Reviewer #1 (Public Review):

Lammer et al. examined the effects of social loneliness, and longitudinal change in social loneliness, on cognitive and brain aging. In a large sample longitudinal dataset, the authors found that both baseline loneliness and an increase in loneliness at follow-up were significantly associated with smaller hippocampal volume, reduced cortical thickness, and worse cognition in healthy older adults. In addition, those older adults with high loneliness at baseline showed even smaller hippocampal volume at follow-up. These results are interesting in identifying the importance of social support to cognitive and brain health in old age. With a longitudinal design, they were able to show that increased loneliness was related to reduced brain structural measures. Such results could help guide clinicians and policymakers in designing social support systems that would benefit the growing aging population.

The strength of the current study lies in the large sample size and longitudinal follow-up design. The multilevel models used to separate within and between subject effects are well constructed. Combining neuroimaging data with behavioral changes provided further evidence that social loneliness may be related to accelerated brain aging. Stringent FDR correction, Bayes factor comparison, and the additional analyses for sensitivity showed the robustness and credibility of the results.

Weaknesses of the study were related to the interpretation and discussion of their findings.

Social loneliness is a relatively little-studied factor in cognitive ageing, and the authors should consider expanding the discussion, with some additional analyses, as to how their results could be used by clinicians and older adults to monitor social behaviors.

The authors examined the interaction between baseline and age change to see if higher baseline loneliness was associated with accelerated decline. The interaction was significant, but the authors did not further explore the interaction effect, which may have clinical significance. The authors should consider identifying a cut-off point in LSNS that suggests persons scoring less than this score on the LSNS may be at greater risk of accelerated brain decline than others. Such a cut-off point is important for clinicians, as well as for future researchers to compare their results.

Although it was not directly tested in the paper, LSNS scores did not seem to change with increasing age (Table 1). This general stability of LSNS scores in older adults should be discussed further. The authors should consider how their relatively healthy and high SES sample may be less vulnerable to loss of family or friends in old age, making this sample sub-optimal for the question they have. The significance of the subject effect suggests that some individuals still experience a loss of social connectedness. The authors may want to elaborate on this and give some explanations of such subject differences in the ageing effect on social loneliness. Although stress was not a significant mediating factor, is it related to baseline loneliness or changes in loneliness in the current sample?

The presentation of longitudinal data (Figure 1) lacks dimensionality. The scatter plots presented here are more suitable for cross-sectional studies and could cause confusion regarding the interpretation of the results. The authors should consider individual growth curves or spaghetti plots in visualizing change within subjects.

Reviewer #1 (Public Review):

Muscle is a major insulin-responsive tissue for the disposal of glucose, a process dependent on the translocation of GLUT4 glucose transporter from intracellular compartments to the plasma membrane. Knudsen and co-workers provide an analysis of the impact of microtubule-based movement on GLUT4 biology in muscle cell lines, and rodent and human muscle fibers ex vivo. A role for microtubules in the control of GLUT4 vesicle dynamics in both unstimulated and insulin-stimulated adipocytes (cultured and primary) has been previously reported by a number of groups. Less is known about the requirement for microtubules for GLUT4 translocation in muscle. A strength of this study is that key aspects of the work were performed in muscle fibers rather than muscle cell lines.

Conclusions that are strongly supported by the data presented include:

1. Demonstration of constitutive GLUT4 movement along microtubule tracks in both unstimulated and insulin-stimulated muscle fibers. GLUT4 dynamics in unstimulated fibers were captured by fluorescence recover after photobleaching (FRAP) and by quantifying vesicle movements by live cell microscopy, whereas in insulin-stimulated cells GLUT4 dynamics were captured by following the movements of GLUT4-containing vesicles. These data support a model in which intracellular GLUT4 is dynamic in both unstimulated and insulin-stimulated muscle fibers rather than being static in unstimulated conditions and only mobilized upon insulin-stimulation.

2. Similar microscopy analyses of GLUT4-containing vesicles demonstrate that depolymerization of microtubules reduced GLUT4 vesicle movement and impacted insulin-stimulated glucose uptake. Short term depolymerization of microtubules (5 min) did not affect insulin-stimulated glucose uptake, whereas insulin-stimulated glucose uptake was blocked after prolonged depolymerization (2 hrs). The use of a muscle on a chip method to monitor glucose uptake in real time was critical for these experiments.

The changes in glucose uptake were accompanied by changes in the morphologies of intracellular GLUT4-containing structures. The differences between short and long term depolymerization of microtubules support a model in which GLUT4 can be translocated to the plasma membrane by insulin stimulation in the absence of microtubules but an intact microtubule cytoskeleton is required to maintain GLUT4 in a "compartment" that can be recruited by insulin. Stated another way, the microtubule-dependent dynamics of GLUT4-containing vesicles in unstimulated cells is permissive for insulin-stimulated GLUT4 translocation.

3. Knockdown of the microtubule motor protein, Kif5b, blunts insulin-stimulated translocation of GLUT4 to the plasma membrane of cultured muscle cells. These findings agree with previously demonstrated role for Kif5b in adipocytes.

4. In an in vitro model of insulin resistance (incubation of muscle fibers with short chain C2 ceramide) unstimulated and insulin-stimulated GLUT4-containing vesicle movement was blunted and unstimulated and insulin-stimulated microtubule polymerization was reduced.

Weakness of the study include:

1. There are no data supporting a role for insulin regulation of microtubule-dependent GLUT4-containg vesicle movement. The data in Fig.2B do not support a differences in the number of "moving" GLUT4 vesicles between basal and insulin-stimulated fibers. The statement on line 103 that they "observed a ~16% but insignificant increase" to be confusing. These data do not support an effect of insulin on the number of moving GLUT4 vesicles that can be detected in an individual experiment. There is also effect of insulin on GLUT4 vesicles in the data reported in Fig.S2D, Fig.S5B, and Fig.S5F. However, the data in Fig. 2C suggest there was a consistent increase in "moving" vesicles in insulin-stimulated conditions in 4 independent experiments (how are these data normalized?). Because the basis of insulin-regulation of glucose uptake is the control of GLUT4 translocation to the plasma membrane, the authors need to clarify their thinking on why they do not detect insulin robust effects on GLUT4 dynamics in the individual experiments. Is it that they are not measuring the correct parameter? That the assay is not sensitive to the changes?

The small (or no effect) of insulin distracts a bit from the findings that there is microtubule-dependent GLUT4 movement in basal and stimulated muscle fibers, and that disruption of this movement by depolymerization of microtubules or Kif5b knockdown blunts GLUT4 translocation. As noted above, the data strongly support microtubule-dependent GLUT4 dynamics as permissive for insulin-stimulated GLUT4 translocation even if this dynamics might not be a target of insulin action.

2. The analyses of GLUT4-containing structures are not particularly informative. Co-localization with other markers (beyond syntaxin6) are needed to understand these structures. Defining structures as small, medium or large is incomplete. In particular, it is important to probe the microtubule nucleation site clusters for other membrane markers. Transferrin receptor? IRAP?

3. The Kinesore data do not support the authors hypothesis. The data show that Kinesore increases the amount of GLUT4 in the plasma membrane of basal cells and that insulin further increases plasma membrane GLUT4 to the same extent as it does in control cells. How does that provide insight into the role microtubules (or kif5b) in GLUT4 biology? Why does Kinesore increase plasma membrane GLUT4? Is it an effect of Kinesin 1 on GLUT4 vesicles? Kinesore is reported to remodel the microtubule cytoskeleton by a mechanism dependent on Kinesin 1. Is that the reason for the change in GLUT4?

4. The analysis of Kif5b is a bit cursory. Depolymerization of microtubules in muscle fibers essentially blocks all GLUT4 movement (only the insulin condition is shown in Fig.2B but I assume basal would be equally inhibited), and fully inhibits insulin-stimulated glucose uptake in muscle fibers. What are the effects of nocodazole in L6 cells (cell used for kif5b studies) and is it similar in magnitude to kif5b knockdown? Those data would identify there are non-Kif5b microtubule-dependent effects.

5. The authors need to show that the fibers isolated from the HFD mice remain insulin-resistant ex vivo by measuring glucose uptake. It is possible that once removed from the mice they "revert" to normal insulin-sensitivity, which might contribute to the differences reported in Fig5.

6. Although it is interesting that the authors have included the insulin-resistance models/experiments, they are not well developed and therefore the conclusions are not particularly strong.

7. The data do not support the title.

Reviewer #1 (Public Review):

The authors study the control of the timing of Q neuroblast migration, through the precisely timed expression of the Wnt receptor MIG-1/Frizzled, which halts migration of the QR.pa cell at its intended position. Understanding the underlying mechanism is important, as similar mechanisms might play a role in controlling the timing of biological processes in development much more broadly. The authors use precise measurements of mig-1 mRNA molecules, fitted to mathematical models of different mechanisms to control the timing of mig-1 expression, and couple this with experimental perturbations of mig-1 expression. In this way, the authors convincingly show that mig-1 dynamics is best explained by a model where mig-1 expression is controlled by the accumulation of an activator, rather than the degradation of a repressor, which is an important result. In addition, they show that the asymmetric division of QR.p into the larger QR.pa and smaller QR.pp cells is important for proper mig-1 expression in Qr.pa, likely by asymmetric inheritance of the activator. In the process, the authors identify novel conserved binding motifs that are responsible for different aspects of mig-1 dynamics, which will potentially allow identifying the putative activator in the future.

In its current form, I find the manuscript has two main weak points: First, the connection between the experiments and models is relatively weak. Now, the model is mostly used to aid the interpretation of experiments, by predicting rough trends. However, even though the model is in principle fitted to the experimental data in some cases, a detailed comparison between experimental results and the model is often lacking. For example, there are multiple occasions where the data appears to not fit the model in some aspects, but the potential origin of these mismatches is typically not discussed. Second, the authors present experimental evidence of an earlier model prediction, that positive feedback loops in mig-1 expression reduce variability in timing. Here, the authors speculate that this feedback loop might be due to the activation of mig-1 expression by mig-1-induced Wnt signaling, which in itself is an interesting idea. However, the genetic perturbation used here - manipulation of the Wnt pathway, rather than perturbing specifically the induction of mig-1 expression by Wnt signaling - likely changes the expression of many genes in the cell, making it difficult to establish whether the increased variability in Qr.pa position is indeed due breaking the proposed feedback loop.

Reviewer #1 (Public Review)

This paper utilizes two well-established mathematical models of colorectal cancer (CRC) screening to estimate the impact of disruptions in screening caused by the COVID-19 pandemic on long-term outcomes related to CRC. For screening, the authors use two recommendations from the US Preventive Services Task Force (USPSTF) (which were informed by the results of these models): screening colonoscopy every 10 years at ages 50, 60, and 70, and annual fecal immunochemical tests (FIT) from ages 50-75. Separate model runs were performed for 8 different cohorts at the time of the pandemic based on age, screening history, and adherence to screening. For each cohort, microsimulations were performed for 3 different scenarios--no disruption, delays in screening, or discontinuation from screening. The primary outcome was life-years gained (LYG) from screening.

In general, severe prolonged disruptions in any screening led to the largest loss of benefit from screening - for example, unscreened 50-year-olds forced to wait until age 65 (Medicare eligibility) had the largest absolute and relative loss in screening-associated LYG compared to shorter delays of 18 months or less. Losses were also higher in those who were semi-adherent to screening recommendations. The prolonged disruption had a consistently much greater impact than short-term reductions, changes in regimen, or assumptions about test sensitivity. The results are consistent between the two models. The authors point out that, since pandemic-induced disruptions in insurance coverage had a greater impact on minority populations already at risk for reduced access to screening and other preventive services, the pandemic may lead to further exacerbations in existing disparities in CRC incidence and mortality.

The strengths of this paper include the use of well-validated models, the consistent results between the models, the relatively intuitive nature of the findings, and the use of LYG, a commonly used metric for screening recommendations. As the authors point out, estimates of the population impact of the pandemic given the current age structure of the US would be helpful, these would be inherently speculative given the lack of empirical data on pandemic effects on screening. Although prioritizing screening individuals with long pandemic-induced delays is clearly the optimal policy approach, how this might be achieved is unclear.

Reviewer #1 (Public Review):

In this study, the authors use open-access datasets of Neuropixel recordings to explore the relationship between ripple strength and propagation in the septal/dorsal hippocampal pole. They found that the ripple strength correlates with the direction of propagation and that the duration of the events is dependent on the site of initiation. Medial pole ripples are longer and engage significantly more neurons than lateral ripples. These findings may have theoretical and practical implications for the study of sharp-wave ripples, a main oscillatory event underlying memory consolidation. While the approach is not entirely novel (e.g. Patel et al., JN 2013; Kumar and Deshmukh 2020), the study provides some additional insights. The strength of evidence of propagation dynamics is solid and claims are broadly supported. Some points however may require revision. In particular, issues regarding the definition of the longitudinal and transversal axes, as well as additional analysis on microcircuit interactions and neuronal dynamics per cell types and hippocampal sectors should be more thoroughly addressed in support of mechanisms.

Reviewer #1 (Public Review):

Nikolaos Koutras et al shed light on potential distinct functions of the Src family kinases (SFKs) Lck and Lyn in lymphoid signal transduction. The authors therefore overexpress Lyn and ectopic Lck in the B lymphoid cell line BJAB in an elegant Dox-inducible manner and compare the SFK's ability to trigger and shape B-lymphoid signal transduction. The findings indicate that ectopic expression of Lck is sufficient to phosphorylate the B cell receptor (BCR) ITAMs in BJAB cells. In these cells, constitutive ITAM and ITIM phosphorylation by both overexpressed Lck and Lyn induces BCR signaling, as demonstrated by phosphorylation of Syk and Akt, as well as CD22 inhibitory signaling, as shown by SHP-1 phosphorylation. In direct comparison, the influence of Lyn on said phosphorylation is stronger when it is (over-)expressed in the same amounts as Lck. This outcome was somewhat expected, since ITIM/ITAM phosphorylation is considered to be the principal function of Lyn in B cells.

The study finds Lyn to be degraded more efficiently via the proteasome and to be more tightly controlled by phosphatases when compared to Lck. However, rather than interpreting the findings as distinct kinase-intrinsic properties, one could attribute the slower degradation and stricter PTP control of Lyn to the fact that Lyn is the principal and predominant SFK in B cells and thus a "standard target" of the B-lymphoid molecular machinery, to which it is better adapted to.

Next, the authors present a RNAseq transcriptome analysis of Lck- and Lyn-expressing B cells and validate selected findings via qPCR. The data show Lyn and Lck to regulate pathways and biological functions of critical importance to B lymphocytes. Generally, most of the Lck/Lyn-regulated biological functions and pathways shown here (antigen presentation, cytokine production, migration, apoptosis, autophagy, etc.) are well known to be controlled by BCR signaling, which the overexpression of SFKs are constitutively activating, as shown earlier. While the authors draw a Venn diagram depicting differentially regulated transcripts between Lck- and Lyn-expressing cells, it does not seem like Lck is able to regulate pathways which are not "canonically" regulated by Lyn. There is also the persisting problem of Lck being expressed to a much higher extent and the effect of the endogenously expressed Lyn, since the model systems are not based on a Lyn-deficient cell line.

Lastly, the authors follow up their finding of deregulated transcripts belonging to the ER/UPR ontology cluster. Flow cytometric analysis indeed shows an influence of Lck and Lyn expression on ER homeostasis, which can be reverted with SFK inhibitors. Alas, additional follow-up experiments to functionally investigate the deregulated pathways suggested by the RNAseq analysis are not included in this study.

While there definitely are implications for the role of ectopic expression of Lck in CLL cells, this work however presents no direct comparison of expression strength or signaling outcomes between the study's BJAB (Burkitt lymphoma) cell line-based model and a model of CLL - be it a mouse model, human patient samples or a CLL cell line. Since the B-lymphoid cell line used, the Burkitt lymphoma line BJAB, is not CLL-derived, the conclusions that can be drawn for the pathophysiology of CLL is limited.

In principal, the authors show that the Src kinase Lck - when ectopically expressed - largely fills out the role of the predominant B-lymphoid Src kinase Lyn, namely phosphorylation of the CD79-ITAMs and induction of constitutive antigen receptor signaling. Given that the established role of Lck is the phosphorylation of ITAMs and activation of the T cell receptor in T cells, where it is predominantly expressed, these findings provide limited advancement of our current understanding of antigen receptor signal transduction. As a distinct functional difference between Lck and Lyn is not established in this work, said SFKs' largely exclusive expression in T and B cells remains enigmatic.

Reviewer #1 (Public Review):

Members of the SLC11/NRAMP family of transporters permit the movement of transition metals across cell membranes in all kingdoms of life. The current study builds off previous structural and mechanistic work on the SLC11/NRAMP family of transporters by Manatschal and colleagues reported in eLife; the current study presents a cryo-EM structure of a plant aluminum (Al3+) transporter that combats aluminum toxicity in soil. The structure was not determined in the presence of added metal ions, so the paper also employs a variety of established functional assays to test the effects of mutating suggested binding site residues. One notable result is the identification of a mutation (S68A) that maintains divalent transport but disrupts trivalent binding/transport. Strengths of the manuscript include the extensive legwork required to identify a combination of plant homologue, cameloid nanobody, and amphipol that is required to provide homogenous protein and interpretable cryo-EM data. The cryo-EM maps are reliable with low orientation bias and clear features. In addition, the authors perform a number of biochemical and transport assays with divalent metals to bolster their structural model.

Reviewer #1 (Public Review):

In this paper the authors are estimating the amount of transmission (via the force of infection) of EV-D^8 in England. The strengths of the study are the use of serological data for understanding underlying transmission, and the assessment of the sensitivity of the conclusions to the seropositivity cut off and the model form used. The weaknesses are the data not being annually and the lack of link to HFMD cases,, but these do not detract from the conclusions that can be drawn from the paper. The results do support the conclusions.

Reviewer #1 (Public Review):

In this paper, the authors developed a method that allows one to test a large number of drug combinations in a single cell culture sample. In principle, the experiments rely on the randomness of drug uptake in individual cells as a tool to create and encode drug treatments. They used a single sample containing thousands of cells treated with a combination of fluorescent barcoded drugs, and created transient drug gradients. They also developed segmentation- free image analysis capable of handling optical fields with a substantial number of cells. The major strength of this work is the demonstration of the feasibility of testing drug combinations in a relatively straightforward manner that could be used by many laboratories. As such this paper could have a significant impact on the early drug discovery of combinatorial therapy. One of the weaknesses in this manuscript is the absence of studies beyond just HeLa cells. In addition, the phenotype tested is cell death, which might limit the application to other drug interactions that might look at other phenotypes; e.g inhibition of cell proliferation or changes in differentiation phenotypes. Finally, there is a basic assumption that drug leakage does not occur or is minimal, but secondary uptake of the drug is likely and may not be homogeneous. Notwithstanding, the approach is feasible and likely will be applied in several laboratories.

Reviewer #1 (Public Review):

In this manuscript, Nocka and colleagues reveal a novel layer of regulation of the Btk tyrosine kinase, a key signaling protein in B lymphocyte signaling and an important drug target with 3 recently FDA-approved drugs, by the SH3-SH2 domain-containing adaptor protein Grb2. The authors nicely demonstrate a critical role of the interaction of the Grb2 SH3 domains with the Pro-rich linker C-terminal to the Btk PH-TH domains on membranes for full kinase activation of Btk. Hence this interaction recruits Btk to scaffold-mediated signaling clusters.

This is a technically sound paper with high-quality experiments. The manuscript is easy to follow and excellently written. The findings are novel and of high relevance towards a complete understanding of Btk regulation and signaling in cancer and normal cells.

Reviewer #1 (Public Review):

The study by Oikawa and colleagues demonstrates for the first time that a descending inhibitory pathway for nociception exists in non-mammalian organisms, such as Drosophila. This descending inhibitory pathway is mediated by a Drosophila neuropeptide called Drosulfakinin (DSK), which is homologous to mammalian cholecystokinin (CCK). The study creates and uses several Drosophila mutants to convincingly show that DSK negatively regulates nociception. They then use several sophisticated transgenic manipulations to demonstrate that a descending inhibitory pathway for nociception exists in Drosophila.

Strengths:

This study creates the possibility of using Drosophila to study descending nociceptive systems.

CRISPR/Cas9 is used to generate mutants of dsk, CCKLR-17D1, and CCKLR-17D3. The authors then use these mutants to clearly show that DSK negatively regulates nociception.

Several GAL4s are used to clearly show that these effects are likely mediated by two sets of neurons in the brain, MP1 and Sv.

RNAi and rescue experiments further show that CCKLR-17D1, a DSK receptor, functions in Goro neurons to negatively regulate nociception.

Thermogenetic experiments nicely show that activation of DSK neurons attenuates the nociceptive response.

Weaknesses:

A minor weakness in the study is that it is unclear how DSK negatively regulates nociception. An earlier study at the Drosophila nmj shows that loss of DSK signaling impairs neurotransmission and synaptic growth. In the current study, loss of CCKLR-17D1 in Goro neurons seems to increase intracellular calcium levels in the presence of noxious heat. An interesting future study would be the examination of the underlying mechanisms for this increase in intracellular calcium.

Reviewer #1 (Public Review):

Chan et al. tried identifying the binding sites or pockets for the KCNQ1-KCNE1 activator mefenamic acid. Because the KCNQ1-KCNE1 channel is responsible for cardiac repolarization, genetic impairment of either the KCNQ1 or KCNE1 gene can cause cardiac arrhythmia. Therefore, the development of activators without side effects is highly demanded. Because the binding of mefenamic acid requires both KCNQ1 and KCNE1 subunits, the authors performed drug docking simulation by using KCNQ1-KCNE3 structural model (because this is the only available KCNQ1-KCNE structure) with substitution of the extracellular five amino acids (R53-Y58) into D39-A44 of KCNE1. That could be a limitation of the work because the binding mode of KCNE1 might differ from that of KCNE3. Still, they successfully identified some critical amino acid residues, including W323 of KCNQ1 and K41 and A44 of KCNE1. They subsequently tested these identified amino acid residues by analyzing the point mutants and confirmed that they attenuated the effects of the activator. They also examined another activator, yet structurally different DIDS, and reported that DIDS and mefenamic acid share the binding pocket, and they concluded that the extracellular region composed of S1, S6, and KCNE1 is a generic binding pocket for the IKS activators.

The data are solid and well support their conclusions, although there are a few concerns regarding the choice of mutants for analysis and data presentation.

Other comments:

1. One of the limitations of this work is that they used psKCNE1 (mostly KCNE3), not real KCNE1, as written above. It is also noted that KCNQ1-KCNE3 is in the open state. Unbinding may be facilitated in the closed state, although evaluating that in the current work is difficult.<br /> 2. According to Figure 2-figure supplement 2, some amino acid residues (S298 and A300) of the turret might be involved in the binding of mefenamic acid. On the other hand, Q147 showing a comparable delta G value to S298 and A300 was picked for mutant analysis. What are the criteria for the following electrophysiological study?<br /> 3. It is an interesting speculation that K41C and W323A stabilize the extracellular region of KCNE1 and might increase the binding efficacy of mefenamic acid. Is it also the case for DIDS? K41 may not be critical for DIDS, however.<br /> 4. Same to #2, why was the pore turret (S298-A300) not examined in Figure 7?

Reviewer #1 (Public Review):

This is a valuable study demonstrating convincingly that PI3K signaling lies downstream of Pdgfra signaling in zebrafish cardiomyocyte progenitors as they undergo latero-medial migration and midline fusion, essential for heart tube formation, likely via chemotaxis. Whereas the authors used both multiple inhibitory drugs and dominant negative transgene expression to interrupt PI3K expression, with findings strongly aligning, the manuscript would have been stronger if genetic approaches were used to complement the above approaches. Nonetheless, the impact of dnPI3K inhibition allowed the authors to suggest that the effects were cell autonomous to migrating cardiomyocytes. The authors used contemporary live imaging techniques allowing quantification of key cell behaviors, and this is a strength of the paper. There are some issues about the inter-study alignment of trajectory data that need to be addressed. Perhaps the most conspicuous weakness is that the authors have not advanced the model for cardiomyocyte migration beyond adding the involvement of PI3K downstream of Pdgfra, which is to a significant degree expected. The recording of cardiomyocyte protrusions biased in their orientation towards the direction of migration, which is lost in the mutants, is an interesting advance, although it was not shown whether protrusions are causally related to migration.

Reviewer #1 (Public Review):

This manuscript addresses the important and understudied issue of circuit-level mechanisms supporting habituation, particularly in pursuit of the possible role of increases in the activity of inhibitory neurons in suppressing behavioral output during long-term habituation. The authors make use of many of the striking advantages of the larval zebrafish to perform whole brain, single neuronal calcium imaging during repeated sensory exposure, and high throughput screening of pharmacological agents in freely moving, habituating larvae. Notably, several blockers/antagonists of GABAA(C) receptors completely suppress habituation of the O-bend escape response to dark flashes, suggesting a key role for GABAergic transmission in this form of habituation. Other substances are identified that strikingly enhance habituation, including melatonin, although here the suggested mechanistic insight is less specific. To add to these findings, a number of functional clusters of neurons are identified in the larval brain that has divergent activity through habituation, with many clusters exhibiting suppression of different degrees, in line with adaptive filtration during habituation, and a single cluster that potentiates during habituation. Further assessment reveals that all of these clusters include GABAergic inhibitory neurons and excitatory neurons, so we cannot take away the simple interpretation that the potentiating cluster of neurons is inhibitory and therefore exerts an influence on the other adapting (depressing) clusters to produce habituation. Rather, a variety of interpretations remain in play.

