Reviewer #1 (Public Review):

In principle a very interesting story, in which the authors attempt to shed light on an intriguing and poorly understood phenomenon; the link between damage repair and cell cycle re-entry once a cell has suffered from DNA damage. The issue is highly relevant to our understanding of how genome stability is maintained or compromised when our genome is damaged. The authors present the intriguing conclusion that this is based on a timer, implying that the outcome of a damaging insult is somewhat of a lottery; if a cell can fix the damage within the allocated time provided by the "timer" it will maintain stability, if not then stability is compromised. If this conclusion can be supported by solid data, the paper would make a very important contribution to the field.

However, the story in its present form suffers from a number of major gaps that will need to be addressed before we can conclude that MASTL is the "timer" that is proposed here. The primary concern being that altered MASTL regulation seems to be doing much more than simply acting as a timer in control of recovery after DNA damage. There is data presented to suggest that MASTL directly controls checkpoint activation, which is very different from acting as a timer. The authors conclude on page 8 "E6AP promoted DNA damage checkpoint signaling by counteracting MASTL", but in the abstract the conclusion is "E6AP depletion promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner". These 2 conclusions are definitely not in alignment. Do E6AP/MASTL control checkpoint signaling or do they control recovery, which is it?<br /> Also, there is data presented that suggest that MASTL does more than just controlling mitotic entry after DNA damage, while the conclusions of the paper are entirely based on the assumption that MASTL merely acts as a driver of mitotic entry, with E6AP in control of its levels. This issue will need to be resolved.

Finally, the authors have shown some very compelling data on the phosphorylation of E6AP by ATM/ATR, and its role in the DNA damage response. But the time resolution of these effects in relation to arrest and recovery have not been addressed.

Reviewer #2 (Public Review):

This is an interesting study from Admin Peng's laboratory that builds on previous work by the PI implicating Greatwall Kinase (the mammalian gene is called MASTL) in checkpoint recovery.

The main claims of this study are:

1) Greatwall stability is regulated by the E6-AP ubiquitin ligase and this is inhibited following DNA damage in an ATM dependent manner.<br /> 2) Greatwall directly interacts with E6-AP and this interaction is suppressed by ATM dependent phosphorylation of E6-AP on S218<br /> 3) E6-AP mediates Greatwall stability directly via ubiqitylation<br /> 4) E6-AP knock out cells show reduced ATM/ATR activation and quicker checkpoint recovery following ETO and HU treatment<br /> 5) Greatwall mediated checkpoint recovery via increased phosphorylation of Cdk substrates

In my opinion, there are several interesting findings presented here but the overall model for a role of the E6-AP -Greatwall axis is not fully supported by the current data and will require further work. Moreover, there are a number of technical issues making it difficult to assess and interpret the presented data.

Major points:

1) The notion that Greatwall is indeed required for checkpoint recovery hinges on two experiments shown in Figures 5A and B where Greatwall depletion blocks the accumulation of HELA cells in mitosis following recovery from ETO treatment and in G2/M following release from HU. An alternative possibility to the direct involvement of Greatwall in checkpoint recovery could be that Greatwall in HeLA cells is required for S-phase progression (as for example Charrasse et al. suggested). A simple control would be to monitor the accumulation of mitotic cells by microscopy or FACS following Greatwall depletion without any further checkpoint activation.

2) The changes in protein levels of Greatwall and the effects of E6-AP on Greatwall stability are rather subtle and depend mostly on a qualitative assessment of western blots. Where quantifications have been made (Figures 2D and 4F) the loading control and the starting conditions for Greatwall (0 timepoints in the right panel) appear saturated making precise quantification impossible. I would argue that the authors should at least quantify the immuno-blots that led them to conclude on changes in Greatwall levels and make sure that the exposure times used are in the dynamic range of the camera (or film). A more precise experiment would be to use the exogenously expressed CFP-Greatwall that is described in Figure 6 and measure the acute changes in protein levels using quantitative fluorescence microscopy in live cells. This is, in my opinion, a lot more trustworthy than quantitative immuno-blots.<br /> I also note here that most experiments linking Greatwall levels to E6-AP were done using siRNA, while the E6-AP ko cells would be a more reliable background for these experiments, especially with reconstituted controls.

3) This study has no data linking the effects of Greatwall to its canonical target PP2A:B55. The model shown in Figure 9 is therefore highly speculative. The possibility that Greatwall could act independently of PP2A:B55 should at least be considered in the discussion given the lack of experimental evidence.

4) The major effect of E6-AP depletion on the checkpoint appears to be a striking reduction in ATM/ATR activation, suggesting that this ubiquitin ligase is involved in checkpoint activation rather than recovery. It is not clear if this phenotype is dependent on Greatwall. If so it would be hard to reconcile with the default model that E6-AP acts via the destabilisation of Greatwall. In the permanent absence of E6-AP, increased Greatwall levels should inactivate B55:PP2A. How would this lead to a decrease in ATM/ATR activation? This is unlikely, and indeed Figure 5E shows that the reduction of MASTL in parallel to E6-AP does not result in elevated levels of ATR/ATM activation. Conversely, the S215A E6-AP mutant does have a strong rescue impact on ATR/ATM (Figure 8D).

5) In summary, I do not think that the presented experiments clearly dissect the involvement of E6-AP and Greatwall in checkpoint activation and recovery. E6-AP depletion has a strong effect on checkpoint activation while Greatwall depletion is likely to have various checkpoint-independent effects on cell cycle progression.

Reviewer #3 (Public Review):

In this manuscript, Li et al. describe the contribution of the ATM-E6AP-MASTL pathway in recovery from DNA damage. Different types of DNA damage trigger an increase in protein levels of mitotic kinase MASTL, also called Greatwall, caused by increased protein stability. The authors identify E3 ligase E6AP to regulate MASTL protein levels. Depletion or knockout of E6AP increases MASTL protein levels, whereas overexpression of E6AP leads to lower MASTL levels. E6AP and MASTL were suggested to interact in conditions without damage and this interaction is abrogated after DNA damage. E6AP was shown to be phosphorylated upon DNA damage on Ser218 and a phosphomimicking mutant does not interact with MASTL. Stabilization of MASTL was hypothesized to be important for recovery of the cell cycle/mitosis after DNA damage.

The identification of this novel pathway involving ATM and E6AP in MASTL regulation in the DNA damage response is interesting. However, is surprising that authors state that not a lot is known about DNA damage recovery while Greatwall and MASTL have been described to be involved in DNA damage (checkpoint) recovery. In addition, PP2A, a phosphatase downstream of MASTL is a known mediator of checkpoint recovery, in addition to other proteins like Plk1 and Claspin. Although some of the publications regarding these known mediators of DNA damage recovery are mentioned, the discussion regarding the relationship to the data in this manuscript are very limited.

The regulation of MASTL stability by E6AP is novel, although the data regarding this regulation and the interaction are not entirely convincing. In addition, several experiments presented in this paper suggest that E6AP is (additionally) involved in checkpoint signalling/activation, whereas the activation of the G2 DNA damage checkpoint was described to be independent of MASTL. Has E6AP multiple functions in the DNA damage response or is ATM-E6AP-MASTL regulation not as straightforward as presented here?

Altogether, in my opinion, not all conclusions of the manuscript are fully supported by the data.

Reviewer #1 (Public Review):

In this report, the authors use what they describe as a novel phenotypic survival screening method to uncover ATP-dependent kinases that may show synthetic lethality (when inhibited) with BRCA2 loss. Interestingly, they find that inhibiting ROCK kinases in BRCA2 deficient cells (but not BRCA1 deficient cells), triggers synthetic lethality. They further show that the synthetic lethality is independent of acute replication stress and is preceded by enhanced M-phase defects (anaphase bridges and abnormal mitotic figures). These data, therefore, suggest a new pathway (ROCK kinases) that may be targeted to induce synthetic lethality in BRCA2 deficient cells.

Reviewer #2 (Public Review):

This paper describes a novel synthetic lethal interaction between BRCA2 loss and the cytokinesis regulators, including ROCK. The SL effects are restricted to short-term in vitro assays, and the underlying mechanisms remain largely elusive. The impact of the work in its current form is limited.

Strengths:<br /> - A novel synthetic lethal (SL) interaction, which appears independent from the -BRCA2 SL interaction that depends on replication fork stalling and DNA damage induction.<br /> - The SL interaction is validated in a panel of genetic models of BRCA2 deficiency.<br /> - The SL interaction can be induced using clinically approved agents.

Weaknesses<br /> - The evidence that this SL interaction is independent of replication defects is not solid.<br /> - The SL interaction is based on chemical inhibitors only, with 6 out of 9 ROCK inhibitors not demonstrating the SL interaction.<br /> - The mechanisms by which ROCKi specifically affects BRCA2-defective cells are elusive.<br /> - It remains unclear what the cause of the multiple mitotic defects is.

Combined, it remains unclear if the identified SL interaction is therapeutically meaningful. Clinical stage inhibitors are available, and various BRCA2-deficient cancer models have been described, allowing the authors to address this in long-term in vitro assays and in vivo assays. Also, the authors describe multiple phenotypic consequences, but the order of events and the reason why the effects are specific to BRCA2 remain largely unclear. Furthermore, the notion that the observed effects are independent of replication defects requires further substantiation.

Reviewer #3 (Public Review):

Martino et al. demonstrated that BRCA2-deficient cells (but not BRCA1-deficient cells) bear additional vulnerabilities (i.e., cytokinesis failure) outside S phase that could represent new synthetic lethality targets. Strengths of the study include the ability of the authors to recapitulate the cell death by ROCK inhibition by inhibiting another key cytokinesis enzyme, CITK. The claims are well supported by the data, and because the study indicates HR failure/replication stress is not the only possible way to achieve synthetic lethality in BRACA mutant cancers the study will be of broad interest to potential readers.

Reviewer #1 (Public Review):

The manuscript by Zheng et al. examined the disease-causing mechanisms of two missense mutations within the homeodomain (HD) of CRX protein. Both mutations were found in humans and can produce severe dominant retinopathy. The authors investigated the two CRX HD mutants via in vitro DNA-binding assay (Spec-seq), in vivo chromatin-binding assay (ChIP-seq), in vivo expression assay of downstream target genes (RNA-seq), and retinal histological and functional assays. They concluded that p.E80A increased the transactivation activity of CRX and resulted in precocious photoreceptor differentiation, whereas p.K88N significantly changed the binding specificity of CRX and led to defects in photoreceptor differentiation and maintenance. The authors performed a significant amount of analyses. The claims are sufficiently supported by the data. The results not only uncovered the underlying disease-causing mechanisms, but also can significantly improve our understanding of the interaction between HD-TF and DNA during development.

Minor concerns:<br /> 1. The E80A, K88N and R90W (previously reported by the same group) mutations are located very close to each other in the homeodomain (Figure 1A), but had distinct effects on the activity of CRX. Has the structure of the homeodomain (of CRX) been resolved? If so, could the authors discuss this phenomenon (mutations close to each other but have distinct effects) based on the HD-DNA structure? In addition, has this phenomenon been observed in other homeodomain TFs?<br /> 2. The authors should briefly summarize the effects/disease-causing-mechanisms of all the reported CRX mutations in the discussion part. The readers can then have a better overview of the topic.<br /> 3. CRX can also function as a pioneer factor (reported by the same group). Would these HD mutations distinctively affect chromatin accessibility (which then leads to ectopic binding on the genome)?<br /> 4. The discussion part can be shortened and simplified.

Reviewer #2 (Public Review):

Zheng et al., investigated the molecular and functional mechanisms of two homeodomain missense mutations causing human retinal photoreceptor degeneration diseases in photoreceptor development regulated by the CRX transcription factor. They analyzed the E80A mutation associated with dominant cone-rod dystrophy (CRD) and the K88N mutation associated with dominant Leber Congenital Amaurosis (LCA). The authors found that E80A CRX binds to the same target DNA sites as WT CRX, but the binding specificity of K88N CRX is altered from that of WT in an in vitro assay. They generated Crx(E80A) and Crx(K88N) KI mice and performed ChIP assay and observed that K88N CRX binds to novel genomic regions from the WT-binding sites, while E80A binds to the WT sites. In addition, using the KI mice, they found that E80A and K88N differently affect the expression of Crx target genes. This study is well executed with proper and solid methodologies, and the manuscript is clearly written. This study gives us the insights how single missense CRX mutations lead to different types of human retinal photoreceptor degeneration diseases.

While the study has strengths in principle, it has a couple of weaknesses. One is how well E80A KI mice function as a pathological model of dominant CRD, in which cones are mainly first affected, is not clearly shown in this study. More data investigating how cones are affected by performing histological, molecular, and physiological analyses will be helpful and useful. For example, in the Discussion, the authors describe that E80A associates with S-cone opsin promoter results is "data now shown". This data must be presented for the readers. In addition, more molecular insights as to how E80A affects cones will strengthen this study. Another point is that it will be very valuable if the authors could show how E80A and K88N differently affect the 3D structure of the CRX homeodomain. Even a simulation model would be valuable.

Reviewer #1 (Public Review):

Zhou et al. investigated the factors that regulate mitotic chromosome size scaling during the early embryo divisions in Xenopus laevis using imaging of intact whole embryos and of embryo extracts with different sources of nuclei. They find that chromosome volume decreases during embryogenesis, and scales with nuclear and spindle volume throughout a broad range of embryo stages (stages 3 to 9) and cell sizes. They show that extracts from stage 3 or stage 8 embryos demonstrate significant differences in chromosome length, mirroring changes to chromosome volume observed in vivo. Using extracts from eggs or stage 8 embryos, and nuclei from sperm or stage 8 embryos, the authors demonstrate that chromosome length is dictated by the chromosomes and not the maternal mitotic environment, and find that the major determining factor is the amount of condensin I loading on mitotic chromosomes, which they correlate to changes in DNA loop size and layering. Interestingly, they find that the prior state of nuclei prior to entry into mitosis dictates mitotic chromosome length. They attribute this phenomenon to the nuclear to-cytoplasmic ratio during the prior interphase and suggest that some factor is titrated on chromatin that sets condensin I loading in mitosis. Notably, they found that chromosome length does not scale with nuclear or spindle size in vitro. In another set of experiments, the authors found that artificially increasing the palmitoylation of importin resulted in decreased chromosome length. However, this scaling effect is not due to condensin I loading differences, but to some unidentified importin cargo that would get released as cell size decreases during development. Overall, the conclusions of this paper are well supported by data, but some aspects of data interpretation and analysis need to be clarified and extended. The approaches used here are quite impressive and creative and provide compelling evidence for factors that regulate chromosome scaling during development in a vertebrate organism.

Reviewer #2 (Public Review):

In this work from Zhou et al., the authors address mechanisms of mitotic chromosome size scaling during development. Their approach, which employs complementary use of in vivo (Xenopus embryos) and in vitro systems (Xenopus extracts), rendered investigation of this relationship experimentally tractable and allowed the authors to convincingly demonstrate that mitotic chromosome scaling is mediated by differential loading of maternal chromatin remodeling factors during interphase. The authors show that this scaling is dependent on an increasing nucleo-cytoplasmic (N/C) and that condensin I is titrated away from chromosomes as the N/C ratio is increased. Interestingly, the authors found that spindle and nuclei did not scale with changes in N/C ratio, suggesting that although mitotic chromosome scaling correlates with spindle and nuclei scaling, it is mechanistically distinct. Complementary Hi-C analyses of chromatin architectures of both larger condensin I-rich chromosomes and smaller condensin I-poor chromosomes support a condensin-based looping model to explain the inverse relationship between chromosome-associated condensin and chromosome length, however, this model seems somewhat contrived due to inherent limitations of the approach. A characterization of an independent importin-α-dependent mitotic chromosome scaling mechanism, though potentially interesting, is too premature to be included and a bit of a non sequitur in terms of the overarching narrative and major findings of the work. Though there is some room for improvement in terms of image analysis and measurements, the work is well-written, comprehensive in scope, and addresses a fundamental biological question. Furthermore, the authors' major conclusions and substantive claims are well-supported by the experimental results.

Reviewer #3 (Public Review):

In the work by Zhou, et.al., the authors pursue a mechanistic understanding of chromosome size scaling in development, a problem first noted in 1912 by Conklin and largely unstudied until very recently. Using the tools available in the Xenopus genus developmental biology system (cell extracts, related species of differing size, etc.), they nicely show that condensin I levels directly correlates with chromosome size. Further, importin levels decrease leading to axial shortening of chromosomes during development. The combined physical outcome is that Mitotic Chromatin looping changes, resulting in axial compression of chromosomes. This work represents a major step in the molecular understanding of how the genome is regulated through development and changing cell size, which also occurs in many other adult tissues and cancers. Further work to understand other contributing factors and understanding how loop structure changes the polymer dynamics of mitotic chromatin will be exciting in the future.

Peer review report

Title: Publication Patterns and Perceptions of Open Science in Indian Scholarly Community: Insights from a Survey

version: 2

Referee: Moumita Koley

Institution: Indian Institute of Science, Bangalore, India

email: moumitakoley@iisc.ac.in

ORCID iD: 0000-0003-2394-0663

General assessment

The author of the article provides a limited perspective on Publication Patterns and Perceptions of Open Science in the Indian Scholarly Community. This article sheds some light on open science practices, considering the scarcity of data on this topic. The main drawbacks, the survey conducted in the article is the only methodology used, and it is limited to a small group of researchers. Understanding publication patterns (Open Access publication and use of preprints) should be made using bibliometric studies. Another drawback of the article is that it primarily focuses on researchers from the agriculture field, which is over-represented and makes it misleading to claim that the study represents the entire Indian scholarly community. This is particularly problematic since the physical and chemical sciences dominate the Indian research community, and data from these fields are entirely absent in the article.

Essential revisions that are required to verify the manuscript

This study is at an early stage; more data points and representations of various fields are necessary to claim validity. Moreover, a mixed-method approach is more suitable.

Other suggestions to improve the manuscript

The introduction section mentions several preprint servers, but some are not operational. For example, ArabiXiv is not accepting new submissions, and IndiaRxiv has few submissions. Since there are limited responses from other South Asian countries, the author has chosen to focus on India. However, it is unclear if the percentages of career levels of professionals are representative of India alone.

One drawback of this article is the over-representation of researchers from the agriculture field. In the Indian ecosystem, agricultural research institutes are separate from the general university system and governed by different funding and governance systems. Therefore, the norms and practices can vary significantly.

In the STEM subjects, Chemical Sciences contributed the most publications in India from 2015-2019, followed by Physical Sciences. Biological Sciences had fewer publications during this period(the method used for this statement: a quick search in the Web of Science). However, this data does not represent the publication behaviour of the major constituents (Physical and Chemical Science) of the Indian academic community. Therefore, it is suggested to shift the narrative towards agricultural science.

The statement "patent and scholarly data website, India has produced 19,76,966 scholarly works till date" lacks a timeframe.

The APC section statements are unclear. As far as current knowledge goes, no study has shown a correlation between JIF and APC. The statement "Was it because of JIFs they must publish in Open Access when there is Green Route to Open Access (depositing in subject or institutional repositories)" is unclear. Moreover, while Indian national funding agencies have green OA mandates, they have not been enforced. Several studies indicate that Indian researchers' adoption of green OA is low, so authors have no obligation to publish OA.

The article concludes that advocacy is needed, but it is essential to understand the research assessment frameworks of Indian academia and funding agencies. Without recognition in assessment, researchers are unlikely to adopt Preprints. Preprint submission is becoming the norm in some disciplines, which may improve the situation.

Decision

Requires revisions: The manuscript contains objective errors or fundamental flaws that must be addressed and/or major revisions are suggested.

Reviewer #3 (Public Review):

International researchers from the International Agency for Research on Cancer and cancer screening program experts from six countries in Latina America (Argentina, Colombia), Asia (Sri Lanka, Bangladesh, Thailand), and Northern Africa (Morocco), provide detailed information on the impact of the COVID Pandemic on cancer screening and diagnostic services.

The authors examine countries that have had screening programs and a surveillance system/registry to see how the volume of screening, diagnostic procedures, and detection of precancers or cancers are impacted. The data are presented as case studies with an explanation of the program, the technologies, and then the impact. They describe no matter how low or high income the country, there was a considerable impact on the volume of screening.

Usually, the impact of the pandemic on cancer screening has been limited to Europe or North America and is usually not quantified. This information will be helpful for these countries to examine the impact on stage distribution and eventually mortality impact through modeling studies. The authors also comment on some interesting hypotheses such that the impact on recovery based on if one is detecting precancers (e.g. colon cancer/cervical cancer) vs. invasive cancers (breast cancer). Strategies that require less frequent screening,self-collection, where screening and treatment can be combined in fewer visits, or where some visits can occur via telehealth are valuable strategies or lessons learned that will allow for quicker recovery time after a pandemic.

The authors acknowledge the limitations and strengths of these case-based studies well.

It is beautiful storytelling with both qualitative and quantitative data.

Reviewer #1 (Public Review):

The impact of the COVID-19 pandemic on cancer screening, diagnosis, referrals, and management has been well documented in high-resourced countries; but such quantitative estimates are rarely available from low- and middle-income countries (LMIC). The authors chose two very high human development index (HDI) category LMICs (Argentina and Thailand), two high HDI category LMICs (Colombia and Sri Lanka), and two medium HDI category LMICs (Bangladesh and Morocco), and looked at available data for cervical, breast, and colorectal cancer screening. The authors demonstrate that the reduction in the test volumes during the pandemic (2020) versus the previous year (2019) was quite comparable to that observed in high-income countries. Additionally, some countries demonstrated resilient catch-up of programmatic performance within a short period of time after the disruptions.

Major strengths include the use of national-level data estimates from key focal points for the CancScreen-5 project, an international data repository of cancer screening programmatic data, the use of appropriately comparable monthly estimates in the pre-pandemic vs. pandemic year, and representation of illustrative case studies from six countries across the medium-to-very high HDI status among LMICs.

Weaknesses include inherent limitations of such real-world outcome/registry data, lack of data across the screening continuum, inability to explore granular-level country-specific factors affecting disruptions as well as catch-up of screening, and high variability of performance of screening tests (especially those with subjective interpretation such as VIA for cervical cancer or clinical breast exam) across the comparison periods such that screen positivity rates may have been affected in unpredictable ways.

The authors have achieved their aims since this descriptive epidemiology analysis provides key estimates from LMICs that have not been explored/evaluated in the literature.

This work will be useful for future studies conducted by health modellers on measuring the impact on late/advanced stage detection and excess case burden and mortality.

Reviewer #2 (Public Review):

The Covid-19 pandemic has had major adverse impacts on cancer screening globally. Despite this, most prior reports have not included observations from LMICs. This paper aims to address this important gap.

Because comparable data were not available across the countries reported here, comparisons would not be appropriate, so the authors chose a case study design, which was a prudent decision and a strength of the work.

The authors make use of data from IARC's CanScreen5 reporting system, which is completely appropriate. In addition, this aspect serves to demonstrate the usefulness of the CanScreen5 system, as it can be used to support this type of study. National data were not available in all countries.

The main findings in the paper describe the early impact of the Covid-19 pandemic on cancer screening participation for the screening programs reported on in the 6 countries that were selected.

I would anticipate that, having demonstrated that this type of case study focusing on cancer screening in LMICs is feasible, this would encourage others to conduct further studies among LMICs, which would be welcomed by the field.

Reviewer #1 (Public Review):

This paper is based on the premise that ketamine exerts antidepressant effects that are rapid by increasing glutamatergic transmission. However, the authors note that how this effect occurs is unclear because ketamine antagonizes the NMDA receptor, a glutamatergic receptor.<br /> Others have suggested a compensatory change in the glutamatergic transmission and the authors suggest how this might occur. The authors should clarify if prior studies suggested a mechanism different from theirs and if so, which might be correct.

There are also other mechanisms, such as the block of NMDA receptors on interneurons and the disinhibition of principal cells. It is important to clarify if this has already been addressed in the literature. Also, if their cultures are primarily glutamatergic neurons or they include interneurons and glia.

The authors show calcineurin is reduced after ketamine exposure and this increases AMPA receptor GluA1 phosphorylation. They also show that Calcium permeable AMPA receptors )CP-AMPARs) increase.

They also use suggest that the CP-AMPARs and other changes lead to enhanced synaptic plasticity, which could lead to antidepressant effects.

Although a lot of work is done in cultured hippocampal neurons, 14 days in vitro, they show effects in vivo that are consistent with the data from cultures. For example, ketamine increases GluA1 phosphorylation. Also, blocking CPAMPARs in vivo reduces anxiety/depressive behaviors such as the open field and tail suspension tests.

Overall the study appears to be done well and the presentation, writing, and references are good. There are important concerns regarding statistics, behavior, and pharmacology and several minor concerns.

Major concerns<br /> 1. Statistics.<br /> What was the stat test if the control was always 1?<br /> Often the control group is 1.00 with no SD but in other tests, the control group is 1.000 with an SD.<br /> e.g., line 145: "(CTRL) (CTRL, 1.000 and ketamine, 1.598 {plus minus} 0.543, p = 145 0.0039), but not GluA2 (CTRL, 1.000 and ketamine, 1.121 {plus minus} 0.464, p = 0.6498"

Line 188:<br /> Here the control group has a SD:<br /> Line 188 CTRL, 1.000 {plus minus} 0.106 and ketamine, 0.942 {plus minus} 0.051, p = 0.0170

2. Behavior.<br /> It is not clear that the open field and tail suspension tests measure antidepressant actions. Why were more standard tests such as forced swim or sucrose preference, novelty-suppressed feeding, etc not used?

3. Pharmacology.<br /> The conclusions rest on the specificity of drugs.<br /> Is 5 uM FK506 specific?<br /> 20 μM 1-naphthyl acetyl spermine (NASPM)?<br /> 10 mg/kg IEM-1460?

Reviewer #2 (Public Review):

The abstract and introduction framework asserts that ketamine's enhancement of excitatory synaptic drive in the hippocampus is presumed to underlie its rapid antidepressant effects. This is not the only, and perhaps not the primary effect mechanism suggested by prior experiments, also strongly implicating disinhibitory effects in the prefrontal cortex as necessary and sufficient to mediate antidepressant effects. Nevertheless, it is valuable to seek mechanistic motifs that provide multiple paths for explaining the seemingly counterintuitive effects where NMDAR blocker enhances excitatory transmission. These need not be conserved across brain regions and cell classes. The primary result of this study demonstrates that 1 hr-long ketamine application to cultured cells reduces calcineurin and GCaMP activity to elevate AMPA receptor subunit GluA1 phosphorylation and enhance the expression of Ca2+-permeable, GluA2-lacking (CP-)AMPARs. These observations are then evaluated in vivo, where calcineurin shows a similar response to ketamine and CP-AMPAR antagonist-abolished ketamine effects on behavior in the open field and tail suspension tests. One significant uncertainty this study helps resolve is whether GluA2-containing AMPARs are removed from synapses or whether GluA2-lacking AMPARs are inserted following ketamine administration. GCaMP imaging, FRET and glutamate uncaging assays provide a strong complement to biochemistry and in vivo data. There are several significant technical and conceptual limitations in this work, which substantially limit the extent of conclusions that can be drawn at this point.

1. The age of neurons in cell culture experiments was 14 days in vitro (DIV), representing developing cultures that are just starting to form synapses. How these effects carry over to more mature cultures or adult animals is unclear.

2. Phosphorylation analyses, forming the foundation of this work, are carried out 1 hr after ketamine treatment. This is prior to the observed clinical effects of ketamine and this point should be acknowledged. Whether and how long this effect lasts remains to be examined. If the goal is to highlight the earliest likely effects of ketamine that should precede potential clinical effects, this should be acknowledged, and in that case, the onset of effects should be clarified. At this point, the temporal features remain undersampled, with a single time-point.

3. A lower dose (50%) treatment was used to evaluate potential sex differences in ketamine effects, which is not sufficiently justified, except post hoc based on behavioral data. The discussion section does consider potential factors that can account for observed differences.

4. The 1-hr timeline to behavioral testing is fast, relative to clinical effects on behavior as well as behavioral effects measured in most studies using mouse models.

5. Tail suspension test is broadly acknowledged as an inadequate model of antidepressant effects.

6. There is no evidence from the in vivo experiments that effects in the hippocampus are due to direct actions of ketamine, as those reported for the cell culture studies. Intraperitoneal injections cannot be used to localize primary effects in vivo to the hippocampus, which would require local delivery.

7. If (MNI)-caged L-glutamate was used at 1 μM concentration, as stated in methods, this is considerably below typical concentrations reported in the literature.

Reviewer #3 (Public Review):

Ketamine has been shown to be effective at producing a rapid-antidepressant effect at low doses, but the underlying molecular mechanism of this effect is still not clear. Previous studies have suggested that the effect of low-dose ketamine may occur by promoting neuronal plasticity in the hippocampus. However, this goes against the findings that ketamine acts as a noncompetitive NMDA receptor antagonist, which should prevent NMDAR-dependent plasticity. Furthermore, a therapeutic dose of ketamine has been shown to increase neuronal Ca2+ signaling, which again does not conform to its antagonistic action on NMDA receptors. In this paper, the authors provide evidence that therapeutic low-dose ketamine increases the expression of Ca2+-permeable AMPA receptors (CP-AMPARs) by increasing phosphorylation of GluA1 subunit of AMPARs and surface expression of GluA1-containing CP-AMPARs. They further provide evidence that this is likely mediated by a decrease in calcineurin activity and that blocking CP-AMPARs prevent the antidepressant effect of ketamine in mice. One interesting finding of this study is that the authors see heightened sensitivity of ketamine in female mice, both at the level of behavioral readout and for molecular correlates. This finding is interesting in light of the different pharmacokinetics of ketamine reported in females and that ketamine metabolites can bind estrogen receptors.

Based on their data and previous findings, the authors outline a plausible molecular signaling mechanism for the antidepressant effect of ketamine. Specifically, the authors propose that reduced neuronal activity, which could be triggered by ketamine-induced NMDAR antagonism, causes homeostatic plasticity to upregulate GluA1-containing CP-AMPARs. Their data would support this idea, as phosphorylation of GluA1 as well as increased surface expression and functional incorporation of CP-AMPARs at synapses have been shown before in models of homeostatic plasticity.

Overall, the study is well-done and the data presented support the main conclusions. One main question is whether the current finding provides a conceptual advancement in our understanding of the molecular signaling involved in ketamine's antidepressant effects. There are previous studies that showed an increase in CP-AMPARs in the nucleus accumbens and an increase in the expression of GluA1 in the hippocampus with low-dose ketamine. In addition, ketamine's antidepressant effect has been shown to require GluA1 phosphorylation. The main contribution of this paper might be that it provides the potential molecular signaling within the same preparation (i.e. hippocampal neurons) and provides a causal link of CP-AMPARs in mediating the behaviorally measured antidepressant effect of ketamine.

Another question is whether the behavioral effect of ketamine is due to molecular changes in the hippocampus as outlined in this paper. A more targeted inhibition of CP-AMPAR function could resolve this issue. With the systemic application of CP-AMPAR antagonist as done in this study, it would be hard to know the role of CP-AMPAR upregulation in the hippocampus in mediating ketamine's effect. Especially, considering that low-dose ketamine has been shown to upregulate CP-AMPARs in the nucleus accumbens. While it would have been nice to know the site of action, this does not alter the conclusion that CP-AMPARs are involved in mediating the antidepressant effect of ketamine on behavioral readouts.

Reviewer #1 (Public Review):

The essentiality of Rv1636 has previously been predicted in numerous genetic studies. Here, the authors provide evidence that Rv1636 is an essential protein in Mtb. The authors report that chromosomal deletion of the gene encoding Rv1636 is only possible when an additional copy of the wild type gene is provided at the L5 integration site in the chromosome. While this is a standard method of demonstrating gene/protein essentiality in this system, the manuscript only provides a PCR reaction with "no amplicon" as proof of a double crossover event in an engineered merodiploid strain (Fig 6C). The authors fail to provide definitive evidence for a double crossover mutation in the merodiploid using primers that amplify a double crossover-dependent amplicon or the authors should a provide a southern blot demonstrating evidence for a bona fide double crossover event. The authors suggest that silencing the gene encoding Rv1636 with a CRISPRi system decreases viability of Mtb when a silencing guide RNA is expressed following Atc addition and spot plated onto agar. These studies lack a "no Atc control" and it is unclear how Mtb colonies appear after 6-7 days in these studies given the slow growth of this bacterium.

A sub-point of the manuscript describes the genetic organization around the gene that encodes Rv1636 in various Mycobacterial spp. Figure 1 also highlights the putative transcriptional start sites for the gene encoding Rv1636. The putative transcriptional start site information is just a summary of work from other groups and this information adds little to the main goals of this manuscript.

Another sub-point of this manuscript is that Rv1636 may be secreted by Mtb in a SecA2 dependent manner. The authors demonstrate that Rv1636 is not present in the culture filtrate of Mtb lacking SecA2 (Fig 2). However, these data are difficult to interpret without a secreted protein "loading control" which is typical for these types of experiments. The authors also report the development of a luciferase-based detection method for quantifying protein secretion in Mtb and use this to support their conclusion. This is a new tool that could be useful in detecting secreted proteins in Mtb. However, this method is not rigorously validated in these studies and do not present controls for cell lysis for example. Additionally, the authors fuse a ~19 kDA luciferase subunit to the C-terminus of CFP10 as a reporter for Esx1-dependent secretion. It is known that this region of CFP10 is critical for interactions with secretory components of the Esx1 system fractionation and it unclear if the CFP10 fusion protein is actually secreted.

The authors explore the idea that Rv1636 may potentially function as a "sink" for cAMP and quantify the molar amounts cAMP, ATP, and Rv1636 in Mtb. These studies demonstrate that the molar amounts of Rv1636 exceeds the levels of cAMP (free or protein-bound) in the cytosol of the Mtb. The authors conclude that the excess of Rv1636 may potentially be a "sink" for unbound cAMP but do not test this idea experimentally in Mtb due to the very low levels of cAMP in this bacteria.

Instead, the authors continue exploring the idea that specific proteins can serve as a cAMP "sink" using M. smegmatis (Msm) since this bacterium produces more cAMP (~25x) in the cytosol compared to Mtb. The authors present data that over expression of Rv1636 in Msm increases the amount of protein-bound cAMP. It is presumed here that the protein-bound cAMP is bound to Rv1636. Alternatively, deleting the Rv1636 homolog in Msm (MSMEG_3811) results in an increase in the amount of "free cAMP". Again, it is presumed that deleting the cAMP binding protein MSMEG_3811 is responsible for the increase in the amount of "free cAMP" in the cell.

Lastly, the authors use two small molecule compounds that may bind Rv1636 and demonstrate some level of bacterial inhibition using a spot plating method. No evidence is provided to demonstrate that these compounds are specifically binding/inhibiting Rv1636. These studies are lacking rigorous demonstration of "on target" inhibition and add very little to the reliable conclusions in this paper.

Reviewer #2 (Public Review):

In this paper by Banerjee et al., the authors described the potential role of two universal stress proteins in M. smegmatis and M. tuberculosis in regulating intracellular free cAMP concentration, which was a unique observation. The experiments were logically designed to prove the expression and interactions; it would have been worthwhile to explore beyond to gain an insight into how the changing levels of free cAMP could modulate any key phenotypes in the bacteria such as virulence, antibiotic resistance, etc. in the content of knockout/knockdown and overexpression of MSMEG_3811 and Rv1636 in individual organisms. The preliminary data of natural inhibitor STOCKIN43384 impacting the survival of M. smegmatis was interesting, but authors need to prove the MOA by using knockdown and overexpression strains of Rv1636.

Reviewer #3 (Public Review):

This paper describes fundamental work which attempts to understand how universal stress proteins Rv1636/Msmeg3811 function as a sink allowing mycobacteria to use intra-bacterial cAMP. Because cAMP is a major second messenger, Rv1636 remains essential to mycobacteria. A compound that inhibits cAMP binding of Rv1636 also can effectively inhibit mycobacterial growth. The major strength of the manuscript is that the authors probed their hypothesis by different approaches. In general, the conclusions from the results are largely justified. However, I find the manuscript quite difficult to follow. Also, the results and functional analyses are inadequate as they rely on a limited set of experiments, thereby making the evidences less than compelling.

Reviewer #2 (Public Review):

The evolution and control of the three-part life history of holometabolous insects have been controversial issues for over a century. While the functioning of broad as a master gene controlling the pupal stage and of E93 as a master gene for the adult stage has been known for about a decade or more, chinmo has only recently been proposed as being the master gene responsible for maintaining the larval stage (Truman & Riddiford, 2022). While the former paper focused on the embryonic and early larval function of Chinmo, this paper explores its metamorphic effects and defines the roles of Broad and E93 in the phenotypes produced by manipulations of Chinmo expression.

Overall, the paper is well presented but in places, readers would be helped if the authors were more explicit about the logic and details of their manipulations. There are a couple of conceptual issues that the authors should address.

The role of Broad in larval tissues:<br /> One intriguing issue relates to the relationship of Chinmo to Broad and E93 in larval versus imaginal tissues prior to metamorphosis. The knock-down of chinmo in imaginal discs results in severe suppression of growth and the lack of metamorphic patterning genes such as cut and wingless. Normal growth and patterning are reestablished though, if broad is also knocked-down, supporting the notion that the effects of the lack of Chinmo are mediated through the premature expression of Broad.<br /> In the salivary glands, by contrast, chinmo knock-down suppresses growth, and this growth suppression is not reversed by simultaneous broad knockdown. They properly conclude that the role of Chinmo in supporting the growth of larval tissues does not involve Broad, but their data on the expression of salivary gland proteins suggest that Broad still plays some role in Chinmo function in salivary glands. Fig. 5E shows the levels of various salivary glue proteins in the glands of Chinmo knock-down larvae. The levels are reduced, as expected by the lack of salivary gland growth, but a significant finding is that they are there at all! The Costantino et al. (2008) paper shows that these genes are only induced in the mid-L3. Ecdysone, acting through Broad isoforms, is necessary for their appearance and these SGS genes can be induced in the L1 and L2 stages by ectopic expression of some Broad isoforms. Their low levels in Fig 5, would be due to the small size of the gland, but the gland's premature expression of Broad likely causes their induction. In larval cells, then, Chinmo may feed into two parallel pathways, one that does not involve broad and regulates growth and the other, utilizing Broad, regulating premetamorphic changes.<br /> It would be useful to look at early larval salivary gland proteins such as ng-1 to -3 that are expressed in salivary glands before the critical weight. Also, it would be interesting if the appearance of the SGS proteins after chinmo knock-down (Fig 5E) is abolished by simultaneous knock-down of broad.

Role of Chinmo and Broad in Hemimetabolous insects:<br /> In the conclusion of their comparative studies on the cockroach (line 342), the authors state that Broad exerts no role in the development of hemimetabolous insects. However, this conclusion is not consistent with the literature. The first study of broad knockdown in a hemimetabolous insect was in the milkweed bug Oncopeltus fasciatus by Erezyilmaz et al. (2006). Surprisingly to Erezyilmaz et al., broad knock-down in early-stage nymphs did not cause premature metamorphosis. However, Broad expression was essential for tissues of the wing pads and dorsal thorax to undergo morphogenetic growth (rather than simple isomorphic growth), and for stage-specific changes in coloration through the nymphal series (but not for the nymph to adult color change). A similar function for Broad on wing growth during the later nymphal stages was later shown in Blattella (Fernandez-Nicolas et al., 2022; Huang et al., 2013). The wing- and genital pads represent "imaginal" tissues in the nymph and the need for Broad in these tissues are the same as seen in imaginal discs as the latter shift from isomorphic growth to morphogenesis at the critical weight checkpoint in the L3.<br /> This would suggest that important roles for Broad and E93 are already established in the hemimetabolous insects with E93 controlling the shift from immature (nymphal) to adult phenotypes and Broad controlling the premetamorphic growth of imaginal tissues in early-stage nymphs. Chinmo might then be needed to keep both in check.

Reviewer #1 (Public Review):

This study demonstrates that Chinmo promotes larval development as part of the metamorphic gene network (MGN), in part by regulating Br-C expression in some tissues (exemplified in the wing disc) and in a Br-C independent manner in other tissues such as the salivary gland. I have included below the following comments on the submitted version of this manuscript:

1. The authors have shown experimentally that Chinmo regulates Br-C expression in the wing disc but not the larval salivary gland. Based on this, they posit that Chinmo promotes larval development in a Br-C-dependent manner in imaginal tissues and a Br-C-independent manner in other larval tissues. This generalization of Chinmo's role in development would be more compelling if the relationship between Chinmo and Br-C were explored in other examples of imaginal/larval tissues.

2. Chinmo, Br-C, and E93 have all been shown to be EcR-regulated in larval tissues, including the brain and wing disc (as in Zhou et al. 2006, Dev Cell; Narbonne-Reveau and Maurange 2019, PLOS Biology; Uyeharu et al. 2017, ). It would be interesting (and I believe relevant to this study) to know whether the roles of these factors in their respective developmental stages are EcR-dependent and whether their regulation by EcR (or lack thereof) depends on whether the tissue is larval or imaginal.

3. In the chinmo qPCR analysis shown in Fig1A, whether animals were sex-matched or controlled was not indicated. Since Chinmo has a published role in regulating sexual identity (Ma et al. 2014, Dev Cell; Grmai et al. 2018, PLOS Genetics), and since growth/body size is known to be a sexually dimorphic trait (Rideout et al. 2015, PLOS Genetics), it seems important to establish whether the requirement of Chinmo for larval development and/or growth. I recommend either 1) controlling for sex by repeating qPCRs in Fig 1A in either males or females, or 2) reporting male/female chinmo levels at each stage side-by-side.

4. In Fig2E, the authors show that salivary gland secretion (sgs) genes are repressed in salivary glands lacking chinmo. Sgs genes are expressed during late larval stages as the animal prepares to pupate. Thus, based on the proposed model where Chinmo promotes larval development and represses the larval-to-pupal transition, one might expect that larval salivary glands lacking chinmo would express higher than normal levels of sgs genes. This expectation directly opposes the observed result - it would be helpful to speculate on this in the interpretation of results.

Reviewer #2 (Public Review):

Like humans, Bengalese finches rely on auditory feedback to maintain the acoustic stability of their learned vocalizations, and deafening causes acoustic degradation of their songs. How disruptions to sensory input alter gene expression in brain regions important for singing and song learning remains relatively unexplored. The authors develop an innovative serial laser capture RNA-sequencing method, which allows them to conduct large-scale analyses of gene expression in spatially defined singing-related regions, as well as in surrounding non-singing-related regions. These methods are used to demonstrate that deafening preferentially alters gene expression in song-related regions relative to surrounding song-related areas, and that deafening reduces correlations in gene expression between connected song-related regions. The authors then compare their findings to a previous single-cell RNA sequencing dataset to determine the cell types whose gene expression is likely to be most strongly affected by deafening and song degradation. Finally, the authors repeat their measurements of gene expression changes in RA following unilateral lesion of LMAN and find that LMAN lesions have the largest effect on groups of genes whose expression was also strongly affected by deafening. The study is elegant and rigorous, and its conclusions are well-supported. This work reveals candidate genes that may play a role in stable vocal performance and whose changes in expression may contribute to the acoustic degradation of vocal performance following deafening.

Reviewer #1 (Public Review):

In this manuscript, the authors seek to define the transcriptional response to deafening in the songbird brain. They compare transcriptional changes in the song regions with changes in the non-singing-associated surrounds, compute a song degradation score against which they can compare gene expression, and they use single-cell sequencing data from these brain regions to map genes to cells. The study is impressively comprehensive for time points, replicates, brain regions, comparisons, and alternative strategies (e.g. the LMAN lesions). This dataset builds nicely upon studies that assessed gene expression changes upon singing and applies a broad and useful series of bioinformatics analyses to get the strongest evidence for function from the data.

I think this dataset will be of great interest to a broad range of researchers who study neuronal plasticity mechanisms.

Reviewer #3 (Public Review):

In this study, the authors probe the molecular changes that occur in a neural circuit for learned behavior that depends on sensory input to maintain stereotypy. Songbirds, as the Bengalese finches used here are, are premier systems in which to ask these questions because they produce a highly stereotyped song that emerges after sensory learning becomes integrated into the function of a sensorimotor neural circuit responsible for singing. By deafening a group of birds (who show a shift in their song structure) and comparing them to hearing birds, clues as to how plasticity in motor output may emerge from genomic changes that alter the function of cells within the various components of the neural circuit.

There are multiple strengths of the paper:<br /> 1. The results may have broad implications because the type of sensorimotor neural circuit (cortico-basal ganglia-thalamic-cortical loop) used for singing is generally necessary for learned behaviors.

2. The methods and analyses are generally rigorous, including the parsing of song elements, and the type of detailed RNA sequencing and analysis that demonstrates the power of a genomic view of neural plasticity as it relates to behavioral plasticity.

