Reviewer #2 (Public Review):

Summary:<br /> The authors investigate the phosphotransfer capacity of Ser/Thr kinase IκB kinase (IKK), a mediator of cellular inflammation signaling. Canonically, IKK activity is promoted by activation loop phosphorylation at Ser177/Ser181. Active IKK can then unleash NF-κB signaling by phosphorylating repressor IκBα at residues Ser32/Ser26. Noting the reports of other IKK phosphorylation sites, the authors explore the extent of autophosphorylation.

Semi-phosphorylated IKK purified from Sf9 cells, exhibits the capacity for further autophosphorylation. Anti-phosphotyrosine immunoblotting indicated unexpected tyrosine phosphorylation. Contaminating kinase activity was tested by generating a kinase-dead K44M variant, supporting the notion that the unexpected phosphorylation was IKK-dependent. In addition, the observed phosphotyrosine signal required phosphorylated IKK activation loop serines.

Two candidate IKK tyrosines were examined as the source of the phosphotyrosine immunoblotting signal. Activation loop residues Tyr169 and Tyr188 were each rendered non-phosphorylatable by mutation to Phe. The Tyr variants decreased both autophosphorylation and phosphotransfer to IκBα. Likewise, Y169F and Y188F IKK2 variants immunoprecipitated from TNFa-stimulated cells also exhibited reduced activity in vitro.

The authors further focus on Tyr169 phosphorylation, proposing a role as a phospho-sink capable of phosphotransfer to IκBα substrate. This model is reminiscent of the bacterial two-component signaling phosphotransfer from phosphohistidine to aspartate. Efforts are made to phosphorylate IKK2 and remove ATP to assess the capacity for phosphotransfer. Phosphorylation of IκBα is observed after ATP removal, although there are ambiguous requirements for ADP.

Strengths:

Ultimately, the authors draw together the lines of evidence for IKK2 phosphotyrosine and ATP-independent phosphotransfer to develop a novel model for IKK2-mediated phosphorylation of IκBα. The model suggests that IKK activation loop Ser phosphorylation primes the kinase for tyrosine autophosphorylation. With the assumption that IKK retains the bound ADP, the phosphotyrosine is conformationally available to relay the phosphate to IκBα substrate. The authors are clearly aware of the high burden of evidence required for this unusual proposed mechanism. Indeed, many possible artifacts (e.g., contaminating kinases or ATP) are anticipated and control experiments are included to address many of these concerns. Taken together, the observations are thought-provoking, and I look forward to seeing this model tested in a cellular system.

Weaknesses:

It seems that the analysis hinges on the fidelity of pan-specific phosphotyrosine antibodies.

The analysis often returns to the notion that tyrosine phosphorylation(s) (and critical active site Lys44) dictate IKK2 substrate specificity, but evidence for this seems diffuse and indirect. This is an especially difficult claim to make with in vitro assays, omitting the context of other cellular specificity determinants (e.g., localization, scaffolding, phosphatases).

Multiple phosphorylated tyrosines in IKK2 were apparently identified by mass spectrometric analyses, but the data and methods are not described. It is common to find non-physiological post-translational modifications in over-expressed proteins from recombinant sources. Are these IKK2 phosphotyrosines evident by MS in IKK2 immunoprecipitated from TNFa-stimulated cells? Identifying IKK2 phosphotyrosine sites from cells would be especially helpful in supporting the proposed model.

Reviewer #3 (Public Review):

Summary:

The authors investigate the kinase activity of IKK2, a crucial regulator of inflammatory cell signaling. They describe a novel tyrosine kinase activity of this well-studied enzyme and a highly unusual phosphotransfer from phosphorylated IKK2 onto substrate proteins in the absence of ATP as a substrate.

Strengths:

The authors provide an extensive biochemical characterization of the processes with recombinant protein, western blot, autoradiography, and protein engineering.

Weaknesses:

The identity and purity of the used proteins is not clear. Since the findings are so unexpected and potentially of wide-reaching interest - this is a weakness. Similar specific detection of phospho-Ser/Thr vs phospho-Tyr relies largely on antibodies which can have varying degrees of specificity.

Reviewer #1 (Public Review):

Summary:

The individual roles of both cosolvents and intrinsically disordered proteins (IDPs) in desiccation have been well established, but few studies have tried to elucidate how these two factors may contribute synergistically. The authors quantify the synergy for the model and true IDPs involved with desiccation and find that only the true IDPs have strong desiccation tolerance and synergy with cosolvents. Using these as model systems, they quantify the local (secondary structure vis-a-vi CD spectroscopy) and global dimensions (vis-a-vi the Rg of SAXS experiments) and find no obvious changes with the co-solvents. Instead, they focus on the gelation of one of the IDPs and, using theory and experiments, suggest that the co-solvents may enable desiccation tolerance, an interesting hypothesis to guide future in vivo desiccation studies. A few minor points that remain unclear to this reviewer are noted.

Strengths:

This paper is quite extensive and has significant strengths worth highlighting. Notably, the number and type of methods employed to study IDPs are quite unusual, employing CD spectroscopy, SAXS measurements, and DSC. The use of the TFE is an exciting integration of the physical chemistry of cosolvents into the desiccation field is a nice approach and a clever way of addressing the gap of the lack of conformational changes depending on the cosolvents. Furthermore, I think this is a major point and strength of the paper; the underlying synergy of cosolvents and IDPs may lie in the thermodynamics of the dehydration process.

Figure S6A is very useful. I encourage readers who are confused about the DSC analysis, interpretation, and calculation to refer to it.

Weaknesses:

Overall, the paper is sound and employs strong experimental design and analysis. However, I wish to point out a few minor weaknesses.

Perhaps the largest, in terms of reader comprehension, focuses on the transition between the model peptides and real IDPs in Figures 1 and 2. Notably, little is discussed with respect to the structure of the IDPs and what is known. Notably, I was confused to find out when looking at Table 1 that many of the IDPs are predicted to be largely unordered, which seemed to contrast with some of the CD spectroscopy data. I wonder if the disorder plots are misleading for readers. Can the authors comment more on this confusion? What are these IDPs structurally?

Related to the above thoughts, the alpha fold structures for the LEA proteins are predicted (unconfidently) as being alpha-helical in contrast to the CD data. Does this complicate the TFE studies and eliminate the correlation for the LEA proteins? Additionally, the notation that the LEA and BSA proteins do not correlate is unclear to this reviewer, aren't many of the correlations significant, having both a large R^2 and significant p-value?

The calculation of synergy seems too simplistic or even problematic to me. While I am not familiar with the standards in the desiccation field, I think the approach as presented may be problematic due to the potential for higher initial values of protection to have lower synergies (two 50%s for example, could not yield higher than 100%). Instead, I would think one would need to really think of it as an apparent equilibrium constant between functional and non-functional LDH (Kapp = [Func]/[Not Func] and frac = Kapp/(1+Kapp) or Kapp = frac/(1-frac) ) Then after getting the apparent equilibrium constants for the IDP and cosolvent (KappIDP and KappCS), the expected additive effect would be frac = (KappIDP+KappCS)/(1+KappIDP+KappCS). Consequently, the extent of synergy could be instead calculated as KappBOTH-KappIDP-KappCS. Maybe this reviewer is misunderstanding. It is recommended that the authors clarify why the synergy calculation in the manuscript is reasonable.

Related to the above, the authors should discuss the utility of using molar concentration instead of volume fraction or mass concentration. Notably, when trehalose is used in concentration, the volume fraction of trehalose is much smaller compared to the IDPs used in Figure 2 or some in Figure 1. Would switching to a different weighted unit impact the results of the study, or is it robust to such (potentially) arbitrary units?

Reviewer #2 (Public Review):

Summary:

The paper aims to investigate the synergies between desiccation chaperones and small molecule cosolutes, and describe its mechanistic basis. The paper reports that IDP chaperones have stronger synergies with the cosolutes they coexist with, and in one case suggests that this is related to oligomerization propensity of the IDP.

Strengths:

The study uses a lot of orthogonal methods and the experiments are technically well done. They are addressing a new question that has not really been addressed previously.

Weaknesses:

The conclusions are based on a few examples and only partial correlations. While the data support mechanistic conclusions about the individual proteins studied, it is not clear that the conclusions can be generalized to the extent proposed by the authors due to small effect sizes, small numbers of proteins, and only partial correlations.

The authors pose relevant questions and try to answer them through a systematic series of experiments that are all technically well-conducted. The data points are generally interpreted appropriately in isolation, however, I am a little concerned about a tendency to over-generalize their findings. Many of the experiments give negative or non-conclusive results (not a problem in itself), which means that the overall storyline is often based on single examples. For example, the central conclusion that IDPs interact synergistically with their endogenous co-solute (Figure 2E) is largely driven by one outlier from Arabidopsis. The rest are relatively close to the diagonal, and one could equally well suggest that the cosolutes affect the IDPs equally (which is also the conclusion in 1F). Similarly, the mechanistic explanations tend to be based on single examples. This is somewhat unavoidable as biophysical studies cannot be done on thousands of proteins, but the text should be toned down to reflect the strength of the conclusions.

The central hypothesis revolves around the interplay between cosolutes and IDP chaperones comparing chaperones from species with different complements of cosolutes. In Table 1, it is mentioned that Arabidopsis uses both trehalose and sucrose as a cosolute, yet experiments are only done with either of these cosolutes and Arabidopsis is counted in the sucrose column. While it makes sense to compare them separately from a biophysical point of view, the ability to test the co-evolution of these systems is somewhat diminished by this. At least it should be discussed clearly.

It would be helpful if the authors could spell out the theoretical basis of how they quantify synergy. I understand what they are doing - and maybe there are no better ways to do it - but it seems like an approach with limitations. The authors identify one in that the calculation only works far from 100%, but to me, it seems there would be an equally strict requirement to be significantly above 0%. This would suggest that it is used wrongly in Figure 6H, where there is no effect of betaine (at least as far as the color scheme allows one to distinguish the different bars). In this case, the authors cannot really conclude synergy or not, it could be a straight non-synergistic inhibition by betaine.

Reviewer #2 (Public Review):

Retroviral integration in general, and HIV integration in particular, takes place in dsDNA, not in R-loops. Although HIV integration can occur in vitro on naked dsDNA, there is good evidence that, in an infected cell, integration occurs on DNA that is associated with nucleosomes. This review will be presented in two parts. First, a summary will be provided giving some of the reasons to be confident that integration occurs on dsDNA on nucleosomes. The second part will point out some of the obvious problems with the experimental data that are presented in the manuscript.

(1) 2017 Dos Passos Science paper describes the structure of the HIV intasome. The structure makes it clear that the target for integration is dsDNA, not an R-loop, and there are very good reasons to think that structure is physiologically relevant. For example, there is data from the Cherepanov, Engelman, and Lyumkis labs to show that the HIV intasome is quite similar in its overall structure and organization to the structures of the intasomes of other retroviruses. Importantly, these structures explain the way integration creates a small duplication of the host sequences at the integration site. How do the authors propose that an R-loop can replace the dsDNA that was seen in these intasome structures?

(2) As noted above, concerted (two-ended) integration can occur in vitro on a naked dsDNA substrate. However, there is compelling evidence that, in cells, integration preferentially occurs on nucleosomes. Nucleosomes are not found in R loops. In an infected cell, the viral RNA genome of HIV is converted into DNA within the capsid/core which transits the nuclear pore before reverse transcription has been completed. Integration requires the uncoating of the capsid/core, which is linked to the completion of viral DNA synthesis in the nucleus. Two host factors are known to strongly influence integration site selection, CPSF6 and LEDGF. CPSF6 is involved in helping the capsid/core transit the nuclear pore and associate with nuclear speckles. LEDGF is involved in helping the preintegration complex (PIC) find an integration site after it has been released from the capsid/core, most commonly in the bodies of highly expressed genes. In the absence of an interaction of CPSF6 with the core, integration occurs primarily in the lamin-associated domains (LADs). Genes in LADs are usually not expressed or are expressed at low levels. Depending on the cell type, integration in the absence of CPSF6 can be less efficient than normal integration, but that could well be due to a lack of LEDGF (which is associated with expressed genes) in the LADs. In the absence of an interaction of IN with LEDGF (and in cells with low levels of HRP2) integration is less efficient and the obvious preference for integration in highly expressed genes is reduced. Importantly, LEDGF is known to bind histone marks, and will therefore be preferentially associated with nucleosomes, not R-loops. LEDGF fusions, in which the chromatin binding portion of the protein is replaced, can be used to redirect where HIV integrates, and that technique has been used to map the locations of proteins on chromatin. Importantly, LEDGF fusions in which the chromatin binding component of LEDGF is replaced with a module that recognizes specific histone marks direct integration to those marks, confirming integration occurs efficiently on nucleosomes in cells. It is worth noting that it is possible to redirect integration to portions of the host genome that are poorly expressed, which, when taken with the data on integration into LADs (integration in the absence of a CPSF6 interaction) shows that there are circumstances in which there is reasonably efficient integration of HIV DNA in portions of the genome in which there are few if any R-loops.

(3) Given that HIV DNA is known to preferentially integrate into expressed genes and that R-loops must necessarily involve expressed RNA, it is not surprising that there is a correlation between HIV integration and regions of the genome to which R loops have been mapped. However, it is important to remember that correlation does not necessarily imply causation.

If we consider some of the problems in the experiments that are described in the manuscript:

(1) In an infected individual, cells are almost always infected by a single virion and the infecting virion is not accompanied by large numbers of damaged or defective virions. This is a key consideration: the claim that infection by HIV affects R-loop formation in cells was done with a VSVg vector in experiments in which there appears to have been about 6000 virions per cell. Although most of the virions prepared in vitro are defective in some way, that does not mean that a large fraction of the defective virions cannot fuse with cells. In normal in vivo infections, HIV has evolved in ways that avoid signaling infected the cell of its presence. To cite an example, carrying out reverse transcription in the capsid/core prevents the host cell from detecting (free) viral DNA in the cytoplasm. The fact that the large effect on R-loop formation which the authors report still occurs in infections done in the absence of reverse transcription strengthens the probability that the effects are due to the massive amounts of virions present, and perhaps to the presence of VSVg, which is quite toxic. To have physiological relevance, the infections would need to be carried out with virions that contain HIV even under circumstances in which there is at most one virion per cell.

(2) Using the Sso7d version of HIV IN in the in vitro binding assays raises some questions, but that is not the real question/problem. The real problem is that the important question is not what/how HIV IN protein binds to, but where/how an intasome binds. An intasome is formed from a combination of IN bound to the ends of viral DNA. In the absence of viral DNA ends, IN does not have the same structure/organization as it has in an intasome. Moreover, HIV IN (even Sso7d, which was modified to improve its behavior) is notoriously sticky and hard to work with. If viral DNA had been included in the experiment, intasomes would need to be prepared and purified for a proper binding experiment. To make matters worse, there are multiple forms of multimeric HIV IN and it is not clear how many HIV INs are present in the PICs that actually carry out integration in an infected cell.

(3) As an extension of comment 2, the proper association of an HIV intasome/PIC with the host genome requires LEDGF and the appropriate nucleic acid targets need to be chromatinized.

(4) Expressing any form of IN, by itself, in cells to look for what IN associates with is not a valid experiment. A major factor that helps to determine both where integration takes place and the sites chosen for integration is the transport of the viral DNA and IN into the nucleus in the capsid core. However, even if we ignore that important part of the problem, the IN that the authors expressed in HeLa cells won't be bound to the viral DNA ends (see comment 2), even if the fusion protein would be able to form an intasome. As such, the IN that is expressed free in cells will not form a proper intasome/PIC and cannot be expected to bind where/how an intasome/PIC would bind.

(5) As in comment 1, for the PLA experiments presented in Figure 5 to work, the number of virions used per cell (which differs from the MOI measured by the number of cells that express a viral marker) must have a high, which is likely to have affected the cells and the results of the experiment. However, there is the additional question of whether the IN-GFP fusion is functional. The fact that the functional intasome is a complex multimer suggests that this could be a problem. There is an additional problem, even if IN-GFP is fully functional. During a normal infection, the capsid core will have delivered copies of IN (and, in the experiments reported here, the IN-GFP fusion) into the nucleus that is not part of the intasome. These "free" copies of IN (here IN-GFP) are not likely to go to the same sites as an intasome, making this experiment problematic (comment 4).

