Reviewer #1 (Public Review):

In the study described in the manuscript, the authors identified Mecp2, a methyl-CpG binding protein, as a key regulator involved in the transcriptional shift during the exit of quiescent cells into the cell cycle. Their data show that Mecp2 levels were remarkably reduced during the priming/initiation stage of partial hepatectomy-induced liver regeneration and that altered Mecp2 expression affected the quiescence exit. Additionally, the authors identified Nedd4 E3 ligase that is required for downregulation of Mecp2 during quiescence exit. This is an interesting study with well-presented data that supports the authors' conclusions regarding the role of Mecp2 in transcription regulation during the G0/G1 transition. However, the significance of the study is limited by a lack of mechanistic insights into the function of Mecp2 in the process. This weakness can be addressed by identifying the signaling pathway(s) that trigger Mecp2 degradation during the quiescence exit.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors reported that miR-199b-5p is elevated in osteoarthritis (OA) patients. They also found that overexpression of miR-199b-5p induced OA-like pathological changes in normal mice and inhibiting miR-199b-5p alleviated symptoms in knee OA mice. They concluded that miR-199b-5p is not only a potential micro target for knee OA, but also provides a potential strategy for future identification of new molecular drugs.

Strengths:

The data are generated from both human patients and animal models. The data presented in this revised manuscript is solid and support their conclusions. The questions from reviewers are also properly addressed and the quality of this manuscript has been significantly improved.

There are no significant weaknesses identified in this revised manuscript.

Reviewer #2 (Public Review):

Summary:

The Authors identified miR-199b-5p is a potential OA target gene using serum exosomal small RNA-seq from human healthy and OA patients. Their RNA-seq results were further compared with publicly available datasets to validate their finding of miR-199b-5p. In vitro chondrocyte culture with miR-199b-5p mimic/inhibitor and in vivo animal models were used to evaluate the function of miR-199b-5p in OA. The possible genes that were potentially regulated by miR-199b-5p were also predicted (i.e., Fzd6 and Gcnt2) and then validated by using Luciferase assays.

Strengths:

(1) Strong in vivo animal models including pain tests.<br /> (2) Validate the binding of miR-199b-5p with Fzd6 and binding of miR-199b-5p with Gcnt2

The authors have addressed my concerns.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Chen et al. investigate the statistical structure of social interactions among mice living together in the ECO-Hab. They use maximum entropy models (MEM) from statistical physics that include individual preferences and pair-wise interactions among mice to describe their collective behavior. They also use this model to track the evolution of these preferences and interactions across time and in one group of mice injected with TIMP-1, an enzyme regulating synaptic plasticity. The main result is that they can explain group behavior (the probability of being together in one compartment) by a MEM that only includes pair-wise interactions. Moreover, the impact of TIMP-1 is to increase the variance of the couplings J_ij, the preference for the compartment containing food, as well as the dissatisfaction triplet index (DTI).

Strengths:

The ECO-Hab is a really nice system to ask questions about the sociability of mice and to tease apart sociability from individual preference. Moreover, combining the ECO-Hab with the use of MEM is a powerful and elegant approach that can help statistically characterize complex interactions between groups of mice -- an important question that requires fine quantitative analysis.

Weaknesses:

However, there is a risk in interpreting these models. In my view, several of the comparisons established in the current study would require finer and more in-depth analysis to be able to establish firmer conclusions (see below). Also, the current study, which closely resembles previous work by Shemesh et al., finds a different result but does not provide the same quantitative model comparison included there, nor a conclusive explanation of why their results are different. In total, I felt that some of the results required more solid statistical testing and that some of the conclusions of the paper were not entirely justified. In particular, the results from TIMP-1 require proper interaction tests (group x drug) which I couldn't find. This is particularly important when the control group has a smaller N than the drug groups.

Reviewer #1 (Public review):

The key discovery of the manuscript is that the authors found that genetically wild type females descended from Khdc3 mutants shows abnormal gene expression relating to hepatic metabolism, which persist over multiple generations and pass through both female and male lineages. They also find dysregulation of hepatically-metabolized molecules in the blood of these wild type mice with Khdc3 mutant ancestry. These data provide solid evidence further support that phenotype can be transmitted to multiple generations without altering DNA sequence, supporting the involvement of epigenetic mechanisms. The authors further performed exploratory studies on the small RNA profiles in the oocytes of Khdc3-null females, and their wild type descendants, suggesting that altered small RNA expression could be a contributor of the observed phenotype transmission, although this has not been functionally validated.

Reviewer #2 (Public review):

Summary:

This manuscript aimed to investigate the non-genetic impact of KHDC3 mutation on the liver metabolism. To do that they analyzed the female liver transcriptome of genetically wild type mice descended from female ancestors with a mutation in the Khdc3 gene. They found that genetically wild type females descended from Khdc3 mutants have hepatic transcriptional dysregulation which persist over multiple generations in the progenies descended from female ancestors with a mutation in the Khdc3 gene. This transcriptomic deregulation was associated with dysregulation of hepatically-metabolized molecules in the blood of these wild type mice with female mutational ancestry. Furthermore, to determine whether small non-coding RNA could be involved in the maternal non-genetic transmission of the hepatic transcriptomic deregulation, they performed small RNA-seq of oocytes from Khdc3-/- mice and genetically wild type female mice descended from female ancestors with a Khdc3 mutation and claimed that oocytes of wild type female offspring from Khdc3-null females has dysregulation of multiple small RNAs.

Finally, they claimed that their data demonstrates that ancestral mutation in Khdc3 can produce transgenerational inherited phenotypes.

However, at this stage and considering the information provided in the paper, I think that these conclusions are too preliminary. Indeed, several controls/experiments need to be added to reach those conclusions.

Additional context you think would help readers interpret or understand the significance of the work<br /> • Line 25: this first sentence is very strong and needs to be documented in the introduction.<br /> • Line 48: Reference 5 is not appropriate since the paper shows the remodeling of small RNA during post-testicular maturation of mammalian sperm and their sensibility to environment. Please, change it<br /> • Line 51: "implies" is too strong and should be replaced by « suggests »<br /> • Line 67: reference is missing<br /> Database, the accession numbers are lacking.<br /> • References showing the maternal transmission of non-genetically inherited phenotypes in mice via small RNA need to be added<br /> • Line 378: All RNA-Seq and small RNA-Seq data are available in the NCBI GEO

Reviewer #1 (Public Review):

Summary:

In their article entitled "Formin-like 1 beta phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse", Javier Ruiz-Navarro and co-workers address the question of the mechanisms regulating the polarization of the microtubule organizing center (MTOC) and of the multivesicular bodies (MVB) at the immunological synapse (IS) in T lymphocytes.

This work is a follow-up of previous studies published by the same team showing that TCR-stimulated protein kinase C delta(PKCdelta) phosphorylates FMNL1beta, which plays a crucial role in cortical actin reorganization at the IS, and controls MTOC/MVB polarization and thus exosome secretion by T lymphocytes at the IS.

The authors first compare the amino acid sequences of FMNL2 and of FMNL1beta, to seek similarities in the DID-DAD auto-inhibition sequences and find that the sequence surrounding S1086 in the arginine-rich DAD of FMNL1beta displays high similarity to that around S1072 in FMNL2 which is phosphorylated by PKCdelta. They then interrogate the role of the phosphorylation of S1086 in the arginine-rich DAD of FMNL1betaby introducing S1086A and S1086D mutations that, respectively, cannot be phosphorylated or mimic the phosphorylated form of FMNL1beta, in cells expressing an FMNL1 shRNA.

Using these tools, they show that:

- FMNL1beta is phosphorylated by PMA an activator of PKCs.

- The S1086A mutant of FMNL1beta does not restore the defect in MTOC and MVB polarization at the IS present in FMNL1 deficient T cells, whereas the phosphomimetic mutant does.

- Although FMNL1betaphosphorylation at S1086 is necessary, it is not sufficient for MTOC polarization, since it does not restore the defect of polarization observed in PKCdelta deficient T cells.

- FMNL1b translocates to the IS. This neither requires PKC expression nor phosphorylation of S1086.

- Phosphorylation of FMNL1betaon S1086 regulates actin remodeling at the immune synapse.

- Phosphorylation of FMNL1betaon S1086 regulates secretion of extracellular vesicles containing CD63 by T lymphocytes.

Strengths:

This work shows for the first time the role of the phosphorylation of FMNL1beta on S1086 on the regulation of the IS formation and secretion of extracellular vesicles by T lymphocytes.

Weaknesses:

Although of interest, this work has several weaknesses. First, all the experiments are performed in Jurkat T cells that may not recapitulate the regulation of polarization in primary T cells. Moreover, all the experiments analyzing the role of PKCdelta are performed in one clone of wt or PKCdelta KO Jurkat cells. This is problematic since clonal variation has been reported in Jurkat T cells. Moreover, the remodeling of F-actin at the IS lacks careful quantification as well as detailed analysis of the actin structure in mutant cells. Finally, although convincing, the defect in the secretion of vesicles by T cells lacking phosphorylation of FMNL1beta on S1086 is preliminary. It would be interesting to analyze more precisely this defect. The expression of the CD63-GFP in mutants by WB is not completely convincing. Are other markers of extracellular vesicles affected, e.g. CD3 positive?

Reviewer #2 (Public Review):

Summary:

The authors have addressed the role of S1086 in the FMNL1beta DAD domain in F-actin dynamics, MVB polarization, and exosome secretion, and investigated the potential implication of PKCdelta, which they had previously shown to regulate these processes, in FMNL1beta S1086 phosphorylation. This is based on:<br /> (1) the documented role of FMNL1 proteins in IS formation;<br /> (2) their ability to regulate F-actin dynamics;<br /> (3) the implication of PKCdelta in MVB polarization to the IS and FMNL1beta phosphorylation;<br /> (4) the homology of the C-terminal DAD domain of FMNL1beta with FMNL2, where a phosphorylatable serine residue regulating its auto-inhibitory function had been previously identified.

They demonstrate that FMNL1beta is indeed phosphorylated on S1086 in a PKCdelta-dependent manner and that S1086-phosphorylated FMNL1beta acts downstream of PKCdelta to regulate centrosome and MVB polarization to the IS and exosome release. They provide evidence that FMNL1beta accumulates at the IS where it promotes F-actin clearance from the IS center, thus allowing for MVB secretion.

Strengths

The work is based on a solid rationale, which includes previous findings by the authors establishing a link between PKCdelta, FMNL1beta phosphorylation, synaptic F-actin clearance, and MVB polarization to the IS. The authors have thoroughly addressed the working hypotheses using robust tools. Among these, of particular value is an expression vector that allows for simultaneous RNAi-based knockdown of the endogenous protein of interest (here all FMNL1 isoforms) and expression of wild-type or mutated versions of the protein as YFP-tagged proteins to facilitate imaging studies. The imaging analyses, which are the core of the manuscript, have been complemented by immunoblot and immunoprecipitation studies, as well as by the measurement of exosome release (using a transfected MVB/exosome reporter to discriminate exosomes secreted by T cells).

Weaknesses

The data on F-actin clearance in Jurkat T cells knocked down for FMNL1 and expressing wild-type FMNL1 or the non-phosphorylatable or phosphomimetic mutants thereof would need to be further strengthened, as this is a key message of the manuscript. Also, the entire work has been carried out on Jurkat cells. Although this is an excellent model easily amenable to genetic manipulation and biochemical studies, the key finding should be validated on primary T cells.

Reviewer #3 (Public Review):

In this important work, the authors show compelling evidence that the Rapid Alkalinisation Factor1 (RALF1) peptide acts as an interlink between pectin methyl esterification status and FERONIA receptor-like kinase in mediating extracellular sensing. Moreover, the RALF1-mediated pectin perception is surprisingly independent of LRX-mediated extracellular sensing in roots. The authors also show that the peptide directly binds demethylated pectin and the positively charged amino acids are required for pectin binding as well as for its physiological activity.

Some present findings are surprising; previously, the FERONIA extracellular domain was shown to bind pectin directly, and the mode of operation in the pollen tube involves the LRX8-RALF4 complex, which seems not the case for RALF1 in the present study. Although some aspects remain controversial, this work is a very valuable addition to the ongoing debate about this elusive complex regulation and signaling.

The authors drafted the manuscript well, so I do not have a lot of criticism or suggestions. The experiments are well-designed, executed, and presented, and they solidly support the authors' claims.

Reviewer #1 (Public Review):

Summary:

Rößling et al., report in this study that the perception of RALF1 by the FER receptor is mediated by the association of RALF1 with deesterified pectin, contributing to the regulation of the cell wall matrix and plasma membrane dynamics. In addition, they report that this mode of action is independent from the previously reported cell wall sensing mechanism mediated by the FER-LRX complex.

This manuscript reproduces and aligns with the results from a recently published study (Liu et al., Cell) where they also report that RALF1 can interact with deesterified pectin, forming coacervates and promoting the recruitment of LLG-FER at the membrane.

Reviewer #2 (Public Review):

Summary:

The study by Rößling et al. addresses the link between the biochemical constitution of the cell wall, in particular the methylesterification state of pectin with signalling induced by the extracellular RALF peptide. The work suggests that only in the presence of demethylesterifies pectin, RALF is able to trigger activation of its receptor FERONIA (FER).

Remarkably, the application of RALF peptides leads to rather dramatic FER-dependent changes in wall integrity and plasma membrane invaginations not observed before. Interestingly, RALF can be out-titrated from the wall by short pectin fragments. In addition, the study provides further evidence for multiple FER-dependent pathways by showing the presence of LRX proteins is not required for the pectin/RALF mediated signalling.

Strengths:

This work provides fundamental insight into a complex emerging pathway, or perhaps several pathways, linking pectin sensing, pectin structure and RALF/FER signalling. The study provides convincing evidence that pectin methylesterase activity is required for RALF sensing, indicating that the physical interaction of RALFs with the cell wall is important for signalling. Beyond that, the study documents very clearly how profoundly RALF signalling can affect cell wall integrity and membrane topology.

Weaknesses:

The genetic material used by the authors to strengthen the connection of RALF signalling and PME activity might not be as suitable as an acute inhibition of PME activity.

The PMEI3ox line generated by Peaucelle et al., 2008 is alcohol-inducible. Was expression of the PMEI induced during the experiments? As ethanol inducible systems can be rather leaky, it would not be surprising if PME activity would be reduced even without induction, but maybe this would warrant testing whether PMEI3 is actually overexpressed and/or whether PME activity is decreased. On a similar note, the PMEI5ox plants do not appear to show the typical phenotype described for this line. I personally don't think these lines are necessary to support the study. Short-term interference with PME activity (such as with EGCG) might be more meaningful than life-long PMEI overexpression, in light of the numerous feedback pathways and their associated potential secondary effects. This might also explain why EGCG leads to an increase in pH, as one would expect from decreased PME activity, while PMEI expression (caveats from above apply) apparently does not (Fig 3A-D).

At least at first sight, the observation that OGs are able to titrate RALF from pectin binding seems at odds with the idea of cooperative binding with low affinity, leading to high avidity oligomers. Perhaps the can provide a speculative conceptual model of these interactions?

I could not find a description of the OG treatment/titration experiments, but I think it would be important to understand how these were performed with respect to OG concentration, timing of the application, etc.

Reviewer #1 (Public Review):

Summary:

The authors report evidence for a microprotein of AtHB2-miP. The authors came across HB2 in a screen for alternative transcription start sites in Arabidopsis in response to white light or a white light followed by a far red light representative of shade. Out of 337 potential microproteins, authors selected AtHB2. At the beginning of the manuscript, it is investigated that an alternative transcription start site of HB2 gene can be used in response to far red light. The resulting shorter protein form seems to interact with HB2 protein forms, altering the localization of HB2 in transient expression assays. The functionality of HB2-miP overexpression has been addressed in transgenic Arabidopsis lines using a 35S promoter. The responses and phenotypes were compared with either WT or various types of athb2 mutant lines with disrupted HB2 gene. Such mutants and the 35S promoter-driven AtHB2-miP line showed various types of phenotypes versus each other that can be classified as mild or none, e.g. small effects on root growth, iron homeostasis gene expression, and iron contents.

Strengths:

The authors performed an interesting screen for alternative transcription start sites which resulted in 337 candidates (Figure 1A). Principally, it can be interesting to find that plants may use alternative start sites for HB2 in response to shading light. The authors provide evidence that alternative transcription start sites of HB2 can be present and used in response to FR. The possibility that potentially resulting small protein may have effects under FR light, causing alteration of root growth and physiology, is an interesting idea.

Weaknesses:

In the present manuscript, there are several signs of incomplete analysis.

(1) The transient expression experiments are not conducted with much detail to demonstrate that indeed HB2 miP is produced and can interact with regular protein. The localization of HB2 was found to be linked with condensates, but perhaps not in the presence of HB2 miP. Clearly, the lack of quantitative and qualitative analysis hampers a clear assessment of this point.

(2) The authors, unfortunately, did not provide the data of the screen to demonstrate which concrete candidates may have miPs and whether there is enrichment of certain functions. There is no supplemental table accompanying Figure 1A.

(3) One of the major unclear points that is also not addressed in the discussion is that the function of miR is studied in overexpression plants (35S promoter::miP). The effects are only compared to wild type and various lines of HB2 knockouts or knockdowns, partly with fairly uncharacterized phenotypes. It can now not be clearly determined whether the miP effects are due to a regular function of miP or due to overexpression of it. A needed control would be a 35S::AtHB2 line, or better at least two different lines (only a single miP overexpression line investigated). Since it has not been assessed by deletion mutant analysis to determine which protein parts of miP are involved in the protein regulation, it cannot be ruled out that the observed miP effects are not naturally occurring but the result of ectopic expression of a protein. Clearly, the effect of miP would be ideally studied in an environment where the levels can be controlled and the resulting phenotypes and protein levels quantified.

(4) It is not shown that the microprotein is generated in Arabidopsis in response to shade, e.g. through Western or fluorescence protein detection. The main idea that authors want to claim, namely that miP binds with regular protein and thereby controls its localization or activity has not been addressed in Arabidopsis. There are no localization experiments of HB2 protein data in the presence of miP in Arabidopsis.

(5) The plants with altered HB2 forms seem to grow well and the recorded phenotypes are rather minor. Photos are not shown. At some point, the authors discuss that there could be redundancy or that HB miP might interact with other HB proteins. However, such protein interactions have not been experimentally investigated.

Reviewer #2 (Public Review):

The first portion of the manuscript centered on identifying and confirming the ATHB2 microprotein (ATHB2miP), which constitutes the core message of this study. Overall, I find no issue with the selection criteria employed for identifying alternative microprotein mRNA transcripts. However, I do have some queries that I hope the authors can address for clarity.

(1) Upon reviewing the supplemental dataset where the authors listed the 377 unique novel miPs, along with those specifically in WL or shade treatments, I sought to comprehend the rationale behind focusing on ATHB2. Have the authors examined the shade response of all 377 potential microprotein candidates? Readers may be intrigued to learn how many of these candidates exhibit induction or repression under shade conditions, and whether such changes correlate positively or negatively with alterations in the full-length TSSs in response to shade. Essentially, I aim to discern the prevalence of microprotein production during shade responses and any shared characteristics among these microprotein transcripts. This inquiry also aims to uncover the existence of a common mechanism regulating microprotein transcription.

(2) To confirm that ATHB2miP stems from an independent transcription event, the authors sequenced full-length cDNAs using PacBio isoseq. However, I find the information regarding isoseq missing from the manuscript. My assumption is that the full-length cDNAs were reverse transcribed from mRNAs isolated from whole seedlings, where mature mRNAs in the cytoplasm predominate, making it challenging to evaluate whether a specific mRNA undergoes post-transcriptional processing. One approach to confirming ATHB2miP as a product of independent transcription involves examining nascent mRNA produced in the nucleus. The authors may need to isolate nascent mRNAs associated with RNA Polymerase II in the nucleus from seedlings treated with shade for 45 min, and then perform reverse transcription and PacBio isoseq.

(3) The authors noted the identification of two potential start codons, TTG and CTG, in the alternative TSS of ATHB2 using TISpredictor. Yet, it's imperative to identify the actual translation initiation site and the full-length sequence of ATHB2miP. I suggest the authors fuse an epitope tag (e.g., 3xFLAG) to the C-terminus of ATHB2 (utilizing the genomic sequence of ATHB2) and generate transgenic lines to be treated with shade to induce ATHB2miP-3xFLAG production. Affinity purification (anti-FLAG beads) and mass spectrometry can then identify the actual start site of ATHB2miP. This step is crucial, as the current ATHB2miP used may not be the exact sequence, and any observed phenotype could be artifacts arising from these lines.

(4) My confusion arose when analyzing the results in Figures 1E - G. The authors didn't specify whether these plants were subjected to shade treatment. What are the sequences within the second intron and third exon excluded from pATHB2control::GUS that promote transcription and translation? Have the authors examined the sequence features? This information is pivotal and related to the above question #1 because it may tell us whether the sequence feature is shared by other miP candidates.

The latter part of the manuscript focused on the functional characterization of ATHB2miP. The approaches adopted by the authors resemble those used in studying antimorphic (dominant negative) alleles. However, I have several concerns regarding the approaches and conclusions.

(5) Firstly, as mentioned in question #3, the authors did not map the actual translation initiation site of ATHB2miP. Therefore, all constructs involving ATHB2miP, such as eGFP-ATHB2miP, BD-ATHB2miP, and mCherry-ATHB2miP in Figure 2, and 35S::miP in Figures 3-5, may contain extra amino acids in the N-terminus, given that epitope tags were all added to the N terminus. These additional amino acids could potentially impact the behavior of ATHB2miP and lead to artifacts. Identifying the translation initiation site in ATHB2miP would facilitate the development of tools to disrupt ATHB2miP expression without affecting full-length ATHB2 expression. For instance, if the "CTG" before the leucine zipper domain is confirmed as the translation initiation site, mutating it to another Leu codon (e.g., TTA) could generate transgenic lines using the genomic sequence of ATHB2, including this mutation, to evaluate the impact of losing ATHB2miP on shade responses.

(6) Another concern pertains to the 35S::miP line utilized in Figures 3-5. The authors only presented results from one 35S::miP line, raising the possibility of T-DNA insertion disrupting an endogenous gene in the transgenic plant genome. It is essential to clarify how many individual T1 plants were generated and how many of them showed the same phenotype as the line used in the manuscript. Additionally, the use of the constitutive CaMV35S promoter could generate artifacts akin to neomorphic mutations. For example, the authors identified Cluster 1 genes that were only induced in 35S::miP, but not in t-athb2 or WT plants (Figure 3B); moreover, they found an overrepresentation of genes involved in root development in this cluster. This observation correlated well with the root phenotype of 35S::miP under the proximity shade (Figure 4D), in which the short-root phenotype was only observed in lines expressing 35S::miP. These data could be artifacts due to the constitutive expression of ATHB2miP in roots but didn't necessarily reflect the natural function of ATHB2miP.

(7) Furthermore, I seek clarification regarding the rationale behind employing different shade conditions, including deep shade, canopy shade, and proximity shade, and the significance of treating plants with these conditions. The results were challenging to interpret, and I have reservations about some statements made. The authors claimed that ATHB2 acts as a growth repressor in deep shade but a growth promoter in the canopy and proximity shade (Lines 366-368). However, it appears that regardless of the shade conditions, most mutant and transgenic lines were not significantly different from WT (Figure 4C). Additionally, the definition of proximity shade in this manuscript (R:FR = 0.06) differs from that in Roig-Villanova & Martinez-Garcia (Front. Plant Sci., 2016; R:FR, 0.5-0.3). Clarity on this disparity would be appreciated.

(8) In Figure 5, no statistical analyses were presented in Figure 5C. It remains unclear whether the differences observed are statistically significant. Moreover, the values appear quite similar among all three genotypes. Even if statistically significant, do these minor differences in Fe concentrations significantly impact plant physiology? Additionally, some statements related to Figure 5 do not align with the data presented. For instance, claims about longer hypocotyls in t-athb2, athb2∆, and atbh2∆LZ mutants compared to wild type under shade conditions on high iron media (lines 453-455) were not supported by the data in Figure 5D. Similarly, statements about the differences between mutants (lines 458-460) were not substantiated by the data.

Reviewer #3 (Public Review):

Summary and Strengths:

In this interesting manuscript, the authors identify a large number of alternative transcription start sites (TSS) and focus their functional analysis on an alternative TSS that is expected to produce a micro-protein (miP) encoding the C-terminus of ATHB2 (ATHB2miP). ATHB2miP is expected to comprise the leucine zipper part of ATHB2 and hence interact with the full-length protein through this dimerization motif. Such interactions are shown using yeast two-hybrid and FRET-FLIM assays. ATHB2 is a well-known shade-induced gene that has been implicated in shade-regulated growth responses. The authors then test the potential role for ATHB2miP genetically by comparing several athb2 loss-of-function (LOF) alleles: one does not express either full-length ATHB2 or the short ATHB2miP (t-ATHB2), two CRISPR alleles give rise to frameshift mutations in the full-length transcript but still express a potentially functional short ATHB2miP (athb2deltaLZ and athb2delta). The authors also use plants that over and ectopically express ATHB2miP (35S:miP). Overall, the results are consistent with the hypothesis that ATHB2miP inhibits the function of ATHB2, which constitutes a novel negative feedback loop. Potentially ATHB2miP may also inhibit the activity of other related HD ZIP proteins (based on 35S:miP). The effects of these genetic alterations on shade-regulated hypocotyl growth are relatively modest. Effects on root growth are also investigated and in one intriguing case, the negative feedback model does not appear to explain the data (Figure 4D, effect on lateral roots, because for this phenotype 35S:miP is very different from the lof alleles). The authors also identify a potentially interesting link between shade-regulated hypocotyl growth and iron uptake. A number of text changes and corrections to the figures would be important for clarity. They primarily concern three issues: names of the alleles, names of the studied shade conditions, and statements about significant differences between genotypes. Also, it would be interesting to know whether the effects of ATHB2 on iron uptake are due to local effects of ATHB2. Is ATHB2 expressed in roots?

Weaknesses:

(1) The naming of the different shade conditions is difficult to follow and not consistent with the way most authors in the field call such conditions. Deep shade is ok (low PAR and low R/FR, WL, PAR 13microE, R/FR 0.13). This condition is clearly defined for experiments in Figure 4. However, data in Figure 1 also use Deep shade (line 174) but PAR is not defined there. I suggest that all light conditions are clearly defined in the figure legends and in the M&M (not the case in this ms). Regarding Canopy shade (WL, PAR 45microE, R/FR 0.15) and proximity shade (WL, PAR 45microE, R/FR 0.06), see lines 355-357, this nomenclature is unclear. First proximity shade has a higher R/FR ratio than canopy shade. Second for canopy shade (compared to the WL control) PAR should decrease which is not what is done here. What is called proximity shade and canopy shade are 2 WL conditions with different R/FR ratios, which are compared to WL controls with the same PAR. It would make more sense to call them proximity shade and indicate the different R/FR ratios. Finally, extensive literature from many plant species and numerous labs has shown that hypocotyl elongation increases with R/FR decreasing. In the data shown in Figure 4, it is the opposite. Hypocotyls in Canopy shade (WL, PAR 45microE, R/FR 0.15) are longer than those in proximity shade (WL, PAR 45microE, R/FR 0.06), while with these R/FR ratios the opposite is expected. Could this be a mistake in the text? Please check.

(2) In several instances (in particular regarding data from Figures 4 and 5), the authors write that 2 genotypes are significantly different while the statistical analysis of the data does not support such statements. For example lines 392-395, the authors write that in WL the t-DNA mutant, both CRISPR mutants and 35S:miP lines all had significantly lower number of lateral roots than the WT. This is true for the t-DNA mutant (group bc, while the WT is in group a), however, all other genotypes are in group ab, hence not significantly different from the WT. Please carefully check all such statements about significant differences.

(3) The naming of the CRISPR mutants is problematic. In particular athb2delta, such a name suggests that the gene is deleted (also suggested by Figure 4A), which is not the case in this CRISPR allele leading to a frameshift early in the coding sequence. This is particularly problematic because in this allele ATHB2miP is still expressed, while based on such a name one would expect that in this mutant both the full length and the miP are lost. Both CRISPR alleles lead to a frameshift and this should be clarified in Figure 4A and in the text.

(4) Overall hypocotyl growth phenotypes of athb2 lof mutants and 35S:miP are similar and consistent with a model according to which ATHB2miP inhibits the full-length protein. However, this is not the case for the root phenotype described in 4D. It would be interesting to discuss this.

(5) The authors propose a role for ATHB2 in the root, in particular linked to iron uptake. Is this due to a local effect of ATHB2 in the roots? Is ATHB2 expressed in roots? It would be very informative if the authors would show such data, e.g. using the reporter lines used in Figure 1. Are both the FL and the miP expressed in roots?

(6) From the description regarding 5'PEAT.seq data presented in Figure 1 (see lines 174-177) it is not clear in which light conditions the seedlings were grown. It appears that samples were collected in 3 conditions. WL and after 45 and 90 minutes of low R/FR treatment. However, then the data is discussed collectively. Does the 12398 TSS correspond to what was found in all three conditions together? Are the authors showing shade-regulation of TSS? This is clearly the case for ATHB2miP. This needs to be clarified.

(7) The way gene expression of low F/FR effects is done might conflate circadian effects and low R/FR effects because the samples from different light conditions are not collected at the same ZT. This is how I understood the text. If I'm wrong please clarify the text. If I am right, this potential problem should be mentioned in the text.

(8) Could the authors envisage a way to genetically test the role of ATHB2miP by using an allele that makes the full length but not the miP? Currently, the authors use lof alleles that either make none of the transcripts (t-DNA) or potentially only the miP (CRISPR alleles). Overall, these alleles do not appear to differ in their phenotypes, suggesting that most of the effect of ATHB2miP is through ATHB2 FL. Having an allele only producing the FL would be nice (but technically I'm not sure how one could do that).

Reviewer #1 (Public Review):

Summary:

In their manuscript entitled 'The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition', Kavaklıoğlu and colleagues delve into the role of L1TD1, an RNA binding protein (RBP) derived from a LINE1 transposon. L1TD1 proves crucial for maintaining pluripotency in embryonic stem cells and is linked to cancer progression in germ cell tumors, yet its precise molecular function remains elusive. Here, the authors uncover an intriguing interaction between L1TD1 and its ancestral LINE-1 retrotransposon.

The authors delete the DNA methyltransferase DNMT1 in a haploid human cell line (HAP1), inducing widespread DNA hypo-methylation. This hypomethylation prompts abnormal expression of L1TD1. To scrutinize L1TD1's function in a DNMT1 knock-out setting, the authors create DNMT1/L1TD1 double knock-out cell lines (DKO). Curiously, while the loss of global DNA methylation doesn't impede proliferation, additional depletion of L1TD1 leads to DNA damage and apoptosis.

To unravel the molecular mechanism underpinning L1TD1's protective role in the absence of DNA methylation, the authors dissect L1TD1 complexes in terms of protein and RNA composition. They unveil an association with the LINE-1 transposon protein L1-ORF1 and LINE-1 transcripts, among others.

Surprisingly, the authors note fewer LINE-1 retro-transposition events in DKO cells than in DNMT1 KO alone.

Strengths:

The authors present compelling data suggesting the interplay of a transposon-derived human RNA binding protein with its ancestral transposable element. Their findings spur interesting questions for cancer types, where LINE1 and L1TD1 are aberrantly expressed.

Weaknesses:

Suggestions for refinement:

The initial experiment, inducing global hypo-methylation by eliminating DNMT1 in HAP1 cells, is intriguing and warrants a more detailed description. How many genes experience misregulation or aberrant expression? What phenotypic changes occur in these cells? Why did the authors focus on L1TD1? Providing some of this data would be helpful to understand the rationale behind the thorough analysis of L1TD1.

The finding that L1TD1/DNMT1 DKO cells exhibit increased apoptosis and DNA damage but decreased L1 retro-transposition is unexpected. Considering the DNA damage associated with retro-transposition and the DNA damage and apoptosis observed in L1TD1/DNMT1 DKO cells, one would anticipate the opposite outcome. Could it be that the observation of fewer transposition-positive colonies stems from the demise of the most transposition-positive colonies? Further exploration of this phenomenon would be intriguing.

Reviewer #2 (Public Review):

In this study, Kavaklıoğlu et al. investigated and presented evidence for the role of domesticated transposon protein L1TD1 in enabling its ancestral relative, L1 ORF1p, to retrotranspose in HAP1 human tumor cells. The authors provided insight into the molecular function of L1TD1 and shed some clarifying light on previous studies that showed somewhat contradictory outcomes surrounding L1TD1 expression. Here, L1TD1 expression was correlated with L1 activation in a hypomethylation-dependent manner, due to DNMT1 deletion in the HAP1 cell line. The authors then identified L1TD1-associated RNAs using RIP-Seq, which displays a disconnect between transcript and protein abundance (via Tandem Mass Tag multiplex mass spectrometry analysis). The one exception was for L1TD1 itself, which is consistent with a model in which the RNA transcripts associated with L1TD1 are not directly regulated at the translation level. Instead, the authors found the L1TD1 protein associated with L1-RNPs, and this interaction is associated with increased L1 retrotransposition, at least in the contexts of HAP1 cells. Overall, these results support a model in which L1TD1 is restrained by DNA methylation, but in the absence of this repressive mark, L1TD1 is expressed and collaborates with L1 ORF1p (either directly or through interaction with L1 RNA, which remains unclear based on current results), leads to enhances L1 retrotransposition. These results establish the feasibility of this relationship existing in vivo in either development, disease, or both.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Thronlow Lamson et al., the authors develop a "beads-on-a-string" or BOAS strategy to link diverse hemagglutinin head domains, to elicit broadly protective antibody responses. The authors are able to generate varying formulations and lengths of the BOAS and immunization of mice shows induction of antibodies against a broad range of influenza subtypes. However, several major concerns are raised, including the stability of the BOAS, that only 3 mice were used for most immunization experiments, and that important controls and analyses related to how the BOAS alone, and not the inclusion of diverse heads, impacts humoral immunity.

Strengths:

Vaccine strategy is new and exciting.

Analyses were performed to support conclusions and improve paper quality.

Weaknesses:

Controls for how different hemagglutinin heads impact immunity versus the multivalency of the BOAS.

Only 3 mice were used for most experiments.

There were limited details on size exclusion data.

Reviewer #2 (Public Review):

Summary:

The authors describe a "beads-on-a-string" (BOAS) immunogen, where they link, using a non-flexible glycine linker, up to eight distinct hemagglutinin (HA) head domains from circulating and non-circulating influenzas and assess their immunogenicity. They also display some of their immunogens on ferritin NP and compare the immunogenicity. They conclude that this new platform can be useful to elicit robust immune responses to multiple influenza subtypes using one immunogen and that it can also be used for other viral proteins.

Strengths:

The paper is clearly written. While the use of flexible linkers has been used many times, this particular approach (linking different HA subtypes in the same construct resembling adding beads on a string, as the authors describe their display platform) is novel and could be of interest.

Weaknesses:

The authors did not compare to individuals HA ionized as cocktails and did not compare to other mosaic NP published earlier. It is thus difficult to assess how their BOAS compare.

Other weaknesses include the rationale as to why these subtypes were chosen and also an explanation of why there are different sizes of the HA1 construct (apart from expression). Have the authors tried other lengths? Have they expressed all of them as FL HA1?

Reviewer #3 (Public Review):

This work describes the tandem linkage of influenza hemagglutinin (HA) receptor binding domains of diverse subtypes to create 'beads on a string' (BOAS) immunogens. They show that these immunogens elicit ELISA binding titers against full-length HA trimers in mice, as well as varying degrees of vaccine mismatched responses and neutralization titers. They also compare these to BOAS conjugated on ferritin nanoparticles and find that this did not largely improve immune responses. This work offers a new type of vaccine platform for influenza vaccines, and this could be useful for further studies on the effects of conformation and immunodominance on the resulting immune response. 

Overall, the central claims of immunogenicity in a murine model of the BOAS immunogens described here are supported by the data. 

Strengths included the adaptability of the approach to include several, diverse subtypes of HAs. The determination of the optimal composition of strains in the 5-BOAS that overall yielded the best immune responses was an interesting finding and one that could also be adapted to other vaccine platforms. Lastly, as the authors discuss, the ease of translation to an mRNA vaccine is indeed a strength of this platform. 

One interesting and counter-intuitive result is the high levels of neutralization titers seen in vaccine-mismatched, group 2 H7 in the 5-BOAS group that differs from the 4-BOAS with the addition of a group 1 H5 RBD. At the same time, no H5 neutralization titers were observed for any of the BOAS immunogens, yet they were seen for the BOAS-NP. Uncovering where these immune responses are being directed and why these discrepancies are being observed would constitute informative future work. 

There are a few caveats in the data that should be noted: 

(1) 20 ug is a pretty high dose for a mouse and the majority of the serology presented is after 3 doses at 20 ug. By comparison, 0.5-5 ug is a more typical range (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380945/, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9980174/). Also, the authors state that 20 ug per immunogen was used, including for the BOAS-NP group, which would mean that the BOAS-NP group was given a lower gram dose of HA RBD relative to the BOAS groups. 

(2) Serum was pooled from all animals per group for neutralization assays, instead of testing individual animals. This could mean that a single animal with higher immune responses than the rest in the group could dominate the signal and potentially skew the interpretation of this data. 

(3) In Figure S2, it looks like an apparent increase in MW by changing the order of strains here, which may be due to differences in glycosylation. Further analysis would be needed to determine if there are discrepancies in glycosylation amongst the BOAS immunogens and how those differ from native HAs.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:<br /> - The authors can do a few additional experiments to test the role of ATG6 in plant immunity.<br /> I recommend the authors to test the interaction between ATGs and other NPR1 homologs (such as NPR2).

-The concentration of SA used in the experiment (0.5-1 mM) seems pretty high. Does a lower concentration of SA induce ATG6 accumulation in the nucleus?

-Does the silencing of ATG6 affect the cell death (or HR) triggered by AvrRPS4?

-SA and NPR1 are also required for immunity and are activated by other NLRs (such as RPS2 and RPM1). Is ATG6 also involved in immunity activated by these NLRs?

Reviewer #2 (Public Review):

Summary:

The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.

However, the overall conclusions of the study are not well supported experimentally. The significance of the findings is low because of their mostly correlational nature, and lack of consistency with earlier reports on the same protein.

Based on the integrity and quality of the data as well as the depth of analysis, it is not yet clear if ATG6 is a specific regulator of NPR1 or if it is affecting NPR1's stability indirectly, through inducing an elevation of SA levels in plants. As such, the current study demonstrates a correlation between overexpression of ATG6, SA accumulation, and NPR1 stability, however, whether and how these components work together is not yet demonstrated.

Based on the provided biochemical data, it is not yet clear if the ATG6 functions specifically through NPR1 or through its paralogs NPR3 and NPR4, which are negative regulators of immunity. It is quite possible that interaction with NPR1 (or any NPR) is not the major regulatory step in the activity of ATG6 in plant immunity. The effect of ATG6 on NPR1 could well be indirect, through a change in the SA level and redox environment of the cell during the immune response. Both SA level and redox state of the cell were reported to induce accumulation of NPR1 in the nucleus and increase in stability.

Another major issue is the poor quality of the subcellular analyses. In contradiction to previous studies, ATG6 in this study is not localized to autophagosome puncta, which suggests that the soluble localization pattern presented here does not reflect the true localization of ATG6. Even if the authors propose a novel, non-canonical nuclear localization for ATG6, they still should have detected the canonical autophagy-like localization of this protein.

for - book review - The Birth and Death of Meaning - Ernest Becker

Reviewer #1 (Public Review):

In this study, the authors reported that disruption of the X-linked ciliary protein OFD in the cranial neural crest-derived cells (CNCCs) leads to a migration defect in the CNCCs and that aberrant CNCCs abnormally differentiate into osteoblasts due to a lack of Hh signal. Furthermore, CNCC defects lead to the failure of mesoderm-derived cells to differentiate into myoblasts and instead result in abnormal differentiation of mesoderm-derived cells into adipocytes. The Ofd cko mouse model has a very striking phenotype and nicely mimics the phenotype of human patients, making it a very valuable model to understand human disease.

Reviewer #2 (Public Review):

In this study, the authors report that both mice and human patients carrying function-disrupting mutations in the OFD1 gene exhibited ectopic brown adipose tissue formation in the malformed tongue. The OFD1 gene is located on the X-chromosome and encodes a protein product required for the formation and function of the primary cilium, which is required for cells to properly receive and activate several signaling pathways, particularly the hedgehog signaling pathway. Loss of OFD1 function causes prenatal lethality of male fetuses and mosaic disruption of tissues in females due to random inactivation of the X-chromosome carrying either the mutant or wildtype allele. Using cell type-specific gene inactivation and genetic lineage labeling, the manuscript shows that the ectopic brown adipose tissue in the mutant tongue was not derived from cranial neural crest cells (CNCCs). Additional genetic and embryological studies led to the conclusion that loss of Ofd1 function in the CNCC cells in the embryonic hypoglossal cord, via which the tongue myoblast precursor cells migrate from anterior somites to the tongue primordia, caused disruption of cell-cell interactions between the CNCCs and migrating muscle precursor cells, resulting in altered differentiation of those myoblast precursor cells into brown adipocytes. The authors provided data that disruption of Smo in a subset of CNCCs also resulted in ectopic adipose tissue formation in the tongue, indicating that this phenotype in the Ofd1 mutant mice was likely caused by disruption of hedgehog signaling in CNCCs. However, no experimental evidence is provided to support a major conclusion of the manuscript regarding altered differentiation of the tongue myoblast precursor cells into brown adipocytes in the Ofd1 mutant mice. Since it is well established that hedgehog signaling in the CNCCs is required for them to direct tongue myoblast cell migration as well as for tongue muscle differentiation/organization after the myoblasts arrived in the tongue primordia, the finding of tongue muscle defects in the Ofd1 mutant mice is not surprising. However, if proven true that disruption of Ofd1 function in CNCCs caused tongue myoblast precursor cells to alter their fate and differentiate into brown adipocytes, it would be an interesting new finding. Further identification of the signals produced by the Ofd1 mutant CNCCs for directing the cell fate switch will be a highly significant new advance in understanding the cellular and molecular mechanisms regulating tongue morphogenesis.