Overall, there is great potential in the approach that has been used here to gain insight into circuit-level mechanisms of habituation. There are many experiments performed by the authors that cannot be achieved currently in other vertebrate systems, so the manuscript serves as a potential methodological platform that can be used to support a rich array of future work. While there are several key observations that one can take away from this manuscript, a clear interpretation of the role of GABAergic inhibitory neurons in habituation has not been established. This potential feature of habituation is emphasized throughout, particularly in the introduction and discussion sections, meaning that one is obliged as a reader to interrogate whether the results as they currently stand really do demonstrate a role for GABAergic inhibition in habituation. Currently, the key piece of evidence that may support this conclusion is that picrotoxin, which acts to block some classes of GABA receptors, prevents habituation. However, there are interpretations of this finding that do not specifically require a role for modified GABAergic inhibition. For instance, by lowering GABAergic inhibition, an overall increase in neural activity will occur within the brain, in this case below a level that could cause a seizure. That increase in activity may simply prevent learning by massively increasing neural noise and therefore either preventing synaptic plasticity or, more likely, causing indiscriminate synaptic strengthening and weakening that occludes information storage. Sensory processing itself could also be disrupted, for instance by altering the selectivity of receptive fields. Alternatively, it could be that the increase in neural activity produced by the blockade of inhibition simply drives more behavioral output, meaning that more excitatory synaptic adaptation is required to suppress that output. The authors propose two specific working models of the ways in which GABAergic inhibition could be implemented in habituation. An alternative model, in which GABAergic neurons are not themselves modified but act as a key intermediary between Hebbian assemblies of excitatory neurons that are modified to support memory and output neurons, is not explored. As yet, these or other models in which inhibition is not required for habituation, have not been fully tested.

This manuscript describes a really substantial body of work that provides evidence of functional clusters of neurons with divergent responses to repeated sensory input and an array of pharmacological agents that can influence the rate of a fundamentally important form of learning.

Reviewer #1 (Public Review):

Rosas et al studied the mechanism/s that enabled carbapenems resistance of a Klebsiella isolate, FK688, which was isolated from an infected patient. To identify and characterize this mechanism, they used a combination of multiple methods. They started by sequencing the genome of this strain by a combination of short and long read sequencing. They show that Klebsiella FK688 does not encode a carbapenemase, and thus looked for other mechanisms that can explain this resistance. They discover that both DHA-1 (located on the mega-plasmid) and an inactivation of the porin OmpK36, are required for carbapenem resistance in this strain. By using experimental evolution, it was shown that resistance is lost rapidly in the absence of antibiotics selection, by a deletion in pNAR1 that removed blaDHA-1. Moreover, their results suggested that it is likely that exposure to other antibiotics selected for the acquisition of the mega-plasmid that carries DHA-1, which then enabled this strain to gain resistance to carbapenemase by a single deletion.

The major strength of this study is the use of various approaches, to tackle an important and interesting problem.

The conclusions of this paper are mostly well supported by data, but one aspect is not clear enough. The description of the evolutionary experiment is not clear. I could not find a clear description of the names of the evolved populations. However, the authors describe strains B3 and A2, but their source is not clear. The legends of the relevant figure (Figure 5) are confusing. For example, the text describing panel B is not related to the image shown in this panel. Moreover, it is shown in panel C (and written in the main text) that the OmpK36+ evolved populations had only translucent colonies, so what is the source of B3(o)?

Reviewer #1 (Public Review):

The glideosome-associated connector is an essential piece of the machinery used by the apicomplexa parasites as they invade host cells. This GAC makes important interactions with the membrane and with actin during this process. Here, Kumar et al present the first structure of the GAC from T. gondii, showing a complex fold in a closed form. This structure was determined at pH 5, and they show that at more physiological pH values the structure is far more open. However, this is not in the context of actin, membrane, or other binding partners, and so the question remains about how open the structure is in its physiological context. The authors next use molecular dynamics, NMR, and mutagenesis to identify the residues involved in membrane binding and also assess actin binding through modelling which is not validated by experiment. This paper presents an important contribution to our understanding of the molecular machinery involved in host cell invasion but leaves many questions remaining about how this protein links to the cytoskeleton and functions during the invasion process.

• The structure of TgGAC provides the first such structure of this complex and is an important contribution to our understanding. The structure presented in Figure 1A is a composite, containing the crystal structure of the majority of the protein, determined at pH 5, to which has been docked the PH domain structure, determined by NMR. It would be good to see more clarity in the figure about what is experimentally determined and what is modelled.<br /> • SAXS data shows that, at pH 8, a substantial fraction of the protein is in a very extended conformation, which differs significantly from the compact structure seen in crystals at pH 5. I would prefer to see the models in Figure 2d represented as spheres or surfaces, to prevent over-interpretation associated with showing models with low-resolution data. However, the SAXS findings are robust and this is clearly a dynamic molecule in solution. It will be interesting to see what the situation is in the context of binding partners.<br /> • Molecular dynamic simulations next indicate the region which binds to a lipid bilayer, with contact residues forming a consistent interaction surface in three independent simulations. This identified the PH domain and neighbouring residues as the membrane interaction surface.<br /> • Switching to Plasmodium falciparum protein, the authors next use NMR to investigate the binding of the PH domain to membrane nanodiscs, and show that the same protein region identified in the MD simulations was found to bind in the NMR experiments.<br /> • These membrane binding assays were then followed up through liposome pelleting assays, using TgGAP, which showed that the protein only pellets in the presence of PA lipid and that mutation of residues identified through NMR abolished liposome binding. The mutations didn't have the same effect on full-length and PH domains (noting KER for example) suggesting that lipid binding is not entirely mediated by the PH domain in the full-length protein.<br /> • The authors next put the mutants into toxoplasma and assay the effect on apical localisation and on invasion percentage. Interestingly the mutants had little effect, perhaps due to the role of other regions of the GAC on lipid binding, suggesting that abolishing PH domain lipid binding is not sufficient. Unfortunately, as the mutations only partly reduced lipid binding in the context of full-length GAC, as shown in liposome experiments, it is hard to come to a firm conclusion about the importance of lipid binding from this data as the protein used in this experiment will still have partial lipid binding properties.<br /> • The authors next investigate actin binding by TgGAC and show that most of the N-terminal half of the protein is required for this function. The authors propose, using AlphaFold2 and similarities to catenins, how GAC might bind to actin. In the absence of any validation from experimental data, caution is needed here, and I would personally not rely on the accuracy of these models.

Reviewer #1 (Public Review):

During the height of the Covid19-pandemic, there was great and widely spread concern about the lowered protection the screening programs within the cancer area could offer. Not only were programs halted for some periods because of a lack of staff or concern about the spreading of SARS CoV2. When screening activities were upheld, participation decreased, and follow-up of positive test results was delayed. Mariam El-Zein and coworkers have addressed this concern in the context of cervical screening in Canada, one of the rather few countries in the world with well organized, population-based, although regionalized, cervical screening program.

Despite the existence of screening registries, they choose to do this in form of a survey on the internet, to different professional groups within the chain of care in cervical screening and colposcopy. The reason for taking this "soft data" approach is somewhat diffuse. The authors claim they want to "capture modifications". However, the suggestions that come from this study are limited and are submitted for publication 2 years after the survey when the height of the pandemic has passed long since, and its burden on the screening program has largely disappeared. The value of the study had been larger if either the conclusions had been communicated almost directly, or if the survey had been done later, to sum up the total effect of the pandemic on the Canadian cervical screening program.

Another major problem with this study is the coverage. The results of persistent activities to get a large uptake is somewhat depressing although this is not expressed by the authors. 510 professionals filled out the survey partially or in total. 10 professions were targeted. The authors make no attempt to assess the coverage or the validity of the sample. They state the method used does not make that possible. But the number of family practicians, colposcopists, cytotechnicians, etc. involved in the program should roughly be known and the proportion of those who answered the survey could have been calculated. My guess is that it is far below 10%. Also, the national distribution seems shewed despite the authors boosting its pan-Canadian character. I am just faintly familiar with the Canadian regions, but, as an example, only 2 replies from Quebec must question the national validity of this survey.

The result section is dominated by quantitative data from the responses to the 61 questions. All questions and their answers are tabulated. As there is no way to assess the selection bias of the answers these quantitative results have no real value from an epidemiological standpoint. The replies to the open-ended questions are summarized in a table and in the text. The main conclusion of the content analysis of the answers to the direct questions, and one of the main conclusions of the study, is that the majority favors HPV self-sampling in light of the pandemic. However, this not-surprising view is taken by only 80 responders while almost as many (n=60) had no knowledge about HPV self-sampling.

The authors conclude that their study identified the need for recommendations and strategies and building resilience in the screening system. No one would dispute the need, but the additional weight this study adds, unfortunately, is low, from a scientific standpoint.

The conclusion I draw from this study is that the authors have done a good job in identifying some possible areas within the Canadian screening programs where the SARS-Cov2 pandemic had negative effects and received some support for that in a survey. Furthermore, they listed a few actions that could be taken to alleviate the vulnerability of the program in a future similar situation, and received limited support for that. No more, no less.

Reviewer #1 (Public Review):

OTOP ion channels are proton-activated, proton-permeable proteins that participate in sour tasting but for which other physiological roles are just beginning to be elucidated. The authors of this manuscript noticed that the isoform OTOP3 shows activation by protons that are potentiated in the presence of Zn2+ and other divalent ions, while other isoforms are not weakly or not at all potentiated. This allowed them to apply a chimeric approach to define which regions of the protein are responsible for the Zn2+ effect. The authors found that a single extracellular loop and a single histidine residue located in it are sufficient to explain the potentiation and propose that this histidine is part of a binding site that allosterically couples to yet undefined proton binding sites(s) responsible for proton gating.

The authors have performed very high-quality experiments and carried out a careful analysis of the data. This characterization of gating behavior of OTOP channels should be a step in elucidating physiological roles and in understanding the dynamics of these proteins. For these reasons, it should be of interest to researchers working in molecular biophysics and the physiological roles of ion channels.

Reviewer #1 (Public Review):

In this study, the authors set out to determine the degree to which early language experience affects neural representations of concepts. To do so, they use fMRI to measure responses to 90 words in adults who are deaf. One group of deaf adults (n=16) were native signers (and thus had early language exposure); a second group (n=21) was exposed to sign language later on. The groups were relatively well-matched in other respects. The primary finding was that the high dimensional representations of concepts in the left lateral anterior temporal lobe (ATL) differed between native and delayed signers, suggesting a role for early language experience in concept representation.

The analyses are carefully conducted and reflect a number of thoughtful choices. These include the "inverted MDS" method for constructing semantic RDMs, a normal hearing comparison group for both behavioral and fMRI data, and care taken to avoid bias in defining functional ROIs. And, comparing early and delayed signing groups is a clever way to study the role of early language experience on adult language representations.

One interesting result that I struggled to put in a broader context relates to the disconnect between behavioral and neural results. Specifically, the behavioral semantic RDMs (Figure 1a) did not differ between any of the groups of participants. This suggests that the representations of the 90 concepts are represented similarly in all of the participants. However, the similarity of the neural RDMs in left lateral ATL differs between the native and delayed signing groups (but not in other regions). Given the similarity of the behavioral semantic RDMs, it is unclear how to interpret the difference in left lateral ATL representations. In other words, the neural differences in left ATL do not affect behavior (semantic representation). The importance of the differences in neural RDMs is therefore questionable.

An important point is that, if I understand correctly, the semantic space is defined by the 90 experimental items. That is, behavioral RDMs were created by having normal hearing participants arrange 90 items spatially, and neural RDMs were created by comparing patterns of responses to these 90 experimental items. This 90-dimensional space is thus both (a) lower dimensional than many semantic space models that include hundreds of directions and (b) constrained by the specific 90 experimental items chosen. On the one hand, this seems to limit the generalizability of the findings for semantic representations more broadly.

The logic behind using a categorical semantic RDM (e.g., Figure 2a) was not clear. The behavioral semantic RDMs (Figure 1a) clearly show gradations in dissimilarity, particularly for the abstract categories. It would seem that using the behavioral semantic RDM would capture a more accurate representation of the semantic space than the categorical one.

Reviewer #1 (Public Review):

In this paper, the authors present a method for discovering response properties of neurons, which often have complex relationships with other experimentally measured variables, like stimuli and animal behaviors. To find these relationships, the authors fit neural data with artificial neural networks, which are chosen to have an architecture that is tractable and interpretable. To interpret the results, they examine the first- and second-order approximations of the fitted artificial neural network models. They apply their method profitably to two datasets.

The strength of this paper is in the problem it is attempting to solve: it is important for the field to develop more useful ways to analyze and understand the massive neural datasets collected with modern imaging techniques.

The weaknesses of this paper lie in its claims (1) to be model free and (2) to distinguish the method from prior methods for systems identification, including spike triggered averaging and covariance (or rather their continuous response equivalents). On the first claim, the systems identification methods are arguably substantially more model free approach. On the second claim, this reviewer would require more evidence that the presented approach is substantially different from or an improvement on systems identification methods in common use applied directly to the data.

Reviewer #1 (Public Review):

Li et al investigated the behavioral response and fMRI activations associated with deep brain stimulation (DBS) of the lateral habenula (LHb) in 2 distinct rodent models of depression. They found that a) LHb DBS reduces depressive and anxiety behaviors using multiple behavioral tests: sucrose preference, forced swim, and open field. These results held across multiple models of depression and multiple tests, and generally restored results of these behavioral tests to parity with controls. Furthermore, fMRI activations of brain regions with known connectivity to LHb strongly correlated with behavioral responses to LHb DBS, particularly in limbic regions. These behavioral responses clearly depended on electrode location, with more medial placements within the LHb producing a more robust behavioral effect.

The conclusions of this paper are generally well supported by the data, with the primary weaknesses of the study being 1) limited novelty due to LHb already being a well-established target for DBS in depression, and 2) the questionable validity of rodent models of depression in general. The authors deal with the first point (novelty) by extending their study to electrode localization and fMRI correlates with the behavioral response, leading to insight into surgical targeting as well as mechanism of effect, respectively. They also partially mitigate fundamental problems with rodent models of depression by using 2 different models and showing consistent responses to LHb DBS across both. The methods used in this study were sound, with high-quality techniques used for electrode implantation, confirmation of electrode placement, fMRI acquisition, anesthesia and physiological monitoring, as well as an appropriate statistical analytic approach.

Reviewer #1 (Public Review):

Chromosomal aneuploidy in humans causes diseases such as Down syndrome associated with changes in cognitive and metabolic activities, but how extra copies of chromosomes cause the changes remains largely unknown. In this important paper, the authors characterized the metabolisms and physiology of the transgenic mouse with most of human chromosome 21 thoroughly and nicely showed the overexpression of sarcolipin which uncouples Ca2+ import with ATP hydrolysis of sarcoplasmic reticulum Ca2+ ATPase (SERCA), which results in heat production and hyperactive mitochondria activity.

Reviewer #1 (Public Review):

This is a very interesting and timely paper and one of very few that crosses species. Linear multielectrode array recordings are rapidly becoming state-of-the-art. This means that there is a greater need for finding motifs and/or reliable markers that characterize activity in different cortical layers.

Reviewer #1 (Public Review):

This important study by Di et al., focuses on the mechanism by which potassium channels are activated prior to NLRP3 inflammasome activation. Using confocal- and electron-microscopy studies the authors demonstrate that the potassium channel, TWIK2, located in the endosomal compartment during basal conditions, is translocated onto the plasmalemma upon ATP stimulation. The authors suggest that this translocation triggers potassium efflux and subsequent NLRP3 inflammasome activation. Using Rab11a-deficient cells, the authors also show an essential role for Rab11a in this process.

This is a well written mechanistic study that has novel findings that are of interest to the inflammasome field. It addresses a long-standing question in the field, the exact mechanism by which potassium channel is activated upon treatment with NLRP3 stimuli. However, to make the conclusions more convincing, the authors should include additional stimuli such as pore-forming toxins, LPS transfection, and/or infections with bacterial pathogens to show that the Rab11a-dependent TWIK2 translocation is a universal requirement for initiation of potassium efflux by multiple stimuli and not specific to ATP. Similarly, the authors should include important controls in their inhibitor/siRNA experiments to show that the cells are still functional and the defects they observe are specific to NLRP3 inflammasome.

Reviewer #1 (Public Review):

In this paper, Liu et al. analyze a dataset of primate retinal ganglion cell responses to visual stimuli in order to find maximally informative dimensions in the inputs. They use models based on these analyses to examine features of early visual processing that influence predictive coding of visual motion in the early retina. This is an important set of questions because it remains unclear what principles drive sensory encoding and how those principles relate to circuit mechanisms found in sensory systems.

The strength in this paper lies in its rigorous analysis of the maximally informative dimensions (MIDs) of primate retinal ganglion cell signals, and the connections it makes between those dimensions and circuit models for retinal function.

The weakness of this paper lies in drawing strong connections between those analyses and predictive coding by these cells. These analyses of predictive coding are interesting but not tightly related to the MID analysis. This paper also does little to address how the structure of the stimuli affect the conclusions they draw about what circuit features contribute to predictive coding of motion.

Reviewer #1 (Public Review):

In this study, Hara and Kuraku identified the genes lost multiple times across the mammalian phylogenetic tree and termed them "elusive genes." They then investigated the features of these elusive genes in the species where they are well preserved. The authors identified several genomic features that drive gene fates toward loss, in addition to the long-presumed functional dispensability. This analysis explains why some genes are more likely to lose during evolution than others.

This study extends the selection-mutation balance theory from nucleotide substitutions to gene losses. In the context of gene losses, functional dispensability determines the selective coefficient, and the genomic features determine the rate of gene loss mutations. While the selective force has been long presumed to be important, the heterogenous genomic features that led to the mutability of gene losses were not carefully investigated in previous studies. This study fills this gap and shows that some genes are intrinsically prone to be lost (and why).

Strengths:<br /> Identification of gene losses across the phylogenetic tree is not trivial, especially when considering the incompleteness of genomes. The authors conducted their bioinformatic analyses carefully and required two independent gene loss events, each supported by multiple species in a monophyletic group. The accuracy in the identification of elusive genes provides a solid basis for the following analyses.

The authors identified genomic features associated with the gene losses in the species where the gene is preserved. This is an important strategy to avoid identifying genomic features that are formed during the gene losses but to identify the genomic features that likely formed before the gene loss. Using this strategy, the authors were able to recognize the intrinsic properties of elusive genes.

Weaknesses:

Gene expression level as a confounding factor was not well controlled throughout the study. Higher gene expression often makes genes less dispensable after gene duplication. Gene expression level is also a major determining factor of evolutionary rates (reviewed in http://www.ncbi.nlm.nih.gov/pubmed/26055156). Some proposed theories explain why gene expression level can serve as a proxy for gene importance (http://www.ncbi.nlm.nih.gov/pubmed/20884723, http://www.ncbi.nlm.nih.gov/pubmed/20485561). In that sense, many genomic/epigenomic features (such as replication timing and repressed transcriptional regulation) that were assumed "neutral" or intrinsic by the authors (or more accurately, independent of gene dispensability) cannot be easily distinguishable from the effect of gene dispersibility.

Ks was used by the authors to indicate mutation rates. However, synonymous mutations substantially affect gene expression levels (https://pubmed.ncbi.nlm.nih.gov/25768907/, https://pubmed.ncbi.nlm.nih.gov/35676473/). Thus, synonymous mutations cannot be simply assumed as neutral ones and may not be suitable for estimating local mutation rates. If introns can be aligned, they are better sequences for estimating the mutability of a genomic region.

The term "elusive gene" is not necessarily intuitive to readers.

Reviewer #1 (Public Review):

The first synapses of the pain pathway are concentrated in the superficial spinal cord dorsal horn. Here peripheral inputs are processed by local interneuron circuitry before ascending to the brain. The spinal dorsal horn is organized into lamina with the resident interneurons differentiated by their anatomy, physiological and molecular properties. Over the past decade, the restricted expression of select genes has been used to assign potential function to dorsal horn neuron "cell types". This type of work has relied on the genesis of Cre-reporter mouse strains and intersectional tools to generate mice where select sets of neurons can be activated, inhibited, or ablated. The picture that has emerged from these types of experiments is murky but favors the model where there exist genetically defined cell-types play distinct roles in the detection of painful, itchy, thermal, and mechanical stimuli under normal and pathological situations. The current work by Boyle and colleagues concerns itself with the dorsal horn neurons expressing the neuropeptide NPY. This study is directly related to previously published work that demonstrated that ablating spinal cord neurons that express Npy, including those who express this gene transiently during development, resulted in mice that had heightened touch-evoked itch that seemed different from the canonical chemical itch pathways previously identified. A major conclusion from this previous work was that other modalities were unaffected. Subsequent work built on these findings to identify the potential touch inputs and spinal neuron expressing the Npy receptor as part of a mechanical itch circuit.

This current work by Boyle and colleagues challenge challenges this view by providing evidence that in adult mice, the dorsal horn neurons expressing Npy function to broadly inhibit pain and itch. The authors use direct injection of viral vectors, chemogenetics and synaptic silencing to probe the behavioral effects of stimulating or silencing Npy-expressing dorsal horn neurons in a variety of assays under normal and pathological conditions known to produce allodynia and hyperalgesia. Overall, this is a rather carefully conducted study with the appropriate controls. The data are clear, the effect sizes robust and the presentation easy to follow. These findings challenge the conclusion that these neurons are involved selectively in mechanical itch and instead reveal a potentially clinical important group of neurons for pain.

Reviewer #1 (Public Review):

Huang C-K. and colleagues in this work address the understudied role of environmental conditions and external forces in cell extrusion as a fundamental part of epithelial homeostasis. They suggest that hydrostatic stress plays a significant role in counteracting cell extrusion forces through the indirect regulation of the focal adhesion kinase (FAK) - protein kinase B (AKT) survival pathway. The team nicely exploits their expertise in fabricating cell culture substrates to control hydrostatic stress on a common epithelial cell model from the kidney (i.e., MDCK). This was done by creating waving surfaces with different lengths from 50µm to 200 µm, thus creating a heterogenous distribution of monolayer forces towards the substrate. Finally, using a specific inhibitor for FAK, they suggest that the survivor pathway FAK-AKT is involved in the observed phenomenon.

In conclusion, the presented data underline the importance of considering external forces and tissue geometry in regulating epithelial homeostasis and the selective transport of water and solutes. These results may have a significant impact on understanding the basic mechanisms of epithelial physiology and pathology, such as in the kidney, intestine, or retina.

Reviewer #1 (Public Review): 

How morphogens spread within tissues remains an important question in developmental biology. Here the authors revisit the role of glypicans in the formation of the Dpp gradient in wing imaginal discs of Drosophila. They first use sophisticated genome engineering to demonstrate that the two glypicans of Drosophila are not equivalent despite being redundant for viability. They show that Dally is the relevant glypican for Dpp gradient formation. They then provide genetic evidence that, surprisingly, the core domain of Dally suffices to trap Dpp at the cell surface (suggesting a minor role for GAGs). They conclude with a model that Dally modulates the range of Dpp signaling by interfering with Dpp's degradation by Tkv. These are important conclusions, but more independent (biochemical/cell biological) evidence is needed.

As indicated above, the genetic evidence for the predominant role of Dally in Dpp protein/signalling gradient formation is strong. In passing, the authors could discuss why overexpressed Dlp has a negative effect on signaling, especially in the anterior compartment. The authors then move on to determine the role of GAG (=HS) chains of Dally. They find that in an overexpression assay, Dally lacking GAGs traps Dpp at the cell surface and, counterintuitively, suppresses signaling (fig 4 C, F). Both findings are unexpected and therefore require further validation and clarification, as outlined in a and b below. 

a) In loss of function experiments (dallyDeltaHS replacing endogenous dally), Dpp protein is markedly reduced (fig 4R), as much as in the KO (panel Q), suggesting that GAG chains do contribute to trapping Dpp at the cell surface. This is all the more significant that, according to the overexpression essays, DallyDeltaHS seems more stable than WT Dally (by the way, this difference should also be assessed in the knock-ins, which is possible since they are YFP-tagged). The authors acknowledge that HS chains of Dally are critical for Dpp distribution (and signaling) under physiological conditions. If this is true, one can wonder why overexpressed dally core 'binds' Dpp and whether this is a physiologically relevant activity. 

b) Although the authors' inference that dallycore (at least if overexpressed) can bind Dpp. This assertion needs independent validation by a biochemical assay, ideally with surface plasmon resonance or similar so that an affinity can be estimated. I understand that this will require a method that is outside the authors' core expertise but there is no reason why they could not approach a collaborator for such a common technique. In vitro binding data is, in my view, essential. 