(6) In the Introduction, the authors state that the site of integration affects the probability that the resulting provirus will be expressed. Although this idea is widely believed in the field, the actual data supporting it are, at best, weak. See, for example, the data from the Bushman lab showing that the distribution of integration sites is the same in cells in which the integrated proviruses are, and are not, expressed. However, given what the authors claim in the introduction, they should be more careful in interpreting enzyme expression levels (luciferase) as a measure of integration efficiency in experiments in which they claim proviruses are integrated in different places.

(7) Using restriction enzymes to create an integration site library introduces biases that derive from the uneven distribution of the recognition sites for the restriction enzymes.

Reviewer #3 (Public Review):

In this manuscript, Park and colleagues describe a series of experiments that investigate the role of R-loops in HIV-1 genome integration. The authors show that during HIV-1 infection, R-loops levels on the host genome accumulate. Using a synthetic R-loop prone gene construct, they show that HIV-1 integration sites target sites with high R-loop levels. They further show that integration sites on the endogenous host genome are correlated with sites prone to R-loops. Using biochemical approaches, as well as in vivo co-IP and proximity ligation experiments, the authors show that HIV-1 integrase physically interacts with R-loop structures.

My primary concern with the paper is with the interpretations the authors make about their genome-wide analyses. I think that including some additional analyses of the genome-wide data, as well as some textual changes can help make these interpretations more congruent with what the data demonstrate. Here are a few specific comments and questions:

(1) I think Figure 1 makes a good case for the conclusion that R-loops are more easily detected HIV-1 infected cells by multiple approaches (all using the S9.6 antibody). The authors show that their signals are RNase H sensitive, which is a critical control. For the DRIPc-Seq, I think including an analysis of biological replicates would greatly strengthen the manuscript. The authors state in the methods that the DRIPc pulldown experiments were done in biological replicates for each condition. Are the increases in DRIPc peaks similar across biological replicates? Are genomic locations of HIV-1-dependent peaks similar across biological replicates? Measuring and reporting the biological variation between replicate experiments is crucial for making conclusions about increases in R-loop peak frequency. This is partially alleviated by the locus-specific data in Figure S3A. However, a better understanding of how the genome-wide data varies across biological replicates will greatly enhance the quality of Figure 1.

(2) I think that the conclusion that R-loops "accumulate" in infected cells is acceptable, given the data presented. However, in line 134 the authors state that "HIV-1 infection induced host genomic R-loop formation". I suggest being very specific about the observation. Accumulation can happen by (a) inducing a higher frequency of the occurrence of individual R-loops and/or (b) stabilizing existing R-loops. I'm not convinced the authors present enough evidence to claim one over the other. It is altogether possible that HIV-1 infection stabilizes R-loops such that they are more persistent (perhaps by interactions with integrase?), and therefore more easily detected. I think rephrasing the conclusions to include this possibility would alleviate my concerns.

(3) A technical problem with using the S9.6 antibody for the detection of R-loops via microscopy is that it cross-reacts with double-stranded RNA. This has been addressed by the work of Chedin and colleagues (as well as others). It is absolutely essential to treat these samples with an RNA:RNA hybrid-specific RNase, which the authors did not include, as far as their methods section states. Therefore, it is difficult to interpret all of the immunofluorescence experiments that depend on S9.6 binding.

(4) Given that there is no clear correlation between expression levels and R-loop peak detection, combined with the data that show increased detection of R-loop frequency in non-genic regions, I think it will be important to show that the R-loop forming regions are indeed transcribed above background levels. This will help alleviate possible concerns that there are technical errors in R-loop peak detection.

(5) In Figures 4C and D the hashed lines are not defined. It is also interesting that the integration sites do not line up with R-loop peaks. This does not necessarily directly refute the conclusions (especially given the scale of the genomic region displayed), but should be addressed in the manuscript. Additionally, it would greatly improve Figure 4 to have some idea about the biological variation across replicates of the data presented 4A.

(6) The authors do not adequately describe the Integrase mutant that they use in their biochemical experiments in Figure 5A. Could this impact the activity of the protein in such a way that interferes with the interpretation of the experiment? The mutant is not used in subsequent experiments for Figure 5 and so even though the data are consistent with each other (and the conclusion that Integrase interacts with R-loops) a more thorough explanation of why that mutant was used and how it impacts the biochemical activity of the protein will help the interpretation of the data presented in Figure 5.

Reviewer #1 (Public Review):

Summary:

The researchers demonstrated that when cytokine priming is combined with exposure to pathogens or pathogen-associated molecular patterns, human alveolar macrophages and monocyte-derived macrophages undergo metabolic adaptations, becoming more glycolytic while reducing oxidative phosphorylation. This metabolic plasticity is greater in monocyte-derived macrophages than in alveolar macrophages.

Strengths:

This study presents evidence of metabolic reprogramming in human macrophages, which significantly contributes to our existing understanding of this field primarily derived from murine models.

Weaknesses:

The study has limited conceptual novelty.

Reviewer #2 (Public Review):

Summary:

The authors aimed to functionally characterize primary human airway macrophages and monocyte-derived macrophages, correlating their glycolytic shift in metabolism. They conducted this macrophage characterization in response to type II interferon and IL-4 priming signals, followed by different stimuli of irradiated Mycobacterium tuberculosis and LPS.

Strengths:

(1) The study employs a thorough measurement of metabolic shift in metabolism by assessing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of differentially polarized primary human macrophages using the Seahorse XFe24 Analyzer.<br /> (2) The effect of differential metabolic shift on the expression of different surface markers for macrophage activation is evaluated through immunofluorescence flow cytometry and cytokine measurement via ELISA.<br /> (3) The authors have achieved their aim of preliminarily characterizing the glycolysis-dependent cytokine profile and activation marker expression of IFN-g and IL-4 primed primary human macrophages.<br /> (4) The results of the study support its conclusion of glycolysis-dependent phenotypical differences in cytokine secretion and activation marker expression of AMs and MDMs.

Weaknesses:

(1) The data are presented in duplicates for cross-analyses.<br /> (2) The data presented supports a distinct functional profile of airway macrophages (AMs) compared to monocyte (blood)-derived macrophages (MDMs) in response to the same priming signals. However, the study does not attempt to explore the underlying mechanism for this difference.<br /> (3) The study is descriptive in nature, and the results validate IFN-g-mediated glycolytic reprogramming in primary human macrophages without providing mechanistic insights.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors explore the contribution of metabolism to the response of two subpopulations of macrophages to bacterial pathogens commonly encountered in the human lung, as well as the influence of priming signals typically produced at a site of inflammation. The two subpopulations are resident airway macrophages (AM) isolated via bronchoalveolar lavage and monocyte-derived macrophages (MDM) isolated from human blood and differentiated using human serum. The two cell types were primed using IFNγ and Il-4, which are produced at sites of inflammation as part of initiation and resolution of inflammation respectively, followed by stimulation with either irradiated Mycobacterium tuberculosis (Mtb) or LPS to simulate interaction with a bacterial pathogen. The authors use human cells for this work, which makes use of widely reported and thoroughly described priming signals, as well as model antigens. This makes the observations on the functional response of these two subpopulations relevant to human health and disease. To examine the relationship between metabolism and functional response, the authors measure rates of oxidative phosphorylation and glycolysis under baseline conditions, primed using IFNγ or IL-4, and primed and stimulated with Mtb or LPS.

Strengths:

• The data indicate that both populations of macrophages increase metabolic rates when primed, but MDMs decrease their rates of oxidative phosphorylation after IL-4 priming and bacterial exposure while AMs do not.<br /> • It is demonstrated that glycolysis rates are directly linked to the expression of surface molecules involved in T-cell stimulation and while secretion of TNFα in AM is dependent on glycolysis, in MDM this is not the case. IL-1β is regulated by glycolysis only after IFN-γ priming in both MDM and AM populations. It is also demonstrated that Mtb and LPS stimulation produces responses that are not metabolically consistent across the two macrophage populations. The Mtb-induced response in MDMs differed from the LPS response, in that it relies on glycolysis, while this relationship is reversed in AMs. The difference in metabolic contributions to functional outcomes between these two macrophage populations is significant, despite acknowledgement of the reductive nature of the system by the authors.<br /> • The observations that AM and MDM rely on glycolysis for the production of cytokines during a response to bacterial pathogens in the lung, but that only MDM shift to Warburg Metabolism, though this shift is blocked following exposure to IL-4, are supported by the data and a significant contribution the study of the innate immune response.

Weaknesses:

• It is unclear whether changes in glycolysis and oxidative phosphorylation in primed cells are due to priming or subsequent treatments. ECAR and OCR analyses were therefore difficult to interpret.<br /> • The data may not support a claim that AM has greater "functional plasticity" without a direct comparison of antigen presentation. Moreover, MDM secrete more IL-1β than AM. The claim that AM "have increased ability to produce all cytokines assayed in response to Mtb stimulation" does not appear to be supported by the data.<br /> • The claim that AM are better for "innate training" via IFNγ may not be consistent with increased IL-1β and a later claim that MDM have increased production and are "associated with optimal training."<br /> • Statistical analyses may not appropriately support some of the conclusions.<br /> • AM populations would benefit from further definition-presumably this is a heterogenous, mixed population.<br /> • The term "functional plasticity" could also be more stringently defined for the purposes of this study.

Conclusion:

Overall, the authors succeed in their goals of investigating how inflammatory and anti-inflammatory cytokine priming contributes to the metabolic reprogramming of AM and MDM populations. Their conclusions regarding the relationship between cytokine secretion and inflammatory molecule expression in response to bacterial stimuli are supported by the data. The involvement of metabolism in innate immune cell function is relevant when devising treatment strategies that target the innate immune response during infection. The data presented in this paper further our understanding of that relationship and advance the field of innate immune cell biology.

Reviewer #1 (Public Review):

Summary:

The authors have developed a zebrafish model of glioblastoma and characterized this, with a particular focus on the role of recruited myeloid cells in the tumours. Microglia/macrophages in the tumours are proposed to have an inflammatory phenotype and are engaged in phagocytosis. Knockout of Irf7 and Irf8 genes enhanced tumour initiation. Depleting mature myeloid cell types with chlodronate also enhanced tumour initiation. It is proposed that in early stage tumours, microglia/macrophages have tumour suppressive activity.

Strengths:

The authors have generated a novel glioblastoma model in zebrafish. Two key strengths of the zebrafish model are that early stage tumours can be studied and in vivo visualization can be readily performed. The authors show video of microglia/macrophages adopting the ameboid phenotype in tumours (as is observed in human tumours) and engaging in phagocytosis. Video 1 was very impressive in my opinion and shows the model is a very useful tool to study microglia/macrophage:glioblastoma cell interactions. The irf7/irf8 knockdown and the chlodronate experiments are consistent with a role for mature myeloid cells in suppressing tumour initiation, suggesting that the model may also be very valuable in understanding immune surveillance in glioblastoma initiation.

Weaknesses:

EGFRvIII is mainly associated with the classical subtype, so the mesenchymal subtype might be unexpected here. This could be commented on. Some more histologic characterization of the tumours would be helpful. Are they invasive, do larger tumours show necrosis and microvascular proliferation? This would help with understanding the full potential of the new model. Current thinking in established human glioblastoma is that the M1/M2 designations for macrophages are not relevant, with microglia macrophage populations showing a mixture of pre- and anti-inflammatory features. Ideally there would be a much more detailed characterization of the intratumoral microglia/macrophage population here, as single markers can't be relied upon. Phagocytosis could have antitumour effects through removal of live cancer cells, or could be cancer promoting if apoptotic cancer cells are being rapidly cleared with concomitant activation of an immunosuppressive phenotype in the phagocytes (i.e. efferocytosis). It may be possible to distinguish between these two types of phagocytosis experimentally. Do the irf7/8 and chlodronate experiments distinguish between effects on microglia/macrophages and dendritic cells?

Update: The more detailed description of the tumour histology is very interesting and the authors have addressed my previous concerns nicely.

Reviewer #2 (Public Review):

Summary:

Glioblastoma is a common primary brain cancer, that is difficult to treat and has a low survival rate. The lack of genetically tractable and immunocompetent vertebrate animal model has prevented discovery of new therapeutic targets and limited efforts for screening of pharmaceutical agents for the treatment of the disease. Here Weiss et al., express oncogenic variants frequently observed in human glioblastoma within zebrafish lacking the tumor suppressor TP53 to generate a patient-relevant in vivo model. The authors demonstrate that loss of TP53 and overexpression of EGFR, PI3KCA, and mScarlet (p53EPS) in neural progenitors and radial glia leads to visible fluorescent brain lesions in live zebrafish. The authors performed RNA expression analysis that uncovered a molecular signature consistent with human mesenchymal glioblastoma and identified gene expression patterns associated with inflammation. Live imaging revealed high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. To define functional roles for regulators of inflammation on specific immune-related responses during tumorigenesis, transient CRISPR/Cas9 gene targeting was used to disrupt interferon regulator factor proteins and showed Inflammation-associated irf7 and irf8 are required to inhibit p53EPS tumor formation. Further, experiments to deplete the macrophages using clodronate liposomes suggest that macrophages contribute to the suppression of tumor engraftment following transplantation. The authors' conclusions are supported by the data and the experiments are thoroughly controlled throughout. Taken together, these results provide new insights into the regulation of glioblastoma initiation and growth by the surrounding microenvironment and provide a novel in vivo platform for the discovery of new molecular mechanisms and testing of therapeutics.

Strengths/Weaknesses:

The authors convincingly show that co-injection of activated human EGFRviii, PI3KCAH1047R, and mScarlet into TP53 null zebrafish promotes formation of fluorescent brain lesions and glioblastoma-like tumor formation. The authors include histological characterization of the tumors, as well as quantifications of p-ERK and p-AKT staining to highlight increased activation of the MAPK/AKT signaling pathways in their tumor model.

The authors use a transplantation assay to further test the tumorigenic potential of dissociated cells from glial-derived tumors in the context of specific manipulations of the tumour microenvironment.

The authors nicely show high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. Quantification of the emergence of macrophages over time in relation to tumor initiation and growth is provided and supports the observations of tumor suppressive activity of the phagocytes. The authors also attempt to delineate if other leukocyte populations are involved and observe tumor formation without significant infiltration of neutrophils.

The authors provide evidence for key genetic regulators of the local microenvironment, showing increased p53EPS tumor initiation following Ifr7 gene knock-down and loss of irf7 expression in the TME.

Reviewer #1 (Public Review):

Summary:

This study demonstrated a novel exciting link between conserved miRNA-target axis of miR-182-Lrp6 in liver metabolism which causatively contributes to type 2 diabetes and NAFLD in mice and, potentially, humans.

Strengths:

The direct interaction and inhibition of Lrp6 by miR-182 is convincingly shown. The effects of miR-182-5p on insulin sensitivity are also credible for the in vivo and in vitro gain-of-function experiments.

Weaknesses:

However, the DIO cohorts lack key assays for insulin sensitivity such as ITT or insulin-stimulated pAKT, as well as histological evidence to support their claims and strengthen the link between miR-182-5p and T2D or NAFLD. Besides, the lack of loss-of-function experiments limits its aptitude as potential therapeutic target.

Reviewer #2 (Public Review):

Summary:

In this study, Christin Krause et al mapped the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, identified miR-182-5p and its target genes LRP6 as potential drivers of dysregulated glucose tolerance and fatty acid metabolism in obese T2-diabetics.

Strengths:

This study contains some interesting findings and are valuable for the understanding of key regulatory role of miRNAs in the pathogenesis of T2D.