Reviewer #3 (Public Review):

The authors observed phenotypes of ciliopathy model mice and they seem to coincide with those in human patients. They used mutants in which cilial function genes are deleted in cranial neural crest cells, and found the mutants exhibit abnormal cell differentiation in both neural crest- and mesoderm-lineage cells. The finding clearly shows the importance of tissue/cell interaction. The authors mainly observed the mouse in which Ofd1 gene that is coded on the X chromosome is deleted, therefore, Ofd1fl/WT;Wnt1Cre(HET) mice show that about one-fourth of neural crest cells can exhibit Ofd1 function whereas Ofd1fl;Wnt1Cre (HM) shows null Ofd1 function and show severer phenotypes than HET.

For ectopic brown adipose tissue in the tongue is derived from mesoderm and the authors tried to show that the hypoglossal cord failed to obtain myogenic lineage after entering branchial arches in HET and HM due to lack of communication with neural crest cells. For ectopic bone formation, they found that it is due to the lack of Hedgehog signaling in neural crest cells, which was consistent with the reports in the Smofl/fl;Wnt1-Cre (Xu et al., 2019) and Ift88fl/fl;Wnt1Cre (Kitamura et al. 2020). The ectopic bone is connected to the original mandibular bone. The authors attribute the ectopic bone formation to the migration of mandibular bone neural crest cells into the tongue-forming area.

For the poor tongue frenum formation, the authors found the importance of cell migration from the lateral sides of the branchial arch to the midline and its formation relies on non-canonical Wnt signaling. The authors observed similar phenotypes in the human patients as those in the mutants. The adipose tissue in the tongue area is normally found in the salivary gland region and intermuscular space, and it is intriguing to find the brown adipose tissue anterior to the cervical area in which the most anterior brown adipose tissue develops. qRT-PCR indicates that some of the marker genes are expressed in the laser micro-dissected sections of the ectopic brown adipose tissue. However, histology does not show the typical brown adipose tissue feature. In addition, brown adipose tissue is normally recognized in the sixth pharyngeal region as the cervical brown tissue from around E14.5 (Schulz and Tseng 2013), not E12 as the authors observe. Although the mutants develop under abnormal conditions, is it possible to say they are brown adipose tissue? The point has to be further investigated with more marker expression by immunohistochemical detection and other methods. Since the mutants seem to show impaired midline formation (which is consistent with the condition of human ciliopathy), is it possible to hypothesize that the adipose-like tissue is derived from the mesoderm of posterior branchial arch levels if the tissue is brown adipose tissue?

Cranial neural crest cells start migrating around E8.0 and reach their destination by E9.5. The authors show the lack of neural crest cells in the midline, the fluorescence is absent from the midline in HM, however, they studied it in the E11 mandible (Fig. 4E), almost more than two days after neural crest migration completes. Since the mandibular arch seems to form at the beginning in the mutants, is there a failure in allocating the neural crest and mesoderm at the beginning of the mandibular arch formation?<br /> The authors tried to disturb the interaction between the hypoglossal cord and neural crest cells by making incisions in the dorsal area of the branchial arches. That area contains both neural crest and mesoderm but not the hypoglossal cord-derived mesoderm. The hypoglossal cord passed through the posterior edge of the caudal (6th) pharyngeal arch, along the lateral side of the pericardium towards the anterior, ventral to branchial arches, and then inside the 2nd and 1st branchial arches (Adachi et al., 2018). It expresses Pax3 before entering the branchial arches, then Myf5 in the branchial arches. It seems that the migration of the hypoglossal cord does not require interaction with neural crest cells but it has to be confirmed as well as neural crest migration into the branchial arches from the beginning. Although the hypoglossal cord migrates mostly in mesoderm-derived mesenchyme, we cannot exclude the possibility that hypoglossal cord migration is affected.

The lack of Myf5 expression in Ofd1fl;Wnt1Cre (HM) was explained as a failure in the differentiation of the hypoglossal cord into myoblasts on entrance into the branchial arches. Most of the cervical brown adipose tissue is derived from either Myf5- or Pax3- expressing lineage (Sanchez-Gurmaches and Guertin, 2014). Although the authors suggest that brown adipose cells are fate-changed mesoderm in the branchial arches, how do they explain the association with Myf5- or Pax3- expression?

In addition, the cervical brown tissue is supposed to be derived from the branchial arch mesoderm (Mo et al., 2017). Is the formation of the cervical brown tissue affected in the Ofd1fl/WT;Wnt1Cre(HET) or Ofd1fl;Wnt1Cre (HM) if dysfunction of neural crest cells results in the cell fate change of mesoderm?

For the tongue frenum development, it is hard to understand to hypothesize that its formation is unlikely to associate with midline formation. Although Lgr5 and Tbx22 are not expressed in the midline, the defect in midline formation could cause unnecessary interaction between the right and left tissues.

Tissue morphogenesis takes place in three dimensions, which were not considered in the data, especially in the labeling experiments. When the authors labelled the cells, which cells in which area were labelled? In the textbook, tongue formation is a result of the fusion of the midline processes derived from the branchial arches, therefore, it is important to identify which cells in which area are labelled.

The weakest point is that the authors demonstrate many interesting phenotypes but fail to show the mechanism of altered cell differentiation and direct evidence of the tissue origin of ectopic brown tissue. Without the data, suggestion from the authors' argument is weak, which is reflected in the conclusion of the abstract.

Reviewer #1 (Public Review):

Recent work reported that the AP2-associated kinase 1 (AAK1) downregulates Wnt signaling by phosphorylating, thus activating, the µ-subunit of the AP2 complex (AP2M1), which recognizes an endocytic signal on the intracellular domain of the Wnt co-receptor LRP6 leading to its internalization (Agajanian, et al., 2018). It has also long been known that DPY-23/AP2M1 and the retromer complex, which controls trafficking between endosomes and the trans-golgi network and recycling from endosomes to the plasma membrane, regulate Wnt signaling in C. elegans, at least in part by modulating trafficking of the Wnt-secretion factor MIG-14/WLS (Pan, et al., 2008; Yan et al., 2008).

Here the authors first set out to ask whether SEL-5/AAK1 plays a conserved role in Wnt signaling via phosphorylation of DPY-23/AP2M1 by assessing the function of SEL-5 in Wnt-regulated morphogenetic events; specifically, the well-characterized migration and polarization of several neurons and the less-understood process of excretory canal cell outgrowth.

The authors found that the simultaneous removal of sel-5 and the retromer complex gene vps-29 resulted in synthetic neuronal and excretory canal outgrowth phenotypes, indicating that sel-5 and the retromer complex function in parallel in these processes. Genetic interactions between sel-5 and Wnt pathway components were also examined, and for QL neuroblast migration, loss of sel-5 exacerbated phenotypes caused by loss of the Wnt receptor LIN-17/FZD, but not those caused by loss of a different receptor, MIG-1/FZD. The authors assessed the site of sel-5 function in neuronal migration defects via tissue-specific rescue and identified the hypodermis, a known source of Wnt ligands, and muscles as sites where sel-5/AAK1 activity is required.

The novelty in this work comes from the discovery of a function for sel-5/AAK1 and the retromer complex in excretory canal outgrowth, identified by phenotypes caused by simultaneous loss of sel-5 and retromer components. This synthetic phenotype is rescued by restoring sel-5 to either the excretory canal cell or the hypodermis, suggesting autonomous and non-autonomous functions for sel-5 in canal outgrowth. The authors also confirmed previous results showing that loss of LIN-17/FZD results in excretory canal overgrowth, and by carrying out an extensive survey of Wnt-pathway mutants they discovered that LIN-44/Wnt is likely the ligand that functions via LIN-17 as a "stop" signal in canal outgrowth. They also implicate a CWN-1/Wnt-CFZ-2/FZD pathway as required for canal outgrowth and find genetic interactions between sel-5/AAK1 and the lamellipodin ortholog mig-10, suggesting that these genes function in parallel to promote excretory canal outgrowth.

The most intriguing claim in this work is the suggestion that neither DPY-23 phosphorylation nor SEL-5 kinase activity is required for their function in Wnt signaling. However, the tools used to support these conclusions are not well-characterized. First, a new dpy-23 phosphorylation site-mutant is not genetically characterized, thus it is difficult to interpret the negative results obtained with this allele. Second, although the mutations introduced into SEL-5 are expected to abolish kinase activity, this is not demonstrated biochemically, nor are the effects, if any, of mutations on protein stability/localization assessed. Finally, experiments testing the function of SEL-5 kinase mutants are reported using only one multi-copy extrachromosomal array per construct. Because these types of transgenes vastly overexpress proteins, it is likely that even proteins with reduced function will rescue, raising concerns regarding the conclusion that kinase activity is not necessary for SEL-5 function.

In conclusion, it is not clear that the findings presented here will be of great general interest, as they mostly support previously-known functions for SEL-5/AAK1, DPY-23/AP2M1, and the retromer complex in Wnt-mediated signaling. Thus, this work will mainly be of interest to researchers studying Wnt-mediated cell outgrowth, and more specifically to those studying the C. elegans excretory canal. Moreover, the study lacks coherence: initially, there is a clear hypothesis testing a role for SEL-5/AAK1 in DPY-23/AP2M1 phosphorylation and how this impinges on Wnt signaling. This model appears to be refuted (although, as noted above the tools used to do this need to be better validated), but the authors do not explore alternative targets or functions for SEL-5/AAK1, nor do they directly assess how SEL-5 or the retromer complex impinge on Wnt signaling in excretory canal outgrowth. Thus, there is little mechanistic insight provided by this work.

Reviewer #2 (Public Review):

Summary<br /> This study by Knop, et al. defines two different developmental roles for the conserved SEL-5/AAK1 protein kinase in Caenorhabditis elegans. In other organisms, AAK1 was known to promote the recycling of the Wntless sorting receptor and endocytosis of Wnt receptors. This study establishes that SEL-5 acts in two roles in C. elegans: in Wnt-producing cells, a role that promotes migration of a neuroblast termed QL.d, and in Wnt-receiving cells, a role that promotes outgrowth of the excretory cell (EXC). Before this study, SEL-5/AAK1 was thought to regulate endocytosis through phosphorylation of AP2M1 and other endocytic adaptor proteins. This study shows convincing data that the SEL-5 makes a partial contribution to AP2M1 phosphorylation, and more surprisingly, that its roles in Wnt-producing and Wnt-receiving cells of C. elegans do not require SEL-5 catalytic activity. Human AAK1 was previously suggested to be a target of drug design efforts due to its roles in neuropathic pain, viral infection, and Alzheimer's disease. The discovery that some roles for SEL-5/AAK1 are independent of catalytic activity will be of broad interest to cell biologists and biochemists.

Strengths<br /> (1) The data establishing the requirement for SEL-5 in QL.d migration and EXC outgrowth (Fig. 1 and Fig. 4) is rigorous and convincing. My assessment of the rigor is based on the following: First, the authors show that two independently derived sel-5 deletion mutations result in defects in QL.d and EXC. Second, the authors show that providing wild-type, GFP-tagged SEL-5 results in significant rescue of both phenotypes. Importantly, they use tissue-specific transgenes to show that the requirement for SEL-5 in QL.d migration is non-cell-autonomous, and the requirement for SEL-5 in EXC outgrowth is cell-autonomous (Fig. 2). For rescue experiments, they show that each tissue-specific transgene is indeed expressed strongly in the tissue of interest. This establishes the roles for SEL-5 in two different roles, in Wnt-producing and Wnt-receiving cells.

(2) The authors present three lines of convincing biochemical and genetic evidence that SEL-5 kinase catalytic activity is not important for its roles in Wnt-producing and Wnt-receiving cells.

Taking a biochemical approach, they use quantitative Westerns to assess the degree of AP2M1 phosphorylation in sel-5 mutants (Fig. 3). Their results show that AP2M1 phosphorylation is diminished, but not absent in mutants. Their results are convincing because they make use of GFP-tagged AP2M1 to probe for total and phospho-AP2M1. I note that they included uncropped Western blots in supplemental data. Furthermore, they make use of a GFP-tagged AP2M1 mutant (T160A) to confirm which residue is phosphorylated. Their results suggest that some mechanism other than AP2M1 phosphorylation may account for the sel-5 mutant phenotypes.

Taking a genetic approach, they make use of a unique allele, dpy-23(mew25), that alters the known AP2M1 phosphorylation site. They show that animals carrying this allele do not display the QL.d and EXC phenotypes (Fig. 3 and Fig. 5). Finally, in a more direct test of whether SEL-5 requires catalytic activity, they make use of GFP-tagged SEL-5 forms mutated at either the active site or the ATP-binding site of the SEL-5 kinase domain. They show that either SEL-5 mutant form successfully rescues the QL.d and EXC defects seen in sel-5 mutants (Fig. 3), suggesting that SEL-5 catalytic activity is unnecessary.

(3) The authors have produced an elegant GFP knock-in allele of the sel-5 gene, allowing analysis of expression and localization in living animals (Fig. 2).

(4) The authors make use of genetic interactions with Wnt signaling mutants to show that SEL-5 acts in a role that promotes Wnt signaling for the QL.d cell (Fig. 1) and counteracts Wnt signaling for the EXC (Fig. 5).

Weaknesses<br /> (1) Some changes to statistical analyses are needed in this study.

Fig. 1B, 1D, 2A, 3E, and 3F report the QL.d phenotype as a percentage of animals scored that were defective in migration. The methods make it clear this data is categorical rather than quantitative. Therefore, a t-test or any test designed for quantitative data is not appropriate. I suggest that the authors should investigate using a chi-squared or Fisher's exact test.

For the reasons mentioned above, the calculation of standard deviation (as shown in error bars) is also not appropriate for Fig. 1B, 1D, 2A, 3E, and 3F. Of course, it is excellent that the authors scored multiple trials. For experiments with mutants, I suggest the authors might combine these trials or show separate results of each trial. For experiments using RNAi (Fig. 1B), each trial should be plotted separately because RNAi effectiveness can vary. If there is not enough space to show multiple trials, then I would ask that a representative trial be shown in the main figure and additional trials in a supplement.

In Fig. 1, 2, 3, and 5, it is not specified whether/how p-values were adjusted for multiple tests.

(2) I felt the author's interpretation of the sel-5 mutant phenotypes in EXC, and the genetic interactions with Wnt signaling mutants, might be improved. The authors show convincing data that the sel-5 mutants display a shortened EXC outgrowth phenotype. Conversely, mutants with reduced Wnt signaling, such as the lin-17 or lin-44 mutants, displayed lengthened EXC outgrowth. The authors show that in double mutants, loss of sel-5 partially suppressed the EXC overgrowth defects of lin-17 or lin-44 mutants (Fig. 5). In my opinion, this data is consistent with a model where SEL-5 acts to inhibit Wnt signaling in EXC. An inhibitory role in a Wnt-receiving cell would be consistent with the known activity for human AAK1 in promoting negative feedback and endocytosis of LPR6. Interestingly, the authors mention in their discussion that a mutant of plr-1, which acts in the internalization of Frizzled receptors, has a shortened EXC phenotype similar to that of sel-5 mutants. These observations all seem consistent with an inhibitory role, yet the authors do not state this as their conclusion. A clarification of their interpretation is needed.

Impact/significance<br /> (1) Among researchers using C. elegans, this study provides a foundation for further investigation of the role of endocytosis, SEL-5, and the retromer, in Wnt trafficking. It is particularly useful that the authors define two different phenotypes that arise from Wnt-producing and Wnt-receiving cells.

(2) Among a broader community of cell biologists and biochemists, this study will be of interest in its finding that SEL-5/AAK1 kinase catalytic activity is unnecessary for the regulation of Wnt signaling.

Reviewer #1 (Public Review):

Salt-inhibited germination and growth in Arabidopsis and other plant species. Here the authors demonstrated that part of that inhibitory effect is caused by the arginine-derived urea hydrolysis, a novel mechanism. They also postulated that urea transport is involved in germination inhibition, but they do not link urea transport from cotyledons to pH changes in roots. At last, they generalized the mechanisms to other glycophytic crops and halophytic plants, but the salt concentration used is the same for the four groups, which are supposed to have very different salt tolerance ranges, questioning the validity of this generalization.<br /> Overall, the authors have provided well-organized genetic and pharmacological evidence to support most of their conclusions.

Reviewer #2 (Public Review):

Urea is widely utilized in agriculture. In this study, the authors the mechanism underlying the adverse impact of urea on seed germination and seedling growth under salt stress conditions. The results show that salt stress induces a pronounced hydrolysis of urea, resulting in an elevation of cytoplasmic pH and subsequent inhibition of seed germination. These findings challenge the previous notion that ammonium accumulation is the primary cause of salt-induced inhibition of germination, thereby offering novel insights into this process.

The authors have provided well-organized genetic or biochemical evidence to support most of their conclusions.

Reviewer #3 (Public Review):

This work submitted by Bu et al. investigated mechanisms of how salt stress-induced arginine catabolism, which is catalyzed by arginase and urease, inhibits seed germination and seedling growth in Arabidopsis using a combination of genetic, biochemical, and live-cell imaging approaches. Their results showed that the two steps for the turnover of arginine into ammonia and the transport of urea from the cotyledon to the root are required for the salt-induced inhibition of seed germination (SISG). Further analysis showed that the cellular accumulation of the end product ammonia is not associated with SISG, but it is the cytoplasmic alkaline stress that primarily causes SISG. Interestingly, they found that the mechanism underlying SISG is conserved in other plant species. In general, this work will be valuable for plant biologists to deeply dissect the complex mechanism that controls salt stress-induced inhibition of plant growth and development in the future.

The conclusions derived from this work are well supported by the data, but some aspects of data analysis need to be clarified and extended.

(1) Inhibition of arginine hydrolysis by enzyme inhibitors (NOHA for arginase and PPD for urease) significantly improved seed germination and seedling growth (Figure 2). It seems that the suppressive effect of NOHA against the salt-induced inhibition of seedling growth is dose-dependent (Figure 2b). Whether NOHA effect on SISG is also dose-dependent and application of a certain level of NOHA can fully rescue the phenotype of SISG remains to be answered. The answers may help to explain the genetic data shown in Figure 3c, where either single (argah1 and argah2) or double (argah1/argah2) mutants partially rescued the phenotype of SISG. However, arginase activity, particularly in argah1 and argah2, is not closely correlated to the phenotype shown in Figure 3c and 3d.

(2) The data shown in Figure 4b and 4e were not fully consistent. The percentage of seed germination rate was about 70% when treated with the highest concentration (7.5 μM) of PPD, but was less than 40% for the aturease mutant.

(3) Cellular pH values detected at the seed germination stage were not convincing. In the text, they did not describe the results showing that the cytoplasmic pH values in hypocotyl and cotyledon cells were alkaline and not affected by NaCl treatment, and PPD treatment only restored the alkaline cytoplasmic pH to that of the control (Figure 7b). This raises two questions: is it true that cytoplasmic pH values are different between root and cotyledon/hypocotyl cells under normal growth conditions? and does PPD treatment alter the cytoplasmic pH only in roots?

Reviewer #1 (Public Review):

HMCV encodes various immunoevasins to inhibit being presented by MHC class I molecules to the cytotoxic cells of the immune system. Here, the authors studied the role and specificity of US10, a relatively uncharacterized immunoevasin from HCMV. They found that US10 differentially affects antigen presentation by different MHC class I allotypes. HLA-A and certain HLA-B and C alleles (so-called "tapasin-independent") were unaffected, while other HLA-B and C alleles (so-called "tapasin-dependent") as well as HLA-G were negatively affected. US10 can bind to different MHC class I allotypes, which inhibits their incorporation into peptide loading complex and slowers maturation. By comparing US10 to the other well-studied immunoevasins from HCMV, US2, US3, and US11, the authors demonstrated only partial overlap between them suggesting the cumulative action of immunoevasins in inhibiting MHC class I antigen presentation of HMCV epitopes. This work contributes to our understanding of the complex immune evasion mechanism by HCMV.

The strengths include using a broad use of available techniques, including overexpression of US10 and US10 siRNA in the infection context that allowed comparison of its net and cumulative effects. Bioinformatic analysis of US10 and US11 to describe how transcription and expression of these two gene products contribute to the control of immunoevasion by HCMV. The conclusions are mostly supported by the experiments.

Reviewer #2 (Public Review):

The manuscript entitled " Multimodal HLA-I genotypes regulation by human cytomegalovirus US10 and resulting surface patterning" by Gerke et al describes the biochemical analysis of US10-mediated down regulation of HLA-I molecules. The authors systemically examine the surface expression of different HLA-I alleles in cells expressing US10 and interactions of US10 with HLA-I and antigen presentation machinery. Further, studies examined genotypic and allotypic differences during expression of US10/US11 transcripts suggest a different allelic class I downregulation. In general, the authors have included data supporting the major claims. Yet, the conclusions and findings of the study only marginally advance the overall understanding of HCMV viral evasion and the mechanism of US10 function.

Strengths:<br /> The studies are well characterized and the studies utilize diverse HLA-I and HCMV viral molecules. The biochemistry is excellent and is of high quality. Importantly, the study describes HLA-I allelic specific HCMV down regulation at the cell surface and molecular levels.

Weaknesses:<br /> (1) The authors use over expressive language such as "strong binding" that does not have a quantitative value and it is relative to the specific assay with only small differences among the factors.<br /> (2) The US10 binding to the HLA-I did not correlate with class I surface levels suggesting that binding to the APC machinery (Figure 1); hence, why does the binding of US10 to the APC define its mechanism of action.<br /> (3) The innovative and significant aspects of the study are limited. The study does not delineate the US10 mechanism of action or show data in which US10-mediated MHC class I down regulation impacts adaptive or innate immune function.

Reviewer #3 (Public Review):

Correlation of the HLA-B effects with previously demonstrated allelic differences in dependence on the peptide loading complex (PLC) component chaperone/editor tapasin and demonstration that US10 does not bind the PLC reflect on possible mechanisms of US10 function. Thus, this paper adds new information that may be integrated into evolving models of the steps of MHC-I dependent antigen presentation and how viruses counter immune recognition for their own benefit. Clearer focus on the proposed models for the function of US10 and its mechanism--i.e. what experiments address the mechanism and what additional finding might clarify the mechanism would be helpful.

Reviewer #2 (Public Review):

Summary:

The interplay between environmental factors and cognitive performance has been a focal point of neuroscientific research, with illuminance emerging as a significant variable of interest. The hypothalamus, a brain region integral to regulating circadian rhythms, sleep, and alertness, has been posited to mediate the effects of light exposure on cognitive functions. Previous studies have illuminated the role of the hypothalamus in orchestrating bodily responses to light, implicating specific neural pathways such as the orexin and histamine systems, which are crucial for maintaining wakefulness and processing environmental cues. Despite advancements in our understanding, the specific mechanisms through which varying levels of light exposure influence hypothalamic activity and, in turn, cognitive performance, remain inadequately explored. This gap in knowledge underscores the need for high-resolution investigations that can dissect the nuanced impacts of illuminance on different hypothalamic regions. Utilizing state-of-the-art 7 Tesla functional magnetic resonance imaging (fMRI), the present study aims to elucidate the differential effects of light on the hypothalamic dynamics and establish a link between regional hypothalamic activity and cognitive outcomes in healthy young adults. By shedding light on these complex interactions, this research endeavors to contribute to the foundational knowledge necessary for developing innovative therapeutic strategies aimed at enhancing cognitive function through environmental modulation.

Strengths:

(1) Considerable Sample Size and Detailed Analysis:<br /> The study leverages a robust sample size and conducts a thorough analysis of hypothalamic dynamics, which enhances the reliability and depth of the findings.

(2) Use of High-Resolution Imaging:<br /> Utilizing 7 Tesla fMRI to analyze brain activity during cognitive tasks offers high-resolution insights into the differential effects of illuminance on hypothalamic activity, showcasing the methodological rigor of the study.

(3) Novel Insights into Illuminance Effects:<br /> The manuscript reveals new understandings of how different regions of the hypothalamus respond to varying illuminance levels, contributing valuable knowledge to the field.

(4) Exploration of Potential Therapeutic Applications:<br /> Discussing the potential therapeutic applications of light modulation based on the findings suggests practical implications and future research directions.

Weaknesses:

(1) Foundation for Claims about Orexin and Histamine Systems:<br /> The manuscript needs to provide a clearer theoretical or empirical foundation for claims regarding the impact of light on the orexin and histamine systems in the abstract.

(2) Inclusion of Cortical Correlates:<br /> While focused on the hypothalamus, the manuscript may benefit from discussing the role of cortical activation in cognitive performance, suggesting an opportunity to expand the scope of the manuscript.

(3) Details of Light Exposure Control:<br /> More detailed information about how light exposure was controlled and standardized is needed to ensure the replicability and validity of the experimental conditions.

(4) Rationale Behind Different Exposure Protocols:<br /> To clarify methodological choices, the manuscript should include more in-depth reasoning behind using different protocols of light exposure for executive and emotional tasks.

Reviewer #1 (Public Review):

Summary:

Campbell et al investigated the effects of light on the human brain, in particular the subcortical part of the hypothalamus during auditory cognitive tasks. The mechanisms and neuronal circuits underlying light effects in non-image forming responses are so far mostly studied in rodents but are not easily translated in humans. Therefore, this is a fundamental study aiming to establish the impact light illuminance has on the subcortical structures using the high-resolution 7T fMRI. The authors found that parts of the hypothalamus are differently responding to illuminance. In particular, they found that the activity of the posterior hypothalamus increases while the activity of the anterior and ventral parts of the hypothalamus decreases under high illuminance. The authors also report that the performance of the 2-back executive task was significantly better in higher illuminance conditions. However, it seems that the activity of the posterior hypothalamus subpart is negatively related to the performance of the executive task, implying that it is unlikely that this part of the hypothalamus is directly involved in the positive impact of light on performance observed. Interestingly, the activity of the posterior hypothalamus was, however, associated with an increased behavioural response to emotional stimuli. This suggests that the role of this posterior part of the hypothalamus is not as simple regarding light effects on cognitive and emotional responses. This study is a fundamental step towards our better understanding of the mechanisms underlying light effects on cognition and consequently optimising lighting standards.

Strengths:

While it is still impossible to distinguish individual hypothalamic nuclei, even with the high-resolution fMRI, the authors split the hypothalamus into five areas encompassing five groups of hypothalamic nuclei. This allowed them to reveal that different parts of the hypothalamus respond differently to an increase in illuminance. They found that higher illuminance increased the activity of the posterior part of the hypothalamus encompassing the MB and parts of the LH and TMN, while decreasing the activity of the anterior parts encompassing the SCN and another part of TMN. These findings are somewhat in line with studies in animals. It was shown that parts of the hypothalamus such as SCN, LH, and PVN receive direct retinal input in particular from ipRGCs. Also, acute chemogenetic activation of ipRGCs was shown to induce activation of LH and also increased arousal in mice.

Weaknesses:

While the light characteristics are well documented and EDI calculated for all of the photoreceptors, it is not very clear why these irradiances and spectra were chosen. It would be helpful if the authors explained the logic behind the four chosen light conditions tested. Also, the lights chosen have cone-opic EDI values in a high correlation with the melanopic EDI, therefore we can't distinguish if the effects seen here are driven by melanopsin and/or other photoreceptors. In order to provide a more mechanistic insight into the light-driven effects on cognition ideally one would use a silent substitution approach to distinguish between different photoreceptors. This may be something to consider when designing the follow-up studies.

Reviewer #3 (Public Review):

Summary:

Campbell and colleagues use a combination of high-resolution fMRI, cognitive tasks, and different intensities of light illumination to test the hypothesis that the intensity of illumination differentially impacts hypothalamic substructures that, in turn, promote alterations in arousal that affect cognitive and affective performance. The authors find evidence in support of a posterior-to-anterior gradient of increased blood flow in the hypothalamus during task performance that they later relate to performance on two different tasks. The results provide an enticing link between light levels, hypothalamic activity, and cognitive/affective function, however, clarification of some methodological choices will help to improve confidence in the findings.

Strengths:

* The authors' focus on the hypothalamus and its relationship to light intensity is an important and understudied question in neuroscience.

Weaknesses:

* I found it challenging to relate the authors' hypotheses, which I found to be quite compelling, to the apparatus used to test the hypotheses - namely, the use of orange light vs. different light intensities; and the specific choice of the executive and emotional tasks, which differed in key features (e.g., block-related vs. event-related designs) that were orthogonal to the psychological constructs being challenged in each task.

* Given the small size of the hypothalamus and the irregular size of the hypothalamic parcels, I wondered whether a more data-driven examination of the hypothalamic time series would have provided a more parsimonious test of their hypothesis.

Reviewer #3 (Public Review):

Summary:

In this study, Han and co-authors showed that implantation of Pik3ca deficient KPC cells (aKO) induced clonal expansion of CD8 T cells in the tumor microenvironment. Using aKO cells, they conducted an in vivo genome-wide gene-deletion screen, which showed that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (p-aKO) leads to immune evasion and tumor progression. Eventually, mice injected with p-aKO but not aKO succumbed to their tumors. Similar to the parental aKO cell line, p-aKO tumors were still infiltrated with clonally expanded CD8+ and CD4+ T cells, as shown by the IHC. Further analyses showed that T cells infiltrating p-aKO tumors expressed high levels of exhaustion markers (PD-1, CTLA-4, TIM3, and TIGIT). Furthermore, PD-1 signaling blockade using PD-1 mAb or genetic depletion of PD-1 reactivated the infiltrated T cells, controlling tumor progression and improving the overall mice survival. Thus, the authors concluded in the abstract that "Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers." Although the data clearly showed that the loss of Pccb facilitated the immune evasion of pancreatic cancer cells, there is no clear evidence provided that Pccb deletion can actually modulate the activity of CD8 T cells. One may argue that the deletion of Pccb reduces the immunogenicity of the p-aKO cancer cells, making them less susceptible to killing by normally functional CD8+ T cells.

Strengths:

In vivo, Crisper-Cas-9 screen using tumor cell lines.

Identify a gene that could reduce the immunogenicity of cancer cells.

Weaknesses:

The IHC technique that was used to stain and characterize the exhaustion status of the tumor-infiltrating T cells.

Reviewer #1 (Public Review):

Summary:

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that does not respond to immunotherapy. This work represents an extension of the authors' prior observation that PI3Ka deletion in an orthotopic KPC pancreatic tumor model confers susceptibility to immune-mediated elimination. The authors' major claims in the present manuscript are as follows:

(1) PI3Ka (Pik3ca) knockout in KPC pancreatic tumor cells induces clonal T cell expansion.

(2) Genome-wide LOF screen in aKPC cells to identify tumor-intrinsic determinants of PI3Ka-KO-enhanced T cell response identified Pccb.

(3) When Pccb is knocked out in the context of Pi3ka knockout KPC, anti-tumor T cell response is reduced as measured by<br /> a. Increased tumor progression<br /> b. Decreased survival<br /> c. T cells are still clonally expanded but less functional

(4) ICB is able to "reactivate" clonally expanded T cells.

(5) Conclusion: Pccb modulates the activity of T cells in PDAC.

Overall, the experiments were appropriately executed and technically sound, albeit underpowered for single-cell analyses. Upon careful consideration of the data, the biggest weakness of the paper is the authors' interpretations of results, particularly for claims 1 and 4 (see below for details). Much of the data is correlative and does not delve into causation, leaving this reviewer wishing for experiments that would clearly demonstrate that Pccb in tumor cells directly impacts T cell anti-tumor activity.

Strengths:

(1) Tumor intrinsic determinants of intratumoral T cell infiltration in PDAC are less commonly evaluated as combination therapies for ICB. This is a point of conceptual innovation and importance.

(2) A sensitized CRISPR screen to identify mutations that rescue KPC/PI3Ka-KO tumors from immune-mediated killing is an elegant method to better understand the molecular mechanisms contributing to KPC immunosurveillance. Further, one screen candidate (Pccb) was experimentally validated.

(3) Single-cell clonotype analyses hold promise for identifying tumor-reactive T cells (though authors never demonstrated that specific clones were tumor antigen specific).

Weaknesses:

(1) "Clonal expansion of cytotoxic T cells infiltrating the pancreatic αKO tumors"<br /> a. Only two tumor-bearing hosts were evaluated by single-cell TCR sequencing, thus limiting conclusions that may be drawn regarding repertoire diversity and expansion.<br /> b. High abundance clones in the TME do not necessarily have tumor specificity, nor are they necessarily clonally expanded. They may be clones which are tissue-resident or highly chemokine-responsive and accumulate in larger numbers independent of clonal expansion. Please consider softening language to clonal enrichment or refer to clone size as clonal abundance throughout the paper.<br /> c. The whole story would be greatly strengthened by cytotoxicity assays of abundant TCR clones to show tumor antigen specificity.

(2) "A genome-wide CRISPR gene-deletion screen to identify molecules contributing to Pik3ca-mediated pancreatic tumor immune evasion"<br /> a. CRISPR mutagenesis yielded outgrowth of only 2/8 tumors. A more complete screen with an increased total number of tumors would yield much stronger gene candidates with better statistical power. It is unsurprising that candidates were observed in only one of the two tumors. Nevertheless, the authors moved forward successfully with Pccb.

(3) T cells infiltrate p-αKO tumors with increased expression of immune checkpoints<br /> a. In Figure 4D, cell counts are not normalized to totalCD8+ T cell counts making it difficult to directly compare aKO to p-aKO tumors. Based on quantifications from Figure 4D, I suspect normalization will strengthen the conclusion that CD8+ infiltrate is more exhausted in p-aKO tumors.<br /> b. Flow cytometric analysis to further characterize the myeloid compartment is incomplete (single replicate) and does not strengthen the argument that p-aKO TME is more immunosuppressive.<br /> c. It could, however, strengthen the argument that TIL has less anti-tumor potential if effector molecule expression in CD8+ infiltrating cells were quantified.

(4) Inhibition of PD1/PD-L1 checkpoint leads to elimination of most p-αKO tumors<br /> a. It is reasonable to conclude that p-aKO tumors are responsive to immune checkpoint blockade. However, there is no data presented to support the statement that checkpoint blockade reactivates an existing anti-tumor CD8+ T cell response and does not instead induce a de novo response.<br /> b. The discussion of these data implies that anti-PD-1 would not improve aKO tumor control, but these data are not included. As such, it is difficult to compare the therapeutic response in aKO versus p-aKO. Further, these data are at best an indirect comparison of the T cell responsiveness against tumor, as the only direct comparison is infiltrating cell count in Figure 4 and there are no public TCR clones with confirmed anti-tumor specificity to follow in the aKO versus p-aKO response.

Reviewer #2 (Public Review):

Summary:

Pancreatic ductal adenocarcinoma is generally considered a "cold" tumor type with little T cell infiltration. This group demonstrated previously that deletion of the PIK3CA isoform of PI3K in the orthotopic pancreatic ductal adenocarcinoma KPC mouse tumor model led to the elimination of tumors by T cells. Here they performed a genome-wide gene-deletion screen in this tumor using CRISPR to determine what was required for this T cell-mediated infiltration and tumor rejection. Deletion of Pccb in the tumors, which encodes propionyl-CoA carboxylase subunit B, allowed for the outgrowth of the PIK3CA-deleted KPC tumors. This was confirmed with the specific deletion of Pccb in the tumor cells. Demonstrating a likely role in tumor progression in human patients as well, high expression of PCCB in pancreatic ductal adenocarcinoma correlated with lower patient survival. T cells still infiltrated these tumors, but had much higher expression of exhaustion markers. Blockade of PD-1 signaling allowed for the rejection of these tumors. While these are intriguing data demonstrating that loss of PCCB by pancreatic ductal adenocarcinoma is a mechanism to escape T cell immunity, the mechanism by which this occurs is not determined. In addition, there are a few issues that suggest the conclusions of the manuscript should be tempered.

Strengths:

In vivo analysis of tumor CRISPR deletion screen.

The study describes a possible novel mechanism by which a tumor maintains a "cold" microenvironment.

Weaknesses:

(1) A major issue is that it seems these data are based on the use of a single tumor cell clone with PIK3CA deleted. Therefore, there could be other changes in this clone in addition to the deletion of PIK3CA that could contribute to the phenotype.

(2) The conclusion that the change in the PCCB-deficient tumor cell line is unrelated to mitochondrial metabolic changes may be incorrect based on the data provided. While it is true that in the experiments performed, there was no statistically significant change in the oxygen consumption rate or metabolite levels, this could be due to experimental error. There is a trend in the OCR being higher in the PCCB-deficient cells, although due to a high standard deviation, the change is not statistically significant. There is also a trend for there being more aKG in this cell line, but because there were only 3 samples per cell line, there is no statistically significant difference.

(3) More data are required to make the authors' conclusion that there are myeloid changes in the PCCB-deficient tumor cells. There is only flow data from shown from one tumor of each type.

(4) The previous published study demonstrated increased MHC and CD80 expression in the PIK3CA-deficient tumors and these differences were suggested to be the reason the tumors were rejected. However, no data concerning the levels of these proteins were provided in the current manuscript.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors delineate the crucial role of the SIRT2-ACSS2 axis in ACSS2 degradation. They demonstrate that SIRT2 acts as an ACSS2 deacetylase specifically under nutrient stress conditions, notably during amino acid deficiency. The SIRT2-mediated deacetylation of ACSS2 at K271 consequently triggers its proteasomal degradation. Additionally, they illustrate that acetylation of ACSS2 at K271 enhances ACSS2 protein levels, thereby promoting De Novo lipogenesis.

Strengths:

The findings presented in this manuscript are clearly interesting.

Weaknesses:

Further support is required for the model put forward by the authors.

Reviewer #2 (Public Review):

Summary:

Karim et al investigated the regulation of ACSS2 by SIRT2. The authors identified a previously undescribed acetylation that they then show is important for the regulation and stability of ACSS2 in cells. The authors show that ACSS2 ubiquitination and degradation by the proteasome is regulated by SIRT2-mediated deacetylation of ACSS2 and that stabilizing ACSS2 by blocking SIRT2 can alter lipid accumulation in adipocytes.

Strengths:

Identification of a novel acetylation site on ACSS2 that regulates its protein stability and that has consequences on its activity in adipocytes. Multiple standard approaches were used to manipulate the expression and function of SIRT2 and ACSS2 (i.e., overexpression, knockdown, inhibitors).

Weaknesses:

The authors do not show direct deacetylation of ACSS2 by SIRT2 in an in vitro biochemical assay.

It would have been nice to have included a bona-fide SIRT2 target as a control throughout the study.

Throughout the manuscript, normalizing the data to 1 and then comparing the fold-change using a t-test is not the best statistical approach in that situation since every normalized value for control is 1 with zero standard deviation. The authors should consider an alternative statistical approach.

Though not necessary, using 13C-acetate or D3-acetate tracing would be better for understanding the impact of acetylation on the activity of ACSS2 and its impact on lipogenesis.

Did the authors also consider investigating SIRT1 in their assays? SIRT1 activates ACSS2 while SIRT2 leads to degradation of ACSS2. They should at least discuss these seemingly opposing roles of SIRT1 and SIRT2 in the regulation of ACSS2 and acetate metabolism in more depth, particularly as it concerns situations (i.e., diseases, pathologies) where either SIRT1, SIRT2, or both sirtuins, are active. This would enhance the significance of the findings to the broader research community.

In Figure 3, the authors should consider immunoblotting for endogenous ACSS2 throughout the differentiation and lipogenesis study since the total ACSS2 levels is the crucial aspect to affecting acetate-dependent promotion of lipogenesis in adipocytes, and to confirm TM-dependent stabilization of ACSS2 in that assay.

Do the authors have any data proving the K271 mutants of ACSS2 are still functional? Or that K271 ACSS2 protein is folded correctly?

Reviewer #3 (Public Review):

Summary:

The manuscript shows SIRT2 can regulate acetylation of ACSS2 at residue 271, acetylation of 271 protects ACSS2 from proteasomal degradation in a SIRT2-dependent manner. Lastly, authors show that ACSS2 acetylation at K271 promotes lipid accumulation.

Strengths:

The author provides solid data showing ACSS2 acetylation can be regulated by targeting SIRT2 and that SIRT2 regulates ACSS2 ubiquitination. They identify K271 as a site of acetylation and show this is a site when mutated alters SIRT2-mediated ubiquitination.

Weaknesses:

However, data for this manuscript seems preliminary as nearly all data is performed in one cell line, some of the conclusions are not well supported by data and the overall role of ACSS2 K271 acetylation is not well characterized.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Bell et al. provide an exhaustive and clear description of the diversity of a new class of predicted type IV restriction systems that the authors denote as CoCoNuTs, for their characteristic presence of coiled-coil segments and nuclease tandems. Along with a comprehensive analysis that includes phylogenetics, protein structure prediction, extensive protein domain annotations, and in-depth investigation of encoding genomic contexts, they also provide detailed hypothesis about the biological activity and molecular functions of the members of this class of predicted systems. This work is highly relevant, it underscores the wide diversity of defence systems that are used by prokaryotes and demonstrates that there are still many systems to be discovered. The work is sound and backed up by a clear and reasonable bioinformatics approach.

Strengths:

The analysis provided by the authors is extensive and covers the three most important aspects that can be covered computationally when analysing a new family/superfamily: phylogenetics, genomic context analysis, and protein-structure-based domain content annotation. With this, one can directly have an idea about the superfamily of the predicted system and infer about their biological role. The bioinformatics approach is sound and makes use of the most current advances in the fields of protein evolution and structural bioinformatics.