In a subsequent set of experiments, the authors assess the activity of a form of Dpp that is expected not to bind GAGs (DppDeltaN). Overexpression assays show that this protein is trapped by DallyWT but not dallyDeltaHS. This is a good first step validation of the deltaN mutation, although, as before, an invitro binding assay would be preferable. Nevertheless, the authors show that DppDeltaN is surprisingly active in a knock-in strain. At face value (assuming that DeltaN fully abrogates binding to GAGs), this suggests that interaction of Dpp with the GAG chains of Dally is not required for signaling activity. This leads to authors to suggest (as shown in their final model) that GAG chains could be involved in mediating the interactions of Dally with Tkv (and not with Dpp. This is an interesting idea, which would need to be reconciled with the observation that the distribution of Dpp is affected in dallyDeltaHS knock-ins (item a above). It would also be strengthened by biochemical data (although more technically challenging than the experiments suggested above). 

In an attempt to determine the role of Dally (GAGs in particular) in the signaling gradient, the paper next addresses its relation to Tkv. They first show that reducing Tkv leads to Dpp accumulation at the cell surface, a clear indication that Tkv normally contributes to the degradation of Dpp. From this they suggest that Tkv could be required for Dpp internalisation although this is not shown directly. The authors then show that a Dpp gradient still forms upon double knockdown (Dally and Tkv). This intriguing observation shows that Dally is not strictly required for the spread of Dpp, an important conclusion that is compatible with early work by Lander suggesting that Dpp spreads by free diffusion. These result show that Dally is required for gradient formation only when Tkv is present. They suggest therefore that Dally prevents Tkv-mediated internalisation of Dpp. Although this is a reasonable inference, internalisation assays (e.g. with anti-Ollas or anti-HA Ab) would strengthen the authors' conclusions especially because they contradict a recent paper from the Gonzalez-Gaitan lab. 

The paper ends with a model suggesting that HS chains have a dual function of suppressing Tkv internalisation and stimulating signaling. This constitutes a novel view of a glypican's mode of action and possibly an important contribution of this paper. As indicated above, further experiments could considerably strengthen the conclusion. Speculation on how the authors imagine that GAG chains have these activities would also be warranted.

Reviewer #1 (Public Review):

This study by Cao et al. demonstrates role of Neutrophil in clearing apoptotic hepatocytes by directly burrowing into the apoptotic hepatocytes and ingesting the effete cells from inside without causing inflammation. The authors applied intravital microscopy, Immunostaining and electron microscopy to visualize perforocytosis of neutrophil in hepatocytes. They also found that neutrophil depletion impairs the clearance of apoptotic hepatocytes causing impaired liver function and generation of autoantibodies, implying a role of defective neutrophil- mediated clearance of apoptotic cells in Autoimmune Liver disease. The experiments were well designed and conducted, the results were reasonably interpreted, and the manuscript was clearly written with logical inputs.

One weak point is that the signals/mechanisms that determine why neutrophil specifically target apoptotic hepatocytes in liver and no other organs or cells is not clearly understood.

Reviewer #1 (Public Review):

In this manuscript, Mastrototaro et al. perform a series of experiments in transgenic murine models assessing the function of Palladin (PALLD) in the heart. Global PALLD KOs are embryonic lethal, precluding the assessment of the roles of this protein in adulthood. To circumvent this limitation, the authors generated a floxed Palld allele and ablated it with two cardiomyocyte-specific Cres: the constitutively active Myh6-Cre and the tamoxifen-inducible aMHC-MerCreMer. Interestingly, ablation with the constitutive Cre (cKO) did not produce any overt phenotype, but ablation in adulthood (cKOi) resulted in compromised cardiac function. These observations suggest a compensation mechanism that takes place when cardiomyocytes develop in the complete absence of this protein but not when cardiomyocytes develop in a wild-type background and are deprived of this protein after achieving full maturation. These experiments were complemented with yeast two-hybrid techniques to identify novel partners that bind to a region of PALLD for each no interactants had been previously identified. Experiments in human samples revealed an upregulation of PALLD transcripts in the hearts of patients.

This manuscript adds important information to our understanding of sarcomeric proteins. Data are generally of good quality and well presented in figures. The numbers of animals in echocardiographic studies are also adequate for proper conclusions. Authors achieve most of their goals, including the identification of novel partners of PALLD and the identification of a requirement for PALLD in cardiomyocytes for normal heart function. However, given that all experiments performed in this study were focused on the loss-of-function of PALLD, it is not clear what is the relevance of the PALLD upregulation observed in human patients. Authors should clearly state this limitation in their results.

Considering that authors have observed evidence for nuclear PALLD, which could hint at potential major gene expression changes when this protein is ablated, it would be interesting to perform an unbiased assessment of transcriptional alterations (RNA-seq) in cardiomyocytes isolated from control and cKOi hearts. In addition, to test if the compensation observed in the embryonic cKO involves mechanisms of transcriptional adaptation, it would be interesting to compare RNA-seq results from cKOi and cKO (genes encoding proteins similar to PALLD that are upregulated in cKO but not cKOi cardiomyocytes would be very strong candidates). However, these transcriptomic data are not essential to support current findings and can be performed in follow-up studies.

Reviewer #1 (Public Review):

The authors set out to analyse the pattern of movement of T cells in different tissues- lymph nodes, villi, and inflamed/infected lungs. The authors are comparing data sets from multiple sites in different studies but acquired using similar instruments, preparations, and imaging conditions.

The more confined movement pattern in the lung that has a turning angle distribution with more incidence of angles near 180 degrees is striking.

T cells in the infected inflamed lung search a smaller volume over time but will explore it more extensively.

The measurements of T cell movement are context-free such that obstacles and tissue boundaries that could account for some of the confined behaviours in the lung parenchyma are not discussed.

Nonetheless, the work will motivate further study of the biological significance of the different T cell movement patterns in the lung, which may also be considered in the context of recent data on changes in B cell motility- a potential interacting cell.

Reviewer #1 (Public Review):

This study presents a resource aiming to unify language and rules used in the literature to describe, curate and assess biology experiments, published or not. Focusing on host-pathogen interactions, the work presents a new ontology and controlled vocabulary, as well as rules to describe 'metagenotypes', a term coined for the joint description of interacting host-pathogen genotypes. 'PHI-Canto' extends a previous resource by also enabling using UniProtKB IDs to curate proteins. Among other important by-products, PHI-Canto could contribute to damping proliferating names and acronyms for genes, processes, and interactions; a chronic annoyance in the biosciences.

The tool does give the impression that, with sufficient time and usage, it could become a rich and robust resource. Just addressing the Uniprot IDs issue is a nice move.

Reviewer #1 (Public Review):

Motivated by the premise that Alzheimer's disease (ADD) and major depressive disorder (MDD) have shared underlying environmental and genetic risk factors, Petrican and Fornito combine non-imaging risk factors and executive task-based functional network change indices into latent variables of resilience to AD and MDD. The authors find two latent variables (LVs): LV1 represents change in network membership over time of distributed nodes during task, which is associated with greater genetic MDD risk, less psychopathology, and more advanced puberty, all while adjusting for age and indices of environmental stressors. LV2 represents occipital lobe nodal flexibility across task and time, decreased AD genetic risk, increased MDD genetic risk and less psychopathology, again adjusted for age and environmental stressors. The authors validate the latent network variables by assessing their overlap with genes for which SNPs have been associated with both depression risk and change in gene expression. Finally, the authors create simple path models in order to break down the relationships between genetic risk, latent variables, and what the authors term "resilience", finding distinct path for MDD and (non-APOE) AD genetic risk. All of these analyses are then re-run using a different brain parcellation. LV2 replicates, while a new LV1 emerges with similar non-imaging variables now being correlated with a different set of distributed network nodes.

The authors conclude from this work that they have identified imaging indices of resilience manifest during adolescent brain development, and that they have found further evidence linking MDD to AD. However, the analyses do not fully support the conclusions. The premise of this work - to examine links between MDD and AD and to try to define indices of resilience during development - is fascinating and will hopefully motivate future work in this direction. However, the impact of this work as currently presented may be limited.

*STUDY STRENGTHS*

There are two premises motivating this study that deserve praise for their innovation and creativity. First, in the introduction the authors present several fairly new papers showing shared environmental and risk factors between AD and MDD. This is a very interesting line of study that certainly deserves more attention. Second, the authors are interested in finding aspects of adolescent brain development that may be helpful to understanding resilience to genetic or environmental risk later in life. The AD resilience community is very interested in contributions of early life experiences and development, but there is still very little research in this domain. I hope the authors continue to conduct research in the direction of these pursuits.

The authors demonstrate great methodological and statistical rigor in some aspects of data preprocessing and analysis. This is particularly salient in null modeling and permutation, graph-based analysis, treatment of motion for functional imaging, using eQTLs to inform disease-relevant genes, statistical considerations in PLS and path modeling, processing of Allen Brain Atlas gene expression data, and validating certain study variables. The methodology of these steps displays great attention to detail and a mastery of certain data types.

The authors reproduce all analyses using a second parcellation and carefully report the results. This type of painstaking analysis is nonetheless important in the context of network-based graph analysis that is reliant on nodal information.

*STUDY LIMITATIONS*

1) The overarching limitation of this study is that the study variables, both independent and dependent, are abstracted to the point where interpretations are challenging. The authors' own interpretations are not sufficiently justified and are often taken at face value rather than supported by analysis. These are further combined into latent variables with weak conceptual foundation, which are then abstracted even further to other analyses with cortical molecular data maps. It is not clear that the conclusions drawn are convincingly supported by this highly abstracted analysis.

2) The other major limitation of this study is that several PLS models are run but, while appropriate null modeling is used to identify "significant" LVs, none of the LVs are cross-validated. Null modeling can help to protect against overfitting to noise in data, but it does not necessarily provide a good index of generalizability nor reliability. Without cross-validation, I question the reliability of the LVs irrespective of how they are interpreted. This is once again partially driven by the fact that changing the atlas resulted in a different imaging LV.

3) The study notes that participants were selected based on "having contributed high-quality data on all measures of interest". This is of course meritorious from a methodological perspective, but the authors should be aware that this may create an important selection bias (10.1007/s11682-022-00665-2, 10.1016/j.ynirp.2022.100085, 10.1016/j.neuroimage.2022.119296)

4) The premise of this paper was interesting, as described in the Strengths section above. However, what was missing was a clear theory or hypothesis as to how resilience to AD and MDD are related, and how the analyses in this study were conducted in order to support that hypothesis. The relevance of the results to AD was not clear; a clear biological model would help put the pieces together.

5) The selection of relevant features involved in LVs was inconsistent. At several points, the authors use an arbitrary threshold of bootstrap ratio (BSR) > 4, which they equated to a p-value. A p-value doesn't make sense in this context, since bootstrap samples are not independent samples. Instead, features should be selected based on 95% CIs that don't cross 0, which the authors do in some places but not in others.

Reviewer #1 (Public Review):

The present study combines quantitative histomorphometry, live cell imaging and tracking, functional analyses, and computational modeling to define potentially pathologic interactions between lung CD8 T cells and fibrocytes in human COPD. The authors use multiple technical approaches to establish the close proximity of CD8 T cells with fibrocytes in peri-bronchial tissue in COPD subjects that notably correlate with functional disease parameters (FEV1/FEV). Their follow-on studies identify specific chemokine pathways and inflammatory consequences of these interactions. Collectively, these seminal data acquired in a unified experimental context, provide support for pathogenic interactions between lung CD8 T cells and fibrocytes and now offer the consideration of mediators and pathways that may be amenable to therapeutic targeting. The strength of the study is the integration of the multi-modality approach, the quality of the quantitative data, and the creation of a tenable model for the interaction role in COPD of CD8 T cells and fibrocytes. While both have been previously implicated in COPD, this new study is more definitive by using this integrated approach.

Reviewer #1 (Public Review):

While the mechanism about arm-races between plant and specialist herbivores has been studied, such as detoxification of specific secondary metabolites, the mechanism of the wider diet breadth, so-called generalist herbivores have been less studied. Since the heterogeneity of host plant species, the experimental validation of phylogenetic generalism of herbivores seemed as hard to be conducted. The authors declared the two major hypotheses about the large diet breadth ("metabolic generalism" and "multi-host metabolic specialism"), and carefully designed the experiment using Drosophila suzukii as a model herbivore species.

By an untargeted metabolomics approach using UHPLC-MS, authors attempted to falsify the hypotheses both in qualitative- and quantitative metabolomic profiles. Intersections of four fruit (puree) samples and each diet-based fly individual samples from the qualitative data revealed that there were few ions that occur as the specific metabolite in each diet-based fly group, which could reject the "multi-host metabolic specialism" hypothesis. Quantitative data also showed results that could support the "metabolic generalism" hypothesis. Therefore, the wide diet breadth of D. suzukii seemed to be derived from the general metabolism rather than the adaptive traits of the diverse host plant species. On the other hand, the reduction of the metabolites (ions) set using GLM seemed logical and 2-D clustering from the reduced ions set showed that quantitative aspects of diet-associated ions could classify "what the flies ate". These interesting results could enhance the understanding of the diet breadth (niche) of herbivorous insects.

The authors' approach seemed clear to falsify the hypotheses based on the appropriate data processing. The intersection of shared ions from the qualitative dataset could distinguish the diet-specific metabolites in flies and commonly occurring metabolites among flies and/or fruits. Also, filtering on the diet-specific ions seemed to be a logical and appropriate way. Meanwhile, the discussion about the results seemed to be focused on different points regarding the research hypotheses which were raised in the introduction part. Discussion about the results mainly focused on the metabolism of D. suzukii itself, rather than the research hypotheses and questions that were raised from the evolution of the wide diet breadth of generalist herbivores. In particular, the conclusion seems to be far from the main context of the authors' research; e.g. frugivory. It makes the implication of the study weaker.

Reviewer #1 (Public Review):

Much experimental work on understanding how the visual system processes optic flow during navigation has involved the use of artificial visual stimuli that do not recapitulate the complexity of optic flow patterns generated by actual walking through a natural environment. The paper by Muller and colleagues aims to carefully document "retinal" optic flow patterns generated by human participants walking a straight path in real terrains that differ in "smoothness". By doing so, they gain unique insights into an aspect of natural behavior that should move the field forward and allow for the development of new, more principled, computational models that may better explain the visual processing taking place during walking in humans.

Strengths:<br /> Appropriate, state-of-the-art technology was used to obtain a simultaneous assessment of eye movements, head movements, and gait, together with an analysis of the scene, so as to estimate retinal motion maps across the central 90 deg of the visual field. This allowed the team to show that walkers stabilize gaze, causing low velocities to be concentrated around the fovea and faster velocities at the visual periphery (albeit more the periphery of the camera used than the actual visual field). The study concluded that the pattern of optic flow observed around the visual field was most likely related to the translation of the eye and body in space, and the rotations and counter-rotations this entailed to maintain stability. The authors were able to specify what aspects of the retinal motion flow pattern were impacted by terrain roughness, and why (concentration of gaze closer to the body, to control foot placement), and to differentiate this from the impact of lateral eye movements. They were also able to identify generalizable aspects of the pattern of retinal flow across terrains by subsampling identical behaviors in different conditions.

Weaknesses:<br /> While the study has much to commend, it could benefit from additional methodological information about the computations performed to generate the data shown. In addition, an estimation of inter-individual variability, and the role of sex, age, and optical correction would increase our understanding of factors that could impact these results, thus providing a clearer estimate of how generalizable they are outside the confines of the present experiments.

Reviewer #1 (Public Review):

Habituation to noxious insults is a conserved mechanism that may act through varying pain-sensitivity thresholds based on previous sensory experience. Impaired regulation of nociceptive habituation may lead to a chronic pain condition. In the current manuscript, the authors identified additional structural elements of the CaM kinase-1 that regulate the protein shuttling between the cytosol and nucleus during nociceptive habituation. Based on the presented findings, we get a more complex regulatory model and a better understanding of the CMK-1 protein redistribution during stimulation-dependent nociceptive plasticity.

The data is carefully planned and results conclusively support the claims of the authors. The performed experiments are easy to follow and the results obtained are robust and statistically well-powered. The complex regulatory model presented in the manuscript is well supported by the reported data. Finally, the presented data presents a complex and dynamic mechanism of nuclear import and export rates of the CMK-1 protein to control nociceptive plasticity.

Reviewer #1 (Public Review):

In this work, the authors propose a phenomenological grounded theoretical framework to explain why microbial taxonomic richness can show positive, unimodal, as well as negative diversity-temperature gradients. They thus propose to introduce a temperature dependence in the form of the Boltzmann-Arrhenius equation in both species' competitive interaction and growth rates. By means of a mean-field-like approximation, they estimate the probability of having N feasible coexisting species as a function of the normalized growth rate, and average competition strength, which in turn depends on temperature. They find that the shape of the microbial community temperature-richness relationship depends on how rapidly the strength of competition between species pairs increases with temperature relative to an increase in the variance of their growth rates. Furthermore, the mean-field result predicts that the position of richness peak depends on the sign of the covariance between the two main parameters of the Boltzmann-Arrhenius law. Finally, they show that the real-world community-level temperature-richness responses observed are qualitatively reproduced by their model.

I found the work interesting and stimulating, surely tackling a relevant research question such as the effect of thermal physiology on biodiversity patterns through a simple, but quantitative model. Overall, I like the proposed approach.

At the same time, the central mathematical results are not clear in my view, some strong approximations are not discussed, but they hold only in very specific conditions. A lot of important details are missing or scattered here and there, the notation is a little sloppy, and in general, it has been difficult for me to reproduce their finding.

The overall structure and flow of the manuscript can be remarkably improved.

Reviewer #1 (Public Review):

The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion within the first intron of the C9ORF72 gene. However, how this repeat contributes to disease pathology is still an active area of research. This study takes a targeted approach to analyzing specifically how the C9ORF72 antisense transcript (C4G2) may be contributing to FTD/ALS.

Using an artificial (C4G2)75 antisense cassette, the authors show in both HEK293T cells and cultured neurons that the C4G2 antisense transcript leads to elevated levels of activated PKR and increased phosphorylated eIF2alpha. This then leads to a decreased level of translation, the formation of stress granules, and decreased survival, phenotypes that can be suppressed through the knockdown of PKR. The authors nicely demonstrate that PKR activation upon transfection with their antisense cassette is independent of toxic dipeptide repeat proteins by using reporter constructs that do not create these dipeptides but are still able to activate PKR. Furthermore, using a construct that expresses both sense and antisense transcripts, the authors show that knockdown of the antisense, but not the sense transcript, abrogates the PKR response (demonstrating the specificity of this stress pathway for the antisense RNA). The authors additionally show the relevance of PKR activation in FTD/ALS through the presence of activated PKR and elevated eIF2alpha in ALS postmortem brain tissue.

This paper shows that, at least in model systems, the C4G2 transcript can have cytotoxic effects through the stimulation of PKR. The experiments are well-controlled and fairly comprehensive. The claim that PKR activation occurs via the antisense RNA, and not the sense, is well supported by the data. However, some limitations exist, some of which the authors explicitly recognize. They are as follows:<br /> 1. It is not clear how the results from these reporter constructs inform on the repeat expansion RNAs produced in disease, which can be significantly longer, and might be expressed at different levels. Perhaps if the C4G2 repeat used in this work were expressed at levels comparable to what the antisense transcript is expressed in an actual disease, or in a similar RNA context, PKR would not be activated. This is important to keep in mind.<br /> 2. It is still unclear how PKR is being activated in the presence of C4G2 (it could be direct or indirect). The authors list a variety of explanations in the discussion. A prior study has shown that a similar repeat expansion leads to the accumulation of cytoplasmic dsRNA inclusions marked by TDP-43 (Rodriguez et al., 2021). It would be interesting to see if these inclusions are present upon expression of the antisense construct.<br /> 3. In the context of C9ORF72 FTD/ALS disease, it is still difficult to say how much of the disease pathology is on account of antisense triggered stress responses as opposed to dipeptide repeat, RBP titration, etc. This study nevertheless provides a new perspective to consider for how the C9ORF72 repeat expansion contributes to the diseased state.

Reviewer #1 (Public Review):

In the manuscript "Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB", Sacco et. al. solved the crystal structure of S. aureus GpsB, an essential cell growth and division protein. The authors also identified its interactions with the master regulator of cell division FtsZ and a penicillin-binding protein PBP4 that is implicated in B-lactam insensitivity. Although GpsB is essential for growth in S. aureus the reason for its essentiality is poorly understood. The authors used biochemical, biophysical, and crystallographic methods to determine the structure of GpsB and characterized its binding with FtsZ and PBP4. The authors also solved the co-crystal structure of GpsB with the C-terminal peptide of PBP4. These results are significant because it details the interactions of an essential growth protein in S. aureus with known cell division proteins. However, the impact of the work could be further enhanced if the authors had more functional studies to demonstrate the importance of the new hinge motif, the binding with FtsZ C-terminal tail, and PBP4.

Reviewer #1 (Public Review):

The authors present a study to test the relationships between a measured dopamine marker in the brain - so-called, dopamine synthesis capacity - and various other measures purported to index dopamine function. These measures include questionnaire answers about behaviour, and measured behaviour. Various studies have used these other measures as indices or proxies of dopamine function with some evidence to support this. However, some of the evidence is in small groups or indirect.

The major strength of this study is the size of the sample (n=66-94) compared to other studies and the three different analytical strategies employed - frequentist, Bayesian, and predictive modelling.

Areas, where the study is more limited, are the use of only one marker of dopamine neurochemistry ([18F]FDOPA) and this does not discount relationships with other markers such as pre-synaptic receptors, post-synaptic receptors, and dynamic release. The authors acknowledge that this study does not speak to the general principle of dopamine relationships with other measures. While the numbers are impressive for this type of study the use of correlation means their power is for correlations of 0.32-0.37 and higher (G*power). It is possible genuine relationships between markers do exist but all studies to date, including this one, are underpowered. The Bayesian analysis conducted speaks to this and is a welcome addition. It is also possible that the conclusions are restricted by the participants recruited as they are limited to the ages of 18-43 and it is not clear how representative they are of the general community from the information provided.

The dopamine system is not one entity in terms of system components (pre-synaptic, post-synaptic, etc), but also in terms of subcortical area with a gradient of input from the brainstem and a distinct connectionist anatomy between the striatum and the cortex (via other structures). Here the authors use a segmentation of the striatum to test the relationships. While this is embedded in the methods and results the introduction's treatment of the subcortical dopamine system is as a single entity. This could be improved.

The results of this work have an important impact in that they strongly suggest one cannot use proxies to estimate endogenous neurochemistry (at least in the dopamine system). However, this implies that any other proxy for any other system needs to be (re-)assessed using similar methods. This is not to say that the proxies are not sensitive to dopamine manipulations, but that they cannot by themselves be used instead of direct measurement. Given the number of studies which suggest that a measure of baseline state may predict the effects of dopaminergic drugs, one must question what the baseline state is being measured.

Despite these limitations, the authors have provided the largest assessment of the relationships between [18F] FDOPA-assessed dopamine synthesis capacity and various markers previously linked to dopamine function. In this respect, it is an important negative. This does mean that the assessments used cannot be used to assess 'baseline' states in relation to dopaminergic drug effects, but the mechanism through which this baseline dependency operates is not well understood.

Reviewer #1 (Public Review):

The manuscript by Lujan and colleagues describes a series of cellular phenotypes associated with the depletion of TANGO2, a poorly characterized gene product but relevant to neurological and muscular disorders. The authors report that TANGO2 associates with membrane-bound organelles, mainly mitochondria, impacting in lipid metabolism and the accumulation of reactive-oxygen species. Based on these observations the authors speculate that TANGO2 function in Acyl-CoA metabolism.

The observations are generally convincing and most of the conclusions appear logical. While the function of TANGO2 remains unclear, the finding that it interferes with lipid metabolism is novel and important. This observation was not developed to a great extent and based on the data presented, the link between TANGO2 and acyl-CoA, as proposed by the authors, appears rather speculative.

1. The data with overexpressed TANGO2 looks convincing but I wonder if the authors analyzed the localization of endogenous TANGO2 by immunofluorescence using the antibody described in Figure S2. The idea that TANGO2 localizes to membrane contact sites between mitochondria and the ER and LDs would also be strengthened by experiments including multiple organelle markers.

2. The changes in LD size in TANGO2-depleted cells are very interesting and consistent with the role of TANGO2 in lipid metabolism. From the lipidomics analysis, it seems that the relative levels of the main neutral lipids in TANGO2-depleted cells remain unaltered (TAG) or even decrease (CE). Therefore, it would be interesting to explore further the increase in LD size for example analyze/display the absolute levels of neutral lipids in the various conditions.

3. Most of the lipidomics changes in TANGO2-depleted cells are observed in lipid species present in very low amounts while the relative abundance of major phospholipids (PC, PE PI) remains mostly unchanged. It would be good to also display the absolute levels of the various lipids analyzed. This is an important point to clarify as it would be unlikely that these major phospholipids are unaffected by an overall defect in Acyl-CoA metabolism, as proposed by the authors.

Reviewer #1 (Public Review):

The manuscript provides a comprehensive analysis of the consequences of a mutation in WDR62 in human pluripotent stem cell-derived progenitor cells and neurons. The experiments are logical and presented well. The data support the conclusion that WDR62 dysfunction causes impaired cell cycle progression and defective neuronal differentiation. The data corroborate previous findings in mouse and human cells and cell lines and extend knowledge to cells that are relevant to the microcephaly characteristic of individuals with WDR62 mutations. The major shortcoming of the data is that it relies on cells from a single donor and so requires additional validation to support the generalization of the conclusions. In addition, limited mechanistic insight is provided.