3. Because the authors assayed the pallial (cortical) areas, as well as the basal ganglia component, of the sensorimotor circuit they were able to creatively compare how different facets of the network contributed to a) unmodified brain properties, b) properties perturbed after the loss of the auditory input that is required to stabilize song structure. As a result, they have added to the known molecular profiles for each of these brain areas, the accounting of how they may be specialized in comparison to the surrounding non-song brain, and what changes occur after deafening. Utilizing some existing single-cell sequencing data, the authors present for the first time some insight into what cell types may be showing the most robust changes, and therefore which may be driving the shift in song structure. The analysis further pushes in new ways to suggest how the molecular properties of a given brain area may relate to those of directly-connected areas. Together, these findings provide valuable clues as to the specific cell types and signaling properties that may be central to the production of stabilized, learned behavior.

4. One of the cortical brain areas, LMAN, was lesioned in a subset of the hearing subjects because it projects to the area that showed the greatest molecular difference between deafened and hearing birds (RA). The idea was to compare how this affected molecular properties with properties after the loss of auditory input; because RA is the output motor area for the song, its properties may be most directly tied to song structure. Using unilateral lesions was a strong choice of experimental design that allowed for rigorous analysis of this idea, and was interpretable because birds do not have a direct inter-hemispheric callosum.

The foundation of the paper is solid, though the results shown raise several questions that are not fully addressed, and limit some of the power of the implications.

The biggest questions arise from the finding that RA shows the largest number of molecular changes after deafening. The analysis and interpretations do not fully incorporate what we know of this circuit, at least from another well-studied songbird, the zebra finch, from which the authors derive other types of information. For example, it is not yet clear if RA is most changed because it is most directly involved in song output or because it receives projections from two areas within the sensorimotor circuit (LMAN and HVC). How do we consider the fact that by adulthood, LMAN and HVC cells project onto the same RA neurons? Are those the cell types being identified here? Would HVC lesions be expected to have the same effect as LMAN lesions? Are the cell types showing the greatest change those that are most involved in song output (e.g. are they projecting to nXIIts)? How do these results relate to the findings of changes in RA after HVC and LMAN lesions reported decades ago? How do these findings compare to an earlier study that also performed sequencing on areas from the sensorimotor circuit in deafened juveniles? Further, RA also receives information from the auditory processing regions of the brain, via the immediate structure RA-cup. It is not yet explicitly addressed how some effects may be from the loss of this more direct access to auditory information, rather than from information and projections originating within the sensorimotor circuit, and reinforces the question of whether or not the number of inputs to a particular brain area is a driving factor in the general pattern of changed RNAs after perturbation.

Importantly, since the LMAN lesions did not create significant changes in the song structure, it is difficult to know how to interpret the meaning of these molecular changes in RA, alone and in combination with the comparison to the RA profiles from deafened birds. Of importance is the question of whether or which molecular profiles are thus signatures of behavioral plasticity or not.

Reviewer #2 (Public Review):

Neurons of the inferior olive exhibit strong subthreshold oscillations, and drive complex spiking through climbing fiber synapses onto Purkinje cells in the cerebellar cortex. This activity plays an essential role in coordinating motor control and the induction of cerebellar plasticity. In this study, the authors make use of optogenetic and electrophysiological approaches to examine the interplay between intrinsic oscillations and two important excitatory and inhibitory input populations to the inferior olive. The authors show that excitation is enhanced when it occurs in the rebound phase of the preceding inhibition. Using a computer model, the authors also show that enhanced excitation can effectively recruit larger populations of neurons, presumably through gap junctional coupling. The strengths of the study include the authors' ability to independently control both excitatory and inhibitory pathways, as well as the rigorous and systematic examination of input timing and amplitude and their effects on spike output. There were some weaknesses; high variability in cell resting potentials raised questions about how cell health impacted the findings, and there needed to be better documentation of recording conditions and parameters. There also needed to be a more extensive discussion about the nature of input timing and frequency under behaviorally relevant conditions. Given these relatively minor issues, the study provides new insight and depth into synaptic integration in the inferior olive and adds to our understanding of how input timing is translated into climbing fiber signals.

Reviewer #1 (Public Review):

The network of neurons of the inferior olive has long been suggested as a timing machine that controls the precise timing of movements, correcting movement and participating in the prediction of movement time. These timing capabilities have been attributed to the unique feature of the neurons to generate subthreshold voltage oscillations that can be used as a timing machine. In this study, the effect of the inhibitory and excitatory synaptic inputs on the oscillatory behavior was examined, demonstrating their different effects as well as the effects of combing the two inputs.

Reviewer #3 (Public Review):

This is an interesting paper but not entirely surprising. Given the known voltage dependency of intrinsic oscillations of IO neurons, the fact that a hyperpolarizing input from a GABAergic synapse or a depolarization from an excitatory input can phase shift an oscillation depending on the timing is not surprising. It could be predicted from what is already known about the underlying conductances of the oscillations in these cells. The authors, however, do provide some quantification for both the inputs and the effects they have on the oscillations. Whether or not this quantification can be extrapolated to in vivo conditions, however, remains to be seen. There are multiple technical issues that the authors need to address.

Reviewer #2 (Public Review):

The authors report a study comparing self-reported stable and unstable knees with total knee arthroplasty. Advanced imaging methods (dynamic fluoroscopy with model-image registration) and analysis of muscle activities were used to characterize the study subjects during three ambulatory activities (level gait, downhill walking, and stair descent).

The subject cohort all received one design of TKA in a similar time period. The unstable subgroup was >60% female, while the stable knee cohort was 70% male, which is a notable limitation. The measurement methods are state-of-the-art and expertly applied.

The results suggest there may be measurable differences in knee kinematics in subjects with unstable knees, but this was not strongly supported across groups. Rather it was highlighted in 3 individuals who self-reported instability during the test session. The muscle activity analysis supports there being differences between the stable and unstable knee groups.

Despite the limitations of a small subject cohort with only a single TKA design, the study highlights important methods that appear suitable to further study the factors contributing to clinical dissatisfaction with TKA as it relates to joint stability and function during ambulatory tasks.

Reviewer #1 (Public Review):

This manuscript seeks a greater understanding of joint movements in recipients of total knee replacements who have symptoms of unstable prosthetic joints. The authors describe the results of a carefully conducted retrospective analysis of joint movements after total knee replacement (TKA) using a recently developed method based on videofluoroscopy. Kinematic data supplemented by electromyography measurements of muscle activation through normal gait. These measurements were conducted while walking on flat ground, down an incline, or down stairs. The kinematics and EMG data provide convincing evidence of altered knee kinematics when symptoms of joint instability occurred that were accompanied by subject-specific changes in patterns of muscle activation. The manuscript raises interesting questions about how patients adapt muscle activation patterns to limit discomfort prior to TKA and to what degree these same defensive strategies influence joint stability post-operatively.

Reviewer #1 (Public Review):

Villalobos-Cantor et al. describe a chemical/genetic strategy to enable cell-type-specific labeling of nascent proteins in living tissues (called POPPi). O-propargyl-puromycin (OPP) is a commonly used compound to label nascent proteins in cells and tissue, however, its application is limited in vivo because it can not be targeted to individual cell types, tissues, or organs. Using Drosophila as a genetically tractable in vivo model organism, Villalobos-Cantor et al. incubate live tissue with a puromycin analog called phenylacetyl-OPP (PhAc-OPP) in combination with cell-type expression of Penicillin G acylase (PGA), which converts PhAc-OPP to OPP. As PGA is under the control of the Gal4/UAS system, a vast library of tissue-specific Gal4 lines can in theory be used to conduct labeling experiments in vivo.

The major strength of the methods and results is the demonstration that labeling can occur in specific cell types of the dissected brain - neurons and glia. For example, protein synthesis in individual dopamine neurons in the brain can be visualized and distinguished from neighboring cells, a remarkable achievement and striking image. These results in dissected brains nicely demonstrate that PhAc-OPP can penetrate into brain tissue, diffuse to internal locations, pass through the cell membrane, and become converted to OPP and label nascent proteins. A major weakness of the methods and results is the lack of exploration of POPPi in tissues other than the brain, as well as in non-dissected living animals. For example, the authors do not test if PhAc-OPP delivery can occur by feeding animals, or if PhAc-OPP can penetrate into various dissected tissues. Results from these experiments would be of great importance to others interested in applying this technique in non-brain tissues, and would properly support the authors' claims in the title and abstract that this is a general method (not only for the brain).

Assuming that PhAc-OPP can penetrate various dissected tissues, this method would have a significant impact on tissue-specific measurements of protein synthesis and could be a valuable new molecular reporter for gene function analysis (e.g. tissue-specific gene knockout + POPPi). If PhAc-OPP could be ingested by flies, perfuse through the body, and label nascent proteins in a cell-type specific manner, then POPPi could be incredibly useful for tissue-specific proteome profiling (i.e. mass spectrometry) in an in vivo living animal (non-dissected), similar to the BioID system.

Reviewer #2 (Public Review):

In this manuscript, Villalobos-Cantor et. al. described a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. PhAc-OPP contains a blocking group that prevents it from incorporating into the growing peptide, and the blocking group can be cleaved off by the enzyme PGA, which is expressed in the cell type of interest.

The authors validated POPPi in different cell types in the Drosophila brain and showed that this method could be used to image general translation or to biochemically enrich nascent peptides in a cell-type-specific manner. They also showed that with an optimized labeling protocol, it is possible to achieve efficient labeling with minimum effect on animal viability and health. The authors further used POPPi to provide independent support for a previously known phenomenon: age-dependent decline in general translation in the neurons. The results of this work are solid, and the main conclusions are well supported by the data presented. The manuscript is very well written with a clear logic flow and is very easy to read.

What is less clear is how generally useful POPPi will be to the community. The authors pointed out two major cell-type specific applications of POPPi, 1) imaging general translation and 2) biochemically purifying nascent peptides. For application #1, although POPPi might be a more desirable method in some cases, a combination of non-cell-type specific labeling using OPP, and marking the cell type of interest by a fluorescent protein might be simpler. Because labeling with OPP eliminates the enzymatic step that converts non-reactive PhAc-OPP to reactive OPP, the labeling kinetics can be improved, and the toxicity associated with PGA expression can be avoided. For application #2, a currently widely used strategy for a similar purpose is various types of ribosome profiling techniques. Ribosome profiling may be easier to perform than POPPi, and because proteins cannot be amplified, a very large quantity of starting materials will be needed if one wants to use POPPi to characterize cell-type specific nascent proteome. In fact, in this manuscript, the authors used western blots to detect candidate proteins and did not use mass spec to characterize the nascent proteome.

Reviewer #3 (Public Review):

In this manuscript, Villalobos-Cantor et al. have implemented the method for monitoring cellular proteome that their lab has established in cell culture models of Drosophila brains. The method uses a puromycin analog (O-propargyl-puromycin, OPP) that is locked by the addition of phenylacetyl group (PhAc-OPP) that can be unlocked by expression of Penicillin G acetylase (PGA) to tag the proteins translated in a specific cell type. When unlocked, OPP can get incorporated into the newly translating nascent peptide, and abort translation while allowing click chemistry addition of various tags, such as fluorophore-azide to visualize or biotin-azide to immunopurify polypeptides. The authors demonstrate the use of the method in adult drosophila brains expressing PGA in neurons or glia, showing that the addition of OPP is indeed PGA dependent and the proteins are only tagged in the cells that express PGA. The authors also show that when fluorophore azide is used to visualize the proteome and the samples are run on a gel, bands of various sizes can be observed to have incorporated OPP, arguing the method labels the proteome indiscriminately. The authors also optimized the protocol by titrating the amount of PhAc-OPP to use to minimize cellular stress. Also, they show that driving the expression of PGA with elav-Gal4 or repo-Gal4 is not toxic and does not cause phenotypes although Actin-Gal4 driven expression causes phenotypes. Finally the authors demonstrate the use of the technique to show that there is an age-induced decrease in total protein synthesis in the fly brain. This is a nice technique to implement in fly but the characterization of the technique is not complete in its current state. It is not clear what percentage of the nascent peptides are tagged, and whether the cells in the tissue are equally represented in the lysates for immunopurification.

Reviewer #1 (Public Review):

This project aimed to understand if decision making impairments commonly observed in older adults arise from working memory (WM) or reinforcement learning (RL) deficits. Evidence in the paper suggests it is the former; they observe poorer task accuracy in older adults that is accompanied by a faster memory decay in older adults using a novel hierarchical instantiation of a previously validated computational model. There were no similar changes in RL in this model. These results are extended using Magnetic Resonance Spectroscopy (MRS) to measure glutamate and GABA levels in striatum, prefrontal and parietal regions. They found that impairments in working memory were linked to reductions of glutamate in PFC, particularly in the older adult group.

The task employed is elegant and has been studied extensively in different populations and is well-validated (though here a hierarchical Bayesian extension is developed and validated). The results however may not be definitive in some respects; the paper did not replicate previously observed RL deficits. It therefore, remains possible that this is due to the sensitivity of the task to this RL component in ageing and future work is needed to fully bridge the gap in the literature.

Although the study is well-executed, there is an obvious limitation in the use of a cross-sectional design to address this question. The authors acknowledge this limitation in the discussion but could go further to highlight the potential confound of cohort effects on gaming, RL and WM tasks more generally. Without within-person change data, the evidence can only be suggestive of potential age-related decline. For this reason, it may be more appropriate to use the terminology "age-related differences' rather than "age-related declines" given the study design.

Reviewer #2 (Public Review):

In this study, Rmus and colleagues contribute to the important open question of whether reinforcement learning deficits observed in older adults are due to impairments in basic learning processes, or can be attributed to a decline in working memory function. The authors present cross-sectional behavioral data from a task designed to assess the role of working memory in reinforcement learning. And they use computational modeling in conjunction with MR spectroscopy to demonstrate a relationship between prefrontal glutamate and age-related impairments in learning specific to working memory decay. I found the overall story compelling, the data novel, and the analysis carefully executed. Below I outline some areas in which the claims of the manuscript could be strengthened.

1. I may have missed this, but does glutamate correlate with other model parameters? Or did the authors only focus on the WM parameters because of the age difference? In support of the specificity argument, it would be important to show that glutamate only predicts WM related parameters regardless of whether there was an age difference or not.<br /> 2. As it is somewhat common with these tasks, it seems like the model does not fully capture the performance deficit in OA (Fig. 2B), even when all the individual difference parameters in WM are allowed to vary. Can the authors say more about the discrepancy? This is an interesting datapoint which may give clues to mechanism.<br /> 3. Relatedly, it may not be possible with these data alone, but can authors discuss what the WM decay parameter captures? In particular for OA, the distinction between generating and maintaining a "task set" has been extensively written about. Older adults tend to have difficulty internally generating and flexibly deploying task sets, but somewhat paradoxically can perform better than YA in certain decision situations (e.g. when reward is dependent on previous choices, see Worthy et. Al. 2011). The task in this study necessarily pushes OA in a regime in which relying on familiar decision strategies is sub-optimal, and task sets must be continuously generated. Is there a type of intervention do authors expect would reverse the observed deficit in WM?<br /> 4. There is a wealth of evidence suggesting striatal DA loss in older adults, which served as the basis for many of the original investigations and hypotheses regarding a simple RL deficit in OA (e.g. work by Shu-Chen Li and others). While the authors do not directly measure DA in this study, it would be helpful to place the results in the context of that literature.<br /> 5. Finally, the main argument of the paper as I read it is that PFC glutamate mediates the performance deficits observed in RL because it reflects a compromised WM system. Sample size permitting, it would be helpful to see a formal test of this mediation relationship.

Reviewer #3 (Public Review):

Aging impacts many cognitive functions, and how these changes affect performance in different tasks is an important question. By testing 42 older and 36 younger healthy adults with a novel learning task and MR spectroscopy, Rmus et al addressed the important question whether age-related declines in learning are driven by WM, or by deficiencies of the RL system. The task varied the role of working memory in learning by asking participants to learn about either 3 or 6 stimulus response associations from feedback (set sizes 3 and 6). The paper combines a detailed computational account of participants behaviour and striatal and prefrontal/parietal MR spectroscopy in order to assess individual glutamate and GABA levels.

The authors report an effect of set-size on learning in both are groups, and show that participant age is associated with (1) worse accuracy, (2) a larger set size performance difference, and (3) a heightened sensitivity to reward. Computational modelling showed that working memory decay differed between age groups, but that reliance on WM to perform the task at hand was similar in both age groups (similarly differing between conditions in both groups). Turning to the MRS results, the paper shows that an aggregate measure of glutamate relates to aggregate task performance, that prefrontal glutamate specifically relates to WM decay observed in the task, and that age was negatively associated with glutamate levels.

While the paper is well worth reading and offers many interesting data points, the title's suggestion that "Age-related decline in prefrontal glutamate predicts failure to efficiently deploy working memory in working memory" is, in my opinion, not fully supported by the evidence. First, the authors don't report clear evidence for any age-related differences in WM reliance in the task overall. Second, the authors find that MFG glutamate relates significantly only to WM decay, not the parameter that captures WM deployment. Third, correlations don't imply predictive relations.

Another important open question relates to the relatively large age difference in the effect of set-size on performance. The authors write that working memory will contribute less to performance in higher set size conditions. Yet, age differences are largest in the set size 6 condition, suggesting that RL-dependent learning is most severely impaired in learning (set size 6 performance), rather than WM dependent learning (set size 3 performance). Finally, a statistically significant age difference in reward sensitivity seems to be hardly integrated into the authors' overall interpretation.

The issues laid out above set aside, the paper has the potential to make an important contribution to the literature on cognitive aging.

Reviewer #2 (Public Review):

The work from Nakajima-Takagi et al describes the phenotypes and study of a PCGF1 mutant mouse model. PCGF1 is a core component of the non-canonical PRC1.1 complex and specific functions of this complex in hematopoiesis. Using somatic inactivation models, the authors demonstrate that the acute deletion of PCGF1 from adult hematopoiesis leads to a progressive myeloid bias in the bone marrow and peripheral blood. This occurs at the expense of the HSPC compartment, with a reduction in all populations and of the lymphoid committed populations. The myeloid bias is cell intrinsic, as competitive transplant of the PGCF1 deficient bone marrow recapitulates the phenotype. The effect is not due to exhaustion or loss of self-renewal of the HSCs.<br /> To understand the basis for the myeloid bias, the authors first assessed transcriptome signatures and see a shift in gene expression programs related to myeloid development and targets of the key myeloid transcription factor C/EBPa. Further analysis demonstrated an increased expression of Cebp1 in the PCGF1-deficient LSK cells. Reducing the expression of Cebpa could modify the myeloid skewing of Pcgf1 deficient cells in culture. This de-repression of Cebpa correlates with changed local H2AK119ub1 levels in the HPSC populations.

Additional studies assessed how the loss of Pcgf1 changed the response to hemoablation, in this instance with a single dose of 5-FU. This study coupled with scRNA-seq suggested that PRC1.1 was important in regulating the GMP populations, potentially through a self-renewal program. This led to a focussed analysis of the GMPs, with evidence for altered Hoxa9 and b-catenin levels contributing to the altered GMP behaviours. Both have been implicated and demonstrated to have functional roles in these programs in other studies.

Finally, ageing of Pcgf1 deficient mice demonstrated that these mice were predisposed to developing T-ALL and MPN. The authors provide a characterisation of these moribund states and their phenotypes are consistent with the diagnosis.

Overall the work demonstrates a specific requirement for Pcgf1, and therefore PRC1.1, in the regulation of hematopoiesis. I think the authors largely achieved the aims and the results are supportive of the conclusions. The work shows myeloid bias, experimental evidence that this is due to a derepression of a myeloid lineage program in the HPSC and associated chromatin changes, and functions for Pcgf1 in both hematopoietic regeneration and malignancy. This suggests a unique role for non-canonical PRC1.1 compared to canonical PRC 1.

Strengths:<br /> - in vivo experiments and evidence;<br /> - multiple lines of evidence supporting the conclusion;<br /> - mechanistic studies provide direct evidence of the proposed mechanism.

Weaknesses:<br /> - can the authors demonstrate normal maturation of the myeloid lineages as this would be important to differentiate between myeloid bias and a block in myeloid differentiation? This is important to distinguish between.<br /> - include analysis of mature myeloid cells and FACS plots to allow assessment of maturation.

Reviewer #3 (Public Review):

The myeloid bias in hematopoietic stem and progenitor cells upon genetic inactivation of Pcgf1, a component of the PRC1 complex is convincingly demonstrated by a Pcgf1 conditional allele crossed with a tamoxifen-inducible Are, combined with transplantation. The overproduction of myeloid cells may be contributed to by the derepression of two PRC target genes Cebpa and Hoxa9 at the multipotent HSPC and lineage-committed GMP levels. The involvement of these two genes is demonstrated by decreased H2AK119ub1, elevated CEBPa expression, and increased CEBPa binding motifs in KO HSCs. Functional rescue by manipulating CEBPa levels on the background of Pcgf1 KO is attempted in vitro. The derepression of Hoxa9 is shown in Pcgf1 KO GMPs, which expanded in the KO mice. Finally, constitutive inactivation of Pcgf1 results in lethal myeloproliferation. Together, this paper demonstrates another HSPC regulator, whose loss of function leads to myeloid-biased hematopoiesis, which in extreme cases could end in myeloid transformation.

Reviewer #1 (Public Review):

This interesting manuscript by Nakajima-Takagi et al describes the roles of the PRC 1.1 member Pcgf1 in myeloid lineage commitment in hematopoiesis and in regulating myeloid differentiation and self-renewal during emergency myelopoiesis. The roles of Pcgf1 have been explored previously in the context of Runx1 depletion or in the context of myelofibrosis together with the JAK2V617F mutation, but this is the first report of the specific roles of Pcgf1 in HSCs and in myelopoiesis. The authors convincingly demonstrate that conditional deletion of Pcgf1 in hematopoietic cells causes a lineage switch in HSCs from lymphoid to myeloid fates and that a key mechanism for this lineage switch is regulation of the H2AK119ub1 chromatin mark, leading to de-repression of CEBPalpha, a key transcription factor that promotes myeloid cell fate. They also perform a single-cell RNAseq experiment and demonstrate an increase in the population of "self-renewing GMPs", and they attribute this increase to an upregulation in HoxA9 expression and beta-catenin activation. They also demonstrate that HoxA9 overexpression promotes beta-catenin activation, which has been observed in emergency myelopoiesis in other studies, though the mechanism for this is unclear. The authors also demonstrate that deletion of Pcgf1 in hematopoietic cells can also lead to deregulated myelopoiesis, leading to a lethal MPN in a subset of animals. They conclude that Pcgf1 plays a critical role to regulate emergency myelopoiesis, and to prevent the malignant transformation of myeloid progenitors.

Overall, the methods are highly rigorous and the results support the authors' conclusions. The only conclusion that would require further clarification is that Pcgf1 promotes emergency myelopoiesis. Emergency myelopoiesis typically starts with a proliferative burst of myeloid progenitors in response to a stress stimulus, followed by enhanced myeloid differentiation into mature functional myeloid cells. In this Pcgf1 KO mouse model, it is clear that there is an increase in the production of myeloid progenitors, and prolonged survival of myeloid progenitors in culture, but there is no demonstration that this results in the generation of mature functional myeloid cells. It appears that there may also be a differentiation block, likely due to the increase in "self-renewing progenitors", which is likely a consequence of HoxA9 upregulation, and possibly the beta-catenin activation in myeloid progenitors. Therefore, if there is also a differentiation block due to Pcgf1 deletion, the statement that emergency myelopoiesis is enhanced may be an oversimplification. What appears to occur is an expansion of a pool of self-renewing transformed or pre-transformed myeloid progenitors, and the relevance of this event to emergency myelopoiesis is not entirely clear. However, there is a clear significance of these findings and this new mouse model for studying the pathogenesis of myeloid malignancies, such as MPN, MDS, or AML, in which mutations in other components of PRC1.1 are frequently mutated, so this study is likely to have a significant impact in the field.

Reviewer #3 (Public Review):

In this study, Cunha et al. examined the role of different oxygen tensions (21%, 5%, and 1% O2) and HIF-1α stabilisation in regulating murine and human CD8+ T cell proliferation and function. The authors find that hypoxia (1% O2) and pharmacological PHD inhibition with FG-4592, enhance murine T cell activation but impair proliferation. Furthermore, adoptive cell transfer (ACT) therapy of CD8+ T cells from both conditions reduced tumour burden in a B16-OVA melanoma model. Short hypoxic conditioning (1% O2) of human CD8+ T cells for 1 day increased HIF-1α stabilisation, with increased activation, glycolysis, and mitochondrial function still observed following 6 days of normoxic cell culture. Short hypoxic conditioning of HER2 and CD19 CAR-T cells improved their activation and cytotoxicity in vitro, while HER2 CAR-T cell counts were increased in vivo, reducing tumour burden, and increasing survival when compared to 21% O2.

Strengths:<br /> The paper convincingly demonstrates that short hypoxic conditioning in a defined window improves CAR-T cell function through in vitro cytotoxicity assays and following adoptive transfer in a preclinical HER2+-SKOV3+ positive tumour model. Thus, the major conclusion of the paper is mostly well supported by the data and could represent a novel strategy to improve CAR-T cell immunotherapy for solid tumours in the future.

Weaknesses:<br /> The extent to which hypoxic conditioning-mediated improvement in CAR-T cell function is dependent on HIF-1a-driven metabolic reprogramming is unclear and other potential mechanisms are not explored. 5FG-4592 and VHL silencing in HER2 CAR-T cells did not phenocopy each other faithfully. In addition, neither approach was as effective as short hypoxic conditioning with 1% O2 in improving CAR-T cell function in vitro or in vivo. Although the authors suggest the temporal dynamics of HIF-1α stabilisation is the key point, this is not convincingly proven, and no metabolic characterisation of these CAR-T cells was performed. It is unclear how changes elicited during short hypoxic conditioning are maintained following continued normoxic cell culture. Hypoxia is known to rapidly regulate histone methylation and chromatin structure in a HIF-independent manner (PMID: 30872525; PMID: 30872526). Are similar epigenetic changes observed in T cells, and if so, could these epigenetic changes underlie improved T cell activation?<br /> Complications may also arise when comparing different oxygen tensions given recent data that suggests standard cell culture conditions can lead to local hypoxia through a combination (https://www.biorxiv.org/content/10.1101/2022.11.29.516437v1) of cellular respiration and poor O2 diffusion. Although it is unclear how this will impact suspension T cells it does beg the question as to whether HIF-1α stability following T cell activation is (at least in part) mediated by pericellular O2 limitations in cell culture over time, even in presumed hyperoxic (21% O2) conditions? Or if T cells subsequently cultured at 21% O2 following short hypoxic conditioning (1% O2) still experience local hypoxia during the 6-day culturing protocol? It would be important to assess this in future work and at least discuss these potential weaknesses.

Reviewer #1 (Public Review):

This study represents an important work in the field of (CAR)T-cell immunotherapy by analyzing the effect of different oxygen tension on the function and differentiation of T-cells (especially CD8+). Although it has been described that low oxygen levels can influence effector function/differentiation of T-cells, as nicely acknowledged by the authors in the introduction, a comprehensive analysis in the context of immunotherapy has been missing so far and this study adds significant findings that will be relevant for patient care in all fields applying (CAR)T-cell immunotherapy.

The strength of the evidence is generally solid although there are some discrepancies between the different ways to induce HIF-1α (i.e. low O2, pharmacological inhibition, shRNA knockdown) that need to be clearly stated and/or discussed.

1) The first section of the results determines the impact of low oxygen and pharmacological HIF-1α stabilization on CD8+ T-cell activation/differentiation. Low oxygen diminishes cell growth but induces T-cell activation and effector cytokines, while HIF-1a stabilization mimics the effects on activation without alterations in expansion. Unfortunately, it remains unclear why effects upon low O2 are more pronounced although pharmacological HIF-1a stabilization is more efficient.<br /> 2) As a next step, in vitro conditioned T-cells are transferred into a subcutaneous B16-OVA model. Although only the low O2 levels increase T-cell numbers in vivo after the transfer, the initial tumor burden was nicely decreased by both low O2 and HIF-1a stabilization. However, only the latter significantly improved survival and it remains unclear and uncommented why.<br /> 3) Next, the authors address whether pre-conditioning of human CART-cells to induce HIF-1α either by pharmacological stabilization or by silencing of VHL shows similar effects. Surprisingly, both ways of HIF-1a stabilization resulted in different effects concerning differential gene expression and cytotoxic capacity of CART-cells. Accordingly, pharmacologically pre-conditioned CART-cells did not have a significant impact on survival in an in vivo model, while the VHL-silenced ones did significantly improve animal survival. This discrepancy between the two modes of HIF-1a stabilization remains uncommented. Unfortunately, it also remains unclear why the pharmacological HIF-1a stabilization significantly improved the survival in animals of the B16-OVA model and not in the human CART-cell model.<br /> 4) After this, the researchers determine how the timing of hypoxic conditioning affects the (CAR)T-cells. Here it is convincingly shown that already a short period of hypoxic conditioning (1 day) with a subsequent expansion phase (additional 6 days) is sufficient to induce HIF-1a mediated alterations (e.g. metabolic changes, calcium flux, intracellular signaling). Although this section is coherent in itself, the switch between different times of hypoxic conditioning, expansion, and analysis is difficult to follow and might lead to confusion. The expression pattern of e.g. HIF-1a on day 1 and day 7 together with the nuclear amounts of NFAT and c-Myc might be misunderstood, like the other presented data as well.<br /> 5) Last, short-term hypoxic conditioning of CART cells is tested in a solid tumor mouse model. The previously identified conditioning protocol also increases CART-cell function against solid tumors (as shown by enhanced cytotoxicity, reduced tumor burden, and prolonged survival). Unfortunately, although both HER2-CART-cells and CD19-CART-cells are shown to have superior cytotoxicity in vitro after the pre-conditioning, only HER2-CART-cells are demonstrated to be superior upon low O2 conditioning in an in vivo adoptive transfer mouse model and CD19-CART-cells remain an open question.

Generally spoken, the limitations of the manuscript are:<br /> 1) The occurring discrepancies of determining effects caused by the different modes of Hif-1a stabilization which certainly are caused by the complex nature of Hif-1a regulatory network, and;<br /> 2) The limitation of detected effects primarily on CD8+ T cells while CART-cells products usually are a mixture of CD4+ and CD8+ ones.

Reviewer #2 (Public Review):

In this work, Cunha et al provide an insightful and exhaustive analysis of the role of hypoxia and HIF-1a for T cell activation and function. The work contributes to the field by showing that transient hypoxia occurring simultaneously with T cell stimulation (antigen recognition) induces an effector program in T cells that results in increased cytotoxicity in vivo and in mouse models. Importantly, the induction of this effector phenotype is not necessarily linked with an increase in proliferation in vitro, and in vitro differences are mostly observed upon antigen re-challenging.

The major strengths of the work are the use of different complementary methods to modulate HIF-1a (low oxygen conditions, inhibition of PDH by FG-4592, and deletion of VHL) and the combination of mouse and human models, especially addressing how to implement the findings to the production of CAR-T cells. Besides, the authors not only evaluate T cell function but also dive into the pathways driving the responses observed, which provides mechanistic insight.

While activation of HIF-1a through the different means mentioned before results in similar signatures in terms of T cell effector phenotype and animal response, there are some aspects that differ between the models. This is probably indicating that low levels of oxygen have other effects beyond the regulation of HIF, and that pharmacological modulation of HIF-1a might not be exactly equivalent to HIF-1a stabilization by real hypoxia.

The work is useful to better understand the discrepancies in the field, where it has been previously shown that hypoxia can have both a pro-inflammatory effect and an immunosuppressive effect on T cells. The answer proposed by the authors is that it´s a matter of timing, and not so much the magnitude of the HIF-1a response. Despite this being relatively easy to control ex vivo, the challenge occurs when considering the role of hypoxia in vivo, which probably lasts longer than the transient hypoxia needed for beneficial effects on T cells, causing T cell exhaustion.

From the translational perspective, the study suggests strategies to improve CAR-T cell therapy but also has some limitations. Despite an improvement of cytotoxicity and survival observed in mouse models upon adoptive cell transfer or injection of CAR-T cells with previously increased HIF1a levels, these approaches do not result in curation and survival is still quite low in all groups. Interestingly, improved survival with HER2 CARs exposed ex vivo to low oxygen conditions for 1 day is clear and more promising.

Reviewer #2 (Public Review):

In this manuscript, Niethamer et al. investigate the role of the transcription factor ATF3 in lung regeneration after H1N1 influenza. They focus on endothelial ATF3 which is present in a subset of lung capillaries in the adult mouse lung. Interestingly, they found that influenza infection upregulates endothelial ATF3 and that endothelial deletion of Atf3 results in impaired regeneration, leading to enlarged airspaces after viral infection. They further show that this effect may be due to an increase in apoptosis and a decrease in proliferation, suggesting that endothelial ATF3 is necessary for pulmonary vascular regeneration, as well as recovery of the alveolar architecture.<br /> Given the recent publications in the field describing lung endothelial heterogeneity, as well as its possible role in injury repair, this work is relevant to the community. It also supports the idea that epithelial-endothelial crosstalk is important for lung regeneration and proposes a potential candidate for this process.

Strengths:<br /> The authors identified and tested the role of endothelial Atf3 in lung regeneration using well-established techniques. They identified this transcription factor as a candidate using state-of-the-art scRNA-seq. They also carefully lineage traced ATF3 expressing cells using an inducible reporter before and after infection and then used a pan-endothelial driver Cdh5 to delete Atf3 specifically in the endothelium. Thus, the authors successfully show significant changes in the alveolar structure after infection in their mutant model.

Weaknesses:<br /> Although there is evidence that the author's claims have biological relevance, this paper would benefit from strengthening and/or clarifying some things:

• The scRNA-seq analysis is performed in two separate objects ("control lung" and "H1N1 infected lung 14dpi"). For these two sets of data to be comparable, the authors should have integrated the objects and analyzed them together. This is not only important for deciding the clusters' identities and making sure that the same clusters are compared between control and infected, but also necessary to compare gene expression.<br /> • ATF3 is not only present in Cap1_B, in the infected lung there seems like Cap1_A express less ATF3. The authors should comment on this difference.<br /> • It is unclear how the clusters Cap1_A and Cap1_B were decided. The manuscript would benefit from clarification.<br /> • It would be beneficial to see via immunofluorescence the morphological and spatial differences between ATF3-expressing and non-expressing endothelial cells since this transcription factor is expressed in multiple endothelial cell types.<br /> • The authors mention ATF3 is not endothelial-specific. Expression of ATF3 in other cell types should be evaluated via immunofluorescence.<br /> • The authors should have shown evidence of the deletion in their Atf3EC-KO mouse and addressed whether they had residual ATF3. If there is no antibody available, RNAscope could be used, or Western Blot or RT-PCR on sorted endothelial cells.<br /> • The authors only show the epithelium as evidence that the alveolar region is altered in their mutant after infection. The endothelium should have also been investigated, especially since their mutant is an endothelial-specific deletion. Within this, the different endothelial cells should have been assessed by a method other than RNAscope such as immunofluorescence, given that this method is unable to show morphology and there are antibodies available.<br /> • Bulk RNA-seq from endothelial cells is used in the manuscript. However, because ATF3 is not specific to Cap1_B cells or even capillaries alone, the downstream gene expression analysis of bulk RNA should be placed into the context of lung endothelial heterogeneity.<br /> • Although the authors mentioned that the infection with H1N1 influenza can have regional differences, they do not show how they picked regions for their analysis and quantification, and whether ATF3 upregulation was found in more severely affected regions. Furthermore, since they quantified via FACS, this heterogeneity in the infection itself could have affected their observations.

Reviewer #1 (Public Review):

Here the authors investigate the mechanisms by which pulmonary endothelial cells (EC) contribute to alveolar repair post-H1N1-mediated acute lung injury and the molecular basis for the heterogeneity of this response among different EC subpopulations. Using single-cell transcriptomic analysis they identify the CREB family factor Atf3 differentially enriched in CAP1B cells, a subpopulation of EC previously known for its proliferative behavior in response to alveolar injury. They report a crucial role for Atf3 in injury repair but not during homeostasis. Using a combination of lineage tracing and loss function approach and an influenza mouse model in vivo, they show that Atf3 inactivation in ECs results in the inability of CAP1B ECs to initiate a proliferative response to repair the vascular compartment and ultimately regenerate the lung. Notably, the decreased number of Atf3 lineage-labeled EC capillaries was shown to correlate with the alveolar regions that failed to repair the post-H1N1 injury. They conclude that Atf3 is an essential factor for repair damaged capillaries in alveolar injury.

The study is carefully designed and the results provide novel important information about a previously undisclosed role of Atf3 in the regeneration of the lung vascular component. The work has many strengths and is supported by impressively coherent data from the analysis of mouse genetic models, single-cell transcriptomic, and phenotypic characterization.

Reviewer #3 (Public Review):

Although the response to stress has been extensively studied in pulmonary epithelium and mesenchyme, the post-injury proliferation and subsequent regeneration of pulmonary capillary endothelial cells remain poorly understood. Following their previous study on identifying mouse lung endothelial cell heterogeneity, Niethamer et al. reported a lung capillary subpopulation, CAP1_B with highly enriched Atf3. This capillary subpopulation expanded and increased the expression of genes involved in vascular regeneration in response to influenza-induced lung injury. Loss of Atf3 in lung endothelial cells led to abnormal alveoli structure and loss of endothelial cells through inhibiting cell proliferation and inducing apoptosis. This manuscript provided strong evidence to demonstrate the importance of Atf3 in mediating endothelial response to lung injury, which is novel to the field.

Reviewer #1 (Public Review):

This manuscript described the role of ALKBH5, an evolutionarily conserved mRNA m6A demethylase as a key regulator of axon regeneration. The authors screened the function of m6A regulators during axon regeneration and found that ALKBH5 limits regenerative growth associated with DRG neurons, by enhancing the stability of Lpin2 mRNA via erasing a single m6A modification in the 3'UTR. The major strength of the manuscript is the convincing importance of ALKDH5 as an attenuator to initially suppress the axon regeneration in the CNS and in the PNS proven by in vivo model system. These findings further suggest the potential use of ALKDH5 inhibitors to enhance neural regeneration upon physical injury.

Reviewer #3 (Public Review):

This is an elegant article that shows the reciprocal actions of Alkbh5 gene modulation and of a putative target, Lpin2. The work focuses on the DRG; it is important to note that the group also examines a retinal ganglion model where changes in Alkbh5 are not as prominent, and perhaps because of that the resulting effects of Alkbh5 modulation are not as pronounced. Thus, the effects of a broadly acting agent such as Alkbh5 might differ depending on the exact biology of the model. This doesn't diminish the finding at all, but the group nicely shows that the efficacy of Alkbh5 gene modulation might vary among different models.

A strength of the manuscript is the paper's use of reciprocal experimental designs to demonstrate effects. For instance, in Fig. 3 the authors show that Alkbh5 knockdown (KD) improves axonal regeneration and in Fig. 4 they show that Alkbh5 KD increases levels of methylated LPIN2, a transcript implicated in promoting axonal regeneration. Conversely, Figs. 5 and 6 also show that WT Alkbh5 over-expression inhibits axonal regeneration while an inactive Alkbh5 mutant has no effect. Alkbh5 KD is expected to increase levels of its target transcripts, and indeed, the group focuses on LPIN2 and shows that Alkbh5 KD increases LPIN2 adenosine methylation which is associated with a reciprocal decrease in total LPIN2 transcript (because m6A promotes degradation of the labeled transcripts). In Fig. 7 they go on to look at a specific target site of m6A on LPIN2. The hypothesis is that methylation of the target m6A site on LPIN2 will lead to a reduction in transcript levels; they show that mutating this target adenosine prevents the resulting effects of Alkbh5, implicating adenosine methylation in the modulation of LPIN2 levels. Finally, they show that over-expressing LPIN2 inhibits axonal regeneration while inhibiting LPIN2 increases regeneration. Thus, the experimental design includes many levels of reciprocal actions that are observed examining regeneration, adenosine methylation, and LPIN2 transcript levels. Taken together this approach is very convincing. The last figure examines the extent to which the KD approach extends to other models; the authors show that Alkbh5 is less active in a retinal ganglion model. The limited efficacy in the retinal ganglion is disappointing but serves to highlight the strength of the actions in the DRG model and provide a warning that the actions of Alkbh5 might vary significantly depending on the particular pathophysiology to which its gene modulation is being applied.

The manuscript does have some weaknesses, but the weaknesses are modest and do not change the overall interpretations of the manuscript. For instance, interpreting the quantitative efficacy in Fig. 1 and 2 depends on knowing the efficiency of uptake of the RNAi for Alkbh5 and subsequent virally transduce shAlkbh5, however, the authors do not show this efficiency. But such weaknesses are quite minor and do not change any of the conclusions.

Reviewer #1 (Public Review):

Baggett C., Murphy K. R., and Sengun E. et al. investigated cell senescence as the basis of pro-arrhythmogenic changes associated with myocardial infarction in the aged heart using the rabbit as a model, with validation of senescence markers on human heart specimens. The study is interesting and addresses a relevant biological and health issue. The authors demonstrate that aged rabbits are prone to arrhythmogenesis associated with higher mortality within 72 h after induction of myocardial infarction. Analysis of scar morphology determined that fibrosis is not sufficient to explain age-associated arrhythmogenesis. Instead, the authors show that senescence, assessed by -galactosidase activity, expression of regulators of the senescence-associated secretory phenotype, and H2AX, is increased in myofibroblasts compared to endothelial cells in infarcted aged rabbit hearts. Accordingly, H2AX was detected in αSMA+ cells in human-aged hearts. The authors tested the influence of myofibroblasts on cardiomyocyte electrophysiology by exposing cardiomyocytes in vitro to conditioned media from fibroblasts in which senescence was induced by treatment with etoposide. Such treatment did not affect action potential duration, leading the authors to conclude that senescent fibroblasts are unlikely to influence cardiomyocytes through paracrine signaling. Instead, the authors propose a possible yuxtacrine effect. To test this, they performed immunofluorescence to infer potential myofibroblast-cardiomyocyte coupling by the presence of connexin 43 in the cell-cell interphase and tested the potential electrophysiological effects of coupling using a computational model.

The analysis of peri-procedure mortality, arrhythmogenesis, and senescence in young and aged rabbits subjected to myocardial infarction is valuable, represents a significant amount of work, and the results support the conclusions drawn. Stronger evidence that senescent myofibroblasts couple with cardiomyocytes in the aged heart is needed to support the proposed model.

The authors conclude a propensity of myofibroblast senescence based on the finding that 80% of αSMA+ cells are also positive for H2AX. Showing the immunofluorescence results on hearts 2 weeks after MI would help to more convincingly illustrate the result. From these immunofluorescence experiments, it is also concluded that most of the persistent senescent cells in the scar correspond to myofibroblasts. The results presented show a continued increase in the proportion of H2AX+ cells in aged hearts up to 12 weeks after myocardial infarction. According to results in Figures 4F and G, these cells do not correspond to either myofibroblasts or endothelial cells. Given that H2AX+ cells are significantly increased in the aged heart, could the results presented suggest that a different cell type might be more important for the aged heart's response to MI? Providing some insight into the identity of these cells would be helpful to better understand the results presented. For example, cardiomyocyte senescence could contribute to arrhythmic phenotypes.

The results presented show that treatment of cardiomyocytes with conditioned media from, and co-cultured with, senescent myofibroblasts did not change action potential duration in cardiomyocytes. This led to the conclusion that paracrine signalling is unlikely to contribute to a pro-arrhythmogenic phenotype. It is possible that cardiomyocytes do couple with myofibroblasts in the in vitro system used. In which case, the results presented would not favor the proposed model. Another important possibility to be considered is that myofibroblasts might not have produced senescence-associated secretory phenotype-mediators at concentrations high enough to alter action potential duration in the conditions tested. Experimental evidence of the levels of selected mediators of the senescence-associated secretory phenotype in conditioned media would help assess a potential paracrine effect.

The evidence of coupling, i.e., the presence of connexin-43 in the interphase between αSMA+ and cardiomyocytes needs to be strengthened. Perhaps analyzing Z-stack 3D reconstructions would help to better define adjacent cells and more precisely reveal the localization of connexin-43.

Reviewer #2 (Public Review):

1) A detailed step-by-step approach to validation of some previously known outcomes.<br /> 2) Useful for more focus to be placed on data from the second half of the paper.<br /> 3) Some reflection on the media used to study paracrine effects is needed - more experiments here would be beneficial.<br /> 4) Path clamp experiments - how does bath solution alter the effect of any limited paracrine effect - we are removing cells from the treatment media and putting them in physiological solutions - an opportunity to recover?