Weaknesses:

It is not clear how coiled-coil segments were assigned if only based on AF2-predicted models or also backed by sequence analysis, as no description is provided in the methods. The structure prediction quality assessment is based solely on the average pLDDT of the obtained models (with a threshold of 80 or better). However, this is not enough, particularly when multimeric models were used. The PAE matrix should be used to evaluate relative orientations, particularly in the case where there is a prediction that parts from 2 proteins are interacting. In the case of multimers, interface quality scores, as the ipTM or pDockQ, should also be considered and, at minimum, reported.

These weaknesses were addressed during revision, and the results provided by the authors support their conclusions. The data resulting from this work will be useful for the general life sciences community, particularly the prokaryotic defense and microbiology communities. It also underscores the high range of functionally unknowns in sequenced genomes that are now much easier to find and interpret due to the success of deep-learning based methods and automated robust bioinformatics pipelines.

Reviewer #2 (Public Review):

Summary:

In this work, using in-depth computational analysis, Bell et al. explore the diverse repertoire of type IV McrBC modification dependent restriction systems. The prototypical two-component McrBC system has been structurally and functionally characterised and is known to act as a defence by restricting phage and foreign DNA containing methylated cytosines. Here, the authors find previously unanticipated complexity and versatility of these systems and focus on detailed analysis and classification of a distinct branch, the so-called CoCoNut, named after its composition of coiled-coil structures and tandem nucleases. These CoCoNut systems are predicted to target RNA as well as DNA and to utilise defence mechanisms with some similarity to type III CRISPR-Cas systems.

Strengths:

This work is enriched with a plethora of ideas and a myriad of compelling hypotheses that now will await experimental verification. The study comes from the group that was amongst the first to describe, characterise, and classify CRISPR-Cas systems. By analogy, the findings described here can similarly promote ingenious experimental and conceptual research that could further drive technological advances. It could also instigate vigorous scientific debates that will ultimately benefit the community.

Weaknesses:

The multi-component systems described here function in the context of large oligomeric complexes similarly to the prototypical McrBC system. While the AlphaFold2 (AF2) multimer predictions are provided in this work, these are not compared with the known McrBC structures. These comparisons could have been helpful not only for providing insights into these multimeric protein systems but also for giving more sound explanations of the differences observed amongst different McrBC types.

Reviewer #1 (Public Review):

Summary:

Major findings or outcomes include a genome for the wasp, characterization of the venom constituents and teratocyte and ovipositor expression profiles, as well as information about Trichopria ecology and parasitism strategies. It was found that Trichopria cannot discriminate among hosts by age, but can identify previously parasitized hosts. The authors also investigated whether superparasitism by Trichopria wasps improved parasitism outcomes (it did), presumably by increasing venom and teratocyte concentrations/densities. Elegant use of Drosophila ectopic expression tools allowed for functional characterization of venom components (Timps), and showed that these proteins are responsible for parasitoid-induced delays in host development. After finding that teratocytes produce a large number of proteases, experiments showed that these contribute to digestion of host tissues for parasite consumption.<br /> The discussion ties these elements together by suggesting that genes used for aiding in parasitism via different parts of the parasitism arsenal arise from gene duplication and shifts in tissue of expression (to venom glands or teratocytes).

Strengths:

The strength of this manuscript is that it describes the parasitism strategies used by Trichopria wasps at a molecular and behavioral level with broad strokes. It represents a large amount of work that in previous decades might have been published in several different papers. Including all of these data in a manuscript together makes for a comprehensive and interesting study.

Weaknesses:

The weakness is that the breadth of the study results in fairly shallow mechanistic or functional results for any given facet of Trichopria's biology. Although none of the findings are especially novel given results from other parasitoid species in previous publications, integrating results together provides significant information about Trichopria biology.

Reviewer #2 (Public Review):

Summary:

Key findings of this research include the sequencing of the wasp's genome, identification of venom constituents and teratocytes, and examination of Trichopria drosophilae (Td)'s ecology and parasitic strategies. It was observed that Td doesn't distinguish between hosts based on age but can recognize previously parasitized hosts. The study also explored whether multiple parasitisms by Td improved outcomes, which indeed it did, possibly by increasing venom and teratocyte levels. Utilizing Drosophila ectopic expression tools, the authors functionally characterized venom components, specifically tissue inhibitors of metalloproteinases (Timps), which were found to cause delays in host development. Additionally, experiments revealed that teratocytes produce numerous proteases, aiding in the digestion of host tissues for parasite consumption. The discussion suggests that genes involved in different aspects of parasitism may arise from gene duplication and shifts in tissue expression to venom glands or teratocytes.

Strengths:

This manuscript provides an in-depth and detailed depiction of the parasitic strategies employed by Td wasps, spanning both molecular and behavioral aspects. It consolidates a significant amount of research that, in the past, might have been distributed across multiple papers. By presenting all this data in a single manuscript, it delivers a comprehensive and engaging study that could help future developments in the field of biological control against a major insect pest.

Weaknesses:

While none of the findings are particularly groundbreaking, as similar results have been reported for other parasitoid species in prior research, the integration of these results into one comprehensive overview offers valuable biological insights into an interesting new potential biocontrol species.

Reviewer #1 (Public Review):

Summary:

This study has as its goal to determine how the structure and function of the circuit that stabilizes gaze in the larval zebrafish depends on the presence of the output cells, the motor neurons. A major model of neural circuit development posits that the wiring of neurons is instructed by their postsynaptic cells, transmitting signals retrogradely on which cells to contact and, by extension, where to project their axons. Goldblatt et al. remove the motor neurons from the circuit by generating null mutants for the phox2a gene. The study then shows that, in this mutant that lacks the isl1-labelled extraocular motor neurons, the central projection neurons have 1) largely normal responses to vestibular input; 2) normal gross morphology; 3) minimally changed transcriptional profiles. From this, the authors conclude that the wiring of the circuit is not instructed by the output neurons, refuting the major model.

Strengths:

I found the manuscript to be exceptionally well-written and presented, with clear and concise writing and effective figures that highlight key concepts. The topic of neural circuit wiring is central to neuroscience, and the paper's findings will interest researchers across the field, and especially those focused on motor systems.

The experiments conducted are clever and of a very high standard, and I liked the systematic progression of methods to assess the different potential effects of removing phox2a on circuit structure and function. Analyses (including statistics) are comprehensive and appropriate and show the authors are meticulous and balanced in most of the conclusions that they draw. Overall, the findings are interesting, and with a few tweaks, should leave little doubt about the paper's main conclusions.

Weaknesses:

The main point is the incomplete characterisation of the effects of removing phox2a on the extra-ocular motor neurons. Are these cells no longer there, or are they there but no longer labelled by isl1:GFP? If they are indeed removed, might they have developed early on, and subsequently lost? These questions matter as the central focus of the manuscript is whether the presence of these cells influences the connectivity and function of their presynaptic projection neurons. Therefore, for the main conclusions to be fully supported by the data, the authors would need to test whether 1) the motor neurons that otherwise would have been labelled by the isl1:GFP line are physically no longer there; 2) that this removal (if, indeed, it is that) is developmental. If these experiments are not feasible, then the text should be adjusted to take this into account. A further point to address is the context of the manipulation. If the phox2a removal does indeed take out the extra-ocular motor neurons, what percentage of postsynaptic neurons to the projection neurons are still present? In other words, how does the postsynaptic nMLF output relate to the motor neurons? If, for instance, the nMLF (which, as the authors state, are likely still innervated by the projection neurons) are the main output of the projections neurons, then this would affect the interpretation of the results.

Reviewer #2 (Public Review):

Summary:

This study was designed to test the hypothesis that motor neurons play a causal role in circuit assembly of the vestibulo-ocular reflex circuit, which is based on the retrograde model proposed by Hans Straka. This circuit consists of peripheral sensory neurons, central projection neurons, and motor neurons. The authors hypothesize that loss of extraocular motor neurons, through CRISPR/Cas9 mutagenesis of the phox2a gene, will disrupt sensory selectivity in presynaptic projection neurons if the retrograde model is correct.

Account of the major strengths and weaknesses of the methods and results:

The work presented is impressive in both breadth and depth, including the experimental paradigms. Overall, the main results were that the loss of function paradigm to eliminate extraocular motor neurons did not 1) alter the normal functional connections between peripheral sensory neurons and central projection neurons, 2) affect the position of central projection neurons in the sensorimotor circuit, or 3) significantly alter the transcriptional profiles of central projection neurons. Together, these results strongly indicate that retrograde signals from motor neurons are not required for the development of the sensorimotor architecture of the vestibulo-ocular circuit.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The results of this study showed that extraocular motor neurons were not required for central projection neuron specification in the vestibulo-ocular circuit, which countered the prevailing retrograde hypothesis proposed for circuit assembly. A concern is that the results presented may be limited to this specific circuit and may not be generalizable to other circuit assemblies, even to other sensorimotor circuits.

Discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

As mentioned above, this study sheds valuable new insights into the developmental organization of the vestibulo-ocular circuit. However, different circuits likely utilize various mechanisms, extrinsic or intrinsic (or both), to establish proper functional connectivity. So, the results shown here, although begin to explain the developmental organization of the vestibulo-ocular circuit, are not likely to be generalizable to other circuits; though this remains to be seen. At a minimum, this study provides a starting point for the examination of patterning of connections in this and other sensorimotor circuits.

Reviewer #3 (Public Review):

In this manuscript by Goldblatt et al. the authors study the development of a well-known sensorimotor system, the vestibulo-ocular reflex circuit, using Danio rerio as a model. The authors address whether motor neurons within this circuit are required to determine the identity, upstream connectivity and function of their presynaptic partners, central projection neurons. They approach this by generating a CRISPR-mediated knockout line for the transcription factor phox2a, which specifies the fate of extraocular muscle motor neurons. After showing that phox2a knockout ablates these motor neurons, the authors show that functionally, morphologically, and transcriptionally, projection neurons develop relatively normally.

Overall, the authors present a convincing argument for the dispensability of motor neurons in the wiring of this circuit, although their claims about the generalizability of their findings to other sensorimotor circuits should be tempered. The study is comprehensive and employs multiple methods to examine the function, connectivity and identity of projection neurons.

Specific comments:

(1) In the introduction the authors set up the controversy on whether or not motor neurons play an instructive role in determining "pre-motor fate". This statement is somewhat generic and a bit misleading as it is generally accepted that many aspects of interneuron identity are motor neuron-independent. The authors might want to expand on these studies and better define what they mean by "fate", as it is not clear whether the studies they are citing in support of this hypothesis actually make that claim.

(2) Although it appears unchanged from their images, the authors do not explicitly quantitate the number of total projection neurons in phox2a knockouts.

(3) For figures 2C and 3C, please report the proportion of neurons in each animal, either showing individual data points here or in a separate supplementary figure; and please perform and report the results of an appropriate statistical test.

(4) In the topographical mapping of calcium responses (figures 2D, E and 3D), the authors say they see no differences but this is hard to appreciate based on the 3D plotting of the data. Quantitating the strength of the responses across the 3-axes shown individually and including statistical analyses would help make this point, especially since the plots look somewhat qualitatively different.

(5) The transcriptional analysis is very interesting, however, it is not clear why it was performed at 72 hpf, while functional experiments were performed at 5 days. Is it possible that early aspects of projection neuron identity are preserved, while motor neuron-dependent changes occur later? The authors should better justify and discuss their choice of timepoint. The inclusion of heterozygotes as controls is problematic, given that the authors show there are notable differences between phox2a+/+ and phox2a+/- animals; pooling these two genotypes could potentially flatten differences between controls and phox2a-/-.

(6) Projection neurons appear to be topographically organized and this organization is maintained in the absence of motor neurons. Are there specific genes that delineate ventral and dorsal projection neurons? If so, the authors should look at those as candidate genes as they might be selectively involved in connectivity. Showing that generic synaptic markers (Figure 4E) are maintained in the entire population is not convincing evidence that these neurons would choose the correct synaptic partners.

Reviewer #1 (Public Review):

Though the Norrin protein is structurally unrelated to the Wnt ligands, it can activate the Wnt/β-catenin pathway by binding to the canonical Wnt receptors Fzd4 and Lrp5/6, as well as the tetraspanin Tspan12 co-receptor. Understanding the biochemical mechanisms by which Norrin engages Tspan12 to initiate signaling is important, as this pathway plays an important role in regulating retinal angiogenesis and maintaining the blood-retina-barrier. Numerous mutations in this signaling pathway have also been found in human patients with ocular diseases. The overarching goal of the study is to define the biochemical mechanisms by which Tspan12 mediates Norrin signaling. Using purified Tspan12 reconstituted in lipid nanodiscs, the authors conducted detailed binding experiments to document the direct, high-affinity interactions between purified Tspan12 and Norrin. To further model this binding event, they used AlphaFold to dock Norrin and Tspan12 and identified four putative binding sites. They went on to validate these sites through mutagenesis experiments. Using the information obtained from the AlphaFold modeling and through additional binding competition experiments, it was further demonstrated that Tspan12 and Fzd4 can bind Norrin simultaneously, but Tspan12 binding to Norrin is competitive with other known co-receptors, such as HSPGs and Lrp5/6. Collectively, the authors proposed that the main function of Tspan12 is to capture low concentrations of Norrin at the early stage of signaling, and then "hand over" Norrin to Fzd4 and Lrp5/6 for further signal propagation. Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Strengths:

• Biochemical reconstitution of Tspan12 and Fzd4 in lipid nanodiscs is an elegant approach for testing the direct binding interaction between Norrin and its co-receptors. The proteins used for the study seem to be of high purity and quality.

• The various binding experiments presented throughout the study were carried out rigorously. In particular, BLI allows accurate measurement of equilibrium binding constants as well as on and off rates.

• It is nice to see that the authors followed up on their AlphaFold modeling with an extensive series of mutagenesis studies to experimentally validate the potential binding sites. This adds credence to the AlphaFold models.

• Table S1 is a further testament to the rigor of the study.

• Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Suggestions for improvement:

• It would be helpful to show Coomassie-stained gels of the key mutant Norrin and Tspan12 proteins presented in Figures 2E and 2F.

• Many Norrin and Tspan12 mutations have been identified in human patients with FEVR. It would be interesting to comment on whether any of the mutations might affect the Norrin-Tspan12 binding sites described in this study.

• Some of the negative conclusions (e.g. the lack of involvement of Tspan12 in the formation of the Norrin-Lrp5/6-Fzd4-Dvl signaling complex) can be difficult to interpret. There are many possible reasons as to why certain biological effects are not recapitulated in a reconstitution experiment. For instance, the recombinant proteins used in the experiment may not be presented in the correct configurations, and certain biochemical modifications, such as phosphorylation, may also be missing.

Reviewer #2 (Public Review):

This is an interesting study of high quality with important and novel findings. Bruguera et al. report a biochemical and structural analysis of the Tspan12 co-receptor for norrin. Major findings are that Norrin directly binds Tspan12 with high affinity (this is consistent with a report on BioRxiv: Antibody Display of cell surface receptor Tetraspanin12 and SARS-CoV-2 spike protein) and a predicted structure of Tspan12 alone or in complex with Norrin. The Norrin/Tspan12 binding interface is largely verified by mutational analysis. An interaction of the Tspan12 large extracellular loop (LEL) with Fzd4 cannot be detected and interactions of full-length Tspan12 and Fzd4 cannot be tested using nano-disc based BLI, however, Fzd4/Tspan12 heterodimers can be purified and inserted into nanodiscs when aided by split GFP tags. An analysis of a potential composite binding site of a Fzd4/Tspan12 complex is somewhat inconclusive, as no major increase in affinity is detected for the complex compared to the individual components. A caveat to this data is that affinity measurements were performed for complexes with approximately 1 molecule Tspan12 and FZD4 per nanodisc, while the composite binding site could potentially be formed only in higher order complexes, e.g., 2:2 Fzd4/Tspan12 complexes. Interestingly, the authors find that the Norrin/Tspan12 binding site and the Norrin/Lrp6 binding site partially overlap and that the Lrp6 ectodomain competes with Tspan12 for Norrin binding. This result leads the authors to propose a model according to which Tspan12 captures Norrin and then has to "hand it off" to allow for Fzd4/Lrp6 formation. By increasing the local concentration of Norrin, Tspan12 would enhance the formation of the Fzd4/Lrp5 or Fzd4/Lrp6 complex.

The experiments based on membrane proteins inserted into nano-discs and the structure prediction using AlphaFold yield important new insights into a protein complex that has critical roles in normal CNS vascular biology, retinal vascular disease, and is a target for therapeutic intervention. However, it remains unclear how Norrin would be "handed off" from Tspan12 or Tspan12/Fzd4 complexes to Fzd4/Lrp6 complexes, as the relatively high affinity of Norrin to Fzd4/Tspan12 dimers likely does not favor the "handing off" to Fzd4/Lrp6 complexes.

Areas that would benefit from further experiments, or a discussion, include:

- The authors test a potential composite binding site of Fzd4/Tspan12 heterodimers for norrin using nanodiscs that contain on average about 1 molecule Fzd4 and 1 molecule Tspan12. The Fzd4/Tspan12 heterodimer is co-inserted into the nanodiscs supported by split-GFP tags on Fzd4 and Tspan12. The authors find no major increase in affinity, although they find changes to the Hill slope, reflecting better binding of norrin at low norrin concentrations. In 293F cells overexpressing Fzd4 and Tspan12 (which may result in a different stoichiometry) they find more pronounced effects of norrin binding to Fzd4/Tspan12. This raises the possibility that the formation of a composite binding requires Fzd4/Tspan12 complexes of higher order, for example, 2:2 Fzd4/Tspan12 complexes, where the composite binding site may involve residues of each Fzd4 and Tspan12 molecule in the complex. This could be tested in nanodiscs in which Fzd4 and Tspan12 are inserted at higher concentrations or using Fzd4 and Tspan12 that contain additional tags for oligomerization.

- While Tspan12 LEL does not bind to Fzd4, the successful reconstitution of GFP from Tspan12 and Fzd4 tagged with split GFP components provides evidence for Fzd4/Tspan12 complex formation. As a negative control, e.g., Fzd5, or Tspan11 with split GFP tags (Fzd5/Tspan12 or Fzd4/Tspan11) would clarify if FZD4/Tspan12 heterodimers are an artefact of the split GFP system.

- Fzd4/Tspan12 heterodimers stabilized by split GFP may be locked into an unfavorable orientation that does not allow for the formation of a composite binding site of FZD4 and Tspan12, this is another caveat for the interpretation that Fzd4/Tspan12 do not form a composite binding site. This is not discussed.

- Mutations that affect the affinity of norrin/fzd4 are not used to further test if Fzd4 and Tspan12 form a composite binding site. Norrin R41E or Fzd4 M105V were previously reported to reduce norrin/frizzled4 interactions and signaling, and both interaction and signaling were restored by Tspan12 (Lai et al. 2017). Whether a Fzd4/Tspan12 heterodimer has increased affinity for Norrin R41E was not tested. Similarly, affinity of FZD4 M105V vs a Fzd4 M105V/Tspan12 heterodimer were not tested.

- An important conclusion of the study is that Tspan12 or Lrp6 binding to Norrin is mutually exclusive. This could be corroborated by an experiment in which LRP5/6 is inserted into nanodiscs for BLI binding tests with Norrin, or Tspan12 LEL, or a combination of both. Soluble LRP6 may remove norrin from equilibrium binding/unbinding to Tspan12, therefore presenting LRP6 in a non-soluble form may yield different results.

- The authors use LRP6 instead of LRP5 for their experiments. Tspan12 is less effective in increasing the Norrin/Fzd4/Lrp6 signaling amplitude compared to Norrin/Fzd4/Lrp5 signaling, and human genetic evidence (FEVR) implicates LRP5, not LRP6, in Norrin/Frizzled4 signaling. The authors find that Norrin binding to LRP6 and Tspan12 is mutually exclusive, however this may not be the case for Lrp5.

- The biochemical data are largely not correlated with functional data. The authors suggest that the Norrin R115L FEVR mutation could be due to reduced norrin binding to tspan12, but do not test if Tspan12-mediated enhancement of the norrin signaling amplitude is reduced by the R115L mutation. Similarly, the impressive restoration of binding by charge reversal mutations in site 3 is not corroborated in signaling assays.

Reviewer #3 (Public Review):

Brugeuera et al present an impressive series of biochemical experiments that address the question of how Tspan12 acts to promote signaling by Norrin, a highly divergent TGF-beta family member that serves as a ligand for Fzd4 and Lrp5/6 to promote canonical Wnt signaling during CNS (and especially retinal) vascular development. The present study is distinguished from those of the past 15 years by its quantitative precision and its high-quality analyses of concentration dependencies, its use of well-characterized nano-disc-incorporated membrane proteins and various soluble binding partners, and its use of structure prediction (by AlphaFold) to guide experiments. The authors start by measuring the binding affinity of Norrin to Tspan12 in nanodiscs (~10 nM), and they then model this interaction with AlphaFold and test the predicted interface with various charge and size swap mutations. The test suggests that the prediction is approximately correct, but in one region (site 1) the experimental data do not support the model. [As noted by the authors, a failure of swap mutations to support a docking model is open to various interpretations. As AlphFold docking predictions come increasingly into common use, the compendium of mutational tests and their interpretations will become an important object of study.] Next, the authors show that Tspan12 and Fzd4 can simultaneously bind Norrin, with modest negative cooperativity, and that together they enhance Norrin capture by cells expressing both Tspan12 and Fzd4 compared to Fzd4 alone, an effect that is most pronounced at low Norrin concentration. Similarly, at low Norrin concentration (~1 nM), signaling is substantially enhanced by Tspan12. By contrast, the authors show that LRP6 competes with Tspan12 for Norrin binding, implying a hand-off of Norrin from a Tspan12+Fzd4+Norrin complex to a LRP5/6+Fzd4+Norrin complex. Thanks to the authors' careful dose-response analyses, they observed that Norrin-induced signaling and Tspan12 enhancement of signaling both have bell-shaped dose-response curves, with strong inhibition at higher levels of Norrin or Tspan12. The implication is that the signaling system has been built for optimal detection of low concentrations of Norrin (most likely the situation in vivo), and that excess Tspan12 can titrate Norrin at the expense of LRP5/6 binding (i.e., reduction in the formation of the LRP5/6+Fzd4+Norrin signaling complex). In the view of this reviewer, the present work represents a foundational advance in understanding Norrin signaling and the role of Tspan12. It will also serve as an important point of comparison for thinking about signaling complexes in other ligand-receptor systems.

Reviewer #1 (Public Review):

Summary:<br /> Bendzunas, Byrne et al. explore two highly topical areas of protein kinase regulation in this manuscript. Firstly, the idea that Cys modification could regulate kinase activity. The senior authors have published some standout papers exploring this idea of late, and the current work adds to the picture of how active site Cys might have been favoured in evolution to serve critical regulatory functions. Second, BRSK1/2 are understudied kinases listed as part of the "dark kinome" so any knowledge of their underlying regulation is of critical importance to advancing the field.

Strengths:<br /> In this study, the author pinpoints highly-conserved, but BRSK-specific, Cys residues as key players in kinase regulation. There is a delicate balance between equating what happens in vitro with recombinant proteins relative to what the functional consequence of Cys mutation might be in cells or organisms, but the authors are very clear with the caveats relating to these connections in their descriptions and discussion. Accordingly, by extension, they present a very sound biochemical case for how Cys modification might influence kinase activity in cellular environs.

Comments on revised version:

The authors have satisfactorily addressed my concerns.

Reviewer #2 (Public Review):

Summary:

In this study by Bendzunas et al, the authors show that the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys at an unusual CPE motif at the end of the activation segment function as repressive regulatory mechanisms in BSK1 and 2. They observed that mutation of the CPE-Cys only, contrary to the double mutation of the pair, increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide-mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family. Understanding the molecular mechanisms underlying kinase regulation by redox-active Cys residues is fundamental as it appears to be widespread in signaling proteins and provides new opportunities to develop specific covalent compounds for the targeted modulation of protein kinases.

The authors demonstrate that intramolecular cysteine disulfide bonding between conserved cysteines can function as a repressing mechanism as indicated by the effect of DTT and the consequent increase in activity by BSK-1 and -2 (WT). The cause-effect relationship of why mutation of the CPE-Cys only increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells is not clear to me. The explanation given by the authors based on molecular modeling and molecular dynamics simulations is that oxidation of the CPE-Cys (that will favor disulfide bonding) destabilizes a conserved salt bridge network critical for allosteric activation. However, no functional evidence of the impact of the salt-bridge network is provided. If you mutated the two main Cys-pairs (aE-CHRD and A-loop T+2-CPE) you lose the effect of DTT, as the disulfide pairs cannot be formed, hence no repression mechanisms take place, however when looking at individual residues I do not understand why mutating the CPE only results in the opposite effect unless it is independent of its connection with the T+2residue on the A-loop.

Strengths:

This is an important and interesting study providing new knowledge in the protein kinase field with important therapeutic implications for the rationale design and development of next-generation inhibitors.

Comments on revised version:

I have one remark related to question number 5 (my question was not clear enough). I meant if the authors did look at the functional relevance of the residues implicated in the identified salt-bridge network/tethers. What happens to the proteins functionally when you mutate those residues? (represented on Fig. 8).

Otherwise, the authors have satisfactorily addressed my concerns.

Reviewer #3 (Public Review):

Distant metastasis is the major cause of death in patients with breast cancer. In this manuscript, Liu et al. show that RGS10 deficiency elicits distant metastasis via epithelial-mesenchymal transition in breast cancer. As a prognostic indicator of breast cancer, RGS10 regulates the progress of breast cancer and affects tumor phenotypes such as epithelial-mesenchymal transformation, invasion, and migration. The conclusions of this paper are mostly well supported by data, but some analyses need to be clarified.

(1) Because diverse biomarkers have been identified for EMT, it is recommended to declare the advantages of using RGS10 as an EMT marker.

(2) The authors utilized databases to study the upstream regulatory mechanisms of RSG10. It is recommended to clarify why the authors focused on miRNAs rather than other epigenetic modifications.

(3) The role of miR-539-5p in breast cancer has been described in previous studies. Hence, it is recommended to provide detailed elaboration on how miR-539-5p regulates the expression of RSG10.

(4) To enhance the clarity and interpretability of the Western blot results, it would be advisable to mark the specific kilodalton (kDa) values of the proteins.

Reviewer #1 (Public Review):

Strengths

The paper has shown the expression of RGS10 is related to the molecular subtype, distant metastasis, and survival status of breast cancer. The study utilizes bioinformatic analyses, human tissue samples, and in vitro and in vivo experiments which strengthen the data. RGS10 was validated to inhibit EMT through a novel mechanism dependent on LCN2 and miR-539-5p, thereby reducing cancer cell proliferation, colony formation, invasion, and migration. The study elaborated the function of RGS10 in influencing the prognosis and biological behavior which could be considered as a potential drug target in breast cancer.

Weakness<br /> The mechanism by which the miR-539-5p/RGS10/LCN2 axis may be related to the prognosis of cancer patients still needs to be elucidated. In addition, the sample size used is relatively limited. Especially, if further exploration of the related pathways and mechanisms of LCN2 can be carried out by using organoid models, as well as the potential of RGS10 as a biomarker for further clinical translation to verify its therapeutic target effect, which will make the data more convincing.

Reviewer #2 (Public Review):

Liu et al., by focusing on the regulation of G protein-signaling 10 (RGS10), reported that RGS10 expression was significantly lower in patients with breast cancer, compared with normal adjacent tissue. Genetic inhibition of RGS10 caused epithelial-mesenchymal transition, and enhanced cell proliferation, migration, and invasion, respectively. These results suggest an inhibitory role of RGS10 in tumor metastasis. Furthermore, bioinformatic analyses determined signaling cascades for RGS10-mediated breast cancer distant metastasis. More importantly, both in vitro and in vivo studies evidenced that alteration of RGS10 expression by modulating its upstream regulator miR-539-5p affects breast cancer metastasis. Altogether, these findings provide insight into the pathogenesis of breast tumors and hence identify potential therapeutic targets in breast cancer.

The conclusions of this study are mostly well supported by data. However, there is a weakness in the study that needs to be clarified.

In Figure 2A, although some references supported that SKBR3 and MCF-7 possess poorly aggressive and less invasive abilities, examining only RGS10 expression in those cells, it could not be concluded that 'RGS10 acts as a tumor suppressor in breast cancer'. It would be better to introduce a horizontal comparison of the invasive ability of these 3 types of cells using an invasion assay.

Reviewer #1 (Public Review):

Summary:

The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation has not been explored.

Strengths:

The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound. The results are clear and support the conclusions.

Weaknesses:

(1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4 bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta-gamma is presented.

(2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

(3) Fig.1B-C: To give a standard deviation from 2 data points has no statistical significance. In this case it would be better to define as range/difference of the 2 data points.

Reviewer #2 (Public Review):

Summary:

In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibit form stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

Strengths:

The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain, and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gβγ subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

Weakness:

The set of experiments with the disulfide bond S235C/M244C caused a bit of confusion for interpretation, it should be moved into the supplement, and the text and Figure 4 were altered accordingly.

Reviewer #3 (Public Review):

Summary:

In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access of the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

Strengths:

The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42 . In their clearly-written discussion, the authors explain how PIP3, the cell membrane and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

Manuscript revision:

The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.

Reviewer #3 (Public Review):

Summary:

Studying evolutionary trajectories provides important insight in genetic architecture of adaptation and provide potential contribution to evaluating the predictability (or unpredictability) in biological processes involving adaptation. While many papers in the field address adaptation to environmental challenges, the number of studies on how genomic contexts, such as large-scale variation, can impact evolutionary outcomes adaptation is relatively low. This research experimentally evolved a genome-reduced strain for ~1000 generations with 9 replicates and dissected their evolutionary changes. Using the fitness assay of OD measurement, the authors claimed there is a general trend of increasing growth rate and decreasing carrying capacity, despite a positive correlation among all replicates. The authors also performed genomic and transcriptomic research at the end of experimental evolution, claiming the dissimilarity in the evolution at the molecular level.

Strengths:

The experimental evolution approach with a high number of replicates provides a good way to reveal the generality/diversity of the evolutionary routes.

The assay of fitness, genome, and transcriptome all together allows a more thorough understanding of the evolutionary scenarios and genetic mechanisms.

Comments on revised version:

5 in the last round of comments: When the authors mentioned no overlapping in single mutation level, I thought the authors would directly use this statement to support their next sentence about no bias of these mutations. As the author's responded, I was suspecting no overlapping for 65 mutation across the entire genome is likely to be not statistically significant. In the revised version, the authors emphasized and specified their simulation and argument in the following sentences, so I do not have questions on this point anymore.

14 in the last round of comments: As what authors responded, "short-term responses" meant transcriptional or physiological changes within a few hours after environmental or genetic fluctuation. "long-term responses" involve new compensatory mutations and selection. The point was that, the authors found that "the transcriptome reorganization for fitness increase triggered by evolution differed from that for fitness decrease caused by genome reduction." That is short vs long-term responses to genetic perturbation. Some other experimental evolution did short vs long-term responses to environmental perturbation and usually also found that the short-term responses are reverted in the long-term responses (e.g., https://academic.oup.com/mbe/article/33/1/25/2579742). I hope this explanation makes more sense. And I think the authors can make their own decisions on whether they would like to add this discussion or not.

Reviewer #1 (Public Review):

In this study, the authors explored how the reduced growth fitness, resulting from genome reduction, can be compensated through evolution. They conducted an evolution experiment with a strain of Escherichia coli that carried a reduced genome, over approximately 1,000 generations. The authors carried out sequencing, and found no clear genetic signatures of evolution across replicate populations. They carry out transcriptomics and a series of analyses that lead them to conclude that there are divergent mechanisms at play in individual evolutionary lineages. The authors used gene network reconstruction to identify three gene modules functionally differentiated, correlating with changes in growth fitness, genome mutation, and gene expression, respectively, due to evolutionary changes in the reduced genome.

I think that this study addresses an interesting question. Many microbial evolution experiments evolve by loss of function mutations, but presumably a cell that has already lost so much of its genome needs to find other mechanisms to adapt. Experiments like this have the potential to study "constructive" rather than "destructive" evolution.

Comments on revised version:

I think the authors have carefully gone through the manuscript and addressed all of my concerns.

Reviewer #2 (Public Review):

This manuscript describes an adaptive laboratory evolution (ALE) study with a previously constructed genome-reduced E. coli. The growth performance of the end-point lineages evolved in M63 medium was comparable to the full-length wild-type level at lower cell densities.

Subsequent mutation profiling and RNA-Seq analysis revealed many changes on the genome and transcriptomes of the evolved lineages. The authors did a great deal on analyzing the patterns of evolutionary changes between independent lineages, such as the chromosomal periodicity of transcriptomes, pathway enrichment analysis, weight gene co-expression analysis, and so on. They observed a striking diversity in the molecular characteristics amongst the evolved lineages, which, as they suggest, reflect divergent evolutionary strategies adopted by the genome-reduced organism.

As for the overall quality of the manuscript, I am rather torn. The manuscript leans towards elaborating observed findings, rather than explaining their biological significance. For this reason, readers are left with more questions than answers. For example, fitness assay on reconstituted (single and combinatorial) mutants was not performed, nor any supporting evidence on the proposed contributions of each mutants provided. This leaves the nature of mutations - be them beneficial, neutral or deleterious, the presence of epistatic interactions, and the magnitude of fitness contribution, largely elusive. Also, it is difficult to tell whether the RNA-Seq analysis in this study managed to draw biologically meaningful conclusions, or instill insight into the nature of genome-reduced bacteria. The analysis primarily highlighted the differences in transcriptome profiles among each lineage based on metrics such as 'DEG counts' and the 'GO enrichment'. However, I could not see any specific implications regarding the biology of the evolved minimal genome drawn. In their concluding remark, 'Multiple evolutionary paths for the reduced genome to improve growth fitness were likely all roads leading to Rome,' the authors observed the first half of the sentence, but the distinctive characteristics of 'all roads' or 'evolutionary paths', which I think should have been the key aspect in this investigation, remains elusive.

Comments on revised version:

I appreciate the author's responses. They responded to most of the comments, but I still think that there is room for improvement. Please refer to the following comments. Quoted below are the author's responses.

"We agree that our study leaned towards elaborating observed findings rather than explaining the detailed biological mechanisms."<br /> - Comment: I doubt if there are scientific merits in merely elaborating observed findings. The conclusion of this study suggests that evolutionary paths in reduced genomes are highly diverse. But if you think about the nature of adaptive evolution, which relies upon the spontaneous mutation event followed by selection, certain degree of divergence is always expected. The problem with current experimental setting is that there are no ways to quantitively assess whether the degree of evolutionary divergence increases as the function of genome reduction, as the authors claimed. In addition, this notion is in direct contradiction to the prediction that genome reduction constraints evolution by reducing the number of solution space. It is more logical to think and predict that genome reduction would, in turn, lead to the loss of evolutionary divergence. We are also interested to know whether solution space to the optimization problem altered in response to the genome reduction. In this regard, a control ALE experiment on non-reduced wild-type seems to be a mandatory experimental control. I highly suggest that authors present a control experiment, as it was done for "JCVI syn3.0B vs non-minimal M. mycoides" (doi: 10.1038/s41586 023 06288 x) and "E. coli eMS57 vs MG1655" (doi: 10.1038/s41467 019 08888 6).<br /> "We focused on the genome wide biological features rather than the specific biological functions."<br /> - Comment: The 'biological features' delivered in current manuscript does not give insight as to which genomic changes translated into strain fitness improvement. Rather than explaining the genotype-phenotype relationships and/or the mechanistic basis of fitness improvement, authors merely elaborated on the observed phenotypes. I question the scientific merits of such 'findings'.<br /> "Although the reduced growth rate caused by genome reduction could be recovered by experimental evolution, it remains unclear whether such an evolutionary improvement in growth fitness was a general feature of the reduced genome and how the genome wide changes occurred to match the growth fitness increase."<br /> - Comment: This response is very confusing to understand. "it remains unclear whether such an evolutionary improvement in growth fitness was a general feature of the reduced genome" - what aspects remain unclear?? What assumption led the authors to believe that reduced genome's fitness cannot be evolutionarily improved?<br /> - Comment: "and how the genome wide changes occurred to match the growth fitness increase" - this is exactly the aspect that authors should deliver, instead of just elaborating the observed findings. Why don't authors select one or two fastest-growing (or the fittest) lineages and specifically analyze underlying adaptive changes (i.e. genotype-phenotype relationships)?

Reviewer #1 (Public Review):

Summary:

Tuberculous meningitis (TBM) is one of the most severe form of extrapulmonary TB. TBM is especially prevalent in people who are immunocompromised (e.g. HIV-positive). Delays in diagnosis and treatment could lead to severe disease or mortality. In this study, the authors performed the largest ever host whole blood transcriptomics analysis on a cohort of 606 Vietnamese participants. The results indicated that TBM mortality is associated with increased neutrophil activation and decreased T and B cell activation pathways. Furthermore, increased angiogenesis was also observed in HIV-positive patients who died from TBM, whereas activated TNF signaling and down-regulated extracellular matrix organisation were seen in the HIV-negative group. Despite similarities in transcriptional profiles between PTB and TBM compared to healthy controls, inflammatory genes were more active in HIV-positive TBM. Finally, 4 hub genes (MCEMP1, NELL2, ZNF354C and CD4) were identified as strong predictors of death from TBM.

Strengths:

This is a really impressive piece of work, both in terms of the size of the cohort which took years of effort to recruit, sample and analyse and also the meticulous bioinformatics performed. The biggest advantage of obtaining a whole blood signature is that it allows an easier translational development into test that can be used in the clinical with a minimally invasive sample. Furthermore, the data from this study has also revealed important insights in the mechanisms associated with mortality and the differences in pathogenesis between HIV-positive and HIV-negative patients, which would have diagnostic and therapeutic implications.

Weaknesses:

The authors have addressed all the weaknesses in the revised version.

Reviewer #2 (Public Review):

Summary:

This manuscript describes the analysis of blood transcriptomic data from patients with TB meningitis, with and without HIV infection, with some comparison to those of patients with pulmonary tuberculosis and healthy volunteers. The objectives were to describe the comparative biological differences represented by the blood transcriptome in TBM associated with HIV co-infection or survival/mortality outcomes, and to identify a blood transcriptional signature to predict these outcomes. The authors report an association between mortality and increased levels of acute inflammation and neutrophil activation, but decreased levels of adaptive immunity and T/B cell activation. They propose a 4-gene prognostic signature to predict mortality.

Strengths:

Biological evaluations of blood transcriptomes in TB meningitis and their relationship to outcomes have not been extensively reported previously.<br /> The size of the data set is a major strength and is likely to be used extensively for secondary analyses in this field of research.<br /> The addition of a new validation cohort to evaluate the generalisability of their prognostic model in the revised manuscript is welcome.

Weaknesses:

The bioinformatic analysis is limited to a descriptive narrative of gene-level functional annotations curated in GO and KEGG databases. This analysis cannot be used to make causal inferences. In addition the functional annotations are limited to 'high-level' terms that fail to define the biology very precisely. As a result, the conclusions about the immunopathogenesis of TBM are not adequately substantiated.<br /> The lack of AUROC confidence intervals and direct comparison to the reference prognostic model in the validation cohort undermines confidence in their conclusion that their new prognostic model combing gene expression data and clinical variables performs better than the reference model.

Reviewer #2 (Public Review):

Summary:

This study from Bamgbose et al. identifies a new and important interaction between H4K20me and Parp1 that regulates inducible genes during development and heat stress. The authors present convincing experiments that form a mostly complete manuscript that significantly contributes to our understanding of how Parp1 associates with target genes to regulate their expression.

Strengths:

The authors present 3 compelling experiments to support the interaction between Parp1 and H4K20me, including:

(1) PR-Set7 mutants remove all K4K20me and phenocopy Parp mutant developmental arrest and defective heat shock protein induction.

(2) PR-Set7 mutants have dramatically reduced Parp1 association with chromatin and reduced poly-ADP ribosylation.

(3) Parp1 directly binds H4K20me in vitro.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public Review):

Summary:

The endocannabinoid system (ECS) regulates many critical functions, including reproductive function. Recent evidence indicates that dysregulated ECS contributes to endometriosis pathophysiology and the microenvironment. Therefore, the authors further examined the dysregulated ECS and its mechanisms in endometriosis lesion establishment and progression using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. The authors presented differential gene expressions and altered pathways, especially those related to the adaptive immune response in CNR1 and CNR2 ko lesions. Interstingly, the T-cell population was dramatically reduced in the peritoneal cavity lacking CNR2, and the loss of proliferative activity of CD4+ T helper cells. Imaging mass cytometry analysis provided spatial profiling of cell populations and potential relationships among immune cells and other cell types. This study provided fundamental knowledge of the endocannabinoid system in endometriosis pathophysiology.

Strengths:

Dysregulated ECS and its mechanisms in endometriosis pathogenesis were assessed using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. Not only endometriotic lesions, but also peritoneal exudate (and splenic) cells were analyzed to understand the specific local disease environment under the dysregulated ECS.

Providing the results of transcriptional profiles and pathways, immune cell profiles, and spatial profiles of cell populations support altered immune cell population and their disrupted functions in endometriosis pathogenesis via dysregulation of ECS.

In line 386: Role of CNR2 in T cells. The finding that nearly absent CD3+ T cells in the peritoneal cavity of CNR2 ko mice is intriguing.

The interpretation of the results is well-described in the Discussion.

Weaknesses:

The study was terminated and characterized 7 days after EM induction surgery without the details for selecting the time point to perform the experiments.

The authors also mentioned that altered eutopic endometrium contributes to the establishment and progression of endometriosis. This reviewer agrees with lines 324-325. If so, DEGs are likely identified between eutopic endometrium (with/without endometriosis lesion induction) and ectopic lesions. It would be nice to see the data (even though using publicly available data sets).