Reviewer #1 (Public Review):

The authors attempted to delete a rhodopsin allele with single-nucleotide mutation seen in a Chinese subpopulation of autosomal dominant retinitis pigmentosa patients, (Rho-T17M). This was done in vitro and in vivo, while keeping the Rho wild type allele intact in vitro and in vivo using CRISPR-SaCAS9 guide RNA-specific approach, a previously established technique. In this study, solid in vitro data was presented showing that one of the tested guide RNAs was effective to specifically delete targeted the Rho-T17M sequence of synthetic DNA as well as in iPSCs from RP patients. However, the in vivo part of this study is incomplete. The issues are: 1. confusing choice of disease animal model (Rho-5m mice that carry 4 additional rhodopsin mutations other than the targeted T17M); 2. no proof of gene editing efficiency at the cellular level of the targeted cell type (i.e. what percentage of rod photoreceptors lose the T17M disease mutation?); and 3) lack of evidence of therapeutic potential (i.e. is there any rescue of vision in the mouse disease model or any toxicity due to the vector itself?).

Reviewer #1 (Public Review):

According to current knowledge, zebrafish neurons maintain the capacity of regenerating with the exception of adult cerebellar Purkinje cells (PC), which are thought to have lost this property. Regeneration instead occurs at larval stages but whether newly generated PC form fully functional circuits is still unclear. This elegant and well-performed study takes advantage of a transgenic zebrafish line that enables inducing apoptosis under a tamoxifen-inducible system and at the same time visualizes PCs morphology through a membrane tagged RFP. Using this line (and other lines that tag radial glial and ventricular progenitors) in combination with morphological and functional analysis, the authors show that ventricular progenitors retain the lifelong ability to regenerate PCs. At larval stages, the newly regenerated PCs form fully functional circuits that lead to normal behavior. In adults, PC regeneration is less efficient (and PCs are also less prone to undergo apoptosis) but sufficient to support exploratory behavior. This study resolves the controversial issue of whether adult PC regeneration is possible and demonstrates that newly formed PCs at larval and adult stages can form functional circuits that support normal behavior.

This is a well-performed and carefully executed and quantified study. There is however a point that needs clarification:

The authors state that acute regeneration occurs between 5-10dpt. However, the graphs in Fig 1D, F, and 2F indicate that most PC generation occurs from 20-30 days. What happens in this period? Does proliferation increase? Can the authors perform BrdU incorporation between 6 days and 1 month? Related to this, as the authors indicate in lines 129-131, the regeneration of new PCs overlaps with normal development. Are other neuronal cell types generated in appropriate numbers?

Reviewer #1 (Public Review):

In this manuscript, Gonzalez et al investigated the dynamics of dopamine signals, measured with optophysiological methods in the lateral shell of the nucleus accumbens (LNAc), in response to different types of visual stimuli. Contrary to most current theories of dopamine signaling, the authors found that LNAcc dopamine transients tracked sensory transitions in visual stimulation rather than any immediately apparent motivational variable. This unorthodox finding is of potential interest to the field, as it suggests that dopamine in this particular area of the striatum supports a very different, albeit unclear behavioral function than what has been previously attributed to this neuromodulator. Many of the approaches used by the authors were very elegant, like the careful selection of visual stimuli parameters and the use of Gnat1/2 KO mice to demonstrate that the dopamine responses were directly dependent on the visual stimulation of rods and cones. That said, the authors did not discuss how their findings relate to much previously published work, many of which offer potential alternative explanations for their results. It is also not clear from the manuscript text which mice were used for which experiments, and how testing history might affect the results.

Reviewer #1 (Public Review):

The endothelin ETB receptor is a G-protein coupled receptor activated by vasoactive peptide endothelins, causing vaslorelaxtion in smooth muscle. By determining the Cryo EM structure of human ETB in complex with the vasoconstricting peptide ET-1 and the inhibitory G-protein (Gi), the study represents a convincing insight into agonist-induced receptor activation and transducer-coupling. The complex structure is solid and will appeal to the GPCR and pharmacology communities.

Strengthens: The authors have managed to obtain the first G-protein complex structure of an ETB receptor by working with a receptor that still retains G-protein coupling (i.e. not a thermostabilized mutant) and by developing new methodologies into how the G-protein is remotely tethered to the GPCR. The Cryo EM structural details highlight clear differences into how the G-protein binds that also includes the more downward movement of TM7.

Weaknesses: While it is technically challenging to obtain an endothelin-1-ETB-Gi complex, the fusion approach means that there is equilibrium is already pushed towards a complex that may otherwise require lipids, such as PIP2. Whilst I don't know what may alter how alpha 5 interacts with ETB, this cannot be ruled out either.

Reviewer #1 (Public Review):

This manuscript describes efforts to understand how independence from ribonucleotide reduction might evolve in obligate intracellular bacterial pathogens using E. coli as a model for this process. The authors successfully deleted the three ribonucleotide reductase (RNR) operons present in E. coli and showed that growth of this knockout strain can be achieved with deoxyribonucleotide supplementation. They also performed evolutionary experiments and analysis of cell growth and morphology under conditions of low nucleotide availability. In this work, they established that certain genes are consistently mutated to compensate for the loss of RNR activity and the low availability of deoxynucleotides. Comparison to genomes of intracellular pathogens that lack RNR genes shows that these patterns are largely conserved.

While the experimental results support the conclusions of the study, the authors do report changes in cell morphology upon the growth of the RNR knockout strains with low concentrations of nucleotides. It would be ideal to note this complication earlier in the manuscript. And to clarify how the possibility of cell elongation might affect the OD measurements in Figure 3 describing the experiments to establish that dC is necessary for growth in the knockout strain. It would also be ideal to provide a more detailed explanation for that observation in the discussion.

Reviewer #1 (Public Review):

This work presents a unification model (of sorts) for explaining how the flow of evidence through networks can be controlled during decision-making. The authors combine two general frameworks previously used as neural models of cortical decision-making, dynamic normalization (that implement value encoding via firing activity) and recurrent network models (which capture winner-take-all selection processes) into a unified model called the local disinhibition-based decision model (LDDM). The simple motif of the LDDM allows for the disinhibition of excitatory cells that represent the engagement of individual actions that happens through a recurrent inhibitory loop (i.e., a leaky competing accumulator). The authors show how the LDDM works effectively well at explaining both decision dynamics and the properties of cortical cells during perceptual decision-making tasks.

All in all, I thought this was an interesting study with an ambitious goal. But like any good study, there are some open issues worth noting and correcting.

MAJOR CONCERNS

1. Big picture

This was a comprehensive and extremely well-vetted set of theoretical experiments. However, the scope and complexity also made the take-home message hard to discern. The abstract and most of the introduction focus on the framing of LDDM as a hybrid of dynamic normalization models (DNM) and recurrent network models (RNMs). This is sold as a unification of value normalization and selection into a novel unified framework. Then the focus shifts to the role of disinhibition in decision-making. Then in the Discussion, the goal is stated as to determine whether the LDDM generates persistent activity and does this activity differ from RNMs. As a reader, it seems like the paper jumps between two high-level goals: 1) the unification of DNM and RNM architectures, and 2) the role of disinhibition. This constant changing makes it hard to focus as the reader goes on. So what is the big picture goal specifically?

Also, the framing of value normalization and WTA as a novel computational goal is a bit odd as this is a major focus of the field of reinforcement learning (both abstractly at the computational level and more concretely in models of the circuits that regulate it). I know that the authors do not think they are the first to unify value judgements with selection criteria. The writing just comes across that way and should be clarified.

2. Link to other models

The LDDM is described as a novel unification of value normalization and winner-take-all (WTA) selection, combining value processing and selection. While the authors do an excellent job of referencing a significant chunk of the decision neuroscience literature (160 references!) the motif they end up designing has a highly similar structure to a well-known neural circuit linked to decision-making: the cortico-basal ganglia pathways. Extensive work over the past 20+ years has highlighted how cortical-basal ganglia loops work via disinhibition of cortical decision units in a similar way as the LDDM (see the work by Michael Frank, Wei Wei, Jonathan Rubin, Fred Hamker, Rafal Bogacz, and many others). It was surprising to not see this link brought up in the paper as most of the framing was on the possibility of the LDDM representing cortical motifs, yet as far as I know, there does not exist evidence for such architectures in the cortex, but there is in these cortical-basal ganglia systems.

3. Model evaluations

The authors do a great job of extensively probing the LDDM under different conditions and against some empirical data. However, most of the time there is no "control" model or current state-of-the-art model that the LDDM is being compared against. In a few of the simulation experiments, the LDDM is compared against the DNM and RNM alone, so as to show how the two components of the LDDM motif compare against the holistic model itself. But this component model comparison is inconsistently used across simulation experiments.

Also, it is worth asking whether the DNM and RNM are appropriate comparison models to vet the LDDM against for two reasons. First, these are the components of the full LDDM. So these tests show us how the two underlying architectural systems that go into LDDM perform independently, but not necessarily how the LDDM compares against other architectures without these features. Second, as pointed out in my previous comment, the LDDM is a more complex model, with more parameters, than either the DNM or RNM. The field of decision neuroscience is awash in competing decision models (including probabilistic attractor models, non-recurrent integrators, etc.). If we really want to understand the utility of the LDDM, it would be good to know how it performs against similarly complex models, as opposed to its two underlying component models.

4. Comparison to physiological data

I quite enjoyed the comparisons of the excitatory cell activity to empirical data from the Shadlen lab experiments. However, these were largely qualitative in nature. In conjunction with my prior point on the models that the LDDM is being compared against, it would be ideal to have a direct measure of model fits that can be used to compare the performance of different competing "control" models. These measures would have to account for differences in model complexity (e.g., AIC or BIC), but such an analysis would help the reader understand the utility of the LDDM in connecting with empirical data much better.

Reviewer #1 (Public Review):<br /> <br /> The pH-dependent conformational change of the envelope protein in flaviviruses is required for the infection process, thus it represents an attractive target for drug development. In this study, the authors conducted extensive atomistic simulations for models for the envelope in six flaviviruses. Using a benzene-mapping approach, they were able to identify several cryptic binding sites that can be targeted for drug development. One of the cryptic binding site was observed in a previous study to be occupied by a detergent molecule, while the other cryptic binding site is located at domain interface. The second binding site involves a cluster of ionizable residues. Using constant pH simulations, the authors suggested that the cluster of ionizable residues contribute to the pH dependent conformational rearrangements. This cluster model helps to explain the inconsistencies reported in the literature regarding the role of several key histidine residues as pH sensors. Overall, the study has provided new mechanistic insights that can be taken advantage of in future drug developments that target flaviviruses. The work also highlights the importance of constant pH simulations to the analysis of pH sensitive biological processes.

Reviewer #1 (Public Review):

This work introduces a new computational model of healthy blood cell formation and chronic myeloid leukemia (CML). By combining data from the literature, animal experiments and patients the authors aim to develop a detailed description of the regulatory mechanisms governing healthy blood cell formation and CML therapy response. The model is used to derive hypotheses explaining why some patients respond to tyrosine kinase inhibitors (TKI) better than others. Based on the model simulations the authors seek predictors of TKI efficacy and for concepts to improve CML therapy.

Strengths:

(1) The authors start from all possible ordinary differential equation models which describe positive and negative regulations of proliferation rates and self-renewal/differentiation probabilities. The models account for hematopoietic stem cells, multipotent progenitors, terminally differentiated myeloid cells, and terminally differentiated lymphoid cells. Using an automated approach referred to as design space analysis (DSA) the authors exclude models with unfeasible qualitative dynamics. Using data from mouse experiments the authors exclude all regulatory configurations except one. This systematic approach combining model analysis and data from various sources is clearly a strength of the work.

(2) The authors consider a large number of parameter sets that are in line with physiological steady-state cell counts and realistic responses to system perturbations. Thus the authors can potentially account for inter-patient differences.

(3) The model predictions are compared to experimental and published data. The proposed predictors of TKI efficacy are tested on retrospective patient data.

Weaknesses:

(1) In my opinion the sub-model of leukemic cells requires a more solid justification. The authors assume that the configuration of regulatory loops and most key parameters are identical for normal and leukemic cells. The only difference the proposed model accounts for is that leukemic cells exhibit a weaker response to the feedback signal acting on stem cell self-renewal. The weaker response of leukemic stem cells is justified by data from the literature supporting differential responses to CCL3. However, the authors propose no justification for the assumption that all other parameters, such as proliferation rates or maximal self-renewal probabilities, are identical or have minor impacts.

(2) The authors come to the conclusion that "a key predictor of refractory response to TKI treatment is an increased probability of self-renewal of normal hematopoietic stem cells" (Abstract). This conclusion is, in my opinion, not fully supported by the model as it is. In the model, it is assumed that normal and leukemic stem cells have the same maximal self-renewal probability. Only the regulation of self-renewal by feedback signals is different. The parameter which is a predictor in the presented analysis (p_{0,max}) is the maximal self-renewal probability of both normal AND leukemic stem cells. Therefore, the conclusion that normal stem cell self-renewal is a predictor of TKI response is, in my opinion, questionable. If I understand the analysis correctly, the authors show the following: Under the assumptions that the maximal self-renewal probability of normal and leukemic stem cells is identical and that the feedback inhibition of self-renewal is weaker in leukemic stem cells compared to normal stem cells, the maximal self-renewal probability of the two stem cell populations is a predictor of TKI response. Notably, if the value of maximal self-renewal probability is increased, the self-renewal probability of leukemic and normal stem cells increases simultaneously at all time points. Therefore, I find it difficult to argue that normal stem cell self-renewal [as opposed to leukemic stem cell self-renewal] is the relevant quantity.

(3) The simulation of differentiation therapy is interesting, however, due to a lack of knowledge in the field, the specific impacts of such therapy on normal versus leukemic cell differentiation have to be rather hypothetical.

(4) The used patient cohort is very small (n = 21).

The proposed model of the regulations governing blood cell formation is a valuable contribution to the fields of computational modeling and experimental hematology. The derived predictors of TKI efficacy are potentially useful.

Reviewer #1 (Public Review):

This is a fascinating effort from the Ryan laboratory, revisiting fundamental issues of calcium-dependent release probability at cultured synapses. The authors point out that our basic understanding of mammalian synapses rests on a foundation of older research that was not acquired at physiological temperature, and represented a statistical interpretation of data acquired electrophysiologically without direct knowledge of release at individual active zones. The authors employ techniques of calcium imaging and glutamate sensing and argue that single synapses can be 'silenced' by a moderate drop in extracellular calcium, a drop that is within the range of calcium channel inhibition following activation of GABAergic signaling. While fascinating, the conclusions are most powerful when the data can be distilled to direct observation of single release sites and this is not uniformly the case.

Reviewer #1 (Public Review):

In this manuscript the authors describe the development and application of hierarchical machine learning model to identify the likely source of S. Enteritidis using whole genome sequence data. The application makes use of a collection of 2,313 genomes from 4 continents, 11 sub-regions and 38 countries. The approach is, to the best of my knowledge, novel and represents a substantial advance over previous approaches. The model is demonstrated to have good performance at the continental level and - where sufficient training data were available - also at the country level.

Strengths of the work include the clear exposition of the methods, application to a large and detailed genomic database of clinical S. Enteritidis isolates, and the use of five independent validation data-sets.

Limitations include lack of validation using post-pandemic data (as the authors state, the model may need retraining in light of changes to the global food network). Also, claimed novelties of the work include greater geographic granularity and faster turnaround time compared to alternative methods, but no explicit comparison to other methods is made.

Overall, the authors achieve their aims in describing a hierarchical machine learning model for source attribution using pathogen whole genome sequences. The approach is likely to be of broad relevance and considerable public health utility.

Reviewer #1 (Public Review):

The author constructed a novel rat model with a clinically relevant PLS3 hemizygous E10-16del mutation (PLS3E10-16del/0), which presents a classic form of early-onset osteoporosis, which recapitulate the osteoporotic phenotypes. Treatment with alendronate and teriparatide significantly improved bone mass and bone microarchitecture. Their results showed alendronate and teriparatide treatment could be a potential treatment for early-onset osteoporosis induced by PLS3 mutation.

This experiment is very interesting and has clinical relevance. The authors used common clinical drugs to treat osteoporosis caused by PLS3 mutation and achieved certain results. This result will give a way to the treatment of osteoporosis induced by PLS3 mutation.

Reviewer #1 (Public Review):

The authors have achieved a demonstration of different stellate ganglion nerve cell functions and transmitter subtypes, of potential cardiac importance. They employ viral tracing techniques. These convincingly make this demonstration. The work will be key to our understanding of sympathetic function at the transmitter and physiological levels.

Reviewer #1 (Public Review):

Pedigo et al, apply statistical modeling to a complete brain nanoscale network - a synaptic connectome of an insect brain: the Drosophila larva. They use a series of approaches to explore the symmetry of the right and left hemispheres. First, they compare network densities and find significant differences between the two hemispheres, with the right hemisphere having a higher density. They further grouped neurons by cell type to determine whether the differences were distributed across the entire brain or to specific connections and find the differences involving neurons in the learning and memory center, the mushroom body. Finally, they explored different definitions of an edge by using different thresholds either based on synaptic counts or proportions of synaptic inputs to a downstream neuron and found that when using the proportion of synaptic inputs, removing fewer edges (compared to when using synaptic count) was necessary to achieve left and right symmetry. The presentation of the methodology and writing is very clear and effective and is accessible to scientists from various backgrounds: both biologists and theoreticians. The methodology and approach used in this paper on the assessment of the degree of bilateral symmetry will serve as a basis for comparing networks and connectomes in general by providing a clear framework for statistical network modeling. This work is particularly timely as an increasing number of synaptic connectomes is being generated giving opportunities for various connectome comparisons. It will be of interest to neuroscientists in order to address various biological questions: to evaluate the degree of inter-individual variability/stereotypy of connectivity in the brain and how it relates to behavioral variability/stereotypy, to characterize changes in network connectivity due to different diseases, etc.

Reviewer #1 (Public Review):

Tornini et al. investigate the function of long non-coding RNAs in vivo. In the manuscript, the authors show that two of these molecules linc-mipep and linc-wrb encode for a micropeptide that regulates zebrafish behavior. In the absence of this peptide, zebrafish larvae show dysregulation of NMDA receptor and glucocorticoid receptor-mediated signaling and immediate early gene induction. Given the homology of linc-mipep and linc-wrb encoded peptides with homology to chromosome binding and chromatin unwinding domain of HMGN1 the authors explore the altered chromatin accessibility in the mutant animals. This analysis revealed a broad dysregulation of 3D chromatin structure with some enrichment at loci regulating the expression of immediate early response genes. Finally, single cell analysis revealed that oligodendrocyte progenitor cells and cerebellar granule cells are more affected in the mutants.

This work represents a technical tour-de-force with extensive genomics data to characterize the molecular phenotype of linc-mipep and linc-wrb loss of function. This data show interesting findings in part consistent with the behavioral phenotype observed.

The manuscript provides compelling evidence that micropeptides encoded by what were previously identified as long non-coding RNAs have a precise biological function.

Reviewer #1 (Public Review):

This study presents a useful study, proposing the modelling of Buruli ulcer occurrence in humans based on detection of M. ulcerans in Australia. The data were collected and analyzed using solid and validated methodology and can be used as a starting point for the elucidation of M. ulcerans transmission in Australia.

Reviewer #1 (Public Review):

Francou et al. examine the dynamics of cell ingression at the primitive streak during mouse gastrulation and correlate this with the localization of elements of the apical Crumbs complex and the actomyosin cytoskeleton. Using time-lapse live imaging, they show that cells at the primitive streak ingress in a stochastic manner, by constricting their apical surface through a ratcheting shrinkage of individual junctions. Meticulous evaluation of immunofluorescent staining for many elements of the actomyosin contractile process as well as junctional and apical domain elements reveals anisotropic localization of Crumbs2, ZO1, and ppMLC. In addition, the localization of two groups of proteins showed a close correlation - actomyosin regulators and apical and junctional components - but there was a lack of correlation of localization of these two groups of proteins to each other. The localization of actomyosin and its activity, was altered and more homogeneous in Crumbs2-/- embryos, and there was a significant decrease in aPKC and Rock1. The authors conclude from these observations that Crumbs2 regulates anisotropic actomyosin contractility to promote apical constriction and cell ingression.

The strengths of this manuscript are the very detailed observations on the process of apical constriction and the meticulous evaluation of the localization of the many proteins likely to be involved in the process. While many of the general observations are not new, Francou et al. provide a much richer understanding of this process, as well as a paradigm with which to evaluate the effects of mutations on the gastrulation process. The figures are beautiful, clear, and informative, and support the conclusions made by the authors. The data provide a very compelling picture of both the dynamics of cell behavior and the anisotropies in protein localization associated with it.

However, much of the Crumbs2 mutant phenotype is not sufficiently explained by the authors' data or conclusions. First, the loss of Crumbs2 does not prevent ingression, as there are mesoderm cells evident between the epiblast and endoderm (Ramkumar et al., 2016, Xiao et al., 2011). There are certainly fewer, and the biggest effect appears to be during the elongation of the axis from E7.75 onward and not during the earlier migratory period (E6.5-E7.75) according to data from both previously published work (Xiao et al., 2011; Ramkumar et al., 2015, 2016) and the data presented here. Nor does the loss of Crumbs2 prevent apical constriction. Ramkumar et al. in their 2016 paper show by live imaging that the major effect of the Crumbs2 mutation is to prevent the cells from detaching from the epithelium, but that the apical domain does undergo constriction, leading to many elongated flask-shaped cells still attached at the apical end. These observations do not fit well with the model proposed by the authors of Crumbs2 regulating anisotropic actomyosin contractility to promote apical constriction and suggest a more complicated story. However, the complications of the Crumbs2 mutant do not detract from the value of the basic observations presented in this manuscript, which are solid and well-documented, and will be a valuable resource for the field.

Reviewer #1 (Public Review):

In this study, Sapiro et al sought to develop technology for a transcriptomic analysis of B. burgdorferi directly from infected ticks. The methodology has exciting implications to better understand pathogen RNA profiles during specific infection timepoints, even beyond the Lyme spirochete. The authors demonstrate successful sequencing of the B. burgdorferi transcriptome from ticks and perform mass spectrometry to identify possible tick proteins that interact with B. burgdorferi. This technology and first dataset will be useful for the field. The study is limited in that no transcripts/proteins are followed-up by additional experiments and no biological interactions/infectious-processes are investigated.

Critiques and Questions:

This study largely develops a method and is a resource article. This should be more directly stated in the abstract/introduction.

Details of the infection experiment are currently unclear and more information in the results section is warranted. State the species of tick and life-stage (larval vs nymphal ticks) used for experiments. For RNA-seq, are mice are infected and ticks are naïve or are ticks infected and transmitting Borrelia to uninfected mice?

What is the limit of detection for this protocol? Experimental data should be provided about the number of B. burgdorferi required to perform this approach.

More information regarding RNA-seq coverage is required. Line 147-148 "read coverage was sufficient"; what defines sufficient? Browser images of RNA-seq data across different genes would be useful to visualize the read coverage per gene. What is the distribution of reads among tRNAs, mRNAs, UTRs, and sRNAs?

My lab group was excited about the data generated from this paper. Therefore, we downloaded the raw RNA-seq data from GEO and ran it through our RNA-seq computational pipeline. Our QC analysis revealed that day 4 samples have a different GC% pattern and that a high percentage of E. coli sequences were detected. This should be further investigated and addressed in the paper: Are other bacteria being enriched by this method? Why would this be unique to day 4 samples? Does this affect data interpretation?

Comprehensive data comparisons of this study and others are warranted. While the authors note examples of known differentially expressed genes (like lines 235-241), how does this global study compare to other global approaches? Are new expression patterns emerging with this RNA-seq approach compared to other methods? What differences emerged from day 1 to day 4 ticks compared to differences observed in unfed to fed ticks or fed ticks to DMC experiments? Directly compare to the following studies (PMID: 11830671; PMID: 25425211; PMID: 36649080).

Details about the categorization of gene functions should be further described. The authors use functional analysis from Drechtrah et al., 2015, but that study also lacks details of how that annotation file was generated. Here, the authors have seemed to supplement the Drechtrah et al., 2015 list with bacteriophage and lipoprotein predictions - which are the same categories they focus their findings. Have they introduced a bias to these functional groups? While it can be noted that many lipoproteins are upregulated (or comment on specific genes classes), there are even more "unknown" proteins upregulated. I argue that not much can be inferred from functional analysis given the current annotation of the B. burgdorferi genome.

Reviewer #1 (Public Review):

In this manuscript, the authors used an unbiased method to identify proteins from porcine oocyte extracts associated with permeabilised boar spermatozoa in vitro. The identification of the proteins is done by mass spectrometry. A previous publication of this lab validated the cell-free extract purification methods as recapitulating early events after sperm entry in the oocyte. This novel method with mammalian gametes has the advantage that it can be done with many spermatozoa at the time and allows the identification of proteins associated with many permeabilised boar spermatozoa at the time. This allowed the authors to establish a list of proteins either enriched or depleted after incubation with the oocytes extract or even only associated with spermatozoa after incubation for 4h or 24h. The total number of proteins identified in their test is around 2 hundred and with very few present in the sample only when spermatozoa were incubated with the extracts.

