurons secrete more Shv. In this context which could then work via integrin signaling as described to control plasticity. They use a glutamate sensor and observe decreased signal (extracellular glutamate) from the sensor in glial Shv KD animals, however, this background has extremely high GluR levels at the synapse which may account for some or all of the decreases in sensor signal in this background. Additional controls to test if increased GluR density alone affects sensor readouts and/or independently modulating GluR levels in the glial KD background would help strengthen this data. In fact, glial-specific shv KD animals have baseline levels of GluR that are potentially high enough to have hit a ceiling of expression or detection that accounts for the inability for these levels to modulate any higher after strong stimulation and such a ceiling effect should be considered when interpreting the data and conclusions of this paper. Several outstanding questions remain-why can't glial derived Shv activate integrin pathways but exogenously applied recombinant Shv protein can? The effects of neuronal specific rescue of shv in a shv mutant are not provided vis-à-vis GluR levels and bouton size to compare to the glial only rescue. Inclusion of this data might provide more insight to outstanding questions of how and why the source of Shv seems to matter for some aspects of the phenotypes but not others despite the fact that exogenous Shv can rescue and in some experimental paradigms but not others.

Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides compelling evidence that glia-derived Shriveled (Shv) modulates activity-dependent synaptic plasticity at the Drosophila neuromuscular junction (NMJ). This mechanism differs from the previously reported function of neuronally released Shv, which activates integrin signaling. They further show that this requirement of Shv is acute and that glial Shv supports synaptic plasticity by modulating neuronal Shv release and the ambient glutamate levels. However, there are a number of conceptual and technical issues that need to be addressed.

Major comments

(1) From the images provided for Fig 2B +RU486, the bouton size appears to be bigger in shv RNAi + stimulation, especially judging from the outline of GluR clusters.<br /> (2) The shv result needs to be replicated with a separate RNAi.<br /> (3) The phenotype of shv mutant resembles that of neuronal shv RNAi - no increased GluR baseline. Any insights why that is the case?<br /> (4) In Fig 3B, SPG shv RNAi has elevated GluR baseline, while PG shv RNAi has a lower baseline. In both cases, there is no activity induced GluR increase. What could explain the different phenotypes?<br /> (5) In Fig 4C, the rescue of PTP is only partial. Does that suggest neuronal shv is also needed to fully rescue the deficit of PTP in shv mutants?<br /> (6) The observation in Fig 5D is interesting. While there is a reduction in Shv release from glia after stimulation, it is unclear what the mechanism could be. Is there a change in glial shv transcription, translation or the releasing machinery? It will be helpful to look at the full shv pool vs the released ones.<br /> (7) In Fig 5E, what will happen after stimulation? Will the elevated glial Shv after neuronal shv RNAi be retained in the glia?<br /> (8) It would be interesting to see if the localization of shv differs based on if it is released by neuron or glia, which might be able to explain the difference in GluR baseline. For example, by using glia-Gal4>UAS-shv-HA and neuronal-QF>QUAS-shv-FLAG. It seems important to determine if they mix together after release? It is unclear if the two shv pools are processed differently.<br /> (9) Alternatively, do neurons and glia express and release different Shv isoforms, which would bind different receptors?<br /> (10) It is claimed that Sup Fig 2 shows no observable change in gross glial morphology, further bolstering support that glial Shv does not activate integrin. This seems quite an overinterpretation. There is only one image for each condition without quantification. It is hard to judge if glia, which is labeled by GFP (presumably by UAS-eGFP?), is altered or not.<br /> (11) The hypothesis that glutamate regulates GluR level as a homeostatic mechanism makes sense. What is the explanation of the increased bouton size in the control after glutamate application in Fig 6?<br /> (12) What could be a mechanism that prevents elevated glial released Shv to activate integrin signaling after neuronal shv RNAi, as seen in Fig 5E?<br /> (13) Any speculation on how the released Shv pool is sensed?

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 #2 (Public review):

Summary:

The authors employ a novel CRISPRi FACS screen and uncover the lysosomal transport complex BORC as a regulator of TDP-43 protein levels in iNeurons. They also find that BORC subunit knockouts impair lysosomal function, leading to slower protein turnover and implicating lysosomal activity in the regulation of TDP-43 levels. This is highly significant for the field given that a) other proteins could also be regulated in this way, b) understanding mechanisms that influence TDP-43 levels are significant given that its dysregulation is considered a major driver of several neurodegenerative diseases and c) the novelty of the proposed mechanism.

Strengths:

The novelty and information provided by the CRISPRi screen. The authors provide evidence indicating that BORC subunit knockouts impair lysosomal function, leading to slower protein turnover and implicating lysosomal activity in the regulation of TDP-43 levels and show a mechanistic link between lysosome mislocalization and TDP-43 dysregulation. The study highlights the importance of localized lysosome activity in axons and suggests that lysosomal dysfunction could drive TDP-43 pathologies associated with neurodegenerative diseases like FTD/ALS. Further, the methods and concepts will have an impact to the larger community as well. The work also sets up for further work to understand the somewhat paradoxical findings that even though the tagged TDP-43 protein is reduced in the screen, it does not alter cryptic exon splicing and there is a longer TDP-43 half-life with BORC KD.

Weaknesses:

While the data is very strong, the work requires some additional clarification.

Reviewer #3 (Public review):

Summary:

In this work, Ryan et al. have performed a state-of-the-art full genome CRISP-based screen of iNEurons expressing a teggd version of TDP-43 in order to determine expression modifiers of this protein. Unexpectedly, using this approach the authors have uncovered a previously undescribed role of the BORC complex in affecting the levels of TDP-43 protein, but not mRNA expression. Taken together, these findings represent a very solid piece of work that will certainly be important for the field.

Strengths:

- BORC is a novel TDP-43 expression modifier that has never been described before and it seemingly acts on regulating protein half life rather than transcriptome level. It has been long known that different labs have reported different half-lives for TDP-43 depending on the experimental system but no work has ever explained these discrepancies. Now, the work of Ryan et al. has for the time identified one of these factors which could account for these differences and play an important role in disease (although this is left to be determined in future studies).<br /> - The genome wide CRISPR screening has demonstrated to yield novel results with high reproducibility and could eventually be used to search for expression modifiers of many other proteins involved in neurodegeneration or other diseases

Weaknesses:

- The fact that TDP-43 mRNA does not change following BORCS6 KD is based on a single qRT-PCR that does not really cover all possibilities. For example, the mRNA total levels may not change but the polyA sites may have switched from the highly efficient pA1 to the less efficient and nuclear retained pA4. There are therefore a few other experiments that could have been performed to make this conclusion more compelling, maybe also performing RNAscope experiments to make sure that no change occurred in TDP-43 mRNA localisation in cells.<br /> - Even assuming that the mRNA does not change, no explanation for the change in TDP-43 protein half life has been proposed by the authors. This will presumably be addressed in future studies: for example, are mutants that lack different domains of TDP-43 equally affected in their half-lives by BORC KD?. Alternatively, can a mass-spec be attempted to see whether TDP-43 PTMs change following BORCS6 KD?

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 #2 (Public review):

Summary:

This study investigates in mice neural mechanisms generating sighs, which are periodic large-amplitude breaths occurring during normal breathing that subserve physiological pulmonary functions and are associated with emotional states such as relief, stress, and anxiety. Sighs are generated by a structure called the preBötzinger complex (preBötC) in the medulla oblongata that generates various forms of inspiratory activity including sighs. The authors have previously described a circuit involving neurons producing bombesin-related peptides Neuromedin B (NMB) and gastrin releasing peptide (GRP) that project to preBötC neurons expressing receptors for NMB (NMBRs) and GRP (GRPRs) and that activation of these preBötC neurons via these peptide receptors generates sighs. In this study the authors further investigated mechanisms of sigh generation by applying optogenetic and chemogenetic strategies to selectively activate the subpopulations of preBötC neurons expressing NMBRs and/or GRPRs, and a separate subpopulation of neurons expressing somatostatin (SST) but not NMBRs and GRPRs. The authors present convincing evidence that sigh-like inspirations can be evoked by photostimulation of the preBötC neurons expressing NMBRs or GRPRs. Photostimulation of SST neurons can independently evoke sighs, and chemogenetic inhibition of these neurons can abolish sighs. The results presented support the authors' conclusion that the preBötC neurons expressing NMBRs or GRPRs produce sighs via pathways to downstream SST neurons. Thus, these studies have identified some of the preBötC cellular elements likely involved in generating sighs.

Strengths:

(1) This study employs an effective combination of electrophysiological, transgenic, optogenetic, chemogenetic, pharmacological, and neuron activity imaging techniques to investigate sigh generation by distinct subpopulations of preBötC neurons in mice.

(2) The authors extend previous studies indicating that there is a peptidergic circuit consisting of NMB and GRP expressing neurons that project from the parafacial (pF) nucleus region to the preBötC and provides sufficient input to generate sighs, since photoactivation of either pF NMB or GRP neurons evoke ectopic sighs in this study.

(3) Solid evidence is presented that sighs can be evoked by direct photostimulation of preBötC neurons expressing NMBRs and/or GRPRs, and also a separate subpopulation of neurons expressing somatostatin (SST) but not NMBRs and GRPRs.

(4) The mRNA-expression data presented from in situ hybridization indicates that most preBötC neurons expressing NMBR, GRPR (or both) are glutamatergic and excitatory.

(5) Measurements in slices in vitro indicate that only the NMBR expressing neurons are normally rhythmically active during normal inspiratory activity and endogenous sigh activity.

(6) Evidence is presented that activation of preBötC NMBRs and/or GRPRs is not necessary for sigh production, suggesting that sighs are not the unique product of the preBötC bombesin-peptide signaling pathway.

(7) The novel conclusion is presented that the preBötC neurons expressing NMBRs and/or GRPRs produce sighs via the separate downstream population of preBötC SST neurons, which the authors demonstrate can independently generate sighs, whereas chemogenetic inhibition of preBötC SST neurons selectively abolishes sighs generated by activating NMBRs and GRPRs.

Weaknesses:

(1) While these studies have identified subpopulations of preBötC neurons capable of episodically evoking sigh-like inspiratory activity, mechanisms producing the normal slow sigh rhythm were not investigated and remain unknown.

(2) The authors have addressed some of the reviewers' main technical concerns and issues relating to interpretation of the results in their rebuttal letter, but have minimally revised the manuscript. Accordingly, there remain important technical and interpretation issues requiring resolution in the revised manuscript.

Comments on revisions:

The authors have clarified in their rebuttal letter the rationale for utilizing two different photostimulation paradigms but have not incorporated any of this explanation in Methods, which would be helpful for readers.

Reviewer #3 (Public review):

Summary:

This manuscript by Cui et al., studies the mechanisms for the generation of sighing, an essential breathing pattern. This is an important and interesting topic, as sighing maintains normal pulmonary function and is associated with various emotional conditions. However, the mechanisms of its generation remain not fully understood. The authors employed different approaches, including optogenetics, chemogenetics, intersectional genetic approach, and slice electrophysiology and calcium imaging, to address the question, and found several neuronal populations are sufficient to induce sighing when activated. Furthermore, ectopic sighs can be triggered without the involvement of neuromedin B (NMB) or gastrin releasing peptide (GRP) or their receptors in the preBötzinger Complex (preBötC) region of the brainstem. Additionally, activating SST neurons in the preBötC region induces sighing, even when other receptors are blocked. Based on these results, the authors concluded that increased excitability in certain neurons (NMBR or GRPR neurons) activates pathways leading to sigh generation, with SST neurons serving as a downstream component in converting regular breaths into sighs.

Strengths:

The authors employed a combination of various sophisticated approaches, including optogenetics, chemogenetics, intersectional genetic approach, and slice electrophysiology and calcium imaging, to precisely pinpoint the mechanism responsible for sigh generation. They utilized multiple genetically modified mouse lines, enabling them to selectively manipulate and observe specific neuronal populations involved in sighing.<br /> Using genetics and calcium imaging, the authors record the neuronal activity of NMBR and GRPR neurons, respectively, and identified their difference in activity pattern. Furthermore, by applying the intersectional approach, the authors were able to genetically target and manipulate several distinct neuronal populations, such as NMBR+, GRPR- neurons and GRPR+, NMBR- neurons, and conducted a detailed characterization of their functions in influencing sighing.

Weaknesses:

(1) The authors employed two conditions for optogenetic activation: long pulse photostimulation (LPP) and short pulse photostimulation (SPP), with durations ranging from 4-10s for LPP and 100-500 ms for SPP. These could generate huge variability in the experiments. The rationale behind the selection of these conditions in each experiment remains unclear in the manuscript. Additionally, it is not explained why these specific durations were chosen. Furthermore, the interpretation for the varied responses observed under these conditions is not provided. Clarification on the rationale and interpretation of these experimental parameters would enhance the understanding of the results. The description of the experiment conditions should be consistent throughout the manuscript.

(2) Regarding the fiber optics, my understanding is that they are placed outside of the brainstem from the ventral side. Given the locations of the pF and preBötC neurons, could the differences in responses be attributed to the varying distances of each population from the ventral surface? In fact, in Figure 8, NMBR is illustrated as being closer to the ventral surface. Does it represent the actual location of these neurons?

(3) The results of recording on NMBR neurons in Figure 4 were compelling. However, I'm curious why the recording of GRPR neurons and their response to the neuropeptide were not presented or examined. Additionally, considering the known cross-reaction between peptides and their receptors, it might be worthwhile to investigate how GRP modulates NMBR neurons and how NMB modulates GRPR neurons.

(4) The authors found that activation of several preBötC populations, including NMBR, GRPR, and SST neurons, despite pharmacological inhibition of NMBR and GRPR, can still induce sighing, and concluded that "activation of preBötC NMBRs and/or GRPRs is not necessary for sigh production". I disagree with this conclusion. Even when the receptors are silenced, artificial (optogenetic or chemogenetic) activation could still activate the same downstream pathways. This cannot be used as evidence to claim that the receptors are not required for sighing in vivo, because it is possible that the receptors are still necessary for the activation of these neurons under natural conditions. For instance, while diaphragm activation induces breathing, it does not negate the crucial role of the nervous system in regulating this process in physiological conditions.

(5) The authors noted varied responses upon activating specific subpopulations of the preBötC neurons, namely NMBR, GRPR, and SST neurons. Could these differences be attributed to variations in viral labeling efficiency among different mouse genetic lines? Are there discrepancies in the number of labeled neurons across the lines? Additionally, the authors did not thoroughly characterize the specificities of AAV targeting in their Cre and Flp lines. It's uncertain whether the AAV-labeled neurons are strictly restricted to the designated population without notable leakage into other populations. This is particularly crucial for the experiments manipulating SST neurons. If there's substantial labeling of NMBR or GRPR neurons, it could undermine the conclusions drawn. Further examination of the precision and selectivity of the labeling techniques is necessary to ensure the accurate interpretation of the experimental findings.

(6) The authors have addressed some of the reviewers' concerns in the revision; however, many important issues remain unaddressed.

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):

Summary:

In this paper, the authors assess the function of Rab10 in dense core vesicle (DCV) exocytosis using RNAi and cultured neurons. The author provides evidence that their knockdown (KD) is effective and provides evidence that DCV is compromised. They also perform proteomic analysis to identify potential pathway that are affected upon KD of Rab10 that may be involved in DCV release. Upon focusing on ER morphology and protein synthesis, the authors conclude that defects in protein synthesis and ER Ca2+ homeostasis contributes to the DVC release defect upon Rab10 KD.

Strengths:

The data related to Rab10's role in DCV release seems to be strong and carried out with rigor. While the paper lacks in vivo evidence that this gene is indeed involved in DCV in a living mammalian organism, I feel the cellular studies have value. The identification of ER defect in Rab10 manipulation is not truly novel but it is a good conformation of studies performed in other systems. The finding that DCV release defect and protein synthesis defect seen upon Rab10 KD can be significantly suppressed by Leucine supplementation is also a strength of this work.

Weaknesses:

The weaknesses mentioned in my previous comments have been addressed through the revision process.

Reviewer #3 (Public review):

In this study, Dong and colleagues set to dissect the role of Rab10 small GTPase on the intracellular trafficking and exocytosis of dense core vesicles (DCVs). While the authors have already shown that Rab3 plays a central role in the exocytosis of DVC in mammalian neurons, the roles of several other Rab-members have been identified genetically, but their precise mechanism of action in mammalian neurons remains unclear. In this study, the authors use a carefully designed and thoroughly executed series of experiments, including live-cell imaging, functional calcium-imaging, proteomics, and electron microscopy, to identify that DCV secretion upon Rab10 depletion in adult neurons is primarily a result of dysregulated protein synthesis and, to a lesser extent, disrupted intracellular calcium buffering. Given that the full deletion of Rab10 has deleterious effect on neurons and that Rab10 has a major role in axonal development, the authors cautiously employed the knock-down strategy from 7 DIV, to focus on the functional impact of Rab10 in mature neurons. The experiments in this study were meticulously conducted, incorporating essential controls and thoughtful considerations, ensuring rigorous and comprehensive results that fully support the conclusions.

Comments on revisions:

The authors have addressed all the comments and suggestions raised by reviewers, making this an excellent and timely study.

Reviewer #2 (Public review):

Summary.