Figure 7 CDEF. The results of the statistical analyses and analyzed sample numbers should be added. Lines 444-450 cannot be reviewed without them.

This reviewer agrees with lines 498-500. In contrast, retrograded menstrual debris is not decidualized. The section could be modified to avoid misunderstanding.

Reviewer #1 (Public Review):

Summary:

The authors want to explore how much two known minibinder protein domains against the Spike protein of SARS-CoV-2 can function as a binding domain of 2 sets of synthetic receptors (SNIPR and CAR); the authors also want to know how some modifications of the linkers of these new receptors affect their activation profile.

Major strengths and weaknesses of the methods and results:

- Strengths include: analysis of synthetic receptor function for 2 classes of synthetic receptors, with robust and appropriate assays for both kinds of receptors. The modifications of the linkers are also interesting and the types of modifications that are often used in the field.

- Weaknesses include: none of the data analysis provides statistical interpretation of the results (that I could find). One dataset is confusing: Figures 5A and C, are said to be the same assay with the same constructs, but the results are 30% in A, and 70% in C.

An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

Given the open-ended nature of the goal (implicit in it being an exploration), it is hard to say if the authors have reached their aims; they have done an exploration for sure; is it big enough an exploration? This reviewer is not sure.

The results are extremely clearly presented, both in the figures and in the text, both for the methods and the results. The claims put forward (with limited exceptions see below) are very solidly supported by the presented data.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

The work may stimulate others to consider minibinders as potential binding domains for synthetic receptors. The modifications that are presented although not novel, do provide a starting point for larger-scale analysis.

It is not clear how much this is generalizable to other binders (the authors don't make such claims though). The claims are very focused on the tested modifications, and the 2 receptors and minibinder used, a scope that I would define as narrow; the take-home message if one wants to try it with other minibinders or other receptors seems to be: test a few things, and your results may surprise you.

Any additional context you think would help readers interpret or understand the significance of the work:

We are at the infancy stage of synthetic receptors optimization and next-generation derivation; there is a dearth of systematic studies, as most focus is on developing a few ones that work. This work is an interesting attempt to catalyze more research with these new minibinders. Will it be picked up based on this? Not sure.

Reviewer #2 (Public Review):

Summary:

Weinberg et al. show that spike LCB minibinders can be used as the extracellular domain for SynNotch, SNIPR, and CAR. They evaluated their designs against cells expressing the target proteins and live virus.

Strengths:

This is a good fundamental demonstration of alternative use of the minibinder. The results are unsurprising but robust and solid in most cases.

Weaknesses:

The manuscript would benefit from better descriptions of the study's novelty. Given that LCB previously worked in SynNotch, what unexpected finding was uncovered by this study? It is well known that the extracellular domain of CAR is amendable to different types of binding domains (e.g., scFv, nanobody, DARPin, natural ligands). So, it is not surprising that a minibinder also works with CAR. We don't know if the minibinders are more or less likely to be compatible with CAR or SNIPR.

The demonstrations are all done using just 1 minibinder. It is hard to conclude that minibinders, as a unique class of protein binders, are generalizable in different contexts. All it can conclude is that this specific Spike minibinder can be used in synNotch, SNIPR, and CAR. The LCB3 minibinder seems to be much weaker.

The sensing of live viruses is interesting, but the output is very weak. It is difficult to imagine a utility for such a weak response.

Reviewer #1 (Public Review):

⍺-synuclein (syn) is a critical protein involved in many aspects of human health and disease. Previous studies have demonstrated that post-translational modifications (PTMs) play an important role in regulating the structural dynamics of syn. However, how post-translational modifications regulate syn function remains unclear. In this manuscript, Wang et al. reported an exciting discovery that N-acetylation of syn enhances the clustering of synaptic vesicles (SVs) through its interaction with lysophosphatidylcholine (LPC). Using an array of biochemical reconstitution, single vesicle imaging, and structural approaches, the authors uncovered that N-acetylation caused distinct oligomerization of syn in the presence of LPC, which is directly related to the level of SV clustering. This work provides novel insights into the regulation of synaptic transmission by syn and might also shed light on new ways to control neurological disorders caused by syn mutations.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors provide evidence that posttranslational modification of synuclein by N-acetylation increases clustering of synaptic vesicles in vitro. When using liposomes the authors found that while clustering is enhanced by the presence of either lysophosphatidylcholine (LPC) or phosphatidylcholine in the membrane, N-acetylation enhanced clustering only in the presence of LPC. Enhancement of binding was also observed when LPC micelles were used, which was corroborated by increased intra/intermolecular cross-linking of N-acetylated synuclein in the presence of LPC.

Strengths:

It is known for many years that synuclein binds to synaptic vesicles but the physiological role of this interaction is still debated. The strength of this manuscript is clearly in the structural characterization of the interaction of synuclein and lipids (involving NMR-spectroscopy) showing that the N-terminal 100 residues of synuclein are involved in LPC-interaction, and the demonstration that N-acetylation enhances the interaction between synuclein and LPC.

Weaknesses:

Lysophosphatides form detergent-like micelles that destabilize membranes, with their steady-state concentrations in native membranes being low, questioning the significance of the findings. Oddly, no difference in binding between the N-acetylated and unmodified form was observed when the acidic phospholipid phosphatidylserine was included. It remains unclear to which extent binding to LPC is physiologically relevant, particularly in the light of recent reports from other laboratories showing that synuclein may interact with liquid-liquid phases of synapsin I that were reported to cause vesicle clustering.

Reviewer #1 (Public Review):

Summary:

The mammalian Shieldin complex consisting of REV7 (aka MAD2L2, MAD2B) and SHLD1-3 affects pathway usage in DSB repair favoring non-homologous endjoining (NHEJ) at the expense of homologous recombination (HR) by blocking resection and/or priming fill-in DNA synthesis to maintain or generate near blunt ends suitable for NHEJ. While the budding yeast Saccharomyces cerevisiae does not have homologs to SHLD1-3, it does have Rev7, which was identified to function in conjunction with Rev3 in the translesion DNA polymerase zeta. Testing the hypothesis that Rev7 also affects DSB resection in budding yeast, the work identified a direct interaction between Rev7 and the Rad50-Mre11-Xrs2 complex by two-hybrid and direct protein interaction experiments. Deletion analysis identified that the 42 amino acid C-terminal region was necessary and sufficient for the 2-hybrid interaction. Direct biochemical analysis of the 42 aa peptide was not possible. Rev7 deficient cells were found to be sensitive to HU only in synergy with G2 tetraplex forming DNA. Importantly, the 42 aa peptide alone suppressed this phenotype. Biochemical analysis with full-length Rev7 and a C-terminal truncation lacking the 42 aa region shows G4-specific DNA binding that is abolished in the C-terminal truncation and with a substrate containing mutations to prevent G4 formation. Rev7 lacks nuclease activity but inhibits the dsDNA exonuclease activity of Mre11. The C-terminal truncation protein lacking the 42 aa region also showed some inhibition suggesting the involvement of additional binding sites besides the 42 aa region. Also, the Mre11 ssDNA endonuclease activity is inhibited by Rev7 but not the degradation of linear ssDNA. Rev7 does not affect ATP binding by Rad50 but inhibits in a concentration-dependent manner the Rad50 ATPase activity. The C-terminal truncation protein lacking the 42 aa region also showed some inhibition but significantly less than the full-length protein.

Using an established plasmid-based NHEJ assay, the authors provide strong evidence that Rev7 affects NEHJ, showing a four-fold reduction in this assay. The mutations in the other Pol zeta subunits, Rev3 and Rev1, show a significantly smaller effect (~25% reduction). A strain expressing only the Rev7 C-terminal 42 aa peptide showed no NHEJ defect, while the truncation protein lacking this region exhibited a smaller defect than the deletion of REV7. The conclusion that Rev7 supports NHEJ mainly through the 42 aa region was validated using a chromosomal NHEJ assay. The effect on HR was assessed using a plasmid:chromosome system containing G4 forming DNA. The rev7 deletion strain showed an increase in HR in this system in the presence and absence of HU. Cells expressing the 42 aa peptide were indistinguishable from the wild type as were cells expressing the Rev7 truncation lacking the 42 aa region. The authors conclude that Rev7 suppresses HR, but the context appears to be system-specific and the conclusion that Rev7 abolished HR repair of DSBs is unwarranted and overly broad.

Strength:

This is a well-written manuscript with many well-executed experiments that suggest that Rev7 inhibits MRX-mediated resection to favor NEHJ during DSB repair. This finding is novel and provides insight into the potential mechanism of how the human Shieldin complex might antagonize resection.

Weaknesses:

The nuclease experiments were conducted using manganese as a divalent cation, and it is unclear whether there is an effect with the more physiological magnesium cation. Additional controls for the ATPase and nuclease experiments to eliminate non-specific effects would be helpful. Evidence for an effect on resection in cells is lacking. The major conclusion about the role of Rev7 in regulating the choice between HR and NHEJ is not justified, as only a highly specialized assay is used that does not warrant the broad conclusion drawn. Specifically, the results that the Rev7 C-terminal truncation lacking the 42 aa region still suppresses HR is unexpected and unexplained. The effect of Rev7 on G4 metabolism is underdeveloped and distracts from the main results that Rev7 modulated MRX activity. The authors should consider removing this part and develop a more complete story on this later.

Reviewer #2 (Public Review):

In this study, Badugu et al investigate the Rev7 roles in regulating the Mre11-Rad50-Xrs2 complex and in the metabolism of G4 structures. The authors also try to make a conclusion that REV7 can regulate the DSB repair choice between homologous recombination and non-homologous end joining.

The major observations of this study are:

(1) Rev7 interacts with the individual components of the MRX complex in a two-hybrid assay and in a protein-protein interaction assay (microscale thermophoresisi) in vitro.<br /> (2) Modeling using AlphaFold-Multimier also indicated that Rev7 can interact with Mre11 and Rad50.<br /> (3) Using a two-hybrid assay, a 42 C terminal domain in Rev7 responsible for the interaction with MRX was identified.<br /> (4) Rev7 inhibits Mre11 nuclease and Rad50 ATPase activities in vitro.<br /> (5) Rev 7 promotes NHEJ in plasmid cutting/relegation assay.<br /> (6) Rev7 inhibits recombination between chromosomal ura3-1 allele and plasmid ura3 allele containing G4 structure.<br /> (7) Using an assay developed in V. Zakian's lab, it was found that rev7 mutants grow poorly when both G4 is present in the genome and yeast are treated with HU.<br /> (8) In vitro, purified Rev7 binds to G4-containing substrates.

In general, a lot of experiments have been conducted, but the major conclusion about the role of Rev7 in regulating the choice between HR and NHEJ is not justified.

(1) Two stories that do not overlap (regulation of MRX by Rev7 and Rev7's role in G4 metabolism) are brought under one umbrella in this work. There is no connection unless the authors demonstrate that Rev7 inhibits the cleavage of G4 structures by the MRX complex.

(2) The authors cannot conclude based on the recombination assay between G4-containing 2-micron plasmid and chromosomal ura3-1 that Rev7" completely abolishes DSB-induced HR". First of all, there is no evidence that DSBs are formed at G4. Why is there no induction of recombination when cells are treated with HU? Second, as the authors showed, Rev7 binds to G4, therefore it is not clear if the observed effects are the result of Rev7 interaction with G4 or its impact on HR. The established HO-based assays where the speed of resection can be monitored (e.g., Mimitou and Symington, 2010) have to be used to justify the conclusion that Rev7 inhibits MRX nuclease activity in vivo.

Reviewer #3 (Public Review):

Summary:

REV7 facilitates the recruitment of Shieldin complex and thereby inhibits end resection and controls DSB repair choice in metazoan cells. Puzzlingly, Shieldin is absent in many organisms and it is unknown if and how Rev7 regulates DSB repair in these cells. The authors surmised that yeast Rev7 physically interacts with Mre11/Rad50/Xrs2 (MRX), the short-range resection nuclease complex, and tested this premise using yeast two-hybrid (Y2H) and microscale thermophoresis (MST). The results convincingly showed that the individual subunits of MRX interact robustly with Rev7. AlphaFold Multimer modelling followed by Y2H confirmed that the carboxy-terminal 42 amino acid is essential for interaction with MR and G4 DNA binding by REV7. The mutant rev7 lacking the binding interface (Rev7-C1) to MR shows moderate inhibition to the nuclease and the ATPase activity of Mre11/Rad50 in biochemical assays. Deletion of REV7 also causes a mild reduction in NHEJ using both plasmid and chromosome-based assays and increases mitotic recombination between chromosomal ura3-01 and the plasmid ura3 allele interrupted by G4. The authors concluded that Rev7 facilitates NHEJ and antagonizes HR even in budding yeast, but it achieves this by blocking Mre11 nuclease and Rad50 ATPase.

Weaknesses:

There are many strengths to the studies and the broad types of well-established assays were used to deduce the conclusion. Nevertheless, I have several concerns about the validity of experimental settings due to the lack of several key controls essential to interpret the experimental results. The manuscript also needs a few additional functional assays to reach the accurate conclusions as proposed.

(1) AlphaFold model predicts that Mre11-Rev7 and Rad50-Rev7 binding interfaces overlap and Rev7 might bind only to Mre11 or Rad50 at a time. Interestingly, however, Rev7 appears dimerized (Figure 1). Since the MR complex also forms with 2M and 2R in the complex, it should still be possible if REV7 can interact with +-*both M and R in the MR complex. The author should perform MST using MR complex instead of individual MR components. The authors should also analyze if Rev7-C1 is indeed deficient in interaction with MR individually and with complex using MST assay.

(2) The nuclease and the ATPase assays require additional controls. Does Rev7 inhibit the other nuclease or ATPase non-specifically? Are these outcomes due to the non-specific or promiscuous activity of Rev7? In Figure 6, the effect of REV7 on the ATP binding of Rad50 could be hard to assess because the maximum Rad50 level (1 uM) was used in the experiments. The author should use the suboptimal level of Rad50 to check if REV7 still does not influence ATP binding by Rad50.

(3) The moderate deficiency in NHEJ using plasmid-based assay in REV7 deleted cells can be attributed to aberrant cell cycle or mating type in rev7 deleted cells. The authors should demonstrate that rev7 deleted cells retain largely normal cell cycle patterns and the mating type phenotypes. The author should also analyze the breakpoints in plasmid-based NHEJ assays in all mutants, especially from rev7 and rev7-C1 cells.

(4) It is puzzling why the authors did not analyze end resection defects in rev7 deleted cells after a DSB. The author should employ the widely used resection assay after a HO break in rev3, rev7, and mre11 rev7 cells as described previously.

(5) Is it possible that Rev7 also contributes to NHEJ as the part of TLS polymerase complex? Although NHEJ largely depends on Pol4, the authors should not rule out that the observed NHEJ defect in rev7 cells is due at least partially to its TLS defect. In fact, both rev3 or rev1 cells are partially defective in NHEJ (Figure 7). Rev7-C1 is less deficient in NHEJ than REV7 deletion. These results predict that rev7-C1 rev3 should be as defective as the rev7 deletion. Additionally, the authors should examine if Rev7-C1 might be deficient in TLS. In this regard, does rev7-C1 reduce TLS and TLS-dependent mutagenesis? Is it dominant? The authors should also check if Rev3 or Rev1 are stable in Rev7 deleted or rev7-C1 cells by immunoblot assays.

(6) Due to the G4 DNA and G4 binding activity of REV7, it is not clear which class of events the authors are measuring in plasmid-chromosome recombination assay in Figure 9. Do they measure G4 instability or the integrity of recombination or both in rev7 deleted cells? Instead, the effect of rev7 deletion or rev7-C1 on recombination should be measured directly by more standard mitotic recombination assays like mating type switch or his3 repeat recombination.

Reviewer #1 (Public Review):

Summary:

Extracellular ATP represents a danger-associated molecular pattern associated to tissue damage and can act also in an autocrine fashion in macrophages to promote proinflammatory responses, as observed in a previous paper by the authors in abdominal sepsis. The present study addresses an important aspect possibly conditioning the outcome of sepsis that is the release of ATP by bacteria. The authors show that sepsis-associated bacteria do in fact release ATP in a growth dependent and strain-specific manner. However, whether this bacterial derived ATP play a role in the pathogenesis of abdominal sepsis has not been determined. To address this question, a number of mutant strains of E. coli has been used first to correlate bacterial ATP release with growth and then, with outer membrane integrity and bacterial death. By using E. coli transformants expressing the ATP-degrading enzyme apyrase in the periplasmic space, the paper nicely shows that abdominal sepsis by these transformants results in significantly improved survival. This effect was associated with a reduction of peritoneal macrophages and CX3CR1+ monocytes, and an increase in neutrophils. To extrapolate the function of bacterial ATP from the systemic response to microorganisms, the authors exploited bacterial OMVs either loaded or not with ATP to investigate the systemic effects devoid of living microorganisms. This approach showed that ATP-loaded OMVs induced degranulation of neutrophils after lysosomal uptake, suggesting that this mechanism could contribute to sepsis severity.

Strengths:

A strong part of the study is the analysis of E. coli mutants to address different aspects of bacterial release of ATP that could be relevant during systemic dissemination of bacteria in the host.

Weaknesses:

As pointed out in the limitations of the study whether ATP-loaded OMVs provide a mechanistic proof of the pathogenetic role of bacteria-derived ATP independently of live microorganisms in sepsis is interesting but not definitively convincing. It could be useful to see whether degranulation of neutrophils is differentially induced by apyrase-expressing vs control E. coli transformants. Also, the increase of neutrophils in bacterial ATP-depleted abdominal sepsis, which has better outcomes than "ATP-proficient" sepsis, seems difficult to correlate to the hypothesized tissue damage induced by ATP delivered via non-infectious OMVs. Are the neutrophils counts affected by ATP delivered via OMVs? A comparison of cytokine profiles in the abdominal fluids of E. coli and OMV treated animals could be helpful in defining the different responses induced by OMV-delivered vs bacterial-released ATP. The analyses performed on OMV treated versus E. coli infected mice are not closely related and difficult to combine when trying to draw a hypothesis for bacterial ATP in sepsis. Also it was not clear why lung neutrophils were used for the RNAseq data generation and analysis.

Reviewer #2 (Public Review):

Summary:

In their manuscript "Released Bacterial ATP Shapes Local and Systemic Inflammation during Abdominal Sepsis", Daniel Spari et al. explored the dual role of ATP in exacerbating sepsis, revealing that ATP from both host and bacteria significantly impacts immune responses and disease progression.

Strengths:<br /> The study meticulously examines the complex relationship between ATP release and bacterial growth, membrane integrity, and how bacterial ATP potentially dampens inflammatory responses, thereby impairing survival in sepsis models. Additionally, this compelling paper implies a concept that bacterial OMVs act as vehicles for the systemic distribution of ATP, influencing neutrophil activity and exacerbating sepsis severity.

Weaknesses:

(1) The researchers extracted and cultivated abdominal fluid on LB agar plates, then randomly picked 25 colonies for analysis. However, they did not conduct 16S rRNA gene amplicon sequencing on the fluid itself. It is worth noting that the bacterial species present may vary depending on the individual patients. It would be beneficial if the authors could specify whether they've verified the existence of unculturable species capable of secreting high levels of Extracellular ATP.

(2) Do mice lacking commensal bacteria show a lack of extracellular ATP following cecal ligation puncture?

(3) The authors isolated various bacteria from abdominal fluid, encompassing both Gram-negative and Gram-positive types. Nevertheless, their emphasis appeared to be primarily on the Gram-negative E. coli. It would be beneficial to ascertain whether the mechanisms of Extracellular ATP release differ between Gram-positive and Gram-negative bacteria. This is particularly relevant given that the Gram-positive bacterium E. faecalis, also isolated from the abdominal fluid, is recognized for its propensity to release substantial amounts of Extracellular ATP.

(4) The authors observed changes in the levels of LPM, SPM, and neutrophils in vivo. However, it remains uncertain whether the proliferation or migration of these cells is modulated or inhibited by ATP receptors like P2Y receptors. This aspect requires further investigation to establish a convincing connection.

(5) Additionally, is it possible that the observed in vivo changes could be triggered by bacterial components other than Extracellular ATP? In this research field, a comprehensive collection of inhibitors is available, so it is desirable to utilize them to demonstrate clearer results.

(6) Have the authors considered the role of host-derived Extracellular ATP in the context of inflammation?

(7) The authors mention that Extracellular ATP is rapidly hydrolyzed by ectonucleotases in vivo. Are the changes of immune cells within the peritoneal cavity caused by Extracellular ATP released from bacterial death or by OMVs?

(8) In the manuscript, the sample size (n) for the data consistently remains at 2. I would suggest expanding the sample size to enhance the robustness and rigor of the results.

Joint Public Review

This work investigates the evolutionary conservation and functional significance of FoxO transcription factors in the response of airway epithelia to diverse stressors, ranging from hypoxia to temperature fluctuations and oxidative stress. Utilizing a comprehensive approach encompassing Drosophila, murine models, and human samples, the study investigates FoxO's role across species. The authors demonstrate that hypoxia triggers a dFOXO-dependent immune response in Drosophila airways, with subsequent nuclear localization of dFOXO in response to various stressors. Transcriptomic analysis reveals differential regulation of crucial gene categories in respiratory tissues, highlighting FoxO's involvement in metabolic pathways, DNA replication, and stress resistance mechanisms.

The study underscores FoxO's importance in maintaining homeostasis by revealing reduced stress resistance in dFOXO Drosophila mutants, shedding light on its protective role against stressors. In mammalian airway cells, FoxO exhibits nuclear translocation in response to hypoxia, accompanied by upregulation of cytokines with antimicrobial activities. Intriguingly, mouse models of asthma show FoxO downregulation, which is also observed in sputum samples from human asthma patients, implicating FoxO dysregulation in respiratory pathologies.

Overall, the manuscript suggests that FoxO signaling plays a critical role in preserving airway epithelial cell homeostasis under stress conditions, with implications for understanding and potentially treating respiratory diseases like asthma. By providing compelling evidence of FoxO's involvement across species and its correlation with disease states, the study underscores the importance of further exploration into FoxO-mediated mechanisms in respiratory health.

Strengths

(1) This study shows that FoxO transcription factors are critical for regulating immune and inflammatory responses across species, and for orchestrating responses to various stressors encountered by airway epithelial cells, including hypoxia, temperature changes, and oxidative stress. Understanding the intricate regulation of FoxO transcription factors provides insights into modulating immune and inflammatory pathways, offering potential avenues for therapeutic interventions against respiratory diseases and other illnesses.

(2) The work employs diverse model systems, including Drosophila, murine models, and human samples, thereby establishing a conserved role for FoxOs in airway epithelium and aiding translational relevance to human health.

(3) The manuscript establishes a strong correlation between FoxO expression levels and respiratory diseases such as asthma. Through analyses of both murine models of asthma and asthmatic human samples, the study demonstrates a consistent reduction in FoxO expression, indicating its potential involvement in the pathogenesis of respiratory disorders. This correlation underscores the clinical relevance of FoxO dysregulation and opens avenues for developing treatments for respiratory conditions like asthma, COPD, and pulmonary fibrosis, addressing significant unmet clinical needs.

(4) The study unveils intriguing mechanistic details regarding FoxO regulation and function. Particularly noteworthy is the observation of distinct regulatory mechanisms governing dFOXO translocation in response to different stressors. The independence of hypoxia-induced dFOXO translocation from JNK signaling adds complexity to our understanding of FoxO-mediated stress responses. Such mechanistic insights deepen our understanding of FoxO biology and pave the way for future investigations into the intricacies of FoxO signaling pathways in airway epithelial cells.

Weaknesses

(1) The manuscript does not distinguish between FoxO expression levels and FoxO activation status. While FoxO nuclear localization is observed in Drosophila and murine models, it remains unclear whether this reflects active FoxO signaling or merely FoxO expression, limiting the mechanistic understanding of FoxO regulation.

(2) The manuscript utilizes various stressors across different experiments without providing a clear rationale for their selection. This lack of coherence in stressor choice complicates the interpretation of results and diminishes the ability to draw meaningful comparisons across experiments.

(3) The manuscript frequently refers to "FoxO signaling" without providing specific signaling readouts. This ambiguity undermines the clarity of the conclusions drawn from the data and hinders the establishment of clear cause-and-effect relationships between FoxO activation and cellular responses to stress.

(4) Many conclusions drawn in the manuscript rely heavily on the quantification of immunostaining images for FoxO nuclear localization. While this is an important observation, it does not provide a sufficient mechanistic understanding of FoxO expression or activation regulation.

(5) The primary weakness in the Drosophila experiments is the analysis of dFoxO in homozygous dFoxO mutant animals, which precludes determining the specific role of dFoxO in airway cells. Despite available tools for tissue-specific gene manipulation, such as tissue-specific RNAi and CRISPR techniques, these approaches were not employed, limiting the precision of the findings.

(6) In mammalian experiments, the results are primarily correlative, lacking causal evidence. While changes in FoxO expression are observed under pathological conditions, the absence of experiments on FoxO-deficient cells or tissues precludes establishing a causal relationship between FoxO dysregulation and respiratory pathologies.

(7) Although the evidence suggests a critical role for FoxO in airway tissues, the precise nature of this role remains unclear. With gene expression changes analyzed only in Drosophila, the extent of conservation in downstream FoxO-mediated pathways between mammals and Drosophila remains uncertain. Additionally, the functional consequences of FoxO deficiency in airway cells were not determined, hindering comparisons between species and limiting insights into FoxO's functional roles in different contexts.

Reviewer #1 (Public Review):

In this manuscript, entitled " Merging Multi-OMICs with Proteome Integral Solubility Alteration Unveils Antibiotic Mode of Action", Dr. Maity and colleagues aim to elucidate the mechanisms of action of antibiotics through combined approaches of omics and the PISA tool to discover new targets of five drugs developed against Helicobacter pylori.

Strengths:

Using transcriptomics, proteomic analysis, protein stability (PISA), and integrative analysis, Dr. Maity and colleagues have identified pathways targeted by five compounds initially discovered as inhibitors against H. pylori flavodoxin. This study underscores the necessity of a global approach to comprehensively understanding the mechanisms of drug action. The experiments conducted in this paper are well-designed and the obtained results support the authors' conclusions.

Weaknesses:

This manuscript describes several interesting findings. A few points listed below require further clarification:

(1) Compounds IVk exhibits markedly different behavior compared to the other compounds. The authors are encouraged to discuss these findings in the context of existing literature or chemical principles.

(2) The incubation time for treating H. pylori with the drugs was set at 4 hours for transcriptomic and proteomic analyses, compared to 20 min for PISA analysis. The authors need to explain the reason for these differences in treatment duration.

(3) The PISA method facilitates the identification of proteins stabilized by drug treatment. DnaJ and Trigger factor (tig), well-known molecular chaperones, prevent protein aggregation under stress. Their enrichment in the soluble fraction is expected and does not necessarily indicate direct stabilization by the drugs. The possibility that their stabilization results from binding to other proteins destabilized by the drugs should be considered. To prevent any misunderstanding, the authors should clarify that their methodology does not solely identify direct targets. Instead, the combination of their findings sheds light on various pathways affected by the treatment.

(4) At the end of the manuscript, the authors conclude that four compounds "strongly interact with CagA". However, detailed molecule/protein interaction studies are necessary to definitively support this claim. The authors should exercise caution in their statement. As the authors mentioned, additional research (not mandated in the scope of this current paper) is necessary to determine the drug's binding affinity to the proposed targets.

(5) The authors should clarify the PISA-Express approach over standard PISA. A detailed explanation of the differences between both methods in the main text is important.

Reviewer #2 (Public Review):

Summary:

This work has an important and ambitious goal: understanding the effects of drugs, in this case antimicrobial molecules, from a holistic perspective. This means that the effect of drugs on a group of genes and whole metabolic pathways is unveiled, rather than its immediate effect on a protein target only. To achieve this goal the authors successfully implement the PISA-Express method (Protein Integral Solubility Alteration), using combined transcriptomics, proteomics, and drug-induced changes in protein stability to retrieve a large number of genes and proteins affected by the used compounds. The compounds used in the study (compound IVa, IVb, IVj, and IVk) were all derived from the precursors compound IV, they are effective against Helicobacter pylori, and their mode of action on clusters of genes and proteins has been compared to the one of the known pylori drug metronidazole (MNZ). Due to this comparison, and confirmed by the diversity of responses induced by these very similar compounds, it can be understood that the approach used is reliable and very informative. Notably, although all compound IV derivatives were designed to target pylori Flavodoxin (Fld), only one showed a statistically significant shift of Fld solubility (compound IVj, FIG S11). For most other compounds, instead, the involvement of other possible targets affecting diverse metabolic pathways was also observed, notably concerning a series of genes with other important functions: CagA (virulence factor), FtsY/FtsA (cell division), AtpD (ATP-synthase complex), the essential GTPase ObgE, Tig (protein export), as well as other proteins involved in ribosomal synthesis, chemotaxis/motility and DNA replication/repairs. Finally, for all tested molecules, in vivo functional data have been collected that parallel the omics predictions, comforting them and showing that compound IV derivatives differently affect cellular generation of reactive oxygen species (ROS), oxygen consumption rates (OCR), DNA damage, and ATP synthesis.

Strengths:

The approach used is very potent in retrieving the effects of chemically active molecules (in this case antimicrobial ones) on whole cells, evidencing protein and gene networks that are involved in cell sensitivity to the studied molecules. The choice of these compounds against H. pylori is perfect, showcasing how different the real biological response is, compared to the hypothetical one. In fact, although all molecules were retrieved based on their activity on Fld, the authors unambiguously show that large unexpected gene clusters may, and in fact are, affected by these compounds, and each of them in different manners.

Impact:

The present work is the first report relying on PISA-Express performed on living bacterial cells. Because of its findings, this work will certainly have a high impact on the way we design research to develop effective drugs, allowing us to understand the fine effects of a drug on gene clusters, drive molecule design towards specific metabolic pathways, and eventually better plan the combination of multiple active molecules for drug formulation. Beyond this, however, we expect this article to impact other related and unrelated fields of research as well. The same holistic approaches might also allow gaining deep, and sometimes unexpected, insight into the cellular targets involved in drug side effects, drug resistance, toxicity, and cellular adaptation, in fields beyond the medicinal one, such as cellular biology and environmental studies on pollutants.

Reviewer #1 (Public Review):

Summary:

This manuscript provides an initial characterization of three new missense variants of the PLCG1 gene associated with diverse disease phenotypes, utilizing a Drosophila model to investigate their molecular effects in vivo. Through the meticulous creation of genetic tools, the study assesses the small wing (sl) phenotype - the fly's ortholog of PLCG1 - across an array of phenotypes from longevity to behavior in both sl null mutants and variants. The findings indicate that the Drosophila PLCG1 ortholog displays aberrant functions. Notably, it is demonstrated that overexpression of both human and Drosophila PLCG1 variants in fly tissue leads to toxicity, underscoring their pathogenic potential in vivo.

Strengths:

The research effectively highlights the physiological significance of sl in Drosophila. In addition, the study establishes the in vivo toxicity of disease-associated variants of both human PLCG1 and Drosophila sl.

Weaknesses:

The study's limitations include the human PLCG1 transgene's inability to compensate for the Drosophila sl null mutant phenotype, suggesting potential functional divergence between the species. This discrepancy signals the need for additional exploration into the mechanistic nuances of PLCG1 variant pathogenesis, especially regarding their gain-of-function effects in vivo.

Overall:

The study offers compelling evidence for the pathogenicity of newly discovered disease-related PLCG1 variants, manifesting as toxicity in a Drosophila in vivo model, which substantiates the main claim by the authors. Nevertheless, a deeper inquiry into the specific in vivo mechanisms driving the toxicity caused by these variants in Drosophila could significantly enhance the study's impact.

Reviewer #2 (Public Review):

The manuscript by Ma et al. reports the identification of three unrelated people who are heterozygous for de novo missense variants in PLCG1, which encodes phospholipase C-gamma 1, a key signaling protein. These individuals present with partially overlapping phenotypes including hearing loss, ocular pathology, cardiac defects, abnormal brain imaging results, and immune defects. None of the patients present with all of the above phenotypes. PLCG1 has also been implicated as a possible driver for cell proliferation in cancer.

The three missense variants found in the patients result in the following amino acid substitutions: His380Arg, Asp1019Gly, and Asp1165Gly. PLCG1 (and the closely related PLCG2) have a single Drosophila ortholog called small wing (sl). sl-null flies are viable but have small wings with ectopic wing veins and supernumerary photoreceptors in the eye. As all three amino acids affected in the patients are conserved in the fly protein, in this work Ma et al. tested whether they are pathogenic by expressing either reference or patient variant fly or human genes in Drosophila and determining the phenotypes produced by doing so.

Expression in Drosophila of the variant forms of PLCG1 found in these three patients is toxic; highly so for Asp1019Gly and Asp1165Gly, much more modestly for His380Arg. Another variant, Asp1165His which was identified in lymphoma samples and shown by others to be hyperactive, was also found to be toxic in the Drosophila assays. However, a final variant, Ser1021Phe, identified by others in an individual with severe immune dysregulation, produced no phenotype upon expression in flies.

Based on these results, the authors conclude that the PLCG1 variants found in patients are pathogenic, producing gain-of-function phenotypes through hyperactivity. In my view, the data supporting this conclusion are robust, despite the lack of a detectable phenotype with Ser1021Phe, and I have no concerns about the core experiments that comprise the paper.

Figure 6, the last in the paper, provides information about PLCG1 structure and how the different variants would affect it. It shows that the His380, Asp1019, and Asp1165 all lie within catalytic domains or intramolecular interfaces and that variants in the latter two affect residues essential for autoinhibition. It also shows that Ser1021 falls outside the key interface occupied by Asp1019, but more could have been said about the potential effects of Ser1021Phe.

Overall, I believe the authors fully achieved the aims of their study. The work will have a substantial impact because it reports the identification of novel disease-linked genes, and because it further demonstrates the high value of the Drosophila model for finding and understanding gene-disease linkages.

Reviewer #3 (Public Review):

Summary:

The paper attempts to model the functional significance of variants of PLCG2 in a set of patients with variable clinical manifestations.

Strengths:

A study attempting to use the Drosophila system to test the function of variants reported from human patients.

Weaknesses:

Additional experiments are needed to shore up the claims in the paper. These are listed below.

Major Comments:

(1) Does the pLI/ missense constraint Z score prediction algorithm take into consideration whether the gene exhibits monoallelic or biallelic expression?

(2) Figure 1B: Include human PLCG2 in the alignment that displays the species-wide conserved variant residues.

(3) Figure 4A:<br /> Given that<br /> (i) sl is predicted to be the fly ortholog for both mammalian PLCγ isozymes: PLCG1 and PLCG2 [Line 62]<br /> (ii) they are shown to have non-redundant roles in mammals [Line 71] and<br /> (iii) reconstituting PLCG1 is highly toxic in flies, leading to increased lethality.<br /> This raises questions about whether sl mutant phenotypes are specifically caused by the absence of PLG1 or PLCG2 functions in flies. Can hPLCG2 reconstitution in sl mutants be used as a negative control to rule out the possibility of the same?

(4) Do slT2A/Y; UAS-PLCG1Reference flies survive when grown at 22{degree sign}C? Since transgenic fly expressing PLCG1 cDNA when driven under ubiquitous gal4s, Tubulin and Da, can result in viable progeny at 22{degree sign}C, the survival of slT2A/Y; UAS-PLCG1Reference should be possible.<br /> and similarly<br /> Does slT2A flies exhibit the phenotypes of (i) reduced eclosion rate (ii) reduced wing size and ectopic wing veins and (iii) extra R7 photoreceptor in the fly eye at 22{degree sign}C?<br /> If so, will it be possible to get a complete rescue of the slT2A mutant phenotypes with the hPLCG1 cDNA at 22{degree sign}C? This dataset is essential to establish Drosophila as an ideal model to study the PLCG1 de novo variants.

(5) Localisation and western blot assays to check if the introduction of the de novo mutations can have an impact on the sub-cellular targeting of the protein or protein stability respectively.

(6) Analysing the nature of the reported gain of function (experimental proof for the same is missing in the manuscript) variants:<br /> Instead of directly showing the effect of introducing the de novo variant transgenes in the Drosophila model especially when the full-length PLCG1 is not able to completely rescue the slT2A phenotype;<br /> (i) Show that the gain-of-function variants can have an impact on the protein function or signalling via one of the three signalling outputs in the mammalian cell culture system: (i) inositol-1,4,5-trisphosphate production, (ii) intracellular Ca2+ release or (iii) increased phosphorylation of extracellular signal-related kinase, p65, and p38.<br /> OR<br /> (ii) Run a molecular simulation to demonstrate how the protein's auto-inhibited state can be disrupted and basal lipase activity increased by introducing D1019G and D1165G, which destabilise the association between the C2 and cSH2 domains. The H380R variant may also exhibit characteristics similar to the previously documented H335A mutation which leaves the protein catalytically inactive as the residue is important to coordinate the incoming water molecule required for PIP2 hydrolysis.

(7) Clarify the reason for carrying out the wing-specific and eye-specific experiments using nub-gal4 and eyless-gal4 at 29˚C despite the high gal4 toxicity at this temperature.

(8) For the sake of completeness the authors should also report other variants identified in the genomes of these patients that could also contribute to the clinical features.

Reviewer #1 (Public Review):

Summary:

Casas-Tinto et al. present convincing data that injury of the adult Drosophila CNS triggers transdifferentiation of glial cells and even the generation of neurons from glial cells. This observation opens up the possibility of getting a handle on the molecular basis of neuronal and glial generation in the vertebrate CNS after traumatic injury caused by Stroke or Crush injury. The authors use an array of sophisticated tools to follow the development of glial cells at the injury site in very young and mature adults. The results in mature adults revealing a remarkable plasticity in the fly CNS and dispels the notion that repair after injury may be only possible in nerve cords which are still developing. The observation of so-called VC cells which do not express the glial marker repo could point to the generation of neurons by former glial cells.

Conclusion:

The authors present an interesting story that is technically sound and could form the basis for an in-depth analysis of the molecular mechanism driving repair after brain injury in Drosophila and vertebrates.

Strengths:

The evidence for transdifferentiation of glial cells is convincing. In addition, the injury to the adult CNS shows an inherent plasticity of the mature ventral nerve cord which is unexpected.

Weaknesses:

Traumatic brain injury in Drosophila has been previously reported to trigger mitosis of glial cells and generation of neural stem cells in the larval CNS and the adult brain hemispheres. Therefore this report adds to but does not significantly change our current understanding. The origin and identity of VC cells is unclear.

Reviewer #2 (Public Review):

Summary:

Casas-Tinto et al., provide new insight into glial plasticity using a crush injury paradigm in the ventral nerve cord (VNC) of adult Drosophila. The authors find that both astrocyte-like glia (ALG) and ensheating glia (EG) divide under homeostatic conditions in the adult VNC and identify ALG as the glial population that specifically ramps up proliferation in response to injury, whereas the number of EGs decreases following the insult. Using lineage-tracing tools, the authors interestingly observe the interconversion of glial subtypes, especially of EGs into ALGs, which occurs independent of injury and is dependent on the availability of the transcription factor Prospero in EGs, adding to the plasticity observed in the system. Finally, when tracing the progeny of differentiated glia, Casas-Tinto and colleagues detect cells of neuronal identity and provide evidence that such glia-derived neurogenesis is specifically favored following ventral nerve cord injury, which puts forward a remarkable way in which glia can respond to neuronal damage.

Strengths:

This study highlights a new facet of adult nervous system plasticity at the level of the ventral nerve cord, supporting the view that proliferative capacity is maintained in the mature CNS and stimulated upon injury.

The injury paradigm is well chosen, as the organization of the neuromeres allows specific targeting of one segment, compared to the remaining intact, and with the potential to later link observed plasticity to behavior such as locomotion.

Numerous experiments have been carried out in 7-day-old flies, showing that the observed plasticity is not due to residual developmental remodeling or a still immature VNC.

By elegantly combining different genetic tools, the authors show glial divisions with mitotic-dependent tracing and find that the number of generated glia is refined by apoptosis later on.

The work identifies Prospero in glia as an important coordinator of glial cell fate, from development to the adult context, which draws further attention to the upstream regulatory mechanisms.

Weaknesses:

Although the authors do use a variety of methods to show glial proliferation, the EdU data (Figure 1B) could be more informative (Figure 1B) by displaying images of non-injured animals and providing quantifications or the mention of these numbers based on results previously acquired in the system.

The experiments relying on the FUCCI cell cycle reporter suggested considerable baseline proliferation for EGs and ALGs, but when using an independent method (Twin Spot MARCM), mitotic marking was only detected for ALGs. This discrepancy could be addressed by assessing the co-localization of the different glia subsets using the identified driver lines with mitotic markers such as PH3.

The data in Figure 1C would be more convincing in combination with images of the FUCCI Reporter as it can provide further information on the location and proportion of glia that enter the cell cycle versus the fraction that remains quiescent.

The analyses of inter-glia conversion in Figure 3 are complicated by the fact that Prospero RNAi is both used to suppress EG - to ALG conversion and as a marker to establish ALG nature. Clarifications if the GFP+ cells still expressed Pros or were classified as NP-like GFP cells are required here.

The conclusion that ALG and EG glial cells can give rise to cells of neuronal lineage is based on glial lineage information (GFP+ cells from glial G-trace) and staining for the neuronal marker Elav. The use of other neuronal markers apart from Elav or morphological features would provide a more compelling case that GFP+ cells are mature neurons.

Although the text discusses in which contexts, glial plasticity is observed or increased upon injury, the figures are less clear regarding this aspect. A more systematic comparison of injured VNCs versus homeostatic conditions, combined with clear labelling of the injury area would facilitate the understanding of the panels.

Context/Discussion

The study finds that glia in the ventral cord of flies have latent neurogenic potential. Such observations have not been made regarding glia in the fly brain, where injury is reported to drive glial divisions or the proliferation of undifferentiated progenitor cells with neurogenic potential.