Reviewer #1 (Public Review):

This manuscript describes the differences in the plasma proteome and metabolome in healthy Tanzanian and healthy Dutch adults. The inflammatory plasma proteome was measured using the Olink 92 Inflammation panel, while the plasma metabolome was analyzed using a mass spectrometry-based untargeted approach. The plasma metabolome was measured only in the Tanzanian cohort. This study aimed to link the pro-inflammatory proteome of Tanzanian and Dutch healthy individuals with environmental factors and dietary lifestyles.

The correlation between the plasma proteome and food-derived metabolome profiles can shed light on the development of non-communicable diseases. This observation stresses the importance of dietary transition and lifestyle changes in expressing inflammation-related molecules. Moreover, this study describes the inflammatory proteome profile in healthy Tanzanian individuals covering a cohort with limited studies. The molecular differences in circulating biomolecules between healthy individuals living in East Africa and individuals living in Western Europe and the correlations with intrinsic and environmental features are novel.

This study lacks a robust and solid validation of some of the differentially regulated circulating proteins and correlations between food-derived metabolites and proteins in a selected cohort. The discovery-driven approach in this manuscript highlights potential findings that need to be supported by a validation phase. According to this reviewer, the lack of such validation impacts the robustness of the results and the hypotheses generated. Due to that, the manuscript should incorporate validation experiments.

Reviewer #2 (Public Review):

This study assessed the inflammatory and metabolic profiles of a healthy sub-Saharan Africa (Tanzania) population versus a healthy population outside Africa (Dutch). Using plasma samples from these cohorts, an O-Link proteomics inflammatory panel and targeted metabolomics platforms were utilised. The study shows that 'healthy' Tanzanians display an enhanced pro-inflammatory phenotype versus Dutch volunteers. Specific pathways and metabolites identified included - increase activation of the Wnt/Beta catenin pathway, and the metabolites 4E-BP1 and FGF21. The study highlights some interesting findings regarding the impact of diet on inflammatory pathway activation.

Major Strengths & Weaknesses - This is an interesting study and approach that aims to address some challenging questions in underrepresented populations. The findings demonstrate the importance of diet and dietary interventions on metabolic health, as well as key inflammatory proteins. It does raise the question whether anti-inflammatory therapies need to be targeted to specific at-risk populations, more so than other populations.

Impact - The study demonstrates the importance of considering differences between populations and the inclusion of underrepresented populations in such studies. The data suggests that lifestyle changes in sub-Saharan Africa are potentially contributing to altered inflammatory and metabolic profiles. Thus, health initiatives advocating traditional diets may alleviate the NCD epidemic in sub-Saharan Africa.

Reviewer #1 (Public Review):

For PRLR, the question being asked is whether and how the intracellular domain (ICD) interacts with the cellular membrane or how the disordered ICD can relay and transmit information. The authors show that PI(4,5)P2 in the membrane localizes around the transmembrane domain (TMD) due to charge interactions and facilitates binding of the ICD to the membrane, even in the absence of the TMD. Furthermore, the ICD and PI(4,5)P2 form a co-structure with JAK2 which locks a disordered part of the ICD into an extended conformation, allowing for signal relay and, through multiple complex conformations, may enable switching signalling on and off.

Strengths:<br /> - NMR paired with MD is a powerful way to probe an interaction especially when peaks disappear and become difficult to probe by NMR.<br /> - Using NMR and MD to formulate hypotheses which are then tested by cell studies is quite informative. The combination of MD, NMR, and cell biology is a strength.<br /> - The authors are diligent in testing MD simulations on systems with and without PIP2.<br /> - The use of Pep1 and Pep2 to differentiate the KxK region that interacts with PIP2 is helpful.<br /> - The four utilized mutants help illustrate the co-dependence of the respective regions in the formation of the co-structure.

Weaknesses:

- In Figure 2G, there is a big change in CSP between 280 and 290, which the authors do not comment about.<br /> - The data in Figure 2 are summarized as indicating the formation of extended structure in the ICD upon binding. It is not clear to me what data show an extended structure.<br /> - No modelling or experiments were done with PIP3 despite conclusions and models which rely on the phosphorylation of PIP2 to PIP3. At the very least, these would be useful as negative controls.<br /> - Only R2 experiments were done when the authors mention investigating dynamics. R1 and -HetNOE dynamics would be useful for creating a complete picture.<br /> - Some of the exciting results are under-emphasized including Fig 3H and 3I.

Reviewer #2 (Public Review):

The authors combine NMR experiments, cell experiments, and molecular simulations to address the question of how lipid interactions of the prolactin receptor contribute to signalling. They assess the interactions of the disordered cytoplasmic tail of the receptor with phosphoinositides among others by chemical shift perturbations from NMR for different PIP2-containing membranes, by coarse-grained simulations, as well as site-directed mutagenesis and subsequent cell signalling experiments to monitor the activation of the mutants. A major result is that PIP2 interactions are functionally important, which so far has not been known for this receptor. Their results are likely relevant for other non-receptor tyrosine kinases.

The hypothesis that the protein complex is regulated by IDR-membrane interactions is very novel. A major strength is the close connection of and feedback between state-of-the-art experiments and simulations.

This is where I see weaknesses:<br /> 1. The motivation of focusing on LID1 is limited.<br /> 2. The data and analysis for the JAK2-PRLR complex appear somewhat superficial, and a connection between conformational states to their functional relevance is lacking. In fact, the majority of the simulation part of the paper is about suggesting different states of the PRLR-JAK2 complex but the states and their hypothesized functional relevance are not further taken up, e.g. by experiments, and yet presented as major results, e.g. in the abstract.<br /> 3. The connection between simulations and mutational study is not very direct.<br /> An open question is if the mutants can distinguish between the effects of PRLR-PIP2 interaction or PRLR-JAK2 interaction, even though this conclusion is still drawn from the data.<br /> 4. The conclusions drawn from the mutagenesis study (lines 547-555) are not directly supported by data. Only a partial correlation between PRLR membrane localisation and STAT5 activation is no reason to attribute the unexplained part of the STAT5 activation to PRLR-JAK2 interactions without further studies.<br /> 5. PIP2 is identified as an important regulator, with very solid support from the presented data. PIP3 is part of the model but not discussed before or as part of the results. The analysis could be similarly applied or the data directly relevant to the understanding of PIP3 plays a similar role, as interactions are likely primarily electrostatically driven.

Reviewer #3 (Public Review):

Araya-Secchi and coauthors present a very interesting study on the role of PIP2 lipids in the potential modulation of prolactin receptor signaling. The study is well-conducted and employs an integrated approach that combines NMR spectroscopy, modeling (primarily coarse-grain MD simulations), and cell biology. This combination of methods is crucial for gaining a deeper understanding of cell receptors, from their biophysical properties to their cellular functions.

The modelling work is mainly based on both coarse grain forcefield versions Martini2.2 and Martini3. These two versions of the forcefield may produce different results. Therefore, depending on the system being modeled, the results presented here should be considered in light of the limitations inherent to each version of the forcefield.

Reviewer #1 (Public Review):

The authors study single and pairs of MDCK cells adherent to an H-shaped geometry on a flat surface. In this pattern, the cells form strong peripheral stress fibers. To a lesser extent, these cells also exhibit stress fibers in the cell interior, which otherwise has a rather homogenous actin distribution. Using a combination of traction force microscopy, from which they infer the stress distribution by monolayer stress microscopy, and "contour analysis" the authors quantify the 'bulk' and the 'surface' stress in these cells. This analysis shows that single cells are mechanically polarized whereas pairs are not.

The authors then go on to optogenetically activate the actomyosin contractility of either one half of a single cell or one cell of a pair. Combining their stress measurements in these situations and using a finite element mechanical model, the authors convincingly show that the mechanical response in the non-activated part is active. By varying the aspect ratio of the adhesion patterns, they also find that the efficacy of active stress propagation depends on the mechanical and structural polarity of the cell. Furthermore, they provide evidence that their results on cell pairs generalize to tissues.

Strengths:

This study uses a nice combination of physical tools to address an important question in tissue mechanics. The data is compelling and fully supports the authors' conclusions.

Weaknesses:

There are no major weaknesses.

In summary, although the fact that mechanical stress propagation in tissues is an active process might not come as a surprise, the study makes substantial contributions to a quantitative contribution of this process. As such it is of fundamental significance in the field. It will be interesting to explore the consequences of this mechanism for mechanical stress propagation in the context of developmental processes. It will be also of great interest to study how this local process can be accounted for in large-scale theories.

Reviewer #2 (Public Review):

In A. Ruppel, et al, the authors study the mechanics of one cell, two cells, and cell monolayers upon a transient local activation of contractility. First, the authors characterize the tractions and stress maps (measured via Traction Force Microscopy and Monolayer Stress Microscopy, resp.) for one and two cells in the absence of contractility activation, and found a correlation between the principal stress direction and actin fiber orientation. Next, the authors use the theory of foams to infer, combining traction force data and cell geometry data, the mechanical parameters of cells like the line tension or the force of adherent fibers. Next, the authors activate contractility by means of optogenetic tools on one half of the system and quantify the response on both halves, concluding that the receiver half response is driven by active processes, increasing contractility for two cells, while fluidizing for one cell. Next, the authors estimate the level of active response in cell doublets by comparing the stress maps to numerical simulations of a thin elastic medium with anisotropic contractility. By varying aspect ratios of the H pattern, the authors find a correlation between the principal stress direction and the orientation of stress fibers and find that the previous active response is in general enhanced when the principal stress direction is perpendicular to the orientation of the fibers. Finally, these features are also found in a cell monolayer for a fixed confinement aspect ratio.

Overall, the manuscript contains a broad characterization of the steady state mechanics and the dynamical response to the activation of contractility for one cell, two cells, and cell monolayers.

Reviewer #1 (Public Review):

Overall, this manuscript by Liu et al. provides a largely convincing mechanism for both how Zfp467 regulates osteoblast differentiation and how PTH1R expression and function in osteoblast-lineage cells is regulated at the transcriptional level, finding that NF-kB (RelB/p50) regulates PTH1R expression downstream of Zfp467. PTH1R expression and activity in turn is enhanced in Zfp467-deficient osteoblasts. In turn, PTH signaling regulates Zfp467 expression through PKA activity. In particular, the new findings on mechanisms of regulating PTH1R expression and evidence that this in turn impacts osteoblast differentiation are felt to be of broad interest and importance. The approach used is felt to be largely sound. Areas of major concern are few and relate mostly to better fleshing out how the NF-kB pathway is impacted as a part of the molecular pathway implicated here and clarifying some confusion regarding uCT data that appears to be discussed but which this reviewer cannot locate in the figure.

Reviewer #2 (Public Review):

The authors hypothesized that PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass. Using a number of methods, they have established a regulatory feedback mechanism of this transcription factor and the PTH receptor in osteoblastic precursors as well as showing that PrrxCre deletion of Zfp467 causes an increase in trabecular bone mass, while AdipoCre does not. Nevertheless, they have not established the actual mechanism of action of the transcription factor nor which gene it acts on in the osteoblast. They have mostly achieved their aims and the results partially support their conclusions. However, the work is descriptive and does not address the central issue of how ZFP467 acts. At present, its impact on the field is limited.

Reviewer #3 (Public Review):

In this paper the authors report a new pathway by which PTH, by activating PTH1R, increases bone mass. Specifically, they describe that PTH1R and the transcription factor Zfp467 are part of a feedback loop that promotes PTH-induced osteogenesis. By using in vivo mouse models, they indicate that deletion of Zfp467 in osteoblast progenitors increases bone mass and osteogenic differentiation whereas deletion of Zfp467 in adipocytes does not cause any bone phenotype, suggesting that deletion of Zfp467 in mesenchymal progenitors is important for bone mass. With a series of in vivo studies, they demonstrate that PTH suppresses Zfp467 expression via the cyclic AMP-PKA pathway and genetic deletion of Zfp467 causes increased PTH1R transcription by increasing nuclear translocation of p50 and activation of the P2 promoter, thus increasing the expression of PTH1R and the cellular responsiveness to PTH.

The strength of the paper are the use of genetically modified animal models, the analysis of both female and male mice and the logic flow of the in vitro data. The weaknesses of the paper are the wrong conclusion that the phenotype of the PrrxCre Zfp467 mice perfectly recapitulates one of the global Zfp467 KO mice, the lack of histomorphometric data showing increased osteoblastogenesis and the missing evidence that the forward feedback loop is relevant to the response to PTH in vivo. The analysis is therefore incomplete. Once those points are addressed, this paper would be of great interest in the bone field.

Reviewer #1 (Public Review):

In this study, the authors sought to develop a measure of Staphylococcus aureus intracellular virulence levels in the lab (the InToxSa assay) that more closely mimics the activity seen in vivo. They then used untargeted approaches (GWAS, homoplasy) on a set of 387 Australasian clinical isolates to identify genes with mutants associated with reduced intracellular toxicity. The authors identified several mutated genes which reduced virulence in the strains chosen for the study, demonstrating that their approach can be used to uncover virulence-related genes in S. aureus.

The study is clearly written, with high-quality figures. The development of the InToxSa assay is carefully described and logical. InToxSa was shown to potentially be more sensitive than the tryptophan blue test in detecting reduced intracellular cytotoxicity phenotype. They also showed evidence for agrA mutants and other transposon mutants with reduced inToxSa cytotoxicity having increased bacterial cell numbers cells compared to wild-type (Fig 2, Fig5GH), which is critical to the argument that bacteremia selects for intracellular persistence as a way to escape the immune system. There was an interesting and thoughtful use of random forest to choose the most appropriate parameters of the kinetic model.

The GWAS studies used publicly deposited genome data and clearly showed lineage effects of reduced intracellular survival of CC239 and CC22, confirming previous results. GWAS also confirmed the well-known pervasive association of agr mutants with reduced toxicity. Using a well-described homoplasy test for convergent evolution to extract more power, several other potential genes associated with enhanced intracellular toxicity were discovered or rediscovered, perhaps most significantly, the ausA gene, with biosynthesizes aureusimines (pyrazinone secondary metabolites) posited to have a role in the phagosomal escape.

There are two main 'weaknesses'. The first is the limited power that comes from only using measuring the phenotype of 387 strains. Whether this is because of the expense/ difficulty of the inToxSa is not discussed, leaving open the question of how much this assay could be scaled up in the future. The second is that the main output of the assay is actually reduced intracellular toxicity (PI uptake AUC), which is inferred to be strongly linked to increased intracellular persistence. The linkage between the phenotypes comes primarily from microscopic studies on a limited number of strains. It may be true of all cases but the possibility exists that for some of the strains, reduced cytotoxicity may be associated with intracellular elimination, which would presumably be a negative outcome for systemic infection.

Overall, the authors achieved their aims in terms of assay development and showing the utility of the pipeline for mutation discovery. This is a waypoint in the larger aim of understanding mutational pathways that lead to increased persistence of systemic S. aureus. Obviously, a lot more data is needed. The InToxSa intracellular screening method is interesting and could be reused/adapted by the community. This research should also spark more interest in the role of ausA and aureusimines in virulence and some of the other genes discovered through the untargeted approach.

Reviewer #2 (Public Review):

This manuscript introduces a novel assay in a 'phenomics' approach to address an important aspect of S. aureus pathogenesis. The authors set out to identify mutations that arise during clinical S. aureus infections that cause a decrease in intracellular host-cell toxicity and increase intracellular persistence. To do this, they use a 'phenomics' approach. For phenotype, they quantify HeLa cell toxicity for each strain in a panel of 387 clinical S. aureus isolates. This is done by measuring HeLa cell death induced by intracellular S. aureus via propidium-iodide uptake. The whole genomes of each of these 387 isolates had previously been sequences. They use the genomic data and phenotype data to carry out a genome-wide association study (GWAS) looking for genetic signatures that correlate with reduced HeLa cell cytotoxicity. As expected, mutations in agr were the strongest locus-level signal, but the study did identify one agr-independent mutation in ausA, which was able to be independently validated, showing that the assay is robust enough to find causal mutations. The analysis is thoughtful, the assay appears robust, and I think the discussion of conclusions and limitations is mostly valid. Thus, my concerns are focused on further understanding the practical utility of the approach and whether or not the HeLa cell model recapitulates what happens in professional phagocytes. For example, it is not clear to me that this system has the statistical power to find novel, biologically relevant rare mutations without first being very mindful in selecting strains that are extremely genetically similar. It is also not clear to me that the toxicity assay captures the important features of the intracellular persistence that occurs in vivo within professional phagocytic cells. Thus, given these practical limitations and a somewhat artificial model system, the impact on the field is likely to be moderate in nature. However, the analysis and approach taken could be re-purposed to any robust quantitative phenotype, and this will certainly be of great interest to others that study bacterial evolution in clinical contexts.

Reviewer #1 (Public Review):

In this paper, Krishnan et al. describe their findings on the genetic architecture of the heart mitochondrial proteome that influences cardiac hypertrophy. They analyzed common genetic variations contributing to mitochondrial and heart functions in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP), by performing whole heart proteomics. The authors have published a number of papers on this panel, which appears to be a powerful system to study various genetic factors. They identified three trans-acting genetic loci, located on chromosome (chr) 7, chr13, and chr17, which control both mitochondrial proteins and heart hypertrophy. High-resolution regional mapping identified NDUFS4, LRPPRC, and COQ7 as the candidate genes for chr13, chr17, and chr7 loci, and variations of these genes were associated with heart mass in isoproterenol-induced heart failure and diet-induced obesity. Using co-expression protein networks using weighted gene co-expression network analysis (WGCNA), they show that the chr13 locus was highly enriched for complex-I proteins, the chr17 locus for mitochondrial ribonucleoprotein complex, and the chr7 locus for ubiquinone biosynthesis. They concluded that "common variations of certain mitochondrial proteins can act in trans to influence mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations."

Although these studies are interesting and provide novel findings in the genetics of cardiac hypertrophy, there are a number of technical and conceptual issues that need to be addressed.

Reviewer #2 (Public Review):

Krishnan, et al describe a unique and powerful approach to assessing the role of genetic variation on mitochondrial and cardiac function and health. Utilizing a panel of inbred mouse strains, on which they performed proteomics on heart samples, they measured 840 mitochondrial proteins and correlated these data to heart function using two heart stress models. This resulted in a number of correlative observations, three of which were explored in more detail to connect three specific genes to cardiac hypertrophy. This is an interesting dataset and there is clearly value in what is presented. The data were largely correlative, however, and there are only a couple of causation-oriented experiments. It's hard to adjudicate between these strengths and weaknesses in determining the overall impact of the manuscript.

Reviewer #3 (Public Review):

The goal of this study is to identify mitochondrial pathways that would have an impact on the process of pathological cardiac hypertrophy. The paper presents a state-of-the-art analysis of the SNP variant and regulatory hot spots associated with quantitative traits, here mitochondrial protein levels and cardiac hypertrophy, using the Hybrid Mouse Diversity Panel. They identify 3 hotspots of trans-acting genetic loci that correlate with the level of proteins involved in complex I assembly, mitochondrial mRNA stability, and CoQ synthesis.

The study is overall very interesting and brings valuable information to the field. However, the impact of each of these loci on cardiac hypertrophy level, even if statistically significant, seems to be rather limited raising questions on the clinical relevance of the findings. It is an interesting study anyway and points to pathways that will deserve to be further explored in the future in clinical studies on human patients.

Reviewer #1 (Public Review):

Mermithid nematodes are ecologically important parasitoids of arthropods, annelids and mollusks today. Their fossil record in amber reaches back into the Early Cretaceous, some 135 million years ago. Luo et al. more than triple this record by presenting, with ample illustrations, exceptionally well preserved new specimens from the beginning of the Late Cretaceous (99 Ma ago) of Myanmar. Their most important finding is that mermithids parasitized a number of insect clades in the Cretaceous that they are not known to infect today or in Cenozoic amber; further, the proportion of holometabolous insects among the hosts is found to be lower in the Cretaceous than in the Cenozoic. The strengths of the paper lie in the specimens, the illustrations of the specimens, and the documentation of when, where and how the specimens were acquired. Certain nomenclatural aspects of the paper require improvement. A potential weakness of the paper could be collection bias: it is not tested whether the collections used to show the shift toward holometabolous hosts from the mid-Cretaceous to the Cenozoic are representative of the fossil record as it is preserved and accessible today.

Reviewer #2 (Public Review):

This manuscript reports on mermithid nematode fossils from amber which dates from the Cretaceous period. The specimens described in the manuscript consist of insects and associated nematodes which have been trapped in amber and fossilised. The nematodes have been identified as belonging to the Mermithidae family, a family of nematode worm that infect insects.

The findings of this manuscript provide an insight into the evolution history of nematodes and parasitism. Despite the ubiquity of both nematodes and parasites in extant ecosystems, fossil records of both are very rare. This is because nematodes and many parasites are soft bodied, and many are located inside their hosts' bodies, thus they rarely become fossilised. Thus, most of what is known about the evolutionary history of nematodes, and evolution of parasitism are based on what could be inferred from extant examples.

The specimens described in this manuscript provides a valuable contribution to our understanding of parasitism in the geological past. These amber specimens are a snapshot of parasite-host interactions - interactions which are commonly found in nature but are rarely captured in fossils. The identification of the specimens as mermithid nematodes are based on sound scientific reasoning. The worms' morphology and position in relation to the insects are consistent with what have been observed with extant mermithid nematodes.

Additionally, one of the values of such parasite fossils is that they provide us with insight into parasite-host combinations or interactions which may have existed throughout the geological past, but no longer exist today or cannot be inferred from extant taxa. It helps fill in major gaps in our understanding of parasitism. This was the case with the amber fossil that contained a bristletail with its nematode parasite.

Reviewer #3 (Public Review):

The authors provide a timely description of new mermithid nematodes from Cretaceous amber and use it to argue an important shift in insect host exploitation. The descriptions are state-of-the-art and will become valid once the appropriate zoobank numbers are used after publication. The authors also compiled crucial and detailed new information on the host exploitation in amber nematodes in the supplementary material. This data is also depicted in pie diagrams and seems at first glance to support their interpretations of a shifts in host exploitation in fossil amber deposits when analysed appropriately and statistically but such an true analysis and depiction should be part of the main manuscript to do the compilation and interpretation justice. For the sake of reproducibility and the field, such fundamental statistical analysis as well as a statistical comparison with modern hosts would make this broad-sweeping claim of a major host shift and importance of amber deposits containing such nematode-insect interactions since the Cretaceous (even) more robust and fundamental.

Reviewer #1 (Public Review):

This paper investigates the neural correlates of noise-induced hearing loss. The authors use an electrode array to capture neural responses across the inferior colliculus to speech and synthetic sounds in both normal-hearing gerbils, and gerbils with noise-induced hearing loss. They use dimensionality reduction to isolate a low-dimensional response subspace that captures most of the information about the speech signals, and find that this low-dimensional representation is altered considerably by hearing loss (evaluated with CCA). To probe the basis of these differences, the authors train an artificial neural network to predict the subspace responses to arbitrary stimuli, for instance to investigate the consequences of frequency-dependent amplification of sound with a hearing aid, or synthetic test stimuli. Using this approach, they find that the representation of sounds in quiet is largely restored by a hearing aid algorithm that amplifies high frequencies to render them audible. However, the representation of sounds in noise also differs between the IC of normal-hearing and hearing-impaired gerbils, and this difference is not eliminated by a hearing aid. Specifically, low-frequency maskers seem to distort the representation of high-frequency sounds (e.g. consonants in speech), even once the high-frequencies have been amplified to compensate for the hearing loss.

Overall, this is a strong paper. The topic is important, the methods are innovative, logical, and rigorous, and the whole thing is exceptionally clearly described. I greatly appreciate the care that clearly went into writing the paper. I have two major concerns. The first seems fairly critical to the paper's conclusions, but I hope can be addressed with some kind of control experiment. The second could potentially be thought of as more of a future direction, but it speaks to the specificity of the conclusions.

1. My main substantive concern is that the conclusions depend critically on believing the predictions of the DNN, and yet it is not clear we should expect it to generalize well to stimuli outside its training distribution. Current artificial neural networks typically work very well for stimuli like those they were trained on, but often do not generalize as well as one might like. The authors recorded responses to speech in quiet and in different noise levels, and show that the trained DNN (trained on these sounds and the associated responses) produces very accurate predictions on held-out sounds from this distribution. But the conclusions depend critically on the DNN predictions for sound processed by a hearing aid, and for synthetic sounds (pure tones, SAM noises) that are quite unlike the training data. The predictions look reasonable in places where we have some prior sense for what to expect (level-dependent frequency tuning to pure tones), which is reassuring, but I am not sure how to be confident that the predictions should be accurate for all of the conditions that are tested, in particular to the results with the simulated hearing aid. I am pretty sure that the predictions will be inaccurate for some types of stimuli (just based on the various pathologies that are known to occur with neural networks). I would hope that this would not be the case for the conditions tested by the authors, but it is hard to be sure, and this makes the conclusions seem a little more vulnerable than I would like.<br /> How do we know that the DNN generalizes beyond its training data well enough to render the conclusions airtight?

2. My second concern is the extent to which the results are specific to a) the IC, and b) noise. The authors assert that similar effects would not be present in the nerve, citing a Heinz paper, but I am not sure how clear this evidence is - it is not described in enough detail here to assess. It would be nice to show this, perhaps by repeating their analysis on a model of the nerve with and without simulated hearing loss. One can similarly wonder about the effects in the cortex, especially given the literature on noise invariance (Rabinowitz, Moore, Khalighinejad, Kell...), which would at least be worth discussing. It is similarly unclear whether the results are specific to additive noise. Would similar conclusions hold for any type of distortion? This could be easily addressed by an additional DNN analysis (e.g. with clipping, or segments of speech intermittently replaced by silence, or reverberation).

Reviewer #2 (Public Review):

This very interesting study uses a combination of high channel count neural recordings and machine learning to characterize neural representations of complex natural and synthetic sounds in the inferior colliculus. The authors use deep neural networks to model sound evoked activity in a large number of IC multiunits with high accuracy in gerbils with normal hearing and hearing loss. They then use the DNNs to simulate activity evoked by a wide range of stimuli and demonstrate systematic differences in latent population representations between normal hearing and hearing-impaired animals. Models for hearing impaired animals show activity consistent with impaired representations of speech in noise. These results lay the groundwork for a potentially valuable approach to improving signal processing in hearing aids and prosthetics.

The large speech dataset and clean hearing loss effects are particularly impressive. While the approach and associated data are novel and likely to be of broad interest, there are some substantial concerns about the study. First, the authors fail to acknowledge substantial previous work on super-threshold activity in cortex of animals with hearing loss, making it appear that they overstate the novelty of the current results. There are also many cases where they fail to clearly report the details of statistics used to support their claims. Finally, while the accuracy of the DNN models is compelling for the speech stimuli in the data set, it is not clear that the comparisons of simulated activity reflect actual neural activity in the stimulus conditions tested.

Reviewer #1 (Public Review):

In this manuscript, Li et al characterize sex differences in the impact of macrophage RELMa in protection against diet-induced obesity [DIO]. This is a key area of interest as obesity studies in mice have generally focused exclusively on male animals, as they tend to gain more weight, faster than female mice. The authors use a combination of flow cytometry, adoptive transfer, and single-cell transcriptomics to characterize the mechanism of action for female-specific DIO protection. They identify a potential role for eosinophils in mediating female DIO protection downstream of RELMa production by macrophage. They also use the transcriptomic characterization of the stromal vascular fraction of the adipose tissue to evaluate molecular and cellular drivers of this sex-specific DIO protection.<br /> Although the authors provide solid evidence for many claims in the manuscript, there is generally not enough information about the studies' methods (especially on the computational/data analysis aspects) for a careful evaluation of the result's robustness at this stage.

Reviewer #2 (Public Review):

In the study by Li et al., the authors hypothesize that RELMa, a macrophage-derived protein, plays a sex-dimorphic role as a protective factor in obesity in females vs males. The authors perform largely in vivo studies utilizing male and female WT and RELMa KO mice on a high-fat diet and perform an in-depth analysis of immune cell composition, gene expression, and single-cell RNA Sequencing. The authors find that WT females are protected from obesity and inflammation vs males, and this protection is lost in female RELMa KO mice. Further analysis by the authors including flow cytometry of the visceral fat SVF in female WT mice showed reduced macrophage infiltration, higher levels of eosinophils, and Th2 cytokine expression compared to WT male mice and female KO mice. The authors show that protection from obesity and inflammation in female RELMa KO mice can be rescued with an injection of eosinophils and recombinant RELMa. Lastly, the authors use single-cell RNA-Sequencing to further analyze SVF cells in WT and KO male and female mice on a high-fat diet.

Overall, we find that the study represents an important finding in the immunometabolism field showing that RELMa is a key myeloid-derived factor that helps influence the macrophage-eosinophil function in female mice and protects from diet-induced obesity and inflammation in a sexually dimorphic manner. Overall, the study provides strong and convincing data supporting the authors' hypothesis and conclusion.

Reviewer #3 (Public Review):

Li, Ruggiero-Ruff et al. examine the role of RELMα, an anti-inflammatory macrophage signature gene, in mediating sex differences in high-fat diet (HFD)-induced obesity in young mice. Specifically, the authors hypothesize that RELMα protects females against HFD-induced obesity. Comparisons between RELMα-knockout (KO) and wildtype (WT) mice of both sexes revealed sex- and RELMα-specific differences in weight gain, immune cell populations, and inflammatory signaling in response to HFD. RELMα-deficiency in females led to increased weight gain, expansion of pro-inflammatory macrophage populations, and eosinophil loss in response to HFD. Female RELMα-deficiency could be rescued by RELMα treatment or eosinophil transfer. Single-cell RNA-sequencing (scRNA-seq) of adipose stromal vascular fraction (SVF) revealed sex- and RELMα-dependent differences under HFD conditions and identified potential "pro-obesity" and "anti-obesity" genes in a cell-type-specific manner. Using trajectory analysis, the authors suggest dysregulation of macrophage-to-monocyte transition in RELMα-deficient mice.

The conclusions of this paper are mostly well supported by the data, but some aspects of the statistical and single-cell analyses will need to be corrected, clarified, and extended to enhance the report.

Strengths:<br /> The authors use several orthogonal approaches (i.e., flow cytometry, immunohistochemistry, scRNA-Seq) and models to support their hypotheses.

The authors demonstrate that phenotypes observed in HFD-fed females with RELMα-deficiency (i.e., weight gain, loss of eosinophils, a gain of M1 macrophages) can be rescued by RELMα treatment or eosinophil transfer.

The authors recognized the complexity of macrophage activation that is beyond the 'M1/M2' paradigm and informed readers in the introduction as to why this paradigm was used in this study. During the scRNA-seq analyses, the authors further sub-cluster macrophages to include more granularity.

Weaknesses:<br /> There are several instances in the text where the authors claim that there is a significant difference between the two groups, but the statistics for these comparisons are not shown in the figure.

It is unfortunate that eosinophils could not be identified in the single-cell analysis since this population of cells was shown to be important in rescuing the RELMα-deficiency in HFD-fed females. The authors should note in the discussion how future scRNA-Seq experiments could overcome this limitation (i.e., enriching immune cells prior to scRNA-Seq).

There are several issues with the scRNA-Seq analysis and interpretation. More details on the steps taken in the single-cell analyses should be included in the methods section. With regards to the 'pseudobulk' analyses presented in Figs. 5-6, several of the differentially expressed genes identified in Fig. 6 are hemoglobin genes (i.e., Hba, Hbb genes). It is not uncommon to filter these genes out of single-cell analysis since their presence usually indicates red blood cell (RBC) contamination (PMID: 31942070, PMID: 35672358). We would recommend assessing RBC contamination as well as removing Fig. 6 from the manuscript and focusing on cell-type-specific analyses. Re-analysis will likely have an impact on the overall conclusions of the study.

Within the text, there are several instances where the authors claim that a pathway is upregulated based on their Gene Ontology (GO) over-representation analysis (ORA). To come to this conclusion, the authors identify genes that are upregulated in one condition and then perform GO-ORA on these genes. However, the authors do not consider negative regulators, whose upregulation would actually decrease the pathway. Authors should either replace their GO-ORA analysis with one that considers the magnitude and direction of differentially expressed genes and provides an activation z-score (i.e., Ingenuity Pathway Analysis) or replace instances of 'upregulated' or 'downregulated' pathways with 'over-represented' pathways.

For Fig.7A, a representative tSNE plot for each group (WT Female, KO Female, WT Male, KO Male) should be shown to ensure there is proper integration of the clusters across groups. There are some instances where the scRNA-Seq data do not appear to be integrated properly (i.e., Supplemental Figure 2C). The authors should explore integration techniques (i.e., Seurat; PMID: 29608179) to correct for potential batch effects within the analysis.

LncRNA Gm47283 is identified as a gene that is differentially expressed by genotype in HFD females (Fig. 7G); however, according to Ensembl this gene is encoded on the Y-chromosome (https://uswest.ensembl.org/Mus_musculus/Gene/Summary?g=ENSMUSG00000096768;r=Y:90796007-90827734). The authors should use the RELMα genotype and sex chromosomally-encoded genes to confirm that their multiplexing was appropriate.

For Fig. 8, samples should be co-clustered and integrated across groups before performing trajectory analysis to allow for direct comparisons between groups.

Since the experiments presented in this report were from young mice using a single diet intervention, the authors should comment on how age and other obesogenic diets may impact the results found here. Also, the authors should expand their discussion as to what upstream regulators (i.e., hormones or genetics) may be driving the sex differences in RELMα expression in response to HFD.

Reviewer #1 (Public Review):

In vertebrates, ciliary motility is important for left-right body patterning, airway clearance, cerebrospinal fluid flow, and the locomotion of spermatozoa. The movement of cilia is powered by the action of dyneins tethered to axonemal doublet microtubules. The largest and most powerful axonemal dynein, OAD, is tethered by a pentameric docking complex (the OAD-DC). Here, Yamaguchi, Morikawa and Kikkawa show convincingly that the Calaxin and Armc4 subunits of the OAD-DC have discrete roles in docking OADs. Using zebrafish mutants, they show that loss of Armc4 causes complete loss of the OAD, whereas mutation of Calaxin causes only partial OAD loss. They demonstrate that Calaxin localization is dependent on Armc4 but independent of the OAD or calcium conditions. Using cryo-ET, they report a higher resolution structure of the wild-type zebrafish sperm axoneme than previously determined (Yamaguchi et al., 2018) and show that the OAD and OAD-DC structures resemble the cryo-EM structures of other organisms. Cryo-ET analysis of calaxin-/- axonemes reveals that without Calaxin, OADs have mostly normal conformations but make fewer connections with the OAD-DC and are less stably bound. The paper is well-written with appropriate methods and conclusions.

Reviewer #2 (Public Review):

Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

Gui et al. (2021) already reported on a cryo-EM observation in bovine tracheal cilia, with the conclusion similar to this paper in the structure of OAD/DC on DMT. Using knockout zebrafish strain, the authors present detailed interaction of calaxin with other DC components. They show that the binding of calaxin induces the changes of conformation in N-terminal region of CCDC151/114. The conformation further changes in the presence of Ca2+; specific conformation of N-terminal region of CCDC151/114 becomes undetectable, instead additional structure appears in the vicinity of calaxin.

1) The authors conclude that the Ca2+-dependent conformational change of DC is subtle and not dynamic. This result is eventually valuable information but may be somewhat unexpected from the point of view that calaxin plays an important role in the regulation of flagellar motility in Ciona sperm. The authors found that calaxin changes the conformation of N-terminal CCDC151/114 region but the core dynein structure shows no dynamic change. What about the changes in the interaction between calaxin, core dynein, and DMT? Is this beyond the resolution of cryo-ET analysis?

2) It would be very helpful if the authors could add the cryo-ET images of calaxin-/- axoneme in the presence of 1 mM EGTA in Figure 7. Although these images are thought to be similar or identical to Figure 4F, it would help to confirm that the conformational changes in CCDC151/114 and additional part of DC are induced in a Ca2+-dependent manner.

3) To clarify the molecular interaction of calaxin with other components, it would also be helpful if the authors add the images rotated 80 degree to Figure 4F and G, in similar way in Figure 7,

4) Despite the molecular phylogenetic difference, there are several similarities between calaxin and Chlamydomonas DC3, not only in the in situ structure and configuration but in the phenotype of mutants; Chlamydomonas mutant lacking DC3 shows OAD loss in the distal part of a flagellum (Casey et al, MBC, 2003). It may be a good reference if the authors add the position of DC3 in Figure 4. A', B', and C.

5) There is a significant difference in sperm motility between WT and calaxin-/- or WT and armc4-/- (Figure 2E). However, it is not clear whether immotile sperm were included in the data for VAP (Figure 2F) or BCF (Figure 2G). For example, WT and calaxin-/- show similar VAP, although both are significantly different in the percent of motile sperm.

6) In calaxin-/- mouse, OAD was clearly detected from the base to two-thirds of a flagellum with unclear border (Figure 2A). Typical distribution of OAD+class and OAD-class are shown in Figure 5 in the ~3 micrometer tomograms. Were these taken from around this unclear border? Are proximal most region of a flagellum occupied with OAD+class only? The authors should clearly indicate the region of a flagellum where the tomograms in Figure 5C and D were selected.

7) Line 229~: It is not clear what the authors meant by "probably reflecting the different distance from the sperm head". In relation to this and the comment 6, does the "proximal" in the sentence "OAD loss occurred even in the proximal part of the flagella" (line 232) indicate the region near the base of a flagellum?

8) In conjugation with comment 7, it would be appreciated to show an authors' idea on why distal region of flagella tends to lack calaxin, if they do not discuss anywhere in the text,

9) Immunofluorescence in twister-/- epithelial cilia showed that the localization of calaxin is independent of OAD (line 271-274). Based on the authors' finding, the localization of calaxin requires Armc4, which is preassembled with calaxin in the cytoplasm. If this is true and the localization of calaxin is NOT resulting from diffusion, Armc4 must be localized with calaxin along the entire length of cilia in twister-/- epithelial cilia (Figure 6D). Although Armc4 is shown localized in cryo-ET images (e.g. Figure 1, Figure 7), authors may provide the immunofluorescence of Armc4 along the entire length of sperm flagella and epithelial cilia.

Reviewer #3 (Public Review):

ODA-DC anchors ODA, the main force generator of ciliary beating, onto the doublet microtubules. Vertebrate ODA-DC contains 5 proteins, including Calaxin and Armc4, whose mutations are associated with defective ciliary motility in animals and human. By generating calaxin-/- and armc4-/- knockout zebrafish lines, this manuscript examined the Kupffer's vesicle cilia and spermatozoa. They showed that calaxin-/- and armc4-/- knockouts both affect ciliary motility but to different degrees. The authors conducted careful structural analyses using cryo-ET and subtomo averaging on both mutants, revealing a partial loss of ODA in calaxin-/- and a complete loss of ODA in armc4-/-. I really like the distribution analysis of calaxin-/- OADs (Figure 5), which emphasizes the strength of cryo-ET in uncovering the molecule distribution of distinct conformational states in situ. Fitting of the atomic models of ODA and ODA-DC into the cryo-ET density maps and Calaxin rescue experiments showed how Calaxin stabilizes ODA at a molecular detail. By using olfactory epithelium, the authors also presented the possible assembly mechanism of ODA-DC proteins, which is also a beautiful experiment. Finally, the authors also investigated how Ca2+ regulate the ODA-DC using cryo-ET.

The thorough structural and functional analyses of Calaxin and Armc4 in WT and gene KO animals could serve as a reference for future study of the detailed function of other ciliary proteins. The experiments are overall well designed and conducted, but some aspects need to be clarified and improved.

The authors interpret the vertebrate ODC-DC to include four linkers (line 193). However, the authors also said that loss of one linker (Calaxin) makes ODA to attach on the DMT through two linkers (line 199 and 246). These descriptions are confusing. It would make more sense to interpret the vertebrate ODC-DC as containing three linkers (CCDC151/114, Armc4/TTC25, Calaxin).

To confirm whether Calaxin directly interacts with β-tubulin (line 213), a control experiment could be needed by incubating WT axoneme with mEGFP-Calaxin followed by IF imaging.

The Immunoblotting experiment should be improved in Figure 5E. Could the authors get the same results in repeating experiments? Why is the Dnah8 signal higher in 50 mM NaCl of the (+)Calaxin group compared to that in 0 NaCl? This makes me doubt if the difference between (-)Calaxin and (+)Calaxin groups are significant.

The authors have covered several important points in the Discussion section. Now that the function of Calaxin in both mouse and zebrafish have been reported, the authors could discuss the similarity and difference of Calaxin function in different species and tissues.

Because of the limited resolution, the authors should be more careful when interpreting the small densities in the difference map, for example, in Figure 4F-G black arrows. Considering that the CCDC151/114 coiled coil is overall poorly resolved both in the WT and mutant cryo-ET maps, the different densities could be due to different map quality or data processing. This makes the following statement suspicious "This structure corresponds to the N-terminus region of CCDC151/114, suggesting that Calaxin affects the conformation of neighboring DC components".

Reviewer #1 (Public Review):

This paper presents a thorough biochemical characterization of inferred ancestral versions of the Dicer helicase function. Probably the most significant finding is that the deepest ancestral protein reconstructed (AncD1D2) has significant double-stranded RNA-stimulated ATPase activity that was lost later, along the vertebrate lineage. These results strongly suggest that the previously known differences in ATPase activity between extant vertebrates and, for example, extant arthropods is due to loss of the ATPase activity over evolutionary time as opposed to gains in specific lineages. Based on their analysis, the authors also "restore" ATPase function in the vertebrate dicer, but they did so by making many (over 40) mutations in the vertebrate protein, and it is not clear which of these many mutations is required for the restoration of the activity. Thus, it is difficult to discern how the results of this experiment relate to the evolutionary history.

A criticism of the paper is the authors' tendency (probably unconscious) to ascribe a purposefulness to evolution. For example, in the introduction, "We speculate that the unique role of the RLR's in the interferon signaling pathway in vertebrates...created an incentive to jettison an active helicase in vertebrates." Although this sentence is clearly labelled as speculation and "incentive" is clearly a metaphor, the implication is that evolution somehow has forethought. (There are other instances of this notion in the paper, for example, in the last line of the abstract). The author's statement also implies that the developing interferon system somehow caused the loss of active helicase, but it seems equally plausible that the helicase function was lost before the interferon system co-opted it.

Reviewer #2 (Public Review):

The manuscript by Aderounmu presents an interesting attempt to reconstruct evolution of the function of the helicase domain in ancestral Dicers, RNase III enzymes producing siRNAs from long double-stranded RNA and microRNAs from small hairpin precursors. The helicase has a role in long dsRNA recognition and processing and this function could have an antiviral role. Authors show on reconstructed ancestral Dicer variants that the helicase was losing dsRNA binding affinity and ATPase activity during evolution of the lineage leading to vertebrates while an early divergent Dicer-2 variant in Arthropods retained high activity and seemed better adapted for blunt ended long dsRNA, which would be consistent with antiviral function.

The work is consistent with apparent adaptation of vertebrate Dicers for miRNA biogenesis and two known modes of substrate loading: "bottom up" dsRNA threading through the helicase domain where the helicase domain recognizes the end of dsRNA and feeds it into the enzyme and "top-down" where the substrate is first anchored in the PAZ domain before it locks into the enzyme. Some extant Dicer variants are known to be adapted for just one of these two modes while Dicer in C. elegans exemplifies an "ambidextrous" variant. The reconstruction of the helicase domain complex enabled authors to test how well would be ancestral helicases supporting the "bottom up" feeding of long dsRNA and whether the helicase would be distinguishing blunt-end dsRNA and 3' 2 nucleotide overhang. Although the reconstruction of an ancestral protein from highly divergent extant sequences yields just a hypothetical ancestor, which cannot be validated, the work provides remarkable data for interpreting evolutionary history of the helicase domain and RNA silencing in more general. While it is not surprising that the ancestral helicase was a functional ATPase stimulated by dsRNA, particularly new and interesting are data that the decline of the helicase function started already at the level of the common deuterostome ancestor and the helicase was essentially dead in the vertebrate ancestor. It has been reported two decades ago that human Dicer carries a helicase, which has highly conserved critical residues in the ATPase domain but it is non-functional (10.1093/emboj/cdf582). Recently published mouse mutants showed that these highly conserved residues are not important in vivo (10.1016/j.molcel.2022.10.010). Aderounmu et al. now suggest that Dicer carried this dead ATPase with conserved residues for over 500 million years of vertebrate evolution.

I do not have any major comments to the biochemical analyses and while I think that the ancestral protein reconstruction could yield hypothetical sequences, which did not exist, I think they represent reasonable reconstructions, which yielded data worth of interpretations. My major criticism of the work concerns clarity for the readership and interpretations of some results where I wish authors would clarify/revise the text. The following three examples are particularly significant:

1) It should be explained to which common ancestor during metazoan evolution belongs the ancestral helicase AncD1D2 or at least what that sequence might represent in terms of common ancestry during metazoan evolution.