Weaknesses:

The authors didn't systemically investigate the function of miR-182 in T2DM or NAFLD.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Krause and colleagues identify miR-182 as diabetes-associated microRNA: miR-182 is increased in bariatric surgery patients with versus without T2D; miR-182 was the only microRNA associated with three metabolic traits; miR-182 levels were associated with increased body weight in mice under different dietary manipulations; overexpression in Hep-G2 led to a decrease in LRP6; and overexpression in HFD fed mice led to increased insulin and liver TG. The manuscript provides a potentially useful resource of microRNA expression in human livers, though the functional importance of miR-182 remains unclear.

Strengths:

The use of human tissues and good sample sizes is strong.

Weaknesses:

The study remains primarily correlative; the in vivo overexpression is non-physiological; and the mechanisms by which miR-182 exerts its effects are not rigorously tested.

Reviewer #1 (Public Review):

Summary:

Wang and co-workers characterise the fossil of Beretella spinosa from the early Cambrian, Yanjiahe Formation, South China. Combining morphological analyses with phylogenetic reconstructions, the authors conclude that B. spinosa is closely related to Saccorhytus, an enigmatic fossil recently ascribed to Ecdysozoa, or moulting animals, as an extinct "basal" lineage. Finding additional representatives of the clade Saccorhytida strengthens the idea that there existed a diversity of body plans previously underappreciated in Ecdysozoa, which may have implications for our understanding of the earliest steps in the evolution of this major animal group.

Strengths:

I'm not a paleobiologist; therefore, I cannot give an expert opinion on the descriptions of the fossils. However, the similarities with Saccorhytus seem evident, and the phylogenetic reconstructions are adequate. Evolutionary interpretations are generally justified, and the consolidation of Saccorhytida as the extinct sister lineage to extant Ecdysozoans will have significant implications for our understanding of this major animal clade.

Weaknesses:

While I generally agree with the author's interpretations, the idea of Saccorhytida as a divergent, simplified off-shot is slightly contradictory with a probably non-vermiform ecdysozoan ancestor. The author's analyses do not discard the possibility of a vermiform ecdysozoan ancestor (importantly, Supp Table 4 does not reconstruct that character), and outgroup comparison with Spiralia (and even Deuterostomia for Protostomia as a whole) indicates that a more or less anteroposteriorly elongated (i.e., vermiform) body is likely common and ancestral to all major bilaterian groups, including Ecdysozoa. Indeed, Figure 4 b depicts the potential ancestor as a "worm". The authors argue that the simplification of Saccorhytida from a vermiform ancestor is unlikely "because it would involve considerable anatomical transformations such as the loss of vermiform organization, introvert and pharynx in addition to that of the digestive system". However, their data support the introvert as a specialisation of Scalidophora (Fig. 4a and Supp Table 4), and a pharyngeal structure cannot be ruled out in Saccorhytida. Likewise, loss of an anus is not uncommon in Bilateria. Moreover, this can easily become a semantics discussion (to what extent can an animal be defined as "vermiform"? Where is the limit?). Therefore, I suggest to leave the evolutionary scenario more open. Supporting Saccorhytida as a true group at the early steps of Ecdysozoa evolution is important and demonstrates that animal body plans are more plastic than previously appreciated. However, with the current data, it is unlikely that Saccorhytida represents the ancestral state for Ecdysozoa (as the authors admit), and a vermiform nature is not ruled out (and even likely) in this animal group. Suggesting that the ancestral Ecdysozoan might have been small and meiobenthic is perhaps more interesting and supported by the current data (phylogeny and outgroup comparison with Spiralia).

Reviewer #2 (Public Review):

Summary:

This work provides important anatomical features of a new species from the Lower Cambrian, which helps advance our understanding of the evolutionary origins of animal body plans. The authors interpreted that the new species possessed a bilateral body covered with cuticular polygonal reticulation and a ventral mouth. Based on cladistic analyses using maximum likelihood, Bayesian, and parsimony, the new species was placed, along with Saccorhytus, in a sister-group ("Saccorhytida") of the Ecdysozoa. The phylogenetic position of Saccorhytida suggests a new scenario of the evolutionary origin of the crown ecdysozoan body plan.

Strengths:

Although the new species reported in this paper show strange morphologies, the interpretation of anatomical features was based on detailed observations of multiple fossil specimens, thereby convincing at the moment. Morphological data about fossil taxa in the Ediacaran and Early Cambrian are quite important for our understanding of the evolution of body plans (and origins of phyla) in paleontology and evolutionary developmental biology, and this paper represents a valuable contribution to such research fields.

Weaknesses:

The preservations of the specimens, in particular on the putative ventral side, are not good, and the interpretation of the anatomical features need to be tested with additional specimens in future. The monophyly of Cycloneuralia (Nematoida + Scalidophora) was not necessarily well-supported by cladistic analyses (Supplementary Figures 7-9), and the evolutionary scenario (Fig. 4) also need to be tested in future works. On the other hand, the revised version provides important contributions from currently available data, and the above-mentioned problems should be studied in a separate paper in future.

Joint Public Review:

Summary:

Bursicon is a key hormone regulating cuticle tanning in insects. While the molecular mechanisms of its function are rather well studied--especially in the model insect Drosophila melanogaster, its effects and functions in different tissues are less well understood. Here, the authors show that bursicon and its receptor play a role in regulating aspects of the seasonal polyphenism of Cacopsylla chinensis. They found that low temperature treatment activated the bursicon signaling pathway during the transition from summer form to winter form and affect cuticle pigment and chitin content, and cuticle thickness. In addition, the authors show that miR-6012 targets the bursicon receptor, CcBurs-R, thereby modulating the function of bursicon signaling pathway in the seasonal polyphenism of C. chinensis. This discovery expands our knowledge of the roles of neuropeptide bursicon action in arthropod biology.

However, the study falls short of its claim that it reveals the molecular mechanisms of a seasonal polyphenism. While cuticle tanning is an important part of the pear psyllid polyphenism, it is not the equivalent of it. First, there are other traits that distinguish between the two morphs, such as ovarian diapause (Oldfield, 1970), and the role of bursicon signaling in regulating these aspects of polyphenism were not measured. Thus, the phenotype in pear psyllids, whereby knockdown bursicon reduces cuticle tanning seems to simply demonstrate the phenotypes of Drosophila mutants for bursicon receptor (Loveall and Deitcher, 2010, BMC Dev Biol) in another species (Fig. 2I, 4H). Second, the study fails to address the threshold nature of cuticular tanning in this species, although it is the threshold response (specifically, to temperature and photoperiod) that distinguishes this trait as a part of a polyphenism. Whereas miR-6012 was found to regulate bursicon expression, there no evidence is provided that this microRNA either responds to or initiates a threshold response to temperature. In principle, miR-6012 could regulate bursicon whether or not it is part of a polyphenism. Thus, the impact of this work would be significantly increased if it could distinguish between seasonal changes of the cuticle and a bona fide reflection of polyphenism.

Strengths:

This study convincingly identifies homologs of the genes encoding the bursicon subunits and its receptor, showing an alignment with those of another psyllid as well as more distant species. It also demonstrates that the stage- and tissue-specific levels of bursicon follow the expected patterns, as informed by other insect models, thus validating the identity of these genes in this species. They provide strong evidence that the expression of bursicon and its receptor depend on temperature, thereby showing that this trait is regulated through both parts of the signaling mechanism.

Several parallel measurements of the phenotype were performed to show the effects of this hormone, its receptor, and an upstream regulator (miR-6012), on cuticle deposition and pigmentation (if not polyphenism per se, as claimed). Specifically, chitin staining and TEM of the cuticle qualitatively show difference between controls and knockdowns, and this is supported by some statistical tests of quantitative measurements (although see comments below). Thus, this study provides strong evidence that bursicon and its receptor play an important role in cuticle deposition and pigmentation in this psyllid.

The study identified four miRNAs which might affect bursicon due to sequence motifs. By manipulating levels of synthetic miRNA agonists, the study successfully identified one of them (miR-6012) to cause a cuticle phenotype. Moreover, this miRNA was localized (by FISH) to the cuticle, body-wide. To our knowledge, this is the first demonstrated function for this miRNA, and this study provides a good example of using a gene of known function as an entry point to discovering others influencing a trait. Thus, this finding reveals another level of regulation of cuticle formation in insects.

Weaknesses:

(1) The introduction to this manuscript does not accurately reflect progress in the field of mechanisms underlying polyphenism (e.g., line 60). There are several models for polyphenism that have been used to uncover molecular mechanisms in at least some detail, and this includes seasonal polyphenisms in Hemiptera. Therefore, the justification for this study cannot be predicated on a lack of knowledge, nor is the present study original or unique in this line of research (e.g., as reviewed by Zhang et al. 2019; DOI: 10.1146/annurev-ento-011118-112448). The authors are apparently aware of this, because they even provide other examples (lines 104-108); thus the introduction seems misleading as framed.

(2) The data in Figure 2H show "percent of transition." However, the images in 2I show insects with tanned cuticle (control) vs. those without (knockdown). Yet, based on the description of the Methods provided, there appears to be no distinction between "percent of transition" and "percent with tanning defects". This an important distinction to make if the authors are going to interpret cuticle defects as a defect in the polyphenism. Furthermore, there is no mention of intermediate phenotypes. The data in 2H are binned as either present or absent, and these are the phenotypes shown in 2I. Was the phenotype really an all-or-nothing response? Instead of binning, which masks any quantitative differences in the tanning phenotypes, the authors should objectively quantify the degree of tanning and plot that. This would show if and to what degree intermediate tanning phenotypes occurred, which would test how bursicon affects the threshold response. This comment also applies to the data in Figures 4G and 6G. Since cuticle tanning is present in more insect than just those with seasonal polyphenism, showing how this responds as a threshold is needed to make claims about polyphenism.

(3) This study also does not test the threshold response of cuticle phenotypes to levels of bursicon, its receptor, or miR-6012. Hormone thresholds are the most widespread and, in most systems where polyphenism has been studied, the defining characteristic of a polyphenism (e.g., Nijhout, 2003, Evol Dev). Quantitative (not binned) measurements of a polyphenism marker (e.g., chitin) should be demonstrated to result as a threshold titer (or in the case of the receptor, expression level) to distinguish defects in polyphenism from those of its component trait.

(4) Cuticle issue:<br /> (a) Unlike Fig. 6D and F, Figs. 2D and F do not correspond to each other. Especially the lack and reduction of chitin in ds-a+b! By fluorescence microscopy there is hardly any signal, whereas by TEM there is a decent cuticle. Additionally, the dsGFP control cuticle in 2D is cut obliquely with a thick and a thin chitin layer. This is misleading.<br /> (b) In Figs. 2F and 3F, the endocuticle appears to be missing, a portion of the procuticle that is produced post-molting. As tanning is also occurring post-molting, there seems to be a general problem with cuticle differentiation at this time point. This may be a timing issue. Please clarify.<br /> (c) To provide background information, it would be useful analyze cuticle formation in the summer and winter morphs of controls separately by light and electron microscopy. More baseline data on these two morphs is needed.<br /> (d) For the TEM study, it is not clear whether the same part of the insect's thorax is being sectioned each time, or if that matters. There is not an obvious difference in the number of cuticular layers, but only the relative widths of those layers, so it is difficult to know how comparable those images are. This raises two questions that the authors should clarify. First, is it possible that certain parts of the thoracic cuticle, such as those closer to the intersegmental membrane, are naturally thinner than other parts of the body? Second, is the tanning phenotype based on the thickness or on the number of chitin layers, or both? The data shown later in Figure 4I, J convincingly shows that the biosynthesis pathway for chitin is repressed, but any clarification of what this might mean for deposition of chitin would help to understand the phenotypes reported. Also, more details on how the data in Fig. 2G were collected would be helpful. This also goes for the data in Fig. 4 (bursicon receptor knockdowns).

(5) Tissue issue:<br /> The timed experiments shown in all figures were done in whole animals. However, we know from Drosophila that Bursicon activity is complex in different tissues. There is, thus, the possibility, that the effects detected on different days in whole animals are misleading because different tissues--especially the brain and the epidermis, may respond differentially to the challenge and mask each other's responses. The animal is small, so the extraction from single tissue may be difficult. However, this important issue needs to be addressed.

(6) No specific information is provided regarding the procedure followed for the rescue experiments with burs-α and burs-β (How were they done? Which concentrations were applied? What were the effects?). These important details should appear in the Materials and Methods and the Results sections.

(7) Pigmentation<br /> (a) The protocol used to assess pigmentation needs to be validated. In particular, the following details are needed: Were all pigments extracted? Were pigments modified during extraction? Were the values measured consistent with values obtained, for instance, by light microscopy (which should be done)?<br /> (b) In addition, pigmentation occurs post-molting; thus, the results could reflect indirect actions of bursicon signaling on pigmentation. The levels of expression of downstream pigmentation genes (ebony, lactase, etc) should be measured and compared in molting summer vs. winter morphs.

(8) L236: "while the heterodimer protein of CcBurs α+β could fully rescue the effect of CcBurs-R knockdown on the transition percent (Figure 4G 4H)". This result seems contradictory. If CcBurs-R is the receptor of bursicon, the heterodimer protein of CcBurs α+β should not be able to rescue the effect of CcBurs-R knockdown insects. How can a neuropeptide protein rescue the effect when its receptor is not there! If these results are valid, then the CcBurs-R would not be the (sole) receptor for CcBurs α+β heterodimer. This is a critical issue for this manuscript and needs to be addressed (also in L337 in Discussion).

(9) Fig. 5D needs improvement (the magnification is poor) and further explanation and discussion. mi6012 and CcBurs-R seem to be expressed in complementary tissues--do we see internal tissues also (see problem under point 2)? Again, the magnification is not high enough to understand and appreciate the relationships discussed.

(10) The schematic in Fig. 7 is a useful summary, but there is a part of the logic that is unsupported by the data, specifically in terms of environmental influence on cuticle formation (i.e., plasticity). What is the evidence that lower temperatures influence expression of miR-6012? The study measures its expression over life stages, whether with an agonist or not, over a single temperature. Measuring levels of expression under summer form-inducing temperature is necessary to test the dependence of miR-6012 expression on temperature. Otherwise, this result cannot be interpreted as polyphenism control, but rather the control of a specific trait.

Reviewer #1 (Public Review):

Most human traits and common diseases are polygenic, influenced by numerous genetic variants across the genome. These variants are typically non-coding and likely function through gene regulatory mechanisms. To identify their target genes, one strategy is to examine if these variants are also found among genetic variants with detectable effects on gene expression levels, known as eQTLs. Surprisingly, this strategy has had limited success, and most disease variants are not identified as eQTLs, a puzzling observation recently referred to as "missing regulation".

In this work, Jeong and Bulyk aimed to better understand the reasons behind the gap between disease-associated variants and eQTLs. They focused on immune-related diseases and used lymphoblastoid cell lines (LCLs) as a surrogate for the cell types mediating the genetic effects. Their main hypothesis is that some variants without eQTL evidence might be identifiable by studying other molecular intermediates along the path from genotype to phenotype. They specifically focused on variants that affect chromatin accessibility, known as caQTLs, as a potential marker of regulatory activity.

The authors present data analyses supporting this hypothesis: several disease-associated variants are explained by caQTLs but not eQTLs. They further show that although caQTLs and eQTLs likely have largely overlapping underlying genetic variants, some variants are discovered only through one of these mapping strategies. Notably, they demonstrate that eQTL mapping is underpowered for gene-distal variants with small effects on gene expression, whereas caQTL mapping is not dependent on the distance to genes. Additionally, for some disease variants with caQTLs but no corresponding eQTLs in LCLs, they identify eQTLs in other cell types.

Altogether, Jeong and Bulyk convincingly demonstrate that for immune-related diseases, discovering the missing disease-eQTLs requires both larger eQTL studies and a broader range of cell types in expression assays. It remains to be seen what fractions of the missing disease-eQTLs will be discovered with either strategy and whether these results can be extended to other diseases or traits.