Some forms of Artificial Intelligence (AI), particularly those based on artificial neural networks (ANNs), draw inspiration from biological brains and neurons. Understanding the functional repertoire and underlying logic of real neurons could, therefore, help improve ANNs. While the cell bodies and axons of neurons produce rapid, high-amplitude action potentials (~100 mV over ~2 ms), dendrites-constituting about 80% of neuronal membrane area-generate smaller but longer-lasting electrical signals, known as glutamate-mediated dendritic plateau potentials (~50 mV over >100 ms). The authors have designed artificial neurons capable of producing these dendritic plateau potentials and, through simulations, demonstrate that such prolonged dendritic signals reduce the negative effects of temporal jitter in real or artificial neural networks. Specifically, they show that in ANNs with neurons capable of dendritic plateau potentials, reliable sparse spiking computation can occur without the need for precise input synchronization. This means that despite fluctuations in network activity (such as delays in the brain circuit responses, for example), neurons can still link related network events. Thus, dendritic plateau potentials enable neurons to retain information longer, connecting events that are not exactly simultaneous. Interestingly, one of the indirect conclusions of the current study is that neurons equipped with dendritic plateau potentials may reduce the total number of cells (nodes, units) required to perform robust computations.

Strengths.

Most studies in neuroscience are descriptive, focusing on observations and measurements. Fewer tackle the more challenging task of explaining the rationale behind specific natural designs. This study does just that, addressing the fundamental problem of asynchrony in neural communication caused by conduction delays and noise. Given that neurons with short membrane time constants can integrate only nearly simultaneous inputs, the authors propose a solution: dendritic plateau potentials. These potentials, generated through glutamate-mediated depolarization within dendritic branches, effectively broaden the temporal integration window, allowing neurons to handle temporal jitter, variability, stochasticity, and maintain reliable computation. Thus, dendritic plateau potentials appear to be an adaptive feature evolved to support rapid, reliable CNS computations.

Weaknesses.

The authors have appropriately revised unsupported statements from previous versions, but the manuscript could benefit from examples of testable hypotheses derived from their findings. For example, what specific experimental questions could be investigated to validate these computational predictions? Providing concrete examples of potential experimental tests would make the work more accessible and actionable for experimentalists, assuming such experiments are feasible.

Additionally, many readers may lack a background in computational modeling or Artificial Neural Networks. To enhance accessibility, key terms and concepts should be explained at a level suitable for first-year graduate students, ensuring clarity for a broader audience.

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 #2 (Public review):

Summary:

The results presented demonstrate AAV2-CFI gene therapy delivers long-term and marginally higher FI protein in vitreous humor that results in a concomitant reduction in the FB activation product Ba. However, the lack of clinical efficacy in the phase I/II study, possibly due to lower in vitro potency when compared to currently approved pegcetacoplan, raise important considerations for the utility of this therapeutic approach. Despite the early termination of the PPY988 clinical development program, the study achieved significant milestones, including the implementation of subretinal gene therapy delivery in older adults, complement biomarker comparison between serial vitreous humor and aqueous humor samples and vitreous humor proteomic assessment via Olink.

Strengths:

Long-term augmentation of FI protein in vitreous humor over 96-weeks and reduction of FB breakdown product Ba in vitreous humor suggests modulation of the complement system. Developed a novel in vitro assay suggesting FI's ability to reduce C3 convertase activity is weaker than pegcetacoplan and FH and may suggest a higher dose of FI will be required for clinical efficacy. Warn of the poor correlation between vitreous humor and aqueous humor biomarkers and suggest aqueous humor may not be a reliable proxy for vitreous humor with regard to complement activation/inhibition studies.

Weaknesses:

The vitrectomy required for subretinal route of administration causes long-term loss of total protein and may influence interpretation of complement biomarker results even with normalization. The modified in vitro assay of complement activation suggests a several hundred-fold increase in FI protein is required to significantly affect C3a levels. Interestingly, the in vitro assay demonstrates 100% inhibition of C3a with pegcetacoplan and FH therapeutics, but only a 50% reduction with FI even at the highest concentrations tested. This observation suggests FI may not be rate-limiting for negative complement regulation under the in vitro conditions tested and potentially in the eye. It is unclear if pharmacokinetic and pharmacodynamic properties in aqueous humor and vitreous humor compartments are a reliable predictor of FI level/activity after subretinal delivery AAV2-CFI gene therapy.

Reviewer #3 (Public review):

Summary:

The manuscript by Hallam et al describes the analysis of various biomarkers in patients undergoing complement factor I supplementation treatment (PPY988 gene therapy) as part of the FOCUS Phase I/II clinical trial. The authors used validated methods (multiplexed assays and OLINK proteomics) for measuring multiple soluble complement proteins in the aqueous humour (AH) and vitreous humour (VH) of 28 patients over a series of timepoints, up to and including 96 weeks. Based on biomarker comparisons, the levels of FI synthesised by PPY988 were believed to be insufficient to achieve the desired level of complement inhibition. Subsequent comparative experiments showed that PPY988-delievred FI was much less efficacious than Pegceptacoplan (FDA approved complement inhibitor under the name SYFORVE) when tested in an artificial VH matrix.

Strengths:

The manuscript is well written with data clearly presented and appropriate statistics used for the analysis itself. It's great to see data from real clinical samples that can help support future studies and therapeutic design. The identification that complement biomarker levels present in the AH do not represent the levels found in the VH is an important finding for the field, given the number of complement-targeting therapies in development and the desperate need for good biomarkers for target engagement. This study also provides a wealth of baseline complement protein measurements in both human AH and VH (and companion measurements in plasma) that will prove useful for future studies.

Weaknesses:

No real weaknesses in the manuscript itself. It is only a shame that it would appear that FI supplementation is not a viable way forward for treating GA secondary to AMD.

Comments on revisions:

I think the authors have done all that they can to present this study in the most robust manner possible.

Reviewer #1 (Public review):

Summary:

In this study, the authors use thermal proteome profiling to capture changes in protein stability following a brief (30 min) treatment of cells with various mitochondrial stressors. This approach identified PEBP1 as a potentiator of Integrated Stress Response (ISR) induction by various mitochondrial stressors, although the specific dynamics vary by stressor. PEBP1 deletion attenuates DELE1-HRI-mediated activation of the ISR, independent of its known role in the RAF/MEK/ERK pathway. These effects can be bypassed by HRI overexpression and do not affect DELE1 processing. Interestingly, in cells, PEBP1 physically interacts with eIF2alpha, but not its phosphorylated form (eIF2alpha-P), leading the authors to suggest that PEBP1 functions as a scaffold to promote eIF2alpha phosphorylation by HRI.

Strengths:

The authors present a clear and well-structured study, beginning with an original and unbiased approach that effectively addresses a novel question. The investigation of PEBP1 as a specific regulator of the DELE1-HRI signaling axis is particularly compelling, supported by extensive data from both genetic and pharmacological manipulations. Including careful titrations, time-course experiments, and orthogonal approaches strengthens the robustness of their findings and bolsters their central claims.

Moreover, the authors skillfully integrate publicly available datasets with their original experiments, reinforcing their conclusions' generality and broader relevance. This comprehensive combination of methodologies underscores the reliability and significance of the study's contributions to our understanding of stress signaling.

Weaknesses:

While the study presents exciting findings, there are a few areas that could benefit from further exploration. The HRI-DELE1 pathway was only recently discovered, leaving many unanswered questions. The observation that PEBP1 interacts with eIF2alpha, but not with its phosphorylated form, suggests a novel mechanism for regulating the Integrated Stress Response (ISR). However, as they note themselves, the authors do not delve into the biochemical or molecular mechanisms through which PEBP1 promotes HRI signaling. Given the availability of antibodies against phosphorylated HRI, it would have been interesting to explore whether PEBP1 influences HRI phosphorylation. Furthermore, since the authors already have recombinant PEBP1 protein (as shown in Figure 1D), additional in vitro experiments such as in vitro immunoprecipitation, FRET, or surface plasmon resonance (SPR) could have confirmed the interaction with eIF2alpha. Future studies might investigate whether PEBP1 directly interacts with HRI, stimulates its auto-phosphorylation or kinase activity, or serves as a template for oligomerization, potentially supported by structural characterization of the complex and mutational validation.

Another point of weakness is the unclear significance of the 1.5-2x enhanced interaction with eIF2alpha upon PEBP1 phosphorylation, as there is little evidence to show that this increase has any downstream effects. The ATF4-luciferase reporter experiments, comparing WT and S153D overexpression, may have reached saturation with WT, making it difficult to detect further stimulation by S153D. Additionally, expression levels for WT and mutant forms are not provided, making it challenging to interpret the results. It would also be interesting to explore whether combined mitochondrial stress and PMA treatment further enhance the ISR.

Lastly, while the authors claim that oligomycin does not significantly alter the melting temperature of recombinant PEBP1 in vitro, the data in Figure S1D suggest a small shift. Without variance measures across replicates or background subtraction, this claim is less convincing. The inclusion of statistical analyses would strengthen the interpretation of these results.

Impact on the field:

The study's relevance is underscored by the fact that overactive ISR is linked to a broad range of neurodegenerative diseases and cognitive disorders, a field actively being explored for therapeutic interventions, with several drugs currently in clinical trials. Similarly, mitochondrial dysfunction plays a well-established role in brain health and other diseases. Identifying new targets within these pathways, like PEBP1, could provide alternative therapeutic strategies for treating such conditions. Therefore, gaining a deeper understanding of the mechanisms through which PEBP1 influences ISR regulation is highly pertinent and could have far-reaching implications for the development of future therapies.

Reviewer #2 (Public review):

Summary:

In this work, Cheng et al use the TPP/MS-CETSA strategy to discover new components for the mitochondria arm of the Integrated Stress Response. By using short exposures of several drugs that potentially induce mitochondrial stress, they find significant CETSA shifts for the scaffold protein PEBP1 both for antimycinA and oligomycin, making PEBP1 a candidate for mitochondrial-induced ISR signaling. After extensive follow-up work, they provide good support that PEBP1 is likely involved in ISR, and possibly act through an interaction with the key ISR effector node EIF2a.

Strengths:

The work adds an important understanding of ISR signaling where PEBP1 might also constitute a druggable node to attenuate cellular stress. Although CETSA has great potential for dissecting cellular pathways, there are few studies where this has been explored, particularly with such an extensive follow-up, also giving the work methodological implications. Together I therefore think this study could have a significant impact.

Weaknesses:

The TPP/MS-CETSA experiment is quite briefly described and might have a too relaxed cut-off. The assays confirming interactions between PEBP1 and EIF2a might not be fully conclusive.

Reviewer #3 (Public review):

Summary:

In this paper, Chang and Meliala et al. demonstrate that PEBP1 is a modulator of the ISR, specifically through the induction of mitochondrial stress. The authors utilize thermal proteome profiling (TPP) by which they identify PEPB1 as a thermally stabilized protein upon oligomycin treatment, indicating its role in mitochondrial stress. Moreover, RNA-sequencing analysis indicated that PEBP1 may be specifically modulating the mitochondrial stress-induced ISR, as PEBP1 knock-out reduces phosphorylation of eIF2α. They also show that PEBP1 function is independent of ER stress specifically tunicamycin treatment and loss of PEBP1 does affect mitochondrial ISR but in an OMA1, DELE1 independent manner. Thus, the authors hypothesized that PEBP1 interacts directly with eIF2α, functioning as a scaffolding protein. However, direct co-immunoprecipitation failed to demonstrate PEBP1 and eIF2α potential interaction. The authors then used a NanoBiT luminescence complementation assay to show the PEBP1-eIF2a interaction and its disruption by S51 phosphorylation.

Strengths:

Taken together, this work is novel, and the data presented suggests PEBP1 has a role as a modulator of the mitochondrial ISR, enhancing the signal to elicit the necessary response.

Weaknesses:

The one major issue of this work is the lack of a mechanism showing precisely how PEBP1 amplifies the mitochondrial integrated stress response. The work, as it is described, presents data suggesting PEBP1's role in the ISR but fails to present a more conclusive mechanism.

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):

The factors that influence the differentiation of EBs and RBs during Chlamydial development are not clearly understood. A previous study had shown a redox oscillation during the Chlamydial developmental cycle. Based on this observation, the authors hypothesize that the bacterial redox state may play a role in regulating the differentiation in Chlamydia. To test their hypothesis, they make knock-down and overexpression strains of the major ROS regulator, ahpC. They show that the knock-down of ahpC leads to a significant increase in ROS levels leading to an increase in the production of elementary bodies and overexpression leads to a decrease in EB production likely caused by a decrease in oxidation. From their observations, they present an interesting model wherein an increase in oxidation favors the production of EBs.

Comments on revisions:

Major concerns have been satisfactorily addressed.

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 #3 (Public review):

Summary:

Suzuki-Okutani and collogues reported a new live-attenuated SARS-CoV-2 vaccine (BK2102) containing multiple deletion/substitution mutations. They show that the vaccine candidate is highly attenuated and demonstrates great safety profile in multiple animal models (hamsters and Tg-Mice). Of importance, their data show that singe intranasal immunization with BK2102 leads to strong protection of hamsters against D614G and BA.5 challenge in both lungs and URT (nasal wash). Both humoral and cellular responses were induced, and neutralization activity remained for >360 after single inoculation.

Strengths:

The manuscript describes a comprehensive study that evaluates safety, immunogenicity, and efficacy of a new live-attenuated vaccine. Strengths of the study include: 1) strong protection against immune evasive variant BA.5 in both lungs and NW; 2) durability of immunity for >360 days; 3) confirmation of URT protection through a transmission experiment.<br /> While first-generation COVID-19 vaccines have achieved much success, new vaccines that provide mucosal and durable protection remain needed. Thus, the study is significant.

Weaknesses:

Lack of a more detailed discussion of this new vaccine approach in the context of reported live-attenuated SARS-CoV-2 vaccines in terms of its advantages and/or weakness<br /> Antibody endpoint titers could be presented.<br /> Lack of elaboration on immune mechanisms of protection at the upper respiratory tract (URT) against an immune evasive variant in the absence of detectable neutralizing antibodies

Comments on revisions:

In the revised submission, the authors have added new data and have modified the manuscript accordingly. They have reasonably addressed my comments raised in the previous round of review. The quality and clarity of the manuscript are improved.

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 #2 (Public Review):

Summary:

In this work, Vivian Salgueiro et al. have comprehensively investigated the role of VirR in the vesicle production process in Mtb using state-of-the-art omics, imaging, and several biochemical assays. From the present study, authors have drawn a positive correlation between cell membrane permeability and vasculogenesis and implicated VirR in affecting membrane permeability, thereby impacting vasculogenesis.

Strengths:

The authors have discovered a critical factor (i.e. membrane permeability) that affects vesicle production and release in Mycobacteria, which can broadly be applied to other bacteria and may be of significant interest to other scientists in the field. Through omics and multiple targeted assays such as targeted metabolomics, PG isolation, analysis of Diaminopimelic acid and glycosyl composition of the cell wall, and, importantly, molecular interactions with PG-AG ligating canonical LCP proteins, the authors have established that VirR is a central scaffold at the cell envelope remodelling process which is critical for MEV production.

Comments on the revision.

Authors have addressed the concerns, specifically regarding the expression of downstream genes. It appears that they are not altered significantly.

Data in Fig 6C shows significantly higher expresssion of VirR compared to control or knock down. In the absence of using a regulatable expression such as nitrile, this is expected from a constitutive promoter.

I have no further questions for the author.

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.

Reviewer #2 (Public review):

This is an interesting manuscript where a CA-only CG model (Mpipi) was used to examine the critical temperature (Tc) of phase separation of a set of 140 variants of prion-like low complexity domains (PLDs). The key result is that Tc of these PLDs seems to have a linear dependence on substitutions of various sticker and space residues. This is potentially useful for estimating the Tc shift when making novel mutations of a PLD.

Comments on revisions: The authors have addressed concerns raised previously.

Reviewer #3 (Public review):

Summary:

"Decoding Phase Separation of Prion-Like Domains through Data-Driven Scaling Laws" by Maristany et al. offers a significant contribution to the understanding of phase separation in prion-like domains (PLDs). The study investigates the phase separation behavior of PLDs, which are intrinsically disordered regions within proteins that have the propensity to undergo liquid-liquid phase separation (LLPS). This phenomenon is crucial in forming biomolecular condensates, which play essential roles in cellular organization and function. The authors employ a data-driven approach to establish predictive scaling laws that describe the phase behavior of these domains.

Strengths:

The study benefits from a robust dataset encompassing a wide range of PLDs, which enhances the generalizability of the findings. The authors' meticulous curation and analysis of this data add to the study's robustness. The scaling laws derived from the data provide predictive insights into the phase behavior of PLDs, which can be useful in the future for the design of synthetic biomolecular condensates.

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 #2 (Public review):

Summary:

The study examined 10 congenitally blind patients who recovered vision through the surgical removal of bilateral dense cataracts, measuring neural activity and neuro chemical profiles from the visual cortex. The declared aim is to test whether restoring visual function after years of complete blindness impacts excitation/inhibition balance in the visual cortex.

Strengths:

The findings are undoubtedly useful for the community, as they contribute towards characterising the many ways in which this special population differs from normally sighted individuals. The combination of MRS and EEG measures is a promising strategy to estimate a fundamental physiological parameter - the balance between excitation and inhibition in the visual cortex, which animal studies show to be heavily dependent upon early visual experience. Thus, the reported results pave the way for further studies, which may use a similar approach to evaluate more patients and control groups.

Weaknesses:

The main methodological limitation is the lack of an appropriate comparison group or condition to delineate the effect of sight recovery (as opposed to the effect of congenital blindness). Few previous studies suggested that Excitation/Inhibition ratio in the visual cortex is increased in congenitally blind patients; the present study reports that E/I ratio decreases instead. The authors claim that this implies a change of E/I ratio following sight recovery. However, supporting this claim would require showing a shift of E/I after vs. before the sight-recovery surgery, or at least it would require comparing patients who did and did not undergo the sight-recovery surgery (as common in the field).