2) This is linked to the first point - authors work with phylogenetic trees reconstructed from a single protein sequence, which are not well aligned with predicted early metazoan divergence (https://doi.org/10.1098/rstb.2015.0036). While their sequence-based trees show early branching of Dicer-2 as if the two Dicers existed in the common ancestor of almost all animals (except of Placozoa), I do not think there is sufficient support for such a statement, especially since antiviral RNAi-dedicated Dicers evolve faster and Dicer-2 is restricted to a few distant taxonomic group, which might be better explained by independent duplications of ambidextrous ancestral Dicers. I would appreciate if authors would discuss this issue in more detail and make readers more aware of the complexity of the problem.

3) Authors should take more into the account existing literature and data when hypothesizing about sequences of events. Some decline of the helicase activity is apparent in AncD1DEUT suggesting that it initiated between AncD1D2 and AncD1DEUT. This implies that a) antiviral role of Dicer was becoming redundant with other cellular protein sensors by then and b) Dicer was already becoming adapted for miRNA biogenesis, which further progressed in the lineage leading to vertebrates to the unique top-down loading with the distinct pre-dicing state where the helicase forms a rigid arm. Authors even cite Qiao et al. (https://doi.org/10.1016/j.dci.2021.103997) who report primitive interferon-like system in molluscs - this places the ancestry of the interferon response upstream of AncD1DEUT and suggests that this ancestral protein-based system was taking over antiviral role of Dicer much earlier. In fact, a bit weaker performance of AncD1LOPH/DEUT combined with the aforementioned interferon-like system and massive miRNA expansion in extant molluscs (10.1126/sciadv.add9938) suggests that molluscs possibly followed a convergent path like mammals. While I am missing this kind of discussion in the manuscript, I think that the model where "interferon appears ..." in AncD1VERT (Fig. 6) is incorrect and misleading.

Reviewer #1 (Public Review):

VO2max is one of the most important gross criteria of peak performance ability and a plethora of studies focused on VO2max prediction. This manuscript provides huge and comprehensive data from male runners and male cyclists. The endurance-trained athletes performed cardiopulmonary exercise testing on a treadmill (n= 3330) or cycle ergometer (n=1094). In contrast to former studies, the authors used machine learning for algorithms and VO2max prediction. Models were derived and internally validated with multiple linear regression. The present study substantially expands current research.

Sadly, the manuscript has an important and relevant main shortcoming as the limitations of the study had not been addressed properly:<br /> - The authors paid no attention to the fact that their results are strongly influenced by the exercise protocol used. It is obvious e.g. that maximal performance attainable in protocols with 2-minute exercise steps will be higher compared to an identical protocol with 3- or 4-minute steps.<br /> - The exercise intensity was kept constant for only 2 minutes before the workload was increased (by 1km/h treadmill or by 20-30 W cycle ergometer). Due to the kinetics of lactate, VO2, etc., it is evident that the short 2-min intervals aggravate the correct determination of aerobic and anaerobic threshold. It is well-known that longer-lasting constant exercise steps (e.g. 4 minutes) are better when the focus is centered on threshold determinations.

The quality of this manuscript will be substantially improved when the authors could implement a comprehensive and blunt paragraph showing the limitations of their study.

Reviewer #1 (Public Review):

The authors provide evidence for chromatin, which in Drosophila muscle cells is peripherally localized in the nucleus, whereas the central region is depleted of chromatin, and is organised such that RNA polymerase II (RNAp) is surrounding dense regions of chromatin. The authors theoretically study the formation of these regions by describing chromatin as a multi-block copolymer, where the blocks correspond to active and inactive chromatin regions. These regions are assumed to phase separately and to have different solvability. The solvability of the active region is regulated by binding RNAp. The authors study the core-shell organization in a layered geometry by analyzing the various contributions to free energy. In this way, they in particular obtain the dependence of the shell-layer thickness, which is described as a polymer brush. From these results, they infer chromatin organization in spherical core-shell chromatin domains and compare these results to Brownian dynamics simulations.

The work is well done and even though it uses standard methods for studying block copolymers and polymer brushes obtains interesting information about local chromatin organization. These findings should be of great interest to researchers in the field of chromatin organization and in general to everybody interested in understanding the physical principles of biological organization.

The work has two main weaknesses: The experimental evidence for RNAp and chromatin micro-organization is weak as only one example is shown. It remains unclear whether the observed organization pattern is common or not. Also, no data is shown concerning the dependence of the extensions of the active and inactive phases on parameters, for example, solvent properties or transcriptional activity. Second, some parts could prove difficult for biologists to assess. For example, the expression for the brush-free energy should be explained in more detail and notions like that of 'mushrooms' need to be introduced. As a second example, biologists might benefit from a better explanation of the concept of a theta solvent and its relevance.

Reviewer #2 (Public Review):

This work formulates a detailed theoretical polymer physics model intended to explain the observed morphology of chromatin in the Drosophila cell nucleus. The model is examined in detail by both analytical calculation and computer simulation. The central premise of the suggested theory is that it is based on equilibrium statistical mechanics. Within this paradigm, authors explore the model that views chromatin fiber as a block copolymer and, most importantly, describes the role of RNA polymerase as it interacts with one of the copolymer blocks and regulates its effective solvent quality. Blocks are assumed to be fixed on the time scale of interest by, e.g., different levels of acetylation or methylation. RNA polymerase is supposed to interact only with one of the chromatin blocks, called active, and assumed interaction is quite peculiar. Namely, RNA polymerase complex may absorb on chromatin fiber and, the model assumes, the fiber decorated with absorbed RNA polymerase molecules is less sticky to itself, or more repulsive than the fiber itself. This peculiar assumption allows authors to make interesting predictions about how proteins can regulate the genome folding architecture.

STRENGTH

The work includes a rather detailed theoretical description of the model and its equilibrium statistical mechanics. As both analytical theory and accompanying simulation indicate, the assumptions put forward in formulating the model do indeed produce the desired morphology, with isolated regions ("micells") of core inactive chromatin surrounded by the less dense shell region in which RNA polymerization may potentially take place. Having such a detailed theory is potentially beneficial for the field and opens up avenues for further exploration.

WEAKNESS

The underlying assumption about the interaction of RNA polymerase complex with the fiber, although important and organic for the model, does not seem easy to justify from a molecular standpoint, especially thinking of the charges and electrostatic interactions.

Reviewer #3 (Public Review):

This theoretical study provides a theoretical explanation for a puzzling question arising from recent experiments: How can chromosomes behave like polymers collapsed in a poor solvent but also contain "open" active chromatin sections? The authors propose that the binding of proteins (e.g. RNAP's) to the active sections can effectively change the solvent quality for these sections and thus open them. They suggest further that chromosomes show micellar structures with inactive blocks forming the cores of the micelles. Protein binding causes swelling of the micellar shells which affects the whole chromosome structure by changing the total number of micelles. This theory fits well to live imaging data of chromatin in Drosophila larvae, like the one shown in the striking Figure 1.

The manuscript is written very clearly.

My only suggestion is that the authors, in both the theory and simulation parts, are more explicit about how the interactions between the various components are modeled. From what I could see, in the theory part, one needs to look closely at Eq. 5 to understand how the influence of the binding of proteins affects the interaction between active monomers, and in the simulation part, one needs to go to the appendix to learn that interaction strengths between monomers within the active blocks and monomers within the inactive blocks have different values. The latter is crucial to understand the micellar structure shown at the top of Fig. 5A.

Reviewer #1 (Public Review):

Marchal-Duval et al studied the role of Prrx1 in lung fibroblasts. Prrx1 is a transcription factor expressed in lung fibroblasts but not in other cell types. The authors showed that Prrx1 gene expression was enhanced in IPF patients. Immunohistochemistry in IPF tissue suggested that Prrx1 was expressed in fibroblasts in fibroblastic foci. The authors then showed that Prrx1 expression was regulated by TGF-b1 stimulation or stiffness of substrate by in vitro experiments using primary human lung fibroblasts from either normal or IPF lungs. The authors also showed that Prrx1 regulated fibroblast proliferation and TGF-b signaling by regulating PPM1A and Tgfbr2 expression. Finally, the authors revealed that Prrx1 knockdown suppressed fibrosis in bleomycin-induced fibrosis or PCLS. This manuscript identified novel molecular roles of Prrx1 in fibroblast activation, which is expressed in not only lung fibroblasts but also in other injured or developing organs. To support the idea that Prrx1 plays a critical role in lung fibrosis, however, some discrepancies between in vitro and in vivo data need to be clarified.

1. Although the authors showed that Prrx1 knockdown in primary fibroblasts reduced Smad2/3 phosphorylation, the reduction of Acta2 or Col1a1 after Prrx1 knockdown and TGF-b1 stimulation was not impressive (Fig. S6), suggesting that the inhibition of TGF-b signaling by Prrx1 knockdown is only partial. In contrast, Prrx1 knockdown by ASO in bleomycin-induced fibrosis showed remarkable fibrosis suppression (Fig. 6, 7). Admittedly there are differences in models and nucleotides used, but this discrepancy needs to be addressed.

2. Fig.6 and 7 lack control groups, where mice are treated with PBS instead of bleomycin and treated with either control ASO or Prrx1 ASO.

3. In Fig. 6F, the hydroxyproline content is shown with ug collagen/ug protein. Total protein in the lung is influenced by infiltration of hematopoietic cells, which are the major population in injured lungs by cell count. Fibrosis should be ideally assessed as ug hydroxyproline/lung (or lobe).

4. Major proliferating populations in bleomycin-treated lungs are not mesenchymal cells but epithelial/endothelial/hematopoietic cells. Mki67+ cells (Fig. 7D) need to be identified by co-staining with mesenchymal markers if the authors claim that Prrx1 knockdown suppresses fibroblast proliferation in vivo.

5. Bleomycin-injured lungs or IPF tissue are patchy and mixed with normal and abnormal areas. Therefore, how areas of interest are chosen for histological quantifications (Fig. 6C, S14D) need to be described in the methods section.

Reviewer #2 (Public Review):

The paper from Marchal-Duval et al reports for the first time the important role played by the transcription factor PRRX1, expressed specifically in the mesenchyme of the lung, in the context of fibrosis. The authors used a combination of human (Donor and IPF) and mouse lungs (saline and bleomycin treated) as well as associated fibroblasts and PCLS to test the functional role of PRRX1 in the context of proliferation and differentiation induced by TGFb1. The work is supported by an impressive amount of data (7 main figures and 14 supplementary figures).

A main weakness in this work is the counterintuitive result that PRRX1 is downregulated in human lung fibroblasts (from both IPF and Donor) treated with TGFb1. Another smaller weakness is the inactivation of Prrx1 in vivo using ASO starting at d7 post bleomycin treatment.

The strengths of this work are the multiple approaches used by the authors to test the role of PRRX1 in lung fibrosis. The results are statistically solid and informative. The results presented are extremely convincing to support their conclusion that PRRX1, downstream of TGFb1 signaling is important for fibrosis.

Joint Public Review:

The manuscript "Monoallelically-expressed Noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression" reports the discovery of a family of ncRNAs they call SNULs for Single NUcleolus Localized RNA and examine their localization with respect to nucleoli and reports that the RNAs they are examining are monoallelically expressed in a mitotically stable manner similar to what happens in X inactivation.

These RNAs come from a screen which is not well described and the descriptions of the sequence analyses are unclear, so it is difficult to know exactly what they are analyzing in the manuscript. If these are RNAs with reasonable abundance, then they should be findable without the extensive PCR amplification they appear to have done for the PacBio sequencing (the methods section is not clear on exactly how many rounds of PCR were performed). Moreover, given the acknowledged sequence similarities of the SNULs with other RNAs, the possibility of chimaera formation during PCR amplification is high. They are clearly detecting RNAs associated with nucleoli but exactly what they are examining is unclear. It is possible that a clear determination of the genomic origin of these RNAs will be complicated by the repetitive sequences in the regions of the genome where they reside.

Note also that the idea of monoallelic expression from rRNA encoding loci is interesting, but has been established in 2009. Title: Allelic inactivation of rDNA loci. Genes Dev. 2009 Oct 15;23(20):2437-47. doi: 10.1101/gad.544509.

Reviewer #1 (Public Review):

Jordan and Keller investigated the possibility that sensorimotor prediction error (mismatch between expected and actual inputs) triggers locus coeruleus (LC) activation, which in turn drives plasticity of cortical neurons that detect the mismatch (e.g. layer 2/3 neurons in V1), thus updating the internal presentation (expected) to match more the sensory input. Using genetic tools to selectively label LC neurons in mice and in vivo imaging of LC axonal calcium responses in the V1 and motor cortex in awake mice in virtual reality training, they showed that LC axons responded selectively to a mismatch between the visual input and locomotion. The greater the mismatch (the faster the locomotion in relation to the visual input), the larger the LC response. This seemed to be a global response as LC responses were indistinguishable between sensory and motor cortical areas. They further showed that LC drove learning (updating the internal model) despite that LC optical stimulation failed to alter acute cellular responses. Responses in the visual cortex increased with locomotion, and this was suppressed following LC phasic stimulation during visuomotor coupled training (closed loop). In the last section, they showed that artificial optogenetic stimulation of LC permitted plasticity over minutes, which would normally take days in non-stimulated mice trained in the visuomotor coupling mode. These data enhance our understanding of LC functionality in vivo and support the framework that LC acts as a prediction error detector and supervises cortical plasticity to update internal representations.

The experiments are well-designed and carefully conducted. The conclusions of this work are in general well supported by the data.

Reviewer #2 (Public Review):

The work presented by Jordan and Keller aims at understanding the role of noradrenergic neuromodulation in the cortex of mice exploring a visual virtual environment. The authors hypothesized that norepinephrine released by Locus Coeruleus (LC) neurons in cortical circuits gates the plasticity of internal models following visuomotor prediction errors. To test this hypothesis, they devised clever experiments that allowed them to manipulate visual flow with respect to locomotion to create prediction errors in visuomotor coupling and measure the related signals in LC axons innervating the cortex using two-photon calcium imaging. They observed calcium responses proportional to absolute prediction errors that were non-specifically broadcast across the dorsal cortex. To understand how these signals contribute to computations performed by V1 neurons in layers 2/3, the authors activated LC noradrenergic inputs using optogenetic stimulations while imaging calcium responses in cortical neurons. Although LC activation had little impact on evoked activity related to visuomotor prediction errors, the authors observed changes in the effect of locomotion on visually evoked activity after repeated LC axons activation that were absent in control mice. Using a clever paradigm where the locomotion modulation index was measured in the same neurons before and after optogenetic manipulations, they confirmed that this plasticity depended on the density of LC axons activated, the visual flow associated with running, and the concurrent visuomotor coupling during LC activation. Based on similar locomotion modulation index dependency on speed observed in mice that develop only with visuomotor experience in the virtual environment, the authors concluded that changes in locomotion modulation index are the result of experience-dependent plasticity occurring at a much faster rate during LC axons optogenetic stimulations.

The study provides very compelling data on a timely and fascinating topic in neuroscience. The authors carefully designed experiments and corresponding controls to exclude any confounding factors in the interpretation of neuronal activity in LC axons and cortical neurons. The quality of the data and the rigor of the analysis are important strengths of the study. I believe this study will have an important contribution to the field of system neuroscience by shedding new light on the role of a key neuromodulator. The results provide strong support for the claims of the study.

Peer review report

Title: Maintained imbalance of triglycerides, apolipoproteins, energy metabolites and cytokines in long-term COVID-19 syndrome (LTCS) patients

version: 1

Referee: Paola Turano

Institution: University of Florence

email: turano@cerm.unifi.it

ORCID iD: 0000-0002-7683-8614

General assessment

This is an integrated study reporting NMR-based metabolomics data and flow cytometry-based cytokine in the blood of 125 individuals (healthy controls (HC; n=73), COVID-19-recovered (n=12), COVID-19 acute (n=7) and LTCS (n=33)).

The main goal appears to be that of demonstrating alterations in the metabolome and immune markers of patients with long COVID. This condition is defined as the continuation or development of new symptoms 3 months after the initial SARS-CoV-2 infection, with these symptoms lasting for at least 2 months with no other explanation.

As admitted by the authors, the 4 groups are very unbalanced in terms of numbers of enrolled subjects; moreover, all numbers are low but those in the recovered groups and even more in the acute phase are extremely low. Therefore, the only reliable comparison appears to be that between HC and LTCS. And this is a pity because the most important comparison to define the signature associated with long-COVID symptoms would have been the one between recovered and LTCS subjects.

Another problem is that there is no information on the status of the LTCS before infection nor during the acute phase. This, combined with the low number of individuals, does not allow to draw a real trajectory of the alterations during the observed time line. It is therefore difficult to be 100% sure that alterations in certain metabolites of lipoproteins are a consequence of LTCS or instead intrinsic characteristics of a group of individual that make them more prone to develop LTCS.

These critical aspects have nothing to do with the experimental approach, which is powerful and carefully performed. Unfortunately, the available cohort is not the best to achieve the goal of a molecular characterization of LTCS.

In any case the present manuscript provides useful hints to be further investigated in future studies and therefore might deserve publication.

Essential revisions that are required to verify the manuscript

If it were possible to enlarge the cohort of patients, confirming the observed trends, this would lead to a significant improvement in the impact of the work. But I understand the practical difficulties in achieving this goal.

Decision

Verified with reservations: The content is academically sound but has shortcomings that could be improved by further studies and/or minor revisions.

Peer review report

Title: Maintained imbalance of triglycerides, apolipoproteins, energy metabolites and cytokines in long-term COVID-19 syndrome (LTCS) patients

version: 1

Referee: Christopher Gerner

Institution: University of Vienna

email: Christopher.gerner@univie.ac.at

ORCID iD: 0000-0003-4964-0642

General assessment

The manuscript of Berezhnoy et al. is a well written report regarding metabolomics and cytokines in long term COVID-19 syndrome patients. The applied methodology is of some interest and I cannot detect methodological errors. However, I have some concerns which need to be addressed before the manuscript should sent for journal publication.

Most importantly, the manuscript did not adhere to good scientific practice regarding literature research. There are papers about LTCS patients published more than a year ago following highly similar research strategies with quite similar results. It is not sufficient to cite them in subordinate clauses in the Discussion, they need to be cited in the Introduction accordingly, as well when discussing the results. Indeed, relevant similarities in the results would deserve some discussion, such as the dysregulation of cytokines in LTCS.

Another weakness is the structure of data interpretation. It is mentioned in the Introduction that several cell types were reported to show altered metabolism after a COVID-19 infection. I cannot see how plasma analysis should allow to verify such observations as it represents a mixture of all cell types in the body. This obvious challenge regarding data interpretation should be addressed.

This is in line with another weak aspect. The numerous findings a reported without a clear structure regarding potential pathomechanisms. As such, the manuscript is sometimes not easy to read.

To sum up, the manuscript reports interesting analysis results largely corroborating previous results, which deserved publication after some essential improvements.

Essential revisions that are required to verify the manuscript

An improved appreciation of existing literature is essential, as well as an improved data interpretation.

Other suggestions to improve the manuscript

A discussion of the pros and cons of NMR-based metabolomics in contrast to other techniques would be helpful.

Decision

Verified with reservations: The content is academically sound but has shortcomings that could be improved by further studies and/or minor revisions.

Reviewer #1 (Public Review):

The authors evaluate a number of stochastic algorithms for the generation of wiring diagrams between neurons by comparing their results to tentative connectivity measured in cell cultures derived from embryonic rodent cortices. They find the best match for algorithms that include a term of homophily, i.e. preference for connections between pairs that connect to an overlapping set of neurons. The trend becomes stronger, the older the culture is (more days in vitro).

From there, they branch off to a set of related results: First, that connectivity states reached by the optimal algorithm along the way are similar to connectivity in younger cultures (fewer days in vitro). Second, that connectivity in a more densely packed network (higher plating density) differs only in terms of shorter-range connectivity and even higher clustering, while other topological parameters are conserved. Third, blocking inhibition results in more unstructured functional connectivity. Fourth, results can be replicated to some degree in cultures of human neurons, but it depends on the type of cell.

The culturing and recording methods are strong and impressive. The connectivity derivation methods use established algorithms but come with one important caveat, in that they are purely based on correlation, which can lead to the addition of non-structurally present edges. While this focus on "functional connectivity" is an established method, it is important to consider how this affects the main results. One main way in which functional connectivity is likely to differ from the structural one is the presence of edges between neurons sharing common innervation, as this is likely to synchronize their spiking. As they share innervation from the same set of neurons, this type of edge is placed in accordance with a homophilic principle. In other words, this is not merely an algorithmic inaccuracy, but a potential bias directly related to the main point of the manuscript. This is not invalidating the main point, which the authors clearly state to be about the correlational, functional connectivity (and using that is established in the field). But it becomes relevant when in conclusion the functional connectivity is implicitly or explicitly equated with the structural one. Specifically, considering a long-range connection to be more costly implies an actual, structural connection to be present. Speculating that the algorithm reveals developmental principles of network formation implies that it is the actual axons and synapses forming and developing. The term "wiring" also implies structural rather than functional connectivity. One should carefully consider what the distinction means for conclusions and interpretation of results.

The main finding is that out of 13 tested algorithms to model the measured functional connectivity, one based on homophilic attachment works best, recreating with a simple principle the distributions of various topological parameters.<br /> First, I want to clear up a potential misunderstanding caused by the naming the authors chose for the four groups of generative algorithms: While the ones labelled "clustering" are based on the clustering coefficient, they do not necessarily lead to a large value of that measure nor are they really based on the idea that connectivity is clustered. Instead, the "homophilic" ones are a form of maximizing the measure (but balanced by the distance term). To be clear, their naming is not wrong, nor needs to be changed, but it can lead to misunderstandings that I wanted to clear up. Also, this means that the principle of "homophilic wiring" is a confirmation of previous findings that neuronal connectivity features increased values of the clustering coefficient. What is novel is the valuable finding that the principle also leads to matching other topological network parameters.

The main finding is based on essentially fitting a network generation algorithm by minimizing an energy function. As such, we must consider the possibility of overfitting. Here the authors provide additional validation by using measures that were not considered in the fitting (Fig 5, to a lesser degree Fig 3e), increasing the strength of the results. Also, for a given generative algorithm, only 2 wiring parameters were optimized. However, with respect to this, I was left with the impression that a different set of them was optimized for every single in-vitro network (e.g. n=6 sets for the sparse PC networks; though this was not precisely explained, I base this on the presence of distributions of wiring parameters in Fig 6c). The results would be stronger if a single set could be found for a given type of cell culture, especially if we are supposed to consider the main finding to be a universal wiring principle. At least report and discuss their variability.

Next, the strength of the finding depends on the strengths of the alternatives considered. Here, the authors selected a reasonably high number of twelve alternatives. The "degree" family places connections between nodes that are already highly connected, implementing a form of rich-club principle, which has been repeatedly found in brain networks. However, I do not understand the motivation for the "clustering" family. As mentioned above, they do not serve to increase the measure of the clustering coefficient, as the pair is likely not part of the same cluster. As inspiration, "Collective dynamics of 'small-world' networks" is cited, but I do not see the relation to the algorithm or results presented in that study. A clearly explained motivation for the alternatives (and maybe for the individual algorithms, not just the larger families) would strengthen the result. 

Related to the interpretation of results, as they are presented in Fig3a, bottom left: What data points exactly go into each colored box? Specifically, into the purple box? What exactly is meant by "top performing networks across the main categories" mean? Compared with Supp Fig S4, it seems as if the authors do not select the best model out of a family and instead pool the various models that are part of the same family, albeit each with their optimized gamma and eta. Otherwise, the purple box at DIV14 in Fig3 would be identical to "degree average" at DIV14 in S4. If true, I find this problematic, as visually, the performance of one family is made to look weaker by including weak-performing models in it. I am sure one could formulate a weak-performing homophily-based rule that drives the red box up. If such pooling is done for the statistical tests in Supp Tables 3-7, this is outright misleading! (for some cases "degree average" seems not significantly worse than the homophily rules).

The next finding is related to the development of connectivity over the days in vitro. Here, the authors compare the connectivity states the network model goes through as the algorithm builds it up, to connectivity in-vitro in younger cultures. They find comparable trajectories for two global topological parameters. <br /> Here, once again it is a strength that the authors considered additional parameters outside the ones used in fitting. However, it should be noted that the values for "global efficiency" at DIV14 (the very network that was optimized!) are clearly below the biological values plotted, weakening the generality of the previous result. This is never discussed in the text.

The conclusion of the authors in this part derives from values of modularity decreasing over time in both model and data, and global efficiency increasing. The main impact of "time" in this context is the addition of more connections, and increasing edge density. And there is a known dependency between edge density and the bounds of global efficiency. I am not convinced the result is meaningful for the conclusion in this state. If one were to work backwards from the DIV14 model, randomly removing connections (with uniform probabilities): Would the resulting trajectory match DIV12, DIV10, and DIV7 equally well? If so, the trajectory resulting from the "matching" algorithm is not meaningful.

Further, the conclusion of the authors implies that connections in the cultures are formed as in the algorithm: one after another over time without pruning. This could be simply tested: How stable are individual connections in vitro over time (between DIV)? 

The next finding is that at higher densities, the connections formed by the neurons still have very comparable structures, only differing in clustering and range; and that the same generative algorithm is optimal for modelling them. I think in its current state, the correlation analysis in Fig. 4a supports this conclusion only partially: Most of these correlations are not surprising. Shortest path lengths feature heavily in the calculation of small worldness and efficiency (in one case admittedly the inverse). Also for example network density has known relations with other measures. The analysis would be stronger if that was taken into account, for example showing how correlations deviate from the ones expected in an Erdos-Renyi-type network of equal sizes.

Yet, overall the results are supported by the depicted data and model fits in Supp. Fig S7. With the caveat that some of the numerical values depicted seem off: <br /> What are the units for efficiency? Why do they take values up to 2000? Should be < 1 as in 4b. Also, what is "strength"? I assume it's supposed to be the value of STTC, but that's not supposed to be >1. Is it the sum over the edges? But at a total degree of around 40, this would imply an average STTC almost three times higher than what's reported in Fig 1i. Also, why is the degree around 40, but between 1000 and 1500 in Fig S2? <br /> Finally, it should be mentioned that "degree average" seems (from the boxplot) to work equally well.

Further, the conclusion of the "matching" algorithm equally fitting both cases would be stronger if we were informed about the wiring parameters (η and γ) resulting in both cases. That way we could understand: Is it the same algorithm fitting both cases or very different variants of the same? It is especially crucial here, because the η and γ parameters determine the interplay between the distance- and topology-dependent terms, and this is the one case where a very different set of pairwise distances (due to higher density) are tested. Does it really generalize to these new conditions?

Conversely, the results relating to GABAa blocking show a case where the distances are comparable, but the topology of functional connectivity is very different. (Here again, the contrast between structural and functional connectivity could be made a bit clearer. How is correlational detection of connections affected by "bursty" activity?) The reduction in tentative inhibition following the application of the block is convincing.

The main finding is that despite of very different connectivities, the "matching" algorithm still holds best. This is adequately supported by applying the previous analyses to this case as well. <br /> The authors then interpret the differences between blocked and control by inspection of the η and γ parameters, finding that the relative impact of the distance-based term is likely reduced, as a lower (less negative) exponent would lead to more equal values for different distances. This is a good example of inspecting the internals of a generative algorithm to understand the modeled system and is confirmed by longer edge lengths in Supp Fig. S12C.

The authors further inspect the wiring probabilities used internally at each step of the algorithm and compare across conditions. They conclude from differences in the distribution of P_ij values that the GABAa-blocked network had a "more random" topology with "less specific" wiring. This is the opposite of the conclusion I would draw, given the depicted data. This may be partially because the authors do not clearly define their concept of "random" vs. "specific". I understand it to be the following: At each time step, one unconnected pair is randomly picked and connected, with probabilities proportional to P_ij, as in Akarca et al., 2021; "randomness" then refers to the entropy of that process. In that case, the "most random" or highest entropy case is given by uniform P_ij values, which would be depicted as a delta peak at 1 / n_pairs in the present plot. A flatter distribution would indicate more randomness if it was the distribution of P_ij over pairs of neurons (x-axis: pairs; y-axis P_ij). The conclusion should be clarified by the use of a mathematical definition and supported by data using that definition.

Next, the methods are repeated for various cultures of human neurons. I have no specific observations there.

In summary, while I think the most important methods are sound, and the main conclusions (reflected in the title of the paper) are supported, the analysis of more specific cases (everything from Fig 3e onwards, except for Fig 5) requires more work as in the current state their conclusions are not adequately supported.

Reviewer #2 (Public Review):

This work presents an exhaustive study of inferred functional networks from in vitro neuronal cultures across several modalities: primary rat cultures (with varying densities and longitudinal points), and human iPSC monolayers from different cell types and organoids. The authors first estimated the functional connectivity of these networks from their spontaneous activity (recorded with high-density MEAs) and then tried to find which wiring principle could better explain the observations. By deploying generative network models (with 13 different wiring principles) they observed that models with homophilic wiring principles were systematically outperforming the other ones. This proposes a universal rule for how neurons connect, which is that they tend to connect with neurons that have many common neighbors.

One of the major strengths of this study is its scope. They analyzed sparse and dense primary rat cultures at 4 different time points during development (from 7 to 28 DIV in total) as well as with the pharmacological application of GABAA blockers; 3 different cell lines: spinal-cord motor neurons, dopaminergic neurons, and glutamatergic neurons, and organoid slices. However, the big scope of this study is also one of its weaknesses, the techniques presented here to analyze the data are used inconsistently; for some preparations, there's much more detail than in others, and the constant jump between preparations and methodologies makes the findings hard to follow.

Similarly, the number of samples used in some preparations (ranging from 6 to 12) appears to be insufficient, since the study relies on multiple comparisons across the results from 13 different generative models. In many cases, it is not possible to identify which results are significant and which aren't. Most of the methodology used in this study has been used before in the context of the human connectome project (Betzel et al, Neuroimage 2016); in there they used data from 380 total participants, which made the comparisons across all the different models much more robust.

Most previous research with generative models for neural connectivity has focused on structural connectivity. In there, the link between wiring principles, energetic costs, and network topology can be made. This study, however, focuses on functional connectivity (measured by the spike time tiling coefficient), where the link between these quantities is unclear. Although the authors highlight this point in the manuscript, the constant comparisons to structural connectivity concepts and studies often lead to confusion. A clear example of this is the section where the authors explore the effect of chronic GABAA receptor blockade. It is unclear whether the authors are trying to claim that this protocol alters the development of the structural network or only the dynamics. The former could have needed additional controls.

The authors have been diligent and thorough with their statistical testing and their claims are commensurate with that. However, given the large number of different types of results and tests being presented, it is often difficult to find the corresponding explanation in the methods.

This is valuable work for experimental and computational neuroscientists studying the development of neuronal networks and the link between structural and functional connectivity. It would greatly benefit from homogenizing the results, methods, and statistics across the different experimental preparations. The conceptual similarities and differences between structural and functional wiring principles also need to be emphasized.

Reviewer #1 (Public Review):

This work deals with courtship behaviour in mice. Authors try to identify the acoustic features that influence the attractivity level of male courtship songs to females. Courtship songs are made of sequences of short ultrasound syllables emitted at a rate of 7-10Hz. Authors manipulated these syllables by changing either the spectrotemporal content of each syllable or the intersyllable intervals. The authors found that it was only when sequences of syllables were irregular (with highly variable intersyllable intervals) that the female was less attracted to the song. The data, therefore, brings evidence that the acoustic features of syllables account less than the song's temporal regularity for the attractivity of courtship songs. The authors suggest that temporal regularity of syllable emission, building on breathing patterns, could reflect male fitness. They also suggest that temporal regularity could be an acoustic cue compressing the complex acoustic information carried by songs.

Strengths:

The study is well-written, very straightforward, and easy to follow. Behavioral tasks are well-designed and many tests, on a large enough set of animals have been done to support the conclusions. Results are clearly presented and provide enough details to see individual points. The discussion makes interesting connections between syllable rhythms and animals' fitness or brain rhythms.

Weaknesses:

Although the study is easy to understand and provides interesting results, the data analysis remains incomplete, and the interpretation of results is not cautious enough.

For instance, Fig. 2 shows a preference for song playback but we cannot determine if it is a general preference for a sound or a specific preference for male songs because only the difference between the presence of song or silence is tested. I acknowledge that the authors did not overstate their results, but the experimental design is incomplete and hard to interpret in that respect. For instance, the expression "preferential approach to song" is ambiguous.

There is no analysis of individual preference across tests and we might have the feeling that the effect shown mostly depends on the preference of only a few animals. Indeed, it seems that roughly one-third of animals showed a strong preference for the intact song while another third showed a strong preference for the modified song, whatever the modification. A few animals are therefore "swing voters". It would have been interesting, if not pertinent, to have a deeper analysis of the behavior of these later animals. Do they choose less (i.e. spend less time close to speakers) or do they swing from one corner to another? What about the animals which always chose the modified song? Are these animals that already showed a weak or strong preference for silence, therefore showing they were not comfortable with the songs played? There is no discussion of these aspects either.

Also, on page 11, it is written "female listeners perceptually compress the high sensory dimensionality of male songs by selectively monitoring a reduced subset of meaningful acoustic features in isolation." This statement or hypothesis is questionable. After all, if someone would change the inter-syllable intervals in human speech, that would become cryptic or at least annoying for the listener. Humans would definitely prefer normal speech. Is this because we compress acoustic features? Not really. It is likely that this modified speech just differs too much from the set of parameters typically encountered and therefore understood/interpreted while learning a language in childhood. Thus, the hypothesis here is rather to determine, for a given acoustic feature, if there is a range within which the perception of the message carried by the song (courtship) is maintained. Interpretation of "compressed acoustic features" with regards to animals' preference seems an overinterpretation. Same remark at the end of the conclusion.

Reviewer #2 (Public Review):

In the present manuscript, Perrodin et al. investigated which properties of ultrasonic vocalizations determine their attractiveness for female mice. They collected a set of male courtship vocalizations and compared their attractiveness for female mice against a number of conditions, including silence, and a number of modified sequences.

The study has a clear design and used insightful modifications on the vocalization sequences, which allow the present results to be linked to previous results. The most interesting outcome of the study is that female mice prefer regularly timed sequences of vocalizations over less regularly timed sequences. This result is novel and adds to our understanding of the determinants of social communication between mice. Overall the study is likely underpowered, which was, however, hard to assess as animal numbers were largely not reported for the individual tests, and statistical analysis was carried out on the level of sessions only.

The study has a very good discussion embedding the current results with the previous literature, although the discussion steps beyond the results in a few respects, in particular when trying to determine the underlying reasons for the preference for regularly spaced sequences.

Methodologically the study is carried out at the appropriate level, although some improvements could be made to the experimental apparatus to avoid reflections.

The study will likely have a substantial impact on the field of mouse communication because the regularity of spacing has not been a focus of previous research. In addition, the confirmation that a lot of other modifications are less determining for the attractiveness of the vocalizations provides solid data on which to base future work.

Reviewer #1 (Public Review):

Tippett et al present whole cell and proteoliposome transport data showing unequivocally that purified recombinant SLC26A6 reconstituted in proteoliposomes mediates electroneutral chloride/bicarbonate exchange, as well as coupled chloride/oxalate exchange unassociated with detectable current. Both functions contrast with the uncoupled chloride conductance mediated by SLC26A9. The authors also present a novel cryo-EM structure of full-length human SLC26A6 chloride/anion exchanger. As part of the structure, they offer the first partial view of the STAS domain previously predicted to be unstructured. They further define a single Arg residue of the SLC26A6 transmembrane domain required for coupled exchange, mutation of which yields apparently uncoupled electrogenic chloride transport mechanistically resembling that of SLC26A9, although of lower magnitude. The authors further apply to proteoliposomes for the first time a still novel approach to the measurement of bicarbonate transport using a bicarbonate-selective Europium fluorophor. The evidence strongly supports the authors' claims and conclusions, with one exception.

The manuscript has numerous strengths.

As a structural biology contribution, the authors extend the range of SLC26 structures to SLC26A6, comparing it in considerable detail to the published SLC26A9 structure, and presenting for the first time the structure of a portion of the STAS IVS domain of SLC26A6 long considered unstructured.

The authors also apply a remarkably extensive range of creative technical approaches to assess the functional mechanisms of anion transport by SLC26A6, among them the first application of the novel, specific bicarbonate sensor Eu-L1+ to directly assess bicarbonate transport in reconstituted proteoliposomes. The authors also present the first (to this reviewer's knowledge) functional proteoliposome reconstitution of chloride-bicarbonate exchange mediated by an SLC26 protein. They define a residue in surrounding the anion binding pocket which explains part of the difference in anion exchange coupling between SLC26A6 and SLC26A9. In the setting of past conflicting results, the current work also contributes to the weight of previous evidence demonstrating that SLC26A6 mediates electroneutral rather than electrogenic Cl-/HCO3- exchange.

Each of these achievements constitutes a significant advance in our understanding.

The paper has only a few weaknesses. One is an incomplete explanation of the mechanistic determinants of anion exchange coupling in SLC26A6 vs. uncoupled anion transport by SLC26A9. A second minor weakness is the inconsistently repeated conclusion that SLC26A6 mediates strictly coupled chloride/oxalate exchange. The data presented do not measure the stoichiometry of Cl-/oxalate exchange. The AMCA proteoliposome assay documented extracellular oxalate-dependent proteoliposomal anion transport that was most simply interpreted as coupled exchange, whereas no stoichiometric coupled exchange was documented in the AMCA experiments as presented.

Overall, the manuscript represents an important advance in our understanding of the SLC26 protein family and of coupled vs uncoupled carrier-mediated anion transport.

Reviewer #2 (Public Review):

The eleven paralogs of SLC26 proteins in humans exhibit a remarkable range of functional diversity, spanning from slow anion exchangers and fast anion transporters with channel-like properties, to motor proteins found in the cochlear outer hair cells. In this study, the authors investigate human SLC26A6, which functions as a bicarbonate (HCO3-)/chloride (Cl-) and oxalate (C2O42-)/Cl- exchanger, combining cryo-electron microscopy, electrophysiology, and in vitro transport assays. The authors provide compelling evidence to support the idea that SLC26A6's exchange anions at equimolar stoichiometry, leading to the electroneutral and electrogenic transport of HCO3-/ Cl- and C2O42-/Cl-, respectively. Furthermore, the structure of SLC26A6 reveals a close resemblance to the fast, uncoupled Cl- transporter SLC26A9, with the major structural differences observed within the anion binding site. By characterizing an amino acid substitution within the SLC26A6 anion binding site (R404V), the authors also show that the size and charge variance of the binding pocket between the two paralogs could, in part, contribute to the differences in their transport mechanisms.

The strength of this work lies in the reductionist, in vitro approach that the authors took to characterize the transport process of SLC26A6. The authors used and developed an array of functional experiments, including two electrogenic transport assays - a fast kinetic (electrophysiology) and a slow-kinetic (fluorescent-based ACMA) - and two electroneutral transport assays, probing for Cl- (lucigenin) and HCO3- (europium), which are well executed and characterized. The structural data is also of high quality and is the first structure of an SLC26 coupled anion exchanger, providing essential information for clarifying our understanding of the functional diversity between the SLC26 family of proteins.

To my knowledge, the outward-facing conformational state has not been determined for any mammalian SLC26 paralog, which limits the mechanistic interpretation of transport and is a weaker point of this manuscript. However, this is a very minor point.

Reviewer #3 (Public Review):

The mechanistically diverse SLC26 transporters play a variety of physiological roles. The current manuscript establishes the SLC26A6 subtype as electroneutral chloride/bicarbonate exchanges and reports its high-resolution structure with chloride bound.

The claims in this manuscript are all well-supported by the data. Strengths include the comprehensive functional analysis of SLC26A6 in reconstituted liposome vesicles. The authors employ an array of assays, including chloride sensors, a newly developed fluorescent probe for bicarbonate, and assays to detect the electrogenicity of anion exchange. With this assortment of assays, the authors are able to establish the anion selectivity and stoichiometry of SLC26A6. Another strength of the manuscript is the functional comparison with SLC26A9, which permits fast, passive chloride transport, in order to benchmark the SLC26A6 activity. The structural analysis, including the assignment of the chloride binding site, is also convincing. The structural details and the chloride binding site are well-conserved among SLC26s. Finally, the authors present an interesting discussion comparing the structures of SLC26A5, SLC26A6, and SLC26A9, and how the details of the chloride binding site might influence the mechanistic distinctions between these similar transporters.

Reviewer #1 (Public Review):

In this study the authors first perform global knockout of the gene coding for the polarity protein Crumbs 3 (CRB3) in the mouse and show that this leads to perinatal lethality and anopthalmia. Next, they create a conditional knockout mouse specifically lacking CRB3 in mammary gland epithelial cells and show that this leads to ductal epithelial hyperplasia, impaired branching morphogenesis and tumorigenesis. To study the mechanism by which CRB3 affects mammary epithelial development and morphogenesis, the authors turn to MCF10A cells and find that CRB3 shRNA-mediated knockdown in these cells impairs their ability to form properly polarized acini in 3D cultures. Furthermore, they find that MCF10A cells lacking CRB3 display reduced primary ciliation frequency compared to control cells, which is in agreement with previous studies implicating CRB3 in primary cilia biogenesis. Using a combination of biochemical, molecular- and imaging approaches the authors then provid evidence indicating that CRB3 promotes ciliogenesis by mediating Rab11-dependent recruitment of gamma tubulin ring complex component GCP6 to the centrosome/ciliary base, and they also show that CRB3 itself is localized to the base of primary cilia. Finally, to assess the functional consequences of CRB3 loss on ciliary signaling function, the authors analyze the effect of CRB3 loss on Hedgehog and Wnt signaling using cell-based assays or a mouse model.

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. However, a major weakness of this study is that quantitative analysis of several key results is either lacking, not done appropriately, or is incompletely described. In addition, some of the cell-based experiments are lacking appropriate controls, and the claim that CRB3 directly binds to Rab11 is not supported by the data provided.

Reviewer #2 (Public Review):

In this work, the authors investigate the role of CRB3 in the formation of the primary cilium both in a mouse model and in human cells. They confirm in a conditional knock-out (KO) mouse model that Crb3 is necessary for the formation of the primary cilium in mammary and renal epithelial tissues and the new-born mice exhibit classical traits of ciliopathies. In the mouse mammary gland, the absence of Crb3 induces hyperplasia and tumorigenesis and in the human mammary tumor cells MCF10A the knock-down (KD) of CBR3 impairs ciliogenesis and the formation of a lumen in 3D-cultures with less apoptosis and spindle orientation defects during cell division.

To determine the subcellular localization of CRB3 the authors have expressed exogenously a GFP-CRB3 in MCF10A and found that this tagged protein localizes in cell-cell junctions and around pericentrin, a centrosome marker, while endogenous CRB3 localizes at the basal body. To dissect the molecular role of CRB3 the authors have performed proteomic analyses after a pull-down assay with the exogenous tagged-CRB3 and found that CRB3 interacts with Rab11 and is present in the endosomal recycling pathway. CRB3 KD also decreases the interactions between components of the γTuRC complex. In addition, the authors showed that CRB3 interacts with a tagged-Rab11 by its extracellular domain and that CRB3 promotes the interaction between Rab11 and CEP290 while CRB3 KD decreased the co-localization of GCP6 with Rab11 and γTub. Finally, the authors showed that CRB3 depletion cannot activate the Hh pathway as opposed to the Wnt pathway.

Reviewer #1 (Public Review):

In "Striatal ensemble activity in an innate behavior", Minkowicz et al. strive to characterize how the striatum, the primary input nucleus of the basal ganglia, represents grooming. Here, grooming is used as a paradigmatic habitual behavior. The pose dynamics of grooming are stereotyped: mice perform it spontaneously and prior work has shown that it is both represented and controlled by the striatum.

The manuscript presents a valuable contribution to the field by shedding light on how ensembles of neurons encode this innate behavior. Additionally, the use of supervised machine learning allowed the authors to collect and precisely align a large number of grooming repetitions, which enabled most of their downstream analysis.

I found the paper to be well-written and the conclusions are mostly well-supported. However, some of the data analysis was a bit opaque, and some more detail and reanalysis could substantially strengthen the authors' claims.

1) The authors identified grooming bouts using empirically defined thresholds and manual tweaking. Next, the boundaries of grooming were used for trial alignment and linear time warping. This is a completely sensible approach; however, in using only the boundaries of grooming episodes, the dynamics of grooming bouts are ignored. I am particularly concerned that pose dynamics of grooming bouts are most stereotyped at the boundaries (e.g. they always begin and end with specific paw movements). To play devil's advocate, if the striatum encodes pose dynamics and not boundaries and pose dynamics are noisy between the beginning and end of these bouts (either due to the dynamics of the behavior or how it was identified), then a "boundary-like" representation may emerge in the average. I strongly recommend re-running a subset of the analysis after accounting for variability in grooming dynamics. A simple thing to try would be to further cluster grooming bouts using 3D keypoint trajectories. Another would be to warp grooming bouts in a manner that accounts for keypoint trajectories (e.g. DTW or other recent time-warping variants).