It should be noted that the problem of "missing regulation" has been investigated and discussed in several recent papers, notably Umans et al., Trends in Genetics 2021; Connally et al., eLife 2022; Mostafavi et al., Nat. Genet. 2023. The results reported by Jeong and Bulyk are not unexpected in light of this previous work (all of which they cite), but they add valuable empirical evidence that mostly aligns with the model and discussions presented in Mostafavi et al.

Reviewer #2 (Public Review):

Summary:

eQTLs have emerged as a method for interpreting GWAS signals. However, some GWAS signals are difficult to explain with eQTLs. In this paper, the authors demonstrated that caQTLs can explain these signals. This suggests that for GWAS signals to actually lead to disease phenotypes, they must be accessible in the chromatin. This implies that for GWAS signals to translate into disease phenotypes, they need to be accessible within the chromatin.

However, fundamentally, caQTLs, like GWAS, have the limitation of not being able to determine which genes mediate the influence on disease phenotypes. This limitation is consistent with the constraints observed in this study.

(1) For reproducibility, details are necessary in the method section.

- Details about adding YRI samples in ATAC-seq: For example, how many samples are there, and what is used among public data? There is LCL-derived iPSC and differentiated iPSC (cardiomyocytes) data , not LCL itself. How does this differ from LCL, and what is the rationale for including this data despite the differences?

- caQTL is described as having better power than eQTL despite having fewer samples. How does the number of ATAC peaks used in caQTL compare to the number of gene expressions used in eQTL?

- Details about RNA expression data: In the method section, it states that raw data (ERP001942) was accessed, and in data availability, processed data (E-GEUV-1) was used. These need to be consistent.

How many samples were used (the text states 373, but how was it reduced from the original 465, and the total genotype is said to be 493 samples while ATAC has n=100; what are the 20 others?), and it mentions European samples, but does this exclude YRI?

(2) Experimental results determining which TFs might bind to the representative signals of caQTL are required.

(3) It is stated that caQTL is less tissue-specific compared to eQTL; would caQTL performed with ATAC-seq results from different cell types, yield similar results?

Interesting commentary here on conversion of African-Americans to Catholicism as well as self-help nature of reading for improvement. Analogizes African-Americans without Catholicism to Mortimer J. Adler as a Jew.

Possible tone of colonialism to assimilate African-Americans into Western Culture here? Though still somehow some space for movement and growth.

Reviewer #1 (Public Review):

Summary:

The authors addressed how long-range interactions between boundary elements are established and influence their function in enhancer specificity. Briefly, the authors placed two different reporters separated by a boundary element. They inserted this construct ectopically ~140 kb away from an endogenous locus that contains the same boundary element. The authors used expression patterns driven by nearby enhancers as an output to determine which enhancers the reporters interact with. They complemented this analysis with 3D DNA contact mapping. The authors found that the orientation of the boundary element determined which enhancers each reporter interacted with. They proposed that the 3D interaction topology, whether being circular or stem configuration, distinguished whether the interaction was cohesin mediated or through an independent mechanism termed pairing.

Strengths:

The transgene expression assays are built upon prior knowledge of the enhancer activities. The 3D DNA contacts confirm that transgene expression correlates with the contacts. Using 4 different orientations covers all combinations of the reporter genes and the boundary placement.

Weaknesses:

The interpretation of the data as a refusal of loop extrusion playing a role in TAD formation is not warranted, as the authors did not deplete the loop extruders to show that what they measure is independent. As the authors show, the single long DNA loop mediated by cohesin loop extrusion connecting the ectopic and endogenous boundary is clearly inconsistent with the results, therefore the main conclusion of the paper that the 3D topology of the boundary elements a consequence of pairing is strong. However, the loop extrusion and pairing are not mutually exclusive models for the formation of TADs. Loop-extruding cohesin complexes need not make a 140 kb loop, multiple smaller loops could bring together the two boundary elements, which are then held together by pairing proteins that can make circular topologies.

Reviewer #2 (Public Review):

In Bing et al, the authors analyze micro-C data from NC14 fly embryos, focusing on the eve locus, to assess different models of chromatin looping. They conclude that fly TADs are less consistent with conventional cohesin-based loop extrusion models and instead rely more heavily on boundary-boundary pairings in an orientation-dependent manner.

Overall, I found the manuscript to be interesting and thought-provoking. However, this paper reads much more like a perspective than a research article. Considering the journal is aimed at the general audience, I strongly suggest the authors spend some time editing their introduction to the most salient points as well as organizing their results section in a more conventional way with conclusion-based titles. It was very difficult to follow the authors' logic throughout the manuscript as written. It was also not clear as written which experiments were performed as part of this study and which were reanalyzed but published elsewhere. This should be made clearer throughout.

It has been shown several times that Drosophila Hi-C maps do not contain all of the features (frequent corner peaks, stripes, etc.) observed when compared to mammalian cells. Considering these features are thought to be products of extrusion events, it is not an entirely new concept that Drosophila domains form via mechanisms other than extrusion. That being said, the authors' analyses do not distinguish between the formation and the maintenance of domains. It is not clear to this reviewer why a single mechanism should explain the formation of the complex structures observed in static Hi-C heatmaps from a population of cells at a single developmental time point. For example, how can the authors rule out that extrusion initially provides the necessary proximity and possibly the cis preference of contacts required for boundary-boundary pairing whereas the latter may more reflect the structures observed at maintenance? Future work aimed at analyzing micro-C data in cohesin-depleted cells might shed additional light on this.

Additional mechanisms at play include compartment-level interactions driven by chromatin states. Indeed, in mammalian cells, these interactions often manifest as a "plume" on Hi-C maps similar to what the authors attribute to boundary interactions in this manuscript. How do the chromatin states in the neighboring domains of the eve locus impact the model if at all?

How does intrachromosomal homolog pairing impact the models proposed in this manuscript (Abed et al. 2019; Erceg et al., 2019). Several papers recently have shown that somatic homolog pairing is not uniform and shows significant variation across the genome with evidence for both tight pairing regions and loose pairing regions. Might loose pairing interactions have the capacity to alter the cis configuration of the eve locus?

In summary, the transgenic experiments are extensive and elegant and fully support the authors' models. However, in my opinion, they do not completely rule out additional models at play, including extrusion-based mechanisms. Indeed, my major issue is the limited conceptual advance in this manuscript. The authors essentially repeat many of their previous work and analyses. The authors make no attempt to dissect the mechanism of this process by modifying extrusion components directly. Some discussion of Rollins et al., 1999 on the discovery of Nipped-B and its role in enhancer-promoter communication should also be made to reconcile their conclusions in the proposed absence of extrusion events.

Reviewer #3 (Public Review):

Bing et al. attempt to address fundamental mechanisms of TAD formation in Drosophila by analyzing gene expression and 3D conformation within the vicinity of the eve TAD after insertion of a transgene harboring a Homie insulator sequence 142 kb away in different orientations. These transgenes along with spatial gene expression analysis were previously published in Fujioka et al. 2016, and the underlying interpretations regarding resulting DNA configuration in this genomic region were also previously published. This manuscript repeats the expression analysis using smFISH probes in order to achieve more quantitative analysis, but the main results are the same as previously published. The only new data are the Micro-C and an additional modeling/analysis of what they refer to as the 'Z3' orientation of the transgenes. The rest of the manuscript merely synthesizes further interpretation with the goal of addressing whether loop extrusion may be occurring or if boundary:boundary pairing without loop extrusion is responsible for TAD formation. The authors conclude that their results are more consistent with boundary:boundary pairing and not loop extrusion; however, most of this imaging data seems to support both loop extrusion and the boundary:boundary models. This manuscript lacks support, especially new data, for its conclusions. Furthermore, there are many parts of the manuscript that are difficult to follow. There are some minor errors in the labelling of the figures that if fixed would help elevate understanding. Lastly, there are several major points that if elaborated on, would potentially be helpful for the clarity of the manuscript.

Major Points:

(1) The authors suggest and attempt to visualize in the supplemental figures, that loop extrusion mechanisms would appear during crosslinking and show as vertical stripes in the micro-C data. In order to see stripes, a majority of the nuclei would need to undergo loop extrusion at the same rate, starting from exactly the same spots, and the loops would also have to be released and restarted at the same rate. If these patterns truly result from loop extrusion, the authors should provide experimental evidence from another organism undergoing loop extrusion.<br /> (2) On lines 311-314, the authors discuss that stem-loops generated by cohesin extrusion would possibly be expected to have more next-next-door neighbor contacts than next-door neighbor contacts and site their models in Figure 1. Based on the boundary:boundary pairing models in the same figure would the stem-loops created by head-to-tail pairing also have the same phenotype? Making possible enrichment of next-next-door neighbor contacts possible in both situations? The concepts in the text are not clear, and the diagrams are not well-labeled relative to the two models.<br /> (3) The authors appear to cite Chen et al., 2018 as a reference for the location of these transgenes being 700nM away in a majority of the nuclei. However, the exact transgenes in this manuscript do not appear to have been measured for distance. The authors could do this experiment and include expression measurements.<br /> (4) The authors discuss the possible importance of CTCF orientation in forming the roadblock to cohesin extrusion and discuss that Homie orientation in the transgene may impact Homie function as an effective roadblock. However, the Homie region inserted in the transgene does not contain the CTCF motif. Can the authors elaborate on why they feel the orientation of Homie is important in its ability to function as a roadblock if the CTCF motif is not present? Trans-acting factors responsible for Homie function have not been identified and this point is not discussed in the manuscript.<br /> (5) The imaging results seem to be consistent with both boundary:boundary interaction and loop extrusion stem looping.<br /> (6) The authors suggest that the eveMa TAD could only be formed by extrusion after the breakthrough of Nhomie and several other roadblocks. Additionally, the overall long-range interactions with Nhomie appear to be less than the interactions with endogenous Homie (Figures 7, 8, and supplemental 5). Is it possible that in some cases boundary:boundary pairing is occurring between only the transgenic Homie and endogenous Homie and not including Nhomie?<br /> (7) In Figure 4E, the GFP hebe expression shown in the LhomieG Z5 transgenic embryo does not appear in the same locations as the LlambdaG Z5 control. Is this actually hebe expression or just a background signal?<br /> (8) Figure 6- The LhomieG Z3 late-stage embryo appears to be showing the ventral orientation of the embryo rather than the lateral side of the embryo as was shown in the previous figure. Is this for a reason? Additionally, there are no statistics shown for the Z3 transgenic images. Were these images analyzed in the same way as the Z5 line images?<br /> (9) Do the Micro-C data align with the developmental time points used in the smFISH probe assays?

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 #2 (Public Review):

Summary:

The tubulin subunits that make up microtubules can be posttranslationally modified and these PTMs are proposed to regulate microtubule dynamics and the proteins that can interact with microtubules in many contexts. However, most studies investigating the roles of tubulin PTMs have been conducted in vitro either with purified components or in cultured cells. Lu et al. use CRISPR/Cas9 genome editing to mutate tubulin genes in C. elegans, testing the role of specific tubulin residues on neuronal development. This study is a real tour de force, tackling multiple proposed tubulin modifications and following the resulting phenotypes with respect to neurite outgrowth in vivo. There is a ton of data that experts in the field will likely reference for years to come as this is one of the most comprehensive in vivo analyses of tubulin PTMs in vivo.

This paper will be very important to the field, however, it would be strengthened if: 1) the authors demonstrated that the mutations they introduced had the intended consequences on microtubule PTMs, 2) the authors explored how the various tubulin mutations directly affect microtubules, and 3) the findings are made generally more accessible to non C. elegans neurobiologist.

(1) The authors introduce several mutations to perturb tubulin PTMs, However, it is unclear to what extent the engineered mutations affecting tubulin in the intended way. i.e. are the authors sure that the PTMs they want to perturb are actually present in C. elegans. Many of the antibodies used did not appear to be specific and antibody staining was not always impacted in the mutant cases as expected. For example, is there any evidence that S172 is phosphorylated in C. elegans, e.g. from available phosphor-proteomic data? Given the significant amount of staining left in the S172A mutant, the antibody seems non-specific in this context and therefore not a reliable readout of whether MTs are actually phosphorylated at this residue. As another example, there is no evidence presented that K252 is acetylated in C. elegans. At the very least, the authors should consider demonstrating the conservation of these residues and the surrounding residues with other organisms where studies have demonstrated PTMs exist.

(2) Given that the authors have the mutants in hand, it would be incredibly valuable to assess the impact of these mutations on microtubules directly in all cases. MT phenotypes are inferred from neurite outgrowth phenotypes in several cases, the authors should look directly at microtubules and/or microtubule dynamics via EBP-2 when possible OR show evidence that the only way to derive the neurite phenotypes shown is through the inferred microtubule phenotypes. For example, the effect of the acetylation or detyrosination mutants on MTs was not assessed.

(3) There is a ton of data here that will be important for experts working in this field to dig into, however, for the more general cell biologist, some of the data are quite inaccessible. More cartoons and better labeling will be helpful as will consistent comparisons to control worms in each experiment. A good example of this issue is demonstrated in Figure 2 and Figure 4:

- Fig. 2: Please label images with what is being probed in each panel<br /> - Fig 2G is very hard to interpret-cartoon diagramming what is being observed would be helpful.<br /> - Line 182-185: is this referring to your data or to Wu et al? It is not clear in this paragraph when the authors are describing published work versus their own data presented here.<br /> - Fig 2!-2K is not well described. What experiment is being done here? What is dlk-1 and why did you look at this mutant?<br /> - Figure 4C: this phenotype is hard to interpret. Where is the wt control? Where is the quantification?<br /> - There are no WT comparison images in Figure 4I, making the quantification difficult to interpret

(4) In addition, I am left unconvinced of the negative data demonstrating that MBK does not phosphorylate tubulin. First, the data described in lines 207-211 does not appear to be presented anywhere. Second, RNAi is notoriously finicky in neurons, thus necessitating tissue specific degradation using either the ZF/ZIF-1 or AID/TIR1 systems which both work extremely well in C. elegans. Third, there appears to be increasing S172 phosphorylation in Figure 3 supplement 2 with added MBK-2, but there is no anti-tubulin blot to show equal loading, so this experiment is hard to interpret.

Reviewer #1 (Public Review):

Summary:

The paper measures the prevalence and mortality of stroke and its comorbidities across geographic regions in order to find differences in risks that may lead to more effective guidance for these subpopulations. It also does a genetic analysis to look for variants that may drive these phenotypic variations.

Strengths:

The data provided here will provide a foundation for a lot of future research into the causes of the observed correlations as well as whether the observed differences in comorbidities across regions have clinically relevant effects on risk management.

The use of data from before COVID-19 is both a strength and a weakness. Because COVID had effects on vascular health and had higher death rates for groups with the comorbidities of interest here, it has likely shifted the demographics in ways that would shift the results in unpredictable ways if the analysis were repeated with current data. This can be a strength in providing a reference point for studying those changes as well as allowing researchers to study differences between regions without the complication of different public health responses adding extra variation to the data. On the other hand, it limits the usefulness of the data in research concerned with the current status of the various populations.

Reviewer #1 (Public Review):

The study by Prieto et al. faces the increasingly serious problem of bacterial resistance to antimicrobial agents. This work has an important element of novelty proposing a new approach to control antibiotic resistance spread by plasmids. Instead of targeting the resistance determinant, plasmid-borne proteins are used as antigens to be bound by specific nanobodies (Nbs). Once bound plasmid transfer was inhibited and Salmonella infection blocked. This in-depth study is quite detailed and complex, with many experiments (9 figures with multiple panels), rigorously carried out. Results fully support the authors' conclusions. Specifically, the authors investigated the role of two large molecular weight proteins (RSP and RSP2) encoded by the IncHI1 derivative-plasmid R27 of Salmonella. These proteins have bacterial Ig-like (Big) domains and are expressed on the cell surface, creating the opportunity for them to serve as immunostimulatory antigens. Using a mouse infection model, the authors showed that RSP proteins can properly function as antigens, in Salmonella strains harboring the IncHI1 plasmid. The authors clearly showed increased levels of specific IgG and IgA antibodies against these RSP proteins proteins in different tissues of immunized animals. In addition, non-immunized mice exhibited Salmonella colonization in the spleen and much more severe disease than immunized ones.