Discussing this different strategy for cell replacement adopted by glia in the VNC and pointing out differences to other modes seems fascinating. Highlighting differences in the<br /> the reactiveness of glia in the VNC compared to the brain also seems highly relevant as they may point to different properties to repair damage.

Based on the assays employed, the study points to a significant amount of glial "identity" changes or interconversions, which is surprising under homeostatic conditions. The significance of this "baseline" plasticity remains undiscussed, although glia unarguably show extensive adaptations during nervous system development.

It would be interesting to know if the "interconversion" of glia is determined by the needs in the tissue or would shift in the context of selective ablation/suppression of a glial type.

Reviewer #3 (Public Review):

In this manuscript, Casas-Tintó et al. explore the role of glial cells in the response to a neurodegenerative injury in the adult brain. They used Drosophila melanogaster as a model organism and found that glial cells are able to generate new neurons through the mechanism of transdifferentiation in response to injury.

This paper provides a new mechanism in regeneration and gives an understanding of the role of glial cells in the process.

Reviewer #1 (Public Review):

In this manuscript, Huang and colleagues explored the role of iron in bacterial therapy for cancer. Using proteomics, they revealed the upregulation of bacterial genes that uptake iron, and reasoned that such regulation is an adaptation to the iron-deficient tumor microenvironment. Logically, they engineered E. Coli strains with enhanced iron-uptake efficiency, and showed that these strains, together with iron scavengers, suppress tumor growth in a mouse model. Lastly, they reported the tumor suppression by IroA-E. Coli provides immunological memory via CD8+ T cells. In general, I find the findings in the manuscript novel and the evidence convincing.

(1) Although the genetic and proteomic data are convincing, would it be possible to directly quantify the iron concentration in (1) E. Coli in different growth environments, and (2) tumor microenvironment? This will provide functional consequence of upregulating genes that import iron into the bacteria.

(2) Related to 1, the experiment to study the synergistic effect of CDG and VLX600 (lines 139-175) is very nice and promising, but one flaw here is a lack of the measurement of iron concentration. Therefore, a possible explanation could be that CDG acts in another manner, unrelated to iron uptake, that synergizes with VLX600's function to deplete iron from cancer cells. Here, a direct measurement of iron concentration will show the effect of CDG on iron uptake, thus complementing the missing link.

(3) Lines 250-268: Although statistically significant, I would recommend the authors characterize the CD8+ T cells a little more, as the mechanism now seems quite elusive. What signals or memories do CD8+ T cells acquire after IroA-E. Coli treatment to confer their long-term immunogenicity?

(4) Perhaps this goes beyond the scope of the current manuscript, but how broadly applicable is the observed iron-transport phenomenon in other tumor models? I would recommend the authors to either experimentally test it in another model, or at least discuss this question.

Reviewer #2 (Public Review):

Summary:

The authors provide strong evidence that bacteria, such as E. coli, compete with tumor cells for iron resources and consequently reduce tumor growth. When sequestration between LCN2 and bacterobactin is blocked by upregulating CDG(DGC-E. coli) or salmochelin(IroA-E.coli), E. coli increase iron uptake from the tumor microenvironment (TME) and restrict iron availability for tumor cells. Long-term remission in IroA-E.coli treated mice is associated with enhanced CD8+ T cell activity. Additionally, systemic delivery of IroA-E.coli shows a synergistic effect with chemotherapy reagent oxaliplatin to reduce tumor growth.

Strengths:

It is important to identify the iron-related crosstalk between E. coli and TME. Blocking lcn2-bacterobactin sequestration by different strategies consistently reduce tumor growth.

Weaknesses:

As engineered E.coli upregulate their function to uptake iron, they may increase the likelihood of escaping from nutritional immunity (LCN2 becomes insensitive to sequester iron from the bacteria). Would this raise the chance of developing sepsis? Do authors think that it is safe to administrate these engineered bacteria in mice or humans?

Reviewer #3 (Public Review):

Summary:

Based on their observation that tumor has an iron-deficient microenvironment, and the assumption that nutritional immunity is important in bacteria-mediated tumor modulation, the authors postulate that manipulation of iron homeostasis can affect tumor growth. This paper uses straightforward in vitro and in vivo techniques to examine a specific and important question of nutritional immunity in bacteria-mediated tumor therapy. They are successful in showing that manipulation of iron regulation during nutritional immunity does affect the virulence of the bacteria, and in turn the tumor. These findings open future avenues of investigation, including the use of different bacteria, different delivery systems for therapeutics, and different tumor types. The authors were also successful in addressing the reviewer's concerns adequately.

Reviewer #2 (Public Review):

The goal of the present study is to better understand the 'control objectives' that subjects adopt in a video-game-like virtual-balancing task. In this task, the hand must move in the opposite direction from a cursor. For example, if the cursor is 2 cm to the right, the subject must move their hand 2 cm to the left to 'balance' the cursor. Any imperfection in that opposition causes the cursor to move. E.g., if the subject were to move only 1.8 cm, that would be insufficient, and the cursor would continue to move to the right. If they were to move 2.2 cm, the cursor would move back toward the center of the screen. This return to center might actually be 'good' from the subject's perspective, depending on whether their objective is to keep the cursor still or keep it near the screen's center. Both are reasonable 'objectives' because the trial fails if the cursor moves too far from the screen's center during each six-second trial.

This task was recently developed for use in monkeys (Quick et al., 2018), with the intention of being used for the study of the cortical control of movement, and also as a task that might be used to evaluate BMI control algorithms. The purpose of the present study is to better characterize how this task is performed. What sort of control policies are used. Perhaps more deeply, what kind of errors are those policies trying to minimize? To address these questions, the authors simulate control-theory style models and compare with behavior. They do in both in monkeys and in humans.

These goals make sense as a precursor to future recording or BMI experiments. The primate motor-control field has long been dominated by variants of reaching tasks, so introducing this new task will likely be beneficial. This is not the first non-reaching task, but it is an interesting one and it makes sense to expand the presently limited repertoire of tasks. The present task is very different from any prior task I know of. Thus, it makes sense to quantify behavior as thoroughly as possible in advance of recordings. Understanding how behavior is controlled is, as the authors note, likely to be critical to interpreting neural data.

From this perspective - providing a basis for interpreting future neural results - the present study is fairly successful. Monkeys seem to understand the task properly, and to use control policies that are not dissimilar from humans. Also reassuring is the fact that behavior remains sensible even when task-difficulty become high. By 'sensible' I simply mean that behavior can be understood as seeking to minimize error: position, velocity, or (possibly) both, and that this remains true across a broad range of task difficulties. The authors document why minimizing position and minimizing velocity are both reasonable objectives. Minimizing velocity is reasonable, because a near-stationary cursor can't move far in six seconds. Minimizing position error is reasonable, because the trial won't fail if the cursor doesn't stray far from the center. This is formally demonstrated by simulating control policies: both objectives lead to control policies that can perform the task and produce realistic single-trial behavior. The authors also demonstrate that, via verbal instruction, they can induce human subjects to favor one objective over the other. These all seem like things that are on the 'need to know' list, and it is commendable that this amount of care is being taken before recordings begin, as it will surely aid interpretation.

Yet as a stand-alone study, the contribution to our understanding of motor control is more limited. The task allows two different objectives (minimize velocity, minimize position) to be equally compatible with the overall goal (don't fail the trial). Or more precisely, there exists a range of objectives with those two at the extreme. So it makes sense that different subjects might choose to favor different objectives, and also that they can do so when instructed. But has this taught us something about motor control, or simply that there is a natural ambiguity built into the task? If I ask you to play a game, but don't fully specify the rules, should I be surprised that different people think the rules are slightly different?

The most interesting scientific claim of this study is not the subject-to-subject variability; the task design makes that quite likely and natural. Rather, the central scientific result is the claim that individual subjects are constantly switching objectives (and thus control policies), such that the policy guiding behavior differs dramatically even on a single-trial basis. This scientific claim is supported by a technical claim: that the authors' methods can distinguish which objective is in use, even on single trials. I am uncertain of both claims.

Consider Figure 8B, which reprises a point made in Figure 1&3 and gives the best evidence for trial-to-trial variability in objective/policy. For every subject, there are two example trials. The top row of trials shows oscillations around the center, which could be consistent with position-error minimization. The bottom row shows tolerance of position errors so long as drift is slow, which could be consistent with velocity-error minimization. But is this really evidence that subjects were switching objectives (and thus control policies) from trial to trial? A simpler alternative would be a single control policy that does not switch, but still generates this range of behaviors. The authors don't really consider this possibility, and I'm not sure why. One can think of a variety of ways in which a unified policy could produce this variation, given noise and the natural instability of the system.

Indeed, I found that it was remarkably easy to produce a range of reasonably realistic behaviors, including the patterns that the authors interpret as evidence for switching objectives, based on a simple fixed controller. To run the simulations, I made the simple assumption that subjects simply attempt to match their hand position to oppose the cursor position. Because subjects cannot see their hand, I assumed modest variability in the gain, with a range from -1 to -1.05. I assumed a small amount of motor noise in the outgoing motor command. The resulting (very simple) controller naturally displayed the basic range of behaviors observed across trials (see Image 1)

Image 1.

Some trials had oscillations around the screen center (zero), which is the pattern the authors suggest reflects position control. In other trials the cursor was allowed to drift slowly away from the center, which is the pattern the authors suggest reflects velocity control. This is true even though the controller was the same on every trial. Trial-to-trial differences were driven both by motor noise and by the modest variability in gain. In an unstable system, small differences can lead to (seemingly) qualitatively different behavior on different trials.

This simple controller is also compatible with the ability of subjects to adapt their strategy when instructed. Anyone experienced with this task likely understands (or has learned) that moving the hand slightly more than 'one should' will tend to shepherd the cursor back to center, at the cost of briefly high velocity. Using this strategy more sparingly will tend to minimize velocity even if position errors persist. Thus, any subject using this control policy would be able to adapt their strategy via a modest change in gain (the gain linking visible cursor position to intended hand position).

This model is simple, and there may be reasons to dislike it. But it is presumably a reasonable model. The nature of the task is that you should move your hand opposite where the cursor is. Because you can't see your hand, you will make small mistakes. Due to the instability of the system, those small mistakes have large and variable effects. This feature is likely common to other controllers as well; many may explicitly or implicitly blend position and velocity control, with different trials appearing more dominated by one versus the other. Given this, I think the study presents only weak evidence that individual subjects are switching their objective on individual trials. Indeed, the more parsimonious explanation may be that they aren't. While the study certainly does demonstrate that the control policy can be influenced by verbal instructions, this might be a small adjustment as noted above.

I thus don't feel convinced that the authors can conclusively tell us the true control policy being used by human and monkey subjects, nor whether that policy is mostly fixed or constantly switching. The data are potentially compatible with any of these interpretations, depending on which control-style model one prefers.

I see a few paths that the authors might take if they chose.<br /> --First, my reasoning above might be faulty, or there might be additional analyses that could rule out the possibility of a unified policy underlying variable behavior. If so, the authors may be able to reject the above concerns and retain the present conclusions. The main scientifically novel conclusion of the present study is that subjects are using a highly variable control policy, and switching on individual trials. If this is indeed the case, there may be additional analyses that could reveal that.<br /> --Second, additional trial types (e.g., with various perturbations) might be used as a probe of the control policy. As noted below, there is a long history of doing this in the pursuit system. That additional data might better disambiguate control policies both in general, and across trials.<br /> --Third, the authors might find that a unified controller is actually a good (and more parsimonious) explanation. Which might actually be a good thing from the standpoint of future experiments. Interpretation of neural data is likely to be much easier if the control policy being instantiated isn't in constant flux.

In any case, I would recommend altering the strength of some conclusions, particularly the conclusion that the presented methods can reliably discriminate amongst objectives/policies on individual trials. This is mentioned as a major motivation on multiple occasions, but in most of these instances, the subsequent analysis infers the objective only across trial (e.g., one must observe a scatterplot of many trials). By Figure 7, they do introduce a method for inferring the control policy on individual trials, and while this seems to work considerably better than chance, it hardly appears reliable.

In this same vein I would suggest toning down aspects of the Introduction and Discussion. The Introduction in particular is overly long, and tries to position the present study as unique in ways that seem strained. Other studies have built links between human behavior, monkey behavior, and monkey neural data (for just one example, consider the corpus of work from the Scott lab that includes Pruszynski et al. 2008 and 2011). Other studies have used highly quantitative methods to infer the objective function used by subjects (e.g. Kording and Wolpert 2004). The very issue that is of interest in the present study - velocity-error-minimization versus position-error-minimization - has been extensively addressed in the smooth pursuit system. That field has long combined quantitative analyses of behavior in humans and monkeys, along with neural recordings. Many pursuit experiments used strategies that could be fruitfully employed to address the central questions of the present study. For example, error stabilization was important for dissecting the control policy used by the pursuit system. By artificially stabilizing the error (position or velocity) at zero, or at some other value, one can determine the system's response. The classic Rashbass step (1961) put position and velocity errors in opposition, to see which dominates the response. Step and sinusoidal perturbations were useful in distinguishing between models, as was the imposition of artificially imposed delays. The authors note the 'richness' of the behavior in the present task, and while one could say the same of pursuit, it was still the case that specific and well-thought through experimental manipulations were pretty critical. It would be better if the Introduction considered at least some of the above-mentioned work (or other work in a similar vein). While most would agree with the motivations outlined by the authors - they are logical and make sense - the present Introduction runs the risk of overselling the present conclusions while underselling prior work.

Reviewer #2 (Public Review):

Summary

The paper concerns the phenomenon of continuous flash suppression (CFS), relevant to questions about the extent and nature of subconscious visual processing. Whereas standard CFS studies only measure the breakthrough threshold-the contrast at which an initially suppressed target stimulus with steadily increasing contrast becomes visible-this study also measures the re-suppression threshold, the contrast at which a visible target with decreasing contrast becomes suppressed. Thus, the authors could calculate suppression depth, the ratio between the breakthrough and re-suppression thresholds. To measure both thresholds, the study introduces the tracking-CFS method, a continuous-trial design that results in faster, better controlled, and lower-variance threshold estimates compared to the discrete trials standard in the literature. The study finds that suppression depths are similar for different image categories, providing an interesting contrast to previous results that breakthrough thresholds differ for different image categories. The new finding calls for a reassessment of interpretations based solely on the breakthrough threshold that subconscious visual processing is category-specific.

Strengths

(1) The tCFS method quickly estimates breakthrough and re-suppression thresholds using continuous trials, which also better control for slowly varying factors such as adaptation and attention. Indeed, tCFS produces estimates with lower across-subject variance than the standard discrete-trial method (Fig. 2). The tCFS method is straightforward to adopt in future research on CFS and binocular rivalry.

(2) The CFS literature has lacked re-suppression threshold measurements. By measuring both breakthrough and re-suppression thresholds, this work calculated suppression depth (i.e., the difference between the two thresholds), which warrants different interpretations from the breakthrough threshold alone.

(3) The work found that different image categories show similar suppression depths, suggesting some aspects of CFS are not category-specific. This result enriches previous findings that breakthrough thresholds vary with image categories. Re-suppression thresholds vary symmetrically, such that their differences are constant.

Weakness

I do not follow the authors' reasoning as to why the suppression depth is a better (or fuller, superior, more informative) indication of subconscious visual processing than the breakthrough threshold alone. To my previous round of comments, the authors replied that 'breakthrough provides only half of the needed information.' I do not understand this. One cannot infer the suppression depth from the breakthrough threshold alone, but *one cannot obtain the breakthrough threshold from the suppression depth alone*, either. The two measures are complementary. (To be sure, given *both* the suppression depth and the re-suppression threshold, one can recover the breakthrough threshold. The discussion concerns the suppression depth *alone* and the breakthrough threshold *alone*.) I am fully open to being convinced that there is a good reason why the suppression depth may be more informative than the breakthrough threshold about a specific topic, e.g., inter-ocular suppression or subconscious visual processing. I only request that the authors make such an argument explicit. For example, in the significance statement, the authors write, 'all images show equal suppression when both thresholds are measured. We *thus* find no evidence of differential unconscious processing and *conclude* reliance on breakthrough thresholds is misleading' (emphasis added). Just what supports the 'thus' and the 'conclude'? Similarly, at the end of the introduction, the authors write, '[...] suppression depth was constant for faces, objects, gratings and visual noise. *In other words*, we find no evidence to support differential unconscious processing among these particular, diverse categories of suppressed images' (emphasis added). I am not sure the statements in the two sentences are equivalent.

The authors' reply included a discussion of neural CRFs, which may explain why the bCFS thresholds differ across image categories. A further step seems necessary to explain why CRFs do not qualify as a form of subconscious processing.

Reviewer #1 (Public Review):

Summary

A new method, tCFS, is introduced to offer richer and more efficient measurement of interocular suppression. It generates a new index, the suppression depth, based on the contrast difference between the up-ramped contrast for the target to breakthrough suppression and the down-ramped contrast for the target to disappear into suppression. A uniform suppression depth regardless of image types (e.g., faces, gratings and scrambles) was discovered in the paper, favoring an early-stage mechanism involving CFS. Discussions about claims of unconscious processing and the related mechanisms.

Strength

The tCFS method adds to the existing bCFS paradigms by providing the (re-)suppression threshold and thereafter the depression depth. Benefiting from adaptive procedures with continuous trials, the tCFS is able to give fast and efficient measurements. It also provides a new opportunity to test theories and models about how information is processed outside visual awareness.

Weakness:

This paper reports the surprising finding of uniform suppression depth over a variety of stimuli. This is novel and interesting. But given the limited samples being tested, the claim of uniformity suppression depth needs to be further examined, with respect to different complexities and semantic meanings.

From an intuitive aspect, the results challenged previous views about "preferential processing" for certain categories, though it invites further research to explore what exactly could suppression depth tell us about unconscious visual processing.

Reviewer #3 (Public Review):

Summary:

In the 'bCFS' paradigm, a monocular target gradually increases in contrast until it breaks interocular suppression by a rich monocular suppressor in the other eye. The present authors extend the bCFS paradigm by allowing the target to reduce back down in contrast until it becomes suppressed again. The main variable of interest is the contrast difference between breaking suppression and (re) entering suppression. The authors find this difference to be constant across a range of target types, even ones that differ substantially in the contrast at which they break interocular suppression (the variable conventionally measured in bCFS). They also measure how the difference changes as a function of other manipulations. Interpretation is in terms of the processing of unconscious visual content, as well as in terms of the mechanism of interocular suppression.

Strengths:

Interpretation of bCFS findings is mired in controversy, and this is an ingenuous effort to move beyond the paradigm's exclusive focus on breaking suppression. The notion of using the contrast difference between breaking and entering suppression as an index of suppression depth is interesting. The finding that this difference is similar for a range of target types that do differ in the contrast at which they break suppression, suggests a common mechanism of suppression across those target types.

Reviewer #2 (Public Review):

Summary:

This study aims to address existing differences in the literature regarding the extent of reward versus aversive dopamine signaling in the prefrontal cortex. To do so, the authors chose to present mice with both a reward and an aversive stimulus during different trials each day. The authors used high spatial resolution two-photon calcium imaging of individual dopaminergic axons in the medial PFC to characterize the response of these axons to determine the selectivity of responses in unique axons. They also paired the reward (water) and an aversive stimulus (tail shock) with auditory tones and recorded across 12 days of associative learning.

The authors find that some axons respond to both reward and aversive unconditioned stimuli, but overall, there is a preference to respond to aversive stimuli consistent with expectations from prior studies that used other recording methods. The authors find that both of their two auditory stimuli initially drive responses in axons, but that with training axons develop more selective responses for the shock associated tone indicating that associative learning led to changes in these axon's responses. Finally, the authors use anticipatory behaviors during the conditioned stimuli and facial expressions to determine stimulus discrimination and relate dopamine axons signals with this behavioral evidence of discrimination. This study takes advantage of cutting-edge imaging approaches to resolve the extent to which dopamine axons in PFC respond appetitive or aversive stimuli. They conclude that there is a bias to respond to the aversive tail shock in most axons and weaker more sparse representation of water reward.

Strengths:

The strength of this study is the imaging approach that allows for investigation of the heterogeneity of response across individual dopamine axons unlike other common approaches such as fiber photometry which provide a measure of the average population activity. The use of appetitive and aversive stimuli to probe responses across individual axons is another strength as it reveals response diversity that is often overlooked in reward-only studies.

Weaknesses:

A weakness of this study is the design of the associative conditioning paradigm. The use of only a single reward and single aversive stimulus makes it difficult to know whether these results are specific to the valence of the stimuli versus the specific identity of the stimuli. Further, the reward presentations are more numerous than the aversive trials making it unclear how much novelty and habituation account for results. Moreover, the training seems somewhat limited by the low number of trials and did not result in strong associative conditioning. The lack of omission responses reported may reflect weak associative conditioning. Finally, the study provides a small advance in our understanding of dopamine signaling in the PFC and lacks evidence for if and what might be the consequence of these axonal responses on PFC dopamine concentrations and PFC neuron activity.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Abe and colleagues employ in vivo 2-photon calcium imaging of dopaminergic axons in the mPFC. The study reveals that these axons primarily respond to unconditioned aversive stimuli (US) and enhance their responses to initially-neutral stimuli after classical association learning. The manuscript is well-structured and presents results clearly. The utilization of a refined prism-based imaging technique, though not entirely novel, is well-implemented. The study's significance lies in its contribution to the existing literature by offering single-axon resolution functional insights, supplementing prior bulk measurements of calcium or dopamine release. Given the current focus on neuromodulator neuron heterogeneity, the work aligns well with current research trends and will greatly interest researchers in the field.

Comment on the revised version:

In my opinion, the authors did a great job with the revision of the manuscript.

Reviewer #3 (Public Review):

Summary:

The authors image dopamine axons in medial prefrontal cortex (mPFC) using microprism-mediated two-photon calcium imaging. They image these axons as mice learn that two auditory cues predict two distinct outcomes, tailshock, or water delivery. They find that some axons show a preference for encoding of the shock and some show a preference for encoding of water. The authors report a greater number of dopamine axons in mPFC that respond to shock. Across time, the shock-preferring axons begin to respond preferentially to the cue predicting shock, while there is a less pronounced increase in the water-responsive axons that acquire a response to the water-predictive cue (these axons also increase non-significantly to the shock-predictive cue). These data lead the authors to argue that dopamine axons in mPFC preferentially encode aversive stimuli.

Strengths:

The experiments are beautifully executed and the authors have mastered an impressively complex technique. Specifically, they are able to image and track individual dopamine axons in mPFC across days of learning. And this technique is used the way it should be: the authors isolate distinct dopamine axons in mPFC and characterize their encoding preferences and how this evolves across learning of cue-shock and cue-water contingencies. Thus, these experiments are revealing novel information about how aversive and rewarding stimuli is encoded at the level of individual axons, in a way that has not been done before. This is timely and important.

Weaknesses:

The overarching conclusion of the paper is that dopamine axons preferentially encode aversive stimuli. However, this is confounded by differences in the strength of the aversive and appetitive outcomes. As the authors point out, the axonal response to stimuli is sensitive to outcome magnitude (Supp Fig 3). That is, if you increase the magnitude of water or shock that is delivered, you increase the change in fluorescence that is seen in the axons. Unsurprisingly, the change in fluorescence that is seen to shock is considerably higher than water reward. Further, over 40% of the axons respond to water early in training [yet just a few lines below the authors write: "Previous studies have demonstrated that the overall dopamine release at the mPFC or the summed activity of mPFC dopamine axons exhibits a strong response to aversive stimuli (e.g., tail shock), but little to rewards", which seems inconsistent with their own data]. Given these aspects of the data, it could be the case that the dopamine axons in mPFC encodes different types of information and delegates preferential processing to the most salient outcome across time. The use of two similar sounding tones (9Khz and 12KHz) for the reward and aversive predicting cues are likely to enhance this as it requires a fine-grained distinction between the two cues in order to learn effectively. That is not to say that the mice cannot distinguish between these cues, rather that they may require additional processes to resolve the similarity, which are known to be dependent on the mPFC.

There is considerable literature on mPFC function across species that would support such a view. Specifically, theories of mPFC function (in particular prelimbic cortex, which is where the axon images are mostly taken) generally center around resolution of conflict in what to respond, learn about, and attend to. That is, mPFC is important for devoting the most resources (learning, behavior) to the most relevant outcomes in the environment. This data then, provides a mechanism for this to occur in mPFC. That is, dopamine axons signal to the mPFC the most salient aspects of the environment, which should be preferentially learnt about and responded towards. This is also consistent with the absence of a negative prediction error during omission: the dopamine axons show increases in responses during receipt of unexpected outcomes but do not encode negative errors. This supports a role for this projection in helping to allocate resources to the most salient outcomes and their predictors, and not learning per se. Below are a just few references from the rich literature on mPFC function (some consider rodent mPFC analogous to DLPFC, some mPFC), which advocate for a role in this region in allocating attention and cognitive resources to most relevant stimuli, and do not indicate preferential processing of aversive stimuli.

References:<br /> 1. Miller, E. K., & Cohen, J. D. (2001). An integrative theory of prefrontal cortex function. Annual review of neuroscience, 24(1), 167-202.<br /> 2. Bissonette, G. B., Powell, E. M., & Roesch, M. R. (2013). Neural structures underlying set-shifting: roles of medial prefrontal cortex and anterior cingulate cortex. Behavioural brain research, 250, 91-101.<br /> 3. Desimone, R., & Duncan, J. (1995). Neural mechanisms of selective visual attention. Annual review of neuroscience, 18(1), 193-222.<br /> 4. Sharpe, M. J., Stalnaker, T., Schuck, N. W., Killcross, S., Schoenbaum, G., & Niv, Y. (2019). An integrated model of action selection: distinct modes of cortical control of striatal decision making. Annual review of psychology, 70, 53-76.<br /> 5. Ridderinkhof, K. R., Ullsperger, M., Crone, E. A., & Nieuwenhuis, S. (2004). The role of the medial frontal cortex in cognitive control. science, 306(5695), 443-447.<br /> 6. Nee, D. E., Kastner, S., & Brown, J. W. (2011). Functional heterogeneity of conflict, error, task-switching, and unexpectedness effects within medial prefrontal cortex. Neuroimage, 54(1), 528-540.<br /> 7. Isoda, M., & Hikosaka, O. (2007). Switching from automatic to controlled action by monkey medial frontal cortex. Nature neuroscience, 10(2), 240-248.

Reviewer #1 (Public Review):

In this paper, the authors evaluate the utility of brain age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative suited to applications of cognitive decline. While this is less accurate overall than brain age, it explains more unique variance in the downstream regression.

REVISED VERSION: while the authors have partially addressed my concerns, I do not feel they have addressed them all. I do not feel they have addressed the weight instability and concerns about the stacked regression models satisfactorily. I also must say that I agree with Reviewer 3 about the limitations of the brain age and brain cognition methods conceptually. In particular that the regression model used to predict fluid cognition will by construction explain more variance in cognition than a brain age model that is trained to predict age. This suffers from the same problem the authors raise with brain age and would indeed disappear if the authors had a separate measure of cognition against which to validate and were then to regress this out as they do for age correction. I am aware that these conceptual problems are more widespread than this paper alone (in fact throughout the brain age literature), so I do not believe the authors should be penalised for that. However, I do think they can make these concerns more explicit and further tone down the comments they make about the utility of brain cognition. I have indicated the main considerations about these points in the recommendations section below.

In this paper, the authors evaluate the utility of brain age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative that explains more unique variance in the downstream regression.

This is a reasonably good paper and the use of a commonality analysis is a nice contribution to understanding variance partitioning across different covariates. I have some comments that I believe the authors ought to address, which mostly relate to clarity and interpretation

First, from a conceptual point of view, the authors focus exclusively on cognition as a downstream outcome. I would suggest the authors nuance their discussion to provide broader considerations of the utility of their method and on the limits of interpretation of brain age models more generally.

Second, from a methods perspective , there is not a sufficient explanation of the methodological procedures in the current manuscript to fully understand how the stacked regression models were constructed. I would request that the authors provide more information to enable the reader to better understand the stacked regression models used to ensure that these models are not overfit.

Please also provide an indication of the different regression strengths that were estimated across the different models and cross-validation splits. Also, how stable were the weights across splits?

Please provide more details about the task designs, MRI processing procedures that were employed on this sample in addition to the regression methods and bias correction methods used. For example, there are several different parameterisations of the elastic net, please provide equations to describe the method used here so that readers can easily determine how the regularisation parameters should be interpreted.

Reviewer #1 (Public Review):

Summary:

Previous work in humans and non-human animals suggests that during offline periods following learning, the brain replays newly acquired information in a sequential manner. The present study uses an MEG-based decoding approach to investigate the nature of replay/reactivation during a cued recall task directly following a learning session, where human participants are trained on a new sequence of 10 visual images embedded in a graph structure. During retrieval, participants are then cued with two items from the learned sequence, and neural evidence is obtained for the simultaneous or sequential reactivation of future sequence items. The authors find evidence for both sequential and clustered (i.e., simultaneous) reactivation. Replicating previous work, low-performing participants tend to show sequential, temporally segregated reactivation of future items, whereas high-performing participants show more clustered reactivation. Adding to previous work, the authors show that an image's reactivation strength varies depending on its proximity to the retrieval cue within the graph structure.

Strengths:

As the authors point out, work on memory reactivation has largely been limited to the retrieval of single associations. Given the sequential nature of our real-life experiences, there is clearly value in extending this work to structured, sequential information. State-of-the-art decoding approaches for MEG are used to characterize the strength and timing of item reactivation. The manuscript is very well written with helpful and informative figures in the main sections. The task includes an extensive localizer with 50 repetitions per image, allowing for stable training of the decoders and the inclusion of several sanity checks demonstrating that on-screen items can be decoded with high accuracy.

Weaknesses:

Of major concern, the experiment is not optimally designed for analysis of the retrieval task phase, where only 4 min of recording time and a single presentation of each cue item are available for the analyses of sequential and non-sequential reactivation. In their revision, the authors include data from the learning blocks in their analysis. These blocks follow the same trial structure as the retrieval task, and apart from adding more data points could also reveal a possible shift from sequential to clustered reactivation as learning of the graph structure progresses. The new analyses are not entirely conclusive, maybe given the variability in the number of learning blocks that participants require to reach criterion. In principal, they suggest that reactivation strength increases from learning (pre-rest) to final retrieval (post-rest).

On a more conceptual note, the main narrative of the manuscript implies that sequential and clustered reactivation are mutually exclusive, such that a single participant would show either one or the other type. With the analytic methods used here, however, it seems possible to observe both types of reactivation. For example, the observation that mean reactivation strength (across the entire trial, or in a given time window of interest) varies with graph distance does not exclude the possibility that this reactivation is also sequential. In fact, the approach of defining one peak time window of reactivation may bias towards simultaneous, graded reactivation. It would be helpful if the authors could clarify this conceptual point. A strong claim that the two types of reactivation are mutually exclusive would need to be substantiated by further evidence, for instance a suitable metric contrasting "sequenceness" vs "clusteredness".

On the same point, the non-sequential reactivation analyses use a time window of peak decodability that is determined based on the average reactivation of all future items, irrespective of graph distance. In a sequential forward cascade of reactivations, it could be assumed that the reactivation of near items would peak earlier than the reactivation of far items. In the revised manuscript, the authors now show the "raw" timecourses of item decodability at different graph distances, clearly demonstrating their peak reactivation times, which show convincingly that reactivation for near and far items occurs at very similar time points. The question that remains, therefore, is whether the method of pre-selecting a time window of interest described above could exert a bias towards finding clustered reactivation.

Reviewer #2 (Public Review):

Summary:

The authors investigate replay (defined as sequential reactivation) and clustered reactivation during retrieval of an abstract cognitive map. Replay and clustered reactivation were analysed based on MEG recordings combined with a decoding approach. While the authors state to find evidence for both, replay and clustered reactivation during retrieval, replay was exclusively present in low performers. Further, the authors show that reactivation strength declined with an increasing graph distance.

Strengths:

The paper raises interesting research questions, i.e., replay vs. clustered reactivation and how that supports retrieval of cognitive maps. The paper is well written, well structured and easy to follow. The methodological approach is convincing and definitely suited to address the proposed research questions.

The paper is a great combination between replicating previous findings (Wimmer et al. 2020) with a new experimental approach but at the same time presenting novel evidence (reactivation strength declines as a function of graph distance).

What I also want to positively highlight is their general transparency. For example, they pre-registered this study but with a focus on a different part of the data and outlined this explicitly in the paper.

The paper has very interesting findings. However, there are some shortcomings especially in the experimental design. These are shortly outlined below but are also openly and in detail discussed by the authors.

Weaknesses:

The individual findings are interesting. However, due to some shortcomings in the experimental design they cannot be profoundly related to each other. For example, the authors show that replay is present in low but not in high performers with the assumption that high performers tend to simultaneously reactivate items. But then, the authors do not investigate clustered reactivation (= simultaneous reactivation) as a function of performance due to a low number of retrieval trials and ceiling performance in most participants.<br /> As a consequence of the experimental design, some analyses are underpowered (very low number of trials, n = ~10, and for some analyses, very low number of participants, n = 14).

Reviewer #1 (Public Review):

Summary:

In this study, Jellinger et al. performed engram-specific sequencing and identified genes that were selectively regulated in positive/negative engram populations. In addition, they performed chronic activation of the negative engram population over 3 months and observed several effects on fear/anxiety behavior and cellular events such as upregulation of glial cells and decreased GABA levels.

Strengths:

They provide useful engram-specific GSEA data and the main concept of the study, linking negative valence/memory encoding to cellular level outcomes including upregulation of glial cells, is interesting and valuable.

Weaknesses:

A number of experimental shortcomings make the conclusion of the study largely unsupported. In addition, the observed differences in behavioral experiments are rather small, inconsistent, and the interpretation of the differences is not compelling.

Major points for improvement:

(1) Lack of essential control experiments

With the current set of experiments, it is not certain that the DREADD system they used was potent and stable throughout the 3 months of manipulations. Basic confirmatory experiments (e.g., slice physiology at 1m vs. 3m) to show that the DREADD effects on these vHP are stable would be an essential bottom line to make these manipulation experiments convincing.

Furthermore, although the authors use the mCherry vector as a control, they did not have a vehicle/saline control for the hM3Dq AAV. Thus, the long-term effects such as the increase in glial cells could simply be due to the toxicity of DREADD expression, rather than an induced activity of these cells.

(2) Figure 1 and the rest of the study are disconnected

The authors used the cFos-tTA system to label positive/negative engram populations, while the TRAP2 system was used for the chronic activation experiments. Although both genetic tools are based on the same IEG Fos, the sensitivity of the tools needs to be validated. In particular, the sensitivity of the TRAP2 system can be arbitrarily altered by the amount of tamoxifen (or 4OHT) and the administration protocols. The authors should at least compare and show the percentage of labeled cells in both methods and discuss that the two experiments target (at least slightly) different populations. In addition, the use of TRAP2 for vHP is relatively new; the authors should confirm that this method actually captures negative engram populations by checking for reactivation of these cells during recall by overlap analysis of Fos staining or by artificial activation.

(3) Interpretation of the behavior data

In Figures 3a and b, the authors show that the experimental group showed higher anxiety based on time spent in the center/open area. However, there were no differences in distance traveled and center entries, which are often reduced in highly anxious mice. Thus, it is not clear what the exact effect of the manipulation is. The authors may want to visualize the trajectories of the mice's locomotion instead of just showing bar graphs.

In addition, the data shown in Figure 4b is somewhat surprising - the 14MO control showed more freezing than the 6MO control, which can be interpreted as "better memory in old". As this is highly counterintuitive, the authors may want to discuss this point. The authors stated that "Mice typically display increased freezing behavior as they age, so these effects during remote recall are expected" without any reference. This is nonsense, as just above in Figure 4a, older mice actually show less freezing than young mice.

Overall, the behavioral effects are rather small and random. I would suggest that these data be interpreted more carefully.

(4) Lack of citation and discussion of relevant study

Khalaf et al. 2018 from Gräff lab showed that experimental activation of recall-induced populations leads to fear attenuation. Despite the differences in experimental details, the conceptual discrepancy should be discussed.

Reviewer #2 (Public Review):

Summary:

Jellinger, Suthard, et al. investigated the transcriptome of positive and negative valence engram cells in the ventral hippocampus, revealing anti- and pro-inflammatory signatures of these respective valences. The authors further reactivated the negative valence engram ensembles to assay the effects of chronic negative memory reactivation in young and old mice. This chronic re-activation resulted in differences in aspects of working memory, and fear memory, and caused morphological changes in glia. Such reactivation-associated changes are putatively linked to GABA changes and behavioral rumination.

Strengths:

Much of the content of this manuscript is of benefit to the community, such as the discovery of differential engram transcriptomes dependent on memory valence. The chronic activation of neurons, and the resultant effects on glial cells and behavior, also provide the community with important data. Laudable points of this manuscript include the comprehensiveness of behavioral experiments, as well as the cross-disciplinary approach.

Weaknesses:

There are several key claims made that are unsubstantiated by the data, particularly regarding the anthropomorphic framing of "rumination" on a mouse model and the role of GABA. The conclusions and inferences in these areas need to be carefully considered.

(1) There are many issues regarding the arguments for the behavioural data's human translation as "rumination." There is no definition of rumination provided in the manuscript, nor how rumination is similar/different to intrusive thoughts (which are psychologically distinct but used relatively interchangeably in the manuscript), nor how rumination could be modelled in the rodent. The authors mention that they are attempting to model rumination behaviours by chronically reactivating the negative engram ("To understand if our experimental model of negative rumination..."), but this occurs almost at the very end of the results section, and no concrete evidence from the literature is provided to attempt to link the behavioural results (decreased working memory, increased fear extinction times) to rumination-like behaviours. The arguments in the final paragraph of the Discussion section about human rumination appear to be unrelated to the data presented in the manuscript and contain some uncited statements. Finally, the rumination claims seem to be based largely upon a single data figure that needs to be further developed (Figure 6, see also point 2 below).

(2) The staining and analysis in Figure 6 are challenging to interpret, and require more evidence to substantiate the conclusions of these results. The histological images are zoomed out, and at this resolution, it appears that only the pyramidal cell layer is being stained. A GABA stain should also label the many sparsely spaced inhibitory interneurons existing across all hippocampal layers, yet this is not apparent here. Moreover, both example images in the treatment group appear to have lower overall fluorescence intensity in both DAPI and GABA. The analysis is also unclear: the authors mention "ROIs" used to measure normalized fluorescence intensity but do not specify what the ROI encapsulates. Presumably, the authors have segmented each DAPI-positive cell body and assessed fluorescence - however, this is not explicated nor demonstrated, making the results difficult to interpret.

(3) A smaller point, but more specific detail is needed for how genes were selected for GSEA analysis. As GSEA relies on genes to be specified a priori, to avoid a circular analysis, these genes need to be selected in a blind/unbiased manner to avoid biasing downstream results and conclusions. It's likely the authors have done this, but explicitly noting how genes were selected is an important context for this analysis.

Reviewer #3 (Public Review):

Summary:

The authors note that negative ruminations can lead to pathological brain states and mood/anxiety dysregulation. They test this idea by using mouse engram-tagging technology to label dentate gyrus ensembles activated during a negative experience (fear conditioning). They show that chronic chemogenetic activation of these ensembles leads to behavioral (increased anxiety, increased fear generalization, reduced fear extinction) and neural (increases in neuroinflammation, microglia, and astrocytes).

Strengths:

The question the authors ask here is an intriguing one, and the engram activation approach is a powerful way to address the question. Examination of a wide range of neural and behavioral dependent measures is also a strength.

Weaknesses:

The major weakness is that the authors have found a range of changes that are correlates of chronic negative engram reactivation. However, they do not manipulate these outcomes to test whether microglia, astrocytes, or neuroinflammation are causally linked to the dysregulated behaviors.

Reviewer #1 (Public Review):

Summary:

This important study investigated the role of oxytocin (OT) neurons in the paraventricular nucleus (PVN) and their projections to the medial prefrontal cortex (mPFC) in regulating pup care and infanticide behaviors in mandarin voles. The researchers used techniques like immunofluorescence, optogenetics, OT sensors, and peripheral OT administration. Activating OT neurons in the PVN reduced the time it took pup-caring male voles to approach and retrieve pups, facilitating pup-care behavior. However, this activation had no effect on females. Interestingly, this same PVN OT neuron activation also reduced the time for both male and female infanticidal voles to approach and attack pups, suggesting PVN OT neuron activity can promote pup care while inhibiting infanticide behavior. Inhibition of these neurons promoted infanticide. Stimulating PVN->mPFC OT projections facilitated pup care in males and in infanticide-prone voles, activation of these terminals prolonged latency to approach and attack. Inhibition of PVN->mPFC OT projections promoted infanticide. Peripheral OT administration increased pup care in males and reduced infanticide in both sexes. However, some results differed in females, suggesting other mechanisms may regulate female pup care.

Strengths:

This multi-faceted approach provides converging evidence, strengthens the conclusions drawn from the study, and makes them very convincing. Additionally, the study examines both pup care and infanticide behaviors, offering insights into the mechanisms underlying these contrasting behaviors. The inclusion of both male and female voles allows for the exploration of potential sex differences in the regulation of pup-directed behaviors. The peripheral OT administration experiments also provide valuable information for potential clinical applications and wildlife management strategies.

Weaknesses:

While the study presents exciting findings, there are several weaknesses that should be addressed. The sample sizes used in some experiments, such as the Fos study and optogenetic manipulations, appear to be small, which may limit the statistical power and generalizability of the results. Effect sizes are not reported, making it difficult to evaluate the practical significance of the findings. The imaging parameters and analysis details for the Fos study are not clearly described, hindering the interpretation of these results (i.e., was the entire PVN counted?). Also, does the Fos colocalization align with previous studies that look at PVN Fos and maternal/ paternal care? Additionally, the study lacks electrophysiological data to support the optogenetic findings, which could provide insights into the neural mechanisms underlying the observed behaviors.