The list of proteins identified using this approach and these criteria provide a list of proteins likely associated with spermatozoa remnants after their entry and either removed or recruited for the transformation of spermatozoa-derived structures.

Using WB and histochemistry labelling of spermatozoa and early embryos using specific antibodies the authors confirmed the association/dissociation of 6 proteins suspected to be involved in autophagy.

While this unique approach provides a list of potential proteins involved in sperm mitochondria clearance it's (only) a starting point for many future studies and does not provide the demonstration that any of these proteins has indeed a role in the processes leading to sperm mitochondria clearance since the protein identified may also be involved in other processes going-on in the oocyte at this time of early development.

Concerning the localisation of the 6 proteins further analysed, the authors must add how much the presented picture represents the observed patterns. They must include the details on the fraction of spermatozoa and embryos displaying the presented pattern.

Reviewer #1 (Public Review):

Mice and humans have two Cylicin genes (X-linked Cylicin 1 and the autosomal Cylicin 2) that encode cytoskeletal proteins. Cylicins are localized in the acrosomal region of round spermatids, yet they resemble a calyx component within the perinuclear theca of mature sperm nuclei. The function of Cylicins during this developmental stage of spermiogenesis (tail formation and head elongation/shaping) was not known. In this study, using CRISPR/Cas genome editing, the authors generated Cylc1-and Cylc2-knockout mouse lines to study the loss-of-function of each Cylicin or all together.

The major strengths of the study are the rigorous and comparative phenotypic analyses of all the combinatorial genotypes from the cross between the two mouse lines (Cylc1-/y, Cylc2-/-, Cylc1-/y Cylc2+/- and Cylc1-/y Cylc2-/-) at the levels of male fertility, cellular, and subcellular levels to support the conclusion of the study. While spermatogenesis appeared undisturbed, with germ cells of all types detected in the testis, low sperm counts in epididymis were observed. Mice were subfertile or infertile in a dose-dependent manner where fewer functional alleles had more severe phenotypes; the loss of Cylc2 was less tolerated than the loss of Cylc1. Thus, loss of Cylc1, and to an even greater extent, loss of Cylc2, leads to sperm structure anomalies and decrease sperm motility. Particularly, the sperm head and sperm head-neck region are affected, with calyx not forming in the absence of Cylicins, the acrosomal region being attached more loosely, and the sperm head itself appearing structurally rounder and shorter. Furthermore, manchette, which disassembles during spermiogenesis, persists in mature sperm of mice missing Cylc2. It is interesting that the study identifies a human male that has mutations in both CYLC1 and CYLC2 genes, and suffers from infertility, with similar motility and sperm structure defects compared to the mouse models. CYLC1 in the sperm from the infertile patient sperm is absent, providing evidence that in both rodents and primates, Cylicins are essential for male fertility.

The major weakness of the study is the less robust or absent of statistical analyses determining the statistical significance of some of the morphological phenotypes observed (e.g., the roundness/shortening of sperm head). Evolutionary analysis of two genes-while interesting- is less congruent with the other parts of the study and disrupts the overall flow of the functional studies. The authors show that the reason for the loss of Cylc2 being more severe is due to the higher conservation of Cylc2 compared to Cylc1 in rodents and primates, however, the conservation of these genes in other species is not discussed.

Overall, the work highlights the relevance and importance of Cylicins in male infertility and advances our understanding of perinuclear theca formation during spermiogenesis.

Reviewer #1 (Public Review):

Gordon-Fennell et al., here present a relatively low-cost, open-source platform for head-fixed operant and consummatory behaviour, called OHRBETS (prounounced Orbitz). This setup provides a great advantage over other systems in that it enables the animal to perform a truly operant response (i.e.one that fulfills the criterion of bidirectionality) whilst head-fixed. The authors provide thorough evidence of the utility of this setup, showing that a number of behavioural paradigms can be performed whilst the animal is head-fixed, as well as consummatory behaviours, optogenetic manipulations, and photometry recordings. These findings will be of broad interest to neuroscientists across multiple fields.

Strengths:<br /> 1. The work presented here is extremely thorough and explores multiple different types of paradigms. There is a huge amount of data that will be immensely useful to individuals who hope to use this setup and build on these findings. The setup is generally well-explained.<br /> 2. The statistics reported are generally quite strong and the sample sizes are sufficient (although strictly speaking ANOVA and Tukey should not be used together - Tukey's 'overall' test is a test of the maximal comparison, if the maximal comparison is not significant then no other pairwise comparison will be).<br /> 3. The open-source nature of the system is a great advantage as the fact that it is relatively low cost (as long as a lab has access to a 3D printer). This and similar endeavours will promote equality throughout the field.<br /> 4. The response here is truly operant as it is bidirectional. In other words, the animal shows that its response is governed by the relation between that response and the outcome, not stimulus-outcome associations like so many other so-called 'operant' responses (e.g. licking, food approach behaviours). Here, the stimuli are kept constant but the animal will either turn the wheel to the left or to the right to receive the food, depending on which direction is reinforced. This means that the animal cannot be governed solely by a stimulus-outcome response as in Pavlovian conditioning, because their response would not flexibly reverse the way that it is shown to here, particularly in Figure 1Q.<br /> 5. The accumbens shell recordings are interesting data in their own right (i.e. not simply to demonstrate the viability of the system), particularly the heterogeneity of the responses in the medial and lateral shells. This could be interesting for future studies to follow up on.<br /> 6. The correlational data between the head-fixed and free-moving versions of paradigms is, for the most part, quite convincing.

Weaknesses<br /> 1. I was curious as to how novel this setup is. Although I do not do head-fixed research myself, I thought there were already some open-source, relatively cheap systems available. I'm not sure how the current setup differs from those already available. Personally, even if this system involves only the wheel turning, as this is a truly operant response, that is novel enough for my liking.<br /> 2. It would be useful to have a bit more detail in the manuscript (not just on the GitHub link - in supplemental material perhaps?) on how to build such a system, just to get a sense of how difficult building such a system might be and how many components it has.<br /> 3. I wasn't sure how to feel about the comparisons across experimental set-ups in Figures 2 and 3. Usually, these sorts of comparisons are not considered statistically valid due to the many variables that differ between set-ups. However, I do see that the intent here is a bit different - i.e. is to show that despite all these alterations in variables the behavioural outputs are still highly correlated. However, without commenting on this intent, I did find these comparisons a little jarring to read.<br /> 4. The only dataset I was not wholly convinced by was that in Figure 3 (real-time place preference and aversion). I think the authors have done the best job that they can of replicating such a procedure in a head-fixed mouse, but the head-fixed version is going to necessarily differ from the freely moving version in a fundamental way when the contextual cues and spatial navigation form part of the RTPT task. Giving a discrete cue, such as a tone, just is not a sufficient substitute for contextual cues, and I think the two types of task would engage fundamentally different brain cells and circuits (e.g. only the free-moving version is likely to engage place cells in the hippocampus).<br /> 5. Personally, I found having the statistics in a separate file confusing.<br /> 6. Line 589-594. Suggesting the medial/lateral shell recording results mean that the medial shell 'tracks value, and the range of values during the multi-spout consumption of gradients of NaCl is greater than the range of values during multi-spout consumption of gradients of sucrose" seems to engage in circular logic to me. That is, the authors should use behavioural data to infer what the animal is experiencing and whether it is a change in value, and/or a greater change in value during NaCl vs. sucrose consumption, and only then should they make an inference about what the larger medial shell response means.

Overall this is a very solid paper in which the authors achieve their aims of demonstrating an open-source system for head-fixed operant and consummatory behavioural assessment, that is successfully employed across a number of different behavioural assays as well as in conjunction with optogenetic manipulations and fibre photometry recordings.

Reviewer #1 (Public Review):

This paper provides new technological approaches to expand adipocytes and aggregate them into structures that resemble fat. The authors use two cell types: a mouse cell line, as well as primary porcine cells. They demonstrate excellent lipid droplet accumulation in the mouse cell line however, this does not have translational relevance. So they go on to also perform those same experiments with the porcine cell line. The results are also encouraging especially if the cultivation is carried out over a period of 97 days.<br /> The authors also demonstrate similar mechanically mechanical properties of their cultivated fat to the native fat as well as the ability to aggregate it using two different approaches.

Overall, I think this is a thorough manuscript in the area of food bioengineering. The limitations remain the ability to fully remove FBS during this production process.

Reviewer #1 (Public Review):

This manuscript represents a substantial and well-executed body of work that contributes new data on 32 hymenopteran genomes, systematically identifies viral endogenization and domestication events, and tests whether this phenomenon is more common in hymenopteran species with specific lifestyles, eg. endoparasitism. The authors developed a pipeline to identify endogenization that improves upon previously described pipelines and is more comprehensive for the identification of endogenization events from a variety of virus types. Significant findings include the identification of previously undocumented cases of viral endogenization in several hymenopteran species and also moderate statistical support for a higher rate of dsDNA virus endogenization and domestication in endoparasitoids.

1. The authors have tested whether the lifestyle of hymenopteran species (endoparasitism, ectoparasitism, or free-living) is related to the incidence of virus endogenization and domestication. Addressing this kind of question has only become possible with the availability of genome sequences from many taxa so that any results can be statistically supported by appropriate sample sizes. It appears that the authors have not included new genomic data from hymenopteran genomes that have been published since 2019, which are of similar or better quality than the data used in this manuscript. A number of taxa with endogenous viruses (and also without) have become available since then. The best solution would be for the authors to use their pipeline to incorporate the new data, which may have an impact on their findings and could even strengthen their conclusions about virus domestication being more common in endoparasitoids. If this is not possible, the authors should at least justify their decision not to include the most recent data and discuss how it could affect their results.

2. Please summarize in the main manuscript (results or discussion) what the limitations of the pipeline to detect EVEs and dEVEs are - what are important factors to consider, including the availability of closely related "free-living" viruses, and of closely related wasp species for dN/dS analyses.

3. In this manuscript, a description of the methods that precede the results would make it much easier to appreciate the results shown. It appears that this is allowed in cases where it makes sense, according to the author's instructions.

4. The sensitivity and specificity of methods analysis are commendable, as is the availability of substantial supplementary data and scripts on GitHub. However, more effort could be made to align numbers reported in the text and in figures so that readers can verify support for the conclusions described.

Reviewer #1 (Public Review):

The article from Salas Lucia et al addresses the distribution and transport of thyroid hormones (TH, including T4 and T3) in the adult brain. This is a complex and important question. Overall, the manuscript is difficult to follow as it jumps from one question (Dio2 polymorphism) to another (Mct8 function in the uptake of TH in neurons, and then the connection between TRH neurons and tanycytes), without deepening any aspect. There are, however, interesting findings in the article, but they should be confirmed by additional experiments.

Part 1: Type 2 deiodinase<br /> T4 entry is easier than T3 entry in the brain. However, type 2 deiodinase (Dio2 expressed mainly in glial cells) converts T4 to T3 and produces around 80% of the brain T3. In the introduction, the authors mention the controversial observation according to which a polymorphism of type 2 deiodinase, Thr92Ala-DIO2, is detrimental to the entry of TH into the brain. One of the associated issues, mentioned by the authors, is that some patients treated with TH have normalized circulating levels of hormones but still complain of fatigue, a typical feature of brain hypothyroidism.<br /> Experiment 1: Hippocampal Responsiveness to L-T4 is Impaired in the Ala92-Dio2 Mouse<br /> This first part is a continuation of a previous study published by the same authors. Here, they use transgenic mice with Ala92-Dio2 and Thr92-Dio2 to address possible differences in the TH response of several areas of the brain. The readout is a reporter mRNA, coming from an additional reporter transgene.<br /> Table I is supposed to clarify and summarize the results but brings confusion. The text says that table I supports the claim that "in the cerebellum Luc-mRNA was lower in the Ala92-Dio2 mice" whereas figure 1G does not show any difference. It is unclear whether Table I and figure 1 report the same data, and what the statistical tests are actually addressing (effect of genotype vs effect of treatment, whereas what matters here is only the interaction between genotype and treatment). Overall, it is not acceptable to present quantitative data without giving numbers, standard deviation, p-value, etc. as in Table I. Also, evaluating T3 signaling by only looking at the luc reporter and the Hprt housekeeping gene is not always sufficient (many T3 responsive genes can be found in the literature and more than one housekeeping gene should be used as a reference).<br /> Another important weakness is that the wild-type mice have a proline at position 92. Why not include them? In absence of structural prediction, one wonders whether the mouse models are relevant to the human situation and whether the absence of the proline reduces the enzymatic activity when substituted for an Ala or Thr. This might have been addressed in previous work, but the authors should explain.<br /> Experiment 2: Ala92-Dio2 Astrocytes Have Limited Ability to Activate T4 to T3<br /> Here, the authors use primary cell cultures from different areas of the brain to measure the in vitro conversion of T4 to T3 by Dio2. They find that hippocampus astrocytes are less active, notably if they come from Ala92-Dio2 mice.<br /> This part has the following weaknesses:<br /> - This result correlates with the results from Fig 1F however the difference between Ala92-Dio2 and Thr92-Dio2 is significant in vitro, but not in vivo. What matters is not the activity/astrocytes, but the total activity of the brain area, which depends on the number of astrocytes x individual activity. This is not measured.<br /> - What the authors called 'primary astrocytes' is an undefined mixed population of glial cells, (including radial glial cells, stem cells, ependymal cells, progenitor cells, etc...) that proliferated differentially for more than a week in culture, among which an unknown ratio expresses Dio2. The cellular model is thus poorly characterized, and the interpretation must be prudent.<br /> - Again, wild-type mice are not included.

Part 2: Neuronal response to T3 Involves MCT8 and Retrograde TH transport<br /> The authors next move to primary neuronal cultures, prepared from the fetal cortex which they grow in the microfluidic chamber to study axonal transport. This is a surprising move: the focus is not on Dio2 anymore, but on the MCT8 transporter, which is known in humans to play an important role to transfer TH into the brain. It is expressed mainly in glia, but also in neurons. They study the influence of endosomes and type 3 deiodinase on the trafficking and metabolism of TH.<br /> It would be useful to perform an experiment, in which radioactive T3 is introduced in the "wrong" side of the chamber, in an attempt to detect a possible anterograde transport. This would address the possibility that Mct8 also promotes efflux and control so that the chamber is not leaking.<br /> The authors use sylichristin as an inhibitor of Mct8, to demonstrate that transport is Mct8 dependent. They do not provide indications or references that would clearly indicate that this drug is a fully selective antagonist of Mct8 (but not of Oatp1c1, Mct10, Lat1, Lat2, etc., the other TH transporters). A good alternative would be to use Mct8 KO mice as controls.<br /> The B27 used in primary neuronal culture might contain TH. This is not easy to know, but at least some batches do.<br /> The presence of astrocytes, probably expressing Mct8 and Dio2 is inevitable in primary neuronal cultures, and is not mentioned, but might interfere with TH metabolism.

Part 3: T3 Transport Triggers Localized TH Signaling in the Mouse Brain<br /> The authors return to in vivo experiments, implanting T3 crystals, labeled or not with radioactive iodine. They do so in the hypothalamus, where they address the retrograde transport of TH in TRH neurons, and in the cortex, looking for contralateral transport.<br /> These data are the most difficult to interpret.<br /> - First, T3 is hydrosoluble and would probably migrate without active transport.<br /> - The authors do not demonstrate that these specific neuronal populations contain Mct8, and that these observations are connected to the previous in vitro observation (which used cortical neurons prepared from the fetus). The possibility that astrocytes are involved, as reported in the literature, is not considered.<br /> - Here again, using Mct8KO mice would greatly help to interpret the data. In particular, the experiments with cold T3 involve a 48h delay which is very long in comparison to the 30 minutes required for long-distance transfer of radioactive T3.<br /> Discussion<br /> Considering the diversity of questions that are addressed in the study, it is not surprising that the discussion is not covering all aspects. The authors implicitly consider that their conclusions can be extended to all neurons, while they use in their experiments a variety of different populations coming from either the fetal cortex, hippocampus, adult cortex, or hypothalamus. The claim that they discovered a mechanism applying to all neurons is not supported by the data. Some highly relevant literature is not cited. In particular:<br /> - Mct8 KO mice do not have a marked brain hypothyroidism (PMID: 24691440) which at least suggests that the pathway discovered by the authors can be efficiently compensated by alternative pathways.<br /> - Dio3 KO only increases T3 signaling in a few areas of the brain and only in the long term (PMID: 20719855).<br /> - Anterograde transport of T3 has been reported for some brainstem neurons (PMID: 10473259)

Reviewer #1 (Public Review):

This article is somewhat far afield from my typical line of research, but, to not bury the lede, I thought that this article makes an important point and is rigorously argued but could use some space to breathe in order to increase its impact.

More precisely, the authors perform a set of detailed calculations and simulations to show that the purported benefits of having non-linear morphogen decays are small near the source and decidedly reversed near the far end. I didn't have any specific concerns with these calculations, but one question I did have was if the typical context of morphogen gradients needs to be taken into account a little more (the paper doesn't really discuss how downstream morphogen gradients' noise might be affected by the structure of noise discussed here).

That said, I think that this is a rigorous submission.

Reviewer #1 (Public Review):

In this study 1458 Enterobacterales isolates, derived from animals, waste-water and human bloodstream infections, were genetically characterized. This also yielded 3697 plasmids and many AMR genes.

All isolates were derived in a restricted geographical region and within a few years time. They defined "groups of near-identical plasmids" with plasmids derived from different genera, species, and clonal background; 8% of these groups contained plasmids from the different ecological niches and 35% of these cross-niche groups plasmids carried AMR genes. This fits with the concept of recent transfer of AMR plasmids between these ecological niches. Through detailed analyses they provide evidence that for E. coli, AMR dissemination between human and livestock-associated niches is most likely not the result of clonal spread but rather that plasmids transit between ecological niches.

Strengths

This is - to the best of my knowledge - one of the largest and most detailed studies elucidating the epidemiology of plasmids and AMR genes in different ecological niches.

Reviewer #1 (Public Review):

The authors conducted an extensive characterization of canine H3N2 influenza viruses. By analyzing gene sequences of canine H3N2 influenza viruses isolated in their laboratory and those that are available in public databases, they identified various genetic clades (also somehow correlate with antigenic groups identified in serological assays) and human-like amino acid substitutions in these viruses, which indicated the evolution of these viruses towards potentially more adaptive to humans. By experiments with several selected canine H3N2 influenza isolates, they found that more recent canine H3N2 influenza viruses have acquired receptor specificity for both avian- and human-like receptors, enhanced low-pH stability and in vitro growth as well as improved replication and transmission in the dog and ferret models. They further identified amino acid substitutions underlying the improved transmissibility of these canine H3N2 influenza viruses. The study was well-designed and the conclusions in the manuscript are in general well supported by the experimental data. Findings from the study will certainly help understand the evolution of canine influenza viruses and assessing the risk posed by these viruses to public health.

Although the authors have identified some properties/molecular markers of canine H3N2 influenza viruses that highlight the potential for infecting humans, it needs to be cautious to emphasize the threat of these viruses to public health. One fact is that despite the increasing prevalence of these viruses in dogs and the close proximity between dogs and humans, there is so far no report of human infection with canine H3N2 influenza viruses. The authors are wished to discuss this in their manuscript so that the readers can have a more comprehensive understanding of their findings and the public health importance of canine influenza viruses.

Reviewer #1 (Public Review):

In this manuscript, the authors have elegantly demonstrated the significance of asking fundamental questions in patient-derived models of patient-derived organoids (PDOs). This is especially relevant for studying complex cancers such as High-Grade Serous Ovarian Carcinoma (HSCOG). In addition to developing patient-derived organoids, this study has comprehensively examined transcriptomic, genomic, and single-cell data. In addition, based on this data, the authors have performed a complex drug sensitivity assay that further stratifies the PDOs into drug-sensitive and resistant categories. This approach would be central to identifying therapeutic regimens for difficult-to-treat cancers in the future.

Reviewer #1 (Public Review):

The basis of this method is to clone guides into a Crispr-based editing plasmid, transfect pools into Leishmania, maintain them as episomes, then look at phenotypes. The guides are designed to cause editing that converts codons to stop codons, and the authors have designed a computational tool that enables the design of guides that work for the first half of each gene. Selection for the episome is necessary and editing efficiencies were variable (99% to 0%) depending on the species, being worst for L. major. The use of premature termination codons also clearly raises issues for false positives and negatives, especially as there is no evidence for nonsense-mediated mRNA decay in Leishmania.

There are already two genome-wide screening options for Leishmania, so the advantages and disadvantages of the method proposed here need to be discussed in a much more detailed and balanced way.<br /> In the "LeishGEM" project (http://www.leishgem.org) all Leishmania mexicana genes will be knocked out and each KO will be bar-coded. At the end, 170 pooled populations of 48 bar-coded mutants will be publicly available. The only real reason the authors of the current paper give for not using this approach is that it is labour-intensive. However, LeishGEM is funded and underway, with several centres involved, so that argument is weak.<br /> There is also a preprint describing RNAi for functional analysis in Leishmania braziliensis.

Reviewer #1 (Public Review):

This umbrella review aims to synthesize the results of systematic reviews of the impact of the COVID-19 pandemic on various dimensions of cancer care from prevention to treatment. This is a challenging endeavour given the diversity of outcomes that can be assessed in cancer care.

Search and review methods are good and are in line with recommendations for umbrella reviews. Perhaps one weakness of the search strategy was that only one database (Pubmed) was searched. The search strategy appears adequate, though perhaps some more search terms related to reviews and cancer could have been included. It is therefore possible that some reviews may have been missed by the search strategy.

It is challenging to perform a good umbrella review that yields novel insights, as it is difficult to combine results from different reviews which themselves combine results from different studies with different methodologies. However, I think perhaps one of the main weaknesses of this study is that it is not clear to me what is the core objective of the umbrella review, and how analyses relate to that core objective. In other words, I do not understand based on the introduction what new information the authors are hoping to learn from their umbrella review that could not be learned from reading the individual systematic reviews, beyond a vague objective of "synthesizing" the literature. Because of this, it is not very clear to me how the data extracted and the analysis fits into the larger objectives, and what the new knowledge generated by this review is. Based on the reported results, it would appear that one of the main goals is to assess the quality of systematic reviews and of the underlying studies in the reviews, but it is hard to tell. I think there are potentially important insights this review could tell us, but the message and implications of current evidence remain for me a little confused in the current manuscript.

Reviewer #1 (Public Review):

The article is a straightforward continuation of their previous 2016 study. The authors demonstrate an organism-level role of intermediate filaments (IFs) in C. elegans with a model highlighting intermediate filament functions in organism development, larval development, oxidative stress-resilience, size, and lifespan.

The study uses endotube morphogenesis in C. elegans as an elegant model to examine the effect of aberrant IF network morphogenesis on endotube morphology and how these effects are reflected in terms of progeny growth and development.

The study identifies the C. elegans IF protein IFB-2 as a core component of IF network morphogenesis where any mutation or dysfunction of IF interacting proteins such as SMA-5, IFO-1, and BBLN1 can be mostly rescued by silencing of IFB-2.

The observed mutations cause a range of systemic and functional defects of which endotube-related defects that include luminal widening and cytoplasmic invaginations are regarded as the key parameters to observe the direct result of IF network perturbation in the study. Based on these parameters authors narrowed down on IFB-2 head domain as a critical interactor in IF network morphogenesis and function.

On the whole, very interesting findings and an elegant study with excellent data that would be of broad interest for cytoskeletal research. The study has clear ramifications also for the understanding of the evolutionary development and roles of IF, both IF aspects that are still very poorly understood.

Reviewer #1 (Public Review):

This carefully done research paper presents a fundamental model of techniques that are useful for the elucidation of kinase substrates. The paper utilizes state-of-the-art approaches to define a kinetic phosphoproteome and how to integrate that data with complementary approaches using a chemical probe (in this case KTPyS, a triphosphate) to find these substrates. Using these approaches TgCDPK1 was demonstrated to affect microneme secretion via a direct interaction with a HOOK complex (defined as a HOOK protein TGG1_289100, an FTS TGGT1_264050 and 2 other proteins TGGT1_316650 and 306920).

This work is carefully controlled and the analysis pathways are logical and provide paradigms for how to approach the question of identifying substrates of kinases using proteomic approaches employing genetic and chemical strategies.

The authors succeeded in the identification of candidate substrates for TgCDPK1. Validation of the results was provided by previous studies in the literature that characterized some of these substrates as well as the experiments in this manuscript on the characterization of the HOOK complex that is phosphorylated by CDPK1.

The HOOK complex (defined as a HOOK protein TGG1_289100, an FTS TGGT1_264050, and 2 other proteins TGGT1_316650 and 306920) was clearly demonstrated to be involved in invasion via its role in microneme trafficking.

Reviewer #1 (Public Review):

This study builds an odorant organization map as estimated by a neural network trained on several odor perceptual classification databases. The authors come up with an attractive hypothesis about the link of odor perception to metabolic connectedness, as opposed to a range of other ways of classifying odorant compounds. There are several interesting implications of this, which the authors touch upon, but could perhaps frame as specific predictions.

The authors clearly have generated a powerful methodology, a useful classifying network, and a well-organized database. The study would be much stronger if the methodology were more thoroughly explained, with open code and data availability as expected for a computational study, and as a resource for further research on the topic.