There are also more technical limitations related to the correlation analyses, which are partly acknowledged in the manuscript. A bland correlation between GLX/GABA and the visual impairment is reported, but this is specific to the patients group (N=10) and would not hold across groups (the correlation is positive, predicting the lowest GLX/GABA ratio values for the sighted controls - opposite of what is found). There is also a strong correlation between GLX concentrations and the EEG power at the lowest temporal frequencies. Although this relation is intriguing, it only holds for a very specific combination of parameters (of the many tested): only with eyes open, only in the patients group.

Conclusions:

The main claim of the study is that sight recovery impacts the excitation/inhibition balance in the visual cortex, estimated with MRS or through indirect EEG indices. However, due to the weaknesses outlined above, the study cannot distinguish the effects of sight recovery from those of visual deprivation. Moreover, many aspects of the results are interesting but their validation and interpretation require additional experimental work.

Reviewer #3 (Public review):

This manuscript examines the impact of congenital visual deprivation on the excitatory/inhibitory (E/I) ratio in the visual cortex using Magnetic Resonance Spectroscopy (MRS) and electroencephalography (EEG) in individuals whose sight was restored. Ten individuals with reversed congenital cataracts were compared to age-matched, normally sighted controls, assessing the cortical E/I balance and its interrelationship and to visual acuity. The study reveals that the Glx/GABA ratio in the visual cortex and the intercept and aperiodic signal are significantly altered in those with a history of early visual deprivation, suggesting persistent neurophysiological changes despite visual restoration.

First of all, I would like to disclose that I am not an expert in congenital visual deprivation, nor in MRS. My expertise is in EEG (particularly in the decomposition of periodic and aperiodic activity) and statistical methods. Although the authors addressed some of the concerns of the previous version, major concerns and flaws remain in terms of methodological and statistical approaches along with the (over)interpretation of the results. Specific concerns include:

(1 3.1) Response to Variability in Visual Deprivation<br /> Rather than listing the advantages and disadvantages of visual deprivation, I recommend providing at least a descriptive analysis of how the duration of visual deprivation influenced the measures of interest. This would enhance the depth and relevance of the discussion.

(2 3.2) Small Sample Size<br /> The issue of small sample size remains problematic. The justification that previous studies employed similar sample sizes does not adequately address the limitation in the current study. I strongly suggest that the correlation analyses should not feature prominently in the main manuscript or the abstract, especially if the discussion does not substantially rely on these correlations. Please also revisit the recommendations made in the section on statistical concerns.

(3 3.3) Statistical Concerns<br /> While I appreciate the effort of conducting an independent statistical check, it merely validates whether the reported statistical parameters, degrees of freedom (df), and p-values are consistent. However, this does not address the appropriateness of the chosen statistical methods.

Several points require clarification or improvement:<br /> (4) Correlation Methods: The manuscript does not specify whether the reported correlation analyses are based on Pearson or Spearman correlation.<br /> (5) Confidence Intervals: Include confidence intervals for correlations to represent the uncertainty associated with these estimates.<br /> (6) Permutation Statistics: Given the small sample size, I recommend using permutation statistics, as these are exact tests and more appropriate for small datasets.<br /> (7) Adjusted P-Values: Ensure that reported Bonferroni corrected p-values (e.g., p > 0.999) are clearly labeled as adjusted p-values where applicable.<br /> (8) Figure 2C<br /> Figure 2C still lacks crucial information that the correlation between Glx/GABA ratio and visual acuity was computed solely in the control group (as described in the rebuttal letter). Why was this analysis restricted to the control group? Please provide a rationale.<br /> (9 3.4) Interpretation of Aperiodic Signal<br /> Relying on previous studies to interpret the aperiodic slope as a proxy for excitation/inhibition (E/I) does not make the interpretation more robust.<br /> (10) Additionally, the authors state:<br /> "We cannot think of how any of the exploratory correlations between neurophysiological measures and MRS measures could be accounted for by a difference e.g. in skull thickness."<br /> (11) This could be addressed directly by including skull thickness as a covariate or visualizing it in scatterplots, for instance, by representing skull thickness as the size of the dots.<br /> (12 3.5) Problems with EEG Preprocessing and Analysis<br /> Downsampling: The decision to downsample the data to 60 Hz "to match the stimulation rate" is problematic. This choice conflates subsequent spectral analyses due to aliasing issues, as explained by the Nyquist theorem. While the authors cite prior studies (Schwenk et al., 2020; VanRullen & MacDonald, 2012) to justify this decision, these studies focused on alpha (8-12 Hz), where aliasing is less of a concern compared of analyzing aperiodic signal. Furthermore, in contrast, the current study analyzes the frequency range from 1-20 Hz, which is too narrow for interpreting the aperiodic signal as E/I. Typically, this analysis should include higher frequencies, spanning at least 1-30 Hz or even 1-45 Hz (not 20-40 Hz).<br /> (13) Baseline Removal: Subtracting the mean activity across an epoch as a baseline removal step is inappropriate for resting-state EEG data. This preprocessing step undermines the validity of the analysis. The EEG dataset has fundamental flaws, many of which were pointed out in the previous review round but remain unaddressed. In its current form, the manuscript falls short of standards for robust EEG analysis. If I were reviewing for another journal, I would recommend rejection based on these flaws.<br /> (14) The authors mention:<br /> "The EEG data sets reported here were part of data published earlier (Ossandón et al., 2023; Pant et al., 2023)." Thus, the statement "The group differences for the EEG assessments corresponded to those of a larger sample of CC individuals (n=38) " is a circular argument and should be avoided."<br /> The authors addressed this comment and adjusted the statement. However, I do not understand, why not the full sample published earlier (Ossandón et al., 2023) was used in the current study?

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 #2 (Public review):

Summary:

The overall question that is addressed in this study is how the S. coelicolor contractile injection system (CISSc) works and affects both cell viability and differentiation, which it has been implicated to do in previous work from this group and others. The CISSc system has been enigmatic in the sense that it is free-floating in the cytoplasm in an extended form and is seen in contracted conformation (i.e. after having been triggered) mainly in dead and partially lysed cells, suggesting involvement in some kind of regulated cell death. So, how do the structure and function of the CISSc system compare to those of related CIS from other bacteria, does it interact with the cytoplasmic membrane, how does it do that, and is the membrane interaction involved in the suggested role in stress-induced, regulated cell death? The authors address these questions by investigating the role of a membrane protein, CisA, that is encoded by a gene in the CIS gene cluster in S. coelicolor. Further, they analyse the structure of the assembled CISSc, purified from the cytoplasm of S. coelicolor, using single-particle cryo-electron microscopy.

Strengths:

The beautiful visualisation of the CIS system both by cryo-electron tomography of intact bacterial cells and by single-particle electron microscopy of purified CIS assemblies are clearly the strengths of the paper, both in terms of methods and results. Further, the paper provides genetic evidence that the membrane protein CisA is required for the contraction of the CISSc assemblies that are seen in partially lysed or ghost cells of the wild type. The conclusion that CisA is a transmembrane protein and the inferred membrane topology are well supported by experimental data. The cryo-EM data suggest that CisA is not a stable part of the extended form of the CISSc assemblies. These findings raise the question of what CisA does.

Weaknesses:

The investigations of the role of CisA in function, membrane interaction, and triggering of contraction of CIS assemblies, are important parts of the paper and are highlighted in the title. However, the experimental data provided to answer these questions appear partially incomplete and not as conclusive as one would expect.

The stress-induced loss of viability is only monitored with one method: an in vivo assay where cytoplasmic sfGFP signal is compared to FM5-95 membrane stain. Addition of a sublethal level of nisin lead to loss of sfGFP signal in individual hyphae in the WT, but not in the cisA mutant (similarly to what was previously reported for a CIS-negative mutant). Technically, this experiment and the example images that are shown give rise to some concern. Only individual hyphal fragments are shown that do not look like healthy and growing S. coelicolor hyphae. Under the stated growth conditions, S. coelicolor strains would normally have grown as dense hyphal pellets. It is therefore surprising that only these unbranched hyphal fragments are shown in Fig. 4ab. Further, S. coelicolor would likely be in a stationary phase when grown 48 h in the rich medium that is stated, giving rise to concern about the physiological state of the hyphae that were used for the viability assay. It would be valuable to know whether actively growing mycelium is affected in the same way by the nisin treatment, and also whether the cell death effect could be detected by other methods.

The model presented in Fig. 5 suggests that stress leads to a CisA-dependent attachment of CIS assemblies to the cytoplasmic membrane, and then triggering of contraction, leading to cell death. This model makes testable predictions that have not been challenged experimentally. Given that sublethal doses of nisin seem to trigger cell death, there appear to be possibilities to monitor whether activation of the system (via CisA?) indeed leads to at least temporally increased interaction of CIS with the membrane. Further, would not the model predict that stress leads to an increased number of contracted CIS assemblies in the cytoplasm? No clear difference in length of the isolated assemblies if Fig. S7 is seen between untreated and nisin-exposed cells, and also no difference between assemblies from WT and cisA mutant hyphae.

The interaction of CisA with the CIS assembly is critical for the model but is only supported by Alphafold modelling, predicting interaction between cytoplasmic parts of CisA and Cis11 protein in the baseplate wedge. An experimental demonstration of this interaction would have strengthened the conclusions.

The cisA mutant showed a similarly accelerated sporulation as was previously reported for CIS-negative strains, which supports the conclusion that CisA is required for function of CISSc. But the results do not add any new insights into how CIS/CisA affects the progression of the developmental life cycle and whether this effect has anything to do with the regulated cell death that is caused by CIS. The same applies to the effect on secondary metabolite production, with no further mechanistic insights added, except reporting similar effects of CIS and CisA inactivations.

Concluding remarks:<br /> The work will be of interest to anyone interested in contractile injection systems, T6SS, or similar machineries, as well for people working on the biology of streptomycetes. There is also a potential impact of the work in the understanding of how such molecular machineries could have been co-opted during evolution to become a mechanism for regulated cell death. However, this latter aspect remains still poorly understood. Even though this paper adds excellent new structural insights and identifies a putative membrane anchor, it remains elusive how the Streptomyces CIS may lead to cell death. It is also unclear what the advantage would be to trigger death of hyphal compartments in response to stress, as well as how such cell death may impact (or accelerate) the developmental progression. Finally, it is inescapable to wonder whether the Streptomyces CIS could have any role in protection against phage infection.

Reviewer #3 (Public review):

Summary:

In this work, Casu et al. have reported the characterization of a previously uncharacterized membrane protein CisA encoded in a non-canonical contractile injection system of Streptomyces coelicolor, CISSc, which is a cytosolic CISs significantly distinct from both intracellular membrane-anchored T6SSs and extracellular CISs. The authors have presented the first high-resolution structure of extended CISSc structure. It revealed important structural insights in this conformational state. To further explore how CISSc interacted with cytoplasmic membrane, they further set out to investigate CisA that was previously hypothesized to be the membrane adaptor. However, the structure revealed that it was not associated with CISSc. Using fluorescence microscope and cell fractionation assay, the authors verified that CisA is indeed a membrane-associated protein. They further determined experimentally that CisA had a cytosolic N-terminal domain and a periplasmic C-terminus. The functional analysis of cisA mutant revealed that it is not required for CISSc assembly but is essential for the contraction, as a result, the deletion significantly affects CISSc-mediated cell death upon stress, timely differentiation, as well as secondary metabolite production. Although the work did not resolve the mechanistic detail how CisA interacts with CISSc structure, it provides solid data and a strong foundation for future investigation toward understanding the mechanism of CISSc contraction, and potentially, the relation between the membrane association of CISSc, the sheath contraction and the cell death.

Strengths:

The paper is well-structured, and the conclusion of the study is supported by solid data and careful data interpretation was presented. The authors provided strong evidence on (1) the high-resolution structure of extended CISSc determined by cryo-EM, and the subsequent comparison with known eCIS structures, which sheds light on both its similarity and different features from other subtypes of eCISs in detail; (2) the topological features of CisA using fluorescence microscopic analysis, cell fractionation and PhoA-LacZα reporter assays, (3) functions of CisA in CISSc-mediated cell death and secondary metabolite production, likely via the regulation of sheath contraction.

Weaknesses:

The data presented are not sufficient to provide mechanistic details of CisA-mediated CISSc contraction, as authors are not able to experimentally demonstrate the direct interaction between CisA with baseplate complex of CISSc (hypothesized to be via Cis11 by structural modeling), since they could not express cisA in E. coli due to its potential toxicity. Therefore, there is a lack of biochemical analysis of direct interaction between CisA and baseplate wedge. In addition, there is no direct evidence showing that CisA is responsible for tethering CISSc to the membrane upon stress, and the spatial and temporal relation between membrane association and contraction remains unclear. Further investigation will be needed to address these questions in future.

Discussion:

Overall, the work provides a valuable contribution to our understanding on the structure of a much less understood subtype of CISs, which is unique compared to both membrane-anchored T6SSs and host-membrane targeting eCISs. Importantly, the work serves as a good foundation to further investigate how the sheath contraction works here. The work contributes to expanding our understanding of the diverse CIS superfamilies.

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 #2 (Public review):

The main strength of this work is the impressive performance of a microscope assembled for a fraction of the cost of a commercial, turnkey system. The authors have created a very clever design that removes everything that is not essential. They show compelling time-lapse data looking at single molecules, tracking particles visible in brightfield mode, and looking at cell division with multiple labels in a live cell preparation.

The weaknesses of the paper include:<br /> (1) the lack of more comprehensive explanations of the microscope and what it takes to build and operate it.<br /> For example, the dimensions of the microscope, how samples are mounted, which lenses are compatible, and whether eduWOSMs have been built by groups other than the authors would be useful information.<br /> (2) the absence of more detailed descriptions of some of the experiments, such as frame rates and Z-stack information.<br /> (3) the lack of standardized measures of performance.<br /> For example, images of subresolution tetraspeck beads and measurements of PSF would provide estimates on resolution in XY, resolution in Z, axial chromatic aberrations and lateral chromatic aberrations. Repeating these measurements on different eduWOSMs will provide an idea of how reliably the performance can be achieved.<br /> If these issues were addressed, it would make it more likely that other groups could build and operate this system successfully.

Overall, the authors have designed and built an impressive system at low cost. Providing a bit more information in the manuscript would make it much more likely that other laboratories could replicate this design in their own environments.

Reviewer #1 (Public review):

Summary:

The authors aim to explore the effects of the electrogenic sodium-potassium pump (Na+/K+-ATPase) on the computational properties of highly active spiking neurons, using the weakly-electric fish electrocyte as a model system. Their work highlights how the pump's electrogenicity, while essential for maintaining ionic gradients, introduces challenges in neuronal firing stability and signal processing, especially in cells that fire at high rates. The study identifies compensatory mechanisms that cells might use to counteract these effects, and speculates on the role of voltage dependence in the pump's behavior, suggesting that Na+/K+-ATPase could be a factor in neuronal dysfunctions and diseases

Strengths:

(1) The study explores a less-examined aspect of neural dynamics-the effects of Na+/K+-ATPase electrogenicity. It offers a new perspective by highlighting the pump's role not only in ion homeostasis but also in its potential influence on neural computation.<br /> (2) The mathematical modeling used is a significant strength, providing a clear and controlled framework to explore the effects of the Na+/K+-ATPase on spiking cells. This approach allows for the systematic testing of different conditions and behaviors that might be difficult to observe directly in biological experiments.<br /> (3) The study proposes several interesting compensatory mechanisms, such as sodium leak channels and extracellular potassium buffering, which provide useful theoretical frameworks for understanding how neurons maintain firing rate control despite the pump's effects.

Weaknesses:

(1) While the modeling approach provides valuable insights, the lack of experimental data to validate the model's predictions weakens the overall conclusions.<br /> (2) The proposed compensatory mechanisms are discussed primarily in theoretical terms without providing quantitative estimates of their impact on the neuron's metabolic cost or other physiological parameters.

Reviewer #2 (Public review):

Summary:

The paper 'The electrogenicity of the Na+/K+-ATPase poses challenges for computation in highly active spiking cells' by Weerdmeester, Schleimer, and Schreiber uses computational models to present the biological constraints under which electrocytes-specialized highly active cells that facilitate electro-sensing in weakly electric fish-may operate. The authors suggest potential solutions these cells could employ to circumvent these constraints.

Electrocytes are highly active or spiking (greater than 300Hz) for sustained periods (for minutes to hours), and such activity is possible due to an influx of sodium and efflux of potassium ions into these cells for each spike. This ion imbalance must be restored after each spike, which in electrocytes, as with many other biological cells, is facilitated by the Na-K pumps at the expense of biological energy, i.e., ATP molecules. For each ATP molecule the pump uses, three positively charged sodium ions from the intracellular space are exchanged for two positively charged potassium ions from the extracellular volume. This creates a net efflux of positive ions into the extracellular space, resulting in hyperpolarized potentials for the cell over time. This does not pose an issue in most cells since the firing rate is much slower, and other compensatory mechanisms and other pumps can effectively restore the ion imbalances. In electrocytes of weakly electric fish, however, that operate under very different circumstances, the firing rate is exceptionally high. On top of this, these cells are also involved in critical communication and survival behaviors, emphasizing their reliable functioning.