The study has several limitations that warrant further discussion. Firstly, the potential effects of manipulating OT neurons on the release of other neurotransmitters (or the influence of other neurochemicals or brain regions) on pup-directed behaviors, especially in females, are not fully explored. Additionally, it is unclear whether back-propagation of action potentials during optogenetic manipulations causes the same behavioral effect as direct stimulation of PVN OT cells. Moreover, the authors do not address whether the observed changes in behavior could be explained by overall increases or decreases in locomotor activity.

The authors do not specify the percentage of PVN->mPFC neurons labeled that were OT-positive, nor do they directly compare the sexes in their behavioral analysis (or if they did, it is not clear statistically). While the authors propose that the sex difference in pup-directed behaviors is due to females having greater OT expression, they do not provide evidence to support this claim from their labeling data. It is also uncertain whether more OT neurons were manipulated in females compared to males. The study could benefit from a more comprehensive discussion of other factors that could influence the neural circuit under investigation, especially in females.

Reviewer #2 (Public Review):

Summary:

This series of experiments studied the involvement of PVN OT neurons and their projection to the mPFC in pup-care and attack behavior in virgin male and female Mandarin voles. Using Fos visualization, optogenetics, fiber photometry, and IP injection of OT the results converge on OT regulating caregiving and attacks on pups. Some sex differences were found in the effects of the manipulations.

Strengths:

Major strengths are the modern multi-method approaches and involving both sexes of Mandarin vole in every experiment.

Weaknesses:

Weaknesses include the lack of some specific details in the methods that would help readers interpret the results. These include:

(1) No description of diffusion of centrally injected agents.

(2) Whether all central targets were consistent across animals included in the data analyses. This includes that is not stated if the medial prelimbic mPFC target was in all optogenetic study animals as shown in Figure 4 and if that is the case, there is no discussion of that subregion's function compared to other mPFC subregions.

(3) How groups of pup-care and infanticidal animals were created since there was no obvious pre-test mentioned so perhaps there was the testing of a large number of animals until getting enough subjects in each group.

(4) The apparent use of a 20-minute baseline data collection period for photometry that started right after the animals were stressed from handling and placement in the novel testing chamber.

(5) A weakness in the results reporting is that it's unclear what statistics are reported (2 x 2 ANOVA main effect of interaction results, t-test results) and that the degrees of freedom expected for the 2 X 2 ANOVAs in some cases don't appear to match the numbers of subjects shown in the graphs; including sample sizes in each group would be helpful because the graph panels are very small and data points overlap.

The additional context that could help readers of this study is that the authors overlook some important mPFC and pup caregiving and infanticide studies in the introduction which would help put this work in better context in terms of what is known about the mPFC and these behaviors. These previous studies include Febo et al., 2010; Febo 2012; Peirera and Morrell, 2011 and 2020; and a very relevant study by Alsina-Llanes and Olazábal, 2021 on mPFC lesions and infanticide in virgin male and female mice. The introduction states that nothing is known about the mPFC and infanticide. In the introduction and discussion, stating the species and sex of the animals tested in all the previous studies mentioned would be useful. The authors also discuss PVN OT cell stimulation findings seen in other rodents, so the work seems less conceptually novel. Overall, the findings add to the knowledge about OT regulation of pup-directed behavior in male and female rodents, especially the PVN-mPFC OT projection.

Reviewer #3 (Public Review):

Summary:

Here Li et al. examine pup-directed behavior in virgin Mandarin voles. Some males and females tend towards infanticide, others tend towards pup care. c-Fos staining showed more oxytocin cells activated in the paraventricular nucleus (PVN) of the hypothalamus in animals expressing pup care behaviors than in infanticidal animals. Optogenetic stimulation of PVN oxytocin neurons (with an oxytocin-specific virus to express the opsin transgene) increased pup-care, or in infanticidal voles increased latency towards approach and attack.

Suppressing the activity of PVN oxytocin neurons promoted infanticide. The use of a recent oxytocin GRAB sensor (OT1.0) showed changes in medial prefrontal cortex (mPFC) signals as measured with photometry in both sexes. Activating mPFC oxytocin projections increased latency to approach and attack in infanticidal females and males (similar to the effects of peripheral oxytocin injections), whereas in pup-caring animals only males showed a decrease in approach. Inhibiting these projections increased infanticidal behaviors in both females and males and had no effect on pup caretaking.

Strengths:

Adopting these methods for Mandarin voles is an impressive accomplishment, especially the valuable data provided by the oxytocin GRAB sensor. This is a major achievement and helps promote systems neuroscience in voles.

Weaknesses:

The study would be strengthened by an initial figure summarizing the behavioral phenotypes of voles expressing pup care vs infanticide: the percentages and behavioral scores of individual male and female nulliparous animals for the behaviors examined here. Do the authors have data about the housing or life history/experiences of these animals? How bimodal and robust are these behavioral tendencies in the population?

Optogenetics with the oxytocin promoter virus is a nice advance here. More details about their preparation and methods should be in the main text, and not simply relegated to the methods section. For optogenetic stimulation in Figure 2, how were the stimulation parameters chosen? There is a worry that oxytocin neurons can co-release other factors- are the authors sure that oxytocin is being released by optogenetic stimulation as opposed to other transmitters or peptides, and acting through the oxytocin receptor (as opposed to a vasopressin receptor)?

Given that they are studying changes in latency to approach/attack, having some controls for motion when oxytocin neurons are activated or suppressed might be nice. Oxytocin is reported to be an anxiolytic and a sedative at high levels.

The OT1.0 sensor is also amazing, these data are quite remarkable. However, photometry is known to be susceptive to motion artifacts and I didn't see much in the methods about controls or correction for this. It's also surprising to see such dramatic, sudden, and large-scale suppression of oxytocin signaling in the mPFC in the infanticidal animals - does this mean there is a substantial tonic level of oxytocin release in the cortex under baseline conditions?

Figure 5 is difficult to parse as-is, and relates to an important consideration for this study: how extensive is the oxytocin neuron projection from PVN to mPFC?

In Figures 6 and 7, the authors use the phrase 'projection terminals'; however, to my knowledge, there have not been terminals (i.e., presynaptic formations opposed to a target postsynaptic site) observed in oxytocin neuron projections into target central regions.

Projection-based inhibition as in Figure 7 remains a controversial issue, as it is unclear if the opsin activation can be fast enough to reduce the fast axonal/terminal action potential. Do the authors have confirmation that this works, perhaps with the oxytocin GRAB OT sensor?

As females and males had similar GRAB OT1.0 responses in mPFC, why would the behavioral effects of increasing activity be different between the sexes?

Reviewer #1 (Public Review):

Summary:

The present paper introduces Oscillation Component Analysis (OCA), in analogy to ICA, where source separation is underpinned by a biophysically inspired generative model. It puts the emphasis on oscillations, which is a prominent characteristic of neurophysiological data.

Strengths:

Overall, I find the idea of disambiguating data-driven decompositions by adding biophysical constrains useful, interesting and worth-pursuing. The model incorporates both a component modelling of oscillatory responses that is agnostic about the frequency content (e.g.. doesn't need bandpass filtering or predefinition of bands) and a component to map between sensor and latent-space. I feel these elements can be useful in practice.

Weaknesses:

Lack of empirical support: I am missing empirical justification of the advantages that are theoretically claimed in the paper. I feel the method needs to be compared to existing alternatives.

Reviewer #3 (Public Review):

The authors delved into an important aspect of abortifacient diseases of livestock in Tanzania. The thoughts of the authors on the topic and its significance are implied, and the methodological approach needs further clarity. The number of wards in the study area, statistical selection of wards, type of questionnaire ie open or close-ended. Statistical analyses of outcomes were not clearly elucidated in the manuscript. Fifteen wards were mentioned in the text but 13 used what were the exclusion criteria. Observations were from pastoral, agropastoral, and smallholder agroecological farmers. No sample numbers or questionnaires were attributed to the above farming systems to correlate findings with management systems. The impacts of the research investigation output are not clearly visible as to warrant intervention methods. What were the identified pathogens from laboratory investigation, particularly with the use of culture and PCR not even mentioning the zoonotic pathogens encountered if any? The public health importance of any of the abortifacient agents was not highlighted.

In conclusion, based on the intent of the authors and the content of this research, and the weight of the research topic, there are obvious weaknesses in the critical data analysis to demonstrate cause, effect, and impact.

Reviewer #2 (Public Review):

The paper "The Value of Livestock Abortion Surveillance in Tanzania: Identifying Disease Priorities and Informing Interventions" provides a comprehensive analysis of the importance of livestock abortion surveillance in Tanzania. The authors aim to highlight the significance of this surveillance system in identifying disease priorities and guiding interventions to mitigate the impact of livestock abortions on both animal and human health.

Summary:

The paper begins by discussing the context of livestock farming in Tanzania and the significant economic and social impact of livestock abortions. The authors then present a detailed overview of the livestock abortion surveillance system in Tanzania, including its objectives, methods, and data collection process. They analyze the data collected from this surveillance system over a specific period to identify the major causes of livestock abortions and assess their public health implications.

Evaluation:

Overall, this paper provides valuable insights into the importance of livestock abortion surveillance as a tool for disease prioritization and intervention planning in Tanzania. The authors effectively demonstrate the utility of this surveillance system in identifying emerging diseases, monitoring disease trends, and informing evidence-based interventions to control and prevent livestock abortions.

Strengths:

(1) Clear Objective: The paper clearly articulates its objective of highlighting the value of livestock abortion surveillance in Tanzania.

(2) Comprehensive Analysis: The authors provide a thorough analysis of the surveillance system, including its methodology, data collection process, and findings as seen in the supplementary files.

(3) Practical Implications: The paper discusses the practical implications of the surveillance system for disease control and public health interventions in Tanzania.

(4) Well-Structured: The paper is well-organized, with clear sections and subheadings that facilitate understanding and navigation.

Suggestions for Improvement:

(1) Data Presentation: While the analysis is comprehensive, the presentation of data could be enhanced with the use of more visual aids such as tables, graphs, or charts to illustrate key findings.

(2) Discussion Section: The paper could benefit from a more in-depth discussion of the implications of the findings for disease control strategies and policy formulation in Tanzania.

(3) Future Directions: Including recommendations for future research or areas for further investigation would add depth to the paper.

Summary:

This paper contains thorough analysis and valuable insights. Overall, it makes a significant contribution to the literature on livestock abortion surveillance and its implications for disease control in Tanzania.

Reviewer #1 (Public Review):

Summary:

The paper examined livestock abortion, as it is an important disease syndrome that affects productivity and livestock economies. If livestock abortion remains unexamined it poses risks to public health.

Several pathogens are associated with livestock abortions across Africa however the livestock disease surveillance data rarely include information from abortion events, little is known about the aetiology and impacts of livestock abortions, and data are not available to inform prioritisation of disease interventions. Therefore the current study seeks to examine the issue in detail and proposes some solutions.

The study took place in 15 wards in northern Tanzania spanning pastoral, agropastoral, and smallholder agro-ecological systems. The key objective is to investigate the causes and impacts of livestock abortion.

The data collection system was set up such that farmers reported abortion cases to the field officers of the Ministry of Livestock and Fisheries livestock.

The reports were made to the investigation teams. The team only included abortion of those that the livestock field officers could attend to within 72 hours of the event occurring.

Also, a field investigation was carried out to collect diagnostic samples from aborted materials. In addition, aborting dams and questionnaires were administered to collect data on herd/flock management. Laboratory diagnostic tests were carried out for a range of abortigenic pathogens

Over the period of the study, 215 abortion events in cattle (n=71), sheep 48 (n=44), and goats (n=100) were investigated. All 49 investigated cases varied widely across wards. The aetiological attribution, achieved for 19.5% of cases through PCR-based diagnostics, was significantly affected by delays in the field investigation.

The result also revealed that vaginal swabs from aborting dams provided a practical and sensitive source of diagnostic material for pathogen detection.

Livestock abortion surveillance can generate valuable information on causes of zoonotic disease outbreaks, and livestock reproductive losses and can identify important pathogens that are not easily captured through other forms of livestock disease surveillance. The study demonstrated the feasibility of establishing an effective reporting and investigation system that could be implemented across a range of settings, including remote rural areas,

Strengths:

The paper combines both science and socio-economic methodology to achieve the aim of the study. The methodology was well presented and the sequence was great. The authors explain where and how the data was collected. Figure 2 was used to describe the study area which was excellently done. The section on the investigation of cases was well written. The sample analysis was also well-written. The authors devoted a section to summarizing the investigated cases and description of the livestock 221-study population. The logit model was well-presented.

Reviewer #1 (Public Review):

In this manuscript, Naseri et al. present a new strategy for identifying human genetic variants with recessive effects on disease risk by the genome-wide association of phenotype with long runs-of-homozygosity (ROH). The key step of this approach is the identification of long ROH segments shared by many individuals (termed "shared ROH diplotype clusters" by the authors), which is computationally intensive for large-scale genomic data. The authors circumvented this challenge by converting the original diploid genotype data to (pseudo-)haplotype data and modifying the existing positional Burrow-Wheeler transformation (PBWT) algorithms to enable an efficient search for haplotype blocks shared by many individuals. With this method, the authors identified over 1.8 million ROH diplotype clusters (each shared by at least 100 individuals) and 61 significant associations with various non-cancer diseases in the UK Biobank dataset.

Overall, the study is well-motivated, highly innovative, and potentially impactful. Previous biobank-based studies of recessive genetic effects primarily focused on genome-wide aggregated ROH content, but this metric is a poor proxy for homozygosity of the recessive alleles at causal loci. Therefore, searching for the association between phenotype and specific variants in the homozygous state is a key next step towards discovering and understanding disease genes/alleles with recessive effects. That said, I have some concerns regarding the power and error rate of the methods, for both identification of ROH diplotype clusters and subsequent association mapping. In addition, some of the newly identified associations need further validation and careful consideration of potential artifacts (such as cryptic relatedness and environment sharing).

(1) Identification of ROH diplotype clusters.<br /> The practice of randomly assigning heterozygous sites to a homozygous state is expected to introduce errors, leading to both false positives and false negatives. An advantage that the authors claim for this practice is to reduce false negatives due to occasional mismatch (possibly due to genotyping error, or mutation), but it's unclear how much the false positive rate is reduced compared to traditional ROH detection algorithm. The authors also justified the "random allele drawing" practice by arguing that "the rate of false positives should be low" for long ROH segments, which is likely true but is not backed up with quantitative analysis. As a result, it is unclear whether the trade-off between reducing FNs and introducing FPs makes the practice worthwhile (compared to calling ROHs in each individual with a standard approach first followed by scanning for shared diplotypes across individuals using BWT). I would like to see a combination of back-of-envelope calculation, simulation (with genotyping errors), and analysis of empirical data that characterize the performance of the proposed method.

In particular, I find the high number of ROH clusters in MHC alarming, and I am not convinced that this can be fully explained by a high density of SNPs and low recombination rate in this region. The authors may provide further support for their hypothesis by examining the genome-wide relationship between ROH cluster abundance and local recombination rate (or mutation rate).

(2) Power of ROH association. Given that the authors focused on long segments only (which is a limitation of the current method), I am concerned about the power of the association mapping strategy, because only a small fraction of causal alleles are expected to be present in long, homozygous haplotypes shared by many individuals. It would be useful to perform a power analysis to estimate what fraction of true causal variants with a given effect size can be detected with the current method. To demonstrate the general utility of this method, the authors also need to characterize the condition(s) under which this method could pick up association signals missed by standard GWAS with recessive effects considered. I suspect some variants with truly additive effects can also be picked up by the ROH association, which should be discussed in the manuscript to guide the interpretation of results.

(3) False positives of ROH association. GWAS is notoriously prone to confounding by population and environmental stratification. Including leading principal components in association testing alleviates this issue but is not sufficient to remove the effects of recent demographic structure and local environment (Zaidi and Mathieson 2020 eLife). Similar confounding likely applies to homozygosity mapping and should be carefully considered. For example, it is possible that individuals who share a lot of ROH diplotypes tend to be remotely related and live near each other, thus sharing similar environments. Such scenarios need to be excluded to further support the association signals.

(4) Validation of significant associations. It is reassuring that some of the top associations are indirectly corroborated by significant GWAS associations between the same disease and individual SNPs present in the ROH region (Tables 1 and 2). However, more sanity checks should be done to confirm consistency in direction of effect size (e.g., risk alleles at individual SNPs should be commonly present in risk-increasing ROH segment, and vice versa) and the presence of dominance effect.

Reviewer #2 (Public Review):

The authors have proposed a computational algorithm to identify runs of homozygosity (ROH) segments in a generally outbred population and then study the association of ROH with self-reported disorders in the UK biobank. The algorithm certainly identifies such segments. However, more work is needed to justify the importance of ROH.

Reviewer #3 (Public Review):

A classic method to detect recessive disease variants is homozygosity mapping, where affected individuals in a pedigree are scanned for the presence of runs of homozygosity (ROH) intersecting in a given region. The method could in theory be extended to biobanks with large samples of unrelated individuals; however, no efficient method was available (to the best of my knowledge) for detecting overlapping clusters of ROH in such large samples. In this paper, the authors developed such a method based on the PBWT data structure. They applied the method to the UK biobank, finding a number of associations, some of them not discovered in single SNP associations.

Major strengths:<br /> • The method is innovative and algorithmically elegant and interesting. It achieves its purpose of efficiently and accurately detecting ROH clusters overlapping in a given region. It is therefore a major methodological advance.<br /> • The method could be very useful for many other researchers interested in detecting recessive variants associated with any phenotype.<br /> • The statistical analysis of the UK biobank data is solid and the results that were highlighted are interesting and supported by the data.

Major weaknesses:<br /> • The positions and IDs of the ROH clusters in the UK biobank are not available for other researchers. This means that other researchers will not be able to follow up on the results of the present paper.<br /> • The vast majority of the discoveries were in regions already known to be associated with their respective phenotypes based on standard GWAS.<br /> • The running time seems rather long (at least for the UK biobank), and therefore it will be difficult for other researchers to extensively experiment with the method in very large datasets. That being said, the method has a linear running time, so it is already faster than a naïve algorithm.

Reviewer #1 (Public Review):

In this study, the authors offer a fresh perspective on how visual working memory operates. They delve into the link between anticipating future events and retaining previous visual information in memory. To achieve this, the authors build upon their recent series of experiments that investigated the interplay between gaze biases and visual working memory. In this study, they introduce an innovative twist to their fundamental task. Specifically, they disentangle the location where information is initially stored from the location where it will be tested in the future. Participants are tasked with learning a novel rule that dictates how the initial storage location relates to the eventual test location. The authors leverage participants' gaze patterns as an indicator of memory selection. Intriguingly, they observe that microsaccades are directed towards both the past encoding location and the anticipated future test location. This observation is noteworthy for several reasons. Firstly, participants' gaze is biased towards the past encoding location, even though that location lacks relevance to the memory test. Secondly, there's a simultaneous occurrence of an increased gaze bias towards both the past and future locations. To explore this temporal aspect further, the authors conduct a compelling analysis that reveals the joint consideration of past and future locations during memory maintenance. Notably, microsaccades biased towards the future test location also exhibit a bias towards the past encoding location. In summary, the authors present an innovative perspective on the adaptable nature of visual working memory. They illustrate how information relevant to the future is integrated with past information to guide behavior.

Reviewer #2 (Public Review):

Summary:

The manuscript by Liu et al. reports a task that is designed to examine the extent to which "past" and "future" information is encoded in working memory that combines a retrocue with rules that indicate the location of an upcoming test probe. An analysis of microsaccades on a fine temporal scale shows the extent to which shifts of attention track the location of the encoded item (past) and the location of the future item (test probe). The location of the encoded grating and test probe were always on orthogonal axes (horizontal, vertical) so that biases in microsaccades could be used to track shifts of attention to one or the other axis (or mixtures of the two). The overall goal here was then to (1) create a methodology that could tease apart memory for the past and future, respectively, (2) to look at the time-course attention to past/future, and (3) to test the extent to which microsaccades might jointly encode past and future memoranda. Finally, some remarks are made about the plausibility of various accounts of working memory encoding/maintenance based on the examination of these time-courses.

Strengths:

This research has several notable strengths. It has a clear statement of its aims, is lucidly presented, and uses a clever experimental design that neatly orthogonalized "past" and "future" as operationalized by the authors. Figure 1b-d shows fairly clearly that saccade directions have an early peak (around 300ms) for the past and a "ramping" up of saccades moving in the forward direction. This seems to be a nice demonstration that the method can measure shifts of attention at a fine temporal resolution and differentiate past from future oriented saccades due to the orthogonal cue approach. The second analysis shown in Figure 2, reveals a dependency in saccade direction such that saccades toward the probe future were more likely also to be toward the encoded location than away from the encoded direction. This suggests saccades are jointly biased by both locations "in memory". The "central contribution" (as the authors characterize it) is that "the brain simultaneously retains the copy of both past and future-relevant locations in working memory, and (re)activates each during mnemonic selection", and that: "... while it is not surprising that the future location is considered, it is far less trivial that both past and future attributes would be retained and (re)activated together. This is our central contribution." The authors provide a nuanced analysis that offers persuasive evidence that past and future representations are jointly maintained in memory.

Reviewer #1 (Public Review):

Using A. carterae as a model system, this work investigates the properties of the trans-spliced SL leader sequences and the dinoflagellate eIF4E protein family members.

Analysis was performed to identify the 5' cap type of the SL leader. Variation in the SL leader sequence and an abundance of modified bases was documented.

Various aspects of the sequence and expression of the eIF4E family members were examined. This included phylogeny, mRNA, and protein expression levels in A. carterae, and the ability of eIF4E proteins to bind cap structures. Differences in expression levels and cap-binding capacity were characterized, leading to the proposition that eIF4E-1a serves as the major cap-binding protein in A. carterae.

A major discussion point is the potential for differential eIF4E binding to specific SL leader sequences as a regulatory mechanism, which is an exciting prospect. However, despite indications of sequence variability and the presence of various nucleotide modifications in the SL, and the several eIF4E variants, direct evidence to support this hypothesis is lacking.

It is an extensive and highly descriptive study. The work is presented clearly, although it is rather lengthy and contains repetition across the introduction, results, and discussion sections. Its style leans more towards a review format. As a non-expert in the field, I appreciated the extensive background however I do believe the paper would benefit from a more concise format.

Reviewer #2 (Public Review):

Summary:

Jones et al. extend their previous work on the translation machinery in Dinoflagellate. In particular, they study the species Amphidium carterae. They characterize the type of cap structure mRNAs possess in this species, as well as the eight eIF4E family members A. carterae possesses and their affinity to the mRNA cap. They also establish the leader sequences of the transpliced mRNAs that A. carterae generates during gene expression.

Strengths:

The authors performed a solid phylogenetic and biochemical study to understand the structure of Dinoflagellate mRNAs at the 5'-UTR as well as the divergence and biochemical features of eIF4Es across Dinoflagellate. They also establish eIF4E-1a as the prototypical paralog of the eIF4E family of proteins. The scientific questions they ask are very relevant to the gene expression field across eukaryotes. The experiments and the phylogenetic analysis are performed with a very high quality. They perform a wide spectrum of experimental approaches and techniques to answer the questions.

Weaknesses:

The authors assume all eIF4E from Dinoflagellate are involved in translation, i.e., mRNA recruitment to the ribosome. Indeed, they think that the diverse biochemical features of all eIF4E in A. carterae have to do with the possible recruitment of different subsets of mRNAs to the ribosome for translation. I think that the biochemical differences among all paralogs also might be due to the involvement of some of them in different processes of RNA metabolism, other than translation. For instance, some of them could be involved only in RNA processing in the nucleus or mRNA storage in cytoplasmic foci.

Reviewer #3 (Public Review):

Summary:

In this article, the authors provide an inventory of the 5' spliced leader sequences, cap structures, and eIF4E isoforms present in the model dinoflagellate species A. carterae. They provide evidence that the 5' cap structure is m7G, as it is in most characterized eukaryotes that do not employ trans-splicing for mRNA maturation, and that there are additional methylated nucleotides throughout the spliced leader RNAs. They then show that of the 8 different eIF4E species in A. carterae, only a subset of eIF4E1 and eIF4E2 proteins are detected and that the levels change according to time of day. Interestingly, while the eIF4E1 proteins bind a canonical cap nucleotide and are able to complement eIF4E-deficiency in yeast, an eIF4E2 paralog does not bind the traditional cap.

Strengths:

A strength of the article is that the authors have clearly presented the findings and by straying away from traditional model organisms, they have highlighted unique and interesting features of an understudied system for translational control. They provide complementary evidence for most findings using multiple techniques. E.g. the evidence that eIF4E1A binds m7GTP is supported by both pulldowns using m7GTP sepharose as well as SPR experiments to directly monitor binding of recombinant protein with affinity measurements. The methods are extremely detailed noting cell numbers, volumes, concentrations, etc. used in the experiments to be easily replicated.

Weaknesses:

While not necessary to support the author's conclusions, the significance of the work would be further enhanced by additional experiments to gain insights into mechanisms for translational control and to link specific SLs to organismal functions or mechanisms of mRNA recruitment.

-Monitoring diel expression of SLs and direct sequencing of mature mRNA would yield insights into whether there is regulated expression of RNAs with different SLs or the SLs themselves. This would also allow the authors to perform gene ontology to link SL expression at different points in the diel cycle to related functions, e.g. photosynthesis.

-In addition, the work would be strengthened by polysome sequencing or ribosome profiling as a function of the diel cycle, with analyses of when various spliced leader sequences are recruited to ribosomes in parallel with western blotting of polysome fractions to determine when various eIF4E isoforms are present on polysomes. This is a substantial expansion though from what the authors focused on in this manuscript, and not having these experiments does not undermine the findings presented. Alternatively, they could attempt to make bioinformatic comparisons with existing ribosome profiling datasets from a related dinoflagellate, Lingulodinium polyedrum, discussed briefly, if there were sufficient overlap between SL RNAs in these organisms.

Reviewer #1 (Public Review):

Summary:

The authors want to determine the role of the sperm hook of the house mouse sperm in movement through the uterus. The authors are trying to distinguish between two hypotheses put forward by others on the role of the sperm hook: (1) the sperm cooperation hypothesis (the sperm hook helps to form sperm trains) vs (2) the migration hypothesis (that the sperm hook is needed for sperm movement through the uterus). They use transgenic lines with fluorescent labels to sperm proteins, and they cross these males to C57BL/6 females in pathogen-free conditions. They use 2-photon microscopy on ex vivo uteri within 3 hours of mating and the appearance of a copulation plug. There are a total of 10 post-mating uteri that were imaged with 3 different males. They provide 10 supplementary movies that form the basis for some of the quantitative analysis in the main body figures. Their data suggest that the role of the sperm hook is to facilitate movement along the uterine wall.

Strengths:

Ex vivo live imaging of fluorescently labeled sperm with 2-photon microscopy is a powerful tool for studying the behavior of sperm.

Weaknesses:

The paper is descriptive and the data are correlations.

The data are not properly described in the figure legends.

When statistical analyses are performed, the authors do not comment on the trend that sperm from the three males behave differently from each other. This weakens confidence in the results. For example, in Figure 1 the sperm from male 3613 (blue squares) look different from male 838 (red circles), but all of these data are considered together. The authors should comment on why sperm across males are considered together when the individual data points appear to be different across males.

Movies S8-S10 are single data points and no statistical analyses are performed. Therefore, it is unclear how penetrant the sperm movements are.

Movies S1B - did the authors also track the movement of sperm located in the middle of the uterus (not close to the wall)? Without this measurement, they can't be certain that sperm close to the uterus wall travels faster.

Movie S5A - is of lower magnitude (200 um scale bar) while the others have 50 and 20 uM scale bars. Individual sperm movement can be observed in the 20 uM (Movie 5SC). If the authors went to prove that there is no upsucking movement of sperm by the uterine contractions, they need to provide a high magnification image.

Movie S8 - if the authors want to make the case that clustered sperm do not move faster than unclustered sperm, then they need to show Movie S8 at higher magnification. They also need to quantify these data.

Movie S9C - what is the evidence that these sperm are dead or damaged?

MovIe S10 - both slow- and fast-moving sperm are seen throughout the course of the movie, which does not support the authors' conclusion that sperm tails beat faster over time.

Reviewer #2 (Public Review):

Summary:

The specific objective of this study was to determine the role of the large apical hook on the head of mouse sperm (Mus musculus) in sperm migration through the female reproductive tract. The authors used a custom-built two-photon microscope system to obtain digital videos of sperm moving within the female reproductive tract. They used sperm from genetically modified male mice that produce fluorescence in the sperm head and flagellar midpiece to enable visualization of sperm moving within the tract. Based on various observations, the authors concluded that the hook serves to facilitate sperm migration by hooking sperm onto the lining of the female reproductive tract, rather than by hooking sperm together to form a sperm train that would move them more quickly through the tract. The images and videos are excellent and inspirational to researchers in the field of mammalian sperm migration, but interpretations of the behaviors are highly speculative and not supported by controlled experimentation.

Strengths:

The microscope system developed by the authors could be of interest to others investigating sperm migration.

The new behaviors shown in the images and videos could be of interest to others in the field, in terms of stimulating the development of new hypotheses to investigate.

Weaknesses:

The authors stated several hypotheses about the functions of the sperm behaviors they saw, but the hypotheses were not clearly stated or tested experimentally.

The hypothesis statements were weakened by the use of hedge words, such as "may".

Reviewer #1 (Public Review):

The authors have performed all-atom MD simulations to study the working mechanism of hsPepT2. It is widely accepted that conformational transitions of proton-coupled oligopeptide transporters (POTs) are linked with gating hydrogen bonds and salt bridges involving protonatable residues, whose protonation triggers gate openings. Through unbiased MD simulations, the authors identified extra-cellular (H87 and D342) and intra-cellular (E53 and E622) triggers. The authors then validated these triggers using free energy calculations (FECs) and assessed the engagement of the substrate (Ala-Phe dipeptide). The linkage of substrate release with the protonation of the ExxER motif (E53 and E56) was confirmed using constant-pH molecular dynamics (CpHMD) simulations and cell-based transport assays. An alternating-access mechanism was proposed. The study was largely conducted properly, and the paper was well-organized. However, I have a couple of concerns for the authors to consider addressing.

(1) As a proton-coupled membrane protein, the conformational dynamics of hsPepT2 are closely coupled to protonation events of gating residues. Instead of using semi-reactive methods like CpHMD or reactive methods such as reactive MD, where the coupling is accounted for, the authors opted for extensive non-reactive regular MD simulations to explore this coupling. Note that I am not criticizing the choice of methods, and I think those regular MD simulations were well-designed and conducted. But I do have two concerns.

a) Ideally, proton-coupled conformational transitions should be modelled using a free energy landscape with two or more reaction coordinates (or CVs), with one describing the protonation event and the other describing the conformational transitions. The minimum free energy path then illustrates the reaction progress, such as OCC/H87D342-  OCC/H87HD342H  OF/H87HD342H as displayed in Figure 3. Without including the protonation as a CV, the authors tried to model the free energy changes from multiple FECs using different charge states of H87 and D342. This is a practical workaround, and the conclusion drawn (the OCCOF transition is downhill with protonated H87 and D342) seems valid. However, I don't think the OF states with different charge states (OF/H87D342-, OF/H87HD342-, OF/H87D342H, and OF/H87HD342H) are equally stable, as plotted in Figure 3b. The concern extends to other cases like Figures 4b, S7, S10, S12, S15, and S16. While it may be appropriate to match all four OF states in the free energy plot for comparison purposes, the authors should clarify this to ensure readers are not misled.

b) Regarding the substrate impact, it appears that the authors assumed fixed protonation states. I am afraid this is not necessarily the case. Variations in PepT2 stoichiometry suggest that substrates likely participate in proton transport, like the Phe-Ala (2:1) and Phe-Gln (1:1) dipeptides mentioned in the introduction. And it is not rigorous to assume that the N- and C-termini of a peptide do not protonate/deprotonate when transported. I think the authors should explicitly state that the current work and the proposed mechanism (Figure 8) are based on the assumption that the substrates do not uptake/release proton(s).

(2) I have more serious concerns about the CpHMD employed in the study.

a) The CpHMD in AMBER is not rigorous for membrane simulations. The underlying generalized Born model fails to consider the membrane environment when updating charge states. In other words, the CpHMD places a membrane protein in a water environment to judge if changes in charge states are energetically favorable. While this might not be a big issue for peripheral residues of membrane proteins, it is likely unphysical for internal residues like the ExxER motif. As I recall, the developers have never used the method to study membrane proteins themselves. The only CpHMD variant suitable for membrane proteins is the membrane-enabled hybrid-solvent CpHMD in CHARMM. While I do not expect the authors to redo their CpHMD simulations, I do hope the authors recognize the limitations of their method.

b) It appears that the authors did not make the substrate (Ala-Phe dipeptide) protonatable in holo-simulations. This oversight prevents a complete representation of ligand-induced protonation events, particularly given that the substrate ion pairs with hsPepT2 through its N- & C-termini. I believe it would be valuable for the authors to acknowledge this potential limitation.

Reviewer #2 (Public Review):

Summary:

This is an interesting manuscript that describes a series of molecular dynamics studies on the peptide transporter PepT2 (SLC15A2). They examine, in particular, the effect on the transport cycle of protonation of various charged amino acids within the protein. They then validate their conclusions by mutating two of the residues that they predict to be critical for transport in cell-based transport assays. The study suggests a series of protonation steps that are necessary for transport to occur in Petp2. Comparison with bacterial proteins from the same family shows that while the overall architecture of the proteins and likely mechanism are similar, the residues involved in the mechanism may differ.

Strengths:

This is an interesting and rigorous study that uses various state-of-the-art molecular dynamics techniques to dissect the transport cycle of PepT2 with nearly 1ms of sampling. It gives insight into the transport mechanism, investigating how the protonation of selected residues can alter the energetic barriers between various states of the transport cycle. The authors have, in general, been very careful in their interpretation of the data.

Weaknesses:

Interestingly, they suggest that there is an additional protonation event that may take place as the protein goes from occluded to inward-facing but they have not identified this residue. Some things are a little unclear. For instance, where does the state that they have defined as occluded sit on the diagram in Figure 1a? - is it truly the occluded state as shown on the diagram or does it tend to inward- or outward-facing? The pKa calculations and their interpretation are a bit unclear. Firstly, it is unclear whether they are using all the data in the calculations of the histograms, or just selected data and if so on what basis was this selection done. Secondly, they dismiss the pKa calculations of E53 in the outward-facing form as not being affected by peptide binding but say that E56 is when there seems to be a similar change in profile in the histograms.

Reviewer #3 (Public Review):

Summary:

Lichtinger et al. have used an extensive set of molecular dynamics (MD) simulations to study the conformational dynamics and transport cycle of an important member of the proton-coupled oligopeptide transporters (POTs), namely SLC15A2 or PepT2. This protein is one of the most well-studied mammalian POT transporters that provides a good model with enough insight and structural information to be studied computationally using advanced enhanced sampling methods employed in this work. The authors have used microsecond-level MD simulations, constant-PH MD, and alchemical binding free energy calculations along with cell-based transport assay measurements; however, the most important part of this work is the use of enhanced sampling techniques to study the conformational dynamics of PepT2 under different conditions.

The study attempts to identify links between conformational dynamics and chemical events such as proton binding, ligand-protein interactions, and intramolecular interactions. The ultimate goal is of course to understand the proton-coupled peptide and drug transport by PepT2 and homologous transporters in the solute carrier family.

Some of the key results include<br /> (1) Protonation of H87 and D342 initiate the occluded (Occ) to the outward-facing (OF) state transition.

(2) In the OF state, through engaging R57, substrate entry increases the pKa value of E56 and thermodynamically facilitates the movement of protons further down.

(3) E622 is not only essential for peptide recognition but also its protonation facilitates substrate release and contributes to the intracellular gate opening. In addition, cell-based transport assays show that mutation of residues such as H87 and D342 significantly decreases transport activity as expected from simulations.

Strengths:

(1) This is an extensive MD-based study of PepT2, which is beyond the typical MD studies both in terms of the sheer volume of simulations as well as the advanced methodology used. The authors have not limited themselves to one approach and have appropriately combined equilibrium MD with alchemical free energy calculations, constant-pH MD, and geometry-based free energy calculations. Each of these 4 methods provides a unique insight regarding the transport mechanism of PepT2.

(2) The authors have not limited themselves to computational work and have performed experiments as well. The cell-based transport assays clearly establish the importance of the residues that have been identified as significant contributors to the transport mechanism using simulations.

(3) The conclusions made based on the simulations are mostly convincing and provide useful information regarding the proton pathway and the role of important residues in proton binding, protein-ligand interaction, and conformational changes.

Weaknesses:

(1) Some of the statements made in the manuscript are not convincing and do not abide by the standards that are mostly followed in the manuscript. For instance, on page 4, it is stated that "the K64-D317 interaction is formed in only ≈ 70% of MD frames and therefore is unlikely to contribute much to extracellular gate stability." I do not agree that 70% is negligible. Particularly, Figure S3 does not include the time series so it is not clear whether the 30% of the time where the salt bridge is broken is in the beginning or the end of simulations. For instance, it is likely that the salt bridge is not initially present and then it forms very strongly. Of course, this is just one possible scenario but the point is that Figure S3 does not rule out the possibility of a significant role for the K64-D317 salt bridge.

(2) Similarly, on page 4, it is stated that "whether by protonation or mutation - the extracellular gate only opens spontaneously when both the H87 interaction network and D342-R206 are perturbed (Figure S5)." I do not agree with this assessment. The authors need to be aware of the limitations of this approach. Consider "WT H87-prot" and "D342A H87-prot": when D342 residue is mutated, in one out of 3 simulations, we see the opening of the gate within 1 us. When D342 residue is not mutated we do not see the opening in any of the 3 simulations within 1 us. It is quite likely that if rather than 3 we have 10 simulations or rather than 1 us we have 10 us simulations, the 0/3 to 1/3 changes significantly. I do not find this argument and conclusion compelling at all.

(3) While the MEMENTO methodology is novel and interesting, the method is presented as flawless in the manuscript, which is not true at all. It is stated on Page 5 with regards to the path generated by MEMENTO that "These paths are then by definition non-hysteretic." I think this is too big of a claim to say the paths generated by MEMENTO are non-hysteretic by definition. This claim is not even mentioned in the original MEMENTO paper. What is mentioned is that linear interpolation generates a hysteresis-free path by definition. There are two important problems here: (a) MEMENTO uses the linear interpolation as an initial step but modifies the intermediates significantly later so they are no longer linearly interpolated structures and thus the path is no longer hysteresis-free; (b) a more serious problem is the attribution of by-definition hysteresis-free features to the linearly interpolated states. This is based on conflating the hysteresis-free and unique concepts. The hysteresis in MD-based enhanced sampling is related to the presence of barriers in orthogonal space. For instance, one may use a non-linear interpolation of any type and get a unique pathway, which could be substantially different from the one coming from the linear interpolation. None of these paths will be hysteresis-free necessarily once subjected to MD-based enhanced sampling techniques.

Reviewer #2 (Public Review):

Summary:

In this article, Kumar et al., report on a previously unappreciated mechanism of translational regulation whereby p130Cas induces LLPS condensates that then traffic out from focal adhesion into the cytoplasm to modulate mRNA translation. Specifically, the authors employed EGFP-tagged p130Cas constructs, endogenous p130Cas, and p130Cas knockouts and mutants in cell-based systems. These experiments in conjunction with various imaging techniques revealed that p130Cas drives assembly of LLPS condensates in a manner that is largely independent of tyrosine phosphorylation. This was followed by in vitro EGFP-tagged p130Cas-dependent induction of LLPS condensates and determination of their composition by mass spectrometry, which revealed enrichment of proteins involved in RNA metabolism in the condensates. The authors excluded the plausibility that p130Cas-containing condensates co-localize with stress granules or p-bodies. Next, the authors determined mRNA compendium of p130Cas-containing condensates which revealed that they are enriched in transcripts encoding proteins implicated in cell cycle progression, survival, and cell-cell communication. These findings were followed by the authors demonstrating that p130Cas-containing condensates may be implicated in the suppression of protein synthesis using puromycylation assay. Altogether, it was found that this study significantly advances the knowledge pertinent to the understanding of molecular underpinnings of the role of p130Cas and more broadly focal adhesions on cellular function, and to this end, it is likely that this report will be of interest to a broad range of scientists from a wide spectrum of biomedical disciplines including cell, molecular, developmental and cancer biologists.

Strengths:

Altogether, this study was found to be of potentially broad interest inasmuch as it delineates a hitherto unappreciated link between p130Cas, LLPS, and regulation of mRNA translation. More broadly, this report provides unique molecular insights into the previously unappreciated mechanisms of the role of focal adhesions in regulating protein synthesis. Overall, it was thought that the provided data sufficiently supported most of the authors' conclusions. It was also thought that this study incorporates an appropriate balance of imaging, cell and molecular biology, and biochemical techniques, whereby the methodology was found to be largely appropriate.

Weaknesses:

Two major weaknesses of the study were noted. The first issue is related to the experiments establishing the role of p130Cas-driven condensates in translational suppression, whereby it remained unclear whether these effects are affecting global mRNA translation or are specific to the mRNAs contained in the condensates. Moreover, some of the results in this section (e.g., experiments using cycloheximide) may be open to alternative interpretation. The second issue is the apparent lack of functional studies, and although the authors speculate that the described mechanism is likely to mediate the effects of focal adhesions on e.g., quiescence, experimental testing of this tenet was lacking.

Reviewer #1 (Public Review):

Summary:

The authors demonstrated the phenomenon of p130Cas, a protein primarily localized at focal adhesions, and its formation of condensates. They identified the constituents within the condensates, which include other focal adhesion proteins, paxillin, and RNAs. Furthermore, they proposed a link between p130Cas condensates and translation.