It would also be valuable to place the current findings in the context of considerable earlier work that has sought to map odor perception and place it in the context of structural and chemical features.

Reviewer #1 (Public Review):

This work focuses on the characterization of neutralizing antibodies from humans survivors of SNV and ANDV hantavirus infections, including the mapping of epitopes located in the Gn and/or Gc glycoproteins, and their mechanism of viral interference blocking receptor binding or membrane fusion. It also confirms previous data on broadly neutralizing epitopes allowing inhibition of different hantavirus species. The work covers for the first time in vivo evidence of cross-protection against HNTV infection by a broadly neutralizing antibody prepared from SNV infection using a prophylaxis animal model and compares the data with protection from ANDV lethal challenge using ANDV-specific neutralizing antibodies. The work provides valuable information for the development of therapeutic measures that cross-protect against several hantavirus species which seems a promising strategy to rise pharmaceutical interest against a group of viruses causing orphan disease.

The strength of the work is based on the impressive amount of work and versatility of methods to identify residues involved in the binding and/or escape from seven different neutralizing antibody clones that allow for important conclusions on species-specific antigenic regions and confirm data on a region that seems broadly conserved among different hantavirus species. At the same time, the weakness of the work is that data processing does not allow for readers data analysis (Figs. 1b, 2a, 2c, Ext. Data Fig. 4).

The authors clearly achieve their aim of characterizing the antigenic sites of neutralizing antibodies. Yet, the presented data on binding to ANDV mutant constructs and negative-staining EM does not allow for the conclusion that the epitope of the broadly neutralizing antibodies ANDV-44 and SNV-53 involved the Gn capping loop. An alternative explanation of the escape mutations in the Gn capping loop could be produced by an allosteric effect on the Gc fusion loop region, and a role in structuring the Gc fusion loop has been previously demonstrated (References 7 and 9). In addition, it is not clear why SNV-24 has no broad neutralizing activity although escape mutations occurred at the highly conserved residues K833 and D822 in Gc domain I.

Finally, concerning the in vivo protection experiments, it would be important to show viral RNA levels in lungs and kidneys in the lethal ANDV animal model (Fig. 7) to allow for comparison with the prophylaxis from HTNV infection (Fig. 6).

Reviewer #1 (Public Review):

Collins et al use mesoscopic two-photon imaging to simultaneously record activity from basal forebrain cholinergic or noradrenergic axons in several distant regions of the dorsal cortex during spontaneous behavior in head-fixed awake mice. They find that activity in axons from both neuromodulatory systems is closely correlated with measures of behavioral state, such as whisking, locomotion and face movements. While axons were globally correlated with these behavioral state-related metrics across the dorsal cortex, they also find evidence of behavioral state independent heterogenous signals.

The use of simultaneous multiarea optical recordings across a large extent of dorsal cortex with single axon resolution for studying the coherence of neuromodulatory afferents across cortical areas is novel and addresses important questions regarding neuromodulation in the neocortex. The manuscript is clearly written, the data is well presented and, for the most part, carefully analyzed. Parts of the manuscript confirm previous results on the influence of behavioral state on norepinephrine and acetylcholine cortical afferents. However, the observation that these modulations are globally broadcasted to the dorsal cortex while behavioral state independent hetetogenous signals are also present in these axons is novel and important for the field.

While the evidence for a behavioral state driven global modulation of activity in both neuromodulatory systems is quite clear, I have concerns that the apparent heterogeneity in axonal responses might be driven by movement-induced artifacts. Moreover, even in the case that the heterogeneity in calcium activity across axons is confirmed, it might not be driven by differences in spiking activity across neuromodulatory axons as concluded, but by other mechanisms that are not explicitly discussed or considered.

1) Motion artifacts are always a concern when imaging from small structures in behaving animals. This issue is addressed in the manuscript in Fig 2A-C by comparing axonal responses to "autofluorescent blebs that did not have calcium-dependent activity" (line 1011). Still, as calcium-dependent activity and motion artifacts can both be locked to behavioral variables the "bleb" selection criterion seems biased and flawed with a circular logic. "Blebs" presenting motion-induced changes in fluorescence that may pass as neural activity will be wrongly excluded when from the "bleb" control group using this criterion. This will result in an underestimation of the extent of the contamination of the GCaMP signals by movement-induced artifacts. This potential confound might generate apparent heterogeneity across axons and regions as some axons and some cortical areas might be more prone to movements artifacts than others.

2) In the case that the heterogeneity is indeed due to differences in calcium activity, it might be not due to modularity in spiking activity within the LC or the BF as interpreted and discussed in the manuscript. As calcium signaling in axons not only relates to spiking activity but can also reflect presynaptic modulations, the observed heterogeneity might be due to local action of presynaptic modulators in a context of global identical broadcasted activity. The current dataset does not allow distinguishing which of the two different mechanisms underlies to the observed signal heterogeneity.

Reviewer #1 (Public Review):

In this study, the authors aim to identify the cell state dynamics and molecular mechanisms underlying melanocyte regeneration in zebrafish. By analyzing thousands of single-cell transcriptomes over regeneration in both wild-type and Kit mutant animals, they provide thorough and convincing evidence of (1) two paths to melanocyte regeneration and (2) that Kit signaling, via the RAS/MAPK pathway, is a key regulator of this process. Finally, the authors suggest that another proliferative subpopulation cells, expressing markers of a separate pigment cell type, constitute an additional population of progenitors with the ability to contribute to melanocytes. The data supporting this claim are not as convincing, and the authors failed to show that these cells did indeed differentiate into melanocytes. Despite the challenges of describing this third cell state, this study offers compelling new findings on the mechanisms of melanocyte regeneration and provides paths forward to understanding why some animals lack this capacity.

The majority of the main conclusions are well supported by the data, but one claim, in particular, should be revisited by the authors.

(1) Provided evidence that the aox5(hi)mitfa(lo) population of cells contributes to melanocyte regeneration is inconclusive and somewhat circumstantial. First, the transcriptional profiles of these cells are much more consistent with the xanthophore lineage. Indeed, xanthophores have been shown to express mitfa (in embryos in Parichy, et al. 2003 (PMID: 10862741), and in post-embryonic cells in Saunders, et al. 2019). Second, while the authors address this possibility in Supplemental figure 7 by showing that interstripe xanthophores fail to divide following melanocyte ablation, they fail to account for the stripe-resident xanthophores/xanthoblasts. The presence and dynamics of aox5+ stripe-resident xanthophores/xanthoblasts are detailed in McMenamin, et al., 2014 (PMID: 25170046) and Eom, et al., 2015 (PMID: 26701906). Without direct evidence that the symmetrically-dividing, aox5+ cells measured in this study do indeed differentiate into melanocytes, it is more likely that these cells are a dividing population of xanthophores/xanthoblasts. The authors should revise their claims accordingly.

Minor revisions

(1) At line 140, it is noted that Xanthophores are pteridine-producing, but they also get their yellow color from carotenoids (especially in adults). This should be noted as well, especially since the authors display the xanthophore marker, scarb1, which plays a key role in xanthophore carotenoid coloration.<br /> [Mapping expression levels onto UMAP space for scarb1 and perhaps other markers of xan, irid, or proliferation would be helpful as a supplement to the dot plot in Fig 1 and could help to clarify the transcriptomic signature of mitfa+ aox5-hi cells and plausibility of the model that they are an McSC population. -Parichy]

(2) The authors should provide the list of genes that comprise their cluster signatures (line 252) as part of the supplementary tables.

(3) The authors should more clearly describe how they performed lineage tracing (line 339). Additionally, for the corresponding figure 4E, the authors should list the number of cells traced. The source data only contains calculated percentages rather than counts for each type of differentiation. My understanding is that the number listed in the figure legend is the number of fish (i.e. n = 4), but this should be clarified as well.<br /> [A supplementary figure of labeled cells is important here with enough context to show that cells can be re-identified unambiguously. Additionally note that "lineage tracing" will typically be assumed to mean single-cell labeling and tracking, so if that is not the case for these experiments it would be preferable to use an alternative descriptor. -Parichy]

(4) Line 321, the authors list the mean regeneration percentages for the kita and kitlga(lf) mutants, but these differences are not significantly different according to Figure 4B. By listing the means (which should be noted), the authors seem to be highlighting the differences but then do not comment on them. The description and integration of this result into the main text should be clarified.

(5) In Figure 6E, the RNA-velocity result is not particularly consistent with the authors' claims. Visually, the arrows seem fairly randomly directed. The data in 6B, showing gene expression associated with the S phase and G2/M phase much more clearly convey the directionality of the loop (S phase, followed by G2/M). I suggest that the authors weaken their claim about the RNA-velocity result or remove it altogether and focus on the cell cycle-related gene expression signatures.

Reviewer #1 (Public Review):

This study addresses the role of the general transcription factor TBP (TATA-binding protein), a subunit of the TFIID complex, in RNA polymerase II transcription. While TBP has been described as a key component of protein complexes involved in transcription by all three RNA polymerases, several previous studies on TBP loss of function and on the function of its TRF2 and TRF3 paralogues have questioned its essential role in RNA polymerase II transcription. This new study uses auxin induced TBP degradation in mouse ES cells to provide strong evidence that its loss does not affect ongoing polymerase II transcription or heat-shock and retinoic acid-induced transcription activation, but severely inhibits polymerase III transcription. The authors coupled TBP degradation with TRF2 knock out to show that it does not account for the residual TBP-independent transcription. Rather the study provides evidence that TFIID can assemble and is recruited to promoters in the absence of TBP.

All together the study provides compelling evidence for TBP-independent polymerase II transcription, but a better characterization of the residual TFIID complex and recruitment of other general transcription factors to promoters would strengthen the conclusions.

Reviewer #1 (Public Review):<br /> <br /> Roberts et al have developed a tool called "XTABLE" for the analysis of publicly available transcriptomic datasets of premalignant lesions (PML) of lung squamous cell carcinoma (LUSC). Detection of PMLs has clinical implications and can aid in the prevention of deaths by LUSC. Hence efforts such as this will be of benefit to the scientific community in better understanding the biology of PMLs.

The authors have curated four studies that have profiled the transcriptomes of PMLs at different stages. While three of them are microarray-based studies, one study has profiled the transcriptome with RNA-seq. XTABLE fetches these datasets and performs analysis in an R shiny app (a graphical user interface). The tool has multiple functionalities to cover a wide range of transcriptomic analyses, including differential expression, signature identification, and immune cell type deconvolution.

The authors have also included three chromosomal instability (CIN) signatures from literature based on gene expression profiles. They showed one of the CIN signatures as a good predictor of progression. However, this signature performed well only in one study. The authors have further utilised the tool XTABLE to identify the signalling pathways in LUSC important for its developmental stages. They found the activation of squamous differentiation and PI3K/Akt pathways to play a role in the transition from low to high-grade PMLs

The authors have developed user-friendly software to analyse publicly available gene expression data from premalignant lesions of lung cancer. This would help researchers to quickly analyse the data and improve our understanding of such lesions. This would pave the way to improve early detection of PMLs to prevent lung cancer.

Strengths:

1. XTABLE is a nicely packaged application that can be used by researchers with very little computational knowledge.<br /> 2. The tool is easy to download and execute. The documentation is extensive both in the article and on the GitLab page.<br /> 3. The tool is user-friendly, and the tabs are intuitively designed for successive steps of analysis of the transcriptome data.<br /> 4. The authors have properly elaborated on the biological interest in investigating PMLs and their clinical significance.

Weaknesses:

The article is focused on the development and the utility of the tool XTABLE. While the tool is nicely developed, the need for a tool focussing only on the investigation of PMLs is not justified. Several shiny apps and online tools exist to perform transcriptomic analysis of published datasets. To list a few examples - i) http://ge-lab.org/idep/ ; ii) http://www.uusmb.unam.mx/ideamex/ ; iii) RNfuzzyApp (Haering et al., 2021); iv) DEGenR (https://doi.org/10.5281/zenodo.4815134); v) TCC-GUI (Su et al., 2019). While some of these are specific to RNA-seq, there are plenty of such shiny apps to perform both RNA-seq and microarray data analysis. Any of these tools could also be used easily for the analysis of the four curated datasets presented in this article. The authors could have elaborated on the availability of other tools for such analysis and provided an explanation of the necessity of XTABLE. Since 3 of the 4 datasets they curated are from microarray technology, another good example of a user-friendly tool is NCBI GEO2R. This is integrated with the NCBI GEO database, and the user doesn't need to download the data or run any tools. iDEP-READS (http://bioinformatics.sdstate.edu/reads/) provide an online user-friendly tool to download and analyse data from publicly available datasets. Another such example is GEO2Enrichr (https://maayanlab.cloud/g2e/). These tools have been designed for non-bioinformatic researchers that don't involve downloading datasets or installing/running other tools.

Secondly, XTABLE doesn't provide a solution to integrate the four datasets incorporated in the tool. One can only analyse one dataset at a time with XTABLE. The differences in terms of methodology and study design within these four datasets have been elaborated on in the article. However, attempts to integrate them were lacking.

The tool also lacks the flexibility for users to add more datasets. This would be helpful when there are more datasets of PMLs available publicly.

Understanding the biology of PML progression would require a multi-omics approach. XTABLE analyses transcriptome data and lacks integration of other omics data. The authors mention the availability of data from whole exome, methylation, etc from the four studies they have selected. However, apart from the CIN scores, they haven't integrated any of the other layers of omics data available.

Lastly, the authors could have elaborated on the limitations of the tool and their analysis in the discussion.

Reviewer #1 (Public Review):

This manuscript builds on data from the same group showing that Lphn2 functions cell-autonomously as a receptor in CA1 pyramidal axons and cell-non-autonomously as a ligand in the neurons of the subiculum. In either case, binding of teneurin-3 to Lphn2 mediates repulsive events, and since different populations of neurons within each region express differing levels of both proteins, this mechanism allows proximal CA1 pyramidal axons to preferentially project to distal subiculum and distal CA1 pyramidal axons to project to proximal subiculum. The authors now ask mechanistic questions about the role of Lphn2 signaling in these wiring processes.

The authors demonstrate that G-protein signaling downstream of Lphn2, which is mediated by the tethered agonist, is necessary for the ability of ectopically expressed Lphn2 to redirect proximal CA1 axons from distal to proximal subiculum. Moreover, the authors show that while autoproteolytic activity of Lphn2 facilitates G-protein signaling, possibly by making the tethered agonist more available for signaling, it is not necessary for axonal mistargeting. Thus, the authors conclude that tethered agonist-dependent G-protein signaling is required for Lphn2-mediated hippocampal neural circuit assembly. Most of the data shown in support of these conclusions are convincing, though I have some concerns about the expression levels and/or effects of the tethered agonist mutants in CA1, which is important since the analyses assume that any defects are in the repulsive interactions described above.

The authors also use heterologous cells to determine that Lphn2 couples to Ga12/13, but not other heteromeric G-proteina-subunits. Within the context of heterologous cells, these experiments are well controlled and exhaustive, as every mutant used in vivo is carefully analyzed. One potential criticism of this work, however, is that perhaps the authors assume too much in simply translating their results in heterologous cells to neurons, especially when one of the most interesting conclusions of this paper (see below) is that Lphn2 signaling is context-dependent. Without further data to confirm the results of these experiments in the neuronal populations studied, these data primarily illustrate possibilities, but don't exclude other possibilities.

Finally, the authors test the role of Lphn2 functioning as a ligand in the subiculum by driving its expression in the normally Lphn2-low dorsal subiculum. As they reported before, this alteration decreases the ability of proximal CA1 axons to project to this area. Interestingly, and in contrast to the role of Lphn2 as a receptor above, neither Lphn2 autoproteolysis nor tethered agonist function are required for this effect.

In summary, this is an interesting paper that addresses timely and pressing issues in the adhesion-GPCR field.

Reviewer #1 (Public Review):

Determination of the biomechanical forces and downstream pathways that direct heart valve morphogenesis is an important area of research. In the current study, potential functions of localized Yap signaling in cardiac valve morphogenesis were examined. Extensive immunostainings were performed for Yap expression, but Yap activation status as indicated by nuclear versus cytoplasmic localization, Yap dephosphorylation, or expression of downstream target genes was not examined. The goal of the work was to determine Yap activation status relative to different mechanical environments, but no biomechanical data on developing heart valves were provided in the study.

There are several major weaknesses that diminish enthusiasm for the study.<br /> 1. The Hippo/Yap pathway activation leads to dephosphorylation of Yap, nuclear localization, and induced expression of downstream target genes. However, there are no data included in the study on Yap nuclear/cytoplasmic ratios, phosphorylation status, or activation of other Hippo pathway mediators. Analysis of Yap expression alone is insufficient to determine activation status since it is widely expressed in multiple cells throughout the valves. The specificity for activated Yap signaling is not apparent from the immunostainings.

2. The specific regionalized biomechanical forces acting on different regions of the valves were not measured directly or clearly compared with Yap activation status. In some cases, it seems that Yap is not present in the nuclei of endothelial cells surrounding the valve leaflets that are subject to different flow forces (Fig 1B) and the main expression is in valve interstitial subpopulations. Thus the data presented do not support differential Yap activation in endothelial cells subject to different fluid forces. There is extensive discussion of different forces acting on the valve leaflets, but the relationship to Yap signaling is not entirely clear.

3. The requirement for Yap signaling in heart valve remodeling as described in the title was not demonstrated through manipulation of Yap activity.

Reviewer #1 (Public Review):

Farahani et al. describe the generation of pYtags, recombinant RTKs, and reporters, that exploit phosphotyrosine/tandem SH2 interaction pairs from immune-specific signaling proteins to allow spatiotemporal monitoring of the activation of different ligand-binding (EGFR and FGFR1) or ligandless (ERBB2) RTKs in living cells stimulated with high and low-affinity ligands (e.g. EGF and EREG or EPGN respectively in the case of EGFR). The study is well-explained and the experiments are clear and clean. Although the authors expanded tool generation to different RTKs and different cells, the potential utility of the approach is limited because the broad concept that different receptor dimers activate different downstream signalling pathways is already well established. Additionally, the results only examine the temporal kinetics of the receptors rather than their spatial organization, e.g. in different vesicular/endosomal compartments. The study also describes the use of CRISPR-Cas9 to generate a pYtag knock-in EGFR-expressing HEK 293T cell line to avoid complications arising from over-expression. There were significant differences in terms of receptor activation dynamics comparing knock-in and over-expressed cell lines.

The study is technologically innovative, yet the analysis of RTK spatial signalling over time in ligand-stimulated cells should be improved.

Reviewer #1 (Public Review):

In this study, Mitterer et al continue their comprehensive investigation of the mechanisms underlying the biogenesis of the eukaryotic large, or 60S, ribosomal subunit. Specifically, they elucidate the roles that the DEAD-box helicase Spb4 and its interaction partner, Rrp17, play in the maturation of nucleolar 60S precursor particles. Using cell biology approaches, the authors demonstrate that Spb4 and Rrp17 are associated with late-stage nucleolar 60S precursor particles and that depletion of these factors arrests 60S biogenesis at a step just prior to nucleolar exit. Cryo-EM imaging of particles carrying Spb4 and Rrp17 (purified using affinity-tagged Spb4 or Rrp17) yielded high-quality structures of Spb4- and Rrp17-bound 60S precursor particles. The structures provide novel insights into the roles of Spb4 and Rrp17 in the maturation of nucleolar 60S precursor particles. In addition, the structures provide novel insights into the Spb4 function that may be of interest and importance to the function of other DEAD-box helicases. The authors then establish an in vitro maturation assay that, although unlikely to exactly recapitulate the in vivo maturation process, provides additional insights, particularly when coupled to cryo-EM structures of the in vitro-matured 60S particles.

A major strength of this work is the combination of cell biology, structural biology, and biochemistry. The cell biology-directed preparation of Spb4- and Rrp17-bound 60S precursor particles is particularly powerful and results in high-quality structures of these precursors. Another strength of the work is the remarkable view of a DEAD-box helicase in action and the interesting finding that the RecA domains of the helicase are in the open conformation while the helicase is likely bound to ADP-this will be an interesting and important observation for researchers working in the broader DEAD-box helicase field. An additional strength of the work is the development and use of an in vitro maturation assay that allowed further details of the activities of Spb4 and Rrp17 in nucleolar maturation of 60S precursor particles to be investigated and visualized.

A minor weakness of this work is a question about the confidence with which the authors can conclude, using just the structural data presented here, that Spb4 is bound to ADP rather than to ATP or ATP-Pi.

The considerable strengths of this work far outweigh the minor weakness, and I expect that this work will have a significant impact on the field.

Reviewer #1 (Public Review):

A quantitative understanding of the mechanisms underlying VDJ recombination is a prerequisite for a better understanding of adaptive immune repertoire generation. Here, Russel et al. study potential sequence-based factors that may drive VDJ trimming, a mechanism involved in VDJ recombination. This work provides a significant advance in the statistical modeling of immune repertoire generation.

Using a previously-published TCR𝛽 repertoire sequencing data set, the authors designed a probabilistic model of nucleotide trimming that allows the exploration of various mechanistically-interpretable sequence-level features. Using this model, they show that local sequence context and the capacity for sequence-breathing, together, can most accurately predict the trimming probabilities of a given V-gene sequence. Their model suggests that double-stranded DNA needs to be able to "breathe" for trimming to occur and provides evidence of a sequence motif that appears to get preferentially trimmed, independent of breathing. Importantly their findings are not dataset-dependent.

So far, there exists no model for VDJ trimming, a major mechanism in the process of VDJ recombination. With this model, we are now in the position to refine modeling tools for VDJ recombination. Importantly, the model developed by Russel et al. enables exploration of what biological sequence-based factors most contribute to VDJ trimming. To support their conclusions, the authors test their approach on multiple model architectures and AIRR datasets.

While I agree that this is important work, the authors might be overstating the mechanistic insight achieved given that solely statistical inference was used in this work. This is something that requires more discussion and support from the authors.

Reviewer #1 (Public Review):

The study employs state-of-art techniques and model-driven fusion of MEG and 7T to characterize the fine spatiotemporal profiles of object recognition in human brains when stimuli are noisy. By using two models, the recognition and the two-state models, to characterize the representational format, the work demonstrates that the ventral visual pathway is more toward two-state representation while the dorsal visual pathway tends to display the recognition-like profile. Overall it is an interesting work addressing an important question. My major concern is on the two selected models and whether they could be fairly compared to address the question. Moreover, some details need more clarification and statistical support.

Reviewer #1 (Public Review):

Gutiérrez-Martínez et al. present a detailed analysis of Siglec-1 nano-distribution on the surface of dendritic cells (DCs) and the role of Siglec-1 in HIV-1 interactions with DCs.

DCs have been proposed as key cellular intermediates in the transmission of HIV and other viruses. Not only can these cells be crucial for the presentation of virus-derived antigens, but, in tissue culture at least, mature DCs (mDC) have been observed to sequester HIV particles into compartments (virus-containing compartment [VCC]) from which the virus can be subsequently transmitted to CD4+ve T cells through cell-cell contacts often termed virological synapses. This so-called trans-infection mechanism is believed to be important in establishing HIV infection and transmission of the virus to immunological tissues. Although there is considerable evidence for this process, the molecular details of how HIV particles are captured by DCs and transferred to VCC are poorly understood. In recent years Siglec-1 (CD169), a plasma membrane-associated sialic acid-binding lectin expressed on monocytic cells has been implicated in the capture of HIV and other viruses. In this paper, the authors have used super-resolution and other imaging methods to perform a detailed quantitative analysis of the cell surface distribution of Siglec-1 on immature and mature DCs, the relationship between this distribution with actin and regulators of actin polymerization, and then how this impacts on the capture of HIV particles and their association with VCCs.

The principal findings, which for the most part are well supported by the data, suggest that small clusters of Siglec-1, which are restricted in their mobility by formin-associated actin, provide platforms with increased avidity for binding virus particles or large unilamellar vesicles through sialic-acid containing gangliosides. In mDCs at least this binding appears to induce the sequestration of bound particles into VCC-like structures. This is a topical and detailed study that addresses important questions of how viral engagement with cell surface receptors leads to events crucial for viral infection and, potentially, pathogenesis. These types of analyses have only recently become feasible with the implementation of super-resolution imaging and few virus-host cell systems have been examined in detail. Thus, this study has relevance not only to HIV but potentially to many other viruses.

Reviewer #1 (Public Review):

In this exciting and well-written manuscript, Alvarez-Buylla and colleagues report a fascinating discovery of an alkaloid-binding protein in the plasma of poison frogs, which may help explain how these animals are able to sequester a diversity of alkaloids with different target sites. This work is a major advance in our knowledge of how poison frogs are able to sequester and even resist such a panoply of alkaloids. Their study also adds to our understanding of how toxic animals resist the effects of their own defenses. Although target site insensitivity and other mechanisms acting to prevent the binding of alkaloids to their targets (often ion channels) are well characterized now in poison frogs, less is known regarding how they regulate the movement of toxins throughout the animal and in blood in particular. In the fugu (pufferfish) a protein binds saxitoxin and tetrodotoxin and in some amphibians possibly the protein saxiphilin has been proposed to be a toxin sponge for saxitoxin. However, little is known about poison frogs in particular and if toxin-binding proteins are involved in their sequestration and auto-resistance mechanisms.