However, the strength of this work is the selection and production of nanobodies (Nbs) that specifically interact with the extracellular domain of RSP proteins. The procedure to obtain Nbs is lengthy and complicated and includes the immunization of dromedaries with purified RPS and the construction of a VHH (H-chain antibody variable region) library in E. coli. As RSP is expressed on the surface of E. coli, specific Nbs were able to agglutinate Salmonella strains harboring the p27 plasmid encoding the RSP proteins.

The authors demonstrated that Nbs-RSP reduced the conjugation frequency of p27 thus limiting the diffusion of the amp resistance harbored by the plasmid. This represents an innovative and promising strategy to fight antibiotic resistance, as it is not blocked by the mechanism that determines, in the specific case, the amp resistance of p27 but it targets an antigen associated with HincHI- derivative plasmids. Thus, RPS vaccination could be effective not only against Salmonella but also against other enteric bacteria. A possible criticism could be that Nbs against RSP proteins reduce the severity of the disease but do not completely prevent the infection by Salmonella.

Reviewer #2 (Public Review):

Summary:

This manuscript aims to tackle the antimicrobial resistance through the development of vaccines. Specifically, the authors test the potential of the RSP protein as a vaccine candidate. The RSP protein contains bacterial Ig-like domains that are typically carried in IncHl1 plasmids like R27. The extracellular location of the RSP protein and its role in the conjugation process makes it a good candidate for a vaccine. The authors then use Salmonella carrying an IncHl plasmid to test the efficacy of the RSP protein as a vaccine antigen in providing protection against infection of antibiotic-resistant bacteria carrying the IncHl plasmid. The authors found no differences in total IgG or IgA levels, nor in pro-inflammatory cytokines between immunized and non-immunized mice. They however found differences in specific IgG and IgA, attenuated disease symptoms, and restricted systemic infection.

The manuscript also evaluates the potential use of nanobodies specifically targeting the RSP protein by expressing it in E. coli and evaluating their interference in the conjugation of IncHl plasmids. The authors found that E. coli strains expressing RSP-specific nanobodies bind to Salmonella cells carrying the R27 plasmid thereby reducing the conjugation efficacy of Salmonella.

Strengths:

- The main strength of this manuscript is that it targets the mechanism of transmission of resistance genes carried by any bacterial species, thus making it broad.

- The experimental setup is sound and with proper replication.

Weaknesses:

- The two main experiments, evaluating the potential of the RSP protein and the effects of nanobodies on conjugation, seem as parts of two different and unrelated strategies.

- The survival rates shown in Figure 1A and Figure 3A for Salmonella pHCM1 and non-immunized mice challenged with Salmonella, respectively, are substantially different. In the same figures, the challenge of immunized mice and Salmonella pHCM1 and mice challenged with Salmonella pHCM1 with and without ampicillin are virtually the same. While this is not the only measure of the effect of immunization, the inconsistencies in the resulting survival curves should be addressed by the authors more thoroughly as they can confound the effects found in other parameters, including total and specific IgG and IgA, and pro-inflammatory cytokines.

- Overall the results are inconsistent and provide only partial evidence of the effectiveness of the RSP protein as a vaccine target.

- The conjugative experiments use very long conjugation times, making it harder to asses if the resulting transconjugants are the direct result of conjugation or just the growth of transconjugants obtained at earlier points in time. While this could be assessed from the obtained results, it is not a direct or precise measure.

- While the potential outcomes of these experiments could be applied to any bacterial species carrying this type of plasmids, it is unclear why the authors use Salmonella strains to evaluate it. The introduction does a great job of explaining the importance of these plasmids but falls short in introducing their relevance in Salmonella.

Joint Public Review:

Chemokines are known to create chemotactic gradients and it is generally recognized that in order to create these gradients they need to bind to glycosaminoglycans (GAGs) on cells and in tissues. However, how the triplicate interaction between chemokines with both GAGs and G protein-coupled receptors (GPCR) works and how gradients are created and potentially maintained in vivo is poorly understood. In their manuscript, Yu et al investigated and showed in detail the ability of soluble and cell-bound GAGs to create gradients of the chemokine CCL5. They show in vitro in a modified leukocyte migration assay that soluble GAGs and GAGs on the tumor cell line THP-1 affect leukocyte migration. This useful work contributes to our in-depth understanding of the role of GAGs in chemokine gradient creation which is important for site-directed leukocyte and potentially tumor cell migration and as such is of potential interest for scientists studying immune responses in infection, inflammation, autoimmunity and tumor biology. In their reply to the comments of both reviewers they indicate that liquid-liquid phase separation (LLPS) was not detected at lower CCL5 concentrations. This is important information since, together with the tendency of CCL5 to form oligomers, it may indicate that oligomerization is crucial for LLPS. This info should at least be added to the discussion of the manuscript.

Reviewer #1 (Public Review):

Summary:

Huang and Luo investigated whether regularities between stimulus features can be exploited to facilitate the encoding of each set of stimuli in visual working memory, improving performance. They recorded both behavioural and neural (EEG) data from human participants during a sequential delayed response task involving three items with two properties: location and colour. In the key condition ('aligned trajectory'), the distance between locations of successively presented stimuli was identical to their 'distance' in colour space, permitting a compression strategy of encoding only the location and colour of the first stimulus and the relative distance of the second and third stimulus (as opposed to remembering 3 locations and 3 colours, this would only require remembering 1 location, 1 colour, and 2 distances). Participants recalled the location and colour of each item after a delay.

Consistent with the compression account, participants' location and colour recall errors were correlated and overall lower compared to a non-compressible condition ('misaligned trajectory'). Multivariate analysis of the neural data permitted decoding of the locations and colours during encoding. Crucially, the relative distance could also be decoded - a necessary ingredient for the compression strategy.

Strengths:

The main strength of this study is a novel experimental design that elegantly demonstrates how we exploit stimulus structure to overcome working memory capacity limits. The behavioural results are robust and support the main hypothesis of compressed encoding across a number of analyses. The simple and well-controlled design is suited to neuroimaging studies and paves the way for investigating the neural basis of how environmental structure is detected and represented in memory. Prior studies on this topic have primarily studied behaviour only (e.g., Brady & Tenenbaum, 2013).

Weaknesses:

The main weakness of the study is that the EEG results could make a clearer case for compression. There is some evidence that distance decoding is present in alpha-band activity in the maintenance delay, but the strongest evidence for this occurs only briefly in the late encoding phase (the re-activation of decoding of the distance between items 1 and 2, Fig. 5A). The link to behaviour (Fig. 5D) seems fairly weak and based on a potentially circular analysis. During location recall, colour decoding re-emerges and is reactivated in sequence, but this finding is consistent both with compression-based and conventional rehearsal mechanisms. Nevertheless, the balance of evidence appears to favour the compression account.

Impact:

This important study elegantly demonstrates that the use of shared structure can improve capacity-limited visual working memory. The paradigm and approach explicitly link this field to recent findings on the role of replay in structure learning and will therefore be of interest to neuroscientists studying both topics.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors intended to prove that gut GLP-1 expression and secretion can be regulated by Piezo1, and hence by mechanistic/stretching regulation. For this purpose, they have assessed Piezo1 expression in STC-1 cell line (a mouse GLP-1 producing cell line) and mouse gut, showing the correlation between Piezo1 level and Gcg levels (Figure S1). They then aimed to generate gut L cell-specific Piezo1 KO mice, and claimed the mice show impaired glucose tolerance and GLP-1 production, which can be mitigated by Ex-4 treatment (Figures 1-2). Pharmacological agents (Yoda1 and GsMTx4) and mechanic activation (intestinal bead implantation) were then utilized to prove the existence of ileal Piezo1-regulated GLP-1 synthesis (Figure 3). This was followed by testing such mechanism in a limited amount of primary L cells and mainly in the STC-1 cell line (Figures 4-7).

While the novelty of the study is somehow appreciable, the bio-medical significance is not well demonstrated in the manuscript. The authors stated (in lines between lines 78-83) a number of potential side effects of GLP-1 analogs, how can the mechanistic study of GLP-1 production on its own be essential for the development of new drug targets for the treatment of diabetes. Furthermore, the study does not provide a clear mechanistic insight on how the claimed CaMKKbeta/CaMKIV-mTORC1 signaling pathway upregulated both GLP-1 production and secretion. This reviewer also has concerns about the experimental design and data presented in the current manuscript, including the issue of how proglucagon expression can be assessed by Western blotting.

Strengths:

The novelty of the concept.

Weaknesses:

Experimental design and key experiment information.

Reviewer #2 (Public Review):

Summary:

The study by Huang and colleagues focuses on GLP-1 producing entero-endocrine (EEC) L-cells and their regulation of GLP-1 production by a mechano-gated ion channel Piezo1. The study describes Piezo1 expression by L-cells and uses an exciting intersectional mouse model (villin to target epithelium and Gcg to target GLP-1-producing cells and others like glucagon-producing pancreatic endocrine cells), which allows L-cell specific Piezo1 knockout. Using this model, they find an impairment of glucose tolerance, increased body weight, reduced GLP-1 content, and changes to the CaMKKbeta-CaMKIV-mTORC1 signaling pathway using a normal diet and then high-fat diet. Piezo1 chemical agonist and intestinal bead implantation reversed these changes and improved the disrupted phenotype. Using primary sorted L-cells and cell model STC-1, they found that stretch and Piezo1 activation increased GLP-1 and altered the molecular changes described above.

Strengths:

This is an interesting study testing a novel hypothesis that may have important mechanistic and translational implications. The authors generated an important intersectional genetics mouse model that allowed them to target Piezo1 L-cells specifically, and the surprising result of impaired metabolism is intriguing.

Weaknesses:

However, there are several critical limitations that require resolution before making the conclusions that the authors make.

(1) A potential explanation for the data, and one that is consistent with existing literature [see for example, PMC5334365, PMC4593481], is that epithelial Piezo1, which is broadly expressed by the GI epithelium, impacts epithelial cell density and survival, and as such, if Piezo1 is involved in L-cell physiology, it may be through regulation of cell density. Thus, it is critical to determine L-cell densities and epithelial integrity in controls and Piezo1 knockouts systematically across the length of the gut, since the authors do not make it clear which gut region contributes to the phenotype they see. Current immunohistochemistry data are not convincing.

(2) Calcium signaling in L-cells is implicated in their typical role of being gut chemo-sensors, and Piezo1 is a calcium channel, so it is not clear whether any calcium-related signaling mechanism would phenocopy these results.

(3) Intestinal bead implantation, while intriguing, does not have clear mechanisms - and is likely to provide a point of intestinal obstruction and dysmotility.

(4) Previous studies, some that are very important, but not cited, contradict the presented results (e.g., epithelial Piezo1 role in insulin secretion) and require reconciliation.

Overall, this study makes an interesting observation but the data are not currently strong enough to support the conclusions.

Reviewer #1 (Public Review):

Summary<br /> This is an interesting paper that concludes that hiring more women will do more to improve the gender balance of (US) academia than improving the attrition rates of women (which are usually higher than men's). Other groups have reported similar findings, i.e. that improving hiring rates does more for women's representation than reducing attrition, but this study uses a larger than usual dataset that spans many fields and institutions so it is a good contribution to the field.

The paper is much improved and far more convincing as a result of the revisions made by the authors.

Strengths<br /> A large data set with many individuals, many institutions and fields of research.<br /> A good sensitivity analysis to test for potential model weaknesses.

Weaknesses<br /> Only a single country with a very specific culture and academic system.<br /> Complex model fitting with many steps and possible places for model bias.

Reviewer #3 (Public Review):

Summary<br /> This study investigates the roles of faculty hiring and attrition in influencing gender representation in U.S. academia. It uses a comprehensive dataset covering tenured and tenure-track faculty across various fields from 2011 to 2020. The study employs a counterfactual model to assess the impact of hypothetical gender-neutral attrition and projects future gender representation under different policy scenarios. The analysis reveals that hiring has a more significant impact on women's representation than attrition in most fields and highlights the need for sustained changes in hiring practices to achieve gender parity.

The revisions made by the authors have improved the paper.

Strengths<br /> Overall, the manuscript offers significant contributions to understanding gender diversity in academia through its rigorous data analysis and innovative methodology.

The methodology is robust, employing extensive data covering a wide range of academic fields and institutions.

Weaknesses<br /> The primary weakness of the study lies in its focus on U.S. academia, which may limit the generalizability of its findings to other cultural and academic contexts. Additionally, the counterfactual model's reliance on specific assumptions about gender-neutral attrition could affect the accuracy of its projections.

Additionally, the study assumes that whoever disappeared from the dataset is attrition in academia. While in reality, those attritions could be researchers who moved to another country or another institution that is not indexed by AA.

Reviewer #3 (Public Review):

In this study, Ruan et al. investigate the role of the IQCH gene in spermatogenesis, focusing on its interaction with calmodulin and its regulation of RNA-binding proteins. The authors examined sperm from a male infertility patient with an inherited IQCH mutation as well as Iqch CRISPR knockout mice. The authors found that both human and mouse sperm exhibited structural and morphogenetic defects in multiple structures, leading to reduced fertility in Ichq-knockout male mice. Molecular analyses such as mass spectrometry and immunoprecipitation indicated that RNA-binding proteins are likely targets of IQCH, with the authors focusing on the RNA-binding protein HNRPAB as a critical regulator of testicular mRNAs. The authors used in vitro cell culture models to demonstrate an interaction between IQCH and calmodulin, in addition to showing that this interaction via the IQ motif of IQCH is required for IQCH's function in promoting HNRPAB expression. In sum, the authors concluded that IQCH promotes male fertility by binding to calmodulin and controlling HNRPAB expression to regulate the expression of essential mRNAs for spermatogenesis. These findings provide new insight into molecular mechanisms underlying spermatogenesis and how important factors for sperm morphogenesis and function are regulated.

The strengths of the study include the use of mouse and human samples, which demonstrate a likely relevance of the mouse model to humans; the use of multiple biochemical techniques to address the molecular mechanisms involved; the development of a new CRISPR mouse model; ample controls; and clearly displayed results. Assays are done rigorously and in a quantitative manner. Overall, the claims made by the authors in this manuscript are well-supported by the data provided.

Reviewer #1 (Public Review):

The role of enteric glial cells in regulating intestinal mucosal functions at a steady state has been a matter of debate in recent years. Enteric glial cell heterogeneity and related methodological differences likely underlie the contrasting findings obtained by different laboratories. Here, Prochera and colleagues used Plp1-CreERT2 driver mice to deplete the majority of enteric glia from the gut. They found that glial loss has very limited effects on the transcriptome of gut cells 11 days after tamoxifen treatment (used to induce DTA expression), and by extension - more specifically, has only minimal impact on cells of the intestinal mucosa. Interestingly, in the colon (where Paneth cells are not present) they did observe transcriptomic changes related to Paneth cell biology. Although no overt gene expression alterations were found in the small intestine - also not in Paneth cells - morphological, ultrastructural, and functional changes were detected in the Paneth cells of enteric glia-depleted mice. In addition, and possibly related to Paneth cell dysfunction, enteric glia-depleted mice also show alterations in intestinal microbiota composition.

In their analyses of enteric glia from existing single-cell transcriptomic data sets, it is stated that these come from 'non-diseased' humans. However, the data on the small intestine is obtained from children with functional gastrointestinal disorders (Zheng 2023). Data on colonic enteric glia was obtained from colorectal cancer patients (Lee 2020). Although here the cells were isolated from non-malignant regions, saying that the large intestines of these patients are non-diseased is probably an overstatement. Another existing dataset including human mucosal enteric glia of healthy subjects is presented in Smillie et al (2019). It would be interesting to see how the current findings relate to the data from Smillie et al.

The time between enteric glia depletion and analyses (mouse sacrifice) must be a crucial determinant of the type of effects, and the timing thereof. In the current study 11 days after tamoxifen treatment was chosen as the time point for analyses, which is consistent with earlier work by the lab using the same model (Rao et al 2017). What would happen when they wait longer than 11 days after tamoxifen treatment? Data, not necessarily for all parameters, on later time points would strengthen the manuscript significantly.