In a computation model, the authors test four increasingly complex solutions to the problem of counteracting the hyperpolarized states that occur due to continuous NaK pump action to sustain baseline activity. First, they propose a solution for a well-matched Na leak channel that operates in conjunction with the NaK pump, counteracting the hyperpolarizing states naturally. Additionally, their model shows that when such an orchestrated Na leak current is not included, quick changes in the firing rates could have unexpected side effects. Secondly, they study the implication of this cell in the context of chirps - a means of communication between individual fishes. Here, an upstream pacemaking neuron entrains the electrocyte to spike, which ceases to produce a so-called chirp - a brief pause in the sustained activity of the electrocytes. In their model, the authors show that it is necessary to include the extracellular potassium buffer to have a reliable chirp signal. Thirdly, they tested another means of communication in which there was a sudden increase in the firing rate of the electrocyte followed by a decay to the baseline. For reliable occurrence of this, they emphasize that a strong synaptic connection between the pacemaker neuron and the electrocyte is warranted. Finally, since these cells are energy-intensive, they hypothesize that electrocytes may have energy-efficient action potentials, for which their NaK pumps may be sensitive to the membrane voltages and perform course correction rapidly.

Strengths:

The authors extend an existing electrocyte model (Joos et al., 2018) based on the classical Hodgkin and Huxley conductance-based models of Na and K currents to include the dynamics of the NaK pump. The authors estimate the pump's properties based on reasonable assumptions related to the leak potential. Their proposed solutions are valid and may be employed by weakly electric fish. The authors explore theoretical solutions that compound and suggest that all these solutions must be simultaneously active for the survival and behavior of the fish. This work provides a good starting point for exploring and testing in in vivo experiments which of these proposed solutions the fish use and their relative importance.

Weaknesses:

The modeling work makes assumptions and simplifications that should be listed explicitly. For example, it assumes only potassium ions constitute the leak current, which may not be true as other ions (chloride and calcium) may also cross the cell membrane. This implies<br /> that the leak channels' reversal potential may differ from that of potassium. Additionally, the spikes are composed of sodium and potassium currents only and no other ion type (no calcium). Further, these ion channels are static and do not undergo any post-translational modifications. For instance, a sodium-dependent potassium pump could fine-tune the potassium leak currents and modulate the spike amplitude (Markham et al., 2013).

This model considers only NaK pumps. In many cell types, several other ion pumps/exchangers/symporters are simultaneously present and actively participate in restoring the ion gradients. It may be true that only NaK pumps are expressed in the weakly electric fish Eigenmannia virescens. This limits the generalizability of the results to other cell types. While this does not invalidate the results of the present study, biological processes may find many other solutions to address the non-electroneutral nature of the NaK pump. For example, each spike could include a small calcium ion influx that could be buffered or extracted via a sodium-calcium exchanger.

Finally, including testable hypotheses for these computational models would strengthen this work.

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 #2 (Public review):

Summary:

The authors present a paper that attempts to tackle an important question, with potential impact far beyond the field of animal behavior research: what are the relative contributions of innate personality traits versus early life experience on individual behavior in the wild? The study, performed on Egyptian fruit bats that are caught in the wild and later housed in an outdoor colony, is solidly executed, and benefits greatly from a unique setup in which controlled laboratory experiments are combined with monitoring of individuals as they undertake undirected, free exploration of their natural environment.

The primary finding of the paper is that there is a strong effect of early life experience on behavior in the wild, where individual bats that were exposed to an enriched environment as juveniles later travelled farther and over greater distances when permitted to explore and forage ad libitum, as compared with individual bats who were subjected to a more impoverished environment. Meanwhile, no prominent effect of innate "personality", as assessed by indices of indoor foraging behavior early on, before the bats were exposed to the controlled environmental treatment, was observed on three metrics of outdoor foraging behavior. The authors conclude that the early environment plays a larger role than innate personality on the behavior of adult bats.

Strengths:

(1) Elegant design of experiments and impressive combination of methods<br /> Bats used in the experiment were taken from wild colonies in different geographical areas, but housed during the juvenile stage in a controlled indoor environment. Bats are tested on the same behavioral paradigm at multiple points in their development. Finally, the bats are monitored with GPS as they freely explore the area beyond the outdoor colony.

(2) Development of a behavioral test that yields consistent results across time<br /> The multiple-foraging box paradigm, in which behavioral traits such as overall activity, levels of risk-taking, and exploratoriness can be evaluated as creative, and suggestive of behavioral paradigms other animal behavior researchers might be able to use. It is especially useful, given that it can be used to evaluate the activity of animals seemingly at most stages of life, and not just in adulthood.

Weaknesses:

(1) Robustness and validity of personality measures<br /> Coming up with robust measures of "personality" in non-human animals is tricky. While this paper represents an important attempt at a solution, some of the results obtained from the indoor foraging paradigm raise questions as to the reliability of this task for assessing "personality".

(2) Insufficient exploitation of data<br /> Between the behavioral measures and the very multidimensional GPS data, the authors are in possession of a rich data set. However, I don't feel that this data has been adequately exploited for underlying patterns and relationships. For example, many more metrics could be extracted from the GPS data, which may then reveal correlations with early measures of personality or further underscore the role of the early environment. In addition, the possibility that these personality measures might in combination affect outdoor foraging is not explored.

(3) Interpretation of statistical results and definition of statistical models<br /> Some statistical interpretations may not be entirely accurate, particularly in the case of multiple regression with generalized linear models. In addition, some effects which may be present in the data are dismissed as not significant on the basis of null hypothesis testing.

Below I have organized the main points of critique by theme, and ordered subordinate points by order of importance:

(1) Assessing personality metrics and the indoor paradigm: While I applaud this effort and think the metrics used are justified, I see a few issues in the results as they are currently presented:<br /> (a) [Major] I am somewhat concerned that here, the foraging box paradigm is being used for two somewhat conflicting purposes: (1) assessing innate personality and (2) measuring changes in personality as a result of experience. If the indoor foraging task is indeed meant to measure and reflect both at the same time, then perhaps this can be made more explicit throughout the manuscript. In this circumstance, I think the authors could place more emphasis on the fact that the task, at later trials/measurements, begins to take on the character of a "composite" measure of personality and experience.

(b) [Major] Although you only refer to results obtained in trials 1 and 2 when trying to estimate "innate personality" effects, I am a little worried that the paradigm used to measure personality, i.e. the stable components of behavior, is itself affected by other factors such as age (in the case of activity, Fig. 1C3, S1C1-2), the environment (see data re trial 3), and experience outdoors (see data re trials 4/5).

Ideally, a study that aims to disentangle the role of predisposition from early-life experience would have a metric for predisposition that is relatively unchanging for individuals, which can stand as a baseline against a separate metric that reflects behavioral differences accumulated as a result of experience.

I would find it more convincing that the foraging box paradigm can be used to measure personality if it could be shown that young bats' behavior was consistent across retests in the box paradigm prior to any environmental exposure across many baseline trials (i.e. more than 2), and that these "initial settings" were constant for individuals. I think it would be important to show that personality is consistent across baseline trials 1 and 2. This could be done, for example, by reproducing the plots in Fig. 1C1-3 while plotting trial 1 against trial 2. (I would note here that if a significant, positive correlation were to be found (as I would expect) between the measures across trial 1 and 2, it is likely that we would see the "habituation effect" the authors refer to expressed as a steep positive slope on the correlation line (indicating that bold individuals on trial 1 are much bolder on trial 2).)

(c) Related to the previous point, it was not clear to me why the data from trial 2 (the second baseline trial) was not presented in the main body of the paper, and only data from trial 1 was used as a baseline.

In the supplementary figure and table, you show that the bats tended to exhibit more boldness and exploratory behavior, but fewer actions, in trial 2 as compared with trial 1. You explain that this may be due to habituation to the experimental setup, however, the precise motivation for excluding data from trial 2 from the primary analyses is not stated. I would strongly encourage the authors to include a comparison of the data between the baseline trials in their primary analysis (see above), combine the information from these trials to form a composite baseline against which further analyses are performed, or further justify the exclusion of data as a baseline.

(2) Comparison of indoor behavioral measures and outdoor behavioral measures<br /> Regarding the final point in the results, correlation between indoor personality on Trial 4 and outdoor foraging behavior: It is not entirely clear to me what is being tested (neither the details of the tests nor the data or a figure are plotted). Given some of the strong trends in the data - namely, (1) how strongly early environment seems to affect outdoor behavior, (2) how strongly outdoor experience affects boldness, measured on indoor behavior (Fig. 1D) - I am not convinced that there is no relationship, as is stated here, between indoor and outdoor behavior. If this conclusion is made purely on the basis of a p-value, I would suggest revisiting this analysis.

(3) Use of statistics/points regarding the generalized linear models<br /> While I think the implementation of the GLMM models is correct, I am not certain that the interpretation of the GLMM results is entirely correct for cases where multivariate regression has been performed (Tables 4s and S1, and possibly Table 3). (You do not present the exact equation they used for each model (this would be a helpful addition to the methods), therefore it is somewhat difficult to evaluate if the following critique properly applies, however...)

The "estimate" for a fixed effect in a regression table gives the difference in the outcome variable for a 1 unit increase in the predictor variable (in the case of numeric predictors) or for each successive "level" or treatment (in the case of categorical variables), compared to the baseline, the intercept, which reflects the value of the outcome variable given by the combination of the first value/level of all predictors. Therefore, for example, in Table 4a - Time spend outside: the estimate for Bat sex: male indicates (I believe) the difference in time spent outside for an enriched male vs. an enriched female, not, as the authors seem to aim to explain, the effect of sex overall. Note that the interpretation of the first entry, Environmental condition: impoverished, is correct. I refer the authors to the section "Multiple treatments and interactions" on p. 11 of this guide to evaluating contrasts in G/LMMS: https://bbolker.github.io/mixedmodels-misc/notes/contrasts.pdf

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 #2 (Public review):

Summary:

In the manuscript, "Prophage regulation of Shewanella fidelis 3313 motility and biofilm formation: implications for gut colonization dynamics in Ciona robusta", the authors are experimentally investigating the idea that integrated viruses (prophages) within a bacterial colonizer of the host Ciona robusta affect both the colonizer and the host. They found a prophage within the Ciona robusta colonizing bacterium Shewanella fidelis 3313, which affected both the bacteria and host. This prophage does so by regulating the phosphodiesterase gene pdeB in the bacterium when the bacterium has colonized the host. The prophage also regulates the activity of the host immune gene VCBP-C during early bacterial colonization. Prophage effects on both these genes affect the precise localization of the colonizing bacterium, motility of the bacterium, and bacterial biofilm formation on the host. Interestingly, VCBP-C expression also suppressed a prophage structural protein, creating a tripartite feedback loop in this symbiosis. This is exciting research that adds to the emerging body of evidence that prophages can have beneficial effects not only on their host bacteria but also on how that bacteria interacts in its environment. This study establishes the evolutionary conservation of this concept with intriguing implications of prophage effects on tripartite interactions.

Strengths:

This research effectively shows that a prophage within a bacterium colonizing a model ascidian affects both the bacterium and the host in vivo. These data establish the prophage effects on bacterial activity and expand these effects to the natural interactions within the host animal. The effects of the prophage through deletion on a suite of host genes are a strength, as shown by striking microscopy.

Weaknesses:

Unfortunately, there are abundant negative data that cast some limitations on the interpretation of the data. That is, examining specific gene expression has its limitations, which could be avoided by global transcriptomics of the bacteria and the host during colonization by the prophage-containing and prophage-deleted bacteria (1 hour and 24 hours). In this way, the tripartite interactions leading to mechanism could be better established.

Impact:

The authors are correct to speculate that this research can have a significant impact on many animal microbiome studies, since bacterial lysogens are prevalent in most microbiomes. Screening for prophages, determining whether they are active, and "curing" the host bacteria of active prophages are effective tools for understanding the effects these mobile elements have on microbiomes. There are many potential effects of these elements in vivo, both positive and negative, this research is a good example of why this research should be explored.

Context:

The research area of prophage effects on host bacteria in vitro has been studied for decades, while these interactions in combination with animal hosts in vivo have been recent. The significance of this research shows that there could be divergent effects based on whether the study is conducted in vitro or in vivo. The in vivo results were striking. This is particularly so with the microscopy images. The benefit of using Ciona is that it has a translucent body which allows for following microbial localization. This is in contrast to mammalian studies where following microbial localization would either be difficult or near impossible.

Reviewer #3 (Public review):

In this manuscript, Natarajan and colleagues report on the role of a prophage, termed SfPat, in the regulation of motility and biofilm formation by the marine bacterium Shewanella fidelis. The authors investigate the in vivo relevance of prophage carriage by studying the gut occupation patterns of Shewanella fidelis wild-type and an isogenic SfPat- mutant derivative in a model organism, juveniles of the marine tunicate Ciona robusta. The role of bacterial prophages in regulating bacterial lifestyle adaptation and niche occupation is a relatively underexplored field, and efforts in this direction are appreciated.

While the research question is interesting, the work presented lacks clarity in its support for several major claims, and, at times, the authors do not adequately explain their data.

Major concerns:

(1) Prophage deletion renders the SfPat- mutant derivative substantially less motile and with a higher biofilm formation capacity than the WT (Fig. 2a-b). The authors claim the mutant is otherwise isogenic to the WT strain upon sequence comparison of draft genome sequences (I'll take the opportunity to comment here that GenBank accessions are preferable to BioSample accessions in Table 1). Even in the absence of secondary mutations, complementation is needed to validate functional associations (i.e., phenotype restoration). A strategy for this could be phage reintegration into the mutant strain (PMID: 19005496).

(2) The authors claim that the downshift in motility (concomitant with an upshift in biofilm formation) is likely mediated by the activity of c-di-GMP turnover proteins. Specifically, the authors point to the c-di-GMP-specific phosphodiesterase PdeB as a key mediator, after finding lower transcript levels for its coding gene in vivo (lines 148-151, Fig. 2c), and suggesting higher activity of this protein in live animals (!)(line 229). I have several concerns here:<br /> (2.1) Findings shown in Fig. 2a-b are in vitro, yet no altered transcript levels for pdeB were recorded (Fig. 2c). Why do the authors base their inferences only on in vivo data?<br /> (2.2) Somewhat altered transcript levels alone are insufficient for making associations, let alone solid statements. Often, the activity of c-di-GMP turnover proteins is local and/or depends on the activation of specific sensory modules - in the case of PdeB, a PAS domain and a periplasmic sensor domain (PMID: 35501424). This has not been explored in the manuscript, i.e., specific activation vs. global alterations of cellular c-di-GMP pools (or involvement of other proteins, please see below). Additional experiments are needed to confirm the involvement of PdeB. Gaining such mechanistic insights would greatly enhance the impact of this study.<br /> (2.3) What is the rationale behind selecting only four genes to probe the influence of the prophage on Ciona gut colonization by determining their transcript levels in vitro and in vivo? If the authors attribute the distinct behavior of the mutant to altered c-di-GMP homeostasis, as may be plausible, why did the authors choose those four genes specifically and not, for example, the many other c-di-GMP turnover protein-coding genes or c-di-GMP effectors present in the S. fidelis genome? This methodological approach seems inadequate to me, and the conclusions on the potential implication of PdeB are premature.

(3) The behavior of the WT strain and the prophage deletion mutant is insufficiently characterized. For instance, how do the authors know that the higher retention capacity reported for the WT strain with respect to the mutant (Fig. 3b) is not merely a consequence of, e.g., a higher growth rate? It would be worth investigating this further, ideally under conditions reflecting the host environment.

(4) Related to the above, sometimes the authors refer to "retention" (e.g., line 162) and at other instances to "colonization" (e.g., line 161), or even adhesion (line 225). These are distinct processes. The authors have only tracked the presence of bacteria by fluorescence labeling; adhesion or colonization has not been assessed or demonstrated in vivo. Please revise.

(5) The higher CFU numbers for the WT after 24 h (line 161) might also indicate a role of motility for successful niche occupation or dissemination in vivo. The authors could test this hypothesis by examining the behavior of, e.g., flagellar mutants in their in vivo model.

(6) The endpoint of experiments with a mixed WT-mutant inoculum (assumedly 1:1? Please specify) was set to 1 h, I assume because of the differences observed in CFU counts after 24 h. In vivo findings shown in Fig. 3c-e are, prima facie, somewhat contradictory. The authors report preferential occupation of the esophagus by the WT (line 223), which seems proficient from evidence shown in Fig. S3. Yet, there is marginal presence of the WT in the esophagus in experiments with a mixed inoculum (Fig. 3d) or none at all (Fig. 3e). Likewise, the authors claim preferential "adhesion to stomach folds" by the mutant strain (line 225), but this is not evident from Fig. 3e. In fact, the occupation patterns by the WT and mutant strain in the stomach in panel 3e appear to differ from what is shown in panel 3d. The same holds true for the claimed "preferential localization of the WT in the pyloric cecum," with Fig. 3d showing a yellow signal that indicates the coexistence of WT and mutant.

(7) In general, and especially for in vivo data, there is considerable variability that precludes drawing conclusions beyond mere trends. One could attribute such variability in vivo to the employed model organism (which is not germ-free), differences between individuals, and other factors. This should be discussed more openly in the main text and presented as a limitation of the study. Even with such intrinsic factors affecting in vivo measurements, certain in vitro experiments, which are expected, in principle, to yield more reproducible results, also show high variability (e.g., Fig. 5). What do the authors attribute this variability to?

(8) Line 198-199: Why not look for potential prophage excision directly rather than relying on indirect, presumptive evidence based on qPCR?