Strengths:

Adhesion components undergo rapid exchange with the cytoplasm for some unclear biological functions. Given that p130Cas is recognized as a prominent mechanical focal adhesion component, investigating its role in condensate formation, particularly its impact on the translation process, is intriguing and significant.

Weaknesses:

The authors identified the disordered region of p130Cas and investigated the formation of p130Cas condensate. They attempted to demonstrate that p130Cas condensates inhibit translation, but the results did not fully support this assertion. There are several comments below:

(1) Despite isolating p130Cas-GFP protein using GFP-trap beads, the authors cannot conclusively eliminate the possibility of isolating p130Cas from focal adhesions. While the characterization of the GFP-tagged pulls can reveal the proteins and RNAs associated with p130Cas, they need to clarify their intramolecular mechanism of localization within p130Cas droplets. Whether the protein condensates retain their liquid phase or these GFP-p130Cas pulls represent protein aggregate remains uncertain.

(2) The authors utilized hexanediol and ammonium acetate to highlight the phenomenon of p130Cas condensates. Although hexanediol is an inhibitor for hydrophobic interactions and ammonium acetate is a salt, a more thorough explanation of the intramolecular mechanisms underlying p130Cas protein-protein interaction is required. Additionally, given that the size of p130Cas condensates can exceed >100um2, classification is needed to differentiate between p130Cas condensates and protein aggregation.

(3) The connection between p130Cas condensates and translation inhibition appears tenuous. The data only suggests a correlation between p130Cas expression and translation inhibition. Further evidence is required to bolster this hypothesis.

Reviewer #1 (Public Review):

Summary:

The manuscript focuses on the role of the deubiquitinating enzyme UPS-50/USP8 in endosome maturation. The authors aimed to clarify how this enzyme drives the conversion of early endosomes into late endosomes. Overall, they did achieve their aims in shedding light on the precise mechanisms by which UPS-50/USP8 regulates endosome maturation. The results support their conclusions that UPS-50 acts by disassociating RABX-5 from early endosomes to deactivate RAB-5 and by recruiting SAND-1/Mon1 to activate RAB-7. This work is commendable and will have a significant impact on the field. The methods and data presented here will be useful to the community in advancing our understanding of endosome maturation and identifying potential therapeutic targets for diseases related to endosomal dysfunction. It is worth noting that further investigation is required to fully understand the complexities of endosome maturation. However, the findings presented in this manuscript provide a solid foundation for future studies.

Strengths:

The major strengths of this work lie in the well-designed experiments used to examine the effects of UPS-50 loss. The authors employed confocal imaging to obtain a picture of the aftermath of the USP-50 loss. Their findings indicated enlarged early endosomes and MVB-like structures in cells deficient in USP-50/USP8.

Weaknesses:

Specifically, there is a need for further investigation to accurately characterize the anomalous structures detected in the ups-50 mutant. Also, the correlation between the presence of these abnormal structures and ESCRT-0 is yet to be addressed, and the current working model needs to be revised to prevent any confusion between enlarged early endosomes and MVBs.

Reviewer #2 (Public Review):

Summary:

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that the localization of USP8 to early endosomes is disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although Rabex5 is accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

Strengths:

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidence to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members of the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

Weaknesses:

- The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion.

- The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient in both factors displayed similar defects in late endosomes/lysosomes. However, the authors didn't confirm whether and/or to which extent USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model.

Reviewer #3 (Public Review):

Summary:

The authors were trying to elucidate the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of "abnormal" enlarged vesicles when USP8 function was lost. At this specific point, it's not clear what the objective of the authors was. What would have been their hypothesis addressing whether the reduced lysosomal structures in USP8 (-) animals were linked to MVB formation? Then they observed that the abnormally enlarged vesicles, marked by the PI3P biosensor YFP-2xFYVE, are bigger but in the same number in USP8 (-) compared to wild-type animals, suggesting homotypic fusion. They confirmed this result by knocking down USP8 in a human cell line, and they observed enlarged vesicles marked by YFP-2xFYVE as well. At this point, there is quite an important issue. The use of YFP-2xFYVE to detect early endosomes requires the transfection of the cells, which has already been demonstrated to produce differences in the distribution, number, and size of PI3P-positive vesicles (doi.org/10.1080/15548627.2017.1341465). The enlarged vesicles marked by YFP-2xFYVE would not necessarily be due to the loss of UPS8. In any case, it appears relatively clear that USP8 localizes to early endosomes, and the authors claim that this localization is mediated by Rabex-5 (or Rabx-5). They finally propose that USP8 dissociates Rabx-5 from early endosomes facilitating endosome maturation.

Weaknesses:

The weaknesses of this study are, on one side, that the results are almost exclusively dependent on the overexpression of fusion proteins. While useful in the field, this strategy does not represent the optimal way to dissect a cell biology issue. On the other side, the way the authors construct the rationale for each approximation is somehow difficult to follow. Finally, the use of two models, C. elegans and a mammalian cell line, which would strengthen the observations, contributes to the difficulty in reading the manuscript.

The findings are useful but do not clearly support the idea that USP8 mediates Rab5-Rab7 exchange and endosome maturation, In contrast, they appear to be incomplete and open new questions regarding the complexity of this process and the precise role of USP8 within it.

Reviewer #1 (Public Review):

Summary:

The authors found that the loss of cell-ECM adhesion leads to the formation of giant monocular vacuoles in mammary epithelial cells. This process takes place in a macropinocytosis-like process and involves PI3 kinase. They further identified dynamin and septin as essential machinery for this process. Interestingly, this process is reversible and appears to protect cells from cell death.

Strengths:

The data are clean and convincing to support the conclusions. The analysis is comprehensive, using multiple approaches such as SIM and TEM. The discussion on lactation is plausible and interesting.

Weaknesses:

As the first paper describing this phenomenon, it is adequate. However, the elucidation of the molecular mechanisms is not as exciting as it does not describe anything new. It is hoped that novel mechanisms will be elucidated in the future. In particular, the molecules involved in the reversing process could be quite interesting. Additionally, the relationship to conventional endocytic compartments, such as early and late endosomes, is not analyzed.

Reviewer #2 (Public Review):

Summary:

The manuscript "Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance" describes an interesting observation and provides initial steps towards understanding the underlying molecular mechanism.

The manuscript describes that the majority of non-tumorigenic mammary gland epithelial cells (MCF-10A) in suspension initiate entosis. A smaller fraction of cells form a single giant unilocular vacuole (hereafter referred to as a GUVac). GUVac appeared to be empty and did not contain invading (entotic) cells. The formation of GUVac could be promoted by disrupting actin polymerisation with LatB and CytoD. The formation of GUVacs correlated with resistance to anoikis. GUVac formation was detected in several other epithelial cells from secretory tissues.

The authors then use electron microscopy and super-resolution imaging to describe the biogenesis of GUVac. They find that GUVac formation is initiated by a micropinocytosis-like phenomenon (that is independent of actin polymerisation). This process leads to the formation of large plasma membrane invaginations, that pinch off from the PM to form larger vesicles that fuse with each other into GUVacs.

Inhibition of actin polymerisation in suspended MCF-10a leads to the recruitment of Septin 6 to the PM via its amphipathic helix. Treatment with FCF (a septin polymerisation inhibitor) blocked GUVac biogenesis, as did pharmacological inhibition of dynamin-mediated membrane fission. The fusion of these vesicles in GUVacs required (perhaps not surprisingly) PI3P.

Strengths:

The authors have made an interesting and potentially important observation. They describe the formation of an endo-lysosomal organelle (a giant unilocular vacuole - GUVac) in suspended epithelial cells and correlate the formation of GUVacs with resistance to aniokis.

Weaknesses:

My major concern is the experimental strategy that is used throughout the paper to induce and study the formation GUVac. Almost every experiment is conducted in suspended cells that were treated with actin depolymerising drugs (e.g. LatB) and thus almost all key conclusions are based on the results of these experiments. I only have a few suggestions that would improve these experiments or change their outcome and interpretation.

Yet, I believe it is essential to identify the endogenous pathway leading to the actin depolymerisation that drives the formation of GUVacs in detached epithelial cells (or alternatively to figure out how it is suppressed in most detached cells). A first step in that direction would be to investigate the polymerization status of actin in MCF-10a cells that 'spontaneously' form GUVacs and to test if these cells also become resistant to anoikis.

Also, it would be great (and I believe reasonably easy) to better characterise molecular markers of GUVacs (LAMP's, Rab's, Cathepsins, etc....) to discriminate them from other endosomal organelles

Reviewer #3 (Public Review):

Summary:

Loss of cell attachment to extracellular matrix (ECM) triggers aniokis (a type of programmed cell death), and resistance to aniokis plays a role in cancer development. However, mechanisms underlying anoikis resistance, and the precise role of F-actin, are not fully known.

Here the authors describe the formation of a new organelle, giant unilocular vacuole (GUVac), in cells whose F-actin is disrupted during loss of matrix attachment. GUVac formation (diameter >500 nm) resulted from a previously unrecognised macropinocytosis-like process, characterized by inwardly curved micron-sized plasma membrane invaginations, dependent on F-actin depolymerization, septin recruitment, and PI(3)P. Finally, the authors show GUVac formation after loss of matrix attachment promotes resistance to anoikis.

From these results, the authors conclude that GUVac formation promotes cell survival in environments where F-actin is disrupted and conditions of cell stress.

Strengths:

The manuscript is clear and well-written, figures are all presented at a very high level.

A variety of cutting-edge cell biology techniques (eg time-lapse imaging, EM, super-resolution microscopy) are used to study the role of the cytoskeleton in GUVac formation. It is discovered that: (i) a macropinocytosis-like process dependent on F-actin depolymerisation, SEPT6 recruitment, and PI(3)P contributes to GUVac formation, and (ii) GUVac formation is associated with resistance to cell death.

Weaknesses:

The manuscript is highly reliant on the use of drugs, or combinations of drugs, for long periods of time (6hr, 18hr..). Wherever possible the authors should test conclusions drawn from experiments involving drugs also using other canonical cell biology approaches (eg siRNA, Crispr). Although suggestive as a first approach, it is not reliable to draw conclusions from experiments where only drug combinations are being advanced (eg LatB + FCF).

F-actin is well known to play a wide variety of roles in cell death and other canonical cell death pathways (PMID: 26292640). The authors show using pharmacological inhibition that F-actin is key for GUVac formation. However, especially when testing for physiological relevance, how can these other roles for F-actin be ruled out?

To test the role of septins in GUVac formation only recruitment studies and no direct functional work is performed. A drug forchlofeneuron (FCF) is used, but this is well known to have off-target effects (PMID: 27473917).

Cells that possess GUVac are resistant to aniokis, but how are these cells resistant? This report is focused on mechanisms underlying GUVac formation and does not directly test for mechanisms underlying aniokis resistance.

Reviewer #1 (Public Review):

The paper 'Structural Analysis of the Dynamic Ribosome-Translocon Complex,' authored by Lewis et al., meticulously explores various conformations and states of the ribosome-translocon complex. Employing advanced techniques such as cryoEM structural determination and AlphaFold modeling, the study delves into the dynamic nature of the ribosome-translocon complex. The findings from these analyses unveil crucial insights, significantly advancing our understanding of the co-translational translocation process in cellular mechanisms.

To begin with, the authors employed a construct comprising the first two transmembrane domains of rhodopsin as a model for studying protein translocation. They conducted in vitro translation, followed by the purification of the ribosome-translocon complex, and determined its cryoEM structures. An in-depth analysis of their ribosome-translocon complex structure revealed that the nascent chain can pass through the lateral gate of translocon Sec61, akin to the behavior of a Signaling Peptide. Additionally, Sec61 was found to interact with 28S rRNA helix 24 and the ribosomal protein uL24. In summary, their structural model aligns with the through-pore model of insertion, contradicting the sliding model.

Secondly, the authors successfully identified RAMP4 in their ribosome-translocon complex structure. Notably, the transmembrane domain of RAMP4 mimics the binding of a Signaling Peptide at the lateral gate of Sec61, albeit without unplugging. Intriguingly, RAMP4 is exclusively present in the non-multipass translocon ribosome-translocon complex, not in those containing multipass translocon. This observation suggests that co-translational translocation specifically occurs in the Sec61 channel that includes bound RAMP4. Additionally, the authors discovered an interaction between the C-tail of ribosomal proteins uL22 and the translocon Sec61, providing valuable insights into the nascent chain's behavior.

Moving on to the third point, the focused classification unveiled TRAP complex interactions with various components. The authors propose that the extra density observed in their novel ribosome-translocon complex can be attributed to calnexin, a major binder of TRAP according to previous studies. Furthermore, the new structure reveals a TRAP-OSTA interaction. This newly identified TRAP-OSTA interaction offers a potential explanation for why patients with TRAP delta defects exhibit congenital disorders of glycosylation.

In conclusion, this paper presents a robust contribution to the field with its thorough structural and modeling analyses. The significance of the findings is evident, providing valuable insights into the intricate mechanisms of protein co-translational translocation. The well-crafted writing, meticulous analyses, and clear figures collectively contribute to the overall strength of the paper.

Major points:

(1) The identification of RAMP4 is a pivotal discovery in this paper. The sophisticated AlphaFold prediction, de novo model building of RAMP4's RBD domain, and sequence analyses provide strong evidence supporting the inclusion of RAMP4 in the ribosome-translocon complex structure.

However, it is crucial to ensure the presence of RAMP4 in the purified sample. Particularly, a validation step such as western blotting for RAMP4 in the purified samples would strengthen the assertion that the ribosome-translocon complex indeed contains RAMP4. This is especially important given the purification steps involving stringent membrane solubilization and affinity column pull-down.

(2) Despite the comprehensive analyses conducted by the authors, it is challenging to accept the assertion that the extra density observed in TRAP class 1 corresponds to calnexin. The additional density in TRAP class 1 appears to be less well-resolved, and the evidence for assigning it as calnexin is insufficient. The extra density there can be any proteins that bind to TRAP. It is recommended that the authors examine the density on the ER lumen side. An investigation into whether calnexin's N-globular domain and P-domain are present in the ER lumen in TRAP class 1 would provide a clearer understanding.

(3) In the section titled 'TRAP competes and cooperates with different translocon subunits,' the authors present a compelling explanation for why TRAP delta defects can lead to congenital disorders of glycosylation. To enhance this explanation, it would be valuable if the authors could provide additional analyses based on mutations mentioned in the references. Specifically, examining whether these mutations align with the TRAP delta-OSTA structure models would strengthen the link between TRAP delta defects and the observed congenital disorders of glycosylation.

Reviewer #2 (Public Review):

Summary:

In the manuscript 'Structural analysis of the dynamic ribosome-translocon complex' Lewis and Hegde present a structural study of the ribosome-bound multipass translocon (MPT) based on re-analysis of cryo-EM single particle data of ribosome-MPTs processing the multipass transmembrane substrate RhoTM2 from a previous publication (Smalinskaité et al, Nature 2022) and AlphaFold2 multimer modeling. Detailed analysis of the laterally open Sec61 is obtained from PAT-less particles.

The following major claims are made:

- TMs can bind similarly to the Sec61 lateral gate as signal peptides.

- Ribosomal H59 is in immediate proximity to basic residues of TMs and signal peptides, suggesting it may contribute to the positive-inside rule.

- RAMP4/SERP1 binds to the Sec61 lateral gate and the ribosome near 28S rRNA's helices 47, 57, and 59 as well as eL19, eL22, and eL31.

- uL22 C-terminal tail binds H24/47 blocking a potential escape route for nascent peptides to the cytosol.

- TRAP and BOS compete for binding to Sec61 hinge.

- Calnexin TM binds to TRAPg.

- NOMO wedges between TRAP and MPT.

Strengths:

The manuscript contains numerous novel new structural analyses and their potential functional implications. While all findings are exciting, the highlight is the discovery of RAMP4/SERP1 near the Sec61 lateral gate. Overall, the strength is the thorough and extensive structural analysis of the different high-resolution RTC classes as well as the expert bioinformatic evolutionary analysis.

Weaknesses:

A minor downside of the manuscript is the sheer volume of analyses and mechanistic hypotheses, which makes it sometimes difficult to follow. The authors might consider offloading some analyses based on weaker evidence to the supplement to maximize impact.

Joint Public Review:

Summary:

This study presents an immunotherapeutic strategy for treating mouse cutaneous squamous cell carcinoma (mCSCC) using serum from mice inoculated with mCSCC. The author hypothesizes that antibodies in the generated serum could aid the immune system in tumor volume reduction. The study results showed a reduction in tumor volume and altered expression of several cancer markers (p53, Bcl-xL, NF-κB, Bax) suggesting the potential effectiveness of this approach.

Strengths:

The approach shows potential effect on preventing tumor progression, from both the tumor size and the cancer biomarker expression levels bringing attention to the potential role of antibodies and B cell responses in cancer therapy.

Weaknesses:

These are some of the specific things that the author could consider to strengthen the evidence supporting the claims in their study.

(1) The study fails to provide evidence of the specific effect of mCSCC-antibodies on mCSCC. The study utilized serum which also contains many immune response factors like cytokines that could contribute to tumor reduction. There is no information on serum centrifugation conditions, which makes it unclear whether immune components like antigen-specific T cells, activated NK cells, or other immune cells were removed from the serum. The study does not provide evidence of neutralizing antibodies through isolation, analysis of B cell responses, or efficacy testing against specific cancer epitopes. To affirm the specific antibodies' role in the observed immune response, isolating antibodies rather than employing whole serum could provide more conclusive evidence. Purifying the serum to isolate mCSCC-binding antibodies, such as through protein A purification, and ELISA would have been more useful to quantify the immune response. It would be interesting to investigate the types of epitopes targeted following direct tumor cell injection. A more thorough characterization of the antibodies, including B cell isolation and/or hybridoma techniques, would strengthen the claim.

(2) In the study design, the control group does not account for the potential immunostimulatory effects of serum injection itself. A better control would be tumor-bearing mice receiving serum from healthy non-mCSCC-exposed mice. Additionally, employing a completely random process for allocating the treatment groups would be preferable. Also, the study does not explain why intravenous injection of tumor cells would produce superior antibodies compared to those naturally generated in mCSCC-bearing mice.

(3) In Figure 2B, it would be more helpful if the author could provide raw data/figures of the tumor than just the bar graph. Similarly in Figure 3, the author should show individual data points in addition to the error bar to visualize the actual distribution.

(4) The author mentioned that different stages of tumor cells have different surface biomarkers. Therefore, experimenting with injecting tumor cells at various stages could reveal the most immunogenic stage. Such an approach would allow for a comparative analysis of immune responses elicited by tumor cells at different stages of development.

(5) In the abstract the author mentioned that using mCSCC is a proof-of-concept for this potential cancer treatment strategy. The discussion session should extend to how this strategy might apply to other cancer types beyond carcinoma.

Reviewer #2 (Public Review):

Summary:

This combined experimental-theoretical paper introduces a novel two-domain statistical thermodynamic model (primarily Equation 1) to study allostery in generic systems but focusing here on the tetracycline repressor (TetR) family of transcription factors. This model, building on a function-centric approach, accurately captures induction data, maps mutants with precision, and reveals insights into epistasis between mutations.

Strengths:

The study contributes innovative modeling, successful data fitting, and valuable insights into the interconnectivity of allosteric networks, establishing a flexible and detailed framework for investigating TetR allostery. The manuscript is generally well-structured and communicates key findings effectively.

Comments on revised version:

I am happy with the changes made by the authors

Reviewer #1 (Public Review):

Summary:

The authors' earlier deep mutational scanning work observed that allosteric mutations in TetR (the tetracycline repressor) and its homologous transcriptional factors are distributed across the structure instead of along the presumed allosteric pathways as commonly expected. Especially, in addition, the loss of the allosteric communications promoted by those mutations, was rescued by additional distributed mutations. Now the authors develop a two-domain thermodynamic model for TetR that explains these compelling data. The model is consistent with the in vivo phenotypes of the mutants with changes in parameters, which permits quantification. Taken together their work connects intra- and inter-domain allosteric regulation that correlate with structural features. This leads the authors to suggest broader applicability to other multidomain allosteric proteins.

Here the authors follow their first innovative observations with a computational model that captures the structural behavior, aiming to make it broadly applicable to multidomain proteins. Altogether, an innovative and potentially useful contribution.

Weaknesses:

None that I see, except that I hope that in the future, if possible, the authors would follow with additional proteins to further substantiate the model and show its broad applicability. I realize however the extensive work that this would entail.

Reviewer #1 (Public Review):

Original review:<br /> The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain. Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

Comments on revision:<br /> I acknowledge the significant changes made by the authors in the revised manuscript. However, I remain puzzled by the data concerning the interaction between BIN1 and PRC1. While I agree with the authors that even weak interactions among proteins can be significant, I am hesitant to accept a priori that the lack of clear evidence of colocalization between proteins can be justified solely by their low affinity.

Moreover, the possibility that other mitotic proteins may be potential partners of BIN1 does not inherently support an interaction between BIN1 and PRC1. I suggest that the authors present the interaction with PRC1 as a potential event and emphasize that further studies are needed to definitively establish it.

Reviewer #2 (Public Review):

Original review:<br /> Summary:<br /> In this paper, Zambo and coworkers use a powerful technique, called native holdup, to measure the affinity of the SH3 domain of BIN1 for cellular partners. Using this assay, they combine data using cellular proteins and proline-containing fragments in these proteins to identify 97 distinct direct binding partners of BIN1. They also compare the binding interactome of the BIN1 SH3 domain to the interactome of several other SH3 domains, showing varying levels of promiscuity among SH3 domains. The authors then use pathway analysis of BIN1 binding partners to show that BIN1 may be involved in mitosis. Finally, the authors examine the impact of clinically relevant mutations of the BIN1 SH3 domain on the cellular interactome. The authors were able to compare the interactome of several different SH3 domains and provide novel insight into the cellular function of BIN1. Generally, the data supports the conclusions, although the reliance on one technique and the low number of replicates in each experiment is a weakness of the study.

Strengths:<br /> The major strength of this paper is the use of holdup and native holdup assays to measure the affinity of SH3 domains to cellular partners. The use of both assays using cell-derived proteins and peptides derived from identified binding partners allows the authors to better identify direct binding partners. This assay has some complexity but does hold the possibility of being used to measure the affinity of the cellular interactome of other proteins and protein domains. Beyond the utility of the technique, this study also provides significant insight into the cellular function of BIN1. The authors have strong evidence that BIN1 might have an undiscovered function in cellular mitosis, which potentially highlights BIN1 as a drug target. Finally, the study provides outstanding data on the cellular binding properties and partners of seven distinct SH3 domains, showing surprising differences in the promiscuity of these proteins.

Weaknesses:<br /> There are three major weaknesses of the study. First, the authors rely completely on a single technique to measure the affinity of the cellular interactome. The native holdup is a relatively new technique that is powerful yet relatively unproven. However, it appears to have the capacity to measure the relative affinity of proteins. Second, the authors appear to use a relatively small number of replicates for the holdup assays. There is no information in the legends about the number of replicates but the materials and methods suggest the native holdup data is from a single experimental replicate with multiple technical replicates. Finally, the authors' data using cellular proteins and fragments show that the affinity of the whole proteins is 5-20 fold lower than individual proline-containing fragments. The authors state that this difference suggests that there is cooperativity between different proline-rich sites of the binding partners of BIN1, yet BIN1 only has one SH3 domain. It is unclear what the molecular mechanism of the cooperative interaction would be exactly since there would be only one SH3 domain to bind the partner. An alternative interpretation would be that the BIN 1 SH3 domain requires sequences outside of the short proline-rich regions for high-affinity interactions with cellular partners, a hypothesis that is supported by other studies.

Comments on revision:<br /> I thank the authors for their thoughtful response. I have additional comments.

I appreciate that this is not a techniques paper and that the authors have done more detailed work in a separate publication. It would be helpful to readers not familiar with this new method to more fully describe this technique in this manuscript.

I also thank the authors for their description of why they performed only 1 biological replicate of the experiment. However, I still believe that multiple biological replicates will provide more rigorous and reproducible data. The data the authors provide actually argues for the inclusion of more biological replicates. They state they performed 2 separate nHU replicates using different mass spectrometers. It is unclear if this data uses the same lysates and protein preparations, but by the data, the two methods detected a total of 207 distinct binding partners. Only 29 of these were significant binders in both replicates and only 90 were detected binders in both replicates. 117 binding partners were found in only one replicate suggesting a significant differences between replicates. Different batches of SH3 domains can have different activities and different replicates of cell lysates can vary, even when made from the same cell line. Thus, there can still be significant differences between replicates in this method. I appreciate the difficulty of performing and analyzing multiple biological replicates, but it is the most rigorous way to identify potential cellular partners.

I also thank the author for including the mechanistic discussion about the differences between peptides and whole proteins. There is literature showing that regions outside of the short PxxP regions drive binding to SH3 domains, especially for the GRB2 family of adaptor proteins.

Joint Public Review:

Zhang et. al. presents compelling results that support the identification of epigenetically mediated control for the recognition of dihydropyrimidine dehydrogenase (DPYD) gene expression that is linked with cancer treatment resistance 5-fluorouracil. The experimental approach was developed and pursued with in vitro and in vivo strategies. Combining molecular, cellular, and biochemical approaches, the authors identify a germline variant with compromised enhancer control. Several lines of evidence were presented that are consistent with increased CEBP recruitment to the DPYD regulatory domain with consequential modifications in promoter-enhancer interactions that are associated with compromised 5-fluorouracil resistance. Functional identification of promoter and enhancer elements was validated by CRISPRi and CRISPRa assays. ChIP and qPCR documented histone marks that can account for the control of DPYD gene expression were established. Consistency with data from patient-derived specimens and direct assessment of 5-fluorouracil sensitivity provides confidence in the proposed mechanisms. The model is additionally supported by genome data from a population with high "compromised allele frequency". It can be informative to directly demonstrate DPYD promoter-enhancer interactions. However, the genetic variants support the integration of regulatory activities.

Reviewer #1 (Public Review):

Gap junction channels establish gated intercellular conduits that allow the diffusion of solutes between two cells. Hexameric connexin26 (Cx26) hemichannels are closed under basal conditions and open in response to CO2. In contrast, when forming a dodecameric gap-junction, channels are open under basal conditions and close with increased CO2 levels. Previous experiments have implicated Cx26 residue K125 in the gating mechanism by CO2, which is thought to become carbamylated by CO2. Carbamylation is a labile post-translational modification that confers negative charge to the K125 side chain. How the introduction of a negative charge at K125 causes a change in gating is unclear, but it has been proposed that carbamylated K125 forms a salt bridge with the side chain at R104, causing a conformational change in the channel. It is also unclear how overall gating is controlled by changes in CO2, since there is significant variability between structures of gap-junction channels and the cytoplasmic domain is generally poorly resolved. Structures of WT Cx26 gap-junction channels determined in the presence of various concentrations of CO2 have suggested that the cytoplasmatic N-terminus changes conformation depending on the concentration of the gas, occluding the pore when CO2 levels are high.

In the present manuscript, Deborah H. Brotherton and collaborators use an intercellular dye-transfer assay to show that Cx26 gap-junction channels containing the K125E mutation, which mimics carbamylation caused by CO2, is constitutively closed even at CO2 concentrations where WT channels are open. Several cryo-EM structures of WT and mutant Cx26 gap junction channels were determined at various conditions and using classification procedures that extracted more than one structural class from some of the datasets. Together, the features on each of the different structures are generally consistent with previously obtained structures at different CO2 concentrations and support the mechanism that is proposed in the manuscript. The most populated class for K125E channels determined at high CO2 shows a pore that is constricted by the N-terminus, and a cytoplasmic region that was better resolved than in WT channels, suggesting increased stability. The K125E structure closely resembles one of the two major classes obtained for WT channels at high CO2. These findings support the hypothesis that the K125E mutation biases channels towards the closed state, while WT channels are in an equilibrium between open and closed states even in the presence of high CO2. Consistently, a structure of K125E obtained in the absence of CO2 appeared to also represent a closed state but at a lower resolution, suggesting that CO2 has other effects on the channel beyond carbamylation of K125 that also contribute to stabilizing the closed state. Structures determined for K125R channels, which are constitutively open because arginine cannot be carbamylated, and would be predicted to represent open states, yielded apparently inconclusive results.

A non-protein density was found to be trapped inside the pore in all structures obtained using both DDM and LMNG detergents, suggesting that the density represents a lipid rather than a detergent molecule. It is thought that the lipid could contribute to the process of gating, but this remains speculative. The cytoplasmic region in the tentatively closed structural class of the WT channel obtained using LMNG was better resolved. An additional portion of the cytoplasmic face could be resolved by focusing classification on a single subunit, which had a conformation that resembled the AlphaFold prediction. However, this single-subunit conformation was incompatible with a C6-symmetric arrangement. Together, the results suggest that the identified states of the channel represent open states and closed states resulting from interaction with CO2. Therefore, the observed conformational changes illuminate a possible structural mechanism for channel gating in response to CO2.

Reviewer #2 (Public Review):

Summary:

The manuscript by Brotherton et al. describes a structural study of connexin-26 (Cx26) gap junction channel mutant K125E, which is designed to mimic the CO2-inhibited form of the channel. In the wild-type Cx26, exposure to CO2 is presumed to close the channel through carbamylation of the redeye K125. The authors mutated K125 to a negatively charged residue to mimic this effect and observed by cryo-EM analysis of the mutated channel that the pore of the channel is constricted. The authors were able to observe conformations of the channel with resolved density for the cytoplasmic loop (in which K125 is located). Based on the observed conformations and on the position of the N-terminal helix, which is involved in channel gating and in controlling the size of the pore, the authors propose the mechanisms of Cx26 regulation.

Strengths:

This is a very interesting and timely study, and the observations provide a lot of new information on connexin channel regulation. The authors use the state of the art cryo-EM analysis and 3D classification approaches to tease out the conformations of the channel that can be interpreted as "inhibited", with important implications for our understanding of how the conformations of the connexin channels controlled.

Weaknesses:

The revised version of the manuscript is improved, and the authors have addressed the review comments/criticisms in a satisfactory manner.

Reviewer #3 (Public Review):

Summary:

The mechanism underlying the well-documented CO2-regulated activity of connexin 26 (Cx26) remains poorly understood. This is largely due to the labile nature of CO2-mediated carbamylation, making it challenging to visualize the effects of this reversible posttranslational modification. This paper by Brotherton et al. aims to address this gap by providing structural insights through cryo-EM structures of a carbamylation-mimetic mutant of the gap junction protein.

Strength:

The combination of the mutation, elevated PCO2, and the use of LMNG detergent resulted in high-resolution maps that revealed, for the first time, the structure of the cytoplasmic loop between transmembrane helix (TM) 2 and 3.

Weaknesses:

While the structure of the TM2-TM3 loop may suggest a mechanism for stabilizing the closed conformation, the EM density is not strong enough to support direct interaction with carbamylated or mutated K125.

Overall, the cryo-EM structures presented in this study support their proposing mechanism in which carbamylation at K125 promotes Cx26 gap junction closure. Through careful control of the pH and PCO2 for each cryo-EM sample, the current study substantiated that the more closed conformation observed in high PCO2 is independent of pH but likely triggered by carbamylation. This was unclear from their prior cryo-EM map of wildtype Cx26 at high PCO2.

While the new structures successfully visualize the TM2-TM3 loop, which likely plays significant roles in CO2-regulated Cx26 activity, further studies are necessary to understand the underlying mechanism. For instance, the current study lacks explanation regarding what propels the movement of the N-terminal helix, how carbamylated K125 interacts with the TM2-TM3 loop, the importance of the lipids visualized in the map, or the reason why gap junctions are constitutively open while hemichannels are closed under normal PCO2 levels

Reviewer #1 (Public Review):

Muscle models are important tools in the fields of biomechanics and physiology. Muscle models serve a wide variety of functions, including validating existing theories, testing new hypotheses, and predicting forces produced by humans and animals in health and disease. This paper attempts to provide an alternative to Hill-type muscle models that includes contributions of titin to force enhancement over multiple time scales. Due to the significant limitations of Hill-type models, alternative models are needed and therefore the work is important and timely.

The effort to include a role for titin in muscle models is a major strength of the methods and results. The results clearly demonstrate the weaknesses of Hill models and the advantages of incorporating titin into theoretical treatments of muscle mechanics. Another strength is to address muscle mechanics over a large range of time scales.

The authors succeed in demonstrating the need to incorporate titin in muscle models, and further show that the model accurately predicts in situ force of cat soleus (Kirsch et al. 1994; Herzog & Leonard, 2002) and rabbit posts myofibrils (Leonard et al. 2010). However, it remains unclear whether the model will be practical for use with data from different muscles or preparations. Several ad hoc modifications were described in the paper, and the degree to which the model requires parameter optimization for different muscles, preparations and experiment types remains unclear.

I think the authors should state how many parameters require fitting to the data vs the total number of model parameters. It would also be interesting for the authors to discuss challenges associated with modeling ex vivo and in vivo data sets, due to differences in means of stimulation vs. model inputs.

Reviewer #2 (Public Review):

This model of skeletal muscle includes springs and dampers which aim to capture the effect of crossbridge and titin stiffness during the stretch of active muscle. While both crossbridge and titin stiffness have previously been incorporated, in some form, into models, this model is the first to simultaneously include both. The authors suggest that this will allow for the prediction of muscle force in response to short-, mid- and long-range stretches. All these types of stretch are likely to be experienced by muscle during in vivo perturbations, and are known to elicit different muscle responses. Hence, it is valuable to have a single model which can predict muscle force under all these physiologically relevant conditions. In addition, this model dramatically simplifies sarcomere structure to enable this muscle model to be used in multi-muscle simulations of whole-body movement.

In order to test this model, its force predictions are compared to 3 sets of experimental data which focus on short-, mid- and long-range perturbations, and to the predictions of a Hill-type muscle model. The choice of data sets is excellent and provide a robust test of the model's ability to predict forces over a range of length perturbations. However, I find the comparison to a Hill-type muscle model to be somewhat limiting. It is well established that Hill-type models do not have any mechanism by which they can predict the effect of active muscle stretch. Hence, that the model proposed here represents an improvement over such a model is not a surprise. Many other models, some of which are also simple enough to be incorporated into whole-body simulations, have incorporated mechanistic elements which allow for the prediction of force responses to muscle stretch. And it is not clear from the results presented here that this model would outperform such models.

The paper begins by outlining the phenomenological vs mechanistic approaches taken to muscle modelling, historically. It appears, although is not directly specified, that this model combines these approaches. A somewhat mechanistic model of the response of the crossbridges and titin to active stretch is combined with a phenomenological implementation of force-length and force-velocity relationships. This combination of approaches may be useful improving the accuracy of predictions of muscle models and whole-body simulations, which is certainly a worthy goal. However, it also may limit the insight that can be gained. For example, it does not seem that this model could reflect any effect of active titin properties on muscle shortening. In addition, it is not clear to me, either physiologically or in the model, what drives the shift from the high stiffness in short-range perturbations to the somewhat lower stiffness in mid-range perturbations.

Reviewer #2 (Public Review):

In this revised manuscript Aguillon and collaborators convincingly demonstrating that CLK is required for free-running behavioral rhythms under constant conditions in the Cnidarian Nematostella. The results also convincingly show that CLK impacts rhythmic gene expression in this organism. This original work thus demonstrate that CLK was recruited very early during animal evolution in the circadian clock mechanism to optimize behavior and gene expression with the time-of-day. The manuscript could still benefit from some improvements so that it is more accessible for a wide readership.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Ruichen Yang et al. investigated the importance of BMP signaling in preventing microtia. Authors showed that Cre recombinase mediated deletion of Bmpr1a using skeletal stem specific Cre Prx1Cre leads to microtia in adult and young mice. In these mice, distal auricle is more affected than middle and proximal. In these Bmpr1a floxed Prx1Cre mice, auricle chondrocyte start to differentiate into osteoblasts through increase in PKA signaling. The authors showed human single-cell RNA-Seq data sets where they observed increased PKA signaling in microtia patient which resembles their animal model experiments.

Strengths:

Although the importance of BMP signaling in skeletal tissues has been previously reported, the importance of its role in microtia prevention is novel and very promising to study in detail. The authors satisfied the experimental questions by performing correct methods and explaining the results in detail.

Reviewer #2 (Public Review):

The authors (Yang et al.) present a well-executed study of a mouse model of Bmpr1a focusing on microtia development and pathogenesis.

The authors report that the generation of the Bmpr1a in Prrx1+ cells in adult mice helps characterize the developmental progression of the external ear.

The authors explain how auricular chondrocytes differ from growth plates or other chondrocytes and BMP-Smd1/5/9 activation, which is required to maintain chondrocyte fate in the distal part of the ear. The authors explain with evidence how BMP signaling actively maintains auricle cartilage in the post-developmental stage.

Elegant immunofluorescence staining, excellent histology preparations and dissections, excellent microscopy, sufficient experimental sample size, and good statistical analyses support the results. The study is well grounded in extensively reviewed and cited existing literature. This report sets the stage for a comprehensive interrogation of Bmpr1a deficiency and ear defects.

Reviewer #1 (Public Review):

In this study, the authors address a fundamental unresolved question in cerebellar physiology: do synapses between granule cells (GCs) and Purkinje cells (PCs) made by the ascending part of the axon (AA) have different synaptic properties from those made by parallel fibers? This is an important question, as GCs integrate sensorimotor information from numerous brain areas with a precise and complex topography.

Summary:<br /> The authors argue that CGs located close to PCs essentially contact PC dendrites via the ascending part of their axons. They demonstrate that joint high-frequency (100 Hz) stimulation of distant parallel fibers and local CGs potentiates AA-PC synapses, while parallel fiber-PC synapses are depressed. On the basis of paired-pulse ratio analysis, they concluded that evoked plasticity was postsynaptic. When individual pathways were stimulated alone, no LRP was observed. This associative plasticity appears to be sensitive to timing, as stimulation of parallel fibers first results in depression, while stimulation of the AA pathway has no effect. NMDA, mGluR1 and GABAA receptors are involved in this plasticity.

Strengths:<br /> Overall, the associative modulation of synaptic transmission is convincing, and the experiments carried out support this conclusion. However, weaknesses limit the scope of the results.

Weaknesses:<br /> One of the main weaknesses of this study is the suggestion that high-frequency parallel-fiber stimulation cannot induce long term potentiation unless combined with AA stimulation. Although we acknowledge that the stimulation and recording conditions were different from those of other studies, according to the literature (e.g. Bouvier et al 2016, Piochon et al 2016, Binda et al, 2016, Schonewille et al 2021 and others), high-frequency stimulation of parallel fibers leads to long-term postsynaptic potentiation under many different experimental conditions (blocked or unblocked inhibition, stimulation protocols, internal solution composition). Furthermore, in vivo experiments have confirmed that high-frequency parallel fibers are likely to induce long-term potentiation (Jorntell and Ekerot, 2002; Wang et al, 2009). This article provides further evidence that long-term plasticity (LTP and LTD) at this connection is a complex and subtle mechanism underpinned by many different transduction pathways. It would therefore have been interesting to test different protocols or conditions to explain the discrepancies observed in this dataset.<br /> Another important weakness is the lack of evidence that the AAs were stimulated. Indeed, without filling the PC with fluorescent dye or biocytin during the experiment, and without reconstructing the anatomical organization, it is difficult to assess whether the stimulating pipette is positioned in the GC cluster that is potentially in contact with the PC with the AAs. According to EM microscopy, AAs account for 3% of the total number of synapses in a PC, which could represent a significant number of synapses. Although the idea that AAs repeatedly contact the same Purkinje cell has been propagated, to the best of the review author's knowledge, no direct demonstration of this hypothesis has yet been published. In fact, what has been demonstrated (Walter et al 2009; Spaeth et al 2022) is that GCs have a higher probability of being connected to nearby PCs, but are not necessarily associated with AAs.

Reviewer #2 (Public Review):

Summary:

The authors describe a form of synaptic plasticity at synapses from granule cells onto Purkinje cells in the mouse cerebellum, which is specific to synapses proximal to the cell body but not to distal ones. This plasticity is induced by the paired or associative stimulation of the two types of synapses because it is not observed with stimulation of one type of synapse alone. In addition, this form of plasticity is dependent on the order in which the stimuli are presented, and is dependent on NMDA receptors, metabotropic glutamate receptors and to some degree on GABAA receptors. However, under all experimental conditions described, there is a progressive weakening or run-down of synaptic strength. Therefore, plasticity is not relative to a stable baseline, but relative to a process of continuous decline that occurs whether or not there is any plasticity-inducing stimulus.

Strengths:

The focus of the authors on the properties of two different synapse-types on cerebellar Purkinje cells is interesting and relevant, given previous results that ascending and parallel fiber synapses might be functionally different and undergo different forms of plasticity. In addition, the interaction between these two synapse types during plasticity is important for understanding cerebellar function. The demonstration of timing and order-dependent potentiation of only one pathway, and not another, after associative stimulation of both pathways, changes our understanding of potential plasticity mechanisms. In addition, this observation opens up many new questions on underlying intracellular mechanisms as well as on its relevance for cerebellar learning and adaptation.