2) The authors should consider if the correlation to grooming is due to (at least in part) a correlation with another aspect of movement, e.g. overall velocity, acceleration, height, or angular velocity. This should be straightforward to analyze with the current dataset. To start, I would simply take the velocity and acceleration of the mouse's centroid (head and body could be considered separately). Next, look at the correlation with DLS spiking. If a clear relationship emerges, then check to see how velocity (or another variable) maps onto grooming. It may be that DLS neurons appear to encode the boundaries of grooming when they (at least partially) encode other variables.

3) The ensemble analysis is potentially critical to our understanding of SPNs. Figure 4A suggests that ensembles encode grooming with a probabilistic code - ensembles appear to be engaged for a small number of grooming bouts in the session. First, a basic question is what is the probability a given ensemble is activated during grooming? Second, the more complex question is whether there is an explanation for why one ensemble is engaged for some trials and not others? Related to point 2, I wonder if another aspect of behavior - e.g. vigor, duration, or speed - determines this. I suggest some analysis to at least rule out some simple explanations.

Reviewer #2 (Public Review):

The manuscript by Minkowicz et al., investigates the presence of neuronal ensembles in the striatum that may encode grooming (as a model of a naturalistic behavior). They implemented a semi-automated detection of grooming, and by recording populations of striatal cells they show that individual neurons in the striatum contain activity modulations around the start, end, or during grooming. Then using this activity they identify ensembles of cells in individual sessions/animals at the start, end or during grooming.

The behavioral tracking and recordings are remarkable, the manuscript is clearly written and the finding mostly sound with the proposed conclusions, providing original findings in the field. Nonetheless some points are raised that need further clarification

1. When claiming that the findings show encoding of transitions into or out of grooming (and duration of grooming) one could expect to see specific regressions between the neuronal activity (of individual cells or ensembles) and the parameters mentioned besides the analysis shown in figure 3 and 5.<br /> 2. Was the detection of ensembles presented in figure 4 sensible to use less than 5 seconds before/after grooming. I am thinking that 5 seconds are times that could contain behaviors that may have their own ensembles. Why 5 seconds?<br /> 3. According to Figure 2-figure supplement 1. The recordings were performed covering the lateral and in some cases the central part of the striatum. Shall it be specified along the text where the specific recordings come from?

Reviewer #1 (Public Review):

In this manuscript by Douglas et al, the investigative team seeks to identify Staphylococcus aureus genes (and associated polymorphisms) that confer altered susceptibility to human serum, with the hypothesis that such genes might contribute to the propensity of a strain to cause bacteremia, invasive disease, and/or death. Using an innovative GWAS-like approach applied to a bank of over 300 well-characterized clinical S. aureus isolates, the authors discover SNPs in seven different staphylococcal genes that confer increased survival in the setting of serum exposure. The authors then mainly focus on one gene, tcaA, and illustrate a potential mechanism whereby modification of peptidoglycan structure and WTA display leads to altered susceptibility to serum, serum-derived antimicrobial compounds, and antibiotics. One particularly significant finding is that the identified tcaA SNP is significantly associated with patient mortality, in that patients infected with the SNP bearing isolate are less likely to die from infection. It is therefore hypothesized that this SNP represents an adaptive mutation that promotes serum survival while decreasing virulence and host mortality. In a murine model of infection, the strain bearing the WT allele of tcaA is significantly more virulent than the tcaA mutant, suggesting that the role of tcaA in bacteremia is infection-phase dependent.

This manuscript has many strengths. The triangulation of genomic analysis, patient outcomes data, and in vitro and in vivo mechanistic testing adds to the significance of the findings in terms of human disease. Testing the impact of mutating tcaA in multiple staphylococcal lineages and backgrounds also increases the rigor of the study. The identification of bacterial loci that impact susceptibility to both host antimicrobial compounds and commonly used antibiotics is also a strength of this work, given the evolutionary and treatment implications for such genes.

One moderate weakness is that the impact of the identified SNP in tcaA is only tested in some of the assays, whereas the majority of the testing is performed with a whole gene knockout. Additionally, for some experiments, rigor is lacking in that statistical measures are not deployed to support the conclusions of biologically meaningful changes based on data with very modest differences between groups. In some cases this results in more speculative conclusions that will require further testing to validate. Finally, there are instances of inter-experiment variability that require further explanation. All in all, this is an exciting manuscript that will be of interest to the broader research communities focused on staphylococcal pathogenesis, bacterial evolution, and host-pathogen interactions, as well as to clinicians who care for patients with invasive staphylococcal infection.

Reviewer #2 (Public Review):

The authors embarked on a study to identify SNPs in clinical isolates of S. aureus that influence sensitivity to serum killing. Through a phenotypic screen of 300 previously sequenced S. aureus bacteremia (SAB) isolates, they identified ~40 SNPs causing altered serum survival. The remainder of the study focuses of tcaA, a gene with unknown function. They show that when tcaA is disrupted, it results in increased resistance to glycopeptides and antimicrobial components of human serum.

They perform an elegant series of experiments demonstrating how a tcaA knockout is more resistant to killing by whole serum. arachadonic acid, LL-37 and HNP-1. They provide compelling evidence that in the absence of tcaA resistance to arachidonic acid is mediated through release of wall teichoic acids from the cell wall, which acts as a decoy and sequesters the fatty acid.

Similarly, they suggest that resistance to cationic antimicrobial peptides is through alteration of the net charge of the cell wall due to loss of negatively charged WTAs based on reduced cytochrome C binding.

They continue to show that tcaA is induced in the presence of human serum, which causes increased resistance to the glycopeptide teichplanin.

They propose that tcaA disruption causes altered cell wall structure based on morphologic changes on TEM and increased sensitivity to lysostaphin and increased autolysis via triton x-100 assay.

5, Finally, they propose that tcaA influences mortality in SAB based on raw differences in 30-day morality. Interestingly they do decreased fitness during murine bacteremia model compared to wild-type.

Strengths:

1. The manuscript is well-written and easy to follow<br /> 2. The identification of SNPs leading to altered serum killing is convincing and valuable data<br /> 3. The mechanism for tcaA-mediated resistance to arachadonic acid and AMPs is compelling and novel<br /> 4. The murine infection data demonstrating that tcaA mutants exhibit reduced virulence is important data

Weaknesses:

1. Some of the conclusions are not supported by the data shown (either missing or incomplete)<br /> 2. The authors conclude that tcaA mutants show reduced peptidoglycan crosslinking. This conclusion is based on qualitative TEM images and increased sensitivity to lysostaphyin/autolysis. While these data are suggestive. it is difficult to draw such a conclusion without analysis of the cell wall by LC-MS (such as http://doi.org/10.1371/journal.ppat.1009468).<br /> 3. The authors conclude "TcaA contributes to increased disease severity in mice and humans". While it seems biologically plausible that a polymorphism known to increase glycopeptide MIC affects mortality, the human data presented is based on raw 30-day mortality numbers. It is misleading to make the association with mortality without adjusting for confounding variables known to influence mortality in SAB (e.g. age, comorbidities, presence of sepsis, endocarditis, duration of bacteremia). Also, with just 12 patients in the SNP group, this is likely underpowered to detect any difference.

Overall, I think this is a good submission and the majority of their conclusions are supported by the data. The mechanism behind the clinically relevant tcaA mutation is important, given its known role in glycopeptide resistance and therefore likely clinical outcomes. This manuscript would benefit with the inclusion of some additional experiments to help support their finding.

Reviewer #3 (Public Review):

In this manuscript by Douglas et al., the authors used a functional genomics approach to understand how Staphylococcus aureus survives in the bloodstream to cause bacteraemia. They identified seven novel genes that affect serum survival. The study focused on tcaA, a gene associated with resistance to the antibiotic teicoplanin and is activated when exposed to serum and plays a role in producing a critical virulence factor called wall teichoic acids (WTA) in the cell envelope. This protein affects the bacteria's sensitivity to cell wall attacking agents, human defense fatty acids, and antibiotics, as well as autolytic activity and lysostaphin sensitivity. The data in this study suggested that TcaA play a role in the ligation or retention of WTA within the cell wall. However, more work is needed to clarify that part. Interestingly, despite making the bacteria more vulnerable to serum killing, tcaA contributes to S. aureus virulence by altering the cell wall architecture, as demonstrated by the wild type strain outcompeting the tcaA mutant in a Mouse Co-infection model. The study raises an important point that TcaA in S. aureus may represent a system balancing two scenarios: it makes the bacteria more susceptible to serum killing, potentially limiting bacteraemia and providing long-term benefits between hosts; however, once established in the bloodstream, the bacteria survive and thrive, causing successful bacteraemia, as per the short-sighted evolution of virulence hypothesis. This duality highlights the complex interplay between within-host and between-host fitness in bacterial evolution. I strongly suggest creating a graphical abstract to illustrate the complex relationship between within-host and between-host fitness scenarios involving TcaA. Having this visual representation in the discussion will enhance comprehension and provide a concise summary of the complex system for the reader.

In this manuscript, the authors achieved their aims, and the results support their conclusions. This work will be fundamental for understanding this complex system and for developing novel therapeutics and vaccines for S. aureus.

Reviewer #1 (Public Review):

The manuscript by Hussein et al. uses cryoEM structure, microscale thermophoresis (MST), and molecular dynamics simulations (conventional and CpHMD) to unravel the Zn2+ and proton role in the function of the Cation Diffusion Facilitator YiiP. First, they generate mutants that abolish each of the three Zn2+ models to study the role of each of them separately, both structurally and functionally. Next, they used a Monte Carlo approach refining the CpHMD data with the MST points to establish the Zn2+ or proton binding state depending on the pH. That predicted a stoichiometry of one Zn2+ to 2 or 3 protons (1:3 under lower pH values). Finally, they proposed a mechanism that involves first the binding of Zn2+ to one low-affinity site and then, after the Zn2+ migrates to the highest affinity site in the transmembrane portion of the protein. The lack of Zn2+ in the low-affinity site might induce occlusion of the transporter.

The manuscript is well-written it is of interest to the field of Cation Facilitator Transporters. It is also an excellent example of a combination of different techniques to obtain relevant information on the mechanism of action of a transporter.

I have only a few comments that might need clarification from the authors:

- If the unbinding of Zn2+ to site B triggers the occlusion (and maybe the OF state) and the external pH does not affect that binding, how is it prevented from being always bound to Zn2+ and thus occluded also while it should be transporting protons (B to C panels in Figure 5)? Are there some other factors that I am missing?<br /> - I am not an expert on experiments, but the results for mutants that abolish site C are difficult to understand. For D287A/H263A, the SEC columns data suggest a population of higher oligomers. Still, for the D70A/D287A/H263A and D51A/D287A/H263A, they showed a native dimer. I understand your suggestion that the Fab induces the domain swap, but how do you explain the double mutant SEC column result? Please elaborate.<br /> - Since in the D287A mutant, you are disrupting the preferred tetrahedral coordination of Zn2+, but it still binds, do you observe any waters that compensate for the missing aspartate? Maybe in the MD simulations?

Reviewer #2 (Public Review):

In this work, the authors reported cryo-EM structures of four types of zinc-binding site mutants of a bacterial Zn2+/H+ antiporter YiiP, and proposed distinct structural/functional roles of each of the binding sites in the intramolecular Zn2+ relay and the integrity of the homodimeric structure of YiiP. MST analysis using the mutants with a single Zn2+-binding site at different pH further clarified the pH dependence of Zn2+ binding affinity of each site. Moreover, the inverse Multibind approach refined the CpHMD pKa values of the key Zn2+-binding residues so that they agreed with the MST data. Consequently, energetic coupling of Zn2+ export to the proton-motive force has been suggested. These findings definitely provide new mechanistic insight into this Zn2+/H+ antiporter.

Regrettably, the resolutions of the cryo-EM structures presented in this work are, overall, not high enough to describe detailed structure of some specific regions including the Zn2+-binding sites, and the density is missing for some important regions including the kinked segment of TM5. Further attempts toward higher resolution cryo-EM maps would be beneficial to corroborate their conclusions. Additionally, it may, in a sense, appear that the MD simulations have been carried out forcibly so that the outcomes are compatible with or nicely explain the experimental data. Although this is not unusual or unacceptable, I am concerned that the determined pKa values of some residues, especially of Asp residues at Site A, are unusually high. These outcomes seem to need careful interpretation and discussion.

Reviewer #3 (Public Review):

This contribution focuses on the zinc(II) transporter YiiP, a widely used model system of the Cation Diffusion Facilitator (CDF) superfamily. CDF proteins function as dimers and are typically involved in the maintenance of homeostasis of transition metal ions in organisms from all kingdoms of life. The system investigated here, YiiP, is a prokaryotic zinc(II)/H+ antiporter that exports zinc(II) ions from the cytosol. The authors addressed multiple crucial questions related to the functioning of YiiP, namely the specific role of the three zinc(II) binding sites present in each protomer, the zinc(II):H+ stoichiometry of antiport, and the impact of protonation on the transport process. Clarity on all these aspects is required to reach a thorough understanding of the transport cycle.

The experimental approach implemented in this work consisted of a combination of site-directed mutagenesis, high-quality 3D structural determination by cryoEM, microscale electrophoresis, thermodynamic modeling and molecular dynamics. The mutants generated in this work removed one (for the structural characterization) or two (for microscale electrophoresis) of the three zinc(II) binding sites of YiiP, allowing the authors to unravel respectively the structural role of metal binding at each site and the metal affinity of every site individually. pH-dependent measurements and constant pH molecular dynamics simulations, together with the metal affinity data, provided a detailed per-site overview of dissociation constants and Ka values of the metal-binding residues, casting light on the interplay between protonation and metal binding along the transport cycle. This thermodynamic modeling constitutes an important contribution, whose impact is however limited by the lack of an evaluation of whether the measured affinities in the various mutants differ significantly vs the affinities in the WT protein. In particular, this is true for the mutations disrupting site C, which cause a large-scale change in the quaternary structure of the protein.

Overall the authors were successful in providing a model of the transport cycle (Figure 5) that is convincing and well supported by the experimental data. The demonstration that two protomers act asymmetrically during the cycle is another nice achievement of this work, confirming previous suggestions. This novel overview of the cycle can constitute a basis for future work on other systems such as human ZnT transporters, also exploiting a methodological approach for the thermodynamic of these proteins similar to the one deployed here. The latter approach may be applicable also to other superfamilies of metal transporters.

Reviewer #3 (Public Review):

This study sought to identify relations between parameters of the diffusion decision model (DDM) and concentration of the neurotransmitters glutamate and GABA, as measured by magnetic resonance spectroscopy, and to evaluate the possibility that age moderates these relations in a developmental sample spanning middle childhood through young adulthood. The authors find a set of age-by-neurotransmitter concentration interaction effects indicating that lower levels of glutamate and greater levels of GABA in the intraparietal sulcus are related to faster non-decision times (lower values of the Ter parameter of the DDM) for "younger" participants but have the opposite relations in "older" participants (although given the way that the results are reported, the reader has little indication of what age group the terms "younger" and "older" refer to). The authors find similar interaction effects regarding relations between neurotransmitter concentration and connectivity in a visuomotor network and between neurotransmitter concentration and a fluid intelligence test. They then test moderated mediation models to determine whether functional connectivity in the visual-motor network mediates relations between neurotransmitter concentration and Ter, and whether Ter mediates the relation between neurotransmitter concentration and intelligence.

Strengths of the study include the relatively large sample size and the unique combination of brain and behavioral measures. The reported bivariate associations indicate an intriguingly consistent pattern of age-related moderation effects on the relation between neurotransmitter concentrations and several variables relevant to cognition (Ter, visuomotor connectivity, intelligence test scores) that could provide valuable insights to the field about the interplay between neurotransmitters and cognitive processes across development. However, the inferences that can be drawn from this work are seriously limited by an array of conceptual and methodological concerns.

A major conceptual issue is that the study is motivated by the premise that the nondecision time parameter (Ter) of the DDM is a major mechanistic underpinning of intelligence during child and adolescent development. There are several reasons why this premise is not well-supported. Although Ter is sometimes found to have weak correlations with scores on intelligence tests, the clearest pattern of findings across multiple studies is instead that individual differences in intelligence are primarily related to the DDM's drift rate (v) parameter (e.g., Schmiedek et al., 2007; Schulz-Zhecheva et al., 2016; Schubert & Frischkorn, 2020). The authors highlight the earlier finding that Ter mediates the effect of age on intelligence in the Krause et al. (2020) paper, but this paper is of questionable relevance to the current study. Krause et al. (2020) investigated cognitive changes in aging (ages 18-62) which are quite different from the current study's focus on development from middle childhood through young adulthood (ages ~ 7-24). Aging in older adults is known to have limited and task-specific effects on drift rate but strong effects on boundary and Ter whereas development from childhood through young adulthood coincides with the rapid maturation of drift rate (as shown in both prior research and in the current study's supplemental plots). Beyond the relatively weak evidence that Ter is a major contributor to intelligence during development, it is important to note that Ter is a nonspecific "residual" parameter (Schubert & Frischkorn, 2020) that is, theoretically, the summation of a wide array of different processes that are difficult to dissociate (perceptual encoding, visual search, motor responding). Therefore, in contrast to the drift rate and boundary parameters, it is difficult to interpret Ter as indexing a unitary mechanistic process, which is consistent with earlier findings that Ter shows limited evidence of psychometric validity as a task-general trait (Schubert et al., 2016). Finally, it is notable that bivariate tests of DDM parameters' relations with intelligence in the current study's sample (Supplemental File 9) suggest that Ter does not show a robust relation with intelligence, whereas drift rate shows relatively strong relations with intelligence in every group except for "older" individuals, who have likely fully matured and may therefore have less variance in both v and intelligence.

There are several opaque and potentially problematic features of the EZ DDM analysis. The tasks have relatively few trials spread across multiple different conditions within each task and it is unclear whether the DDM parameters were estimated separately in each condition or were estimated from trial-level data that were collapsed across conditions. This is especially concerning for the ANT, which has 96 trials distributed across 12 conditions, or apparently only 8 trials per design cell. Given the relatively low number of trials (both per design cell and overall) it is also concerning that parameter recovery studies do not appear to have been completed to ensure that this number of trials is sufficient to reliably estimate DDM parameters. In addition, accuracy rates and other behavioral summary statistics are not reported for any of the tasks. As ceiling levels of accuracy (i.e., few error RTs) can also cause prevent accurate estimation of parameters, this is another indication that assessing parameter recovery could be critical for inferences in this study.

A broader concern related to the measurement of DDM parameters is that they are each assumed to reflect the same mechanistic process across the three different tasks, but this assumption is not explicitly modeled (e.g., as a latent factor). Although the fact that Ter parameters across all three tasks have similar patterns of results is consistent with this assumption, constructing a latent factor using EFA or CFA would provide an explicit, and critical, test of the assumption. Latent factors formed from DDM parameters across the three tasks would also have several key methodological advantages over single-task measures, including separating variance in the cross-task mechanism of interest from task-specific "method variance", increasing statistical power by improving measurement of the latent mechanism, and reducing the number of multiple comparisons that need to be corrected for. The last two points are particularly critical for this study because it is possible that poor (i.e., single task) measurement and the large number of comparisons that were corrected for may have resulted in a consequential Type II error, such as a failure to detect effects involving DDM parameters other than Ter (drift rate and boundary). Related to these points, it is generally difficult for readers to judge the study's claims about the cross-task relevance of each DDM parameter or about dissociations between the different parameters because no intercorrelations between the parameters (within and between tasks) are reported or discussed.

Although the setup of the bivariate and moderation tests of relations between neurotransmitter concentrations and other variables is generally rigorous, it is concerning that only linear effects of age appear to have been considered. There appears to be clear evidence of nonlinear age-related trends in the scatterplots of parameter values displayed in Supplementary File 2.

The mediation analyses are central to the study's claims, but their results are particularly difficult to draw conclusions from due to several problematic methodological details. First, the confidence interval (CI) used to evaluate the significance of effects in these analyses was a 90% CI, essentially changing the alpha level for these tests from the conventional p<.05 to p<.10. Although this is claimed, in the Methods section, to be justified because these were "follow-up analyses based on the significant results obtained in advance", it is a highly unusual change that appears likely to have been made post hoc. Another apparent post hoc change is also mentioned in Methods: "we additionally removed cases that fell beyond three standard deviations after running the multiple regression models". It is not clear what variable or residual score this statement refers to and it is also not clear why this outlier trimming step was carried out only at the mediation stage, and not prior to the earlier bivariate analyses. The combination of an unusually high effective alpha level and potential post hoc adjustments to researcher degrees of freedom seriously undermines confidence in the mediation results. Even if the statistical hypothesis tasks are taken at face value, though, the evidence for mediation still appears very weak because the standardized effects are small and only significant in one age group (either "older" or "younger" but not both).

A broader challenge for readers is that neither the absolute ages nor the general developmental period of participants is mentioned anywhere in the main text or main plots of the paper. "Younger" and "older" mean very different things when referring to an aging sample (e.g., in the cited Klaus et al. study) versus a developing sample that spans middle childhood through young adulthood. Even if the range of the current study's sample is known, plots that split groups up by plus or minus one SD of age obscure age-related trends because the ages of the subgroups are not known. It may be easier to interpret findings if groups are instead split by neurotransmitter levels and plotted by absolute age.

Reviewer #2 (Public Review):

Zacharopoulos et al. investigated the relationship between MR spectroscopy-detected neurotransmitter concentrations (GABA/glutamate) in the intra-parietal sulcus (IPS) and middle frontal gyrus (MFG), behaviourally measured indices of sensory and cognitive processing, and fMRI measured functional connectivity within the frontoparietal network. They find that increased IPS glutamate concentration is related to poorer visuomotor processing in younger participants and better performance in older participants, while IPS GABA predicts the opposite pattern. They further show that these relationships are mediated by frontoparietal functional connectivity. Finally, they show that IPS GABA and glutamate concentration are related to fluid intelligence and that this relationship is mediated by visuomotor processing and moderated by the developmental stage. These data add to our understanding of the dynamic role of excitatory and inhibitory neurotransmitter systems in cognitive processes throughout development.

Strengths:

The study employs an impressively large cross-sectional, multimodal, dataset, with almost 300 participants ranging from 6 to 18+ years old.

The main finding (i.e., the interaction between GABA/Glu, visuomotor processing, and age) is found across three behavioural tasks and replicated in a second dataset collected 1.5 years after the first.

The authors extensively report the results of the numerous analyses performed in the supplementary material.

Weaknesses:

Pre-registration of experimental and analytical plans should be the norm, e.g., to reduce so-called 'p-hacking'. I am by no means asserting that this behaviour has occurred in the current study; however, it is disappointing that there is no reported pre-registration for such a large-scale study, where the selection and order of analyses (and the subsequent corrections applied) can meaningfully influence the pattern of results.

Many tests were performed in the study using frequentist statistics, and the way the results are reported makes it difficult to discern how distinguishable those that were reported as meaningful are from those that were disregarded.

Insufficient analyses were conducted to describe the relationships between a) GABA and glutamate, b) repeated behavioural measures, and c) test-retest reliability. This reduces the strength of the claims, some of which could be accounted for by simpler, potentially less interesting, explanations.

Reviewer #1 (Public Review):

Zacharopoulos et al. present a multi-modal investigation into the developmental trajectories of cognitive processing, decision-making processing, and visuomotor processing in children and young adults, and attempt to relate them to neuroimaging measures of functional brain connectivity and neurotransmitter concentrations in two distinct brain regions.

Results suggest specific interactions between neurotransmitter concentrations and visuomotor task performance. Interestingly, GABA and Glu levels appear to have different relationships with task performance if the participant group is trichotomized into older, 'mean-age', and younger participants. These findings appear consistent across three different visuomotor processing tasks and replicate well between two time points at which task performance and MRS measures were established for each participant (1.5 years apart). Visuomotor connectivity (assessed with resting-state-fMRI) also showed age-group-specific relationships with neurotransmitter levels. Finally, the authors present evidence that visuomotor processing mediates the relationship between neurochemical levels and scores of fluid intelligence, but only for older participants.

STRENGTHS

The study has an astonishing sample size in the context of MRS research, a field that has historically struggled to aggregate large datasets because of a severe lack of methodological standardization. Longitudinal MRS data from close to 300 participants means that this is one of the largest MRS datasets to date, enabling the group to add another exciting piece of work to their six previously published manuscripts on relationships between cognitive performance and neurochemical measures from this powerful resource. MRS data quality appears excellent, owed to state-of-the-art acquisition and raw data processing. The authors are further to be commended for making the raw MRS data publicly available - they will serve as a fantastic resource for method developers and applied researchers in the field.

WEAKNESSES

There is generally little to no consideration or discussion concerning age trajectories of MRS-derived metabolite estimates during childhood and early adulthood, which are not clearly established at all. There is evidence for increasing GABA+macromolecules during childhood (Porges et al, eLife 2021, https://elifesciences.org/articles/62575), although it may be ascribed to macromolecules rather than GABA itself (Bell et al, Sci Rep 2021, https://pubmed.ncbi.nlm.nih.gov/33436899/). The findings should at least be discussed in the context of this literature, but I suggest going a step further. The authors have all the data to make a major contribution to the scarce body of evidence on metabolite changes between 6 and 18 years by examining whether GABA and Glu estimates actually appear to change systematically across the age range of their dataset (especially exciting since they have longitudinal data)! It would be immensely valuable to see an analysis like this.

With that said, a methodological weakness concerns the computation of neurochemical concentrations presented here. Firstly, the authors can provide more detail about the acquisition and data processing/modeling decisions. Secondly, and more importantly, MRS-derived estimates of concentration can never be absolute, and always require several assumptions about the relative contributions of tissue classes (GM, WM, CSF) to the measurement volume, tissue water content, water and metabolite MR relaxation times, MR visibility, etc. Quantitative MRS estimates therefore need to be interpreted with caution, especially when these confounding factors are likely to vary between observed groups, or with age, pathology, etc. - there is plenty of reason to assume that cortical maturation, iron accumulation, etc. contribute to changes in relative GM/WM/CSF fractions or relaxation time changes. The authors present two different correction methods to account for some of these aspects, but only present the results of one, stating that "The results showed the same general pattern across all quantification methods.", which is insufficient to assess what changed and what didn't. Interestingly, the authors have presented no less than *four* different quantification methods in a similar manuscript using the same dataset (Zacharopoulos et al, Human Brain Mapp 2021; https://onlinelibrary.wiley.com/doi/10.1002/hbm.25396), but they do not mention normalization to the internal creatine signal in this present work, or whether it yielded different results (which might indicate that their method of tissue correction introduces a confounder rather than correcting for it). There is no mention of whether any further analysis of the water T2 relaxation time estimates was performed, but it would be vital to understand whether they themselves change with age, since this would establish that they are likely to confound GABA and Glu estimation. Generally, the choice to perform additional subject-specific acquisitions to allow corrections for water T2 relaxation is understandable, but not clearly motivated or explained in the experimental section. The authors should further clarify whether the relative tissue volume fractions of GM, WM, and CSF are stable across the age range, or whether there is a systematic tissue composition change with age that may also confound the Glu and GABA estimation.

Finally, I am surprised to find no discussion of limitations at all. It is important to point out the methodological limitations of MRS, which are widely discussed in the MRS literature, but probably less obvious to those readers less intimately familiar with it. This concerns not only the confounding factors for quantification that I described above but also the challenges of the comparably low spectral resolution at 3 Tesla. Even with high-quality data as presented here, it remains unclear whether the small GABA signal can be reliably separated from glutamate, glutamine, and glutathione, all of which exhibit substantial spectral overlap with each other and other strong signals as well as the underlying macromolecular background. The limitations (and how they impact interpretation) ought to be mentioned and discussed in the context of the vast amount of literature. They should provide the reader with the appropriate context and the awareness that all MRS measures are extremely sensitive to many different experimental factors and modeling decisions.

Reviewer #2 (Public Review):

The mechanisms of action potential firing were studied by whole-cell patch-clamp recordings in acute brain slices of the zebra finch. The study builds on the initial finding by Zemel et al. (2021) that the action potentials of robustus arcopallialis projection neurons (RAPNs) have an exceptional small half-duration of about 0.2 ms at 40C. The authors, therefore, set out to investigate the mechanisms of action potential repolarization. They use an impressive set of complementary techniques including voltage clamp and current clamp recordings, pharmacological interventions with classical and novel subunit-specific blockers, in situ hybridization, and comparative genomics of the KCNC/Kv3 potassium channel genes. The data convincingly demonstrate that the Kv3.1 but not Kv3.2-Kv3.4 nor Kv1.1/1.2/1.6, Kv7, or BK channels mediate the rapid repolarization. The manuscript is clearly written and the data and the presentation of the data are of the highest scientific quality. The study is of interest to a broad readership because the zebra finch is a fascinating and novel model to investigate the mechanisms of rapid motor control. The similarities of these neurons of the zebra finch with the specialized Betz cells in the motor cortex of humans and other primates demonstrates the exciting advantages of this animal model in comparison with well-established rodent models to investigate the mechanisms of complex sensory-motor control in vertebrates.

Reviewer #3 (Public Review):

This work describes intracellular recordings from motor neurons of the zebrafinch. The authors use isolated brain slices allowing careful analysis of both voltage- and current-clamp recordings to document differences in action potentials in two motor cortical areas. RAPN neurons are associated with vocal commands that generate bird song, while Ald neurons are also motor neurons, but are not involved in song.

RAPN neurons are found to have much faster action potentials than Ald neurons, and pharmacological experiments provide evidence for the involvement of a particular class of voltage-gated potassium channels, Kv3, in RAPNs that presumably contributes to a faster rate of action potential repolarization and a concomitant narrowing of the action potential width. A set of experiments is included to verify that the findings obtained under normal ex vivo recording conditions (23 deg C) are retained under more physiological conditions (40 deg C). Consistent with the role of Kv3, the action potentials, and underlying potassium currents, are modified by imperfect pharmacological tools TEA, 4AP, and AUT5. Even though imperfect, together they provide support for the role of Kv3. Examination of transcripts for Kv3 family members documents that Kv3.1 is more highly expressed in RAPN than Ald neurons.

The experiments are adequately replicated and the paper is written very clearly. The authors claim that these cells are similar to Betz cells, highly specialized pyramidal neurons mainly found in the primate motor cortex and that they may play a similar role in primates and birds in generating fine motor behavior.

Some weaknesses include missing controls such as reversibility of pharmacological effects and improved statistical analysis. In addition, the linkage of RAPN neurons to Betz cells is not very strong.

Reviewer #1 (Public Review):

This manuscript presents evidence for Kv3 subunits being involved in shaping fast action potentials (APs) within the high-precision circuitry of the zebra finch song circuitry. The authors compare and contrast the morphology of Robustus Arcopallialis (RA) neurons with those in the adjacent intermediate arcopallium (AId) and compare their passive properties, action potential waveforms, and voltage-gated outward currents. Data using pharmacological agents known to interact with Kv3 channels reinforce their other observations.

Strengths:<br /> 1. Interesting avian model of cortical molecular mechanisms.<br /> 2. Comparative study at the level of cortical motoneurons showing those involved in fine motor control for vocalizations express high levels of Kv3.1.<br /> 3. Makes a case for convergent evolutionary utilization of Kv3.1 supporting fast spiking.<br /> 4. Clearly shows other Kv3 subunits are present in the nuclei under study.<br /> 5. Employs well-characterised pharmacological tools to support the physiology.

Weaknesses:<br /> 1. Comparison with Betz Cells comes across as of secondary importance and is perhaps a discussion point rather than the first introductory paragraph.<br /> 2. Fails to adequately quantify the absolute levels of Kv3 mRNA or protein in the zebra finch brain nuclei.<br /> 3. The comparison of % or fold differences between the two avian nuclei (RA and AId neuron) masks important quantitative evidence and the contribution of multiple subunits to functional channels is not well developed.<br /> 4. The voltage-clamp data suggests that the large TEA-sensitive current is too slow to dominantly contribute to the repolarization of a single AP (but would require sustained or cumulative depolarization to be activated), while the fast transient current which could contribute to single APs, is not sufficiently characterised.<br /> 5. It is not possible to conclude that the pharmacology is specific for Kv3.1, it is at best indicative, and the absence of more precise molecular tools (e.g. knockout or gene-edited animals) undermines the authors' justification of the zebra finch as an accessible model.<br /> 6. Although the authors acknowledge the presence of other Kv3 subunits, the report fails to explain whether they are functional, but focuses on Kv3.1 as being dominant, without sufficiently addressing how other subunits contribute (perhaps as heteromeric assemblies of subunits).

Reviewer #2 (Public Review):

This paper is an interesting and novel addition to our understanding of the link between ER stress and lipid homeostasis. Utilizing a genetic screen to determine modulators of the UPRER, Garcia, G., et al., determine C. elegans cannot activate the UPRER as strongly with knockdown of the putative hydroxysteroid dehydrogenase let-767. Additionally, let-767 knockdown results in smaller lipid droplets and changes to ER morphology. Both lipid droplet size and ER morphology size can be restored with supplementation of lipids, while the defect to UPRER activation persists. The authors elegantly show that one impact of let-767 knockdown on UPR is downstream of XBP1 splicing. The authors then go on to show that in mammalian cells, the lipid precursor 3-oxoacyl-CoA can cause a similar reduction to UPRER activation to that seen in C. elegans with let-767 knockdown. Some limitations of this study are that let-767 exact role in lipid metabolism is not well understood and it is unclear what the impact of let-767 knockdown in C. elegans has on lipid composition. It is also unclear mechanistically how let-767 is able to effect UPRER, as the authors show one potential mechanism is by blocking activation of the UPR downstream of XBP1 splicing. While the authors demonstrate that high levels of 3-oxoacyl-CoA can cause a reduction in the UPR response in mammalian cells, this finding is not recapitulated in C. elegans, nor does the study determine whether this compound accumulates in a let-767 knockdown.

Reviewer #3 (Public Review):

Here, the authors identify and characterize the role of C. elegans putative hydroxysteroid dehydrogenase gene let-767 to be essential for both lipid and endoplasmic reticulum (ER) homeostasis. They demonstrated that plays a role in lipid storage, maintaining ER morphology and that the lack of let-767 inhibits the unfolded protein response (UPR) upon proteotoxic stress, presumably by the accumulation of the predicted metabolite directly upstream of LET-767, 3-oxoacyl.

Strengths of the manuscript<br /> The complementary data in human cell line huh-7 that support the authors findings in C. elegans. The ablation of let-767 in C. elegans render the animal incapable of mounting a UPR response upon proteotoxic stress (tunicamycin). Similarly, supplementing the media of huh-7 cells with LET-767 precursor, 3-oxoacyl, attenuates the UPR activation by tunicamycin.

Overall, the experiments are well designed and in logical order throughout the manuscript.

Weakness of the manuscript<br /> The biggest weakness of this manuscript is the difficulty to appreciate the differences reported by the authors from the images provided. Providing images of higher quality or highlighting the differences to note within the figure panels will make the interpretation of data easier.

Additionally, many of the reported data are from biological duplicates. The lack of additional biological replicate might undermine the authors' findings.

Reviewer #1 (Public Review):

The underlying principle of the experimental system described here is to test potential candidate genes that intersect with the proteotoxic-induced UPR by screening an siRNA pool that diminishes the UPR transcription reporter activated by sec-11 RNAi-mediated ER proteotoxic stress. The authors specifically focused on genes reported to play roles in LD biology, instead of general lipid synthesis genes. Systematic evaluation of the LD genes with respect to the induction of the UPR provides important insights into the overall functions and mechanisms of the UPR.

Using this set-up, the authors identified the hydroxysteroid dehydrogenase gene let-767/HSD17B12. Subsequent analyses revealed that let-767-mediated signaling is a key component that establishes the orchestration of both ER lipid and protein homeostasis and ER organismal functions, including ER lipid storage and ER structural changes. In addition, the authors found that acs-1i, knockdown of a gene involved in metabolism of lipids such as LCFA and mmBCFA, also diminished UPRE-GFP levels induced by sec-11i, albeit to a lesser extent than let-767i. Supplementation of lipid metabolites such as LCFA and mmBCFA recovered not only the sec-11-induced UPRE-GFP reporter phenotypes in acs-1i worms, but also the ER size and morphology and the LD and body sizes.

In contrast, the UPRE-GFP reporter phenotype in let-767i worms was not recovered by exogenously added LCFA or mmBCFA, although it was recovered by spb-1 RNAi, knockdown of a major lipogenic enzyme/pathway. The system established by the authors allowed them to quantitatively dissect the involvement of the Ire1-Xbp1 splicing UPR signaling branch. Finally, the authors demonstrated similar effects in mammalian tissue culture cells, suggesting conservation of the mechanisms.

The conclusions of this manuscript are generally in agreement with the data and the authors' interpretations are reasonable. However, at this point, the work remains descriptive and does not provide a mechanistic understanding. Overall contributions/advances towards providing new insights into how the UPR pathway is wired with respect to lipid-associated perturbations remain somewhat limited.

Reviewer #2 (Public Review):

Aimon et al. used fast whole-brain imaging to investigate the relationship between walking and neural activity in adult fruit flies. They find that increases in brain-wide activity are tightly correlated with walking behavior, and not with grooming or flailing, and are independent of visual input. They reveal that excitatory, inhibitory, and neuromodulatory neurons all contribute to brain-wide increases in neural activity during walk. Aimon et al. extend their observations of brain-wide activity to reveal that activity in some inferior brain regions is more correlated with walk than in other brain regions. The authors further analyzed their imaging dataset to identify candidate brain regions and cell types that may be important for walking behavior, which will be useful in hypothesis generation in future studies. Finally, the authors show that brain-wide activity is similar between spontaneous and forced walk and that severing the connection between the ventral nerve cord and central brain abolishes walk-related increases in brain activity. These results suggest that increases in brain-wide activity during walking may be largely attributed to sensory and proprioceptive feedback ascending to the central brain from the ventral nerve cord rather than to top-down executive and motor control programs. The observations presented in this study suggest hypotheses that may be tested in future studies.

Strengths: This paper presents a rich imaging dataset that is well-analyzed and cataloged, which will be valuable for researchers who use this paper for future hypothesis generation. The comparison of many different reagents, imaging speeds, and behavioral conditions suggests that the observed increases in brain-wide activity during walking are quite robust to imaging methods in adult fruit flies.

Weaknesses: This study is largely observational, and the few experimental manipulations presented are insufficient to support the author's broad claims about the generation of brain-wide neural activity.

Notably, the authors suggest that their image analysis can reveal individual cell types that are important for walking by matching their morphologies to registered components from whole-brain imaging experiments. While these predictions are a useful starting point for future experiments, they have not convincingly shown that their method can identify individual cell types in genetic reagents with more restricted expression patterns. Adding further validation to show that genetically subtracting the candidate neurons from the overall expression pattern of the calcium indicator abolishes that component from the response would strengthen this claim. Furthermore, imaging the matched candidate neuronal cell type to show that it recapitulates the activity dynamics of the proposed component would add additional evidence.

In addition, increases in neural activity prior to walk onset in specific brain regions are intriguing but insufficient to demonstrate the neurons in these regions trigger walking. This claim should await further studies that employ targeted and acute manipulation of neural activity, as noted by the authors. Furthermore, that activity in these brain regions is significantly increased prior to walk onset awaits more rigorous statistical testing, as do the authors' claims that spontaneous versus forced walking alters these dynamics. The suggestion that walking increases brain-wide activity via feedback from the ventral nerve cord is an interesting possibility and would also benefit from additional experimental validation. Activating and silencing neurons that provide proprioceptive feedback from the legs and determining the effect of this manipulation on brain-wide neural activity would be a good starting point.

Reviewer #1 (Public Review):

This paper uses light field microscopy to measure calcium signals across the fly brain while it is walking and turning, and also while the fly is externally driven to walk and turn, using a treadmill. The authors drive calcium indicator expression using pan-neuronal drivers, as well as drivers specific to individual neurotransmitters and neuromodulators. From their experiments, the authors show that inhibitory and excitatory neurons in the brain are activated in similar patterns by walking and that neurons expressing machinery for different neuromodulatory amines tend to show differentially strong calcium signals during walking. By examining spontaneous and forced walking and turning, the authors identify brain regions that activate before spontaneous turning and that activate asymmetrically in concert with spontaneous or forced turning.

Strengths: Overall, the strength of this paper is in its careful descriptions and analyses of whole brain activation patterns that correlate with spontaneous and forced behaviors. Showing how the pattern of activity relates to broad classes of cells is also useful for understanding brain activation. Especially in brain regions identified as preceding spontaneous walking and in being asymmetrically involved in spontaneous and forced turning, it provides a wealth of potential hypotheses for new experiments. Overall, it contributes to a coarse-grained understanding of broad changes in brain activity during behavior.

Weaknesses: The primary weakness of this paper is that it presents some speculative interpretations and conclusions too strongly. Most importantly, average activity in a neuropil can represent the calcium activity of hundreds or thousands of neurons, and it is hard to know what fraction is active, for instance, or how expression pattern differences might play into calcium signals. Calcium signals also do not reliably indicate hyperpolarization, so a net increase in the average Ca++ indicator signal does not necessarily reflect that the average neuron is becoming more active, just that some labeled neurons are becoming more active, while others may be inactive or hyperpolarized. The conclusions about regions triggering walk (rather than just preceding it) are too strong for the manipulations in this paper, as are some of the links with individual neuron types. Thus, more presenting substantial caveats is required for the conclusions being drawn from the data presented here.

Reviewer #3 (Public Review):

Aimon and colleagues investigated brain activity in flies during spontaneous and forced walking. They used light-field microscopy to image calcium activity in the brain at high temporal resolution as the animal walked on a ball and they used the statistical inference methods PCA and ICA to tease out subregions of the brain that had distinct patterns of activity. They then sought to relate those patterns to walking. Most interesting are the experiments they performed comparing forced walking to spontaneous walking because this provides a framework to generate hypotheses about which aspects of neural activity are reporting the animal's movements versus generating those movements. The authors identify subregions and neuron types that may be involved in generating vs reporting walking. Their analysis is reasonable but could be further strengthened with a more powerful statistical framework that explicitly considered the multiple hypotheses being tested. More broadly, the work serves as a starting point to investigate the role of different regions in the brain and should spur follow-up investigations that involve more perturbative approaches in addition to the correlative approaches presented here.

Reviewer #1 (Public Review):

Tomasi et al. performed a combination of bioinformatic, next-generation tRNA sequencing experiments to predict the set of tRNA modifications and their corresponding genes in the tRNAs of the pathogenic bacteria Mycobacterium tuberculosis. Long known to be important for translation accuracy and efficiency, tRNA modifications are now emerging as having regulatory roles. However, the basic knowledge of the position and nature of the modifications present in a given organism is very sparse beyond a handful of model organisms. Studies that can generate the tRNA modification maps in different organisms along the tree of life are good starting points for further studies. The focus here on a major human pathogen that is studied by a large community raises the general interest of the study. Finally, deletion of the gene mnmA responsible for the insertion of s2U at position 34 revealed defects in in growth in macrophage but in test tubes suggesting regulatory roles that will warrant further studies. The conclusions of the paper are mostly supported by the data but the partial nature of the bioinformatic analysis and absence of Mass-Spectrometry data make it incomplete. The authors do not take advantage of the Mass spec data that is published for Mycobacterium bovis (PMID: 27834374) to discuss what they find.

Important points to be considered:

1) The authors say they took a list of proteins involved in tRNA modifications from Modomics and added manually a few but we do not know the exact set of proteins that were used to search the M. mycobacterium genome.

2) The absence of mnmGE genes in TB suggested that the xcm5U derivatives are absent. These are present in M. bovis (PMID: 27834374). Are the MnmEG gene found in M. bovis? If yes, then the authors should perform a phylogenetic distribution analysis in the Mycobacterial clade to see when they disappeared. If they are not present in M. bovis then maybe a non-orthologous set of enzymes do the same reaction and then the authors really do not know what modification is present or not at U34 without LC-MS. The exact same argument can be given for the xmo5U derivatives that are also found in M.bovis but not predicted by the authors in M. tuberculosis.

3) Why is the Psi32 predicted by the authors because of the presence of the Rv3300c/Psu9 gene not detected by CMC-treated tRNA seq while the other Psi residues are? Members of this family can modify both rRNA and tRNA. So the presence of the gene does not guarantee the presence of the modification in tRNAs

4) What are tsaBED not essential but tsaC (called sua5 by the authors) essential?

Reviewer #2 (Public Review):

In this study, Tomasi et al identify a series of tRNA modifying enzymes from Mtb, show their function in the relevant tRNA modifications and by using at least one deleted strain for MnmA, they show the relevance of tRNA modification in intra-host survival and postulate their potential role in pathogenesis.