The authors found transcriptional dysregulation related to Paneth cell biology in the colon, where Paneth cells are normally not present. Given the bulk RNA sequencing approach, the cellular identity in which this shift is taking place cannot be determined. However, it would be useful if the authors could speculate on which colonic cell type they reckon this is happening in. On the other hand, enteric glia depletion was found to affect Paneth cells structurally and functionally in the small intestine, where transcriptional changes were initially not identified. Only when performing GSEA with the in silico help of cell type-specific gene profiles, differences in Paneth cell transcriptional programs in the small intestine were uncovered. A comment on this discrepancy would be helpful, especially for the non-bioinformatician readers among us.

From looking at Figure 3B it is clear that Paneth cells are not the only epithelial cell type affected (after less stringent in silico analyses) by enteric glial cell depletion. Although the authors show that this does not translate into ultrastructural or numerical changes of most of these cell types, this makes one wonder how specific the enteric glia - Paneth cell link is. Besides possible indirect crosstalk (via neurons), it is not clear if enteric glia more closely associate with Paneth cells as compared to these other cell types. Immunofluorescence stainings of some of these cells in the Plp1-GFP mice would be informative here. The authors mention IL-22 as a possible link, but do Paneth cells express receptors for transmitters commonly released by enteric glia? Maybe they can have a look at putative cell-cell interactions by mapping ligand-receptor pairs in the scRNAseq datasets they used.

Previously the authors showed that enteric glia regulation of intestinal motility is sex-dependent (Rao et al 2017). While enteric glia depletion caused dysmotility in female mice, it did not affect motility in males. For this reason, most experiments in the current study were conducted in male mice only. However, for the experiments focusing on the effect of enteric glia depletion on host-microbiome interactions and intestinal microbiota composition both male and female mice were used. In Figure 8A male and female mice are distinctly depicted but this was not done for Figure 8C. Separate characterization of the microbiome of male and female mice would have helped to figure out how much intestinal dysmotility (in females) contributes to the effect on gut microbial composition. This is an important exercise to confirm that the effect on the microbiome is indeed a consequence of altered Paneth cell function, as suggested by the authors (in the results and discussion, and in the abstract). In this context, it would also be interesting to compare the bulk sequencing data after enteric glia depletion between female and male mice.

Reviewer #2 (Public Review):

This is an excellent and timely study from the Rao lab investigating the interactions of enteric glia with the intestinal epithelium. Two early studies in the late 1990s and early 2000s had previously suggested that enteric glia play a pivotal role in control of the intestinal epithelial barrier, as their ablation using mouse models resulted in severe and fatal intestinal inflammation. However, it was later identified that these inflammatory effects could have been an indirect product of the transgenic mouse models used, rather than due to the depletion of enteric glia. In previous studies from this lab, the authors had identified expression of PLP1 in enteric glia, and its use in CRE driver lines to label and ablate enteric glia.

In the current paper, the authors carefully examine the role of enteric glia by first identifying that PLP1-creERT2 is the most useful driver to direct enteric glial ablation, in terms of the number of glial cells targeted, their proximity to the intestinal epithelium, and the relevance for human studies (GFAP expression is rather limited in human samples in comparison). They examined gene expression changes in different regions of the intestine using bulk RNA-seq following ablation of enteric glia by driving expression of diphtheria toxin A (PLP1-creERT2;Rosa26-DTA). Alterations in gene expression were observed in different regions of the gut, with specific effects in different regions. Interestingly, while there were gene expression changes in the epithelium, there were limited changes to the proportions of different epithelial cell types identified using immunohistochemistry in control vs glial-ablated mice. The authors then focused on the investigation of Paneth cells in the ileum, identifying changes in the ultrastructural morphology and lysozyme activity. In addition, they identified alterations in gut microbiome diversity. As Paneth cells secrete antimicrobial peptides, the authors conclude that the changes in gut microbiome are due to enteric glia-mediated impacts on Paneth cell activity.

Overall, the study is excellent and delves into the different possible mechanisms of action, including the investigation of changes in enteric cholinergic neurons innervating the intestinal crypts. The use of different CRE drivers to target enteric glial cells has led to varying results in the past, and the authors should be commended on how they address this in the Discussion.

Reviewer #3 (Public Review):

In this study, Prochera, et al. identify PLP1+ cells as the glia that most closely interact with the gut epithelium and show that genetic depletion of these PLP1+ glia in mice does not have major effects on the intestinal transcriptome or the cellular composition of the epithelium. Enteric glial loss, however, causes dysregulation of Paneth cell gene expression that is associated with morphological disruption of Paneth cells, diminished lysozyme secretion, and altered gut microbial composition. Overall, the authors need to first prove whether the Plp1CreER Rosa26DTA/+ mice system is viable. Also, most experimental systems have been evaluated by immunohistochemistry, scRNAseq, and electron microscopy, but need quantitative statistical processing. In addition, the value of the paper would be enhanced if the significance of why the phenotype appeared in the large intestine rather than the small intestine when PLP1 is deficient for Paneth cells is clarified.

Weaknesses:

Major:

(1) Supplementary Figure 2; Cannot be evaluated without quantification.

(2) Figure 2A; Is Plp1CreER Rosa26DTA/+ mice system established correctly? S100B immunohistology picture is not clear. A similar study is needed for female Plp1CreER Rosa26DTA/+ mice. What is the justification for setting 5 dpt, 11 dpt? Any consideration of changes to organs other than the intestine? Wouldn't it be clearer to introduce Organoid technology?

3) Figure 2B; Need an explanation for the 5 genes that were altered in the colon. Five genes should be evaluated by RT-qPCR. Why was there a lack of change in the duodenum and ileum?

(4) Supplementary Figure 3; Top 3 genes should be evaluated by RT-qPCR.

(5) Supplementary Figure 4B, C, and D; Why not show analysis in the small intestine?

(6) Supplementary Figure 4D; Cannot be evaluated without quantification.

(7) Figure 3D; Cannot be evaluated without quantification.

(8) Supplementary Figure 5B and C; Top 3 genes should be evaluated by RT-qPCR.

(9) Supplementary Figure 6; Top 3 genes should be evaluated by RT-qPCR.

(10) Figure 4A; Cannot be evaluated without quantification.

(11) Figure 4D; Cannot be evaluated without quantification.

(12) Additional experiments on in vivo infection systems comparing Plp1CreER Rosa26DTA/+ mice and controls would be great.

Reviewer #1 (Public Review):

Summary:

This is a very nice paper in which the authors addressed the potential for NK cell cellular therapy to treat and potentially eliminate previously established metastases after surgical resections, which are a major cause of death in human cancer patients. To do so they developed a model using the EO771 breast cancer cell line, in which they establish and then resect tumors and the draining lymph node, after which the majority of mice eventually succumb to metastatic disease. They found that when the initiating tumors were resected when still relatively small, adoptive transfers of IL-15/12-conditioned NK cells substantially enhanced the survival of tumor-bearing animals. They then delved into the cellular mechanisms involved. Interestingly and somewhat unexpectedly, the therapeutic effect of the transferred NK cells was dependent on the host's CD8+ T cells. Accordingly, the NK cell therapy contributed to the formation of tumor-specific CD8+ T cells, which protected the recipient animals against tumor re-challenge and were effective in protecting mice from tumor formation when transferred to naive mice. Mechanistically, they used Ifng knockout NK cells to provide evidence that IFNgamma produced by the transferred NK cells was crucial for the accumulation and activation of DCs in the metastatic lung, including expression of CD86, CD40, and MHC genes. In turn, IFNgamma production by NK cells was essential for the induced accumulation of activated CD8 effector T cells and stem cell-like CD8 T cells in the metastatic lung. The authors then expanded their findings from the mouse model to a small clinical trial. They found that inoculations of IL-15/12-conditioned autologous NK cells in patients with various malignancies after resection were safe and showed signs of efficacy.

Strengths:

- Monitoring of long-term metastatic disease and survival after resection used in this paper is a physiological model that closely resembles clinical scenarios more than the animal models usually used, a great strength of the approach.

- Previous literature focused on the notion that NK cells clear metastatic lesions directly, within a short period. The authors' use of a more relevant model and time frame revealed the previously unexplored T cell-dependent mechanism of action of infused NK cells for long-term control of metastatic diseases.

- Also important, the paper provides solid evidence for the contribution of IFNgamma produced by NK cells for activation of dendritic cells and T cells. This is an interesting finding that provokes additional questions concerning the action of the interferon-gamma in this context.

- The results from the clinical trial in cancer patients based on the same type of IL-15/12-conditioned NK cell infusions, were encouraging with respect to safety and showed signals of efficacy, which support the translatability of the author's findings.

Weaknesses:

- Having demonstrated that NK cell IFNgamma is important for recruiting and activating DCs and T cells in their model, one is left to wonder whether it is important for the therapeutic effect, which was not tested.

- Relatedly, previous studies, cited by the authors, reported that NK cells promote T cell activation by producing the chemokines CCL5 and XCL1, and FLT3 ligand, which respectively recruit and activate dendritic cells that can subsequently mobilize a T cell response. The present study demonstrates an important role for NK cell-produced IFNgamma in these processes. One is left wondering whether the model used by the authors is also dependent on CCL5, XCL1, and FLT3 production by NK cells, and if so whether IFNgamma plays a role in that or acts in parallel. The issue could be discussed by the authors, even if they cannot easily resolve it.

- The authors do not address whether the IL-12 in their cocktail is essential for the effects they see. Relatedly, it was of interest that despite the effectiveness of the transferred IL-15/IL-12 cultured NK cells, the cells failed to persist very long after transfer. Published studies have reported that so-called memory-like NK cells, which are pre-activated with a cocktail of IL-12, IL-18 and IL-15, persist much longer in lympho-depleted mice and patients than IL-2 cultured NK cells. It would be illuminating to compare these two types of NK cell products in the author's model system, and with, or without, lymphodepletion, to identify the critical parameters. If greater persistence occurred with the memory-like NK cell product, it is possible that the NK cells might provide greater benefit, including by directly targeting the tumor.

- It was somewhat difficult to gauge the clinical trial results because the trial was early stage and therefore not controlled. Evaluation of the results therefore relies on historical comparisons. To evaluate how encouraging the results are, it would be valuable for the authors to provide some context on the prognoses and likely disease progression of these patients at the time of treatment.

Reviewer #2 (Public Review):

Summary:

The authors show convincing data that increasing NK cell function/frequency can reduce the development and progression of metastatic disease after primary tumor resection.

Strengths:

The inclusion of a first-in-human trial highlighting some partial responses of metastatic patients treated with in vitro expanded NK cells is tantalising. It is difficult to perform trials in preventing further metastasis since the timelines are very protracted. However, more data like these that highlight the role of NK cells in improving local cDC1/T cells anti-tumor immunity will encourage deeper thinking around therapeutic approaches to target endogenous NK cells to achieve the same.

Weaknesses:

As always, more patient data would help increase confidence in the human relevance of the approach.

Reviewer #1 (Public Review):

Summary:

The use of a multi-omics approach to elucidate the regulatory mechanism underlying parturition and myometrial quiescence adds novelty to the study. The identification of myometrial cis-acting elements and their association with gene expression, particularly the regulation of the PLCL2 gene by PGR opens the door to further investigate the impact of PGR and other regulators.

Strengths:

(1) Multi-Omic Approach: The paper employs a comprehensive multi-omic approach, combining ChIP-Seq, RNA-Seq, and CRISPRa-based Perturb-Seq assays, which allow for a thorough investigation of the regulatory mechanisms underlying myometrial gene expression.

(2) Clinical Relevance: Investigating human myometrial specimens provides direct clinical relevance, as understanding the molecular mechanisms governing parturition and myometrial quiescence can have significant implications for the management of pregnancy-related disorders.

(3) Functional work: For functional screening, They have used CRISPRa-based screening of PLCL2 gene regulation using immortalized human cell-line hTERT-HM and T-hESC to add more dimension to the work which strengthens their finding of PGR-dependent regulation of the PLCL2 gene in the human myometrial cells.

Weaknesses:<br /> (1) Variability in epigenomic mapping: The significant variations in the number and location of H3K27ac-positive intervals across different samples and studies suggest potential challenges in accurately mapping the myometrial epigenome. This variability may introduce uncertainty and complicate the interpretation of results.

(2) Sample specificity: The study focuses on term pregnant nonlabor myometrial specimens, limiting the generalizability of the findings to other stages of pregnancy or labor.

(3) Limited Understanding of Regulatory Mechanisms: While the study identifies potential regulatory programs within super-enhancers, the exact mechanisms by which these enhancers regulate gene expression and cellular functions in the myometrium remain unclear. Further mechanistic studies are needed to elucidate these processes.

(4) Discordant analysis: Why are regular enhancers being understood in terms of motif enrichment of transcription factors and super-enhancers in terms of pathways enriched for active genes? This needs a clear reason.

Reviewer #2 (Public Review):

Summary:

In "Assessment of the Epigenomic Landscape in Human Myometrium at Term Pregnancy" the authors generate a number of genome-wide data sets to investigate epigenomic and transcriptomic regulation of the myometrium at term pregnancy. These data provide a useful resource for further evaluation of gene regulatory mechanisms in the myometrium and include the first Hi-C data published for this tissue. There is a comprehensive comparison to previously published histone modification data and integration with RNA-seq to highlight potential enhancer-gene regulatory relationships. The authors further investigate putative enhancers upstream of the PLCL2 gene and identify a candidate region that may be regulated by the PGR (progesterone receptor) signaling.

Strengths:

The strengths of this study are in the multi-omics nature of the design as several genome-wide data sets are generated from the same patient samples. Extending this type of approach in the future to a larger number of samples will allow for additional investigation into gene regulation as the correlation between epigenomic features and gene expression across a larger number of samples can reveal regulatory relationships.

Weaknesses:

One of the most interesting aspects of this study is the generation of the first Hi-C data for the human pregnant myometrium, however, there is a minimal description in the results section of the Hi-C data analysis and the only data shown are the number of loops identified and one such loop that includes the PLCL2 promoter shown in Figure 3A. The manuscript would benefit from a more extensive analysis of the Hi-C data, for example, the analysis of TADs (topological associating domains) would be interesting to add and could be used to evaluate to what extent H3K27ac domains and putative regulated genes fall within the same TAD.

The authors present some convincing evidence on the transcriptional regulation of the PLCL2 gene using Perturb-Seq to identify putative upstream enhancer regions and PGR over-expression showing PGR can act as an activator. These two experiments on their own are interesting, however, they are not as mechanistically integrated as they could be to clarify the molecular mechanisms. Deletion of the putative enhancer upstream of PLCL2 followed by over-expression of PGR would clarify the mechanistic relationship between the proposed enhancer, PGR, and PLCL2 expression. Does PGR act through the proposed enhancer? In addition, reporter assays using this proposed enhancer region with and without increased expression of PGR and mutation of any PRE sequences would also provide mechanistic insight. Although CRISPRa and Perturb-Seq can be used to identify potential regulatory regions, the best approach to verify the requirement for a particular enhancer in regulating a specific gene is a deletion approach.

Reviewer #3 (Public Review):

In this manuscript, Wu et al. investigate active H3K27ac and H3K4me1 marks in term pregnant nonlabor myometrial biopsies, linking putative-enhancers and super-enhancers to gene expression levels. Through their findings, they reveal the PGR-dependent regulation of the PLCL2 gene in human myometrial cells via a cis-acting element located 35-kilobases upstream of the PLCL2 gene. By targeting this region using a CRISPR activation system, they were able to elevate the endogenous PLCL2 mRNA levels in immortalized human myometrial cells.

This research offers novel insights into the molecular mechanisms governing gene expression in myometrial tissues, advancing our understanding of pregnancy-related processes.

Major comments:

(1) A more comprehensive analysis of the epigenetic and transcriptomic data would have strengthened the paper, moving beyond basic association studies. Currently, it is challenging to assess the quality and significance of the data as much of the information is lacking.