Weaknesses and suggested improvements:

A concern with this study is that all recordings demonstrate "rundown", a progressive decrease in the amplitude of the EPSC, starting during the baseline period and continuing after the plasticity-induction stimulus. In the absence of a stable baseline, it is hard to know what changes in strength actually occur at any set of synapses. Moreover, the issues that are causing rundown are not known and may or may not be related to the cellular processes involved in synaptic plasticity. This concern applies in particular to all the experiments where there is a decrease in synaptic strength.<br /> The authors should consider changes in the shape of the EPSC after plasticity induction, as in Fig 1 (orange trace) as this could change the interpretation.<br /> In addition, the inconsistency with previous results is surprising and is not explained; specifically, that no PF-LTP was induced by PF-alone repeated stimulation.<br /> The authors test the role of NMDARs, GABAARs and mGluRs in the phenotype they describe. The data suggest that the form of plasticity described here is dependent on any one of the three receptors. However, the location of these receptors varies between the Purkinje cells, granule cells and interneurons. The authors do not describe a convincing hypothetical model in which this dependence can be explained. They suggest that there is crosstalk between AA and PF synapses via endocannabinoids downstream of mGluR or NO downstream of NMDARs. However, it is not clear how this could lead to the long-term potentiation that they describe. Also, there is no long-lasting change in paired-pulse ratio, suggesting an absence of changes in presynaptic release.<br /> Is the synapse that undergoes plasticity correctly identified? In this study, since GABAergic inhibition is not blocked for most experiments, PF stimulation can result in both a direct EPSC onto the Purkinje cell and a disynaptic feedforward IPSC. The authors do address this issue with Supplementary Fig 3, where the impact of the IPSC on the EPSC within the EPSC/IPSC sequence is calculated. However, a change in waveform would complicate this analysis. An experiment with pharmacological blockade will make the interpretation more robust. The observed dependence of the plasticity on GABAA receptors is an added point in favor of the suggested additional experiments.<br /> A primary hypothesis of this study is that proximal, or AA, and distal, or PF, synapses are different and that their association is specifically what drives plasticity. The alternative hypothesis is that the two synapse-types are the same. Therefore, a good control for pairing AA with PF would be to pair AA with AA and PF with PF, thereby demonstrating that pairing with each other is different from pairing with self.<br /> It is hypothesized that the association of a PF input with an AA input is similar to the association of a PF input with a CF input. However, the two are very different in terms of cellular location, with the CF input being in a position to directly interact with PF-driven inputs. Therefore, there are two major issues with this hypothesis: 1) how can sub-threshold activity at one set of synapses affect another located hundreds of micrometers away on the same dendritic tree? 2) There is evidence that the CF encodes teaching/error or reward information, which is functionally meaningful as a driver of plasticity at PF synapses. The AA synapse on one set of Purkinje cells is carrying exactly the same information as the PF synapses on another set of Purkinje cells further up and down the parallel fiber beam. It is suggested that the two inputs carry sensory vs. motor information, which is why this form of plasticity was tested. However, the granule cells that lead to both the AA and PF synapses are receiving the same modalities of mossy fiber information. Therefore, one needs to presuppose different populations of granule cells for sensory and motor inputs or receptive field and contextual information. As a consequence, which granule cells lead to AA synapses and which to PF synapses will change depending on which Purkinje cell you're recording from. And that's inconsistent with there being a timing dependence of AA-PF pairing in only one direction. Overall, it would be helpful to discuss the functional implications of this form of plasticity.

Reviewer #3 (Public Review):

Granule cells' axons bifurcate to form parallel fibers (PFs) and ascending axons (AAs). While the significance of PFs on cerebellar plasticity is widely acknowledged, the importance of AAs remains unclear. In the current paper, Conti and Auger conducted electrophysiological experiments in rat cerebellar slices and identified a new form of synaptic plasticity in the AA-Purkinje cell (PC) synapses. Upon simultaneous stimulation of AAs and PFs, AA-PC EPSCs increased, while PFs-EPSCs decreased. This suggests that synaptic responses to AAs and PFs in PCs are jointly regulated, working as an additional mechanism to integrate motor/sensory input. This finding may offer new perspectives in studying and modeling cerebellum-dependent behavior. Overall, the experiments are performed well. However, there are two weaknesses. First, the baseline of electrophysiological recordings is influenced significantly by run-down, making it difficult to interpret the data quantitatively. The amplitude of AA-EPSCs is relatively small and the run-down may mask the change. The authors should carefully reexamine the data with appropriate controls and statistics. Second, while the authors show AA-LTP depends on mGluR, NMDA receptors, and GABA-A receptors, which cell types express these receptors and how they contribute to plasticity is not clarified. The recommended experiments may help to improve the quality of the manuscript.

Reviewer #1 (Public Review):

Summary:

The manuscript proposes a series of steps using the FIJI environment, the authors have created a plugin for the initial steps of the process, merging images into an RGB stack, conversion to HSV, and then using brightness for reference and hue to distinguish the phases of the cycle. Then, the well-known Trackmate plugin was used to identify single cells and extract intensities. The data was further post-processed in R, where a series of steps, smoothing, scaling, and addressing missing frames were used to train a random forest. Hard-coded values of hue were used to distinguish G1, S, and G2/M. The process was validated with a score comparing the quality of the tracks and the authors reported the successful measure of the cell cycles.

Strengths:

The implementation of the pipeline seems easy, although it requires two separate platforms: Fiji and R. A similar approach could be implemented in a single programming environment like Python or Matlab and there would not be any need to export from one to the other. However, many labs have similar setups and that is not necessarily a problem.

Weaknesses:

I found two important weaknesses in the proposal:

(1) The pipeline relies on a large number of hard-coded conditions: size of Gaussian blur (Gaussian should be written in uppercase), values of contrast, size of filters, levels of intensity, etc. Presumably, the authors followed a heuristic approach and tried values of these and concluded that the ones proposed were optimal. A proper sensitivity analysis should be performed. That is, select a range of values of the variables and measure the effect on the output.

(2) Linked to the previous comments. Other researchers that want to follow the pipeline would have either to have exactly the same acquisition conditions as the manuscript or start playing with values and try to compensate for any difference in their data (cell diameter, fluorescent intensity, etc.) to see if they can match the results of the manuscript.

Reviewer #2 (Public Review):

Summary:

This paper presents an automated method to track individual mammalian cells as they progress through the cell cycle using the FUCCI system and applies the method to look at different tumor cell lines that grow in suspension and determine their cell cycle profile and the effect of drugs that directly affect the cell cycles, on progression through the cell cycle for a 72 hour period.

Strengths:

This is a METHODS paper. The one potentially novel finding is that they can identify cells that are at the G1-S transition by the change in color as one protein starts to go up and the other one goes down, similar to the change seen as cells enter G2/M.

Weaknesses:

They did not clearly indicate whether the G1/S cells are identified automatically or need to be identified by the person reviewing the data. In Figures 1 and S1, the movie shows cells with no color at a time corresponding to what is about the G1/S transition. Their assigned cell cycle phase is shown in Figure 1 but not in Figure S1. None of these pictures show the G1/S cells that they talk about being able to detect with a different color.

Reviewer #2 (Public Review):

In this study, Wang et al., report the significance of XAP5L and XAP5 in spermatogenesis, involved in transcriptional regulation of the ciliary gene in testes. In previous studies, the authors demonstrate that XAP5 is a transcription factor required for flagellar assembly in Chlamydomonas. Continuing from their previous study, the authors examine the conserved role of the XAP5 and XAP5L, which are the orthologue pair in mammals.

XAP5 and XAP5L express ubiquitously and testis specifically, respectively, and their absence in the testes causes male infertility with defective spermatogenesis. Interestingly, XAP5 deficiency arrests germ cell development at the pachytene stage, whereas XAP5L absence causes impaired flagellar formation. RNA-seq analyses demonstrated that XAP5 deficiency suppresses ciliary gene expression including Foxj1 and Rfx family genes in early testis. By contrast, XAP5L deficiency abnormally remains Foxj1 and Rfx genes in mature sperm. From the results, the authors conclude that XAP5 and XAP5L are the antagonistic transcription factors that function upstream of Foxj1 and Rfx family genes.

This reviewer thinks the overall experiments are performed well and that the manuscript is clear. However, the current results do not directly support the authors' conclusion. For example, the transcriptional function of XAP5 and XAP5L requires more evidence. In addition, this reviewer wonders about the conserved XAP5 function of ciliary/flagellar gene transcription in mammals - the gene is ubiquitously expressed despite its functional importance in flagellar assembly in Chlamydomonas. Thus, this reviewer thinks authors are required to show more direct evidence to clearly support their conclusion with more descriptions of its role in ciliary/flagellar assembly.

Reviewer #1 (Public Review):

Summary:

Wang et al. generate XAP5 and XAP5L knockout mice and find that they are male infertile due to meiotic arrest and reduced sperm motility, respectively. RNA-Seq was subsequently performed and the authors concluded that XAP5 and XAP5L are antagonistic transcription factors of cilliogenesis (in XAP5-KO P16 testis: 554 genes were unregulated and 1587 genes were downregulated; in XAP5L-KO sperm: 2093 genes were unregulated and 267 genes were downregulated).

Strengths:

Knockout mouse models provided strong evidence to indicate that XAP5 and XAP5L are critical for spermatogenesis and male fertility.

Weaknesses:

The key conclusions are not supported by evidence. First, the authors claim that XAP5 and XAP5L transcriptionally regulate sperm flagella development; however, detailed molecular experiments related to transcription regulation are lacking. How do XAP5 and XAP5L regulate their targets? Only RNA-Seq is not enough. Second, the authors declare that XAP5 and XAP5L are antagonistic transcription factors; however, how do XAP5 and XAP5L regulate sperm flagella development antagonistically? Only RNA-Seq is not enough. Third, I am concerned about whether XAP5 really regulates sperm flagella development. XAP5 is specifically expressed in spermatogonia and XAP5-cKO mice are in meiotic arrest, indicating that XAP5 regulates meiosis rather than sperm flagella development.

Reviewer #1 (Public Review):

• A summary of what the authors were trying to achieve.

The authors cultured pre- and Post-vaccine PBMCs with overlapping peptides encoding S protein in the presence of IL-2, IL-7, and IL-15 for 10 days, and extensively analyzed the T cells expanded during the culture; by including scRNAseq, scTCRseq, and examination of reporter cell lines expressing the dominant TCRs. They were able to identify 78 S epitopes with HLA restrictions (by itself represents a major achievement) together with their subset, based on their transcriptional profiling. By comparing T cell clonotypes between pre- and post-vaccination samples, they showed that a majority of pre-existing S-reactive CD4+ T cell clones did not expand by vaccinations. Thus, the authors concluded that highly-responding S-reactive T cells were established by vaccination from rare clonotypes.

• An account of the major strengths and weaknesses of the methods and results.

Strengths

• Selection of 4 "Ab sustainers" and 4 "Ab decliners" from 43 subjects who received two shots of mRNA vaccinations.<br /> • Identification of S epitopes of T cells together with their transcriptional profiling. This allowed the authors to compare the dominant subsets between sustainers and decliners.

Weaknesses were adequately addressed in the revised manuscript, and I do not have any additional concerns.

Reviewer #3 (Public Review):

The paper aims to investigate the relationship between anti-S protein antibody titers with the phenotypes & clonotypes of S-protein-specific T cells in people who receive SARS-CoV2 mRNA vaccines. The paper recruited a cohort of COVID-19 naive individuals who received the SARS-CoV2 mRNA vaccines and collected sera and PBMCs samples on different time points. Then, three sets of data were generated: 1). Anti-S protein antibody titers on all time points. 2) Single-cell RNAseq/TCRseq analysis for divided T cells after in vitro stimulation by S-protein. 3) Peptide epitopes for each expanded TCR clone. Based on these, the paper reports two major findings: A) Individuals having more sustained anti-S protein antibody response also have more Tfh-featured S-specific cells in their blood after 2nd-dose vaccination. B). S-specific cross-reactive T cells exist in COVID-19 naive individuals, but most of these T cell clones are not expanded after SARS-CoV-2 vaccination.

The paper's strength is that it uses a very systemic strategy trying to dissect the relationship between antibody titers, T cell phenotypes, TCR clonotypes and corresponding epitopes. The conclusion is solid in general. However, the weaknesses include the relatively small sample size (4 sustainers vs. 4 decliners) and the use of in vitro stimulated cells for analysis, which may 'blur' the classification of T cell subsets. Nevertheless, it may have great impact on future vaccine design because it demonstrated that promoting Tfh differentiation is crucial for the longevity of antibody response. Additionally, this paper nicely showed that most cross-reactive clones that are specific to environmental/symbiotic microbes did not expand post- vaccination, providing important fundamental insights into the establishment of T-cell responses after SARS-CoV-2 vaccination.

Reviewer #2 (Public Review):

Summary:

This work follows previous work from the group where they have demonstrated the role of TASK1 in the regulation of glucose stimulated insulin secretion. Moreover, a recent study links a mutation in KCNK16, the gene encoding TALK-1 channels to MODY. Here the authors have constructed a mouse model with the specific mutation (TALK-1 L114P mutation) and investigated the phenotype. They have to perform a couple of breeding tricks to find a model that is lethal in adult which might complicate the conclusions, however, the phenotype of the heterozygote model used have a MODY-like phenotype. The study is convincing and solid.

Strengths:

(1) The work is a natural follow-up from previous studies from the groups.<br /> (2) The authors present convincing and solid data that in the long perspective will help patients with this mutations.<br /> (3) Both in vivo and in vitro data are presented to give the full picture of the phenotype.<br /> (4) Data from both female and male mice are presented.

Weaknesses:

The authors have answered all my comments in the revised version and I find no more weaknesses. Some questions still remain but have been clearly discussed in the new version of the manuscript.

Reviewer #1 (Public Review):

Summary:

This paper focuses on the effects of a L114P mutation in the TALK-1 channel on islet function and diabetes. This mutation is clinically relevant and a cause of MODY diabetes. This work employs a mouse model with heterozygous and homozygous mutants. The homozygous mice are homozygous lethal from severe hyperglycemia. The work shows that the mutation increases K+ currents and inhibits insulin secretion. This is a very nice paper with mechanistic insight and clear clinical importance. It is generally well written and the data is well presented.

Comments on revision:

I have no further comments to add at this time. The authors have adequately addressed my concerns.

Reviewer #3 (Public Review):

Summary

The L114P gain of function mutation in the K2P channel TALK-1 encoded by KCNJ16 has been associated with maturity-onset diabetes of the young (MODY). In this study, Nakhe et al. generated mice carrying L114P TALK-1 and evaluated the impact of the mutation on pancreatic islet functions and glucose homeostasis. The authors report that the mutation increases neonatal lethality, owing to hyperglycemia caused by a lack of glucose-stimulated Ca2+ influx and insulin secretion. Adult mutant mice showed glucose intolerance and fasting hyperglycemia, which is attributed to blunted glucose-stimulated insulin secretion as well as increased glucagon secretion. Interestingly, male mice were more affected than female mice. Islets from adult mutant mice were found to have reduced Ca2+ entry upon glucose stimulation but also enhanced IP3-induced ER Ca2+ release, consistent with previous studies from the group showing a role of TALK-1 in ER Ca2+ homeostasis. Finally, comparison of bulk RNA sequencing results from WT and mutant islets revealed altered expression of genes involved in β-cell identify, function and signaling, which also contributes to the observed islet dysfunction.

Strengths

This is a well-executed and rigorous study that will be of great interest to the diabetes and islet biology communities. The findings provide convincing evidence supporting a causal role of the L114P gain of function TALK-1 mutation in glucose-stimulated insulin secretion defects and diabetes. The neonatal diabetes phenotype and the gender difference uncovered by the study have important clinical implications. The complexity of TALK-1 expression and hormone secretion in different endocrine cell types and how it impacts glucose homeostasis is elegantly illustrated in the L114P TALK-1 mouse model. The authors carefully and thoroughly addressed limitations of their study and discussed future directions. The importance of TALK-1 in β-cell and islet function demonstrated by this study will prompt future efforts targeting this important channel for diabetes treatment.

Reviewer #1 (Public Review):

Summary:

Babosha et al. deeply investigate the N-terminal region of the Drosophila dosage compensation protein MSL1. Much of the prior research into the dosage compensation complex has focused on the male-specific MSL2 protein. However, the authors point out prior evidence that the N-terminus of MSL1 is important for protein function, including interaction with MSL2. Through a series of transgenic deletions and substitutions, the authors pinpoint two regions: N-terminal amino acids 3-7 and 41-65, which are critical for the binding of MSL1 to the X-chromosome and recruitment of MSL2. To deepen these observations, the authors perform well-controlled immunoprecipitation experiments to test the interaction of mutant MSL1 proteins with the lncRNA roX2, which is critical for the stability and localization of the dosage compensation complex. Through immunoprecipitation, the authors discover that the interaction of their mutant MSL1 proteins with roX2 is compromised. They suggest that the roX-MSL1 interaction is mediated by the N-terminal amino acids and is also critical for interaction with MSL2 and X-specific localization. This agrees with previous models that MSL1 and MSL2 directly interact through other regions.

This work lays the foundation for future investigations into the overall structure of the dosage compensation machinery, which allows this unique complex to specifically target the X-chromosome through still unclear mechanisms.

Strengths:

The data provided by the authors is of high quality and supports the authors' conclusions, which are nicely contextualized in the text with previous models. The novelty of this study is specifically pinpointing the amino acid regions of MSL1 that interact with roX. The authors point out that, surprisingly, the N-terminal region of MSL1 is not particularly well conserved, indicating that the interactions outlined in this study might be Drosophila/Diptera-specific.

The major strength of this study is that the authors find agreement between multiple dimensions of experimentation: the regions of MSL1 that are required for roX2 interaction (immunoprecipitation experiments) are also the regions that are critical for MSL1 localization to polytene chromosomes in an artificial female in vivo system, which are also critical for male-specific survival. The authors later suggest that it is the roX2 interaction that is responsible for the latter observations, although there is no direct evidence for this suggestion.

Weaknesses:

A minor weakness of the study is that it largely supports, and incrementally expands, the existing model in the field: that roX RNAs mediate the assembly of the complex on chromatin. I hesitate to call this a weakness, as supporting an existing model is still strong scientifically. However, the current study does not dramatically push the model forward.

Reviewer #2 (Public Review):

Summary:

A deletion analysis of the MSL1 gene to assess how different parts of the protein product interact with the MSL2 protein and roX RNA to affect the association of the MSL complex with the male X chromosome of Drosophila was performed.

Strengths:

The deletion analysis of the MSL1 protein and the tests of interaction with MSL2 are adequate.

Weaknesses:

This reviewer does not adhere to the basic premise of the authors that the MSL complex is the primary mediator of dosage compensation of the X chromosome of Drosophila. Several lines of evidence from various laboratories indicate that it is involved in sequestering the MOF histone acetyltransferase to the X chromosome but there is a constraint on its action there. When the MSL complex is disrupted, there is no overall loss of compensation but there is an increase in autosomal expression. Sun et al (2013, PNAS 110: E808-817) showed that ectopic expression of MSL2 does not increase expression of the X and indeed inhibits the effect of acetylation of H4Lys16 on gene expression. Aleman et al (2021, Cell Reports 35: 109236) showed that dosage compensation of the X chromosome can be robust in the absence of the MSL complex. Together, these results indicate that the MSL complex is not the primary mediator of X chromosome dosage compensation. The authors use sex-specific lethality as a measure of disruption of dosage compensation, but other modulations of gene expression are the likely cause of these viability effects.

A detailed explanation was provided by Birchler and Veitia (2021, One Hundred Years of Gene Balance: How stoichiometric issues affect gene expression, genome evolution, and quantitative traits. Cytogenetics and Genome Research 161: 529-550). The relevant portions of that article that pertain to Drosophila are quoted below. The cited references can be found in that publication.

"In Drosophila, the sex chromosomes consist of an X and a Y. The Y in this species contains only a few genes required for male fertility (Zhang et al., 2020). The X consists of approximately 20% of the genome. Thus, females have two X chromosomes and males have one. Muller (1932) found that the expression of genes between the two sexes was similar but when individual genes on the X were varied in dosage they exhibited a proportional dosage effect. Each copy in a male was expressed at about twice the level as each copy in a female. Females with three X chromosomes are highly inviable but when they do survive to the adult stage, Stern (1960) found that they too exhibited dosage compensation in that the expression in the triple X genotype was similar to normal females and males. Studies in triploid flies found that dosage compensation also occurred among X; AAA, XX; AAA, and XXX; AAA genotypes via upregulation of the Xs, where X indicates the dosage of the X and A indicates the triploid nature of the autosomes (see Birchler, 2016 for further discussion). Diploid and triploid females have a similar per-gene expression but the other five genotypes each must modulate gene expression by different amounts equivalent to an inverse relationship between the X versus autosomal dosage to achieve a balanced expression between the X and the A (Birchler, 1996).

Some years ago, mutations were sought in Drosophila that were lethal to males but viable in females. A number of such mutations were found and termed Male Specific Lethal (MSL) loci (Belote and Lucchesi, 1980). Once the products of these genes were identified, they were found to be at high concentrations on the male X chromosome (Kuroda et al., 1991). One of these genes encodes a histone acetyltransferase that acetylates Lysine16 of Histone H4 (Bone et al., 1994; Hilfiker et al., 1997). The recognition of the MSL complex and its association with the male X was an important set of contributions to an understanding of sex chromosome evolution in Drosophila (Kuroda et al., 2016). Thus, the hypothesis arose that the MSL complex accumulated this chromatin modifier on the male X to activate the expression about two-fold to bring about dosage compensation. Other data that contributed to this hypothesis were that when autoradiography of nascent transcription on salivary gland polytene chromosomes was examined in the MSL maleless mutation, the ratio of the number of grains over the X versus an autosomal region was reduced compared to the normal ratio (Belote and Lucchesi, 1980).

It has been pointed out (Hiebert and Birchler, 1994; Bhadra et al., 1999; Pal Bhadra et al., 2005; Sun et al., 2013a; Birchler, 2016), however, that the grain counts over the X and the autosomes when considered in absolute terms rather than as a ratio show that the X more or less retained dosage compensation and the autosomal numbers are about doubled, i.e. exhibit an inverse dosage effect. The same situation occurs with the msl3 mutation (Okuno et al., 1984), another MSL gene, in that the autoradiographic grain numbers as an absolute measure show retention of X dosage compensation and an autosomal increase. The data treatment to produce an X to A ratio seemed reasonable in the context of the time when all regulation in eukaryotes was considered positive. However, when studies were conducted in such a manner as to assay the absolute effect on gene expression in the maleless mutation, in adults (Hiebert and Birchler, 1994), larvae (Hiebert and Birchler, 1994; Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005), and embryos (Pal Bhadra et al., 2005), the trend was for retention of dosage compensation of X linked genes and an increase in expression of autosomal genes.

In global studies, if the X to autosomal expression does not change between mutant and normal, one can conclude that dosage compensation is operating. However, a lower X to A ratio could be a loss of compensation or an increased transcriptome size from the increase of the autosomes, as suggested by the absolute data of Belote and Lucchesi (1980) and Okuno et al (1984) and was visualized directly in embryos (Pal Bhadra et al., 2005). The transcriptome size in aneuploids can change, which cannot be detected in RNA-seq analyses alone (Yang et al., 2021), so it is an important consideration for studies of dosage compensation. It was recently acknowledged that in MSL2 knockdowns the relative X expression is decreased and a moderate autosomal increase is found (Valsecchi et al., 2021b). A similar trend is evident in the microarray data on MSL2 knockdown in SL2 tissue culture cells (Hamada et al., 2005) and in the roX RNA (noncoding RNAs essential for MSL localization on the male X) mutants (Deng and Meller, 2006). This trend is in fact consistent with the absolute data that suggest an increase in the transcriptome size (Figure 7). A global change in transcriptome size can cause a generalized dosage compensation of a single chromosome to appear as a proportional dosage effect (loss of compensation) to some degree (Figure 7).

Examination of expression in triple X metafemales, where there is no MSL complex, found that X-linked genes generally show dosage compensation but there is a generalized inverse effect on the autosomes, which could account for the detrimental effects of metafemales (Birchler et al., 1989; Sun et al., 2013b). An examination in metafemales of alleles of the white eye color gene that do or do not exhibit dosage compensation in males, showed the same response, namely, increased expression if there was no dosage compensation in males and no difference from normal females for the male dosage-compensated alleles (Birchler, 1992). This experiment demonstrated a relationship between the mechanism of dosage compensation in males and metafemales and implicated the inverse dosage effect in both. An involvement of the inverse effect in Drosophila dosage compensation provides an explanation for how the five levels of gene expression can be explained (Birchler, 1996), whereas an all-or-none presence of a complex on the X does not. The stoichiometric relationship of regulatory gene products provides a means to read the relative dosage at multiple doses to produce the appropriate inverse level.

What then is the function of the MSL complex? It was discovered that the MSL complex will actually constrain the effect of H4 lysine16 acetylation to prevent it from causing overexpression of genes (Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005; Sun and Birchler 2009; Sun et al., 2013a). Indeed, in the chromatin remodeling Imitation Switch (ISWI) mutants, the male X chromosome was specifically overexpressed suggesting that its normal function is needed for the constraint to occur (Pal Bhadra et al., 2005). Independently, the Mtor nuclear pore component shows a similar specific male X upregulation when Mtor is knocked down and this effect was shown to operate on the transcriptional level (Aleman et al., 2021). Interestingly, the increased expression of the X in the Mtor knockdown is accompanied by an inverse modulation of a substantial subset of autosomal genes, illustrating why the constraining process evolved to counteract male X overexpression. The constraining effect might involve a number of gene products (Birchler, 2016) and is an interesting direction for further study.

Furthermore, when the H4Lys16 acetylase was individually targeted to reporter genes, there was an increase in expression (Sun et al., 2013a). However, when other members of the MSL complex were present in normal males or ectopically expressed, this increase did not occur (Sun et al., 2013a). It thus appears that the function of the MSL complex is to sequester the acetylase from the autosomes and constrain it on the X (Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005; Sun and Birchler, 2009; Sun et al., 2013a). Indeed, in the Mtor knockdowns, the X-linked genes with the greatest upregulation were those with the greatest association with the acetylase and the H4K16ac histone mark (Aleman et al 2021), supporting the idea of a constraining activity that becomes released in the Mtor knockdown. When the MSL complex is disrupted, there is an inverse effect on the autosomes that occurs but in normal circumstances the sequestration mutes this effect. The MSL complex disruption releases the acetylase to be uniformly distributed across all chromosomes as determined cytologically (Bhadra et al., 1999) or via ChIPseq for H4Lys16ac (Valsecchi et al., 2021a). Indeed, the quantity of the H4Lys16ac mark only has a proportional effect on gene expression when the constraining activity is disrupted (Aleman et al., 2021) or when the MSL complex is not present (Sun et al., 2013a). Thus, in normal flies, there is a more or less equalized expression of the X and autosomes despite the monosomy for 20% of the genome.

The component of the complex that is expressed in males and thought to organize the complex to the male X, MSL2, was recently found to also be associated with autosomal dosage-sensitive regulatory genes (Valsecchi et al., 2018). MSL2 was found to modulate these autosomal dosage-sensitive genes in various directions, which illustrates that MSL2 has a role in dosage balance that goes beyond the X chromosome. This finding is consistent with the evolutionary scenario that the initial attraction of the complex to the X chromosome was to upregulate dosage-sensitive genes in hemizygous regions as the progenitor Y became deleted for them, with the constraining activity evolving to prevent an overexpression as the amount of acetylase on the male X increased with time (Birchler, 2016).

The MSL hypothesis takes an X-centric view that does not accommodate what is now known about dosage effects across the whole genome. The idea that dissolution of the MSL complex would cause a reduction in expression of the male X-linked genes without any consequences for the autosomes is not consistent with current knowledge of gene regulatory networks and their dosage sensitivity. Indeed, the finding of dosage compensation in large autosomal aneuploids that operates on the transcriptional level (Devlin et al., 1982; 1984; Birchler et al., 1990; Sun et al., 2013c), as well as a predominant inverse effect by the same (Devlin, et al., 1988; Birchler et al., 1990), argues that one must consider the inverse effect for an understanding of the evolution of dosage compensation in Drosophila (and other species). Further discussion of models of Drosophila compensation has been published (Birchler, 2016).

What is likely to be the most critical issue with sex chromosome evolution is the consequences for dosage-sensitive regulatory genes. This fact is nicely illustrated by the retention of these types of genes in different independent vertebrate sex chromosome evolutions (Bellott and Page, 2021). In Drosophila, by contrast, dosage compensation is more of a blanket effect on most but not all X-linked genes despite the fact that many genes on the X are unlikely to have dosage detrimental effects, although dosage-sensitive genes might have played a role as noted above. The particularly large size of the X in Drosophila compared to the whole genome is potentially a contributing factor because such a large genomic imbalance is likely to modulate most genes across the genome. Also, there is no evidence of a WGD in Drosophila as there is in other species for which the inverse effect has been documented (maize, Arabidopsis, yeast, mice, human). These other species have various numbers of retained duplicate dosage-sensitive regulatory genes from WGDs. Thus, the relative change of regulatory genes in aneuploids in these species will not be as great compared to some of their interactors in the remainder of the genome, which could result in lesser magnitudes of some trans-acting effects, similar to how aneuploids in ascending ploidies have fewer effects as described above. The absence of duplicate regulatory genes in Drosophila would predict a stronger inverse effect in general and that could have been capitalized upon to produce dosage compensation of most genes on the X chromosome despite many of them not being dosage critical. While sex chromosome evolution must accommodate dosage-sensitive genes for proper development and viability, it could also be capitalized upon to evolve sexual dimorphisms in expression (Sun et al., 2013c)."

Reviewer #1 (Public Review):

Summary:

In this study the authors demonstrated that ablation of astrocytes in lumbar spinal cord not only reduced neuropathic pain but also caused microglia activation. Furthermore, RNA sequencing and bioinformatics revealed an activation of STING/type I IFNs signal pathway in spinal cord microglia after astrocyte ablation.

Strengths:

The findings are novel and interesting and provide new insights into astrocyte-microglia interaction in neuropathic pain. This study may also offer a new therapeutic strategy for the treatment of debilitating neuropathic pain in patients with SCI.

Weaknesses:

More details are needed to justify the sample size, statistics, and sex of animals.

Reviewer #2 (Public Review):

Summary:

In the manuscript, Zhao et al. have carried out a thorough examination of the effects of targeted ablation of resident astrocytes on behavior, cellular responses, and gene expression after spinal cord injury. Employing transgenic mice models alongside pharmacogenetic techniques, the authors have successfully achieved the selective removal of these resident astrocytes. This intervention led to a notable reduction in neuropathic pain and induced a shift in microglial cell reactivation states within the spinal cord, significantly altering transcriptome profiles predominantly associated with interferon (IFN) signaling pathways.

Strengths:

The findings presented add considerable value to the current understanding of the role of astrocyte elimination in neuropathic pain, offering convincing evidence that supports existing hypotheses and valuable insights into the interactions between astrocytes and microglial cells, likely through IFN-mediated mechanisms. This contribution is highly relevant and suggests that further exploration in this direction could yield meaningful results.

Weaknesses:

The methodology and evidence underpinning the study are solid, yet some areas would benefit from further clarification, particularly concerning methodological details and the choice of statistical analyses. Additionally, the manuscript's organization and clarity could be improved, as certain figures and schematics appear inconsistent or misleading.

Reviewer #1 (Public Review):

Summary:

This work revealed an important finding that the blood-brain barrier (BBB) functionality changes with age and is more pronounced in males. The authors applied a non-invasive, contrast-agent-free approach of MRI called diffusion-prepared arterial spin labeling (DP-pCASL) to a large cohort of healthy human volunteers. DP-pCASL works by tracking the movement of magnetically labeled water (spins) in blood as it perfuses brain tissue. It probes the molecular diffusion of water, which is sensitive to microstructural barriers, and characterizes the signal coming from fast-moving spins as blood and slow-moving spins as tissue, using different diffusion gradients (b-values). This differentiation is then used to assess the water exchange rates (kw) across the BBB, which acts as a marker for BBB functionality. The main finding of the authors is that kw decreases with age, and in some brain regions, kw decreases faster in males. The neuroprotective role of the female sex hormone, estrogen, on BBB function is discussed as one of the explanations for this finding, supported by literature. The study also shows that BBB function remains stable until the early 60s and remarkably decreases thereafter.

Strengths:

The two main strengths of the study are the MRI method used and the amount of data. The authors employed a contrast-agent-free MRI method called ASL, which offers the opportunity to repeat such experiments multiple times without any health risk - a significant advantage of ASL. Since ASL is an emerging field that requires further exploration and testing, a study evaluating blood-brain barrier functionality is of great importance. The authors utilized a large dataset of healthy humans, where volunteer data from various studies were combined to create a substantial pool. This strategy is effective for statistically evaluating differences in age and gender.

Weaknesses:

Gender-related differences are only present in some brain regions, not in the whole brain or gray matter - which is usually the assumption unless stated otherwise. From the title, this was not clear. Including simulations could increase readers' understanding related to model fitting and the interdependence of parameters, if present. The discussion follows a clear line of argument supported by literature; however, focusing solely on AQP4 channels and missing a critical consideration of other known/proven changes in transport mechanisms through the BBB and their effects substantially weakens the discussion.

Reviewer #2 (Public Review):

Summary:

This study used a novel diffusion-weighted pseudo-continuous arterial spin labelling (pCASL) technique to simultaneously explore age- and sex-related differences in brain tissue perfusion (i.e., cerebral blood flow (CBF) & arterial transit time (ATT) - a measure of CBF delivery to brain tissue) and blood-brain barrier (BBB) function, measured as the water exchange (kw) across the BBB. While age- and sex-related effects on CBF are well known, this study provides new insights to support the growing evidence of these important factors in cerebrovascular health, particularly in BBB function. Across the brain, the decline in CBF and BBB function (kw) and elevation in ATT were reported in older adults, after the age of 60, and more so in males compared to females. This was also evident in key cognitive regions including the insular, prefrontal, and medial temporal regions, stressing the consideration of age and sex in these brain physiological assessments.

Strengths:

Simultaneous assessment of CBF with BBB along with transit time and at the voxel-level helped elucidate the brain's vulnerability to age and sex-effects. It is apparent that the investigators carefully designed this study to assess regional associations of age and sex with attention to exploring potential non-linear effects.

Weaknesses:

It appears that no brain region showed concurrent CBF and BBB dysfunction (kw), based on the results reported in the main manuscript and supplemental information. Was an association analysis between CBF and kw performed? There is a potential effect of the level of formal education on CBF (PMID: 12633147; 15534055), which could have been considered and accounted for as well, especially for a cohort with stated diversity (age, race, sex).

Reviewer #1 (Public Review):

Updated summary:

Glenn et al. present solid evidence that both lab and clinical Salmonella enterica serovars rapidly migrate towards human serum using an exciting approach that combines microfluidics, structural biology and genotypic analysis. The authors succeed in bringing to light a novel context for the role of serine as a bacterial chemoattractant as well as documenting what is likely to be a key step in bloodstream entry for some of the main sepsis-associated pathogens during gastrointestinal bleeding. They illustrate the generality of their findings through phylogenetic analysis, testing additional species within the Enterobacteriaceae family and showing attraction towards swine and equine serum. Their interdisciplinary approach here greatly increases the scope of their findings.<br /> I would also like to note that, whilst I enjoyed the interdisciplinary scope of this study, I am personally not well placed to review the protein structural aspects of this work.

Additional strengths of the revised manuscript:

All weaknesses raised in my review of the original manuscript have been satisfactorily addressed in the revised manuscript. It is interesting to note that the accumulation pattern of the bacteria 50-75 um from the source of serum could, as the author's now note, be due to the avoidance of bactericidal serum elements. Alternative explanations, however, could include chemoreceptor saturation (i.e. close to the serum source, high ligand concentrations could saturate chemoreceptors preventing further chemotaxis) or Weber's Law considerations (cell's ability to detect a given change in chemical concentrations diminishes with increasing background concentrations - thus, as cells get closer to the serum source, their ability to chemotax decreases).

The authors have also added new experimental data and analyses and these constitute major new strengths of the revised manuscript:<br /> - The authors show that the competitive advantage of WT cells relative to a tsr mutant is removed when serum is treated with serine-racemase and this provides strong evidence that chemotaxis towards serine is responsible for the reduced attraction of the tsr mutant towards serum (i.e. rather than any possible pleiotropic effects).<br /> - New experimental data showing Salmonella enterica is also attracted to swine and equine serum (including an ex vivo swine model) is a useful addition that hints at the potential generality of the response reported here.<br /> - The authors now include additional data to back up the intriguing lack of a movement response towards norepinephrine and DHMA reported here.

Additional weaknesses of the revised manuscript:

- The addition of an ex vivo swine model is an exciting new inclusion in the updated manuscript. However, information regarding biological and technical replication here is currently unclear or missing.

Reviewer #3 (Public Review):

Summary:

This manuscript characterizes a chemoattractant response to human serum by pathogenic bacteria, focusing on pathogenic stratins of Salmonella enterica Se. The researchers conduct the chemotaxis assays using a micropipette injection method that allows real-time tracking of bacterial population densities. They found that clinical isolates of several Se strains present a chemoattractant response to human serum. The specific chemoattractant within the serum is identified as L-serine, a highly characterized and ubiquitous chemoattractant, that is sensed by the Tsr receptor. They further show that chemoattraction to serum is impaired with a mutant strain devoid of Tsr. X-ray crystallography is then used to determine the structure of L-serine in the Se Tsr ligand binding domain, which differs slightly from a previously determine structure of a homologous domain. They went on to identify other pathogens that have a Tsr domain through a bioinformatics approach and show that these identified species also present a chemoattractant response to serum.

Strengths and Weaknesses:

This study is well executed and the experiments are clearly presented. These novel chemotaxis assays provide advantages in terms of temporal resolution and ability to detect responses from small concentrations. That said, it is perhaps not surprising these bacteria respond to serum as it is known to contain high levels of known chemoattractants, serine certainly, but also aspartate. In fact, the bacteria are shown to respond to aspartate and the tsr mutant is still chemotactic. The authors do not adequately support their decision to focus exclusively on the Tsr receptor. Tsr is one of the chemoreceptors responsible for observed attraction to serum, but perhaps, not the receptor. Furthermore, the verification of chemotaxis to serum is a useful finding, but the work does not establish the physiological relevance of the behavior or associate it with any type of disease progression. I would expect that a majority of chemotactic bacteria would be attracted to it under some conditions. Hence the impact of this finding on the chemotaxis or medical fields is uncertain.

The authors also state that "Our inability to substantiate a structure-function relationship for NE/DHMA signaling indicates these neurotransmitters are not ligands of Tsr." Both norepinephrine (NE) and DHMA have been shown previously by other groups to be strong chemoattractants for E. coli (Ec), and that this behavior was mediated by Tsr (e.g. single residue changes in the Tsr binding pocket block the response). Given the 82% sequence identity between the Se and Ec Tsr, this finding is unexpected (and potentially quite interesting). To validate this contradictory result the authors should test E. coli chemotaxis to DHMA in their assay. It may be possible that Ec responds to NE and DHMA and Se doesn't. However, currently the data is not strong enough to rule out Tsr as a receptor to these ligands in all cases. At the very least the supporting data for Tsr being a receptor for NE/DHMA needs to be discussed.

The authors also determine a crystal structure of the SeTsr periplasmic ligand binding domain bound to L-Ser and note that the orientation of the ligand is different than that modeled in a previously determined structure of lower resolution. I agree that the SeTsr ligand binding mode in the new structure is well-defined and unambiguous, but I think it is too strong to imply that the pose of the ligand in the previous structure is wrong. The two conformations are in fact quite similar to one another and the resolution of the older structure, is, in my view, insufficient to distinguish them. It is possible that there are real differences between the two structures. The domains do have different sequences and, moreover, the crystal forms, and cryo-cooling conditions are different in each case. It's become increasingly apparent that temperature, as manifested in differential cooling conditions here, can affect ligand binding modes. It's also notable that full-length MCPs show negative cooperativity in binding ligands, which is typically lost in the isolated periplasmic domains. Hence ligand binding is sensitive to the environment of a given domain. In short, the current data is not convincing enough to say that a previous "misconception" is being corrected.

Reviewer #1 (Public Review):

Summary:

The presented study by Centore and colleagues investigates the inhibition of BAF chromatin remodeling complexes. The study is well-written, and includes comprehensive datasets, including compound screens, gene expression analysis, epigenetics, as well as animal studies. This is an important piece of work for the uveal melanoma research field, and sheds light on a new inhibitor class, as well as a mechanism that might be exploited to target this deadly cancer for which no good treatment options exist.

Strengths:

This is a comprehensive and well-written study.

Weaknesses:

There are minimal weaknesses.

Reviewer #2 (Public Review):

Summary:

The authors generate an optimized small molecule inhibitor of SMARCA2/4 and test it in a panel of cell lines. All uveal melanoma (UM) cell lines in the panel are growth-inhibited by the inhibitor making the focus of the paper. This inhibition is correlated with the loss of promoter occupancy of key melanocyte transcription factors e.g. SOX10. SOX10 overexpression and a point mutation in SMARCA4 can rescue growth inhibition exerted by the SMARCA2/4 inhibitor. Treatment of a UM xenograft model results in growth inhibition and regression which correlates with reduced expression of SOX10 but not discernible toxicity in the mice. Collectively the data suggest a novel treatment of uveal melanoma.

Strengths:

There are many strengths of the study including the strong challenge of the on-target effect, the assays used, and the mechanistic data. The results are compelling as are the effects of the inhibitor. The in vivo data is dose-dependent and doses are low enough to be meaningful and associated with evidence of target engagement.

Weaknesses:

The authors introduce the field stating that SMARCA4 inhibitors are more effective in SMARCA2 deficient cancers and the converse. Since the desirable outcome of cancer therapy would be synthetic lethality it is not clear why a dual inhibitor is desirable. Wouldn't this be associated with more side effects? It is not known how the inhibitor developed here impacts normal cells, in particular T cells which are essential for any durable response to cancer therapies in patients. Another weakness is that the UM cell lines used do not molecularly resemble metastatic UM. These UM most frequently have mutations in the BAP1 tumor suppressor gene. It is not clear if the described SMARCA2/4 inhibitor is efficacious in BAP1 mutant UM cell lines in vitro or BAP1 mutant patient-derived xenografts in vivo.

Reviewer #3 (Public Review):