Conceptually it is a wonderful study, given that tRNA modifications are so fundamental to all life forms, showing their role in Mtb growth in the host is significant. However, the authors have not thoroughly analyzed the phenotype. The growth defect aspect or impact on pathogenesis needs to be adequately addressed.

- The authors show that ΔmnmA grows equally well in the in vitro cultures as the WT. However, they show attenuated growth in the macrophages. Is it because Glu1_TTC and Gln1-TTG tRNAs are not the preferred tRNAs for incorporation of Glu and Gln, respectively? And for some reason, they get preferred over the alternate tRNAs during infection? What dictates this selectivity?

- As such the growth defect shown in macrophages would be more convincing if the authors also show the phenotype of complementation with WT mnmA.

An important consideration here is the universal nature of these modifications across the life forms. Any strategy to utilize these enzymes as the potential therapeutic candidate would have to factor in this important aspect.

Reviewer #3 (Public Review):

The work presented in the manuscript tries to identify tRNA modifications present in Mycobacterium tuberculosis (Mtb) using reverse transcription-derived error signatures with tRNA-seq. The study identified enzyme homologs and correlates them with presence of respective tRNA modifications in Mtb. The study used several chemical treatments (IAA and alkali treatment) to further enhance the reverse transcription signals and confirms the presence of modifications in the bases. tRNA modifications by two enzymes TruB and MnmA were established by doing tRNA-seq of respective deletion mutants. Ultimately, authors show that MnmA-dependent tRNA modification is important for intracellular growth of Mtb. Overall, this report identifies multiple tRNA modifications and discuss their implication in Mtb infection.

Important points to be considered:

- The presence of tRNA-based modifications is well characterised across life forms including genus Mycobacterium (Mycobacterium tuberculosis: Varshney et al, NAR, 2004; Mycobacterium bovis: Chionh et al, Nat Commun, 2016; Mycobacterium abscessus: Thomas et al, NAR, 2020). These modifications are shown to be essential for pathogenesis of multiple organisms. A comparison of tRNA modification and their respective enzymes with host organism as well as other mycobacterium strains is required. This can be discussed in detail to understand the role of common as well as specific tRNA modifications implicated in pathogenesis.

- Authors state in line 293 "Several strong signatures were detected in Mtb tRNAs but not in E. coli". Authors can elaborate more on the unique features identified and their relevance in Mtb infection in the discussion or result section.

- Deletion of MnmA is shown to be essential for E. coli growth under oxidative stress (Zhao et al, NAR, 2021). In similar lines, MnmA deleted Mtb suffers to grow in macrophage. Is oxidative stress in macrophage responsible for slow Mtb growth?

- Authors state in line 311-312 "Mtb does not contain apparent homologs of the tRNA modifying enzymes that introduce the additional modifications to s2U". This can be characterised further to rule out the possibility of other enzyme specifically employed by Mtb to introduce additional modification.

Reviewer #1 (Public Review):

Muller glia function as retinal stem cells in the adult zebrafish retina. Following retinal injury, Muller glia are reprogramned (reactive Muller glia), and then divide to produce a progenitor that amplifies and differentiates into retinal neurons. Previous scRNAseq analysis used total retinal RNA from uninjured and injured retinas isolated at time points when Muller glia are quiescent, being reprogrammed, and proliferating to reveal genes and gene regulatory networks underlying these events (Hoang et al., 2020). The manuscript by Celotto et al., used double transgenic zebrafish that allow them to purify by FACS quiescent and reactive Muller glia, Muller glia-derived progenitors, and their differentiating progeny at different times post retinal damage. RNA from these cell populations was used in scRNAseq studies to identify the transcriptomes associated with these cell populations. Importantly, they report two quiescent and two reactive Muller glia populations. These results raise the interesting possibility that Muller glia are a heterogenous population whose members may exhibit different regenerative responses to retinal injury. However, without further experimentation, the validity and significance of this result remain unclear. In addition to putative Muller cell heterogeneity, Celotto et al., identified multiple progenitor classes, some of which are specified to regenerate specific retinal neuron types. Because of its focus on Muller glia and Muller glia-derived progenitors at mid to late stages of retina regeneration, this new scRNAseq data will be a useful resource to the research community for further interrogation of gene expression changes underlying retina regeneration.

Major concerns:

1) The identification of multiple populations of Muller glia, reactive Muller glia, and progenitors is interesting, but beyond a few in situ hybridization studies to validate injury-dependent gene inductions, there are no experiments that confirm that multiple cell populations exist in vivo, and no experiments examining the significance of these different populations in the regenerative process. It would be helpful to discuss how the peripheral to the central gradient of Muller cell maturation influences the scRNAseq-based cell clustering results.

2) While the reliance on transient GFP and mCherry expression may be sufficient, the final population used for the scRNAseq analysis is only partial in nature. Permanently marking the MG through a Cre-Lox system is more ideal. The authors mention the possibility of missing highly proliferative populations of MG/RPC through the dilution of fluorescent proteins; a transgenic system that allows for true lineage tracing may then capture more appropriate MG/RPC populations. The lack of gating for a pure GFP population also confounds this problem which the authors do mention in the discussions; this oversight was not explained.

3) Much time was taken to identify each cell cluster and to list the differentially expressed genes, but no functional significance for these genes was probed. While a lot of work has gone into the analysis shown, altering some of the MG/RPC trajectories through differentially expressed genes would go a long way to making this study more impactful.

4) The data presented in this paper has significant overlap with scRNAseq data presented by Hoang et al., 2020 in Science where Muller glia, reactive Muller glia, and Muller glia-derived progenitors were carefully analyzed. How does their data fit with the data presented here? The authors could have used that paper as a jumping-off point and offered more time points for comparison, especially as progenitors differentiate.

5) A major conclusion of the paper is that neurogenic progenitors in the injured retina differentiate into neurons with a similar order as that taking place during development. This analysis is based on two time points, and while the trends stay true to the authors' model, two time points are too few to make such a conclusion. In addition, because of the time points chosen for this analysis, many mature neuronal markers are lacking. Including additional time points so mature neuronal markers are detected in the dataset would enhance the trajectory proposed.

Reviewer #2 (Public Review):

In this publication, the authors provide a comprehensive trajectory of transcriptional changes in Müller glia cells (MG) in the regenerating retina of zebrafish. These resident glia cells of the retina can differentiate into all neural cell classes following injury, providing full regenerative capabilities of the zebrafish retina. The authors achieved this by using single-cell RNA sequencing of Müller glia, progenitors, and regenerated progeny, comparing uninjured and light-lesioned retinae.

The isolation strategy involves using two transgenic strains, one labelling dividing cells and their immediate progeny, and the other Müller glia cells. This allowed them to separate injury-induced proliferating and non-reactive Müller glia cells. Subsequent single-cell transcriptomics showed that MG could be non-reactive under both uninjured and lesioned conditions and reactive MG give rise to a cell population that both replenishes the pool of MG and replenish neurogenic retinal precursor cells. These precursor cells produce regenerated neurons in a developmental time series with ganglion cells being born first and bipolar cells being born last. Interestingly hybrid populations have been detected that co-share characteristics of photoreceptor precursors and reactive glia.

This is the first study of its kind following the dynamic changes of transcriptional changes during retinal regeneration, providing a rich data source of genes involved in regeneration. Their finding of transcriptionally separable MG populations is intriguing.

This study focuses on the light-lesioned retina and leaves open the question if the observed transcriptional trajectories of regenerating neurons are generalizable to other lesion models (e.g. chemical or mutational lesions) or are specific to the light-damaged retina.

Reviewer #1 (Public Review):

Ciampa et al. investigated the role of the hypoxia-inducible factor 1 (HIF-1) pathway in placental aging. They performed transcriptomic analysis of prior data of placental gene expression over serial timepoints throughout gestation in a mouse model and identified increased expression of senescence and HIF-1 pathways and decreased expression of cell cycle and mitochondrial transcripts with advancing gestational age. These findings were confirmed by RT-PCR, Western blot, and mitochondrial assessment from mouse placental tissues from late gestation time points. Studies of human placental samples at similar late gestational ages showed similar trends in increased HIF-1 targets and decreased mitochondrial abundance with increasing gestation, but were not significantly significant due to the limited availability of uncomplicated preterm placenta samples. The authors demonstrated that stabilization of HIF-1 in vitro using primary trophoblasts and choriocarcinoma cell lines recapitulated the gene and mitochondrial dysfunction seen in the placental tissues and were consistent with senescence. Interestingly, cell-conditioned media from HIF-1 stabilized placenta cell lines induced myometrial cell contractions in vitro and correspondingly, induction of HIF-1 in pregnant mice was associated with preterm labor in vivo. These data support the role of the HIF-1 pathway in the process of placental senescence with increasing gestational age and highlight this pathway as a potentially important contributor to gestational length and a potential target for therapeutics to reduce preterm birth.

Overall, the conclusions of this study are mostly well supported by the data. The concept of placental aging has been controversial, with several prior studies with conflicting viewpoints on whether placental aging occurs at all, is a normal process during gestation, or rather only a pathologic phenomenon in abnormal pregnancies. This has been rather difficult to study given the difficulty of obtaining serial placental samples in late gestation. The authors used both a mouse model of serial placental sampling and human placental samples obtained at preterm, but non-pathologic deliveries, which is an impressive accomplishment as it provides insight into a previously poorly understood timepoint of pregnancy. The data clearly demonstrate changes in the HIF-1 pathway and cellular senescence at increasing gestational ages in the third trimester, which is consistent with the process of aging in other tissues.

Weaknesses of this study are that although the authors attribute alterations in HIF-1 pathways in advanced gestation to hypoxia, there are no experiments directly assessing whether the changes in HIF-1 pathways are due to hypoxia in either in vitro or in vivo experiments. HIF-1 has both oxygen-dependent and oxygen-independent regulation, so it is unclear which pathways contribute to placental HIF-1 activity during late gestation, especially since the third-trimester placenta is exposed to significantly higher oxygen levels compared to the early pregnancy environment. Additionally, the placenta is in close proximity to the maternal decidua, which consists of immune and stromal cells, which are also significantly affected by HIF-1. Although the in vitro experimental data in this study demonstrate that HIF-1 induction leads to a placenta senescence phenotype, it is unclear whether the in vivo treatment with HIF-1 induction acts directly on the placenta or rather on uterine myometrium or decidua, which could also contribute to the initiation of preterm labor.

Reviewer #2 (Public Review):

The authors sought to characterize normal placental aging to better understand how the molecular and cellular events that trigger the labor process. An understanding of these mechanisms would not only provide insight into term labor, but also potential triggers of preterm labor, a common pregnancy complication with no effective intervention. Using bulk transcriptomic analysis of mouse and human placenta at different gestational timepoints, the authors determined that stabilization of HIF-1 signaling accompanied by mitochondrial dysfunction and cellular senescence are molecular signatures of term placenta. They also used in vitro trophoblast (choriocarcinoma) and a uterine myocyte culture system to further validate their findings. Lastly, using chemically induced HIF-1 induction in vivo in mice, the authors showed that stabilization of HIF-1 protein in the placenta reduced the gestational length significantly.

The major strength of this study is the use of multiple model systems to address the question at hand. The consistency of findings between mouse and human placenta, and the validation of mechanisms in vitro and in vivo modeling are strong support for their conclusions. The rationale for studying the term placentas to understand the abnormal process of preterm birth is clearly explained. Although the idea that hypoxic stress and placental senescence are triggers for labor is not novel, the comprehensiveness of the approach and idea to study the normal aging process are appreciated.

There are some areas of the manuscript that lack clarity and weaknesses in the methodology worth noting. The rationale for focusing on senescence and HIF-1 is not clearly given that other pathways were more significantly altered in the WGCNA analysis. The placental gene expression data were from bulk transcriptomic analyses, yet the authors do not explicitly discuss the limitations of this approach. Although the reader can assume that the authors attribute the mRNA signature of aging to trophoblasts - of which, there are different types - clarification regarding their interpretation of the data and the relevant cell types would strengthen the paper. Additionally, while the inclusion of human placenta data is a major strength, the differences between mouse and human placental structure and cell types make highlighting the specific cells of interest even more important; although there are correlations between mouse and human placenta, there are also many differences, and the comparison is further limited when considering the whole placenta rather than specific cell populations.

Additional details regarding methods and the reasons for choosing certain readouts are needed. Trophoblasts are sensitive to oxygen tension which varies according to gestational age, and it is unclear if this variable was taken into consideration in this study. Many of the cellular processes examined are well characterized in the literature yet the rationale for choosing certain markers is unclear (e.g., Glb1 for senescence; the transcripts selected as representative of the senescence-associated secretory phenotype; mtDNA lesion rate).

Overall, the findings presented are a valuable contribution to the field. The authors provide a thoughtful discussion that places their findings in the context of current literature and poses interesting questions for future pursuit. Their efforts to be comprehensive in the characterization of placental aging is a major strength; few placental studies attempt to integrate mouse and human data to this extent, and the validation and presentation of a potential mechanism by which fetal trophoblasts signal to maternal uterine myocytes are exciting. Nevertheless, a clear discussion of the methodologic limitations of the study would strengthen the manuscript.

Reviewer #3 (Public Review):

In this study, Ciampa and colleagues demonstrate that HIF-1α activity is increased with gestation in humans and mice placentas and use several in vitro models to indicate that HIF activation in trophoblasts may release factors (yet to be identified) which promote myometrial contraction. Previous studies have linked placental factors to the preparation of the myometrium for labour (e.g. prostaglandins), but HIF-1α has not been implicated. Due to several issues regarding the experimental design, the results do not currently support the conclusions.

Major concerns:

1) The hypothesis states that placental aging promotes parturition via HIF-1a activation, the study does not provide any evidence of an aged placenta. Aging is considered a progressive and irreversible loss of functional capacity, inability to maintain homeostasis, and decreased ability to repair the damage. The placenta retains all these abilities throughout pregnancy [PMID: 9462184], and there's no evidence that the placenta functionally declines between 35-39 weeks, otherwise, it wouldn't be able to support fetal development. However, there is evidence of a functional decline in post-term placentas (i.e. >40 weeks in humans) but the authors compare preterm placentas with E17.5 mice placentas or 39-week human placentas, both these gestational periods are prior to the onset of parturition in most pregnancies (human = 40wkGA, mice=E18.5).

2) While the authors provide evidence that HIF-1α activity increases in both the human and mice placenta as gestation progresses, the mechanistic link between placental HIF-1α and parturition is not strongly supported. For example, most of the evidence is based on in vitro studies showing that conditioned media from trophoblasts treated with CoCl2 increased the contraction of myometrial cells. The specific factor responsible was not identified but the authors allude to pro-inflammatory factors such as cytokines. It was therefore interesting to note that the conditioned media had undergone a filtration step that removes all substances >10kDa, which includes the majority of cytokines and hormones.

3) An alternative explanation is that CoCl2 treatment-induced trophoblast differentiation and the effects on myometrial contraction may be related to differences in secreted factors produced by cytotrophoblasts versus syncytiotrophoblast. Although JAR cells do not spontaneously differentiate, they can be induced to syncytialise upon cAMP stimulation. Ref 39 the authors cite shows this. Indeed, the morphology of the cells in Fig5F that were exposed to CoCl2 indicates that they may be syncytialised. Syncytialised trophoblasts also express markers of senescence including increased SA-β-gal activity and reductions in mitochondrial activity.

4) The in vivo experiment showing reduced gestation length in pregnant mice receiving DMOG injection is interesting. However, we cannot exclude the effects of DMOG on non-placental tissues (both maternal and fetal) or the non-specific effects of DMOG (i.e. HIF-1α independent). Furthermore, previous studies using a more direct approach to alter HIF-1α activity in the placenta using trophoblast-specific overexpression of HIF-1α in mice do not lead to changes in gestation length [PMID: 30808910].

5) Lack of appropriate experimental models. E.g. JAR choriocarcinomas are not an ideal model of the human trophoblast as they are malignant. Much better models are available e.g. primary human trophoblasts from term placentas or human trophoblast stem cells from first-trimester placentas. Similarly, the mouse model is also not specific as discussed above.

6) Lack of cohesion between the different experimental models. E.g. CoCl2 was used to induce hypoxia/HIF1α in mouse TBs, but DMOG was used in vivo in mice. SA-β Gal staining was carried out in cells but not in mouse or human tissues.

7) Evidence of senescence and mitochondrial abundance could be strengthened by providing additional markers. E.g. only GLB1 mRNA expression is provided as evidence of senescence, and COX IV protein for mitochondrial abundance in mouse and human placentas.

8) Given that the main goal of this study was to investigate the role of hypoxia, hypoxia (i.e. low oxygen) was never directly induced and the results were based on chemical inducers of HIF-1α which have multiple off-target effects.

Peer review report

Title: If it’s there, could it be a bear?

version: 2

Referee: Rahul Raveendran

Institution: Biodiversity Institute, University of Kansas

email: rahulravi777@gmail.com

General assessment

The manuscript needs to be revised thoroughly.

Essential revisions that are required to verify the manuscript

I feel that the introduction can be a little more elaborative. My suggestions are as follows:

• In the first paragraph, the author can give more details about ‘hominology’ by citing the works of Dmitry Bayanov. This is to give a historical account of ‘hominid research’ to the readers who are unfamiliar to this topic.

• Second paragraph has information related to the ‘misdeeds’ of the ‘proponents of hominology’. According to me, there must be continuous flow of information from paragraph to paragraph. Currently, I do not see a proper chronological flow of details in 1st and 2nd paragraph. I request the learned author to check this in such a way that 1st para must provide details about ‘hominids’, ‘hominology’, and the 2nd para must give the scientific explanation about these ‘controversial findings’.

• Line numbers 43-44: This paragraph must be expanded, and possibly include more information about ‘American black bear’ being misrecognized as ‘bigfoot sightings’ with references. If available, provide details regarding the molecular/clinical test results (i.e., references).

• A separate paragraph has to be incorporated to detail the methods adopted by scientists/researchers to link the population density of American black bear and bigfoot sightings.

• Line numbers 49-51: The sentence “No positive correlation between……a small proportion of all sightings” has to be re-written as I think that it does not convey its meaning properly.

• Provide the fundamentals of ecological niche modeling. How such a concept can be adopted in this sort of a study with a strong emphasis on the results of Lozier et al. (2009) would be helpful for the readers.

• In the last paragraph of the introduction, although not in detail, the author should state clearly the approach that was taken to execute the study. For example, details related to the chosen statistical methods with references. And state your hypothesis clearly.

Materials and Methods

• Line numbers 67-77: Please make these sentences more lucid. I feel that this paragraph lacks coherence.

• Line numbers 90-94: Please make these sentences more understandable.

• Line numbers 113-116: Please re-write these sentences to make them more understandable. Results

• Line numbers 121-123: The article states that both the sasquatch sighting and black bear population maps are strongly coloured in the Pacific Northwest area……”. BUT, in PNW, I do not think that bigfoot sightings in British Columbia are proportional to the black bear population.

• Presentation of results is a bit confusing for me. I would suggest to rewrite the results with a view to make everyone who reads this article understands the results properly.

Discussion

• Discussion must be vastly improved

a) It is difficult to understand the very first sentence of the discussion that starts with “The present study regressed ………………..”. Please re-write it.

b) Results of the present study should be discussed in detail, linking previous published reports.

c) The models employed must be discussed in detail with the support of previous reports to substantiate the conceptual correctness of the methodological framework.

Decision

Requires revisions: Major revisions are suggested.

Peer review report

Title: If it’s there, could it be a bear?

version: 2

Referee: Julie Sheldon

Institution: University of Tennessee

email: jsheldo3@tennessee.edu

ORCID iD: https://orcid.org/0000-0003-2813-3027

General assessment

This manuscript is a collection of statistical analyses attempting to show that sasquatch sightings correlate with black bear populations, and humans may be mistaking black bears for sasquatch.

The author effectively introduces the topic, provides adequate background on sasquatch, but does not provide much on black bear populations, natural history, or human-bear interactions.

The author performs several statistical tests to support the findings. I am not a statistician, but the tests seem valid. The data used for the statistical analyses, however, are not ideal. The resource (Hristienko and McDonald) provided for obtaining black bear populations was published in 2007 and the data was from 2001 via “subjective extrapolations” and “expert opinions”. Thus, this resource is outdated and suboptimal as black bear populations have changed over time. A more updated resource with more scientific methods in data collection would improve this manuscript since having as accurate as possible bear population estimates is very important for the goal of this study. The author notes this briefly in the limitations. If the human population and sasquatch sighting data matched up with the dates of bear population estimates, it would be more valid (just outdated), but there are no date ranges of human or sasquatch data provided in the manuscript.

In the results, the maps of bigfoot sightings and black bear population do not appear to correlate visually, which downplays the value of the statistical analysis. The stats should support the visual data and vice versa if the study is sound. Perhaps more updated bear population data will improve this.

The discussion is short and briefly brings up important points that can invalidate the study without much discussion or argument supporting the findings of this study.

Essential revisions that are required to verify the manuscript

I recommend the following to improve the manuscript enough to consider it valid:

Date-match the bear population, human population, and bigfoot sightings to improve the validity of the data analysis. One way to do this is to use data from the same 10-year period only.

Improve the sources of bear population information.

Expand the discussion to include reasons and ideas the maps don’t line up like the statistical analyses do – ie bears in Florida and the southeast.

Other suggestions to improve the manuscript

I recommend provide some information on black bear population/natural history in the introduction – ie what sort of habitats do black bears live in. Consider the possibility that sasquatch sightings may correlate with a type of habitat (ie forest), which happen to also correlate with black bear habitat. This may support the idea that sasquatch sightings are bears, or that sasquatch also likes to live in similar habitats as bears.

The author reports that black bears are not prominent in Florida; however, there are > 4,000 black bears bears in Florida, that are reportedly large, and it may be worth considering this as a reason for the concentration of sasquatch sightings in Florida as seen on the map. More accurate black bear data as discussed above may help improve this aspect. Experientially, there is also a high concentration of black bears in the southeastern US, where there is also a high concentration of humans and human-bear encounters. The author does not discuss this along with the number of sasquatch sightings in this region as seen on the map.

Decision

Verified with reservations: The content is academically sound but has shortcomings that could be improved by further studies and/or minor revisions.

Reviewer #1 (Public Review):

Summary

Favate et al. measure the relative levels of metabolites in 12 Escherichia coli strains isolated from different replicate populations after 50,000 generations of the Lenski long-term laboratory evolution experiment. They use untargeted LC/MS methods that include standards and report both positive and negative ionization mode measurements. They initially use principal component analysis (PCA) to broadly compare how the metabolomes of these strains are similar and different. Then, they describe several instances where the changes in metabolite abundance they see in specific pathways correlate with mutations that lead to changes in the expression of genes that encode enzymes in those pathways.

Strengths

The statistical analyses and presentation of the high-throughput data are excellent. The most compelling results are communicated in wonderful figures that integrate their measurements of metabolite levels in this study with results from a prior study they conducted looking at changes in gene expression levels in the same bacterial strains. These sections include the ones describing large increases in NAD(P) pools due to mutations in nadR, changes in the levels of arginine and related compounds due to mutations in argR, and changes in metabolites from glycolysis and the TCA cycle related to iclR and arcB.

Weaknesses

Showing that A-2 and especially A-3 are outliers in the PCA analysis is useful, but it may be hiding other interesting signals in the data. The other strains are remarkably colinear on these plots, hinting that if the outliers were removed, one main component would emerge along which they are situated. It also seems possible that this additional analysis step would allow the second dimension to better differentiate them in a way that is interesting with respect to their mutator status or mutations in key metabolic or regulatory genes.

There is a missed opportunity to connect some key results to what is known about LTEE mutations that reduce the activity of pykF (pyruvate kinase I). This gene is mutated in all 12 LTEE populations, and often these mutations are frameshifts or transposon insertions that should completely knock out its activity. At first glance, inactivating an enzyme for a step in glycolysis does not make sense when the nutrient source in the growth medium is glucose, even though PykF is only one of two isozymes E. coli encodes for this reaction. There has been speculation that inactivating pykF increases the concentration of phosphoenolpyruvate (PEP) in cells and that this can lead to increased rates of glucose import because PEP is used by the phosphotransferase system of E. coli to import glucose (see https://doi.org/10.1002/bies.20629). The current study has confirmed the higher PEP levels, which is consistent with this model.

In the introduction, the papers cited to show the importance of changes in metabolism for adaptation do not seem to fit the focus of this study very well. They stress production of toxins and secondary metabolites, which do not seem to be mechanisms that are at work in the LTEE. I can think of two areas of background that would be more relevant: (1) studies of how bacterial metabolism evolves in adaptive laboratory evolution (ALE) experiments to optimize metabolic fluxes toward biomass production (for example, https://doi.org/10.1038/nature01149 ), and (2) discussions of how cross-feeding, metabolic niche specialization, and metabolic interdependence evolve in microbial communities, including in other evolution experiments (for example, https://doi.org/10.1073/pnas.0708504105 and https://doi.org/10.1128/mBio.00036-12).

Impact and Significance

While there has been past speculation about the effects of LTEE mutations on metabolism, this study measures changes in the levels of metabolites in related metabolic pathways for the first time. Therefore, it provides useful information about how metabolism evolves, in general, and will also be a useful resource for those studying other aspects of the LTEE related to metabolism, such as contingency in the evolution of citrate utilization.

Reviewer #2 (Public Review):

This preprint presents a compelling study examining the relationship between genotypic changes and phenotypic traits in bacteria over an extended period using the Long-Term Evolution Experiment (LTEE) as a model. The primary advances in methodology include employing high-resolution mass spectrometry for comprehensive metabolic profiling and combining it with previous gene expression and DNA sequencing datasets. This approach provides insight into how specific genetic mutations can alter metabolic pathways over 50,000 generations, enabling a deeper understanding of how genetic changes lead to observed differences in evolved bacterial strains. The findings reveal that evolved bacteria possess more diverse metabolic profiles compared to their ancestors, suggesting that these populations have uniquely adapted to their environment. The work also attempts to uncover the molecular basis for this adaptive evolution, demonstrating how specific genetic changes have influenced the bacteria's metabolic pathways.

Overall, this is a significant and well-executed research study. It offers new insights into the complex relationship between genetic changes and observable traits in evolving populations and utilizes metabolomics in the LTEE, a novel approach in combination with RNA-seq and mutation datasets.

However, the paper's overall clarity is lacking. It is spread too thin and covers many topics without a clear focus. I strongly recommend a substantial rewrite of the manuscript, emphasizing structure and readability. The science is well executed, but the current writing does not do it justice.

Reviewer #1 (Public Review):

The study examines how hemocytes control whole-body responses to oxidative stress. Using single cell sequencing they identify several transcriptionally distinct populations of hemocytes, including one subset that show altered immune and stress gene expression. They also find that knockdown of DNA Damage Response (DDR) genes in hemocytes increases expression of the immune cytokine, upd3, and that both upd3 overexpression in hemocytes and hemocyte knockdown of DDR genes leads to increased lethality upon oxidative stress.

Strengths

1, The single cell analyses provide a clear description of how oxidative stress can cause distinct transcriptional changes in different populations of hemocytes. These results add to the emerging them in the field that there functionally different subpopulations of hemocytes that can control organismal responses to stress.<br /> 2, The discovery that DDR genes are required upon oxidative stress to limit cytokine production and lethality provides interesting new insight into the DDR may play non-canonical roles in controlling organismal responses to stress.

Weaknesses

1, In some ways the authors interpretation of the data - as indicated, for example, in the title, summary and model figure - don't quite match their data. From the title and model figure, it seems that the authors suggest that the DDR pathway induces JNK and Upd3 and that the upd3 leads to tissue wasting. However, the data suggest that the DDR actually limits upd3 production and susceptibility to death as suggested by several results:<br /> a) PQ normally doesn't induce upd3 but does lead to glycogen and TAG loss, suggesting that upd3 isn't connected to the PQ-induced wasting.<br /> b) knockdown of DDR upregulates upd3 and leads to increased PQ-induced death. This would suggest that activation of DDR is normally required to limit, rather than serve as the trigger for upd3 production and death.<br /> c) hemocyte knockdown of either JNK activity or upd3 doesn't affect PQ-induced death, suggesting that they don't contribute to oxidative stress-induced death. Its only when DDR is impaired (with DDR gene knockdown) that an increase in upd3 is seen (although no experiments addressed whether JNK was activated or involved in this induction of upd3), suggesting that DDR activation prevents upd3 induction upon oxidative stress.

2, The connections between DDR, JNK and upd3 aren't fully developed. The experiments show that susceptibility to oxidative stress-induced death can be caused by a) knockdown of DDR genes, b) genetic overexpression of upd3, c) genetic activation of JNK. But whether these effects are all related and reflect a linear pathway requires a little more work. For example, one prediction of the proposed model is that the increased susceptibility to oxidative stress-induced death in the hemocyte DDR gene knockdowns would be suppressed (perhaps partially) by simultaneous knockdown of upd3 and/or JNK. These types of epistasis experiments would strengthen the model and the paper.

3, The (potential) connections between DDR/JNK/UPD3 and the oxidative stress effects on depletion of nutrient (lipids and glycogen) stores was also not fully developed. However, it may be the case that, in this paper, the authors just want to speculate that the effects of hemocyte DDR/upd3 manipulation on viability upon oxidative stress involve changes in nutrient stores.

Reviewer #2 (Public Review):

Hersperger et al. investigated the importance of Drosophila immune cells, called hemocytes, in the response to oxidative stress in adult flies. They found that hemocytes are essential in this response, and using state-of-the-art single-cell transcriptomics, they identified expression changes at the level of individual hemocytes. This allowed them to cluster hemocytes into subgroups with different responses, which certainly represents very valuable work. One of the clusters appears to respond directly to oxidative stress and shows a very specific expression response that could be related to the observed systemic metabolic changes and energy mobilization. However, the association of these transcriptional changes in hemocytes with metabolic changes is not well established in this work. Using hemocyte-specific genetic manipulation, the authors convincingly show that the DNA damage response in hemocytes regulates JNK activity and subsequent expression of the JAK/STAT ligand Upd3. Silencing of the DNA damage response or excessive activation of JNK and Upd3 leads to increased susceptibility to oxidative stress. This nicely demonstrates the importance of tight control of JNK-Upd3 signaling in hemocytes during oxidative stress. However, it would have been nice to show here a link to systemic metabolic changes, as the authors conclude that it is tissue wasting caused by excessive Upd3 activation that leads to increased susceptibility, but metabolic changes were not analyzed in the manipulated flies. The overall conclusion of this work, as presented by the authors, is that Upd3 expression in hemocytes under oxidative stress leads to tissue wasting, whereas in fact it has been shown that excessive hemocyte-specific Upd3 activation leads to increased susceptibility to oxidative stress (whether due to increased tissue wasting remains a question). The DNA damage response ensures tight control of JNK-Upd3, which is important. However, what role naturally occurring Upd3 expression plays in a single hemocyte cluster during oxidative stress has not been tested. What if the energy mobilization induced by this naturally occurring Upd3 expression during oxidative stress is actually beneficial, as the authors themselves state in the abstract - for potential tissue repair? It would have been useful to clarify in the manuscript that the observed pathological effects are due to overactivation of Upd3 (an important finding), but this does not necessarily mean that the observed expression of Upd3 in one cluster of hemocytes causes the pathology.

Reviewer #3 (Public Review):

In this study, Kierdorf and colleagues investigated the function of hemocytes in oxidative stress response and found that non-canonical DNA damage response (DDR) is critical for controlling JNK activity and the expression of cytokine unpaired3. Hemocyte-mediated expression of upd3 and JNK determines the susceptibility to oxidative stress and systemic energy metabolism required for animal survival, suggesting a new role for hemocytes in the direct mediation of stress response and animal survival.

Strength of the study:<br /> 1. This study demonstrates the role of hemocytes in oxidative stress response in adults and provides novel insights into hemocytes in systemic stress response and animal homeostasis.<br /> 2. The single-cell transcriptome profiling of adult hemocytes during paraquat treatment, compared to controls, would be of broad interest to scientists in the field.

Weakness of the study:<br /> 1. The authors claim that the non-canonical DNA damage response mechanism in hemocytes controls the susceptibility of animals through JNK and upd3 expression. However, the link between DDR-JNK/upd3 in oxidative stress response is incomplete and some of the descriptions do not match their data.<br /> 2. The schematic diagram does not accurately represent the authors' findings and requires further modifications.

Reviewer #1 (Public Review):

In this study, Muronova et al., demonstrate the physiological importance of a centriole and microtubule-associated protein, CCDC146, in sperm flagellar formation and male reproduction. In a previous study, the authors identified two loss-of-function mutations in CCDC146 from the sterile males with multiple morphological abnormalities in flagellar (MMAF) phenotype. To further test physiological significance of the CCDC146, the authors generate its knockout mouse model. The knockout males share the MMAF phenotypes with severely impaired flagellar morphology due to the defective sperm generation in testes. Using CCDC146 knock-in mouse model and expansion microscopy techniques, the authors observed CCDC146 localizes at human and mouse sperm flagella, which is different from the somatic cells. The authors also observed impaired manchette and head-tail coupling apparatus in developing spermatid lacking CCDC146 and address CCDC146 loss-of-function induces molecular and structural defects at axoneme in developing male germ cells, which finally causes MMAF phenotype and male infertility.

This reviewer agrees that identifying and analyzing new pathogenic molecules and variants is hugely valuable to establish male infertility in genetic level. As the authors have done, this study also enlarges the genetic causality underlying MMAF and male infertility. In addition, this study applies new techniques, expansion microscopy, which is also an innovative approach. Although many approaches are used, unfortunately, this study misses the molecular mechanisms to explain pathogenicity to cause MMAF by the CCDC146. Only intracellular localization of the molecule is heavily examined. Although the authors show defective intracellular localization of the centriole and manchette, how CCDC146 loss-of-function and the developmental defects are linked is not examined. These limits provide the impression that this study could be simply another identification of the MMAF-causing gene, which were heavily performed by the authors. Also, in many parts, the results do not clearly support the authors claim. Therefore, this reviewer thinks the current manuscript requires additional results to clearly explain molecular mechanisms underlying the pathogenicity by CCDC146 loss-of-function.

Reviewer #2 (Public Review):

In whole exome sequencing of two patients suffering from MMAF syndrome, mutations of CCDC146 gene that result in premature stop codons were identified. The position of mutations could result in a truncated form of protein, thus whether these patients do indeed lack CCDC146 protein or if present, whether the truncated protein is functional, is unanswered by showing the CCDC146 protein localization only in the sperm from healthy donors. The main claim that CCDC146 protein is microtubule associated protein in the axoneme is well supported imaging expanded sperm flagellum to increase spatial resolution. However, the author's claim that the signal in the mid-piece is not specific is less supported by experimental evidence. The detection of CCDC146 in the sperm head is not further explored while TEM images show spermatogenesis defects in the manchette and acrosome formation. Increased detection of the CCDC146 protein in mouse sperm with sarkosyl supports its association with microtubules but does not exclude its potential role in the formation of sperm head. Overall, this study provides valuable information on CCDC146 function in male germ cells during spermatogenesis.

Reviewer #3 (Public Review):

Male infertility is an important health problem. Among pathologies with multiple morphological abnormalities of the flagellum (MMAF), only 50% of the patients have no identified genetic causes. It is thus primordial to find novel genes that cause the MMAF syndrome. In the current work, the authors follow up the identification of two patients with MMAF carrying a mutation in the CCDC146 gene. To understand how mutations in CCDC146 lead to male infertility, the authors generated two mouse models: a CCDC146-knockout mouse, and a knockin mouse in which the CCDC146 locus is tagged with an HA tag. Male CCDC146-knockout mice are infertile, which proves the causative role of this gene in the observed MMAF cases. Strikingly, animals develop no other obvious pathologies, thus underpinning the specific role of CCDC146 in male fertility.

The authors have carefully characterised the subcellular roles of CCDC146 by using a combination of expansion and electron microscopy. They demonstrate that all microtubule-based organelles, such as the sperm manchette, the centrioles, as well as the sperm axonemes are defective when CCDC146 is absent. Their data show that CCDC146 is a microtubule-associated protein, and indicate, but do not prove beyond any doubt, that it could be a microtubule-inner protein (MIP).<br /> This is a solid work that defines CCDC146 as a novel cause of male infertility. The authors have performed comprehensive phenotypic analysis to define the defects in CCDC146 knockout mice. Surprisingly, the authors provide virtually no information on the penetrance of those defects - in most cases they simply show descriptive micrographs. The message of this manuscript would have been more convincing if the key phenotypes of the CCDC146 knockout mice were quantified, in particular those shown in Fig. 2E, 7A, 11B, 13.

The manuscript text is well written and easy to follow also for non-specialists. The introduction and discussion chapters contain important background information that allow putting the current work into the greater context of fertility research. The figures could have been designed more carefully, with additional information on the genotype and other details such as the antibodies used etc. directly added to the figure panels, which would improve their readability. The author might also consider pooling small figures with complementary content into one bigger figure in order to group related information together, and again facilitate the reading of the manuscript.

Overall, this manuscript provides convincing evidence for CCDC146 being essential for male fertility, and illustrates this with a large panel of phenotypic observations, which however mostly lack quantification in order to judge their penetrance. Together, the work provides important first insights into the role of a so-far unexplored proteins, CCDC146, in spermatogenesis, thereby broadening the spectrum of genes involved in male infertility.

Reviewer #1 (Public Review):

This research article by Watabe T and colleagues characterizes PKA waves triggered by prostaglandin E2 (PGE2). What the author discovered is that waves of PKA occur both in vitro, in MDCK epithelial monolayers, and in vivo, in the ear epidermis in mice. The PKA waves are the consequence PGE2 discharge, that in turn is triggered by Calcium bursts. Calcium level and ERK activity intensity control that mechanism by acting at different levels.

This article is a technological tour de force using different biosensors and optogenetic actuators. What makes this article interesting is the combination of these tools together to dissect a complex, highly dynamic signaling pathway at the single-cell level. For this reason, this paper represents the essence of modern cell biology and paves the way for the cell biology of the future. However, we think that the paper in this stage is still partly descriptive in its nature, and more measurements are needed to increase the strength of the mechanistic insights. Also, the work is not conclusive, some results are over-interpreted, and more work has to be done if the authors want to support all their claims.

Reviewer #2 (Public Review):

This study visualizes a specific localized form of cell-to-cell communication and conveys very well with what dynamics and sensitivity this biological phenomenon occurs.

Using a FRET-based PKA biosensor, the authors observed that radial localized kinase activity changes spontaneously occur in adjacent cells of certain cell density. This phenomenon of radial propagation of PKA activity changes in groups of cells was further mechanistically elucidated and characterized. Interestingly, the authors found that individual cells in the cell groups form spontaneous Ca2+ transients, which at a certain strength can trigger the biosynthesis and release of prostaglandin E2 (PGE2). PGE2 then acts on the neighboring cells and triggers the increase of cAMP levels and the associated activation of the PKA via G-protein-coupled receptors (EP2 and EP4). In systematic, well-structured experiments, it was then found that the frequency of occurrence of such radial activations depends not only on the cell density but also on the activation state of the ERK MAP kinase pathway. The authors skillfully used various modern genetically encoded biosensors and other tools such as optogenetic tools to visualize and characterize an interesting biological phenomenon of cell-to-cell communication. The insights gained with these investigations produce a better understanding of the dynamics, sensitivity, and spatial extent with which such communications can occur in a cell network. It is also worth noting that the authors have not limited the studies to 2D cell culture in vitro, but were also able to confirm the findings in an animal model.

Reviewer #3 (Public Review):

The manuscript entitled "Calcium transients trigger switch-like discharge of prostaglandin E2 (PGE2) in an ERK-dependent manner" by Watabe et al. investigates the interaction between PGE2, PKA, calcium and ERK signaling in MCDK cells and in mouse epidermis. By expressing PKA, calcium and ERK activity reporters, the authors conclude that calcium transients trigger release of PGE2 that signals through GPCR receptors EP2 and EP4 to recruit PKA in neighboring cells. Determining the dynamics of signaling molecules and their interrelationships is important to fully identify the spatiotemporal aspects of signaling mechanisms. This study addresses some aspects of the calcium-PGE2-GPCR-Erk-PKA signaling pathway in a cell line and in mouse skin ex vivo.

However, the sequential recruitment of the different signaling molecules has been described in previous studies. Hence, the novelty of the findings is limited.<br /> Additionally, the interpretation of some of the data is too speculative, more likely explanations are not considered, or not well supported by the data presented. The main conclusions the authors present are potentially artifactual (ie, cell density-dependent phenomena) and the authors need to either do further experiments to better support their conclusions or re-interpret the physiological significance of their findings.

Reviewer #1 (Public Review):

The manuscript by Huang et al. examines the potential "self-policing" of Bacillus cells within a biofilm. The authors first discover the co-regulation of lethal extracellular toxins (BAs) and the self-immunity mechanisms; the global regulator spoA controls both. The authors further show that a subpopulation of cells co-express these genes and speculate that these cells engage in preferential cooperation for biofilm formation (over cells that produce neither). Based on previous literature, the authors then evaluate the relative fitness of the wild-type strain compared to mutants locked into either constantly exporting the toxins or permanently immune to these poisons. The wild-type exhibited increased fitness (compared to the mutants) for the tested biofilm conditions. The manuscript raises interesting ideas and provides a potential model to probe questions of cooperatively in Bacillus biofilms.

Strengths:<br /> - The authors use fluorescence-producing reporter strains to discern the spatial expression patterns within biofilms. This real-time imaging provides striking confirmation of their conclusions about shared co-regulation.<br /> - The authors also nicely deploy genetic constructs in microbiological assays to show how toxin production and immunity can influence biofilm phenotypes, including resilience to stress.

Concerns:<br /> - My biggest concern is that the claim of policing on a single-cell level needs more quantitive microscopy, particularly of the xylose-induced strain. The data support a more tempered consideration of self-policing via BAs and self-resistance in this Bacillus species. It seems sufficient that this manuscript opens the door for a novel and readily examinable system for examining potential cooperation and its molecular controls (without making broader claims).<br /> - The discussion is more speculative than the presented data warrants. For example, the speculation in lines 289 - 310 is not anchored in the results. It is hard for this reviewer to imagine how one would use the genetic framework and tools developed in this manuscript to address the ideas proposed in lines 289 - 310.<br /> - Some conclusions (in the results section) are more decisive than the data supports. For example, the microscopy of the PI staining (as presented in Figure 2 and the supplemental movies) does not prove that only non-expressing cells die. Yet the conclusion in line 143 states that "ECM and BAs producers selectively punish the nonproducing siblings." Also, the presented data shows many non-labeled cells without PI; why do some nearby non-gfp-expressing cells remain alive?

Reviewer #2 (Public Review):

In this study, Huang et al. investigated Bacillus velezensis, a species that colonizes plant roots as part of the rhizosphere. They showed that clone of B. velezensis SQR9 retains a division of labor of motile, planktonic subpopulation that do not produce extracellular matrix (ECM) and biofilm-forming sessile subpopulation that do produce ECM. Specifically, the sessile subpopulation secret toxins named bacillunoic acids (BAs) to kill some, but not all, of the planktonic subpopulation. The killing mechanism is mediated by a global regulator Spo0A, which co-activates BAs production and immunity, as well as ECM production. A strain that has a disrupted policing system revealed reduced biofilm formation, lower resistance to environmental stresses and alleviated ability to colonize plant roots. Overall, the toxin-mediated policing system is important for B. velezensis to mediate division of labor for enhancing population stability and ecological fitness when required (e.g., cell transition from a planktonic style to a multicellular style).

Reviewer #3 (Public Review):

In this manuscript, Huang et al. use a variety of experimental approaches to investigate division of labor and cheater "policing" during biofilm formation in Bacillus velezensis SQR9. The authors show that SQR9 cells differentiate into two populations during biofilm development - one cell type produces extracellular matrix (ECM) and the other does not (referred to as "cheaters"). The authors go on to demonstrate that the ECM producing cells utilize a bacillunoic acid toxin system to selectively kill cheaters, keeping the cheater population in check which maintains the stability of the community. Further, the authors demonstrate that coordination of ECM production and synthesis of bacillunoic acid/immunity via acetyl-CoA carboxylase is mediated in part by Spo0A. I find the work as a whole to be compelling and thorough, and I expect it to be of broad interest to various fields of research.

Reviewer #1 (Public Review):

The authors show that TrafE, which is one of the five Dictyostelium discoideum TRAF proteins, is recruited to the Mycobacterium-containing vacuoles (MCVs) and is required for membrane damage repair and xenophagy. They propose that the TrafE-Ub-ALIX axis is important for the regulation of Vps4, and, thereby, for the normal function of ESCRT. They also suggest that TrafE is involved in phagophore sealing.