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 #2 (Public review):

The article titled "Identification of neurodevelopmental organization of the cell populations of juvenile Huntington's disease using dorso-ventral HD organoids and HD mouse embryos" analyses an in vitro human brain organoid model containig dorsal and ventral telencephalum structures derived from human iPSC from Huntington's disease patients or control subjects.

The authors describe differences in the pattern of expression of genes related to proliferation and neuronal maturation, with a slower pattern of differentiation present in HD cells. Moreover, the authors described a higher differentiation capacity of HD cells to generate choroid plexus identity following dorsal telencephalon prime protocol differentiation when compared to control cells. Whereas the claims related to Choroid plexus identity are intriguing, most of the claims made through the manuscript are not sustained by quantitative data or consistent results in the different conditions analysed, or many experiments seem to be missing to reach final conclusions.

In addition, the quality of the organoids used for experiments does not seem to have been assessed or satisfactorily presented in the figures of this paper. Many important details related to the experimental execution are missing in the current version of this manuscript.

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 #2 (Public review):

The manuscript by Sarkar et al has demonstrated the infection of liver cells/hepatocytes with Mtb and the significance of liver cells in the replication of Mtb by reprogramming lipid metabolism during tuberculosis. Besides, the present study shows that similar to Mtb infection of macrophages (reviewed in Chen et al., 2024; Toobian et al., 2021), Mtb infects liver cells but with a greater multiplication owing to consumption of enhanced lipid resources mediated by PPARg that could be cleared by its inhibitors. The strength of the study lies in the clinical evaluation of the presence of Mtb in human autopsied liver samples from individuals with miliary tuberculosis and the presence of a clear granuloma-like structure. The interesting observation is of granuloma-like structure in liver which prompts further investigations in the field.

The modulation of lipid synthesis during Mtb infection, such as PPARg upregulation, appears generic to different cell types including both liver cells and macrophage cells. It is also known that infection affect PPARγ expression and activity in hepatocytes. It is also known that this can lead to lipid droplet accumulation in the liver and the development of fatty liver disease (as shown for HCV). This study is in a similar line for M.tb infection. As the liver is the main site for lipid regulation, the availability of lipid resources is greater and higher is the replication rate. In short, the observations from the study confirm the earlier studies with these additional cell types. It is known that higher the lipid content, the greater are Lipid Droplet-positive Mtb and higher is the drug resistance (Mekonnen et al., 2021). The DMEs of liver cells add further to the phenotype.

Reviewer #3 (Public review):

This manuscript by Sarkar et al. examines the infection of the liver and hepatocytes during M. tuberculosis infection. They demonstrate that aerosol infection of mice and guinea pigs leads to appreciable infection of the liver as well as the lung. Transcriptomic analysis of HepG2 cells showed differential regulation of metabolic pathways including fatty acid metabolic processing. Hepatocyte infection is assisted by fatty acid synthesis in the liver and inhibiting this caused reduced Mtb growth. The nuclear receptor PPARg was upregulated by Mtb infection and inhibition or agonism of its activity caused a reduction or increase in Mtb growth, respectively, supporting data published elsewhere about the role of PPARg in lung macrophage Mtb infection. Finally, the authors show that Mtb infection of hepatocytes can cause upregulation of enzymes that metabolize antibiotics, resulting in increased tolerance of these drugs by Mtb in the liver.

Overall, this is an interesting paper on an area of TB research where we lack understanding. However, some additions to the experiments and figures are needed to improve the rigor of the paper and further support the findings. Most importantly, although the authors show that Mtb can infect hepatocytes in vitro, they fail to describe how bacteria get from the lungs to the liver in an aerosolized infection. They also claim that "PPARg activation resulting in lipid droplets formation by Mtb might be a mechanism of prolonging survival within hepatocytes" but do not show a direct interaction between PPARg activation and lipid droplet formation and lipid metabolism, only that PPARg promotes Mtb growth. Thus, the correlations with PPARg appear to be there but causation, implied in the abstract and discussion, is not proven.

The human photomicrographs are important and overall well done (lung and liver from the same individuals is excellent). However, in lines 120-121, the authors comment on the absence of studies on the precise involvement of different cells in the liver. In this study there is no attempt to immunophenotype the nature of the cells harboring Mtb in these samples (esp. hepatocytes). Proving that hepatocytes specifically harbor the bacteria in these human samples would add significant rigor to the conclusions made.

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 #2 (Public review):

Summary:

This paper extends a Bayesian perception/action model of habituation behavior (RANCH) to infant-looking behavior. The authors test the model predictions against data from several groups of infants and adults tested in habituation paradigms that vary the number of familiarisation stimuli and the nature of the test stimuli. Model sampling was taken as a proxy for looking times. The predictions of the model generally resemble the empirical data collected, though there are some potentially important differences.

Strengths:

This study addresses an important question, given the fundamental nature of habituation to learning and memory. Previous explanations of infant habituation have typically not been in the form of formal models, making falsification difficult. This Bayesian model is relatively simple but also incorporates a CNN to which the actual stimulus image can be presented, which enables principled predictions about image similarity to be derived.

The paper contains data from a relatively large number of adults and infants, allowing parameter differences across age to be probed.

The data suggests that the noise prior parameter is higher in infants, suggesting one mechanism through which infant and adult habituation is different, though of course, this depends on whether there is sufficient empirical evidence that other explanations can be ruled out, which isn't clear in the manuscript currently.

Weaknesses:

There are no formal tests of the predictions of RANCH against other leading hypotheses or models of habituation. This makes it difficult to evaluate the degree to which RANCH provides an alternative account that makes distinct predictions from other accounts. I appreciate that because other theoretical descriptions haven't been instantiated in formal models this might be difficult, but some way of formalising them to enable comparison would be useful.

The justification for using the RMSEA fitting approach could also be stronger - why is this the best way to compare the predictions of the formal model to the empirical data? Are there others? As always, the main issue with formal models is determining the degree to which they just match surface features of empirical data versus providing mechanistic insights, so some discussion of the level of fit necessary for strong inference would be useful.

The difference in model predictions for identity vs number relative to the empirical data seems important but isn't given sufficient weight in terms of evaluating whether the model is or is not providing a good explanation of infant behavior. What would falsification look like in this context?

For the novel image similarity analysis, it is difficult to determine whether any differences are due to differences in the way the CNN encodes images vs in the habituation model itself - there are perhaps too many free parameters to pinpoint the nature of any disparities. Would there be another way to test the model without the CNN introducing additional unknowns?

Related to that, the model contains lots of parts - the CNN, the EIG approach, and the parameters, all of which may or may not match how the infant's brain operates. EIG is systematically compared to two other algorithms, with KL working similarly - does this then imply we can't tell the difference between an explanation based on those two mechanisms? Are there situations in which they would make distinct predictions where they could be pulled apart? Also in this section, there doesn't appear to be any formal testing of the fits, so it is hard to determine whether this is a meaningful difference. However, other parts of the model don't seem to be systematically varied, so it isn't always clear what the precise question addressed in the manuscript is (e.g. is it about the algorithm controlling learning? or just that this model in general when fitted in a certain way resembles the empirical data?)

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 #2 (Public review):

Summary:

This important theoretical and computational study by Burger and Gerland attempts to set environmental, compositional, kinetic, and thermodynamic constraints on the proposed virtual circular genome (VCG) model for the early non-enzymatic replication of RNA. The authors create a solid kinetic model using published kinetic and thermodynamic parameters for non-enzymatic RNA ligation and (de)hybridization, which allows them to test a variety of hypotheses about the VCG. Prominently, the authors find that the length (longer is better) and concentration (intermediate is better) of the VCG oligos have an outsized impact on the fidelity and yield of VCG production with important implications for future VCG design. They also identify that activation of only RNA monomers, which can be achieved using environmental separation of the activation and replication, can relax the constraints on the concentration of long VCG component oligos by avoiding the error-prone oligo-oligo ligation. Finally, in a complex scenario with multiple VCG oligo lengths, the authors demonstrate a clear bias for the extension of shorter oligos compared to the longer ones. This effect has been observed experimentally (Ding et al., JACS 2023) but was unexplained rigorously until now. Overall, this manuscript will be of interest to scientists studying the origin of life and the behavior of complex nucleic acid systems.

Strengths:

- The kinetic model is carefully and realistically created, enabling the authors to probe the VCG thoroughly.<br /> - Fig. 6 outlines important constraints for scientists studying the origin of life. It supports the claim that the separation of activation and replication chemistry is required for efficient non-enzymatic replication. One could easily imagine a scenario where activation of molecules occurs, followed by their diffusion into another environment containing protocells that encapsulate a VCG. The selective diffusion of activated monomers across protocell membranes would then result in only activated monomers being available to the VCG, which is the constraint outlined in this work. The proposed exclusive replication by monomers also mirrors the modern biological systems, which nearly exclusively replicate by monomer extension.<br /> - Another strength of the work is that it explains why shorter oligos extend better compared to the long ones in complex VCG mixtures. This point is independent of the activation chemistry used (it simply depends on the kinetics and thermodynamics of RNA base-pairing) so it should be very generalizable.

Weaknesses:

- Most of the experimental work on the VCG has been performed with the bridged 2-aminoimidazolium dinucleotides, which are not featured in the kinetic model of this work. Oher studies by Szostak and colleagues have demonstrated that non-enzymatic RNA extension with bridged dinucleotides have superior kinetics (Walton et al. JACS 2016, Li et al. JACS 2017), fidelity (Duzdevich et al. NAR 2021), and regioselectivity (Giurgiu et al. JACS 2017) compared to activated monomers, establishing the bridged dinucleotides as important for non-enzymatic RNA replication. Therefore, the omission of these species in the kinetic model presented here can be perceived as problematic. The major claim that avoidance of oligo ligations is beneficial for VCGs may be irrelevant if bridged dinucleotides are used as the extending species, because oligo ligations (V + V in this work) are kinetically orders of magnitude slower than monomer extensions (F + V in this work) (Ding et al. NAR 2022). Formally adding the bridged dinucleotides to the kinetic model is likely outside of the scope of this work, but perhaps the authors could test if this should be done in the future by simply increasing the rate of monomer extension (F + V) to match the bridged dinucleotide rate without changing rate of V + V ligation?<br /> - The kinetic and thermodynamic parameters for oligo binding appear to be missing two potentially important components. First, base-paired RNA strands that contain gaps where an activated monomer or oligo can bind have been shown to display significantly different kinetics of ligation and binding/unbinding than complexes that do not contain such gaps (see Prywes et al. eLife 2016, Banerjee et al. Nature Nanotechnology 2023, and Todisco et al. JACS 2024). Would inclusion of such parameters alter the overall kinetic model? Second, it has been shown that long base-paired RNA can tolerate mismatches to an extent that can result in monomer ligation to such mismatched duplexes (see Todisco et al. NAR 2024). Would inclusion of the parameters published in Todisco et al. NAR 2024 alter the kinetic model significantly?

Reviewer #1 (Public review):

Summary:

Gene transfer agent (GTA) from Bartonella is a fascinating chimeric GTA that evolved from the domestication of two phages. Not much is known about how the expression of the BaGTA is regulated. In this manuscript, Korotaev et al noted the structural similarity between BrrG (a protein encoded by the ror locus of BaGTA) to a well-known transcriptional anti-termination factor, 21Q, from phage P21. This sparked the investigation into the possibility that BaGTA cluster is also regulated by anti-termination. Using a suite of cell biology, genetics, and genome-wide techniques (ChIP-seq), Korotaev et al convincingly showed that this is most likely the case. The findings offer the first insight into the regulation of GTA cluster (and GTA-mediated gene transfer) particularly in this pathogen Bartonella. Note that anti-termination is a well-known/studied mechanism of transcriptional control. Anti-termination is a very common mechanism for gene expression control of prophages, phages, bacterial gene clusters, and other GTAs, so in this sense, the impact of the findings in this study here is limited to Bartonella.

Strengths:

Convincing results that overall support the main claim of the manuscript.

Weaknesses:

A few important controls are missing.

Reviewer #2 (Public review):

Summary:

In this study, the authors identified and characterized a regulatory mechanism based on transcriptional anti-termination that connects the two gene clusters, capsid and run-off replication (ROR) locus, of the bipartite Bartonella gene transfer agent (GTA). Among genes essential for GTA functionality identified in a previous transposon sequencing project, they found a potential antiterminatior of phage origin within the ROR locus. They employed fluorescence reporter and gene transfer assays of overexpression and knockout strains in combination with ChiPSeq and promoter-fusions to convincingly show that this protein indeed acts as an antiterminator counteracting attenuation of the capsid gene cluster expression.

Impact on the field:

The results provide valuable insights into the evolution of the chimeric BaGTA, a unique example of phage co-domestication by bacteria. A similar system found in the other broadly studied Rhodobacterales/Caulobacterales GTA family suggests that antitermination could be a general mechanism for GTA control.

Strengths:

Results of the selected and carefully designed experiments support the main conclusions.

Weaknesses:

It remains open why overexpression of the antiterminator does not increase the gene transfer frequency.

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 #2 (Public review):

Summary:

The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.

Strengths:

The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.

Weaknesses:

The work can be improved in two major ways. First, word-choice decisions could better conform to the published literature. Alternatively, novel definitions could be included. In particular, the data support the concept of phenotypic tolerance, not persistence. Second, two of the novel observations could be explored more extensively to provide mechanistic explanations for the phenomena.

Overall impact: Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These are field-solidifying observations.

Reviewer #3 (Public review):

Summary:

The manuscript demonstrates that starvation induces persister formation in M. abscesses. They also utilized Tn-Seq for the identification of genes involved in persistence. They identified the role of catalase-peroxidase KatG in preventing death from translation inhibitors Tigecycline and Linezolid. They further demonstrated that a combination of these translation inhibitors leads to the generation of ROS in PBS-starved cells.

Strengths:

The authors used high-throughput genomics-based methods for identification of genes playing a role in persistence.

Weaknesses:

The findings could not be validated in clinical strains.

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 #2 (Public review):

Summary:

This is a strong and well-described study showing for the first time the use and publicly available resources to use a specific PET tracer to track proliferating transplanted cells in vivo, in a full murine immunecompetent environment.

In this study the authors described a previously developed set of VHH-based PET tracers to track transplants (cancer cells, embryo's) in a murine immune-competent environment.

Strengths:

Unique set of PET tracer and mouse strain to track transplanted cells in vivo without genetic modification of the transplanted cells. This is a unique asset, and a first-in-kind.

Weaknesses:

-some methodological aspects and controls are missing

-no clinical relevance?

Reviewer #1 (Public review):

SARS-CoV-2 encodes a macrodomain (Mac1) within the nsp3 protein that removes ADP-ribose groups from proteins. However, its role during infection is not well understood. Evidence suggests that Mac1 antagonizes the host interferon response by counteracting the wave of ADP ribosylation that occurs during infection. Indeed, several PARPs are interferon-stimulated genes. While multiple targets have been proposed, the mechanistic links between ADP ribosylation and a robust antiviral response remain unclear.

Genetic inactivation of Mac1 abrogates viral replication in vivo, suggesting that small-molecule inhibitors of Mac1 could be developed into antivirals to treat COVID-19 and other emerging coronaviruses. The authors report a potent and selective small molecule inhibitor targeting Mac1 (AVI-4206) that demonstrates efficacy in human airway organoids and animal models of SARS-CoV-2 infection. While these results are compelling and provide proof of concept for the therapeutic targeting of Mac1, I am particularly intrigued by the potential of this compound as a probe to elucidate the mechanistic connections between infection-induced ADP ribosylation and the host antiviral response.

The precise function of Mac1 remains unclear. Given its presence in multiple viruses, it likely acts on a fundamental host immune pathway(s). AVI-4206, while promising as a lead compound for the development of antivirals targeting coronaviruses, could also be a valuable tool for uncovering the function of the Mac1 domain. This may lead to fundamental insights into the host immune response to viral infection.

Reviewer #2 (Public review):

Summary:

The authors describe the development of a novel inhibitor (AVI-4206) for the first macrodomains of the nsp3 protein of SARS-CoV-2 (Mac1). This involves both medical chemical synthesis, structural work as well as biochemical characterisation. Subsequently, the authors present their findings of the efficacy of the inhibitor both on cell culture, as well as animal models of SARS-CoV-2 infection. They find that despite high affinity for Mac1 and the known replicatory defects of catalytically inactive Mac1 only moderate beneficial effects can be observed in their chosen models.

Strengths:

The authors employ a variety of different assay to study the affinity, selectivity and potency of the novel inhibitor and thus the in vitro data are very compelling.<br /> Similarly, the authors use several cell culture and in vivo models to strengthen their findings.

Weaknesses:

(a) The selection of Targ1 and MacroD2 as off-target human macrodomains is poor as several studies have shown that the first macrodomains of PARP9 and PARP14 are much closer related to coronaviral macrodomains and both macrodomains are implicated in antiviral defence and immunity.

(b) The authors utilize only replication efficiency and general infection markers as read out for their Mac1 inhibitor. It would be good if they could show impact on the ADP-ribosylation of a known Mac1 target such as PARP14.

Reviewer #3 (Public review):

Summary:

The authors were trying to validate SARS-CoV-2 Mac1 as a drug discovery target and by extension other viral macrodomains.

Strengths:

The medicinal chemistry and structure based optimization is exemplary. Macrodomains and ADPribosyl hydrolases have a reputation for being undruggable, yet the authors managed to optimize hits from a fragment screen using structure based approaches and fragment linking to make a 20nM inhibitor as a tool compound to validate the target.<br /> In addition, the in vivo work is also a strength. The ability to reduce the viral count at a rate comparable to nirmatrelvir is impressive. Tracking the cytokine expression levels also supports much of the genetic data and mechanism of action for macrodomains.