(2) The rationale for and connections between experiments, as well as results, could be bolstered to underscore the significance of this research.

Strengths:

- The combination of ChIP-Seq, RNA-Seq, and CRISPRa Perturb-Seq approaches to investigate gene regulation and expression in myometrial cells.

- The use of CRISPR activation system to specifically target cis-acting elements.

Weaknesses:

- The manuscript would strongly benefit from a deeper analysis of the Omic datasets. Furthermore, expanding figures/graphs to effectively contextualize these datasets would be greatly beneficial and would add more value to this research. Currently, it is difficult for us to assess and appreciate the quality of these data sets across the manuscript, which is mostly correlative.

- Limited sample size, coupled with variability in results and overall lack of details, compromises the robustness of result interpretation.

- For most parts of the results section, a better description is needed, including rationale, approach, and presentation of data. As it stands, it is challenging to assess the quality of the data and appreciate the results.

- Additional efforts are needed to dissect the proposed regulatory mechanisms.

- While the discussion provided helpful context for understanding some of the experiments performed, it lacked interpretation of the results in relation to the existing literature.

Reviewer #1 (Public Review):

Summary:

The authors sought to investigate the associations of age at breast cancer onset with the incidence of myocardial infarction (MI) and heart failure (HF). They employed a secondary data analysis of the UK Biobank. They used descriptive and inferential analysis including Cox proportional hazards models to investigate the associations. Propensity score matching was also used. They found that Among participants with breast cancer, younger onset age was significantly associated with elevated risks of MI (HR=1.36, 95%CI: 1.19 to 1.56, P<0.001) and HF (HR=1.31, 95% CI: 1.18 to 1.46, P<0.001). the reported similar findings after propensity matching.

Strengths:

The use of a large dataset is a strength of the study as the study is well-powered to detect differences. Reporting both the unmatched and the propensity-matched estimates was also important for statistical inference.

Weaknesses:

Despite the merits of the paper, readers may get confused as to whether authors are referring to "age at breast cancer onset" or "age at breast cancer diagnosis". I suppose the title refers to the latter, in which case it will be best to be consistent in using "age at breast cancer diagnosis" throughout the manuscripts. I would recommend a revision to the title to make it explicit that the authors are referring to, "age at breast cancer diagnosis".

Reviewer #2 (Public Review):

This is a well-presented large analysis from the UK Biobank of nearly 250,000 female adults. The authors examined the associations of breast cancer diagnosis with incident myocardial infarction and heart failure by different onset age groups. Based on results from a series of statistical analyses, the authors concluded that younger onset age of breast cancer was associated with myocardial infarction and heart failure, highlighting the necessity of careful monitoring of cardiovascular status in women diagnosed with breast cancer, especially those younger ones.

Comments to consider:

(1) It's thoughtful for the authors to have included and adjusted for menopausal status, breast cancer surgery, and hormone replacement therapy in their sensitivity analysis. It would be informative if the authors presented the number and percentages of menopause and cancer treatments.

(2) The analytical baseline used for follow-up should be pointed out in the methods section. It's confusing whether the analytic baseline was defined as the study baseline or the time at breast cancer diagnosis.

(3) Did the older onset age group have a longer follow-up duration? Could the authors provide information on the length of follow-up by age of onset in Supplementary Table S4? It would give the readers more information regarding different age groups.

Reviewer #1 (Public Review):

Summary:

The manuscript by Raices et al., provides novel insights into the role and interactions between SPO-11 accessory proteins in C. elegans. The authors propose a model of meiotic DSBs regulation, critical to our understanding of DSB formation and ultimately crossover regulation and accurate chromosome segregation. The work also emphasizes the commonalities and species-specific aspects of DSB regulation.

Strengths:

This study capitalizes on the strengths of the C. elegans system to uncover genetic interactions between a large number of SPO-11 accessory proteins. In combination with physical interactions, the authors synthesize their findings into a model, which will serve as the basis for future work, to determine mechanisms of DSB regulation.

Weaknesses:

The methodology, although standard, lacks quantification. This includes the mass spectrometry data, along with the cytology. The work would also benefit from clarifying the role of the DSB machinery on the X chromosome versus the autosomes.

Reviewer #2 (Public Review):

Summary:

Meiotic recombination initiates with the formation of DNA double-strand break (DSB) formation, catalyzed by the conserved topoisomerase-like enzyme Spo11. Spo11 requires accessory factors that are poorly conserved across eukaryotes. Previous genetic studies have identified several proteins required for DSB formation in C. elegans to varying degrees; however, how these proteins interact with each other to recruit the DSB-forming machinery to chromosome axes remains unclear.

In this study, Raices et al. characterized the biochemical and genetic interactions among proteins that are known to promote DSB formation during C. elegans meiosis. The authors examined pairwise interactions using yeast two-hybrid (Y2H) and co-immunoprecipitation and revealed an interaction between a chromatin-associated protein HIM-17 and a transcription factor XND-1. They further confirmed the previously known interaction between DSB-1 and SPO-11 and showed that DSB-1 also interacts with a nematode-specific HIM-5, which is essential for DSB formation on the X chromosome. They also assessed genetic interactions among these proteins, categorizing them into four epistasis groups by comparing phenotypes in double vs. single mutants. Combining these results, the authors proposed a model of how these proteins interact with chromatin loops and are recruited to chromosome axes, offering insights into the process in C. elegans compared to other organisms.

Weaknesses:

This work relies heavily on Y2H, which is notorious for having high rates of false positives and false negatives. Although the interactions between HIM-17 and XND-1 and between DSB-1 and HIM-5 were validated by co-IP, the significance of these interactions was not tested, and cataloging Y2H interactions does not yield much more insight. Moreover, most experiments lack rigor, which raises serious concerns about whether the data convincingly supports the conclusions of this paper. For instance, the XND-1 antibody appears to detect a band in the control IP; however, there was no mention of the specificity of this antibody. Additionally, epistasis analysis of various genetic mutants is based on the quantification of DAPI bodies in diakinesis oocytes, but the comparisons were made without statistical analyses. For cytological data, a single representative nucleus was shown without quantification and rigorous analysis. The rationale for some experiments is also questionable (e.g. the rescue by dsb-2 mutants by him-5 transgenes in Figure 2), making the interpretation of the data unclear. Overall, while this paper claims to present "the first comprehensive model of DSB regulation in a metazoan", cataloging Y2H and genetic interactions did not yield any new insights into DSB formation without rigorous testing of their significance in vivo. The model proposed in Figure 4 is also highly speculative.

Reviewer #3 (Public Review):

During meiosis in sexually reproducing organisms, double-strand breaks are induced by a topoisomerase-related enzyme, Spo11, which is essential for homologous recombination, which in turn is required for accurate chromosome segregation. Additional factors control the number and genome-wide distribution of breaks, but the mechanisms that determine both the frequency and preferred location of meiotic DSBs remain only partially understood in any organism.

The manuscript presents a variety of different analyses that include variable subsets of putative DSB factors. It would be much easier to follow if the analyses had been more systematically applied. It is perplexing that several factors known to be essential for DSB formation (e.g., cohesins, HORMA proteins) are excluded from this analysis, while it includes several others that probably do not directly contribute to DSB formation (XND-1, HIM-17, CEP-1, and PARG-1). The strongest claims seem to be that "HIM-5 is the determinant of X-chromosome-specific crossovers" and "HIM-5 coordinates the actions of the different accessory factors sub-groups." Prior work had already shown that mutations in him-5 preferentially reduce meiotic DSBs on the X chromosome. While it is possible that HIM-5 plays a direct role in DSB induction on the X chromosome, the evidence presented here does not strongly support this conclusion. It is also difficult to reconcile this idea with evidence from prior studies that him-5 mutations predominantly prevent DSB formation on the sex chromosomes, while the protein localizes to autosomes. The one experiment that seems to elicit the conclusion that HIM-5 expression is sufficient for breaks on the X chromosome is flawed (see below). The conclusion that HIM-5 "coordinates the activities of the different accessory sub-groups" is not supported by data presented here or elsewhere.

Like most other studies that have examined DSB formation in C. elegans, this work relies on indirect assays, here limited to the cytological appearance of RAD-51 foci and bivalent chromosomes, as evidence of break formation or lack thereof. Unfortunately, neither of these assays has the power to reveal the genome-wide distribution or number of breaks. These assays have additional caveats, due to the fact that RAD-51 association with recombination intermediates and successful crossover formation both require multiple steps downstream of DSB induction, some of which are likely impaired in some of the mutants analyzed here. This severely limits the conclusions that can be drawn. Given that the goal of the work is to understand the effects of individual factors on DSB induction, direct physical assays for DSBs should be applied; many such assays have been developed and used successfully in other organisms.

Throughout the manuscript, the writing conflates the roles played by different factors that affect DSB formation in very different ways. XND-1 and HIM-17 have previously been shown to be transcription factors that promote the expression of many germline genes, including genes encoding proteins that directly promote DSBs. Mutations in either xnd-1 or him-17 result in dysregulation of germline gene expression and pleiotropic defects in meiosis and fertility, including changes in chromatin structure, dysregulation of meiotic progression, and (for xnd-1) progressive loss of germline immortality. It is thus misleading to refer to HIM-17 and XND-1 as DSB "accessory factors" or to lump their activities with those of other proteins that are likely to play more direct roles in DSB induction. For example, statements such as the following sentence in the Introduction should be omitted or explained more clearly: "xnd-1 is also unique among the accessory factors in influencing the timing of DSBs; in the absence of xnd-1, there is precocious and rapid accumulation of DSBs as monitored by the accumulation of the HR strand-exchange protein RAD-51." The evidence that HIM-17 promotes the expression of him-5 presented here corroborates data from other publications, notably the recent work of Carelli et al. (2022), but this conclusion should not be presented as novel here. The other factors also fall into several different functional classes, some of which are relatively well understood, based largely on studies in other organisms. The roles of RAD-50 and MRE-11 in DSB induction have been investigated in yeast and other organisms as well as in several prior studies in C. elegans. DSB-1, DSB-2, and DSB-3 are homologs of relatively well-studied meiotic proteins in other organisms (Rec114 and Mei4) that directly promote the activity of Spo11, although the mechanism by which they do so is still unclear. Mutations in PARG-1 (a Poly-ADP ribose glycohydrolase) likely affect the regulation of poly-ADP-ribose addition and removal at sites of DSBs, which in turn are thought to regulate chromatin structure and recruitment of repair factors; however, there is no convincing evidence that PARG-1 directly affects break formation. CEP-1 is a homolog of p53 and is involved in the DNA damage response in the germline, but again is unlikely to directly contribute to DSB induction. HIM-5 and REC-1 do not have apparent homologs in other organisms and play poorly understood roles in promoting DSB induction. A mechanistic understanding of their functions would be of value to the field, but the current work does not shed light on this. A previous paper (Chung et al. G&D 2015) concluded that HIM-5 and REC-1 are paralogs arising from a recent gene duplication, based on genetic evidence for a partially overlapping role in DSB induction, as well as an argument based on the genomic location of these genes in different species; however, these proteins lack any detectable sequence homology and their predicted structures are also dissimilar (both are largely unstructured but REC-1 contains a predicted helical bundle lacking in HIM-5). Moreover, the data presented here do not reveal overlapping sets of genetic or physical interactions for the two genes/proteins. Thus, this earlier conclusion was likely incorrect, and this idea should not be restated uncritically here or used as a basis to interpret phenotypes.

DSB-1 was previously reported to be strictly required for all DSB and CO formation in C. elegans. Here the authors test whether the expression of HIM-5 from the pie-1 promoter can rescue DSB formation in dsb-1 mutants, and claim to see some rescue, based on an increase in the number of nuclei with one apparent bivalent (Figure 2C). This result seems to be the basis for the claim that HIM-5 coordinates the activities of other DSB proteins. However, this assay is not informative, and the conclusion is almost certainly incorrect. Notably, a substantial number of nuclei in the dsb-1 mutant (without Ppie-1::him-5) are reported as displaying a single bivalent (11 DAPI staining bodies) despite prior evidence that DSBs are absent in dsb-1 mutants; this suggests that the way the assay was performed resulted in false positives (bivalents that are not actually bivalents), likely due to inclusion of nuclei in which univalents could not be unambiguously resolved in the microscope. A slightly higher level of nuclei with a single unresolved pair of chromosomes in the dsb-1; Ppie-1::him-5 strain is thus not convincing evidence for rescue of DSBs/CO formation, and no evidence is presented that these putative COs are X-specific. The authors should provide additional experimental evidence - e.g., detection of RAD-51 and/or COSA-1 foci or genetic evidence of recombination - or remove this claim. The evidence that expression of Ppie-1::him-5 may partially rescue DSB abundance in dsb-2 mutants is hard to interpret since it is currently unknown why C. elegans expresses 2 paralogs of Rec114 (DSB-1 and DSB-2), and the age-dependent reduction of DSBs in dsb-2 mutants is not understood.

Several of the factors analyzed here, including XND-1, HIM-17, HIM-5, DSB-1, DSB-2, and DSB-3, have been shown to localize broadly to chromatin in meiotic cells. Co-immunoprecipitation of pairs of these factors, even following benzonase digestion, is not strong evidence to support a direct physical interaction between proteins. Similarly, the super-resolution analysis of XND-1 and HIM-17 (Figure 1EF) does not reveal whether these proteins physically interact with each other, and does not add to our understanding of these proteins' functions, since they are already known to bind to many of the same promoters. Promoters are also likely to be located in chromatin loops away from the chromosome axis, so in this respect, the localization data are also confirmatory rather than novel.

The phenotypic analysis of double mutant combinations does not seem informative. A major problem is that these different strains were only assayed for bivalent formation, which (as mentioned above) requires several steps downstream of DSB induction. Additionally, the basis for many of the single mutant phenotypes is not well understood, making it particularly challenging to interpret the effects of double mutants. Further, some of the interactions described as "synergistic" appear to be additive, not synergistic. While additive effects can be used as evidence that two genes work in different pathways, this can also be very misleading, especially when the function of individual proteins is unknown. I find that the classification of genes into "epistastasis groups" based on this analysis does not shed light on their functions and indeed seems in some cases to contradict what is known about their functions.

The yeast two-hybrid (Y2H) data are only presented as a single colony. While it is understandable to use a 'representative' colony, it is ideal to include a dilution series for the various interactions, which is how Y2H data are typically shown.

Additional (relatively minor) concerns about these data:

(1) Several interactions reported here seem to be detected in only one direction - e.g., MRE-11-AD/HIM-5-BD, REC-1-AD/XND-1-BD, and XND-1-AD/HIM-17-BD - while no interactions are seen with the reciprocal pairs of fusion proteins. I'm not sure if some of this is due to pasting "positive" colony images into the wrong position in the grid, but this should be addressed.

(2) DSB-3 was only assayed in pairwise combinations with a subset of other proteins; this should be explained; it is also unclear why the interaction grids are not symmetrical about the diagonal.

(3) I don't understand why the graphic summaries of Y2H data are split among 3 different figures (1, 2, and 3).

Reviewer #1 (Public Review):

Summary and Strengths:

The ability of Wolbachia to be transmitted horizontally during parasitoid wasp infections is supported by phylogenetic data here and elsewhere. Experimental analyses have shown evidence of wasp-to-wasp transmission during coinfection (eg Huigins et al), host to wasp transmission (eg Heath et al), and mechanical ('dirty needle') transmission from host to host (Ahmed et al). To my knowledge this manuscript provides the first experimental evidence of wasp to host transmission. Given the strong phylogenetic pattern of host-parasitoid Wolbachia sharing, this may be of general importance in explaining the distribution of Wolbachia across arthropods. This is of interest as Wolbachia is extremely common in the natural world and influences many aspects of host biology.

Weaknesses:

The first observation of the manuscript is that the Wolbachia strains in hosts are more closely related to those in their parasitoids. This has been reported on multiple occasions before, dating back to the late 1990s. The introduction cites five such papers (the observation is made in other studies too that could be cited) but then dismisses them by stating "However, without quantitative tests, this observation could simply reflect a bias in research focus." As these studies include carefully collected datasets that were analysed appropriately, I felt this claim of novelty was rather strong. It is unclear why downloading every sequence in GenBank avoids any perceived biases, when presumably the authors are reanalysing the data in these papers.