Overall, the parts of membrane damage repair and xenophagy induction are convincing. Although mammalian TRAF6 was already reported to be involved in the ubiquitination of Chlamydia and Toxoplasma-containing vacuoles (Haldar et al. PNAS, 2015, https://www.pnas.org/doi/epdf/10.1073/pnas.1515966112), how TRAF6 is recruited to pathogen-containing vacuoles remained unknown. This study reveals that the recruitment of TrafE to MCVs is dependent on membrane damage or reduced membrane tension. This is novel. However, the part of phagophore closure is too preliminary. The evidence that TrafE is involved in the phagophore closure is mostly indirect and weak.

Reviewer #2 (Public Review):

Raykov et al. reported that TrafE, a member of the E3 ubiquitin ligase family similar to the TRAF proteins in mammalian cells, is essential for Dictyostelium discoideum to effectively respond to endolysosomal damage and defend itself against Mycobacterium marinum infection. First, the authors demonstrate that TrafE is recruited to the site of Mycobacterium-Containing Vacuole (MCV) damage along with ubiquitin molecules. This recruitment is necessary for the effective suppression of M. marinum growth in the cells. They also found that this response was not limited to the damage caused by M. marinum, but was also triggered by sterile damage caused by chemical compounds. Furthermore, the authors revealed that TrafE plays a role in the recruitment of Vps4 to sites of membrane damage and regulates the disassembly of ESCRT subunits. While TRAF6 has been previously implicated in ubiquitination in response to invaded bacteria in mammalian cells, this study provides solid data that furthers our understanding of the mechanism behind xenophagy. The authors conducted a thorough analysis to contribute to this field of research.

Reviewer #1 (Public Review):

This manuscript reports on a rapid and precise CRISPR/Cas9-mediated knock-in approach in the African turquoise killifish, an emerging vertebrate animal and gerontology model. More specifically, it describes an easily adoptable method to efficiently insert fluorescent reporters of different sizes at various genomic loci and to drive cell-type- and tissue-specific expression. This methodology will allow the development of humanized disease models and of cell-type specific molecular probes to study complex vertebrate biology, including aging biology, in the killifish. While this knock-in methodology is already widely used in common vertebrate animal models, the efficient generation of stable lines with germline transmission has been missing in killifish. As killifish have the shortest generation time of vertebrate animal models in laboratory conditions, show a rapid sexual maturity, and a short lifespan, the established method enables the generation of stable lines of homozygous transgenic vertebrate animals in 2-3 months. Overall, we believe this first report on efficient long (1.8kb) construct knock-in using CRISPR/Cas9 in the killifish establishes the killifish as a system for precise genetic engineering at scale, which has been challenging so far in vertebrates.

The establishment of this methodology will have a major impact in the field and be of extreme use within the scientific community. It will allow the development of scalable human disease models and integrate both genetics and age as risk factors, thus having the potential to identify future therapeutic targets for age-related diseases. It also has a generic character as the generated protocol can serve as a template for knock-in approaches in other emerging model organisms.

Although the reported data are of major interest and relevance to the scientific field, they are, as yet not sufficiently shown in convincing figures. The methodology is state-of-the-art and entails an extensive set of molecular, biochemical, and morphological/imaging technologies. While most of the data are nicely presented and accompanied by illustrative figures, the manuscript would benefit from the inclusion of a more detailed material and methods section, and a little more elaboration on morphometrical expression data in the results section, e.g, expression shown for all the studied genes in the larval fish, and a more critical discussion, that also highlights a few of the limitations, e.g., those related to the fast generation of homozygous F1 fish.

Reviewer #2 (Public Review):

The manuscript "Rapid and precise genome engineering in a naturally short-lived vertebrate" describes the development of a CRISPR- based knock-in technology in Nothobranchius furzeri, or the African turquoise killifish, an innovative model species for studying aging and age-related disorders. While Tol2 systems had been demonstrated to be successful in generating reporter killifish lines, endogenous reporters via knock-in had not been reported so far. The major strength of the paper is that the authors show that they have been successful in developing 5 different knock-in fish lines with large inserts (up to 1.8kb) with high efficiency. They have inserted single or dual fluorescent reporters and demonstrated expression in line with the expected pattern. This is a breakthrough in the field and this method can be instrumental for many researchers working with unusual model species, and in particular, will expand the killifish community toolbox.

While this is very promising, the paper would benefit from a more rigorous validation of the KI lines that were generated. The authors did not show a co-localisation of the target gene expression with the reporter to prove bona fide reporting. In addition, it was not clear whether the KI affects the endogenous expression level of the target genes. The targeting efficiency of the method is high, but the quantifications are based on rather limited numbers of animals, which might not yet be very robust. A larger number of animals would have strengthened the efficiency conclusion.

The figures of the manuscript are well designed and support the conclusions, but several contain information that is not discussed in the main text, such as (un)expected bands on gels, reporter staining in WT animals, and unusual staining patterns. The body text seems to ignore these and only discusses findings that are in line with the story. A key point to the efficiency of the method seems to be a chemical modification of the repair template, which was not disclosed in the method section which at the moment hampers replication.

Finally, the discussion is brief and does not benchmark the method to other CRISPR-based KI methods in Xenopus or more typical model species such as mouse.

In conclusion, this paper describes a breakthrough method for a rising animal model that would benefit from a more thorough validation. Full disclosure of the methodology will boost the generation of genetically edited killifish lines and aid in the establishment of this promising animal model.

Reviewer #3 (Public Review):

In this manuscript, the authors detail an exciting protocol to knock-in c-terminal tags at the endogenous locus of a gene of interest in the short-lived vertebrate, the African Turquoise Killifish. The technique is clearly explained and will be a significant advancement for the field. The method relies on the injection of a cocktail containing cas9 protein, proprietary-modified gRNA and dsDNA ordered from IDT, and a chemical enhancer of HDR.

I believe the authors demonstrate that killifish is a tractable emerging system to integrate stable fluorescent tags at desired loci. The method should be easy to reproduce since the components are all commercially available, but the proprietary nature of the modifications could make it a pricey technique for smaller labs. A protospacer adjacent motif (PAM) sequence near the desired insertion site is still a restriction using this method. That being said, I think this represents a significant advancement in knock-in methods that could be adopted in other systems. This manuscript is rather simple and straightforward, and I do not have additional criticisms or critiques.

As a side note, I think the brain sections look very professional, but since I am not a neurobiologist I will defer to the other reviewers about the accuracy and claims about the regions labeled.

For additional context, I suggest reading Wierson et al. 2020 and Seleit et al. 2022, which can both be found in the reference section.

Reviewer #1 (Public Review):

The work in this study builds on previous studies by some of the same authors and aims to test whether the heartbeat evoked response was modulated by the local/global auditory regularities and whether this differed in post-comatose patients with different contagiousness diagnosis. The authors report that during the global effect there were differences between the MCS and UWS patients.

The study is well constructed and analysed and has data from 148 participants (although the maximum in anyone group was 59). The reporting of the results is excellent and the conclusions are supported by the results presented. This study and the results presented are discussed as evidence that EEG based techniques maybe a low cost diagnostic tool for consciousness in post-comatose patients, although it should be stressed that here no classification of diagnostics was performed on the EEG data.

One potential weakness was the relationship between the design of the experiment and the analysis pathway for the results. If I have understood correctly the experimental design the auditory regularity changed on whether the local/global regularity was standard/deviant. In the analysis the differences between all conditions in which the local or global regularity were compared between the standard and deviant trials. This difference was then compared between MCS and UWS patient groups. For these analyses the results for the health and emerging MCS were not included. If this is correct it would be interesting to understand the motivation for this. Relatedly, it would be good to clarify if the effects reported were corrected for the multiple planned contrasts and if not why they should not be corrected.

Reviewer #2 (Public Review):

The goal of this study was to determine whether heartbeat-evoked responses measured at the scalp level with EEG, which followed regularity violations, could potencial help inform the diagnosis of patients with altered states of consciousness.

The authors use high density EEG and an oddball paradigm that probes violations of both local and global regularities. Four groups were considered including unresponsive wakefulness syndrome patents, minimally consciousness patients, emerging minimally consciousness patients and healthy controls. A difference was found between unresponsive and minimally conscious patients in the amplitude of the heartbeat evoked responses measure with EEG following a sound that violated a global regularity. Similarly, differences were found between the variance of these responses between the two above mentioned groups (N=58 and N=59), but no differences were found in relation to the healthy control group, which appear to be "in between" the two other groups (at least for global effect of HER). I thought this was a little counterintuitive and raises some questions about what this neural signature can tell us about the state of consciousness. Having said that, the healthy control sample was very small, more than 5 times smaller (only N=11).

In general, I thought the Discussion section was a little light on the implications of the findings, what they tell us about the brain mechanisms of consciousness and their different levels/states. A question is raised about whether it is necessary to lock EEG to heartbeats to find differences between patients. The data appeared to say that this is not the case but the discussion does not appear to reflect that very clearly.

Reviewer #3 (Public Review):

I found the results very interesting but wondered why the ERP results for the global vs. local effects are not reported. This analysis is mentioned in the methods section, but I do not find it in the results. Is this what is shown in the mid row in panel D? If yes, it should be made clearer. Is there a significant local and global deviant response in each patient group?

Additionally, eyeballing Figure 1, there are a few potential issues that may be affecting the conclusion re HER:

(1) Panel D top: it seems that the orange trace (MCS) is largely the same in both the "Local" and "global" condition. But the blue trace (UWS) shows a larger negative going deflection in the "global" case. Put differently, the UWS, but not MCS patients appear to generate a different response to the Global effect relative to the local effect. Is this the case?<br /> (2) There are some MCS subjects that appear to show a global effect that is larger than that observed in EMCS and healthy controls. How do you interpret these data?<br /> (3) How do you interpret the negative average HER data shown by many UWS patients?

Reviewer #1 (Public Review):

Kazrin appears to be implicated in many diverse cellular functions, and accordingly, localizes to many subcellular sites. Exactly what it does is unclear. The authors perform a fairly detailed analysis of Kazrin in-cell function, and find that it is important for the perinuclear localization of TfN, and that it binds to members of the AP-1 complex (e.g., gamma-adaptin). The authors note that the C-terminus of Kazrin (which is predicted to be intrinsically disordered) forms punctate structures in the cytoplasm that colocalize with components of the endosomal machinery. Finally, the authors employ co-immunoprecipitation assays to show that both N and C-termini of Kazrin interacts with dynactin, and the dynein light-intermediate chain.

Much of the data presented in the manuscript are of fairly high quality and describe a potentially novel function for Kazrin C. However, I had a few issues with some of the language used throughout, the manner of data presentation, and some of their interpretations. Most notably, I think in its current form, the manuscript does not strongly support the authors' main conclusion: that Kazrin is a dynein-dynactin adaptor, as stated in their title. Without more direct support for this function, the authors need to soften their language. Specific points are listed below.

Major comments:<br /> 1) I agree with the authors that the data provided in the manuscript suggest that Kazrin may indeed be an endosomal adaptor for dynein-dynactin. However, without more direct evidence to support this notion, the authors need to soften their language stating as much. For example, the title as stated would need to be changed, as would much of the language in the first paragraph of the discussion. Alternatively, the manuscript could be significantly strengthened if the authors performed a more direct assay to test this idea. For example, the authors could use methods employed previously (e.g., McKenney et al., Science 2014) to this end. In brief, the authors can simply use their recombinant Kazrin C (with a GFP) to pull out dynein-dynactin from cell extracts and perform single molecule assays as previously described.<br /> 2) I'm not sure I agree with the use of the term 'condensates' used throughout the manuscript to describe the cytoplasmic Kazrin foci. 'Condensates' is a very specific term that is used to describe membraneless organelles. Given the presumed association of Kazrin with membrane-bound compartments, I think it's more reasonable to assume these foci are quite distinct from condensates.<br /> 3) The authors note the localization of Tfn as perinuclear. Although I agree the localization pattern in the kazKO cells is indeed distinct, it does not appear perinuclear to me. It might be useful to stain for a centrosomal marker (such as pericentrin, used in Figure 5B) to assess Tfn/EEA1 with respect to MT minus ends.<br /> 4) "Treatment with the microtubule depolymerizing drug nocodazole disrupted the perinuclear localization of GFP-kazrin C, as well as the concomitant perinuclear accumulation of EE (Fig. 5C & D), indicating that EEs and GFP-kazrin C localization at the pericentrosomal region required minus end-directed microtubule-dependent transport, mostly affected by the dynactin/dynein complex (Flores-Rodriguez et al., 2011)."<br /> - I don't agree that the nocodazole experiment indicates that minus end-directed motility is required for this perinuclear localization. In the absence of other experiments, it simply indicates that microtubules are required. It might, however, "suggest" the involvement of dynein. The same is true for the subsequent sentence ("Our observations indicated that kazrin C can be transported in and out of the pericentriolar region along microtubule tracks...").<br /> 5) Although I see a few examples of directed motion of Tfn foci in the supplemental movies, it would be more useful to see the kymographs used for quantitation (and noted by the authors on line 272). Also related to this analysis, by "centripetal trajectories", I assume the authors are referring to those moving in a retrograde manner. If so, it would be more consistent with common vernacular (and thus more clear to readers) to use 'retrograde' transport.<br /> 6) The error bars on most of the plots appear to be extremely small, especially in light of the accompanying data used for quantitation. The authors state that they used SEM instead of SD, but their reasoning is not stated. All the former does is lead to an artificial reduction in the real deviation (by dividing SD by the square root of whatever they define as 'n', which isn't clear to me) of the data which I find to be misleading and very non-representative of biological data. For example, the error bars for cell migration speed in Figure 2B suggest that the speeds for WT cells ranged from ~1.7-1.9 µm/sec, which I'm assuming is largely underrepresenting the range of values. Although I'm not a statistician, as someone that studies biochemical and biological processes, I strongly urge the authors to use plots and error bars that more accurately describe the data to your readers (e.g., scatter plots with standard deviation are the most transparent way to display data).

Reviewer #2 (Public Review):

A distinguishing feature of live cells is that intracellular organelles move powered by molecular motors. However, the arsenal of molecular motors is limited relative to the vast variety of cargoes and processes involving long-distance movement. Cells cope with this mismatch by using adaptors that "bridge" a given molecular motor with a specific cargo, whose identity is dictated by peripheral membrane proteins, such RABs, or identity-determining lipids, such as PtdIns3P. Cytoplasmic dynein walks towards the minus end of the microtubules. A score of cellular processes is dependent on dynein, such that deficient regulation of the motor has deep consequences in cellular homeostasis, and the identification of new adapters is of broad interest, both basic and, potentially, clinical.

Dynein adaptors usually stabilize the binding of dynactin to dynein using coiled-coil regions to longitudinally embrace dynactin, holding it to the elongated dynein cap of the super-complex. Not only do they adapt cargo but additionally increase the processivity and speed of the motor. In this manuscript, Julie and collaborators present evidence that a protein denoted kazrin, which is involved in a variety of processes, is actually an adaptor connecting endosome domains specialized in recycling cargo back to the surface of the cell by way of the RAB11 perinuclear recycling endosome. The topic is important, experiments have been carefully conducted and well controlled and display items faithfully guide readers through the main findings. However, I feel that the evidence that kazrin is a dynein adaptor is somewhat thin and that it could be improved with relatively little additional work. The manuscript would also benefit from better integration of the conclusions in the current state of the art in the dynein field.

Reviewer #3 (Public Review):

The authors sought to define a role for the Kazrin protein in the endosomal pathway. This effort is built on past observations of the impact of Kazrin over-expression on clathrin-mediated endocytosis. However, new Kazrin depletion experiments revealed no impact on endocytosis but a defect in the movement of early endosomes towards the nucleus. This observation that Kazrin depletion results in the dispersion of early endosomes is supported by shRNA knockdowns, CRISPR KO experiments, and the rescue of the phenotype by restoring Kazrin expression. The generalizability of the findings is supported by experiments in 2 different cell types (COS7 and MEFs). A direct role for Kazrin in linking early endosomes to dynein-dynactin is supported by observations that Kazrin is early present on endosomes and interacts with proteins of endosomes as well as with dynein-dynactin. A possible interaction with PI3P (a lipid enriched on early endosomes) is supported by a lipid binding assay. However, definitive results on this interaction would require validation by additional methods. With respect to the dynein-dynactin interactions, the authors strengthen confidence in this interaction and its putative functional relevance by identifying sequence homology between Kazrin and BICDR1 and hook3, 2 proteins with well-characterized functionally relevant roles in linking dynein-dynactin to cargos. The methods that were used to establish these functions for Kazrin were well aligned with the goals of this research and with the conclusions that were drawn. Efforts were made to quantify key observations and to provide statistical tests to establish the significance of differences that were observed. While these quantitative efforts are generally sufficient to support the major claims of the study, the data presentation would be stronger if the authors could better define the experimental sample size and the number of replicates that were performed for each experiment. Furthermore, the idea that the C-terminal region of Kazrin helps to promote the formation of "condensates" was not thoroughly supported by experimental data even if the presence of an intrinsically disordered region is supportive of this interpretation of the formation of Kazrin puncta on or near endosomes.

Reviewer #1 (Public Review):

Nicotine preference is highly variable between individuals. The paper by Mondoloni et al. provided some insight into the potential link between IPN nAchR heterogeneity with male nicotine preference behavior. They scored mice using the amount of nicotine consumption, as well as the rats' preference of the drug using a two-bottle choice experiment. An interesting heterogeneity in nicotine-drinking profiles was observed in adult male mice, with about half of the mice ceasing nicotine consumption at high concentrations. They observed a negative association of nicotine intake with nicotine-evoked currents in the antiparticle nucleus (IPN). They also identified beta4-containing nicotine acetylcholine receptors, which exhibit an association with nicotine aversion. The behavioral differentiation of av vs. n-avs and identification of IPN variability, both in behavioral and electrophysiological aspects, add an important candidate for analyzing individual behavior in addiction.

The native existence of beta4-nAchR heterogeneity is an important premise that supports the molecules to be the candidate substrate of variabilities. However, only knockout and re-expression models were used, which is insufficient to mimic the physiological state that leads to variability in nicotine preference.

Reviewer #2 (Public Review):

In the current study, Mondoloni and colleagues investigate the neural correlates contributing to nicotine aversion and its alteration following chronic nicotine exposure. The question asked is important to the field of individual vulnerability to drug addiction and has translational significance. First, the authors identify individual nicotine consumption profiles across isogenic mice. Further, they employed in vivo and ex vivo physiological approaches to defining how antiparticle nuclei (IPn) neuronal response to nicotine is associated with nicotine avoidance. Additionally, the authors determine that chronic nicotine exposure impairs IPn neuronal normal response to nicotine, thus contributing to higher amounts of nicotine consumption. Finally, they used transgenic and viral-mediated gene expression approaches to establish a causal link between b4 nicotine receptor function and nicotine avoidance processes.

The manuscript and experimental strategy are well designed and executed; the current dataset requires supplemental analyses and details to exclude possible alternatives. Overall, the results are exciting and provide helpful information to the field of drug addiction research, individual vulnerability to drug addiction, and neuronal physiology. Below are some comments aiming to help the authors improve this interesting study.

1. The authors used a two-bottle choice behavioral paradigm to investigate the neurophysiological substrate contributing to nicotine avoidance behaviors. While the data set supporting the author's interpretation is compelling and the experiments are well-conducted, a few supplemental control analyses will strengthen the current manuscript.<br /> a. The bitter taste of nicotine might generate confounds in the data interpretation: are the mice avoiding the bitterness or the nicotine-induced physiological effect? To address this question, the authors mixed nicotine with saccharine, thus covering the bitterness of nicotine. Additionally, the authors show that all the mice exposed to quinine avoid it, and in comparison, the N-Av don't avoid the bitterness of the nicotine-saccharine solution. Yet it is unclear if Av and N-Av have different taste discrimination capacities and if such taste discrimination capacities drive the N-Av to consume less nicotine. Would Av and N-Av mice avoid quinine differently after the 20-day nicotine paradigm? Would the authors observe individual nicotine drinking behaviors if nicotine/quinine vs. quinine were offered to the mice?<br /> b. Metabolic variabilities amongst isogenic mice have been observed. Thus, while the mice consume different amounts of nicotine, changes in metabolic processes, thus blood nicotine concentrations, could explain differences in nicotine consumption and neurophysiology across individuals. The authors should control if the blood concentration of nicotine metabolites between N-Av and Av are similar when consuming identical amounts of nicotine (50ug/ml), different amounts (200ug/ml), and in response to an acute injection of a fixed nicotine quantity.

2. Av mice exposed to nicotine_200ug/ml display minimal nicotine_50ug/ml consumption, yet would Av mice restore a percent nicotine consumption >20 when exposed to a more extended session at 50ug/kg? Such a data set will help identify and isolate learned avoidance processes from dose-dependent avoidance behaviors.

3. The author should further investigate the basal properties of IPn neuron in vivo firing rate activity recorded and establish if their spontaneous activity determines their nicotine responses in vivo, such as firing rate, ISI, tonic, or phasic patterns. These analyses will provide helpful information to the neurophysiologist investigating the function of IPn neurons and will also inform how chronic nicotine exposure shapes the IPn neurophysiological properties.

Reviewer #3 (Public Review):

The manuscript by Mondoloni et al characterizes two-bottle choice oral nicotine consumption and associated neurobiological phenotypes in the antiparticle nucleus (IPN) using mice. The paper shows that mice exhibit differential oral nicotine consumption and correlate this difference with nicotine-evoked inward currents in neurons of the IPN. The beta4 nAChR subunit is likely involved in these responses. The paper suggests that prolonged exposure to nicotine results in reduced nAChR functional responses in IPN neurons. Many of these results or phenotypes are reversed or reduced in mice that are null for the beta4 subunit. These results are interesting and will add a contribution to the literature. However, there are several major concerns with the nicotine exposure model and a few other items that should be addressed.

Strengths:<br /> Technical approaches are well-done. Oral nicotine, electrophysiology, and viral re-expression methods were strong and executed well.<br /> The scholarship is strong and the paper is generally well-written. The figures are high-quality.

Weaknesses:<br /> Two bottle choice (2BC) model. 2BC does not examine nicotine reinforcement, which is best shown as a volitional preference for the drug over the vehicle. Mice in this 2BC assay (and all such assays) only ever show indifference to nicotine at best - not preference. This is seen in the maximal 50% preference for the nicotine-containing bottle. 2BC assays using tastants such as saccharin are confounded. Taste responses can very likely differ from primary reinforcement and can be related to peripheral biology in the mouth/tongue rather than in the brain reward pathway. Moreover, this assay does not test free choice, as nicotine is mixed with water which the mice require to survive. Since most concentrations of nicotine are aversive, this may create a generalized conditioned aversion to drinking water - detrimental to overall health and a confounding factor. What plasma concentrations of nicotine are achieved by 2BC? When nicotine is truly reinforcing, rodents and humans titrate their plasma concentrations up to 30-50 ng/mL. The Discussion states that oral self-administration in mice mimics administration in human smokers (lines 388-389). This is unjustified and should be removed. Similarly, the paragraph in lines 409-423 is quite speculative and difficult or impossible to test. This paragraph should be removed or substantially changed to avoid speculation. Overall, the 2BC model has substantial weaknesses, and/or it is limited in the conclusions it will support.

Statistical testing on subgroups. Mice are run through an assay and assigned to subgroups based on being classified as avoiders or non-avoiders. The authors then perform statistical testing to show differences between the avoiders and non-avoiders. It is circular to do so. When the authors divided the mice into avoiders and non-avoiders, this implies that the mice are different or from different distributions in terms of nicotine intake. Conducting a statistical test within the null hypothesis framework, however, implies that the null hypothesis is being tested. The null hypothesis, by definition, is that the groups do NOT differ. Obviously, the authors will find a difference between the groups in a statistical test when they pre-sorted the mice into two groups, to begin with. Comparing effect sizes or some other comparison that does not invoke the null hypothesis would be appropriate.

Decreased nicotine-evoked currents following passive exposure to nicotine in minipumps are inconsistent with published results showing that similar nicotine exposure enhances nAChR function via several measures (Arvin et al, J Neurosci, 2019). The paper does acknowledge this previous paper and suggests that the discrepancy is explained by the fact that they used a higher concentration of nicotine (30 uM) that was able to recruit the beta4-containing receptor (whereas Arvin et al used a caged nicotine that was unable to do so). This may be true, but the citation of 30 uM nicotine undercuts the argument a bit because 30 uM nicotine is unlikely to be achieved in the brain of a person using tobacco products; nicotine levels in smokers are 100-500 nM. It should be noted in the paper that it is unclear whether the down-regulated receptors would be active at concentrations of nicotine found in the brain of a smoker. The statement in lines 440-41 ("we show that concentrations of nicotine as low as 7.5 ug/kg can engage the IPN circuitry") is misleading, as the concentration in the water is not the same as the concentration in the CSF since the latter would be expected to build up over time. The paper did not provide measurements of nicotine in plasma or CSF, so concluding that the water concentration of nicotine is related to plasma concentrations of nicotine is only speculative.

The results in Figure 2E do not appear to be from a normal distribution. For example, results cluster at low (~100 pA) responses, and a fraction of larger responses drive the similarities or differences.

10 mg/kg/day in mice or rats is likely a non-physiological exposure to nicotine. Most rats take in 1.0 to 1.5 mg/kg over a 23-hour self-administration period (O'Dell, 2007). Mice achieve similar levels during SA (Fowler, Neuropharmacology 2011). Forced exposure to 10 mg/kg/day is therefore 5 to 10-fold higher than rodents would ever expose themselves to if given the choice. This should be acknowledged in a limitations section of the Discussion.

Are the in vivo recordings in IPN enriched or specific for cells that have a spontaneous firing at rest? If so, this may or may not be the same set/type of cells that are recorded in patch experiments. The results could be biased toward a subset of neurons with spontaneous firing. There are MANY different types of neurons in IPN that are largely intermingled (see Ables et al, 2017 PNAS), so this is a potential problem.

Related to the above issue, which of the many different IPN neuron types did the group re-express beta4? Could that be controlled or did beta4 get re-expressed in an unknown set of neurons in IPN? There is insufficient information given in the methods for verification of stereotaxic injections.

Data showing that alpha3 or beta4 disruption alters MHb/IPN nAChR function and nicotine 2BC intake is not novel. In fact, some of the same authors were involved in a paper in 2011 (Frahm et al., Neuron) showing that enhanced alpha3beta4 nAChR function was associated with reduced nicotine consumption. The present paper would therefore seem to somewhat contradict prior findings from members of the research group.

Sex differences. All studies were conducted in male mice, therefore nothing was reported regarding female nicotine intake or physiology responses. Nicotine-related biology often shows sex differences, and there should be a justification provided regarding the lack of data in females. A limitations section in the Discussion section is a good place for this.

Reviewer #1 (Public Review):

Cerebellar parallel fiber to Purkinje cell synapses display multiple forms of long-term plasticity, expressed in both presynaptic and postsynaptic compartments. At this synapse, a prominent form of presynaptic LTP was once thought to operate through cAMP-dependent activation of PKA, and subsequent phosphorylation of RIM1a. However, recent studies have questioned this hypothesis. LTP is not blocked by selective inhibitors of PKA, or by mutations in Rim1a designed to block PKA-dependent serine phosphorylation. In this study, Wang and colleagues use a wide array of pharmacology and genetics to elucidate a potential signaling cascade for presynaptic LTP in parallel fibers, where cAMP activates EPAC, leading to PKCε-dependent phosphorylation of RIM1α. Presynaptic ablation of either EPAC or PKCε leads to loss of presynaptic LTP and forskolin-induced potentiation. The experiments are generally well conceived and executed. The findings provide a new framework for understanding how presynaptic cAMP elevations can alter vesicle release machinery and drive synaptic plasticity, and open new avenues for exploration at synapses throughout the CNS. The manuscript could be improved by better a more transparent citation of previous studies and a more open discussion of the unknown steps in the newly-elucidated signaling cascade.

Reviewer #2 (Public Review):

The authors successfully show that how EPAC and PKCε work together to recruit presynaptic proteins for neurotransmitter release instead of synaptic vesicle formation since the absence of EPAC and PKCε does not affect the number of synaptic vesicles. In addition, the data clearly demonstrate that EPAC and PKCε function specifically at the presynaptic terminals and thus is required for induction of presynaptic LTP. Their suggested EPAC- PKCε module is also essential for proper cerebellar motor performance and motor learning.

Furthermore, the order of data analysis perfectly matches the logical explanation of the entire story. The authors first prove that EPAC and PKCε, together with RIM1a, are necessary for neurotransmitter release at the presynaptic terminal. Then, by using specific knockdown mice of presynaptic granule cells, both proteins contribute to the release of synaptic vesicles in that only the frequencies of EPSC have changed. In addition, presynaptic LTP is only induced with the presence of EPAC and PKCε, highlighting the important role of the EPAC- PKCε module. Ultimately, the impact of EPAC and PKCε is shown by conducting the behavior tasks including OKR, VOR, and VVOR.

The authors suggest the missing link between EPAC and RIM1 is PKCε. Phosphorylation of RIM1 by PKCε is a novel signaling cascade found in this paper. The authors' data from the heterologous expression system and cerebellar granule cell-specific PKCε KO mice indicate that PKCε can regulate RIM1Threonine phosphorylation.<br /> The EPAC-PKCε unit is essential to both presynaptic neurotransmitter release and presynaptic LTP in parallel fiber-Purkinje cell synapse. Future work is necessary to dissect which is responsible for cerebellar motor performance and motor learning.

The study provides the necessity of exploring the new part of the motor learning circuit since the significant focus of cerebellar motor learning has been only confined to postsynaptic plasticity. Generally, postsynaptic plasticity is affected by the presynaptic properties, such as presynaptic vesicle release and recycling of neurotransmitters at the synapse. Also, the presynaptic terminal, which can be referred to as an inducing force of the postsynaptic plasticity, does not merely release the neurotransmitters at a constant rate; they also change as a result of incoming stimuli. Such change is called presynaptic plasticity. Therefore, it should be further scrutinized how presynaptic plasticity is conducted and determined.

Reviewer #3 (Public Review):

The manuscript by Wang et al. investigates the mechanisms and physiological consequences of presynaptic plasticity at parallel fiber synapses of the cerebellum. Using a wide range of molecular, cellular, and genetic approaches, they show that a signaling pathway involving cAMP, EPAC, and PKCε leads to phosphorylation of RIM1α in parallel fiber terminals. Using EM and electrophysiology, they show that RIM1α (by forming a protein complex with Rab3A and Munc13) promotes docking of synaptic vesicles and increased vesicle release probability. The authors demonstrated that EPAC/PKCε are necessary for the induction of presynaptic LTP at parallel fiber synapses. The authors then extend this work to the behavioral level by showing the mice lacking EPAC or PKCε expression in cerebellar granule cells lack presynaptic LTP at parallel fiber synapses and display motor learning deficits during adaptation of the vestibular ocular reflex, a common test of cerebellum-dependent learning. The mechanisms of synaptic plasticity at parallel fiber synapses have been long investigated, but still remain unclear. This work makes a significant and convincing contribution to understanding presynaptic plasticity mechanisms. Likewise, the relative contribution of various pre- and postsynaptic forms of plasticity to cerebellar learning has long been debated but remains unsettled. This work provides novel evidence that presynaptic plasticity contributes to motor learning, possibly complimenting postsynaptic forms of plasticity. However, given the experimental conditions, it is difficult to extrapolate the slice electrophysiology findings to mechanisms of motor learning in vivo (see detailed comments below).

This manuscript provides compelling evidence for the role of EPAC and PKCε in regulating RIM1α and vesicle release. The authors use an impressive range of cellular, molecular, and genetic approaches to establish each link in the chain of the cAMP/EPAC/PKC signaling. In general, the conclusions are well supported by the data, often with multiple approaches used to address each question. In a few cases, the conclusions are overstated or not well supported by the data.

Specific comments:

1. While the data are generally very convincing, the authors overstated the conclusions in several instances. For example, the authors state that EPAC and PKCε are "required" or "essential" for vesicle docking and release. However, the author's own data show that both vesicle docking and release are clearly present (though reduced) in the absence of EPAC and PKCε, demonstrating they are not absolutely required. The language could be toned down without diminishing the impact of the excellent work.

2. The authors used analysis of cumulative EPSCs to estimate release probability (Pr) and the readily releasable pool (RRP) size. Unfortunately, this approach is likely not suited for low release probability synapses such as parallel fibers (the authors estimate Pr to be 0.04-0.06). Thanawala and Regehr (2016) extensively investigated the validity of cumulative EPSC analysis under a variety of conditions. They found that this analysis produces large errors in Pr and RRP at synapses with a Pr below ~0.2. In addition, 20 Hz EPSC stimulation (as was used here) produces much larger errors compared to the more commonly used 100 Hz stimulation. Between the low Pr at parallel fiber synapses and the low stimulus frequency used, it is likely that the cumulative EPSC analysis provides a poor estimate of Pr and RRP in this case.

3. Using a combination of genetic knockouts and pharmacology, this paper convincingly shows that presynaptic EPAC/PCKε are necessary for presynaptic LTP, but do not alter postsynaptic LTP/ LTD. However, given the experimental conditions in the slice experiments, it is difficult to extrapolate from the slice data to in vivo plasticity during motor learning. Synaptic plasticity in the cerebellar cortex is quite complex and can depend significantly on age, temperature, location, and ionic conditions. Unfortunately, these were not well matched between slice and in vivo experiments. Slice experiments used P21 mice, while in vivo experiments were performed at P60. Slice experiments were performed in the vermis, while VOR expression/adaptation generally requires the vestibulo-cerebellum/flocculus. Slice experiments were performed at room temperature, not physiological temperature. Lastly, slice experiments used 2 mM Ca2+ in the ACSF, somewhat high compared to the physiological extracellular fluid. Each of these factors can significantly affect the induction and expression of plasticity. These differences leave one wondering how well the slice data translate into understanding plasticity in the in vivo context.

4. Many experiments use synaptosomal preparation. The authors identify excitatory synapses by VGLUT labelling, but it is unclear how, or if, the authors distinguish between parallel fiber, climbing fiber, and mossy fiber synaptosomes. These synapses likely have very different properties and molecular composition, some quantification or estimation of how many synaptosomes are derived from each type of synapse would be helpful.

5. The math1-cre mouse line is used to selectively knockout EPAC or PKCε expression in cerebellar granule cells. This line also expresses Cre in unipolar brush cells (UBCs) of the cerebellum (Wang et al., 2021). This is likely not a factor in the molecular/slice studies of EPAC/PKC signaling, but UBC dysfunction could play a role in motor/learning deficits observed in vivo. This possibility is not considered in the text.

Joint Public Review:

This paper presents two new tools for investigating GLP-1 signaling. The genetically encoded sensor GLPLight1 follows the plan for other GPCR-based fluorescent sensors, inserting a circularly permuted GFP into an intracellular loop of the GPCR. The light-uncaged agonist peptide, photo-GLP1, has no detectable agonist activity (as judged by the GLPLight1 sensor) until it is activated by light. However, based on the current characterization, it is unclear how useful either of these tools will be for investigating native GLP-1 signaling.

The GLPLight1 sensor has a strong fluorescent response to GLP-1 with an EC50 of ~10 nM, and its specificity is high, as shown by lack of response to ligands of related class B GPCRs. However, the native GLP1R enables biological responses to concentrations that are ~1000-fold lower than this (as shown, for instance, in a supplemental figure of this paper). This makes it difficult to see how the sensor will be useful for in vivo detection of GLP-1 release, as claimed; although there may be biological situations where the concentration is adequate to stimulate the sensor, this is not established. Data using a GLP-1 secreting cell line suggest that the sensor has bound some of the released GLP-1, but it is difficult to have confidence without seeing an actual fluorescence response to stimulated release.

Alternatively, the sensor might be used for drug screening, but it is unclear that this would be an improvement over existing high-throughput methods using the cAMP response to GLP1R activation (since those are much more sensitive and also allow detection of signaling through different downstream pathways).

The utility of the caged agonist PhotoGLP1 is similarly unclear. The data demonstrate a substantial antagonism of GLP-1 binding by the still-caged compound, and it is therefore unclear whether the kinetics of the response to PhotoGLP1 itself would mimic the normal activation by GLP-1 in the absence of the caged compound. A further concern is that the light-dependence of the agonist effect of PhotoGLP1 was evaluated only with the GLPLight1 sensor and not with GLP1R signaling itself, which is 1000x more sensitive and which would be the presumed target of the tool. In addition, PhotoGLP1 is based upon native GLP-1, which is rapidly truncated and inactivated by the peptidase DPPIV, expressed in most cell types, and expressed at very high levels in the plasma. The utility of PhotoGLP1 is therefore limited to acute (minutes) in vitro experiments.

Reviewer #1 (Public Review):

Using a combination of structural biology methods, this report aims at describing the auto-inhibited architecture of kinesin 1 either as homodimers or hetero-tetramers. Hence, the multiple contacts between the protein domains and their folding pattern is addressed using cross-linking mass spectrometry (XL-MS), negative stain electron microscopy and Alpha Fold based structure prediction. Based on the existing literature, the key domains and amino acids responsible for kinesin 1 inhibited state were not clearly deciphered. The synergetic use of different methods now seems to describe in detail the molecular cues which could induce kinesin-1 refolding and opening. Multiple interactions between the different domains seem to induce the folded conformation.

The combination of methodologies is an efficient way to unravel details that could not be addressed previously. The paper is well written. However, the methodology is sometimes not sufficiently detailed and the paper would benefit from additional explanations and demonstrations. The methods for generating the electron microscopy data and its relevance and quality, for instance, are barely described. In addition, the conclusions drawn would be more convincing if similar investigations would be carried out similarly for all isoforms (KIF5B and FIF5C) in parallel.

This article raises the potential strength and power of deep learning structure prediction methods combined simultaneously with other structural biology methods to answer specific questions. In the present context, this study will certainly be helpful to reveal and understand the activation mechanism of kinesin motor proteins.

Reviewer #2 (Public Review):

The authors sought to define the molecular structure of autoinhibited Kinesin-1, which is the major kinesin providing plus-end directed transport on microtubules. The paper reports a structural model of full-length kinesin-1 which builds on the known folded conformation of kinesin-1 and describes its autoinhibitory mechanism using cryo-EM, alphafold structural predictions, cross-linking and mass spectrometry. The authors study the conformation of dimeric Kinesin Heavy Chain (KHC) and tetrameric KHC bound to the Kinesin Light Chains (KLCs), where KLC stabilize the autoinhibited conformation. The combination of these various approaches leads to an integrated molecular model of autoinhibited Kinesin-1. Until now, there was some debate over the role of the small coiled coil 3 (a and b) and where the hinge region of Kinesin-1. The authors resolve this question and present data indicating the hinge is between cc3a and cc3b.

In some places the absence of crosslinks is reported as a lack of interaction, however it could also be that there are no residues that can be crosslinked in this region. The distance is also not reported in the figures so we do not know how valid these model are. For example for TRAP binding to KHC, there are not many crosslinks but it is not clear if there was an issue with the complex assembly or crosslinking reaction-as there is no EM data of this complex. There is also a structural model of KHC and KLC (Fig 4) where the domains are too far apart for the crosslinks to be allowed, raising a question about whether that model is correct or not. The structural data are supported by single molecule motility assays with various mutants of Kinesin-1, which greatly help characterising the domains functionally.

Overall there are some interesting novel data on the autoinhibitory mechanism of Kinesin-1, with well performed and analyzed data with KLC and TRAP. The topic and paper will be of interest to many.

Reviewer #2 (Public Review):

To date, only a handful of studies have addressed the importance of AGS3, a paralog of the relatively well-characterized spindle orientation factor LGN. The authors now show that AGS3 acts as a negative regulator of LGN and propose that this activity could work through competition for binding partner(s). Remarkably, regulation is temporally restricted in such a way that the conserved role played by LGN in metaphase spindle orientation is unaffected. Instead, AGS3 regulates a post-metaphase function for LGN, namely Telophase Correction.

The article is well-written, the experiments are performed at a high level, and the claims are generally supported by the data. Two main points of confusion are raised in the current version. 1) The authors show that AGS3 regulates cortical localization of LGN, but would need to clarify how LGN is being affected. 2) The authors propose in the discussion that AGS3 might exert its regulatory effect through competition for NuMA, an important binding partner for LGN, but would need to clarify how and why NuMA would be involved in Telophase Correction.

Reviewer #1 (Public Review):

The model put forward by the authors in this manuscript is a simple and exciting one, explaining the function of AGS3 as a negative regulator of LGN, acting as a 'dominant-negative' version of LGN. Overall, the results support the model very well, and the results shown in Fig 6, which clearly reveal the functional relevance of AGS3, add strength to the paper.

In Figures 3A and B, the authors claim that AGS3 overexpression leads to depolarization of LGN in epidermal stem cells. However, in the example provided in Figure 3A, the LGN signal appears to be stronger than the control, with more LGN still on the apical side (many would categorize this as 'apically polarized'). In the scoring shown in Figure 3B, I am not sure if 'eyeballing' is the right way to decide whether it is polarized/depolarized/absent. The authors should come up with a bit more quantitative method to quantify the localization/amount of LGN and explain the method well in the manuscript. A similar concern regarding the determination of the LGN localization pattern applies to the rest of figure 3 as well.

Reviewer #3 (Public Review):

This paper examines the mechanisms that control division orientation in the basal layers of the epidermis. Previous work established LGN as a key promoter of divisions where one of the siblings populates the differentiated layers (perpendicular). This work addresses two important, related issues - the mechanisms that determine whether a particular division is planar vs perpendicular, and the function of AGS3, and LGN paralog that has been enigmatic. A central finding is that AGS3 is required for the normal distribution of planar and perpendicular divisions (roughly equal) such that in its absence the distribution is skewed towards the perpendicular. Interestingly, however, the authors find that AGS3 has no detectable effect on orientation if the orientation is measured at anaphase. This timing aspect builds upon previous work from this group demonstrating a phenomenon they term "telophase correction" in which the orientation changes at the latest phases of division (and possibly post division?). Thus AGS3 seems to exert its effect using these later mechanisms and this is supported by further analysis by the authors. Importantly, the authors show that AGS3 acts through LGN, based on localization data and an epistasis analysis. The function of AGS3 has been highly enigmatic so resolving this issue while providing a useful step towards understanding how the division orientation decision is made, makes for exciting progress towards an important problem. I found the overall narrative and presentation to be quite good and especially appreciated the thoughtful discussion section that did an excellent job of putting the results in context and speculating how unknown aspects of the mechanism might work based on current clues. With that said, I think there are some important issues that should be resolved.

Regarding the orientation measurements, the authors should specify how the midbody marker was used to mark sibling cells, especially given the midbody can move following division. For example, how can the authors be confident that the siblings in the middle panel of 1A are correct and not an adjacent cell?

Regarding quantification, it would be useful for the authors to comment on how the following would influence their measurements: 1) movements along the z-axis, and 2) movement of the nucleus within the cell.

A similar question is how much telophase correction really happens in telophase. How confident are the authors that the process actually occurs during division and not subsequent to it? What is drawn in their previous paper and in Figure 7A implies that post-division movements may be important. It would be useful for the authors to comment on whether they can make the distinction and whether or not it might be important.

Does the division angle in the AGS3 OE experiment (Figure 1D) correlate with AGS3 levels within the cell?

I found the localization data to be the weakest part of the paper and feel that some reconsideration and reanalysis are warranted.

First, the quantifications in Figures 2C, 3B, and 3F are unnecessarily vague scoring-based metrics. In 2C, "Localization pattern" should be replaced with membrane/cytoplasm ratio or an equivalent quantification. In 3B "LGN localization" should be replaced with apical/cytoplasmic and apical/basal ratios or equivalents. In 3F, "Polarized LGN frequency" should be replaced with apical/basal ratio or equivalent. It seems to me that non-AI processed data would be most appropriate for these quantifications unless such processing can be justified.

Second, it is important to note that the cytoplasmic localization of AGS3 does not allow one to conclude that AGS3 is not on the membrane. Unfortunately, high cytoplasmic signal can preclude the determination of membrane-bound signal.

Finally, I had difficulty reconciling the images of LGN shown in Figure 3 with the conclusions made by the authors.

The challenge of the localization data is troubling because an important conclusion of the paper is that AGS3 acts via LGS. The localization data provided one leg of support for this conclusion and the other is provided by an epistasis analysis. Unfortunately, this data seems to be right on the edge because it is based on the difference between the solid and dashed blue lines in Figure 5B not being significant. However, we can see how close this is by comparing the solid and dashed red lines in the adjacent 5C, which are significantly different. Between the localization data, which doesn't seem clear cut, and the epistasis experiment, which is on the razor's edge, I'm concerned that the conclusion that AGS3 acts through LGN may be going beyond what the data allows.