Weaknesses:

The main compound AVI-4206, while being very potent and selective is not appreciably orally bioavailable. The fact that they have to use high doses of the compound IP to see in vivo effects may lead to questions regarding off target effects.

The cellular models are not as predictive of antiviral activity as one would expect. However, the authors had enough chutzpah to test the compound in vivo knowing that cellular models might not be an accurate representation of a living system with a fully functional immune system all of which is most likely needed in an antiviral response to test the importance of Mac1 as a target.

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.

Reviewer #2 (Public review):

Summary:

In this manuscript, Espejo et al describe a method, SICKO, that allows for long-term longitudinal examination of bacterial colonization in the gut of C. elegans. SICKO utilizes a well-plate format where single worms are housed in each well with a small NGM pad surrounded by an aversive palmitic acid barrier to prevent worms from fleeing the well. The main benefit of this method is that it captures longitudinal data across individual worms with the ability to capture tens to hundreds of worms at once. The output data of SICKO in the heatmap is also very clear and robustly shows bacterial colonization in the gut across a large sample size, which is far superior to the current gold standard of imaging 10-20 worms in a cross-sectional matter at various timepoints of aging. They then provide a few examples of how this method can be applied to understand how colonization correlates with animal health.

Strengths:

-The method presented in this manuscript is sure to be of great utility to the host-pathogen field of C. elegans. The method also allows for utilization of large sample sizes and a way to present highly transparent data, both of which are excellent for promoting rigor and reproducibility of science.<br /> -The manuscript also does a great job in describing the limitations of the system, which is always appreciated.<br /> -The methods section for the SICKO data analysis pipeline and the availability of the code on Github are strong pluses.

Weaknesses:

-There are minor weaknesses in the methods that could be addressed relatively easily by expanding the explanation of how to set up the individual worm chambers (see comment 1 below).

I am making all my comments and suggestions to the reviewers public, as I believe these comments can be useful to the general readership as well. Comment 1 is important to make the methods more accessible and comment 2 is important to make the data presentation more accessible to a broader audience. However, comments 3-4 are things/suggestions that should be considered by the authors and future users of SICKO for interpretation of all the data presented in the manuscript.

(1) The methods section needs to be described in more detail. Considering that this is a methods development paper, more detailed explanation is required to ensure that readers can actually adapt these experiments into their labs.<br /> (a) What is the volume of lmNGM in each well?<br /> (b) Recommended volume of bacteria to seed in each well?<br /> (c) A file for the model for the custom printed 3D adaptor should be provided.<br /> (d) There should be a bit more detail on how the chambers should be assembled with all the components. After reading this, I am not sure I would be able to put the chamber together myself.<br /> (e) What is the recommended method to move worms into individual wells? Manual picking? Pipetting in a liquid?<br /> (f) Considering that a user-defined threshold is required (challenging for non-experienced users), example images should be provided on what an acceptable vs. nonacceptable threshold would look like.

(2) The output data in 1e is very nice - it is a very nice and transparent plot, which I like a lot. However, since the data is complex, a supplemental figure to explain the data better would be useful to make it accessible for a broader audience. For example, highlighting a few rows (i.e., individual worms) and showing the raw image data for each row would be useful. What I mean is that it would be useful to show what does the worm actually look like for a "large colony size" or "small colony size"? What is the actual image of the worm that represents the yellow (large), versus dark blue (small), versus teal (in the middle)? And also the transition from dark blue to yellow would also be nice to be shown. This can probably also just be incorporated into Fig. 1d by just showing what color each of those worm images from day 1 to day 8 would represent in the heat map (although I still think a dedicated supplemental figure where you highlight a few rows and show matching pictures for each row in image files would be better).

(3) I am not sure that doing a single-time point cross-sectional data is a fair comparison since several studies do multi-timepoint cross-sectional studies (e.g., day 1, day 5, day 9). This is especially true for using only day 1 data - most people do gut colonization assays at later timepoints since the gut barrier has been shown to break down at older ages, not day 1. The data collected by SICKO is done every day across many individuals worms and is clearly superior to this type of cross-sectional data (even with multiple timepoints), and I think this message would be further strengthened by comparing it directly to cross-sectional data collected across more than 1 timepoint of aging.

(4) The authors show that SICKO can detect differences in wild-type vs. pmk-1 loss of function and between OP50 and PA14. However, these are very dramatic conditions that conventional methods can easily detect. I would think that the major benefit of SICKO over conventional methods is that it can detect subtle differences that cross-sectional methods would fail to visualize. It might be useful to see how well SICKO performs for these more subtle effects (e.g., OP50 on NGM vs. bacteria-promoting media; OP50 vs. HT115; etc.).<br /> (a) Similar to the above comment, the authors discuss how pmk-1 has colonization-independent effects on host-pathogen interactions. Maybe using a more direct approach to affect colonization (e.g., perturbing gut actin function like act-5) would be better.

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:

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 #2 (Public review):

Summary:

The inferior colliculus (IC) has been explored for its possible functions in behavioral tasks and has been suggested to play more important roles rather than simple sensory transmission. The authors show us two major findings based on their experiments. The first one is climbing effect, which means that neurons' activities continue to increase along time course. The second one is reward effect, which refers to sudden increase of IC neurons' activities when the rewarding is given. Climbing effect is a surprising finding, but reward effect has not been explored clearly here.

Strengths:

Complex cognitive behaviors can be regarded as simple ideals of generating output based on information input, which depends on all kinds of input from sensory systems. The auditory system has hierarchic structures no less complex than those areas in charge of complex functions. Meanwhile, IC receives projections from higher areas, such as the auditory cortex, which implies IC is involved in complex behaviors. Experiments in behavioral monkeys are always time-consuming work with hardship, and this will offer more approximate knowledge of how the human brain works.

Weaknesses:

These findings are more about correlation but not causality of IC function in behaviors.

About 'reward effect', it is still unknown if the true nature of reward effect is the simple response to the sound elicited by the electromagnetic valve of rewarding system. The authors claimed the testing space is sound-proofed and believed this is enough to support their opinion. Since the electromagnetic valve was connected to the water tube, and the water tube was attached to a monkey-chair or even in monkey's mouth, the click sound may transmit to the monkey independently on air. There are simple ways to test what happens. One is to add a few trials without reward and see what happens, or to vary the latency between sound sequence and reward.

Only one of the major findings is convincing, this definitely reduces the credibility of the authors' statements.

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 #2 (Public review):

Summary:

More and more genes and genetic loci are being linked to bone fragility disorders like osteoporosis and osteogenesis imperfecta through GWAS and clinical sequencing. In this study, the authors seek to develop a pipeline for validating these new candidate genes using crispant screening in zebrafish. Candidates were selected based on GWAS bone density evidence (4 genes) or linkage to OI cases plus some aspect of bone biology (6 genes). NGS was performed on embryos injected with different gRNAs/Cas9 to confirm high mutagenic efficacy, and off-target cutting was verified to be low. Bone growth, mineralization, density, and gene expression levels were carefully measured and compared across crispants using a battery of assays at three different stages.

Strengths:

(1) The pipeline would be straightforward to replicate in other labs, and the study could thus make a real contribution towards resolving the major bottleneck of candidate gene validation.

(2) The study is clearly written and extensively quantified.

(3) The discussion attempts to place the phenotypes of different crispant lines into the context of what is already known about each gene's function.

(4) There is added value in seeing the results for the different crispant lines side by side for each assay.

(5) Caveats to the interpretability of crispant data and limitations of their gene-focused analyses and RT-PCR assays are discussed.

Weaknesses:

(1) The study uses only well-established methods and is strategy-driven rather question/hypothesis-driven. This is in line with the researchers' primary goal of developing a workflow for rapid in vivo functional screening of candidate genes. However, this means that less attention is paid to what the results obtained for a given gene may mean regarding potential disease mechanisms, and how contradictions with prior reports of mouse or fish null mutant phenotypes might be explained.

(2) Normalization to body size was not performed. Measurements of surface area covered by osteoblasts or mineralized bone (Fig. 1) are typically normalized to body size - especially in larvae and juvenile fish - to rule out secondary changes due to delayed or accelerated overall growth. This was not done here; the authors argue that "variations in growth were considered as part of the phenotypic outcome." This is reasonable, but because standard length was reported only for fish at 90 dpf (not significantly different in any line), the reader is not given the opportunity to consider whether earlier differences in, e.g. surface area covered by osteoblasts at 14 dpf, could be accounted for by delayed or accelerated overall growth. Images in Figure S5 were not taken at the same magnification, further confounding this effort.

Comments on latest version:

The authors have largely addressed my comments by making changes to the text.

However, in response to one of my original comments ("It would be helpful to note the grouping of candidates into OI vs. BMD GWAS throughout the figures"), they added a sentence to this effect to the legends. However, because two of the lines were larval-lethal, the legends to Figs. S6-8 are now incorrect in referring to ten genes when only eight mutants are shown.

Reviewer #3 (Public review):

The manuscript describes the use of CRISPR gene editing coupled with phenotyping mosaic zebrafish larvae to characterize functions of genes implicated in heritable fragile bone disorders (FBDs). Authors targeted six high-confident candidate genes implicated in severe recessive forms of FBDs and four Osteoporosis GWAS-implicated genes and observe varied developmental phenotypes across all crispants, in addition to adult skeletal phenotypes. While the study lacks insight on underlying mechanisms that contribute to disease phenotypes, a major strength of the paper is the streamlined method that produced significant phenotypes for all candidate genes tested. It also represents a significant increase in number of candidate genes tested using their crispant approach beyond the single mutant that was previously published.

One weakness was the variability of developmental phenotypes, addressed by authors in the Discussion. This might be a product of maternal transcripts not targeted by CRISPR or genetic compensation, which authors have not fully explored. Overall, the paper was well-written and easy to read.

Comments on latest version:

The authors have addressed many concerns in this revision. Figure 1 and Table 2 are much improved.

While details of orthologous gene expression profiles of target genes is a welcome addition, other features of target genes remain unaddressed. For example, do genes with maternally deposited transcript exhibit dampened phenotypes? Or does genetic compensation impact certain genes more than others? Since authors state that the study represents a methods paper, it will be important for users to understand the caveats of gene selection to effectively implement and interpret results of the approach.

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 #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Comments on revised version:

This version has effectively addressed most of my concerns. However, one key issue remains unresolved regarding the mechanism of NopT in regulating nodule symbiosis. Specifically, the explanation of how NopT catabolizes NFR5 to regulate symbiosis is still not convincing within the current framework of plant-microbe interaction, where plants are understood to genetically control rhizobial colonization.

While alternative regulatory mechanisms in plant-microbe interactions are plausible, the notion that the NRG234-secreted effector NopT could reduce its own infection by either suppressing plant immunity or degrading the symbiosis receptor remains unsubstantiated. I believe further revisions are needed in the discussion section to more clearly address and clarify these findings and any lingering uncertainties.

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 #3 (Public review):

This revision and the accompanying rebuttal indicates the authors want to publish their studies without providing several of the reviewer requested additional experiments (such as determining the impact of other Myc family members on metastatic behavior and expression characteristics compared to overexpression of c-Myc), and determining whether the tumors were responsive or not to standard clinically used therapies. Their argument is the author team has moved on to other endeavors, it is important to communicate their findings to the research field, and they have indicated these issues in the Discussion. All of these things are reasonable. However, there two things that would help. The first is to have the authors clearly state in the Discussion section "Limitations of the current study" and then list these out. In the current format the indication that the authors recognize the "limitations" is not clearly stated. An example - of such a limitation is how well their model now provides a human SCLC like tumor that metastasizes. We know that in patients SCLC is widely metastatic, but in SCLC patient derived xenografts with subcutaneous injection that is not seen, so if their model now generated widely metastatic behavior like that seen in patients, this report and the associated resources would be a significant advance to the field. However, their data shows that using their model the subcutaneous tumors don't metastasize, and even with renal capsule models metastases are not common and do not go to important sites (e.g. brain). Second, a major reason for publishing this paper is that their model system would be available as a resource for the field to study. However, I could not find in the paper or the Methods section any statement as to the availability of this presumable important resource. If the resources will not be easily available in a format that others can readily study (e.g. with instructions on how to handle the cells which would seem to be more complicated than other patient derived SCLC models) then of course the value of this paper to the field as a whole is dramatically reduced. I would assume the authors want their model to be used by other investigators and thus a clear statement of model availability and how to routinely handle their model is important to include in their manuscript.

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 #2 (Public review):

Summary:

In this study the authors have used pull-down experiments in a cell line overexpressing tagged SERPINE1 mRNA binding protein 1 (SERBP1) followed by mass spectrometry-based proteomics, to establish its interactome. Extensive analyses are performed to connect the data to published resources. The authors attempt to connect SERBP1 to stress granules and Alzheimer's disease associated tau pathology. Based on the interactome, the authors propose a cross-talk between SERBP1 and PARP1 functions.

Strengths:

The main strength of this study lies in the extensive proteomics data analysis, and its effort to connect the data to published studies.

Weaknesses:

Support for the proposed model: While the authors propose a feedback regulatory model for SERBP1 and PARP1 function, strong evidence for PARylation modulating SERBP1 functions is lacking. PARP inhibition decreasing the amount of PARylated proteins associated with SERBP1 and likely all other PARylated proteins is expected.<br /> Evidence from autopsy brain tissue: This study shows unexplained round, punctate staining for SERBP1 in immunohistochemistry (IHC) staining. This may be due to poor preservation of cellular structures in frozen autopsy brain tissue. SERBP1 and pTau co-staining lacks an age matched non-AD control. Most quantifications of human IHC staining and co-localization do not indicate the number of cases and what data points are shown.<br /> The link to stress granules (SGs): G3BP1 staining indicates cytoplasmic mislocalization and perhaps aggregation pathology, but not necessarily SGs. It is not clear whether physiological transient stress granules are preserved in autopsy brain tissue. The co-localization of abundant cytoplasmic G3BP1 and SERBP1 under normal conditions does not indicate association with SGs. Stress granule proteins assemble phase-separated granules in the cytoplasm under cellular stress, whereas here it is shown that normally cytoplasmic SERBP1 has a nucleocytoplasmic distribution in the presence of H2O2, with no evidence for SG formation.

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 #2 (Public review):

Summary:

The authors use a combination of techniques including viral genetics, in vitro reporters, and purified proteins and RNA to interrogate how the Japanese encephalitis virus maintains translation of its RNA to produce viral proteins after the host cell has shut down general translation as a means to block viral replication. They report a role for the RNA helicase DDX3 in promoting virus translation in a cap-independent manner through binding a dumbbell RNA structure in the 3' untranslated region previously reported to drive Japanese encephalitis virus cap-independent translation and a stem-loop at the viral RNA 5' end.

Strengths:

The authors clearly show that the Japanese encephalitis virus does not possess an IRES activity to initiate translation using a range of mono- and bi-cistronic mRNAs. Surprisingly, using a replicon system, the translation of a capped or uncapped viral RNA is reported to have the same translation efficiency when transfected into cells. The authors have applied a broad range of techniques to support their hypotheses.

Weaknesses:

(1) The authors' original experiments in Figure 1 where the virus is recovered following transfection of in vitro transcribed viral RNA with alternative 5' ends such as capped or uncapped ignore that after a single replication cycle of that transfected RNA, the subsequent viral RNA will be capped by the viral capping proteins making the RNA in all conditions the same.

(2) The authors report that deletion of the dumbbell and the large 3' stem-loop RNA reduce replication of a Japanese encephalitis virus replicon. These structures have been reported for other flaviviruses to be important respectively for the accumulation of short flaviviral RNAs that can regulate replication and stability of the viral RNA that lacks a polyA tail. The authors don't show any assessment of RNA stability or degradation state.

(3) The authors propose a model for DDX3 to drive 5'-3' end interaction of the Japanese encephalitis virus viral genome but no direct evidence for this is presented.

(4) The authors' final model in Figure 10 proposes a switch from a cap-dependent translation system in early infection to cap-independent DDX3-driven translation system late in infection. The replicon data that measures translation directly however shows identical traces for capped and uncapped RNAs in all untreated conditions so that which mechanism is used at different stages of the infection is not clear.

Reviewer #3 (Public review):

Summary:

This work is a valuable study that aims to decipher the molecular mechanisms underlying the translation process in Japanese encephalitis virus (JEV), a relevant member of the genus Flavivirus. The authors provide evidence that cap-independent translation, which has already been demonstrated for other flaviviruses, could also account in JEV. This process depends on the genomic 3' UTR, as previously demonstrated in other flaviviruses. Further, the authors find that cellular proteins such as DDX3 or PABP1 could contribute to JEV translation in a cap-independent way. Both DDX3 and PABP1 had previously been described to have a role in cellular protein synthesis and also in the translation step of other flaviviruses distinct from JEV; therefore, this work would expand the cap-independent translation in flaviviruses as a general mechanism to bypass the translation repression exerted by the host cell during viral infection. Further, the findings can be relevant for the development of specific drugs that could interfere with flaviviral translation in the future. Nevertheless, the conclusions are not fully supported by the provided results.

Strengths:

The results provide a good starting point to investigate the molecular mechanism underlying the translation in flaviviruses, which even today is an area of knowledge with many limitations.