The authors use a clever approach wherein a fluorescently labeled probe of a pumiliotoxin analog (an alkaloid toxin sequestered by some poison frogs) is able to be crosslinked to proteins to which it binds. The authors then use sophisticated mass spectroscopy to identify the proteins and find an outlier 'hit' that is a serpin protein. A competition assay, as well as mutagenesis studies, revealed that this ~50-60 kDa plasma protein is responsible for binding much of the pumiliotoxin and a few other alkaloids known to be sequestered in the in vivo assay, but not nicotine, an alkaloid not sequestered by these frogs.

In general, their results are convincing, their methods and analyses robust and the writing excellent. Their findings represent a major breakthrough in the study of toxin sequestration in poison frogs. Below, a more detailed summary and both major and minor constructive comments are given on the nature of the discoveries and some ways that the manuscript could be improved.

Detailed Summary

The authors functionally characterize a serine-protease inhibitor protein in Oophaga sylvatica frog plasma, which they name O. sylvatica alkaloid-binding globulin (OsABG), that can bind toxic alkaloids. They show that OsABG is the most highly expressed serpin in O. sylvatica liver and that its expression is higher than that of albumin, a major small molecule carrier in vertebrates. Using a toxin photoprobe combined with competitive protein binding assays, their data suggest that OsABG is able to bind specific poison frog toxins including the two most abundant alkaloids in O. sylvatica skin. Their in vitro isolation of toxin-bound OsABG shows that the protein binds most free pumiliotoxin in solution and suggests that OsABG may play an important role in its sequestration. The authors further show that mutations in the binding pocket of OsABG remove its ability to bind toxins and that the binding pocket is structurally similar to that of other vertebrate serpins.

These results are an exciting advance in understanding how poison frogs, which make and use alkaloids as chemical defenses, prevent self-intoxication. The authors provide convincing evidence that OsABG can function as a toxin sponge in O. sylvatica which sets a compelling precedent for future work needed to test the role of OsABG in vivo.

The study could be improved by shifting the focus to O. sylvatica specifically rather than the convergent evolution of sequestration among different dendrobatid species. The reason for this is that most of the results (aside from some of the photoprobe binding results presented in Fig. 1 and Fig. 4) and the proteomics identification of OsABG itself are based on O. sylvatica. It's unclear whether ABG proteins are major toxin sponges in D. tinctorius or E. tricolor since these frogs may contain different toxin cocktails. The competitive binding results suggest that putative ABG proteins in D. tinctorius and E. tricolor have reduced binding affinity at higher toxin concentrations than ABG proteins in O. sylvatica. Although molecular convergence in toxin sponges may be at play in the dendrobatid poison frogs, more work is needed in non-O. sylvatica species to determine the extent of convergence.

Major constructive comments:

Although the protein gels in Fig.1-2 show clearly the role of ABG, a ~50 kDa protein, it's unclear whether transferrin-like proteins, which are ~80 kDa, may also play a role because the gels show proteins between 39-64 kDa (Fig.1). The gel in Fig.2A is specific to one O. sylvatica and extends this range, but the gel does not appear to be labeled accordingly, making it unclear whether other larger proteins could have been detected in addition to ABG. Clarifying this issue would facilitate the interpretation of the results.

There is what seems to be a significant size difference between the O. sylvatica bands and bands from the other toxic frog species, namely D. tinctorius and E. tricolor. Could the photoprobe be binding to other non-ABG proteins of different sizes in different frog species? Given that O. sylvatica bands are bright and this species was the only one subject to proteomics quantification, a possible conclusion may be that the ABG toxin sponge is a lineage-specific adaptation of O. sylvatica rather than a common mechanism of toxin sequestration among multiple independent lineages of poison frogs. It would be helpful if the authors could address this observation of their binding data and the hypothesis flowing from that in the manuscript.

Figure 1B: The species names should be labeled alongside the images in the phylogeny. In addition, please include symbols indicating the number of times toxicity has evolved (for example, once in the ancestors of O. sylvatica and D. tinctorius frogs and once in the ancestors of E. tricolor frogs).

Figure 4B-C: Photoprobe binding results in the presence of epi and nicotine appear to be missing for D. tinctorius and those in the presence of PTX and nicotine are missing for D. tricolor. Adding these results would make for a more complete picture of alkaloid binding by ABG in non-O. sylvatica species.

Using recombinant proteins with mutations at residues forming the binding pocket of O. sylvatica ABG (as inferred from docking simulations), the authors found that all binding pocket mutations disrupted photoprobe binding completely in vitro (L221-222, Fig. 4E). However, there is no information presented on non-binding pocket mutations. Mutations outside of the binding pocket would presumably maintain photoprobe binding - barring any indirect structural changes that might disrupt binding pocket interactions with the photoprobe. This result is important for the conclusion that the binding pocket itself is the sole mediator of toxin interactions. The authors do show that one binding pocket mutation (D383A) results in some degree of photoprobe binding (Fig. 4E) but more detail on the mutations in the binding pocket per se being causal would be helpful.

Please include concentrations in the descriptions of gel lanes in the main figures. The relative concentrations of the photoprobe and other toxins (eg., PTX, DHQ, epi, and nic) are essential for interpreting the competitive binding images. For example, this was done in Fig. S1 (e.g., PB + 10x PTX).

For clarity, the section "OsABG sequesters free PTX in solution with high affinity" could be presented directly after the section titled "Proteomic analysis identifies an alkaloid-binding globulin". The former highlights in vitro experiments confirming the binding affinity of the ABG protein identified in the latter.

Fig. 6E-F should be included as part of Fig. 1 or 2. Although complementary to the RNA sequencing data, these protein results are more closely related to the results in the first two figures which show the degree of competitive binding affinity of PB in the presence of different toxins. The expanded competitive binding results for total skin alkaloids and the two most abundant skin alkaloids from wild samples are most appropriate here.

Reviewer #1 (Public Review):

Elbaz-Hayoun et al. investigate the role of macrophages in the gliotic response of retinal Müller glia and photoreceptor cell death. Monocytes (a precursor of macrophages) were isolated from age-related macular degeneration (AMD) patients. When injected into light-damaged retinas, a reduction in the number of photoreceptors and ERG b-wave strength (evidence of abnormal photoreceptor function) was observed. The authors reasoned that macrophages generated from the injected monocytes might be responsible for the retinal damage. To test this hypothesis, macrophage subtypes were generated from AMD-derived human monocytes and injected into light-damaged mouse eyes. Interstingly, only the human hM2a macrophage subclass mimicked the retinal degeneration of monocyte injection in mouse retinas. Similarly, human M2a (hM2a) cells cultured on mouse retinal explants and even serum-free hM2a culture supernatant were sufficient to induce photoreceptor apoptosis. These effects were not observed with hM1 cells. To identify possible diffusible factors responsible, proteins present in hM2a and hM1 culture supernatants were identified. Nine cytokines were found at higher levels in the hM2a supernatant, and three of these were ligands for the C-C chemokine receptor CCR1. The authors confirmed CCR1 expression in the retina, which was predominantly detected in Müller glia. Importantly, Müller cell expression of CCR1 in the mouse retina was significantly increased following light damage. In contrast, CCR2 and CCR5 levels were unchanged in Müller cells. The increase in CCR1 expression, gliosis, and photoreceptor death was also observed in the rd10 mouse model of retinitis pigmentosa. Inhibiting CCR1 activity in light-damaged eyes using the drug BX471 had impressive effects. Müller activation and photoreceptor cell death were reduced and ERG b-wave levels were partially recovered - clearly indicating a role for CCR1 in retinal degeneration. Additional evidence was provided suggesting that CCR1 activation in M2a macrophages might also play a role in stimulating the movement of other macrophages into the retina and activating retinal microglia, which migrate to the ONL. These data identify a new link between cells of the immune system and those within the retina which contribute to the progression of retinal degeneration.

The data mostly support the conclusions of this paper. However, additional controls need to be added to some experiments.

Concerns:

1) To determine the effect of diseased monocytes on retinal health, light-injured mouse retinas were injected with monocytes isolated from AMD patients (Figure 1 - figure supplement 1). This resulted in a reduction in photoreceptor number and ERG b-wave amplitude. However, the light-injured control eye was injected with PBS only, so no cells were present. The reasoning for using this control was not provided. The appropriate injection control would include monocytes isolated from non-AMD patients. This control should be performed side-by-side with cells from AMD patients.

2) The authors hypothesize, from the experiments presented in Figure 1 - figure supplement 1, that the injected monocytes generated macrophages in the retina, which were responsible for the observed neurotoxicity (Lines 143-145). However, no direct evidence was presented. This idea should be tested in vivo. This could be done by injecting tracer-labeled human AMD-derived monocytes into light-injured mouse retinas. If the authors' hypothesis is true, collected retinas should contain tracer-labeled cells that express macrophage markers. Tracer-labeled M2a macrophage cells should be present since subsequent experiments identify this subclass as being associated with retinal cell death.

3) Photoreceptor number and b-wave amplitudes were measured in light-injured retinas injected with one of four macrophage cell types generated from human AMD-derived monocytes. The authors conclude that only injection of M2a cells reduced photoreceptor number and b-wave amplitudes (Figure 1C, E). This may be true, but it is difficult for the reader to make a conclusion (especially in Fig. 1E) due to the large error bars and five different traces overlapping each other. To make these results easier to interpret, graph control cells with only one experimental sample (cell type) at a time.

4) Most injected macrophages were located in the vitreous. In the case of M2a cells, the authors note that "several of the cells migrated across the retinal layers reaching the subretinal space" (Lines 167,168). One possible explanation for why M0, M1, and M2c macrophages did not induce retinal degeneration is that they did not migrate to the subretinal space and around the optic nerve head. Supplementary figures should be added to demonstrate that this is not the case.

5) Figure 1 - figure supplement 2: Panel A, B cells were stained with CD206 to demonstrate the presence of M2a macrophages (panel B). The authors conclude that panel A contains M1 and panel B contains M2a cells. The lack of CD206 expression illustrates that panel A cells are not M2a macrophages but do not demonstrate they are M1 macrophages. A control using an M1 cell marker is necessary to show that panel A cells are M1 and M1 cells are not detected in M2a cultures.

6) Ex vivo, apoptotic photoreceptor and RPE cells are observed when cultured with M2a macrophages (Figure 2). Do injected M2a cells also induce apoptosis of RPE cells in vivo? This is important to establish that retinal explants are a good model for in vivo experiments.

7) Reactive oxygen species (ROS) production was measured to determine if M2a cell-mediated neurotoxicity was due to oxidative stress. It is concluded that a ROS increase is partly responsible (Line 218). The data do not support this conclusion. ROS was detected in cultured M2a macrophages. More importantly, however, there was no increase in oxidative damage in vivo. The in vivo and cell culture results contradict each other so no conclusion can be made. The lack of in vivo confirmation weakens the argument that ROS drives M2a neurotoxicity. Text suggesting a role for ROS in neurotoxicity should be appropriately edited (Lines including 218, 244, 401,406,481).

8) The authors ask if the photoreceptor cell death is cytokine-mediated. Multiple cytokines were enriched in M2a-conditioned media. Of particular interest were CCR1 ligands MPIF1 and MCP4. The implication is that these two ligands mediate the M2a macrophages to photoreceptor cell death through CCR1. However, there is no attempt to show that either MPIF1 or MCP4 are present in vivo, or are sufficient to induce the retinal response observed. This could be demonstrated by injection of MPIF1 or MCP4. Evidence that either ligand phenocopies M2a macrophage injection would be direct evidence that CCR1 ligands activate the retinal response. Furthermore, co-injection with BX174 should block the effect of these ligands if they work through CCR1.

Joint Public Review:

The authors employ a range of microscopy, biochemical, and virologic techniques to evaluate the efficacy of CRISPR-nuPin to relocalize DNA and the subsequent impact of HSV-1 replication. There are many compelling experiments that utilize solid approaches to HSV-1 transcription, replication, and histone association. The microscopy images are particularly stunning, strongly supported by biochemical evaluation, and consistent with most of the authors' interpretations. Overall, the manuscript presents data that suggests the dCas9-emerin fusion protein can be used to manipulate the nuclear localization of smaller DNA elements like the HSV-1 viral genome. Chromosomal DNA, as tested by telomere targeting, reveal reduced capacity and elongated kinetics for retargeting. Using this system, authors find differing effects on HSV-1 replication based on the timing of sgRNA electroporation post-infection. Further experiments support that the transcriptional effects of either inhibitory or enhancing treatments may be related to chromatin modifications and expression of the viral protein ICP0.

There are many strengths to both the methodology and analysis in this work. That said, there are several areas where a more expansive explanation of methods and data analysis combined with tempered interpretations and language will greatly improve the manuscript.

Reviewer #1 (Public Review):

In this manuscript, the authors set out to identify the energy-generating protein responsible for powering heme transport through the Isd system of Staphylococcus aureus.

The manuscript convincingly demonstrates that FhuC is required for heme iron utilization and presents strong data to implicate FhuC in binding to IsdF. The authors report that IsdF localizes to functional membrane microdomains in S. aureus. These experiments would benefit from controls showing that the DRM fraction contains the functional membrane microdomains and that the fractionation was successful.

The authors also present strong data demonstrating that loss of floA prevents IsdF incorporation into the membrane although these data would also benefit from genetic complementation.

In a surprising result, the authors report that the IsdA protein is not localized in the functional membrane microdomains which are confounding since IsdA is modeled to work in concert with IsdF. These data suggest there is much more to learn regarding the spatial distribution of this transport system.

Finally, the authors report that FMMs are required for heme transport in the related organism Staphylococcus lugdunensis demonstrating the conservation of this localization across the genus.

Taken together, these exciting and significant data reveal how the canonical heme transporter of S. aureus is regionally localized and acquires energy for heme transport across the membrane.

Reviewer #1 (Public Review):

Idiosyncratic drug-induced liver injury is a disease that appears to be linked to mitochondrial DNA (mtDNA), but there is a lack of model cell lines for the study of this link. To help address this problem, the authors developed ten cybrid HepG2 cell lines that have had their mitochondrial DNA replaced with the mitochondrial DNA of ten human donors. Analysis of single nucleotide polymorphisms in all of the patients' mtDNA allowed the authors to assign the donors to two haplogroups (H and J) with five patients each. The authors also present the results of several assays (e.g. oxygen consumption, ATP production) performed on all ten cell lines in the absence and presence of five clinically-relevant drugs (or drug metabolites). Significant attention was paid to differences observed between the cell lines in the H and J haplogroups. The work is methodologically and scientifically rigorous, ethically conducted, and objectively presented according to the appropriate community standards.

While I feel that the manuscript will be useful to the research field and is an important step towards improving patient outcomes, I feel that the work lacks a broad interest. Much of the paper is spent discussing small and/or statistically insignificant differences between haplogroups H and J. While some interesting interpretations and suggestions are presented in the discussion, the authors didn't perform follow-up experiments to try to nail down any particular mechanistic insights that would be useful to the broader community. I also didn't feel a strong sense that the paper produced any specific suggestions for how clinical outcomes could be improved. Accordingly, any clear insights that would be interesting to a broad scientific community would probably require follow-up studies. The structure of the paper is also not friendly to a broad audience; the results are presented without interspersed commentary that could help the reader understand the meaning or utility of the results as they are being presented. Accordingly, I often felt unsure about how the results being presented were relevant to solving the broader problem established nicely in the introduction. Finally, it wasn't clear that the generated cell lines were made available for anyone to purchase through a cell bank (perhaps the authors did do this, but I don't recall seeing a mention of it). As these cell lines appear to be the primary output of this work, it seems important to better highlight the extent to which they are being made accessible to the scientific community.

Reviewer #1 (Public Review):

Utilizing mouse models as well as in-vitro studies, the authors demonstrate that cardiac cell mapping provides novel insights into intercellular communication drivers underlying pathological extracellular matrix remodeling during diabetic myocardial fibrosis.The work provides new perspectives to help understanding the cellular and molecular mechanisms of diabetes-induced cardiac pathology.

Reviewer #1 (Public Review):

Han et al use sophisticated genetic approaches to investigate leptin-responsive neural circuits. Overall, this is an impressive series of studies that provide fairly convincing evidence for a key inhibitory pathway downstream of AGRP neurons. A few data sets require additional validation or explanation.

Reviewer #1 (Public Review):

Demographic inference is a notoriously difficult problem in population genetics, especially for non-model systems in which key population genetic parameters are often unknown and where the reality is always a lot more complex than the model. In this study, Rose et al. provided an elegant solution to these challenges in their analysis of the evolutionary history of human specialization in Ae. aegypti mosquitoes. They first applied state-of-the-art statistical phasing methods to obtain haplotype information in previously published mosquito sequences. Using this phased data, they conducted cross-coalescent and isolation-with-migration analyses, and they innovatively took advantage of a known historical event, i.e., the spread of Ae. aegypti to South America, to infer the key model parameters of generation time and mutation rate. With these parameters, they were able to confirm a previous hypothesis, which suggests that human specialists evolved at the end of the African Humid Period around 5,000 years ago when Ae. aegypti mosquitoes in the Sahel region had to adapt to human-derived water storage as their breeding sites during intense dry seasons. The authors further carried out an ancestry tract length analysis, showing that human specialists have recently introgressed into Ae. aegypti population in West African cities in the past 20-40 years, likely driven by rapid urbanization in these cities.

Given all the complexities and uncertainties in the system, the authors have done outstanding jobs coming up with well-informed research questions and hypotheses, carrying out analyses that are most appropriate to their questions, and presenting their findings in a clear and compelling fashion. Their results reveal the deep connections between mosquito evolution and past climate change as well as human history and demonstrate that future mosquito control strategies should take these important interactions into account, especially in the face of ongoing climate change and urbanization. Methodologically, the analytical approach presented in this paper will be of broad interest to population geneticists working on demographic inference in a diversity of non-model organisms.

In my opinion, the only major aspect that this paper can still benefit from is more explicit and in-depth communication and discussion about the assumptions made in the analyses and the uncertainties of the results. There is currently one short paragraph on this in the discussion section, but I think several other assumptions and sources of uncertainties could be included, and a few of them may benefit from some quantitative sensitivity analyses. To be clear, I don't think that most of these will have a huge impact on the main results, but some explicit clarification from the authors would be useful. Below are some examples:

1. Phasing accuracy: statistical phasing is a relatively new tool for non-model species, and it is unclear from the manuscript how accurate it is given the sample size, sequencing depth, population structure, genetic diversity, and levels of linkage disequilibrium in the study system. If authors would like to inspire broader adoption of this workflow, it would be very helpful if they could also briefly discuss the key characteristics of a study system that could make phasing successful/difficult, and how sensitive cross-coalescent analyses are to phasing accuracy.

2. Estimation of mutation rate and generation time: the estimation of these important parameters is made based on the assumption that they should maximize the overlap between the distribution of estimated migration rate and the number of enslaved people crossing the Atlantic, but how reasonable is this assumption, and how much would the violation of this assumption affect the main result? Particularly, in the MSMC-IM paper (Wang et al. 2020, Fig 2A), even with a simulated clean split scenario, the estimated migration rate would have a wide distribution with a lot of uncertainty on both sides, so I believe that the exact meaning and limitations of such estimated migration rate over time should be clarified. This discussion would also be very helpful to readers who are thinking about using similar methods in their studies. Furthermore, the authors have taken 15 generations per year as their chosen generation time and based their mutation rate estimates on this assumption, but how much will the violation of this assumption affect the result?

3. The effect of selection: all analyses in this paper assume that no selection is at play, and the authors have excluded loci previously found to be under selection from these analyses, but how effective is this? In the ancestry tract length analysis, in particular, the authors have found that the human-specialist ancestry tends to concentrate in key genomic regions and suggested that selection could explain this, but doesn't this mean that excluding known loci under selection was insufficient? If the selection has indeed played an important role at a genome-wide level, how would it affect the main results (qualitatively)?

Reviewer #1 (Public Review):

In this manuscript Sugatha et al. present a comprehensive study on sorting nexin 32 (SNX32) with a wide-spectrum of methodologies and model systems. Authors investigate binding to other sorting nexins involved in the same pathways (SNX1 and SNX4) as well as to its cargo in biochemical and cell-based experiments. They show the importance and explore mechanisms of SNX32 in Transferrin Receptor and Cation Independent Mannose-6-Phosphate Receptor trafficking. Moreover, this work also demonstrates the role of SNX32 in concert with Basigin in neuron differentiation.

Authors with the help of structure modelling and subsequent biochemical experiments find specific residues within the BAR domain of SNX32 that are crucial for heterodimer formation with its interaction partners on endosomal membranes: SNX1 and SNX4. Moreover, this study, by using various microscopy techniques, also demonstrates localization of SNX32 to early endosomes as well as its co-trafficking with Rab11 and Golgi marker. Furthermore, authors with knock-down and rescue experiments investigate the role of SNX32 in Transferrin Receptor and Cation Independent Mannose-6-Phosphate Receptor trafficking. With co-immunoprecipitation they show that the cargo interaction occurs via the conserved stretch in the PX domain and that single amino acid substitution can disrupt this binding. This feature is utilized in a subsequent neuroblastoma cell-based SILAC screen for SNX32 interactome that identifies Basigin (a transmembrane receptor belonging to the superfamily of immunoglobulins) as one of the most prominent interactors in these cells. Finally, authors identify SNX32 and Basigin as crucial factors involved in neurite outgrowth and network formation. Experiments demonstrate that SNX32, but not its homolog SNX6, assists in the surface localization of Basigin where this protein could potentially interact with monocarboxylate transporters crucial for neuro-glial coordination.

Reviewer #1 (Public Review):

This is a carefully written manuscript describing the structure of a low-light inducible PSI complex from Ostreococcus tauri. The work expands our knowledge of how photosynthetic systems react to changes in light conditions and shows how this ecologically important green alga utilizes its unique antenna, Lhcp.

In general, I find that the work described in the manuscript is of high quality. The cryoEM maps obtained by the authors clearly show the addition of lhcp trimers to PSI under low light conditions and the distinction between lhcp1 and lhcp2 appears sound together with the identification of the phosphorylation site and its binding in the PSI complex.

Reviewer #1 (Public Review):

In this article, Sanz Perl and colleagues set out to use a computational whole-brain model to simulate the patterns of functional connectivity (as observed from functional MRI) that characterise different forms of dementia, namely Alzheimer's Disease (AD) and behavioural variant frontotemporal dementia (bvFTD). To overall goal is to develop a paradigm to model a specific disorder, and then develop an in silico assessment of the effects of different interventions. They show that superior fitting of the simulated data to the empirical data of both pathologies can be achieved when a Hopf model of brain activity is informed by patterns of combined AD and bvFTD atrophy, or by the intrinsic organisation of brain regions into canonical resting-state networks. They also show that regional differences in the fitted parameters pertain to AD and bvFTD, both in terms of location, and in terms of dynamical regime. They then use a machine learning algorithm, the variational auto-encoder (VAE), to compress functional connectivity patterns into a 2-dimensional space (given by the relative activation of the VAE's two hidden neurons). This space reveals that AD and bvFTD perturb brain connectivity along two distinct dimensions, further stratifying sub-categories of AD. Finally, through visualisation in this latent space, the authors can assess the effects of different simulated interventions on the models previously fitted to AD and bvFTD: namely, stimulation of different regions and with different dynamical regimes, to evaluate whether the resulting model is moved closer to the region occupied by healthy controls.

A strength of this work is its creative combination of different modelling approaches, combining the more biologically-informed Hopf model, which incorporates atrophy maps and connectivity, with the VAE for the purpose of dimensionality reduction and visualisation. Another strength is the use of different controls, such as an atrophy map from a different disorder (Parkinson's) or the use of randomised heterogeneities, showing that the improved fit is not just due to increased degrees of freedom: an important concern for high-dimensional models, which the authors lay to rest.

Admittedly, the stimulation paradigm shows limited success at bringing the disorder-fitted models back to the region occupied by controls - except for the AD- sub-category, which is the least affected and shows the most promise in the authors' in-silico trial. The limited success of this approach in this specific context does not invalidate the framework's promise. This may also be attributed to the fact that the authors do not use disease-specific atrophy maps to model AD and bvFTD: rather, they use a single atrophy map obtained by combining the two and use this joint atrophy map both to model AD, and to model bvFTD. Likewise, the connectivity of the model is the same for all conditions.

A weakness of this work is that, as the authors themselves acknowledge, the brain regions whose stimulation pushes the model to be least far from controls in the latent space did not match with those presenting different bifurcation parameters. In fact, it is not clear whether this is because stimulation fails to reverse the regional alterations of the dynamical regime, or whether it does succeed, but introduces new alterations - although it should be possible to extract this information from the model, to provide additional insight. This raises the intriguing question of the biological meaning of the latent space. Although the authors do show what kinds of FC correspond to the different values of the VAE hidden neurons' activation, the latent space effectively acts as a 2-dimensional goodness-of-fit - raising the question of how much of the stimulation results could be captured by simply evaluating the stimulated model's GOF against controls (while acknowledging that this would conflate the two distinct dimensions along which AD and bvFTD differ from controls).

Since stimulation is intended to mimic the effects of different real-life interventions such as tACS and tDCS, it would be helpful to see whether the regions that are suggested as most promising for stimulation, do in fact match the regions that have shown the most success in actual clinical trials that have already been carried out. This would be a powerful validation from model to real applicability.