Reviewer #1 (Public Review):

This work is a follow-up of the work from the same group where the authors showed that Lactiplantibacillus plantarum can enhance juvenile growth by activating the expression of an intestinal protease. They previously showed that this process was mediated by the dlt operon which is involved in the D-Alanylation of teichoic acid.

In the present study, the authors characterized the structure and enzymatic activity of the first protein encoded by this operon and show that the first gene of this operon encodes for an esterase releasing D-Ala from D-Ala lipoteichoic acids (LTA) and renamed it here DltE. The gene encoding this protein was previously uncharacterized and annotated as a peptidoglycan-binding protein putatively involved in peptidoglycan maturation. With the structure and enzymatic characterization of this protein, this study revealed that this protein does not act as peptidoglycan, but instead releases D-Ala from D-alanylated-LTA.

The authors use a Drosophila mutant impaired in response to mDAP-Peptidoglycan fragments (affected in the IMD pathway) to show that this mutant still responds to D-Ala-LTAs. This result is important to show that D-Ala-LTAs act as additional cues sensed by Drosophila independent of m-DAP-peptidoglycan by a still unknown sensory pathway. The study convincingly shows that D-Ala-LTA from the gut microbe L. plantarum leads to increase intestinal peptidase expression (intestinal activity) and enhance juvenile larva growth.

Reviewer #2 (Public Review):

The authors conduct a structure-function analysis of an uncharacterized gene, DltE, which was found by a genetic screen to be involved in the growth promotion of Drosophila larvae by Lactiplantibacillus plantarum, a bacterium that is consistently associated with Drosophila. They find that DltE is a D-Ala carboxylesterase that removes D-Ala from lipoteichoic acids in the cell envelope and that D-alanylated lipoteichoic acids stimulate Drosophila larval growth. The result that D-Ala LTA stimulates larval growth is compelling, although some minor experimental details to do with biological replicates are not shown and the tracking of bacterial abundances should be addressed to make the conclusions more solid. Additionally, I think the use of the terms "direct" and "symbiotic" is inappropriate in the manuscript, but this can be resolved by removing them or performing additional experiments.

The authors make these claims:<br /> - DltE is not a carboxypeptidase modifying Lp peptidoglycan;<br /> - DltE is a D-Ala esterase acting upon D-Ala-LTA;<br /> - only LTAs but not WTAs are D-alanylated in LpNC8 cell envelopes;<br /> - D-Ala-LTAs, in addition to PG, are direct symbiotic cues supporting<br /> (1) intestinal peptidase expression and<br /> (2) juvenile growth in Drosophila.<br /> I find all of the claims to be well supported by data except the suggestion that these are "direct symbiotic" cues. I think the authors provide the support that D-Ala LTAs are nutritional cues, not symbiotic ones.

Overall, I find the work compelling.

Reviewer #3 (Public Review):

This work by Nikolopoulos et al. expands on prior studies demonstrating the ability of a member of the Drosophila melanogaster gut microbiome, Lactiplantibacillus plantarum, to support juvenile development in nutrient-limiting conditions. Previously, the authors identified the pbpX2-dltXABCD operon of L. plantarum that when mutated eliminated the growth-promoting ability of the bacterium to flies experiencing malnutrition (protein starvation). To better understand the bacterial components that support this larval development, the authors used a combination of structural, biochemical, and mutational analysis to describe the physiological role of the DltE, a previously uncharacterized gene within the pbpX2-dltXABCD operon. Although annotated as a serine-type D-Ala-D-Ala-carboxypeptidase, this work supports its role instead as a D-ala esterase that acts upon D-alanylated lipoteichoic acids, which are directly sensed by the host to induce peptidase expression and support juvenile growth in flies.

Overall, the data is compelling, and the conclusions are well-supported. The multiple methods used to examine and support their findings - the combination of structural and biochemical analyses, and the use of both bacterial and fly mutants to substantiate and demonstrate physiological relevance was elegant in execution.

The identification of a role for this bacterial cell component is exciting as it has not previously been appreciated as a bacterial-derived signal in fly immunity and/or metabolism. This work adds to the growing evidence for the breadth and diversity of bacterial metabolites and products that underlie fly-microbiome interactions and may have implications in other animal-microbe interactions, especially L. plantarum-mediated host growth promotion in other models including mammals.

An intriguing aspect of the work is the evidence of a bifurcation of this bacterial signal on immunity and metabolism, with the pathway regulating the latter yet unknown. Likewise, determining how these cell components are sensed by the host will also be of future interest. Another unknown that may limit the implications of this study is the ubiquity of D-ala LTA production among D. melanogaster-associated L. plantarum strains and whether this is a common or rare signal/role.

Reviewer #1 (Public Review):

This manuscript by Walker et. al. explores the interplay between the global regulators HapR (the QS master high cell density (HDC) regulator) and CRP. Using ChIP-Seq, the authors find that at several sites, the HapR and CRP binding sites overlap. A detailed exploration of the murPQ promoter finds that CRP binding promotes HapR binding, which leads to repression of murPQ. The authors have a comprehensive set of experiments that paints a nice story providing a mechanistic explanation for converging global regulation. I did feel there are some weak points though, in particular the lack of integration of previously identified transcription start sites, the lack of replication (at least replication presented in the manuscript) for many figures, some oddities in the growth curve, and not reexamining their HapR/CRP cooperative binding model in vivo using ChIP-Seq.

Reviewer #2 (Public Review):

This manuscript by Walker et al describes an elegant study that synergizes our knowledge of virulence gene regulation of Vibrio cholerae. The work brings a new element of regulation for CRP, notably that CRP and the high density regulator HapR co-occupy the same site on the DNA but modeling predicts they occupy different faces of the DNA. The DNA binding and structural modeling work is nicely conducted and data of co-occupation are convincing. The work could benefit from doing a better job in the manuscript preparation to integrate the findings into our current state of knowledge of HapR and CRP regulated genes and to elevate the impact of the work to address how bacteria are responding to the nutritional environment. Importantly, the focus of the work is heavily based on the impact of use of GlcNAc as a carbon source when bacteria bind to chitin in the environment, but absent the impact during infection when CRP and HapR have known roles. Further, the impact on biological events controlled by HapR integration with the utilization of carbon sources (including biofilm formation) is not explored. The rigor and reproducibility of the work needs to be better conveyed.

Specific comments to address:

1) Abstract. A comment on the impact of this work should be included in the last sentence. Specifically, how the integration of CRP with QS for gene expression under specific environments impacts the lifestyle of Vc is needed. The discussion includes comments regarding the impact of CRP regulation as a sensor of carbon source and nutrition and these could be quickly summarized as part of the abstract.<br /> 2) Line 74. This paper examines the overlap of HapR with CRP, but ignores entirely AphA. HapR is repressed by Qrrs (downstream of LuxO-P) while AphA is activated by Qrrs. WithLuxO activating AphA, it has a significant sized "regulon" of genes turned on at low density. It seems reasonable that there is a possibility of overlap also between CRP and AphA. While doing an AphA CHIP-seq is likely outside the scope of this work, some bioinformatic or simply a visual analysis of the promoters known AphA regulated genes would be interest to comment on with speculation in the discussion and/or supplement.<br /> 3) Line 100. Accordingly with the above statement, the focus here on HapR indicates that the focus is on gene expression via LuxO and HapR, at high density. Thus the sentence should read "we sought to map the binding of LuxO and HapR of V. cholerae genome at high density".<br /> 4) Line 109. The identification of minor LuxO binding site in the intergenic region between VC1142 and VC1143 raises whether there may be a previously unrecognized sRNA here. As another panel in figure S1, can you provide a map of the intergenic region showing the start codons and putative -10 to -35 sites. Is there room here for an sRNA? Is there one known from the many sRNA predictions / identifications previously done? Some additional analysis would be helpful.<br /> 5) Line 117. This sentence states that the CHIP seq analysis in this study includes previously identified HapR regulated genes, but does not reveal that many known HapR regulated genes are absent from Table 1 and thus were missed in this study. Of 24 HapR regulated investigated by Tsou et al, only 1 is found in Table 1 of this study. A few are commented in the discussion and Figure S7. It might be useful to add a Venn Diagram to Figure 1 (and list table in supplement) for results of Tsou et al, Waters et al, Lin et al, and Nielson et al and any others). A major question is whether the trend found here for genes identified by CHIP-seq in this study hold up across the entire HapR regulon. There should also be comments in the discussion on perhaps how different methods (including growth state and carbon sources of media) may have impacted the complexity of the regulon identified by the different authors and different methods.<br /> 6) The transcription data are generally well performed. In all figures, add comments to the figure legends that the experiments are representative gels from n=# (the number of replicate experiments for the gel based assays). Statements to the rigor of the work are currently missing.<br /> 7) Line 357-360. The demonstration of lack of growth on MurNAc is a nice for the impact of the work. However, more detailed comments are needed for M9 plus glucose for the uninformed reader to be reminded that growth in glucose is also impaired due to lack of cAMP in glucose replete conditions and thus minimal CRP is active. But why is this now dependent of hapR? A reminder also that in LB oligopeptides from tryptone are the main carbon source and thus CRP would be active.<br /> 8) A great final experiment to demonstrate the model would have been to show co-localization of the promoter by CRP and HapR from bacteria grown in LB media but not in LB+glucose or in M9+glycerol and M9+MurNAc but not M9+glucose. This would enhance the model by linking more directly to the carbon sources (currently only indirect via growth curves)<br /> 9) Discussion. Comments and model focus heavily on GlcNAc-6P but HapR has a regulator role also during late infection (high density). How does CRP co-operativity impact during the in vivo conditions? Does the Biphasic role of CRP play a role here (PMID: 20862321)?

Reviewer #3 (Public Review):

Bacteria sense and respond to multiple signals and cues to regulate gene expression. To define the complex network of signaling that ultimately controls transcription of many genes in cells requires an understanding of how multiple signaling systems can converge to effect gene expression and ensuing bacterial behaviors. The global transcription factor CRP has been studied for decades as a regulator of genes in response to glucose availability. It's direct and indirect effects on gene expression have been documented in E. coli and other bacteria including pathogens including Vibrio cholerae. Likewise, the master regulator of quorum sensing (QS), HapR), is a well-studied transcription factor that directly controls many genes in Vibrio cholerae and other Vibrios in response to autoinducer molecules that accumulate at high cell density. By contrast, low cell density gene expression is governed by another regulator AphA. It has not yet been described how HapR and CRP may together work to directly control transcription and what genes are under such direct dual control.

Using both in vivo methods with gene fusions to lacZ and in vitro transcription assays, the authors proceed to identify the smaller subset of genes whose transcription is directly repressed (7) and activated (2) by HapR. Prior work from this group identified the direct CRP binding sites in the V. cholerae genome as well as promoters with overlapping binding sites for AphA and CRP, thus it appears a logical extension of these prior studies is to explore here promoters for potential integration of HapR and CRP. Inclusion of this rationale was not included in the introduction of CRP protein to the in vitro experiments.

Seven genes are found to be repressed by HapR in vivo, the promoter regions of only six are repressed in vitro with purified HapR protein alone. The authors propose and then present evidence that the seventh promoter, which controls murPQ, requires CRP to be repressed by HapR both using in vivo and vitro methods. This is a critical insight that drives the rest of the manuscripts focus.

The DNase protection assay conducted supports the emerging model that both CRP and HapR bind at the same region of the murPQ promoter, but interpret is difficult due to the poor quality of the blot. There are areas of apparent protection at positions +1 to +15 that are not discussed, and the areas highlighted are difficult to observe with the blot provided.

The model proposed at the end of the manuscript proposes physiological changes in cells that occur at transitions from the low to high cell density. Experiments in the paper that could strengthen this argument are incomplete. For example, in Fig. 4e it is unclear at what cell density the experiment is conducted. The results with the wild type strain are intermediate relative to the other strains tested. Cell density should affect the result here since HapR is produced at high density but not low density. This experiment would provide important additional insights supporting their model, by measuring activity at both cell densities and also in a luxO mutant locked at the high cell density. Conducting this experiment in conditions lacking and containing glucose would also reveal whether high glucose conditions mimicking the crp results.

Throughout the paper it was challenging to account for the number of genes selected, the rationale for their selection, and how they were prioritized. For example, the authors acknowledged toward the end of the Results section that in their prior work, CRP and HapR binding sites were identified (line 321-22). It is unclear whether the loci indicated in Table 1 all from this prior study. It would be useful to denote in this table the seven genes characterized in Figure 2 and to provide the locus tag for murPQ. Of the 32 loci shown in Table 1, five were selected for further study using EMSA (line 322), but no rationale is given for studying these five and not others in the table.

Since prior work identified a consensus CRP binding motif, the authors identify the DNA sequence to which HapR binds overlaps with a sequence also predicted to bind CRP. Genome analysis identified a total of seven sites where the CRP and HapR binding sites were offset by one nucleotide as see with murPQ. Lines 327-8 describe EMSA results with several of these DNA sequences but provides no data to support this statement. Are these loci in Table 1?

Using structural models, the authors predict that HapR repression requires protein-protein interactions with CRP. Electromobility shift assays (EMSA) with purified promoter DNA, CRP and HapR (Fig 5d) and in vitro transcription using purified RNAP with these factors (Figure 5e) support this hypothesis. However, the model proports that HapR "bound tightly" and that it also had a "lower affinity" when CRP protein was used that had mutations in a putative interaction interface. These claims can be bolstered if the authors calculate the dissociation constant (Kd) value of each protein to the DNA. This provides a quantitative assessment of the binding properties of the proteins. The concentrations of each protein are not indicated in panels of the in vitro analysis, but only the geometric shapes denoting increasing protein levels. Panel 5e appears to indicate that an intermediate level of CRP was used in the presence of HapR, which presumably coincides with levels used in lane 4, but rationale is not provided. How well the levels of protein used in vitro compare to levels observed in vivo is not mentioned.

The authors are commended for seeking to connect the in vitro and vivo results obtained under lab conditions with conditions experienced by V. cholerae in niches it may occupy, such as aquatic systems. The authors briefly review the role of MurPQ in recycling of the cell wall of V. cholerae by degrading MurNAc into GlcNAc, although no references are provided (lines 146-50). Based on this physiology and results reported, the authors propose that murPQ gene expression by these two signal transduction pathways has relevance in the environment, where Vibrios, including V. cholerae, forms biofilms on exoskeleton composed of GlcNAc.

The conclusions of that work are supported by the Results presented but additional details in the text regarding the characteristics of the proteins used (Kd, concentrations) would strengthen the conclusions drawn. This work provides a roadmap for the methods and analysis required to develop the regulatory networks that converge to control gene expression in microbes. The study has the potential to inform beyond the sub-filed of Vibrios, QS and CRP regulation.

Reviewer #1 (Public Review):

Germe and colleagues have investigated the mode of action of bacterial DNA gyrase, a tetrameric GyrA2GyrB2 complex that catalyses ATP-dependent DNA supercoiling. The accepted mechanism is that the enzyme passes a DNA segment through a reversible double-stranded DNA break formed by two catalytic Tyr residues-one from each GyrA subunit. The present study sought to understand an intriguing earlier observation that gyrase with a single catalytic tyrosine that cleaves a single strand of DNA, nonetheless has DNA supercoiling activity, a finding that led to the suggestion that gyrase acts via a nicking closing mechanism. Germe et al used bacterial co-expression to make the wild-type and mutant heterodimeric BA(fused). A complexes with only one catalytic tyrosine. Whether the Tyr mutation was on the A side or BA fusion side, both complexes plus GyrB reconstituted fluoroquinolone-stabilised double-stranded DNA cleavage and DNA supercoiling. This indicates that the preparations of these complexes sustain double strand DNA passage. Of possible explanations, contamination of heterodimeric complexes or GyrB with GyrA dimers was ruled out by the meticulous prior analysis of the proteins on native Page gels, by analytical gel filtration and by mass photometry. Involvement of an alternative nucleophile on the Tyr-mutated protein was ruled unlikely by mutagenesis studies focused on the catalytic ArgTyrThr triad of residues. Instead, results of the present study favour a third explanation wherein double-strand DNA breakage arises as a consequence of subunit (or interface/domain) exchange. The authors showed that although subunits in the GyrA dimer were thought to be tightly associated, addition of GyrB to heterodimers with one catalytic tyrosine stimulates rapid DNA-dependent subunit or interface exchange to generate complexes with two catalytic tyrosines capable of double-stranded DNA breakage. Subunit exchange between complexes is facilitated by DNA bending and wrapping by gyrase, by the ability of both GyrA and GyrB to form higher order aggregates and by dense packing of gyrase complexes on DNA. By addressing a puzzling paradox, this study provides support for the accepted double strand break (strand passage) mechanism of gyrase and opens new insights on subunit exchange that may have biological significance in promoting DNA recombination and genome evolution.

The conclusions of the work are mostly well supported by the experimental data.

Strengths:

The study examines a fundamental biological question, namely the mechanism of DNA gyrase, an essential and ubiquitous enzyme in bacteria, and the target of fluoroquinolone antimicrobial agents.

The experiments have been carefully done and the analysis of their outcomes is comprehensive, thoughtful and considered.

The work uses an array of complementary techniques to characterize preparations of GyrA, GyrB and various gyrase complexes. In this regard, mass photometry seems particularly useful. Analysis reveals that purified GyrA and GyrB can each form multimeric complexes and highlights the complexities involved in investigating the gyrase system.

The various possible explanations for the double-strand DNA breakage by gyrase heterodimers with a single catalytic tyrosine are considered and addressed by appropriate experiments.

The study highlights the potential biological importance of interactions between gyrase complexes through domain-or subunit-exchange

Weaknesses:

The mutagenesis experiments described do not fully eliminate the perhaps unlikely participation of an alternative nucleophile.

Reviewer #2 (Public Review):

DNA gyrase is an essential enzyme in bacteria that regulates DNA topology and has the unique property to introduce negative supercoils into DNA. This enzyme contains 2 subunits GyrA and GyrB, which forms an A2B2 heterotetramer that associates with DNA and hydrolyzes ATP. The molecular structure of the A2B2 assembly is composed of 3 dimeric interfaces, called gates, which allow the cleavage and transport of DNA double stranded molecules through the gates, in order to perform DNA topology simplification.<br /> The article by Germe et al. questions the existence and possible mechanism for subunit exchange in the bacterial DNA gyrase complex.

The complexes are purified as a dimer of GyrA and a fusion of GyrB and GyrA (GyrBA), encoded by different plasmids, to allow the introduction of targeted mutations on one side only of the complex. The conclusion drawn by the authors is that subunit exchange does happen, favored by DNA binding and wrapping. They propose that the accumulation of gyrase in higher-order oligomers can favor rapid subunit exchange between two active gyrase complexes brought into proximity.<br /> The authors are also debating the conclusions of a previous article by Gubaev, Weidlich et al 2016 (https://doi.org/10.1093/nar/gkw740). Gubaev et al. originally used this strategy of complex reconstitution to propose a nicking-closing mechanism for the introduction of negative supercoils by DNA gyrase, an alternative mechanism that precludes DNA strand passage, previously established in the field. Germe et al. incriminate in this earlier study the potential subunit swapping of the recombinant protein with the endogenous enzyme, that would be responsible for the detected negative supercoiling activity.

Accordingly, the authors also conclude that they cannot completely exclude the presence of endogenous subunits in their samples as well.

Strengths

The mix of gyrase subunits is plausible, this mechanism has been suggested by Ideka et al, 2004 and also for the human Top2 isoforms with the formation of Top2a/Top2b hybrids being identified in HeLa cells (doi: 10.1073/pnas.93.16.8288).<br /> Germe et al have used extensive and solid biochemical experiments, together with thorough experimental controls, involving :<br /> - the purification of gyrase subunits including mutants with domain deletion, subunit fusion or point mutations.<br /> - DNA relaxation, cleavage and supercoiling assays<br /> - biophysical characterization in solution (size exclusion chromatography, mass photometry, mass spectrometry)

Together the combination of experimental approaches provides solid evidence for subunit swapping in gyrase in vitro, despite the technical limitations of standard biochemistry applied to such a complex macromolecule.

Weaknesses

The conclusions of this study could be strengthened by in vivo data to identify subunit swapping in the bacteria, as proposed by Ideka et al, 2004. Indeed, if shown in vivo, together with this biochemical evidence, this mechanism could have a substantial impact on our understanding of bacterial physiology and resistance to drugs.

Joint Public Review

The molecular composition of synaptic vesicles (SVs) has been defined in substantial detail, but the function of many SV-resident proteins are still unknown. The present study focused on one such protein, the 'orphan' SV-resident transporter SLC6A17. By utilizing sophisticated and extensive mouse genetics and behavioral experiments, the authors provide convincing support for the notion that certain SLC6A17 variants cause intellectual disability (ID) in humans carrying such genetic variations. This is an important and novel finding. Furthermore, the authors propose, based on LC-MS analyses of isolated SVs, that SLC6A17 is responsible for glutamine (Gln) transport into SVs, leading to the provocative idea that Gln functions as a neurotransmitter and that deficits in Gln transport into SVs by SLC6A17 represents a key pathogenetic mechanism in human ID patients carrying variants of the SLC6A17 gene.

This latter aspect of the present paper is not adequately supported by the experimental evidence so that the main conceptual claims of the study appear insufficiently justified at this juncture. Key weaknesses are as follows:

A. Detection of Gln, along with classical neurotransmitters such as glutamate, GABA, or ACh, in isolated SV fractions does not prove that Gln is transported into SVs by active transport. Gln is quite abundant in extracellular compartments. Its appearance in SV samples can therefore also be explained by trapping in SVs during endocytosis, presence in other - contaminating - organelles, binding to membrane surfaces, and other processes. Direct assays of Gln uptake into SVs, which have the potential to stringently test key postulates of the authors, are lacking.

B. The authors generated multiple potentially very useful genetic tools and models. However, the validation of these models is incomplete. Most importantly, it remains unclear whether the different mutations affect SLC6A17 expression levels, subcellular localization, or the expression and trafficking of other SV and synapse components.

C. Apart from the caveats mentioned above regarding Gln uptake into SVs, the data interpretation provided by the authors lacks stringency with respect to the biophysics of plasma membrane and SV transporters.

Reviewer #1 (Public Review):

Abdellahi et al. used targeted memory reactivation (TMR) and machine learning tools to look for evidence that waking neural activity is reinstated during subsequent REM sleep. Prior work has demonstrated that learning content is successfully decoded following TMR cues during NREM sleep, but a direct link between patterns of brain activity recorded during wakefulness and subsequent REM sleep in humans has never been reported. In this paper, the authors report that an LDA classifier detects wake-like neural activity (specifically, neural activity recorded while imaging performing a trained serial reaction time task) approximately one second after TMR cues are presented during REM sleep. Decoding performance is better when the classifier is trained on sleep trials with high theta compared to low theta power, and classifier performance was correlated with overnight improvement on the task.

Finding evidence of reinstated waking neural activity during REM sleep is an exciting result, and the authors present a promising method that holds implications for advancing our understanding of how memories are reprocessed during REM sleep. I think it is a particular strength of the paper that the authors trained on sleep data and tested in wake data, which is analogous to prior rodent studies that found evidence of replay during REM. I also thought playing sounds during the adaptation night, prior to SRTT training, provided a nice control.

The conclusions of this paper are mostly supported by the results presented, but it is not always clear how those results were obtained. Some aspects of the experimental and data analytic methods need to be clarified and expanded, both for a better understanding of how the results of this study were obtained, as well as for future reproducibility.

Reviewer #2 (Public Review):

I believe the authors succeeded in finding neural evidence of reactivation during REM sleep. This is their main claim, and I applaud them for that. I also applaud their efforts to explore their data beyond this claim, and I think they included appropriate controls in their experimental design. However, I found other aspects of the paper to be unclear or lacking in support. I include major and medium-level comments:

Major comments, grouped by theme with specifics below:<br /> Theta.<br /> Overall assessment: the theta effects are either over-emphasized or unclear. Please either remove the high/low theta effects or provide a better justification for why they are insightful.

Lines ~ 115-121: Please include the statistics for low-theta power trials. Also, without a significant difference between high- and low-theta power trials, it is unclear why this analysis is being featured. Does theta actually matter for classification accuracy?

Lines 123-128: What ARE the important bands for classification? I understand the point about it overlapping in time with the classification window without being discriminative between the conditions, but it still is not clear why theta is being featured given the non-significant differences between high/low theta and the lack of its involvement in classification. REM sleep is high in theta, but other than that, I do not understand the focus given this lack of empirical support for its relevance.

Line 232-233: "8). In our data, trials with higher theta power show greater evidence of memory reactivation." Please do not use this language without a difference between high and low theta trials. You can say there was significance using high theta power and not with low theta power, but without the contrast, you cannot say this.

Physiology / Figure 2.<br /> Overall assessment: It would be helpful to include more physiological data.

It would be nice, either in Figure 2 or in the supplement, to see the raw EEG traces in these conditions. These would be especially instructive because, with NREM TMR, the ERPs seem to take a stereotypical pattern that begins with a clear influence of slow oscillations (e.g., in Cairney et al., 2018), and it would be helpful to show the contrast here in REM. Also, please expand the classification window beyond 1 s for wake and 1.4 s for sleep. It seems the wake axis stops at 1 s and it would be instructive to know how long that lasts beyond 1 s. The sleep signal should also go longer. I suggest plotting it for at least 5 seconds, considering prior investigations (Cairney et al., 2018; Schreiner et al., 2018; Wang et al., 2019) found evidence of reactivation lasting beyond 1.4 s.

Temporal compression/dilation.<br /> Overall assessment: This could be cut from the paper. If the authors disagree, I am curious how they think it adds novel insight.

Line 179 section: In my opinion, this does not show evidence for compression or dilation. If anything, it argues that reactivation unfolds on a similar scale, as the numbers are clustered around 1. I suggest the authors scrap this analysis, as I do not believe it supports any main point of their paper. If they do decide to keep it, they should expand the window of dilation beyond 1.4 in Figure 3B (why cut off the graph at a data point that is still significant?). And they should later emphasize that the main conclusion, if any, is that the scales are similar.

Line 207 section on the temporal structure of reactivation, 1st paragraph: Once again, in my opinion, this whole concept is not worth mentioning here, as there is not really any relevant data in the paper that speaks to this concept.

Behavioral effects.<br /> Overall assessment: Please provide additional analyses and discussion.

Lines 171-178: Nice correlation! Was there any correlation between reactivation evidence and pre-sleep performance? If so, could the authors show those data, and also test whether this relationship holds while covarying our pre-sleep performance? The logic is that intact reactivation may rely on intact pre-sleep performance; conversely, there could be an inverse relationship if sleep reactivation is greater for initially weaker traces, as some have argued (e.g., Schapiro et al., 2018). This analysis will either strengthen their conclusion or change it -- either outcome is good.

Unlike Schönauer et al. (2017), they found a strong correspondence between REM reactivation and memory improvement across sleep; however, there was no benefit of TMR cues overall. These two results in tandem are puzzling. Could the authors discuss this more? What does it mean to have the correlation without the overall effect? Or else, is there anything else that may drive the individual differences they allude to in the Discussion?

Medium-level comments<br /> Lines 63-65: "We used two sequences and replayed only one of them in sleep. For control, we also included an adaptation night in which participants slept in the lab, and the same tones that would later be played during the experimental night were played."

I believe the authors could make a stronger point here: their design allowed them to show that they are not simply decoding SOUNDS but actual memories. The null finding on the adaptation night is definitely helpful in ruling this possibility out.

Lines 129-141: Does reactivation evidence go down (like in their prior study, Belal et al., 2018)? All they report is theta activity rather than classification evidence. Also, I am unclear why the Wilcoxon comparison was performed rather than a simple correlation in theta activity across TMR cues (though again, it makes more sense to me to investigate reactivation evidence across TMR cues instead).

Line 201: It seems unclear whether they should call this "wake-like activity" when the classifier involved training on sleep first and then showing it could decode wake rather than vice versa. I agree with the author's logic that wake signals that are specific to wake will be unhelpful during sleep, but I am not sure "wake-like" fits here. I'm not going to belabor this point, but I do encourage the authors to think deeply about whether this is truly the term that fits.

Reviewer #3 (Public Review):

The authors investigated whether reactivation of wake EEG patterns associated with left- and right-hand motor responses occurs in response to sound cues presented during REM sleep.

The question of whether reactivation occurs during REM is of substantial practical and theoretical importance. While some rodent studies have found reactivation during REM, it has generally been more difficult to observe reactivation during REM than during NREM sleep in humans (with a few notable exceptions, e.g., Schonauer et al., 2017), and the nature and function of memory reactivation in REM sleep is much less well understood than the nature and function of reactivation in NREM sleep. Finding a procedure that yields clear reactivation in REM in response to sound cues would give researchers a new tool to explore these crucial questions.

The main strength of the paper is that the core reactivation finding appears to be sound. This is an important contribution to the literature, for the reasons noted above.

The main weakness of the paper is that the ancillary claims (about the nature of reactivation) may not be supported by the data.

The claim that reactivation was mediated by high theta activity requires a significant difference in reactivation between trials with high theta power and trials with low theta, but this is not what the authors found (rather, they have a "difference of significances", where results were significant for high theta but not low theta). So, at present, the claim that theta activity is relevant is not adequately supported by the data.

The authors claim that sleep replay was sometimes temporally compressed and sometimes dilated compared to wakeful experience, but I am not sure that the data show compression and dilation. Part of the issue is that the methods are not clear. For the compression/dilation analysis, what are the features that are going into the analysis? Are the feature vectors patterns of power coefficients across electrodes (or within single electrodes?) at a single time point? or raw data from multiple electrodes at a single time point? If the feature vectors are patterns of activity at a single time point, then I don't think it's possible to conclude anything about compression/dilation in time (in this case, the observed results could simply reflect autocorrelation in the time-point-specific feature vectors - if you have a pattern that is relatively stationary in time, then compressing or dilating it in the time dimension won't change it much). If the feature vectors are spatiotemporal patterns (i.e., the patterns being fed into the classifier reflect samples from multiple frequencies/electrodes / AND time points) then it might in principle be possible to look at compression, but here I just could not figure out what is going on.

For the analyses relating to classification performance and behavior, the authors presently show that there is a significant correlation for the cued sequence but not for the other sequence. This is a "difference of significances" but not a significant difference. To justify the claim that the correlation is sequence-specific, the authors would have to run an analysis that directly compares the two sequences.

Reviewer #1 (Public Review):

Most previous studies investigating the phenomenon of crowding in depth use small stereoscopic differences in depth. Taken together their results suggest that a depth difference between target and flankers reduces crowding. A potential problem is that stereo displays can reduce depth perception. The studies that have used a real-depth display have provided some inconsistent findings. The present study investigated larger differences, representative of those among many objects in the real world. These larger differences increased crowding, even in the absence of diplopia (double vision).

This study is likely to be impactful in the field as it shows that crowding occurs in-depth and strengthens the importance of crowding in natural 3D environments. All existing models of crowding would need to be modified to explain this experimental finding.

The novel multi-depth plane display that the authors used enables measurements of depth differences that are more likely to correspond to differences in the real world, and could be used by others to further investigate crowding in-depth or other perceptual processes (e.g., visual search).

In general, there are some interactions that were reported and others that were not reported, but it would be important to know if they are significant. (pages 15-16) For example, when the target is at fixation and the target is at a variable flanker depth: In Experiment 1, was there a significant interaction between (a) target-fixation depth and flanker depth (in front versus behind) and (b) target-fixation depth and target-flanker spacing? In Experiment 3, it is reported that perceptual error was higher when the target was in from or behind the flanker ring and fixation and that the greatest perceptual error occurred when the target was behind, but it is not reported if this interaction was significant. Its presence is important to know whether the data should be independently analyzed for 'in front' and 'behind'. In Experiment 5, was the interaction between target-flanker spacing and depth significant?

The findings are clear but the explanation(s) for the findings is not. The authors state that large interocular disparity differences likely induce diplopia, which could increase perceptual error by increasing the number of features. The authors should explain what they mean by features and how an increased perceived number of features would increase crowding. Moreover, the authors acknowledge that only a few observers reported experiencing diplopia; however, they speculate that observers may have experienced diplopia but not noticed it consciously given the short stimulus presentation time.

Reviewer #2 (Public Review):

The authors reexamine the effects of depth on crowding, using a clever display that presents at three depths at once, and find that placing the target or flanker at far depth greatly increases crowding, contrary to what might have been expected by prior work with small depth differences. These stimuli avoid creating conflicting cues to depth and are thus the most relevant to viewing in daily life, indicating more crowding than was expected.

Reviewer #3 (Public Review):

Smithers et al. examine the effects of large differences in target-flanker depth on peripheral visual crowding. To investigate this, they developed a novel real-depth display and measured the perceptual errors caused by the presence of flanker objects that were presented at different distances and at either the same or at different depths from a target object that the participants had to recognize.

Their primary result is that large depth differences between flanking and target objects increase the magnitude of crowding. Interestingly, it appears to be a two-faced finding: when the target is at fixation depth, crowding is more pronounced if the flankers are behind the target as opposed to in front of it. Yet, when the flankers are at fixation depth, crowding is more pronounced if the target is behind the flankers. They explain their finding in terms of increased clutter in areas outside the limits of binocular fusion. This conclusion of the study is well supported by the data and experiments. The work provides compelling evidence that real depth may affect peripheral crowding under the specific circumstances of their experiment. Whether this finding would also apply to more natural viewing conditions, in which there is much more clutter, to begin with, remains to be determined.

Strengths:<br /> By introducing a novel multi-depth plane display authors contribute to future research on the effect of real depth differences on several visual functions and increase the potential ecological validity of their results.<br /> By using perceptual error as their dependent variable and linear mixed models to analyze their data, authors improve their ability to represent the variability in the data.<br /> The authors explain the discrepancies between their results and previous research with sufficient additional experiments and data.<br /> The inclusion of a large number of participants, which is fairly uncommon in this type of experiment.

Weaknesses:<br /> 1. At several points in the paper authors refer to the 'natural three dimensional scenes'. Indeed, the authors increase the ecological validity of their experiment by introducing actual depth differences, therefore allowing for depth cues such as accommodation, vergence and defocus blur. This is indeed a significant improvement over previous studies. However, they still use relatively impoverished visual stimuli in a tightly controlled psychophysical experiment requiring head stabilization by means of a chin rest. So, their experiment is still far removed from deploying actual, ecologically valid, conditions. Consequently, their stimuli mostly lack the complexity and associated clutter of natural stimuli as well as other potential depth cues that an observer might gain from parallax, aerial perspective, lighting, or shading. Therefore, their suggestion "that crowding has a more significant impact on our perception of natural three-dimensional environments than previously estimated with 2D displays." is stretching what can be concluded from their present work.<br /> 2. The inclusion of a large number of participants, in which none of the participants seemed to have performed all the conditions, is both a strength and a potential weakness. Their current approach of including (presumably) naive participants and having each do a portion of the experiments in itself is valid. But it also adds to the complexity of their study and presumably adds variability to their data.

Reviewer #1 (Public Review):

Pelentritou and colleagues investigated the brain's ability to infer temporal regularities in sleep. To do so, they measured the effect on brain and cardiac activity to the omission of an expected sound. Participants were presented with three different categories of sounds: fixed sound-to-sound intervals (isochronous), fixed heartbeat-to-sound intervals (synchronous), and a control condition without any regularity (asynchronous). When omitting a sound, they observed a difference in the isochronous and synchronous conditions compared to the control condition, in both wakefulness and sleep (NREM stage 2). Furthermore, in the synchronous condition, sounds were temporally associated with sleep slow waves suggesting that temporal predictions could influence ongoing brain dynamics in sleep. Finally, at the level of cardiac activity, the synchronous condition was associated with a deceleration of cardiac frequency across vigilance states. Overall, this work suggests that the sleeping brain can learn temporal expectations and responds to their violation.

Major strengths and weaknesses:<br /> The paradigm is elegant and robust. It represents a clever way to investigate an important question: whether the sleeping brain can form and maintain predictions during sleep. Previous studies have so far highlighted the lack of evidence for predictive processes during sleep (e.g. (Makov et al., 2017; Strauss et al., 2015; Wilf et al., 2016)). This work shows that at least a certain type of prediction still takes place during sleep.

However, there are some important aspects of the methodology and interpretations that appear problematic.<br /> (1) The methodology and how it compares to previous articles would need to be clarified. For example, the Methods section indicates that the authors used a right earlobe electrode as a reference. This is quite different from the nose reference used by SanMiguel et al. (2013) or in Dercksen et al. (2022). This could affect the polarity and topographies of the OEP or AEP and thus represents a very significant difference. Likewise, SOs are typically detected in a montage reference to the mastoids. Perhaps the left/right asymmetries present in many plots (e.g. Figure 3) could be due to the right earlobe reference used. Also, the authors did not use the same filters in wakefulness and sleep, which could introduce an important bias when comparing sleep and wake results or sleep results with previous wake papers.<br /> (2) The ERP to sound omission shows significant differences between the isochronous and asynchronous conditions in wakefulness (Figure 3A and Supp. Fig.) but this difference is very different from previous reports in wakefulness. Topographies are also markedly different, which questions whether the same phenomenon is observed. For example, SanMiguel and colleagues observed an N1 in response to omitted but expected sounds. The authors argue that they observe a similar phenomenon in the iso vs baseline contrast, but the timing and topography of their effect are very different from the typical N1. The authors also mention that, within their study, wake and N2 OEPs were "largely similar" but they differ in terms of latencies and topographies (Figure 3A-B). It would be better to have a more objective way to explore differences and similarities across the different analyses of the paper or with the literature.<br /> (3) The authors applied a cluster permutation to identify clusters of significant time points. However, some aspects of this analysis are puzzling. Indeed, the authors restricted the cluster permutation to a temporal window of 0 to 350ms in wake (vs. -100 to 500ms in sleep). This can be misleading since the graphs show a larger temporal window (-100 to 500ms). Consequently, portions of this time window could show no cluster because the analysis revealed an absence of significant clusters but because the cluster permutation was not applied there. Besides, some of the reported clusters are extremely brief (e.g. l. 195, cluster's duration: 62ms), which could question their physiological relevance or raise the possibility that some of these clusters could be false positives (there was no correction for multiple comparisons across the many cluster permutations performed). Finally, there seems to be a duplication of the bar graphs showing the number of significant electrodes in the positive and first negative cluster for Figure 2 Supp. Fig. 1.<br /> (4) More generally, regarding statistics, the absence of exact p-values can render the interpretation of statistical outputs difficult. For example, the authors report a significant modulation of the sound-to-SO latency across conditions (p<0.05) but no significant effect of heartbeat peak-to-SO latency (p>0.05). They interpret this pattern of results rather strongly as evidence that the "readjustment of SOs was specific to auditory regularities and not to cardiac input". Yet, examining the reported chi-square values show very close values between the two analyses (7.9 vs. 7.4). It seems thus difficult to argue for a real dissociation between the two effects. Providing exact p-values for all statistical tests could help avoid this pitfall.

Reviewer #2 (Public Review):

This study was designed to study the cortical response to violations in auditory temporal sequences during wakefulness and sleep. To this end, the study had three levels of temporal sequence, a regular temporal sequence, an auditory tone that was yoked to the cardiac signal, and an irregular tone. The authors show significant EEG differences to an omitted tone when the auditory tone was predictable both during wakefulness and sleep.

The authors analyze the ERP to the omitted tone as well as when aligned to the R-peak of the HEP. The analysis was comprehensive and the effects reported align with the interpretation given. Of particular interest was the fact that a deceleration of the heart rate was present for omissions when the auditory tone was yoked to the R-peak (synch) in all stages of wakefulness and sleep.

However, one weakness was the rationale for the current study and how the results link to current theoretical frameworks for the role of interoception in perception and cognition. This was in contrast to the clear background and explanation to study the response to omissions for a predictable auditory sequence in wakefulness and sleep. It was unclear why the authors selected the cardiac signal to yoke their auditory stimuli. What is the specific motivation for the cardiac signal rather than the respiratory signal? This was not clear.

Reviewer #1 (Public Review):

Kozol et al adapt an important tool, in the form of the atlas, to the Astyanax research community. While broadly the atlas appears to correctly identify large brain regions, it is unclear what is the significance of the finer divisions. The external confirmations are restricted to just a few large brain regions (by independent human observer: e.g., optic tectum, hypothalamus. By molecular marker: hypothalamus only.). As such, interpretations of results from as many as 180 small subregions should be interpreted sceptically.<br /> The authors also suggest that some brain regions have increased in size during cavefish evolution (e.g., hypothalamus, subpallium). The analysis of progeny from a genetic cross of cave and surface morphs suggest a complex genetic program has evolved to control this variant set of brain structures. With the development of genetic manipulation tools in this species, an exciting series of experiments may link causal variants with brain development differences.

MAJOR ISSUES<br /> Line 85+. Segmentation accuracy is not well established by the authors.<br /> For example, Figure S2 states that the pixel correlation is high between Astyanax populations. But the details of how this cross-correlation was done are sparse. Is the Y-axis here showing the fraction of pixels that are shared in the morphs? While the annotation appears to function similarly across morphs, the 80% machine:human correlation is difficult to put into context. On the one hand, this seems low. For what values should one strive? Are there common "mistakes" or differences in human & machine annotations that lead to certain regions being excluded? A discussion of these is warranted and will be useful to others who wish to use this approach.

Line 87. "such as" is misleading since these were the only two antibodies used to confirm molecular definitions of regions.<br /> But more to the point, additional markers should be used to confirm more than just the ISL+ hypothalamic divisions.<br /> This is particularly warranted, as Fig 1d is not convincing. I believe that the yellow label is ISL; this is difficult to see in the figures. ISL is not ideal since this is widespread in the hypothalamus. There are no ISL-negative regions depicted, which would be necessary to demonstrate that the resolution of this subregion labeling tool is high. A complementary approach would be to find molecular markers that are more restricted than ISL which label only subsets of hypothalamic regions.<br /> Finally, do the mid/hindbrain ISL labeled regions correspond to known ISL+ subregions?

The molecular and human-observed confirmations of brain regions suggests that the annotated borders of gross anatomical regions are correctly identified by the algorithm. However, data is not presented that indicates whether the smaller regions correspond to biologically meaningful compartments.

Parameters used in CobraZ to perform the segmentation are not defined. More transparency is required here for others to replicate.

Reviewer #2 (Public Review):

The authors tackled a longstanding question for brain evolution: if the brain regions change based on functional constraints or developmental constraints.

The strength of this study is that the authors introduced an automated method for brain segmentation based on the zebrafish tool, which is a highly advanced technology. They also performed the volume and landmark-based shape analyses in a surface, cave and their F1 and F2 hybrid, highlighting genetic regulations, and revealed 3 genetically correlating clusters of brain regions, which are brand new as far as I know. This study needs intense effort, fine skills to conduct, and intellectual efforts to summarize the vast dataset. I simply admire how the authors achieve their study at this level.

The weakness of this study is that the method/approach used in this study is difficult to test the functional constraint hypothesis although the authors nicely tested the developmental constraint hypothesis, which was highlighted in their correlation studies (volumetric and shape: Fig 4 and 5). I am also a little concerned with the accuracy of the automated segmentation algorithm shown in Figure 1-figure supplement 2. The original zebrafish paper (CobraZ) showed a similar accuracy (cross-correlation as 80%). If this level of accuracy is accepted in the field, I am OK with it.<br /> Their data support the conclusion 'brain-wide evolution occurs in distinct developmental modules' because of their correlation study. However, I am not so positive at the point that one of two central hypotheses were directly tested in this study: the functional constraint hypothesis - to test it, for example, the authors need to address the functional connectivities (calcium imaging, etc) and then test if the correlation between calcium-transients and the size/shape of each pair of brain regions.

Reviewer #3 (Public Review):

In this manuscript the authors use novel techniques and analytical methods on an up and coming animal model for brain evolution. The manuscript utilizes the cavefish Astyanax mexicanus, which can provide future important insights into the field of neurobiology and in evolution in general.<br /> The authors however, only argue that Astyanax is a powerful system for functionally determining basic principles of brain evolution (which clearly it will be), but fail to actually describe what brain evolution insights Astyanax gives. The data is in the paper, but the interpretation needs refinement. This would be a much more valuable paper with a thorough evolutionary context based on the already existing, extensive literature. I believe this manuscript has the potential to be extremely impactful.

Reviewer #1 (Public Review):

The authors of this manuscript aimed to systematically evaluate the pleiotropic effects of MCR-1-mediated colistin resistance. They evaluated the effect of MCR-1 and MCR-3 carried on different plasmids on antimicrobial peptides (AMPs) and assessed their ultimate effect on virulence. The authors find that MCR-1-mediated colistin resistance correlates with increased resistance against some host AMPs, but also increased sensitivity to others. The authors also find that MCR-1 alone is associated with resistance to human serum and to elements of the complement system. This highlights a potential selective advantage for MCR-1-mediated resistance to host immune factors and a potential for enhanced virulence.