Weaknesses:

The main limit of the work is related to the fact that the role of the 3' UTR structural elements and DDX3 is not only circumscribed to translation, but also to replication and encapsidation. In fact, some of the provided results suggest this idea. Particularly, it is intriguing why the virus titer can be completely abrogated while the viral protein levels are only partially affected by the knockdown of DDX3. This points to the fact that many of the drawn conclusions could be overestimated or, at least, all the observed effect cannot be attributed only to the DDX3 effect on translation. Finally, it is noteworthy that the use of uncapped transcripts could be misleading, since this is not the natural molecular context of the viral genome.

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 #2 (Public review):

This manuscript submitted by Takagi et al. details the molecular characterization of the FT-expressing cell at a single-cell level. The authors examined what genes are expressed specifically in FT-expressing cells and other phloem companion cells by exploiting bulk nuclei and single-nuclei RNA-seq and transgenic analysis. The authors found the unique expression profile of FT-expressing cells at a single-cell level and identified new transcriptional repressors of FT such as NIGT1.2 and NIGT1.4.

Although previous researchers have known that FT is expressed in phloem companion cells, they have tended to neglect the molecular characterization of the FT-expressing phloem companion cells. To understand how FT, which is expressed in tiny amounts in phloem companion cells that make up a very small portion of the leaf, can be a key molecule in the regulation of the critical developmental step of floral transition, it is important to understand the molecular features of FT-expressing cells in detail. In this regard, this manuscript provides insight into the understanding of detailed molecular characteristics of the FT-expressing cell. This endeavor will contribute to the research field of flowering time.

Here are my comments on how to improve this manuscript.

(1) The most noble finding of this manuscript is the identification of NTGI1.2 as the upstream regulator of FT-expressing cluster 7 gene expression. The flowering phenotypes of the nigtQ mutant and the transgenic plants in which NIGT1.2 was expressed under the SUC2 gene promoter support that NIGT1.2 functions as a floral repressor upstream of the FT gene. Nevertheless, the expression patterns of NIGT1.2 genes do not appear to have much overlap with those of NIGT1.2-downstream genes in the cluster 7 (Figs S14 and F3). An explanation for this should be provided in the discussion section.<br /> (2) To investigate gene expression in the nuclei of specific cell populations, the authors generated transgenic plants expressing a fusion gene encoding a Nuclear Targeting Fusion protein (NTF) under the control of various cell type-specific promoters. Since the public audience would not know about NTF without reading reference 16, some explanation of NTF is necessary in the manuscript. Please provide a schematic of constructs the authors used to make the transformants.

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 #2 (Public review):

Summary:

The manuscript by Chabukswar et al. describes a comprehensive attempt to identify and describe the diversity of retroviral envelope (env) gene sequences present in primate genomes in the form of ancient endogenous retrovirus (ERV) sequences.

Strengths:

The focus on env can be justified because of the role the Env proteins likely played in determining viral tropism and host range of the viruses that gave rise to the ERV insertions, and to a lesser extent, because of the potential for env ORFs to be coopted for cellular functions (in the rare cases where the ORF is still intact and capable of encoding a functional Env protein). In particular, these analyses can reveal the potential roles of recombination in giving rise to novel combinations of env sequences. The authors began by compiling env sequences from the human genome (from human endogenous retrovirus loci, or "HERVs") to build consensus Env protein sequences, and then they use these as queries to screen other primate genomes for group-specific envs by tBLASTn. The "groups" referred to here are previously described, as unofficial classifications of endogenous retrovirus sequences into three very broad categories - Class I, Class II and Class III. These are not yet formally recognized in retroviral taxonomy, but they each comprise representatives of multiple genera, and so would fall somewhere between the Family and Genus levels. The retrieved sequences are subject to various analyses, most notably they are screened for evidence of recombination. The recombinant forms appear to include cases that were probably viral dead-ends (i.e. inactivating the env gene) even if they were propagated in the germline.<br /> The availability of the consensus sequences (supplement) is also potentially useful to others working in this area.

Weaknesses:

The weaknesses are largely in presentation. Discussions of ERVs are always complicated by the lack of a formal and consistent nomenclature and the confusion between ERVs as loci and ERVs as indirect information about the viruses that produced them. For this reason, additional attention needs to be paid to precise wording in the text and/or the use of illustrative figures.

Reviewer #3 (Public review):

Summary:

Retroviruses have been endogenized into the genome of all vertebrate animals. The envelope protein of the virus is not well conserved and acquires many mutations hence can be used to monitor viral evolution. Since they are incorporated into the host genome, they also reflect the evolution of the hosts. In this manuscript the authors have focused their analyses on the env genes of endogenous retroviruses in primates. Important observations made include the extensive recombination events between these retroviruses that were previously unknown and the discovery of HML species in genomes prior to the splitting of old and new world monkeys.

Strengths:

They explored a number of databases and made phylogenetic trees to look at the distribution of retroviral species in primates. The authors provide a strong rationale for their study design, they provide a clear description of the techniques and the bioinformatics tools used.

Weaknesses:

The manuscript is based on bioinformatics analyses only. The reference genomes do not reflect the polymorphisms in humans or other primate species. The analyses thus likely under estimates the amount of diversity in the retroviruses. Further experimental verification will be needed to confirm the observations.<br /> Not sure which databases were used, but if not already analyzed, ERVmap.com and repeatmesker are ones that have many ERVs that are not present in the reference genomes. Also, long range sequencing of the human genome has recently become available which may also be worth studying for this purpose.

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 #2 (Public review):

Summary:

In this manuscript, the authors aim to identify genetic determinants associated with the invasion profile of Streptococcus pyogenes strains of the emm89 type, which has been increasingly linked to invasive infections. The study leverages both in-house sequenced genomes and publicly available genomic data. Several GWAS approaches are applied to these datasets, leading to the identification of potential genetic targets. For these targets, the authors conduct additional analyses, including three-dimensional structural modeling of the encoded proteins, as well as the development of mutant strains. The functional impact of these mutations is further explored through transcriptomic comparisons between the mutants and wild-type strains

Strengths:

The strengths of this manuscript include the large amount of data analyzed and the various methodologies applied. The identification of CovS, a gene known to influence the invasion profile, as a significant variation further validates the methodology employed in this study. Then, the gene fhuD is an intriguing target, identified through both bioinformatics and wet lab approaches.

Weaknesses:

I do not identify any additional weaknesses in the manuscript, beyond those already acknowledged by the authors themselves.

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 #2 (Public review):

To fuse, differentiated muscle cells must rearrange their cytoskeletaon and assemble actin-enriched cytoskeletal structures. These actin foci are proposed to generate mechanical forces necessary to drive close membrane apposition and fusion pore formation.

While the study of these actin-rich structures has been conducted mainly in drosophila, the present manuscript presents clear evidence this mechanism is necessary for the fusion of adult muscle stem cells in vivo, in mice.

However, the authors need to tone down their interpretation of their findings and remember that genetic proof for cytoskeletal actin remodeling to allow muscle fusion in mice has already been provided by different labs (Vasyutina E, et al. 2009 PMID: 19443691; Gruenbaum-Cohen Y, et al., 2012 PMID: 22736793; Hamoud et al., 2014 PMID: 24567399). In the same line of thought, the authors write they "demonstrated a critical function of branched actin-propelled invasive protrusions in skeletal muscle regeneration". I believe this is not a premiere, since Randrianarison-Huetz V, et al., previously reported the existence of finger-like actin-based protrusions at fusion sites in mice myoblasts (PMID: 2926942) and Eigler T, et al., live-recorded said "fusogenic synapse" in mice myoblasts (PMID: 34932950).

Hence, while the data presented here clearly demonstrate that ARP2/3 and SCAR/WAVE complexes are required for differentiating satellite cell fusion into multinucleated myotubes, this is an incremental story, and the authors should put their results in the context of previous literature.

Reviewer #3 (Public review):

The manuscript by Lu et al. explores the role of the Arp2/3 complex and the actin nucleators N-WASP and WAVE in myoblast fusion during muscle regeneration. The results are clear and compelling, effectively supporting the main claims of the study. However, the manuscript could benefit from a more detailed molecular and cellular analysis of the fusion synapse. Additionally, while the description of macrophage extravasation from ghost fibers is intriguing, it seems somewhat disconnected from the primary focus of the work.

Despite this, the data are robust, and the major conclusions are well supported. Understanding muscle fusion mechanism is still a widely unexplored topic in the field and the authors make important progress in this domain.

I have a few suggestions that might strengthen the manuscript as outlined below.

(1) Could the authors provide more detail on how they defined cells with "invasive protrusions" in Figure 4C? Membrane blebs are commonly observed in contacting cells, so it would be important to clarify the criteria used for counting this specific event.

(2) Along the same line, please clarify what each individual dot represents in Figure 4C. The authors mention quantifying approximately 83 SCMs from 20 fibers. I assume each dot corresponds to data from individual fibers, but if that's the case, does this imply that only around four SCMs were quantified per fiber? A more detailed explanation would be helpful.

(3) Localizing ArpC2 at the invasive protrusions would be a strong addition to this study. Furthermore, have the authors examined the localization of Myomaker and Myomixer in ArpC2 mutant cells? This could provide insights into potential disruptions in the fusion machinery.

(4) As a minor curiosity, can ArpC2 WT and mutant cells fuse with each other?

(5) The authors report a strong reduction in CSA at 14 dpi and 28 dpi, attributing this defect primarily to failed myoblast fusion. Although this claim is supported by observations at early time points, I wonder whether the Arp2/3 complex might also play roles in myofibers after fusion. For instance, Arp2/3 could be required for the growth or maintenance of healthy myofibers, which could also contribute to the reduced CSA observed, since regenerated myofibers inherit the ArpC2 knockout from the stem cells. Could the authors address or exclude this possibility? This is rather a broader criticism of how things are being interpreted in general beyond this paper.

Reviewer #1 (Public review):

Summary:

This manuscript details the results of a small pilot study of neoadjuvant radiotherapy followed by combination treatment with hormone therapy and dalpiciclib for early-stage HR+/HER2-negative breast cancer.

Strengths:

The strengths of the manuscript include the scientific rationale behind the approach and the inclusion of some simple translational studies.

Weaknesses:

The main weakness of the manuscript is that overly strong conclusions are made by the authors based on a very small study of twelve patients. A study this small is not powered to fully characterize the efficacy or safety of a treatment approach, and can, at best, demonstrate feasibility. These data need validation in a larger cohort before they can have any implications for clinical practice, and the treatment approach outlined should not yet be considered a true alternative to standard evidence-based approaches.

I would urge the authors and readers to exercise caution when comparing results of this 12-patient pilot study to historical studies, many of which were much larger, and had different treatment protocols and baseline patient characteristics. Cross-trial comparisons like this are prone to mislead, even when comparing well powered studies. With such a small sample size, the risk of statistical error is very high, and comparisons like this have little meaning.

Reviewer #2 (Public review):

The author and his team explored a novel neoadjuvant strategy of radiotherapy followed by CDK4/6 inhibitor and exemestane for HR+/HER2- breast cancer. This strategy interestingly reached an ORR of 91.7% and RCB 0-I of 16.7%, with satisfying tolerance.

There are several questions for your further consideration.

Firstly, as this is a single-arm preliminary study, we are curious about the order of radiotherapy and the endocrine therapy. Besides, considering the radiotherapy, we also concern about the recovery of the wound after the surgery and whether related data were collected.

Secondly, in the methodology, please describe the sample size estimation of this study and follow up details.

Thirdly, in Table 1, the item HER2 expression, it's better to categorise HER2 into 0, 1+, 2+ and FISH-.

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 #2 (Public review):

Summary:

Makarova et al. provide the first complete cellular-level reconstruction of an insect eye. They use the extremely miniaturized parasitoid wasp, Megaphragma viggiani, and apply improved and optimized volumetric EM methods they can describe, the size, volume, and position of every single cell in the insect compound eye.

This data has previously only been inferred from TEM cross-sections taken in different parts of the eye, but in this study and in the associated 3d datasets video and stacks, one can observe the exact position and orientation in 3D space.

The authors have made a very rigorous effort to describe and assess the variation in each cell type and have also compared two different classes of the dorsal rim and non-dorsal rim ommatidia and the associated visual apparatus for each, confirming previous known findings about the distribution and internal structure that assists in polarization detection in these insects.

Strengths:

The paper is well written and strives to compare the data with previous literature wherever possible and goes beyond cell morphology, calculating the optical properties of the different ommatidia and estimating light sensitivity and spatial resolution limits using rhabdom diameter, focal length and showing how this varies across the eye.

Finally, the authors provide very informative and illustrative videos showing how the cones, lenses, photoreceptors, pigment cells, and even the mitochondria are arranged in 3D space, comparing the structure of the dorsal rim and non-dorsal rim ommatidia. They also describe three 'ectopic' photoreceptors in more anatomical detail providing images and videos of them.

Reviewer #3 (Public review):

Summary:

The article presents a meticulous and quantitative anatomical reconstruction of the compound eye of a tiny wasp at the level of subcellular structures, and cellular and optical organization of the ommatidia and reveals the ectopic photoreceptors, which are decoupled from the eye's dioptrical apparatus.

Strengths:

The graphic material is of very high quality, beautifully organized, and presented in a logical order. The anatomical analysis is fully supported by quantitative numerical data at all scales, from organelles to cells and ommatidia, which should be a valuable source for future studies in cellular biology and visual physiology. The 3D renders are highly informative and a real eye candy.

Weaknesses:

The claim that the large dorsal part of the eye is the dorsal rim area (DRA), supported by anatomical data on rhabdomere geometry and connectomics in authors' earlier work, would eventually greatly benefit from additional evidence, obtained by immunocytochemical staining, that could also reveal a putative substrate for colour vision. The cell nuclei that are located in the optical path in the DRA crystalline cone have only a putative optical function, which may be either similar to pore canals in hymenopteran DRA cornea (scattering) or to photoreceptor nuclei in camera-type eyes (focussing), both explanations being mutually exclusive.

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 #2 (Public review):

Summary:

The authors aimed to determine whether a cryptic pocket in the VP35 protein of Zaire ebolavirus has a functional role in RNA binding and, by extension, in immune evasion. They sought to address whether this pocket could be an effective therapeutic target resistant to evolutionary evasion by studying its role in dsRNA binding among different filovirus VP35 homologs. Through simulations and experiments, they demonstrated that cryptic pocket dynamics modulate the RNA binding modes, directly influencing how VP35 variants block RIG-I and MDA5-mediated immune responses.

The authors successfully achieved their aim, showing that the cryptic pocket is not a random structural feature but rather an allosteric regulator of dsRNA binding. Their results not only explain functional differences in VP35 homologs despite their structural similarity but also suggest that targeting this cryptic pocket may offer a viable strategy for drug development with reduced risk of resistance.

This work represents a significant advance in the field of viral immunoevasion and therapeutic targeting of traditionally "undruggable" protein features. By demonstrating the functional relevance of cryptic pockets, the study challenges long-standing assumptions and provides a compelling basis for exploring new drug discovery strategies targeting these previously overlooked regions.

Strengths:

The combination of molecular simulations and experimental approaches is a major strength, enabling the authors to connect structural dynamics with functional outcomes. The use of homologous VP35 proteins from different filoviruses strengthens the study's generality, and the incorporation of point mutations adds mechanistic depth. Furthermore, the ability to reconcile functional differences that could not be explained by crystal structures alone highlights the utility of dynamic studies in uncovering hidden allosteric features.

Weaknesses:

While the methodology is robust, certain limitations should be acknowledged. For example, the study would benefit from a more detailed quantitative analysis of how specific mutations impact RNA binding and cryptic pocket dynamics, as this could provide greater mechanistic insight. This study would also benefit from providing a clear rationale for the selection of the amber03 force field and considering the inclusion of volume-based approaches for pocket analysis. Such revisions will strengthen the robustness and impact of the study.

Reviewer #3 (Public review):

Summary:

The authors suggest a mechanism that explains the preference of viral protein 35 (VP35) homologs to bind the backbone of double-stranded RNA versus blunt ends. These preferences have a biological impact in terms of the ability of different viruses to escape the immune response of the host.<br /> The proposed mechanism involves the existence of a cryptic pocket, where VP35 binds the blunt ends of dsRNA when the cryptic pocket is closed and preferentially binds the RNA double-stranded backbone when the pocket is open.<br /> The authors performed MD simulation results, thiol labelling experiments, fluorescence polarization assays, as well as point mutations to support their hypothesis.

Strengths:

This is a genuinely interesting scientific question, which is approached through multiple complementary experiments as well as extensive MD simulations. Moreover, structural biology studies focused on RNA-protein interactions are particularly rare, highlighting the importance of further research in this area.

Weaknesses:

- Sequence similarity between Ebola-Zaire (94% similarity) explains their similar behaviour in simulations and experimental assays. Marburg instead is a more distant homolog (~80% similarity relative to Ebola/Zaire). This difference is sequence and structure can explain the propensities, without the need to involve the existence of a cryptic pocket.<br /> - No real evidence for the presence of a cryptic pocket is presented, but rather a distance probability distribution between two residues obtained from extensive MD simulations. It would be interesting to characterise the modelled RNA-protein interface in more detail.

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 #2 (Public review):

Summary:

The authors have previously shown that the mouse neonatal cerebellum can regenerate damage to granule cell progenitors in the external granular layer, through reprogramming of gliogenic nestin-expressing progenitors (NEPs). The mechanisms of this reprogramming remain largely unknown. Here the authors used scRNAseq and ATACseq of purified neonatal NEPs from P1-P5 and showed that ROS signatures were transiently upregulated in gliogenic NEPs ve neurogenic NEPs 24 hours post injury (P2). To assess the role of ROS, mice transgenic for global catalase activity were assessed to reduce ROS. Inhibition of ROS significantly decreased gliogenic NEP reprogramming and diminished cerebellar growth post-injury. Further, inhibition of microglia across this same time period prevented one of the first steps of repair - the migration of NEPs into the external granule layer. This work is the first demonstration that the tissue microenvironment of the damaged neonatal cerebellum is a major regulator of neonatal cerebellar regeneration. Increased ROS is seen in other CNS damage models including adults, thus there may be some shared mechanisms across age and regions, although interestingly neonatal cerebellar astrocytes do not upregulate GFAP as seen in adult CNS damage models. Another intriguing finding is that global inhibition of ROS did not alter normal cerebellar development.