In its essence, the work makes progress towards the authors' goal of modelling different pathologies by incorporating biologically-derived information, highlighting their differences, and enabling the evaluation of different stimulation strategies. This computational framework is widely applicable to a variety of pathological (and even non-pathological) conditions, combining evaluation and intervention in a single workflow.

Reviewer #1 (Public Review):

In this manuscript the authors use single nucleus sequencing together with in situ to profile neurons from the paraventricular nucleus of the thalamus. The PVT has been implicated in diverse functions and here the authors use snRNAseq to try to assign those functions to distinct cell types within the structure. They first use punches of PVT and iterative clustering and filtering to find neuronal clusters with known PVT markers. Other cell types and neurons from surrounding brain regions were also present in the dataset. These data both support the previous division of PVT neurons into Drd2+/- cells and suggest these two groups can be further subdivided into 5 distinct clusters. In a nice in situ experiment the authors assessed top marker gene expression for each cluster across the anterior-posterior axis of the PVT. This showed that the five types were largely in distinct spatial locations. Follow-up in situ with an additional set of marker genes supported the same conclusion but also showed that expression of single genes even within a cell "type" can vary. The authors discuss how the transcriptomes of the cell types could map onto known function of anterior and posterior PVT neurons. Finally, the authors integrate their sequencing data with a dataset of thalamic neurons with specific known projection patterns. Of the cells that co-cluster between the datasets, they identify specific transcriptomic populations of cells that best overlap different cortical projection patterns. The authors identify Col12a1 as a marker of one particular population of PFC-projecting cells.

The idea of spatial gradients of transcription in brain regions rather than discrete cell "types" has been shown in a number of recent studies that combine transcriptomics and in situ hybridization. Application of this idea to other important functional areas of the brain like the PVT generally enhances understanding of the parcellation of neuronal function. Combining these data with mapping of projection patterns by a lab interested in the function of this region, will be of interest to other researchers who study PVT and its role in brain circuits. The data appear to be of high quality and the discussion is scholarly.

Reviewer #1 (Public Review):

Marjaneh et al. studied the atrial septal variation through QTL mapping of inbred mouse strains which show extremes of septal phenotypes. The analysis discovered many interesting septal QTLs. Furthermore, the authors identified high-confidence candidate deleterious variants through whole genome sequencing of parental strains and analyzed variant architecture across gene features.

Overall, this is a comprehensive study that will provide a useful reference for the field. It will be a useful tool for hypothesis generation, which could lead to research on therapies that target atrial septal or common congenital heart disease.

Reviewer #1 (Public Review):

In this study, single neurons were recorded, using tetrodes, from the parahippocampal cortex of 5 rats navigating a double-Y maze (in which each arm of a Y-maze forks again). The goal was located at any one of the 4 branch terminations, and rats were given partial information in the form of a light cue that indicated whether the reward was on the right or left side of the maze. The second decision point was uncued and the rat had no way of knowing which of the two branches was correct, so this phase of the task was more akin to foraging. Following the outbound journey, with or without reward, the rat had to return (inbound journey) to the maze and start to begin again.

Neuronal activity was assessed for correlations with multiple navigation-relevant variables including location, head direction, speed, reward side, and goal location. The main finding is that a high proportion of neurons showed an increase in firing rate when the animal made a wrong turn at the first branch point (the one in which the correct decision was signalled). This increase, which the authors call rate remapping, persisted throughout the inbound journey as well. It was also found that head direction neurons (assessed by recording in an open field arena) in the same location in the room were more likely to show the rate change. The overall conclusion is that "during goal-directed navigation, parahippocampal neurons encode error information reflective of an animal's behavioral performance" or are "nodes in the transmission of behaviorally relevant variables during goal-directed navigation."

Overall I think this is a well-conducted study investigating an important class of neural representation: namely, the substrate for spatial orientation and navigation. The analyses are very sophisticated - possibly a little too much so, as the basic findings are relatively straightforward and the analyses take quite a bit of work to understand. A difficulty with the study is that it was exploratory (observational) rather than hypothesis-driven. Thus, the findings reveal correlations in the data but do not allow us to infer causal relationships. That said, the observation of increased firing in a subset of neurons following an erroneous choice is potentially interesting. However, the effect seems small. What were the actual firing rate values in Hz, and what was the effect size?

I also feel we are lacking information about the underlying behavior that accompanies these firing rate effects. The authors say "one possibility is that the head-direction signal in the parahippocampal region reflects a behavioral state related to the navigational choice or the lack of commitment to a particular navigational route" which is a good thought and raises the possibility that on error trials, rats are more uncertain and turn their heads more (vicarious trial and error) and thus sample the preferred firing direction more thoroughly. Another possibility is that they run more slowly, which is associated with a higher firing rate in these cells. I think we, therefore, need a better understanding of how behavior differed between error trials in terms of running speed, directional sampling, etc. A few good, convincing raw-data plots showing a remapping neuron on an error trial and a correct trial on the same arm would also be helpful (the spike plots were too tiny to get a good sense of this: fewer, larger ones would be more helpful). It would be useful to know at what point the elevated response returned to baseline, how - was it when the next trial began, and was the drop gradual (suggesting perhaps a more neurohumoral response) or sudden.

Reviewer #1 (Public Review):

In this paper, the authors present evidence from studies of biopsies from human subject and muscles from young and older mice that the enzyme glutathione peroxidase 4 (GPx4) is expressed at reduced levels in older organisms associated with elevated levels of lipid peroxides. A series of studies in mice established that genetic reduction of GPx4 and hindlimb unloading each elevated lipid peroxide levels and reduced muscle contractility in young animals. Overexpression of GPx4 or N-acetylcarnosine blocked atrophy and loss of force generating capacity resulting from hindlimb unloading in young mice. Cell culture experiments in C2C12 myotubes were used to develop evidence linking elevated lipid peroxide levels to atrophy using genetic and pharmacologic approaches. Links between autophagy and atrophy were suggested.

Experiments on GPx4 expression levels, lipid peroxide levels, muscle mass and muscle force generating capacity were internally consistent and convincing. I thought the experiments supporting the view that autophagy contributed to atrophy were convincing. The hypothesis that altered lipidation of autophagy factors contributed was tested or supported in my view. Evidence for muscle atrophy in response to genetic or pharmacologic manipulations is a bit inconsistent throughout the paper, possibly because the small N of some experiments does not provide sufficient power to detect observed numeric differences in the means. The pattern of muscle fiber atrophy by fiber type is consistent throughout the paper but there is variability in which comparisons reached the threshold for significance, again, possibly because of the small N of the experiments. I agree with the authors that altered activity of enzymes in the contractile apparatus provides one explanation for the observed weakness but respectfully wish to point out there are others such as impaired excitation-contraction coupling which is well known to occur in aging.

Reviewer #1 (Public Review):

In this study, the authors characterize the impact of histone deacetylation on spatial regulation of gene expression in the early gastrula embryo. They utilize Xenopus tropicalis as a vertebrate model embryo and focus on maternal HDAC1 and HDAC2 deacetylases to characterize the regulatory role of histone acetylation on zygotic transcription. In particular, they are interested in whether this epigenetic mark positively or negatively regulates gene expression for the presumptive germ layer and contributes spatially to cell lineage integrity in gastrulation.

Using gene expression analysis, they find that HDAC1 and HDAC2 are present maternally in the egg and throughout blastula and gastrula stages. By performing HDAC1 ChIP-Seq, they find that the deacetylase is already bound as early as the Stage 8 blastula - time of genome activation - and that HDAC1 peaks located within promoter regions generally increase over time from blastula to early gastrula, Stage 10.5. Interestingly, the binding of HDAC1 is not dependent on the zygotic transcript, as HDAC1 ChIP-seq peaks show little difference upon alpha-amanitin treatment. Many of the HDAC1 peaks correlate with peaks of both FoxH1 and Sox3, suggesting their role in its deacetylase recruitment to the genome. Examination of epigenetic signatures of HDAC1 bound regions using previously published datasets identifies distinct chromatin binding categories: authors find a strong correlation with H3K27-Ac and pan-H3Kac, and that HDAC1 generally binds to regions free of repressive marks such as H3K9-me3. The authors find that a majority of HDAC1 peaks contain H3K27Ac but not H3K37me3 peaks and approximately ten percent of HDAC1 loci have both activating and repressive marks.

The authors investigate a functional role for histone deacetylation by inhibiting it, using the broad inhibitor TSA, and HDAC1 specific inhibitor VPA. Importantly, they spatially characterize pan-H3K acetylation and gene expression changes in animal cag (AC) and vegetal mass (VG) regions on the embryo. These are very useful datasets that provide new insights into how histone acetylation is tied to the maintenance of lineage integrity. At a global level, they find that TSA inhibition leads to gastrulation arrest and leads to widespread upregulation of H3K acetylation (pan-H3Kac); suggesting that proper regulation of histone acetylation is required for development. Further, they find that previously repressed regions, marked by H3K27me3 show the most upregulation of pan-H3Kac upon TSA treatment. Regionally, they find a number of interesting results upon inhibition of histone acetylation. First, TSA treatment causes dysregulation - upregulation - of the animal cap (AC) pan-H3Kac peaks in vegetal mass (VG), and upregulation of VG peaks in the animal cap. This suggests that lineage specifically is likely maintained in part by HDAC-mediated de-acetylation of germ layer genes. Gene expression characterization in AC and VG explants +/- TSA treatment supports this conclusion as inappropriate upregulation of VG gene expression is found in AC and inappropriate upregulation of AC genes is found in VG. Somewhat surprisingly, HDACs also appears to play a positive regulatory role in germ layer expression. Focusing on genes near HDAC1 peaks containing H3K27Ac, the authors show that genes downregulated upon TSA treatment tend to be spatially restricted; downregulated genes in AC tended to be AC genes and downregulated genes in VG tended to be VG genes. This suggests that HDACs play both positive and negative roles in regulating germ layer expression in the gastrula.

Strengths of the work include the demonstration that histone deacetylase HDAC1 binds to the genome by the onset of genome activation, accumulates in promoters as the embryo develops through early gastrula, and that inhibition of histone deacetylation disrupts germ layer lineage integrity. New datasets include ChIP-seq of HDAC1 from blastula to gastrula, panH3Kac ChIP-seq within animal and vegetal regions of the embryo, and regional RNA-seq of embryos with and without TSA inhibition of histone acetylation. This study helps demonstrate and clarify that HDAC enzymes play both a positive and negative role in gene expression regulation, and that histone acetylation is required to maintain spatial specificity of germ layer expression in gastrula. Some of the weaknesses of the work include the correlative nature of the experiments and missing analysis. Overall, the research is interesting and impactful, contributing to a growing body of work about the role of histone acetylation in the spatial regulation of earliest cell fate decisions in the embryo.

Reviewer #1 (Public Review):

In this work, the authors investigated the mechanism by which ions are selected in ATP-gated P2X receptor channels using patch-clamp electrophysiology. P2X receptors are known to be cation-selective channels, but one of them (the P2X5) also displays anion permeability through a molecular mechanism that is unclear. Here, the authors identify in P2X2 a glutamate residue (E17) which plays a critical role in determining ion selectivity. This residue is localized in the intracellular side of the permeating pathway and is part of three large intracellular lateral fenestrations that are thought to be potential exit/entry pathways for ions. The authors elegantly show that when the side chain of E17 was substituted for cysteine, it became accessible to water-soluble, thiol-reactive methanethiosulfonate (MTS) derivatives that were applied from both sides of the membrane. By mutating E17 into lysine, which reverts the charge, they show that mutated channels displayed increased anion permeability, although channels still remained largely cation selective. However, reverting the charge in the mouse P2X5 (K17E and K17D), they provide evidence for a complete ion selectivity switch (that is mutated P2X5 became cation selective). Therefore, although the mechanism by which P2X2 selects cation versus anion still remains incompletely understood, it seems that K17 is a key determinant for P2X5 anion permeability.

The conclusions of this paper are well supported by data. The work should advance our understanding of ion selectivity in P2X receptors and will likely provide the foundation for further studies.

Reviewer #1 (Public Review):

Authors aimed to decode signatures linked to tremor, slowness and effective motor control using different types of signals acquired from a group of Parkinson's disease patients during deep brain stimulation surgery. They were able to identify distinct frequency bands which corresponded to different symptoms and conclude that multi frequency band and cortical decoding surpass single frequency band and subthalamic nucleus-based decoding.

The main strength of the study is the recording types used to decode symptoms emerging during the same experimental task: authors leveraged micro and macro level recordings from the subthalamic nucleus and ECoG recordings from the motor cortex, enabling them to provide decoding performance across distinct recording scales and from two critical structures linked to Parkinson's pathophysiology. This allowed the authors to contrast rhythm-based signatures and timescales of Parkinson's disease motor symptoms.

The primary weakness is the level of description used to describe key methods which makes it difficult to unpack the results: authors should pay particular attention to validating and justifying metrics used for assessing behaviour (e.g., tremor, slowness, and effective motor control). Also, the relationship between behavioural measures and UPDRS scores should be further justified. For instance, (1) what is the definition of tremor amplitude probability density in the absence of tremor and what is its relationship to relevant subcategories of clinical tremor severity?; (2) why did the authors link tremor while performing a task to UPDRS rest tremor scores? ; (3) why did the authors opt for normalised cursor speed as a metric for slowness?; (4) Are there any implications of this normalisation when exploring slowness across participants? Authors consider cortical and subthalamic recordings separately: if these recordings were acquired simultaneously, analysing the relationship between the two signals (i.e., envelope, phase, phase-amplitude) would significantly improve the paper.

Authors aimed to decode signatures linked to different symptoms of Parkinson's disease. Results support their primary conclusions that cortical decoding performs better than subthalamic decoding and that using a multi-dimensional feature space improves the performance of the decoder. The paper and data generated will contribute to movement control, movement disorders, and brain stimulation fields.

Reviewer #1 (Public Review):

Temporal patterning allows a neural stem cell to generate different neural identities through the course of development. While this relationship has been demonstrated in many instances of stem cells and/or neurons, it is unclear how birth order translates to target specification. In this manuscript, the authors use live imaging and new tools generated from single-cell RNA sequencing data to address this issue.

They find that neurons born from a given time window (at the resolution of early>middle>late) innervate together - and distinctly from - those born at different temporal windows, though the specifics of the innervations differ between neural stem cell lineages. They also find that neurons achieve this by extending their dendrites in exploratory directions and selectively stabilising the ones in the appropriate direction. This process likely occurs at the sub-second timescales. Finally, they also demonstrate that embryonic-born (larval-specific) neurons that remodel to integrate into adulty-specific circuitry simultaneously perform pruning and dendrite extension to integrate into the circuitry at the appropriate time.

This is a valuable description of how developmental programmes imparted to neurons at the time of their birth might translate to their targetting and connectivity. It lays down a framework for understanding the defects in these processes.

Reviewer #1 (Public Review):

Previous studies from this group reported that PEG10 is increased in the spinal cord from Ubiquilin 2-/- mice as well as PEG10 being elevated in models of Ubiquilin 2-mediated ALS. In this study, the authors provide evidence supporting the concept that the proteasome factor Ubiquilin 2 regulates the activity of the Gag-pol retrotransposon gene (PEG10). Mutations in Ubiquilin 2 underlie a portion of the familial forms of ALS. It is found that in spinal cord tissue from sporadic ALS patients PEG10-pol levels are elevated, leading to the conclusion that altered regulation of PEG10 levels by Ubiquilin 2 and a subsequent alteration in genes regulating axon remodeling may contribute to all forms of ALS pathogenesis. Strengths of this work include the extensive analyses and direct data on the mechanism by which Ubiquilin 2 regulates PEG10 levels in human cells resulting in less PEG10. They further show that peptides generated by the self-cleavage of PEG10 alter the expression of genes involved in axon function. The major weakness of this study is the complete absence of data that directly show that an alteration of PEG10 by Ubiquilin 2 is critical for ALS pathogenesis. As noted by the authors, multiple pathways have been broadly implicated in genetics and sporadic forms of ALS. Thus while the study provides interesting data on the regulation of PEG10 by Ubiquilin 2, the extent to which this pathway underlies ALS pathogenesis and/or progression is speculative.

Reviewer #1 (Public Review):

In this study, Jigo et al. measured the entire contrast sensitivity function and manipulated eccentricity and stimulus size to assess changes in contrast sensitivity and acuity for different eccentricities and polar angles. They found that CSFs decreased with eccentricity, but to a lesser extent after M scaling while compensating for striate-cortical magnification around the polar angle of the visual field did not equate to contrast sensitivity.

In this article, the authors used classic psychophysical tests and a simple experimental design to answer the question of whether cortical magnification underlies polar angle asymmetries of contrast sensitivity. Contrast sensitivity is considered to be the most fundamental spatial vision and is important for both normal individuals and clinical patients in ophthalmology. The parametric contrast sensitivity model and the extraction of key CSF attributes help to compare the comparison of the effect of M scaling at different angles. This work can provide a new reference for the study of normal and abnormal space vision.

The conclusions of this paper are mostly well supported by data, but some aspects of data collection and analysis need to be clarified and extended. 1) In addition to the key CSF attributes used in this paper, the area under the CSF curve is a common, global parameter to figure out how contrast sensitivity changes under different conditions. An analysis of the area under the CSF curve is recommended. 2) In Figure 2, CRFs are given for several SFs, but were the CRFs at the cutof-sf well-fitted? The authors should have provided the CRF results and corresponding fits to make their results more solid. 3) The authors suggested that the apparent decrease in HVA extent at high SF may be due to the lower cutoff-SF of the perifoveal VM. Analysis of the correlation between the change in HVA and cutoff SF after M scaling may help to draw more comprehensive conclusions. 4) In Figure 6, it would be desirable to add panels of exact values of HVA and VMA effects for key CSF attributes at different eccentricities, as shown in Figures 4B, D, and F, to make the results more intuitive.

More discussions are needed to interpret the results. 1) Due to the different testing distances in VM and HM, their retinae will be in a different adaptation state, making any comparison between VM and HM tricky. The author should have added a discussion on this issue. 2) In Figure 4, the HVA extent appears to change after M-scaling, although the analysis shows that M-scaling only affects the HVA extent at high SF. In contrast, the range of VMA was almost unchanged. The authors could have discussed more how the HVA and VMA effects behave differently after M-scaling. 3) The results in Figure 4 also show that at 11.3 cpd, the measurement may be inaccurate. This might lead to an inaccurate estimate of the M scaling effect at 11.3 cpd. The authors should discuss this issue more. 4) The different neural image-processing capabilities among locations, which is referred to as the "Qualitative hypothesis", is the main hypothesis explaining the differences around the polar angle of the visual field. To help the reader better understand this concept, the author should provide further discussions.

The authors should also provide more details about their measures. For example, high grayscale is crucial in contrast sensitivity measurements, and the authors should clarify whether the monitor was calibrated with high grayscale or only with 8-bit. Since the main experiment was measuring CS at different locations, it should also be clarified whether the global uniformity of the display was calibrated. In addition, their method of data analysis relies on parametric contrast sensitivity model fitting. One of the concerns is whether there are enough trials for each SF to measure the threshold. The authors should have included in their method the number of trials corresponding to each SF in each CSF curve.

Reviewer #1 (Public Review):

Pasquereau and Turner investigated the encoding of reward and delay information in subthalamic (STN) neurons in behaving macaques. They record during a forced-choice task with three levels of reward and two levels of delay, using rejection rates to model subjective value. Task-dependent neurons, those which encoded reward and/or delay, were identified with a sliding-window regression model. They then investigated the time course of reward and delay information using a principal component analysis approach. They find that the strength of the first and four principal components varies systematically along the anteroposterior axis of the STN, suggesting a spatial distribution of value coding. These data, recorded in a controlled task, add to the understanding of STN function.

The data, collected from a well-defined brain area and with appropriate motor and oculomotor controls included during a straight-forward task, are a good foundation for investigating STN function. However, the statistical procedures used are not completely described and may not be appropriate, particularly in the sliding window analysis. Given this analysis underlies some of the further analyses, it must be clarified or corrected for the conclusions to stand. Further, the analysis only explores the encoding of delay at the time of a cue and does not consider how the value of delay may change over time.

The sliding window analysis, a common approach in investigating time-course data, necessitates multiple comparisons (188 time-bins here) and so requires a controlling procedure to keep the family-wise error-rate low. The authors describe, not completely, how the pre-instruction period was used to establish the boundaries for significance for each coefficient. The pre-instruction period, by the authors' own account, is a period of lower variance and so it would be expected that the boundaries for significance would be lower and the number of task-dependent neurons is therefore an overestimate. The shuffling process the authors use when they determine significance in their principal components analysis is a more appropriate method.

The task design and analysis provide a limited test of delay encoding. As only two levels of delay were tested, it is not possible to directly test whether the subjective discounting function is hyperbolic or exponential and hence whether the delay is encoded subjectively or objectively. Further, the task has several variable interval lengths (hold in: 1.2-2.8 s, short delay: 1.8-2.3 s, long delay: 3.5-4s) that frustrate interpretation. The distribution of these delays is not described, for example as it reads it seems possible that some long delay rewards are delivered with shorter latency between cue and reward than some short delay rewards (1.2 + 3.5 = 4.7s vs. 2.8+2.3 = 5.1 s). The authors have not considered that if the delay value is encoding, then the value, both objectively and subjectively, may be changing as the delay elapses. The variation of these task intervals may have an effect on the value of delay.

The principal components analysis is an interesting way to explore patterns of encoding and the spatial distribution of these patterns. In particular, the finding that Discounting- neurons, those whose firing rate increases with increasing reward cues and decreases with increasing delay cues, are preferentially found in the posterior STN, which the authors demonstrate with both the principal component analysis and the sliding-window classification analysis, challenges previous ideas of STN organization.

Reviewer #1 (Public Review):

In this work, Pan et al. investigate the properties of the underexplored snake venom phosphodiesterase (svPDE) from a genomic, transcriptomic, and structural perspective. These analyses are complemented by comparisons with similar ENPP proteins to better understand the elements that may underline the specific role of svPDE in envenomation. The data support a role for svPDE that may be related to its interactions with partner proteins or due to its phosphodiesterase activity to enhance the cytotoxic effects of other venoms present in the environment.

Overall, the authors have done a good job of investigating the origins and function of svPDE. The evolutionary analyses are adequate and informative, which are expanded by further experiments to determine the structure and interactions of svPDE. The protein-protein interaction experiments and the svPDE activity experiments with different substrate types shed light on the possible role of the protein in the context of its cellular environment and point to the potential role of glycosylation as part of the mode of action of svPDE. These results will pose a good prelude for further research into the mechanism and interactions of svPDE from other species. Further, the mechanistic insights from this work may also help the development of antivenom compounds that target svPDE.

Reviewer #1 (Public Review):

ARL15 forms a complex with the TRPM7 channel and CNNM transporters and is involved in the regulation of the TRPM7 function. To understand the regulatory mechanism, the authors performed biochemical and structural characterizations. In this work, they determined the crystal structure of ARL15 in complex with CNNM2 CBS domain, performed the mutational analysis based on the structure, and successfully revealed the binding mechanism between ARL15 and CNNM.

However, the detailed mechanism of TRPM7 inhibition by ARL15 remains unclear because the structure of TRPM7 in complex with ARL15 is still unknown. Furthermore, despite the structure determination of ARL15 in complex with CNNM, the effect of ARL15 on CNNM function is still unclear.

Nevertheless, the structural information on the ARL15-CNNM complex provided by the authors is valuable for the related research field, and the structure-based CNNM mutants specifically targeting disruption of binding to either ARL15 or PRL would also be useful.

Reviewer #1 (Public Review):

These authors use a mouse model of gestational intermittent hypoxia (GIH), a component of sleep apnea during pregnancy, to test the hypothesis that GIH induces inflammation in the central nervous system that impairs respiratory functions, in a sex-dependent manner. The major finding of this work is that spinal cord inflammation, mainly driven by activated microglia cells, impairs inactivity-induced inspiratory motor facilitation (iMF). The authors successfully test this hypothesis and their results support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model (GIH that sets on later in pregnancy), complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies, and sufficient power to evaluate sex differences. In particular, the authors note the upregulation of several NF-kB regulates genes and increased concentration expression of inflammatory markers in the spinal cords of male mice. These mice also have deficits in the iMF response. By depleting microglia and blocking Ik-kinases, the authors convincingly demonstrate that the increased spinal inflammation is causative in the disruption of respiratory plasticity.

The major limitation as the manuscript is currently written is a clear rationale for evaluating the iMF response as a primary endpoint. One of the corresponding authors is an expert in iMF, but there is no rationale for why it is expected that this aspect of plasticity might be disrupted. The authors discuss breathing and respiratory function in the introduction, but these have not been measured here. It is not known whether GIH impacts respiratory response or baseline breathing in a spontaneous breathing model, including baseline frequency and tidal volume and the ventilatory responses to hypoxia and hypercapnia. Shortening the introduction to offer a clear rationale would be beneficial, given the wide audience of this journal. The limitations of this model, including vagotomy, mechanical ventilation, hyperoxic ventilation, and recording from the phrenic nerve in lieu of respiratory measures, should also be discussed in the discussion for readers not familiar with this model outside of the respiratory control field.