Strengths:

This paper presents a beautiful example of using single cell data to generate biologically relevant, testable hypotheses of mechanisms driving important biological processes. The scRNAseq and ATACseq analyses are rigorously conducted and conclusive. Data is very clearly presented and easily interpreted supporting the hypothesis next tested by reduce ROS in irradiated brains.

Analysis of whole tissue and FAC sorted NEPS in transgenic mice where human catalase was globally expressed in mitochondria were rigorously controlled and conclusively show that ROS upregulation was indeed decreased post injury and very clearly the regenerative response was inhibited. The authors are to be commended on the very careful analyses which are very well presented and again, easy to follow with all appropriate data shown to support their conclusions.

Weaknesses:

The authors also present data to show that microglia are required for an early step of mobilizing gliogenic NEPs into the damaged EGL. While the data that PLX5622 administration from P0-P5 or even P0-P8 clearly shows that there is an immediate reduction of NEPs mobilized to the damaged EGL, there is no subsequent reduction of cerebellar growth such that by P30, the treated and untreated irradiated cerebella are equivalent in size. There is speculation in the discussion about why this might be the case, but there is no explanation for why further, longer treatment was not attempted nor was there any additional analyses of other regenerative steps in the treated animals. The data still implicate microglia in the neonatal regenerative response, but how remains uncertain.

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 #2 (Public review):

Summary:

Gylemo et al. present a manuscript focused on identifying the X-inactivation or X-inactivation escape status for 380 genes across 30 normal human tissues. X-inactivation status of X-linked genes across tissues is important for understanding sex-specific differences in X-linked gene expression and therefore traits, and the likely effect of X-linked pathogenic variants in females. These new data are significant as they double the number of genes that have been classified in the human, and double the number of tissues studied previously.

Strengths:

The strengths of this work are that they analyse 3 individuals from the GTex dataset (2 newly identified, 1 previously identified and published) that have highly/ completely skewed X inactivation, which allows the study of escape from X inactivation in bulk RNA-sequencing. The number of individuals and breadth of tissues analysed add significantly to both the number of genes that have been classified and the weight of evidence for their claims. The additional 198 genes that have been classified and the reclassification of genes that previously had only limited support for their status is useful for the field.

In analysing the data they find that tissue-specific escape from X inactivation appears relatively rare. Rather, if genes escape, even variably, it tends to occur across tissues. Similarly, if a gene is inactivated, it is stable across tissues.

Weaknesses:

In my view there are only minor weaknesses in this work, that tend to come about due to the requirement to study individuals with highly skewed X inactivation. I wonder whether the cause of the highly skewed X inactivation may somehow influence the likelihood of observing tissue-specific escape from X inactivation. In this light, it would be interesting to further understand the genetic cause for the highly skewed X inactivation in each of these three cases in the whole exome sequencing data. Future additional studies may validate these findings using single-cell approaches in unrelated individuals across tissues, where there is normal X inactivation.

Reviewer #3 (Public review):

Summary:

Nestor and colleagues identify genes escaping X chromosome inactivation (XCI) in rare individuals with non-mosaic XCI (nmXCI) whose tissue-specific RNA-seq datasets were obtained from the GTEX database. Because XCI is non-mosaic, read counts representing a second allele are tested for statistically significant escape, in this case > 2.5% of active X expression. Whereas a prior GTEX analysis found only one nmXCI female, this study finds two additional donors in GTEX, therefore expanding the number of assessed X-linked genes to 380. Although this is fewer than half of X-linked genes, the study demonstrates that although rare, nmXCI females are represented in RNA-seq databases such as GTEX. Therefore this analytical approach is worthwhile pursuing in other (larger) databases as well, to provide deeper insight into escape from XCI which is relevant to X-linked diseases and sex differences.

Strengths:

The analysis is well-documented, straightforward, and valuable. The supplementary tables are useful, and the claims in the main text well-supported.

Weaknesses:

There are very few, except that this escape catalogue is limited to 3 donors, based on a single (representative) tissue screen in 285 female donors, mostly using muscle samples. However, if only pituitary samples had been screened, nmXCI-1 would have been missed. Additional donors in the 285 representative samples cross a lower threshold of AE = 0.4. It would be worthwhile to query all tissues of the 285 donors to discover more nmXCI cases, as currently fewer than half of X-linked genes received a call using this very worthwhile approach.

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 #2 (Public review):

The major conclusion of the manuscript is expressed in the title: "NR2F2 is required in the embryonic testis for Fetal Leydig Cell development" and also at the end of the introduction and all along the result part. All the authors' assertions are supported by very clear and statistically validated results from ISH, IHC, precise cell counting and gene expression levels by qPCR. The authors used two different conditional Nr2f2 gene ablation systems that demonstrate the same effects at the FLC level. They also showed that the haplo-insufficiency of Wt1 in the first system (knock-in Wt1-cre-ERT2) aggravated the situation in FLC differentiation by disturbing the differentiation of Sertoli cells and their secretion of pro-FLC factors, which had a confounding effect and encouraged them to use the second system. This demonstrates the great rigor with which the authors interpreted the results. In conclusion, all authors' claims and conclusions are justified by their high-quality results.

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 #2 (Public review):

This study addresses the question of how UBCs transform synaptic input patterns into spiking output patterns and how different glutamate receptors contribute to their transformations. The first figure utilizes recorded patterns of mossy fiber firing during eye movements in the flocculus of rhesus monkeys obtained from another laboratory. In the first figure, these patterns are used to stimulate mossy fibers in the mouse cerebellum during extracellular recordings of UBCs in acute mouse brain slices. The remaining experiments stimulate mossy fiber inputs at different rates or burst durations, which is described as 'mossy-fiber like', although they are quite simpler than those recorded in vivo. As expected from previous work, AMPA mediates the fast responses, and mGluR1 and mGluR2/3 mediate the majority of longer-duration and delayed responses. The manuscript is well organized and the discussion contextualizes the results effectively.

Comments on revisions:

The authors have adequately addressed my concerns.

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 #2 (Public review):

Summary:

Conceptually, this study is interesting and is the first attempt to account for the potentially interactive effects of seasonality and blood source on mosquito fitness, which the authors frame as a possible explanation for previously observed host-switching of Culex quinquefasciatus from birds to mammals in the fall. The authors hypothesize that if changes in fitness by blood source change between seasons, higher fitness on birds in the summer and on mammals in the autumn could drive observed host switching. To test this, the authors fed individuals from a colony of Cx. quinquefasciatus on chickens (bird model) and mice (mammal model) and subjected each of these two groups to two different environmental conditions reflecting the high and low temperatures and photoperiod experienced in summer and autumn in Córdoba, Argentina (aka seasonality). They measured fecundity, fertility, and hatchability over two gonotrophic cycles. The authors then used generalized linear mixed models to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and hatchability. The authors were trying to test their hypothesis by determining whether there was an interactive effect of season and host species on mosquito fitness. This is an interesting hypothesis; if it had been supported, it would provide support for a new mechanism driving host switching. While the authors did report an interactive impact of seasonality and host species, the directionality of the effect was the opposite from that hypothesized. The authors have done a very good job of addressing many of the reviewer's concerns, especially by adding two additional replicates. Several minor concerns remain, especially regarding unclear statements in the discussion.

Strengths:

(1) Using a combination of laboratory feedings and incubators to simulate seasonal environmental conditions is a good, controlled way to assess the potentially interactive impact of host species and seasonality on the fitness of Culex quinquefasciatus in the lab.<br /> (2) The driving hypothesis is an interesting and creative way to think about a potential driver of host switching observed in the field.

Weaknesses:

(1) The methods would be improved by some additional details. For example, clarifying the number of generations for which mosquitoes were maintained in colony (which was changed from 20 to several) and whether replicates were conducted at different time points.<br /> (2) The statistical analysis requires some additional explanation. For example, you suggest that the power analysis was conducted a priori, but this was not mentioned in your first two drafts, so I wonder if it was actually conducted after the first replicate. It would be helpful to include further detail, such as how the parameters were estimated. Also, it would be helpful to clarify why replicate was included as a random effect for fecundity and fertility but as a fixed effect for hatchability. This might explain why there were no significant differences for hatchability given that you were estimating for more parameters.<br /> (3) A number of statements in the discussion are not clear. For example, what do you mean by a mixed perspective in the first paragraph? Also, why is the expectation mentioned in the second paragraph different from the hypothesis you described in your introduction?<br /> (4) According to eLife policy, data must be made freely available (not just upon request).

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 #2 (Public review):

The authors developed the TaG-EM system to address challenges in multiplexing Drosophila samples for behavioral and transcriptomic studies. This system integrates DNA barcodes upstream of the polyadenylation site in a UAS-GFP construct, enabling pooled behavioral measurements and cell type tracking in scRNA-seq experiments. The revised manuscript expands on the utility of TaG-EM by demonstrating its application to complex assays, such as larval gut motility, and provides a refined analysis of its limitations and cost-effectiveness.

Strengths

(1) Novelty and Scope: The study demonstrates the potential for TaG-EM to streamline multiplexing in both behavioral and transcriptomic contexts. The additional application to labor-intensive larval gut motility assays highlights its scalability and practical utility.

(2) Data Quality and Clarity: Figures and supplemental data are mostly clear and significantly enhanced in the revised manuscript. The addition of Supplemental Figures 18-21 addresses initial concerns about scRNA-seq data and driver characterization.

(3) Cost-Effectiveness Analysis: New analyses of labor and cost savings (e.g., Supplemental Figure 8) provide a practical perspective.

(4) Improvements in Barcode Detection and Analysis: Enhanced enrichment protocols (Supplemental Figures 18-19) demonstrate progress in addressing limitations of barcode detection and increase the detection rate of labeled cells.

Weaknesses

(1) Barcode Detection Efficiency: While improvements are noted, the low barcode detection rate (~37% in optimized conditions) limits the method's scalability in some applications, such as single-cell sequencing experiments with complex cell populations.

(2) Sparse Labeling: Sparse labeling of cell populations, particularly in scRNA-seq assays, remains a concern. Variability in driver strength and regional expression introduces inconsistencies in labeling density.

(3) Behavioral Applications: The utility of TaG-EM in quantifying more complex behaviors remains underexplored, limiting the generalizability of the method beyond simpler assays like phototaxis and oviposition.

(4) Driver Line Characterization: While improvements in driver line characterization were made, variability in expression patterns and sparse labeling emphasize the need for further refinement of constructs and systematic backcrossing to standardize the genetic background.

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 #2 (Public review):

Summary:

The authors analyzed Xenopus oocytes at different stages of meiosis using quantitative phosphoproteomics. Their advanced methods and analyses revealed changes in protein abundances and phosphorylation states to an unprecedented depth and quantitative detail. In the manuscript they provide an excellent interpretation of these findings putting them in the context of past literature in Xenopus as well as in other model systems.

Strengths:

High quality data, careful and detailed analysis, outstanding interpretation in the context of the large body of the literature.

Weaknesses:

Merely a resource, none of the findings are tested in functional experiments.

I am very impressed by the quality of the data and the careful and detailed interpretation of the findings. In this form the manuscript will be an excellent resource to the cell division community in general, and it presents a very large number of hypotheses that can be tested in future experiments.

Xenopus has been and still is a popular and powerful model system that led to critical discoveries around countless cellular processes, including the spindle, nuclear envelope, translational regulation, just to name a few. This also includes a huge body of literature on the cell cycle describing its phosphoregulation. It is indeed somewhat frustrating to see that these earlier studies using phospho-mutants and phospho-antibodies were just scratching the surface. The phosphoproteomics analysis presented here reveals much more extensive and much more dynamic changes in phosphorylation states. Thereby, in my opinion, this manuscript opens a completely new chapter in this line of research, setting the stage for more systematic future studies.

Reviewer #3 (Public review):

Summary:

The authors performed time-resolved proteomics and phospho-proteomics in Xenopus oocytes from prophase I through the MII arrest of the unfertilized egg. The data contains protein abundance and phosphorylation sites of a large number set of proteins at different stages of oocyte maturation. The large sets of the data are of high quality. In addition, the authors discussed several key pathways critical for the maturation. The data is very useful for the researchers not only researchers in Xenopus oocytes but also those in oocyte biology in other organisms.

Strengths:

The data of proteomics and phospho-proteomics in Xenopus oocyte maturation is very useful for future studies to understand molecular networks in oocyte maturation.

Weaknesses:

Although the authors offered molecular pathways of the phosphorylation in the translation, protein degradation, cell cycle regulation, and chromosome segregation. The author did not check the validity of the molecular pathways based ontheir proteomic data by the experimentation.

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 #2 (Public review):

The work by Gupta et al. addresses the role of tissue compressibility as a driver of cell competition. The authors use a planar epithelial monolayer system to study cell competition between wild type and transformed epithelial cells expressing HRasV12. They combine imaging and traction force measurements from which the authors propose that wild type cells generate compressive forces on transformed epithelial cells. The authors further present a novel setup to directly measure the compressibility of adherent epithelial tissues. These measurements suggest a higher compressibility of transformed epithelial cells, which is causally linked to a reduction in cell-cell adhesion in transformed cells. The authors support their conclusions by theoretical modelling using a self-Propelled Voronoi model that supports differences in tissue compressibility can lead to compression of the softer tissue type.

The experimental framework to measure tissue compressibility of adherent epithelial monolayers establishes a novel tool, however additional controls of this measurement appear required. Moreover, the experimental support of this study is mostly based on single representative images and would greatly benefit from additional data and their quantitative analysis to support the authors' conclusions. Specific comments are also listed in the following:

Major points:

It is not evident in Fig2A that traction forces increase along the interface between wild type and transformed populations and stresses in Fig2C also seem to be similar at the interface and surrounding cell layer. Only representative examples are provided and a quantification of sigma_m needs to be provided.

In Figure 1-3 only panel 2G and 2H provide a quantitative analysis, but it is not clear how many regions of interest and clusters of transform cells were quantified.

Several statements appear to be not sufficiently justified and supported by data.<br /> For example the statement on pg 3. line 38 seems to lack supportive data 'This comparison revealed that the thickness of HRasV12-expressing cells was reduced by more than 1.7-fold when they were surrounded by wild type cells. These observations pointed towards a selective, competition-dependent compaction of HRasV12-expressing transformed cells but not control cells, in the intestinal villi of mice.'<br /> Similarly, the statement about a cell area change of 2.7 fold (pg 3 line 47) lacks support by measurements.

What is the rationale for setting 𝐾p = 1 in the model assumptions if clear differences in junctional membranes of transformed versus wild type cells occur, including dynamic ruffling? This assumption does not seem to be in line with biological observations.

The novel approach to measure tissue compressibility is based on pH dependent hydrogels. As the pH responsive hydrogel pillar is placed into a culture medium with different conditions, an important control would be if the insertion of this hydrogel itself would change the pH or conditions of the culture assays and whether this alters tissue compressibility or cell adhesion. The authors could for example insert a hydrogel pillar of a smaller diameter that would not lead to compression or culture cells in a larger ring to assess the influence of the pillar itself.

The authors focus on the study of cell compaction of the transformed cells, but how does this ultimately lead to a competitive benefit of wild type cells? Is a higher rate of extrusion observed and associated with the compaction of transformed cells or is their cell death rate increased? While transformed cells seem to maintain a proliferative advantage it is not clear which consequences of tissue compression ultimately drive cell competition between wild type and transformed cells.

The argumentation that softer tissues would be more easily compressed is plausible. However, which mechanism do the authors suggest is generating the actual compressive stress to drive the compaction of transformed cells? They exclude a proliferative advantage of wild type cells, which other mechanisms will generate the compressive forces by wild type cells?

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 #2 (Public review):

The work by Gupta et al. addresses the role of tissue compressibility as a driver of cell competition. The authors use a planar epithelial monolayer system to study cell competition between wild type and transformed epithelial cells expressing HRasV12. They combine imaging and traction force measurements from which the authors propose that wild type cells generate compressive forces on transformed epithelial cells. The authors further present a novel setup to directly measure the compressibility of adherent epithelial tissues. These measurements suggest a higher compressibility of transformed epithelial cells, which is causally linked to a reduction in cell-cell adhesion in transformed cells. The authors support their conclusions by theoretical modelling using a self-Propelled Voronoi model that supports differences in tissue compressibility can lead to compression of the softer tissue type.

The experimental framework to measure tissue compressibility of adherent epithelial monolayers establishes a novel tool, however additional controls of this measurement appear required. Moreover, the experimental support of this study is mostly based on single representative images and would greatly benefit from additional data and their quantitative analysis to support the authors' conclusions. Specific comments are also listed in the following:

Major points:

It is not evident in Fig2A that traction forces increase along the interface between wild type and transformed populations and stresses in Fig2C also seem to be similar at the interface and surrounding cell layer. Only representative examples are provided and a quantification of sigma_m needs to be provided.

In Figure 1-3 only panel 2G and 2H provide a quantitative analysis, but it is not clear how many regions of interest and clusters of transform cells were quantified.