I do not doubt the observation that host-parasitoid pairs tend to share related Wolbachia, as it is corroborated by other studies, the effect size is large, and the case study of whitefly is clearcut. It is also novel to do this analysis on such a large dataset. However, the statistical analysis used is incorrect as the observations are pseudo-replicated due to phylogenetic non-independence. When analysing comparative data like this it is essential to correct for the confounding effects of related species tending to be similar due to common ancestry. In this case, it is well-known that this is an issue as it is a repeated observation that related hosts are infected by related Wolbachia. However, the authors treat every pairwise combination of species (nearly a million pairs) as an independent observation. Addressing this issue is made more complex because there are both the host and symbiont trees to consider. The additional analysis in lines 123-124 (including shuffling species pairs) does not explicitly address this issue.

The sharing of Wolbachia between whitefly and their parasitoids is very striking, although this has been reported before (eg the authors recently published a paper entitled "Diversity and Phylogenetic Analyses Reveal Horizontal Transmission of Endosymbionts Between Whiteflies and Their Parasitoids"). In Lines 154-164 it is suggested that from the tree the direction of transfer between host and parasitoid can be inferred from the data. This is not obvious to me given the poor resolution of the tree due to low sequence divergence. There are established statistical approaches to test the direction of trait changes on a tree that could have been used (a common approach is to use the software BEAST).

Reviewer #2 (Public Review):

The paper by Yan et al. aims to provide evidence for horizontal transmission of the intracellular bacterial symbiont Wolbachia from parasitoid wasps to their whitefly hosts. In my opinion, the paper in its current form consists of major flaws.

Weaknesses:

The dogma in the field is that although horizontal transmission events of Wolbachia occur, in most systems they are so rare that the chances of observing them in the lab are very slim.<br /> For the idea of bacteria moving from a parasitoid to its host, the authors have rightfully cited the paper by Hughes, et al. (2001), which presents the main arguments against the possibility of documenting such transmissions. Thus, if the authors want to provide data that contradict the large volume of evidence showing the opposite, they should present a very strong case.

In my opinion, the paper fails to provide such concrete evidence. Moreover, it seems the work presented does not meet the basic scientific standards.

My main reservations are:

- I think the distribution pattern of bacteria stained by the probes in the FISH pictures presented in Figure 4 looks very much like Portiera, the primary symbiont found in the bacterium of all whitefly species. In order to make a strong case, the authors need to include Portiera probes along with the Wolbachia ones.

- If I understand the methods correctly, the phylogeny presented in Figure 2a is supposed to be based on a wide search for Wolbachia wsp gene done on the NCBI dataset (p. 348). However, when I checked the origin of some of the sequences used in the tree to show the similarity of Wolbachia between Bemisia tabaci and its parasitoids, I found that most of them were deposited by the authors themselves in the course of the current study (I could not find this mentioned in the text), or originated in a couple of papers that in my opinion should not have been published to begin with.

- The authors fail to discuss or even acknowledge a number of published studies that specifically show no horizontal transmission, such as the one claimed to be detected in the study presented.

Reviewer #3 (Public Review):

This is a very ordinary research paper. The horizontal of endosymbionts, including Wolbachia, Rickettsia etc. has been reported in detail in the last 10 years, and parasitoid vectored as well as plant vectored horizontal transmission is the mainstream of research. For example, Ahmed et al. 2013 PLoS One, 2015 PLoS Pathogens, Chiel et al. 2014 Enviromental Entomology, Ahmed et al. 2016 BMC Evolution Biology, Qi et al. 2019 JEE, Liu et al. 2023 Frontiers in Cellular and Infection Microbiology, all of these reported the parasitoid vectored horizontal transmission of endosymbiont. While Caspi-Fluger et al. 2012 Proc Roy Soc B, Chrostek et al. 2017 Frontiers in Microbiology, Li et al. 2017 ISME Journal, Li et al. 2017 FEMS, Shi et al. 2024 mBio, all of these reported the plant vectored horizontal transmission of endosymbiont. For the effects of endosymbiont on the biology of the host, Ahmed et al. 2015 PLoS Pathogens explained the effects in detail.

Weaknesses:

In the current study, the authors downloaded the MLST or wsp genes from a public database and analyzed the data using other methods, and I think the authors may not be familiar with the research progress in the field of insect symbiont transmission, and the current stage of this manuscript lacking sufficient novelty.

Reviewer #1 (Public Review):

Summary:

In "Changes in wing morphology..." Roy et al investigate the potential allometric scaling in wing morphology and wing kinematics in 8 different hoverfly species. Their study nicely combines different new and classic techniques, investigating flight in an important, yet understudied alternative pollinator. I want to emphasize that I have been asked to review this from a hoverfly biology perspective, as I do not work on flight kinematics. I will thus not review that part of the work.

Strengths:

The paper is well-written and the figures are well laid out. The methods are easy to follow, and the rationale and logic for each experiment are easy to follow. The introduction sets the scene well, and the discussion is appropriate. The summary sentences throughout the text help the reader.

Weaknesses:

The ability to hover is described as useful for either feeding or mating. However, several of the North European species studied here would not use hovering for feeding, as they tend to land on the flowers that they feed from. I would therefore argue that the main selection pressure for hovering ability could be courtship and mating. If the authors disagree with this, they could back up their claims with the literature. On that note, a weakness of this paper is that the data for both sexes are merged. If we agree that hovering may be a sexually dimorphic behaviour, then merging flight dynamics from males and females could be an issue in the interpretation. I understand that separating males from females in the movies is difficult, but this could be addressed in the Discussion, to explain why you do not (or do) think that this could cause an issue in the interpretation.

The flight arena is not very big. In my experience, it is very difficult to get hoverflies to fly properly in smaller spaces, and definitely almost impossible to get proper hovering. Do you have evidence that they were flying "normally" and not just bouncing between the walls? How long was each 'flight sequence'? You selected the parts with the slowest flight speed, presumably to get as close to hovering as possible, but how sure are you that this represented proper hovering and not a brief slowdown of thrust?

Your 8 species are evolutionarily well-spaced, but as they were all selected from a similar habitat (your campus), their ecology is presumably very similar. Can this affect your interpretation of your data? I don't think all 6000 species of hoverflies could be said to have similar ecology - they live across too many different habitats. For example, on line 541 you say that wingbeat kinematics were stable across hoverfly species. Could this be caused by their similar habitat?

Reviewer #2 (Public Review):

Summary

Le Roy et al quantify wing morphology and wing kinematics across eight hoverfly species that differ in body mass; the aim is to identify how weight support during hovering is ensured. Wing shape and relative wing size vary significantly with body mass, but wing kinematics are reported to be size-invariant. On the basis of these results, it is concluded that weight support is achieved solely through size-specific variations in wing morphology and that these changes enabled hoverflies to decrease in size throughout their phylogenetic history. Adjusting wing morphology may be preferable compared to the alternative strategy of altering wing kinematics, because kinematics may be under strong evolutionary and ecological constraints, dictated by the highly specialised flight and ecology of the hoverflies.

Strengths

The study deploys a vast array of challenging techniques, including flight experiments, morphometrics, phylogenetic analysis, and numerical simulations; it so illustrates both the power and beauty of an integrative approach to animal biomechanics. The question is well motivated, the methods appropriately designed, and the discussion elegantly and convincingly places the results in broad biomechanical, ecological, evolutionary, and comparative contexts.

Weaknesses

(1) In assessing evolutionary allometry, it is key to identify the variation expected from changes in size alone. The null hypothesis for wing morphology is well-defined (isometry), but the equivalent predictions for kinematic parameters remain unclear. Explicit and well-justified null hypotheses for the expected size-specific variation in angular velocity, angle-of-attack, stroke amplitude, and wingbeat frequency would substantially strengthen the paper, and clarify its evolutionary implications.

(2) By relating the aerodynamic output force to wing morphology and kinematics, it is concluded that smaller hoverflies will find it more challenging to support their body mass - a scaling argument that provides the framework for this work. This hypothesis appears to stand in direct contrast to classic scaling theory, where the gravitational force is thought to present a bigger challenge for larger animals, due to their disadvantageous surface-to-volume ratios. The same problem ought to occur in hoverflies, for wing kinematics must ultimately be the result of the energy injected by the flight engine: muscle. Much like in terrestrial animals, equivalent weight support in flying animals thus requires a positive allometry of muscle force output. In other words, if a large hoverfly is able to generate the wing kinematics that suffice to support body weight, an isometrically smaller hoverfly should be, too (but not vice versa). Clarifying the relation between the scaling of muscle force input, wing kinematics, and weight support would resolve the conflict between these two contrasting hypotheses, and considerably strengthen the biomechanical motivation and interpretation.

(3) The main conclusion - that evolutionary miniaturization is enabled by changes in wing morphology - is only weakly supported by the evidence. First, although wing morphology deviates from the null hypothesis of isometry, the difference is small, and hoverflies about an order of magnitude lighter than the smallest species included in the study exist. Including morphological data on these species, likely accessible through museum collections, would substantially enhance the confidence that size-specific variation in wing morphology occurs not only within medium-sized but also in the smallest hoverflies, and has thus indeed played a key role in evolutionary miniaturization. Second, although wing kinematics do not vary significantly with size, clear trends are visible; indeed, the numerical simulations revealed that weight support is only achieved if variations in wing beat frequency across species are included. A more critical discussion of both observations may render the main conclusions less clear-cut, but would provide a more balanced representation of the experimental and computational results.

In many ways, this work provides a blueprint for work in evolutionary biomechanics; the breadth of both the methods and the discussion reflects outstanding scholarship. It also illustrates a key difficulty for the field: comparative data is challenging and time-consuming to procure, and behavioural parameters are characteristically noisy. Major methodological advances are needed to obtain data across large numbers of species that vary drastically in size with reasonable effort, so that statistically robust conclusions are possible.

Reviewer #3 (Public Review):

The paper by Le Roy and colleagues seeks to ask whether wing morphology or wing kinematics enable miniaturization in an interesting clade of agile flying insects. Isometry argues that insects cannot maintain both the same kinematics and the same wing morphology as body size changes. This raises a long-standing question of which varies allometrically. The authors do a deep dive into the morphology and kinematics of eight specific species across the hoverfly phylogeny. They show broadly that wing kinematics do not scale strongly with body size, but several parameters of wing morphology do in a manner different from isometry leading to the conclusion that these species have changed wing shape and size more than kinematics. The authors find no phylogenetic signal in the specific traits they analyze and conclude that they can therefore ignore phylogeny in the later analyses. They use both a quasi-steady simplification of flight aerodynamics and a series of CFD analyses to attribute specific components of wing shape and size to the variation in body size observed. However, the link to specific correlated evolution, and especially the suggestion of enabling or promoting miniaturization, is fraught and not as strongly supported by the available evidence.

The aerodynamic and morphological data collection, modeling, and interpretation are very strong. The authors do an excellent job combining a highly interpretable quasi-steady model with CFD and geometric morphometrics. This allows them to directly parse out the effects of size, shape, and kinematics.

Despite the lack of a relationship between wing kinematics and size, there is a large amount of kinematic variation across the species and individual wing strokes. The absolute differences in Figure 3F - I could have a very large impact on force production but they do indeed not seem to change with body size. This is quite interesting and is supported by aerodynamic analyses.

The authors switch between analyzing their data based on individuals and based on species. This creates some pseudoreplication concerns in Figures 4 and S2 and it is confusing why the analysis approach is not consistent between Figures 4 and 5. In general, the trends appear to be robust to this, although the presence of one much larger species weighs the regressions heavily. Care should be taken in interpreting the statistical results that mix intra- and inter-specific variation in the same trend.

The authors based much of their analyses on the lack of a statistically significant phylogenetic signal. The statistical power for detecting such a signal is likely very weak with 8 species. Even if there is no phylogenetic signal in specific traits, that does not necessarily mean that there is no phylogenetic impact on the covariation between traits. Many comparative methods can test the association of two traits across a phylogeny (e.g. a phylogenetic GLM) and a phylogenetic PCA would test if the patterns of variation in shape are robust to phylogeny.

The analysis of miniaturization on the broader phylogeny is incomplete. The conclusion that hoverflies tend towards smaller sizes is based on an ancestral state reconstruction. This is difficult to assess because of some important missing information. Specifically, such reconstructions depend on branch lengths and the model of evolution used, which were not specified. It was unclear how the tree was time-calibrated. Most often ancestral state reconstructions utilize a maximum likelihood estimate based on a Brownian motion model of evolution but this would be at odds with the hypothesis that the clade is miniaturizing over time. Indeed such an analysis will be biased to look like it produces a lot of changes towards smaller body size if there is one very large taxa because this will heavily weight the internal nodes. Even within this analysis, there is little quantitative support for the conclusion of miniaturization, and the discussion is restricted to a general statement about more recently diverged species. Such analyses are better supported by phylogenetic tests of directedness in the trait over time, such as fitting a model with an adaptive peak or others.

Setting aside whether the clade as a whole tends towards smaller size, there is a further concern about the correlation of variation in wing morphology and changes in size (and the corresponding conclusion about lack of co-evolution in wing kinematics). Showing that there is a trend towards smaller size and a change in wing morphology does not test explicitly that these two are correlated with the phylogeny. Moreover, the subsample of species considered does not appear to recapitulate the miniaturization result of the larger ancestral state reconstruction.

Given the limitations of the phylogenetic comparative methods presented, the authors did not fully support the general conclusion that changes in wing morphology, rather than kinematics, correlate with or enable miniaturization. The aerodynamic analysis across the 8 species does however hold significant value and the data support the conclusion as far as it extends to these 8 species. This is suggestive but not conclusive that the analysis of consistent kinematics and allometric morphology will extend across the group and extend to miniaturization. Nonetheless, hoverflies face many shared ecological pressures on performance and the authors summarize these well. The conclusions of morphological allometry and conserved kinematics are supported in this subset and point to a clade-wide pattern without having to support an explicit hypothesis about miniaturization.

The data and analyses on these 8 species provide an important piece of work on a group of insects that are receiving growing attention for their interesting behaviors, accessibility, and ecologies. The conclusions about morphology vs. kinematics provide an important piece to a growing discussion of the different ways in which insects fly. Sometimes morphology varies, and sometimes kinematics depending on the clade, but it is clear that morphology plays a large role in this group. The discussion also relates to similar themes being investigated in other flying organisms. Given the limitations of the miniaturization analyses, the impact of this study will be limited to the general question of what promotes or at least correlates with evolutionary trends towards smaller body size and at what phylogenetic scale body size is systematically decreasing.

In general, there is an important place for work that combines broad phylogenetic comparison of traits with more detailed mechanistic studies on a subset of species, but a lot of care has to be taken about how the conclusions generalize. In this case, since the miniaturization trend does not extend to the 8 species subsample of the phylogeny and is only minimally supported in the broader phylogeny, the paper warrants a narrower conclusion about the connection between conserved kinematics and shared life history/ecology.

Reviewer #1 (Public Review):

Summary:

This technical report by Kugler at al., expands the application of a fluorescence-based reporter to study the conformational state of various kinases. This reporter, named KinCon (Kinase Conformation), interrogates the conformational state of a kinase (i.e., active vs. inactive) based on engineering complementary fusion proteins that fluoresce upon interaction. This assay has several advantages as it allows studying full-length kinases, that is, the kinase domain and regulatory domains, inside the cell and under various experimental conditions such as the presence of inhibitors or activator proteins, and in wildtype and mutants involved in disease states.

Strengths:

One major strength of this study is that it is quite comprehensive. The authors use KinCon for four different kinases, BRAF, LKB1, RIP and CDK4/6. These kinases have very different regulatory elements and associated proteins, which the authors explore to study their conformational state. Moreover, they use small molecule inhibitors or mutations to further dissect how the conformational state of the kinase in disease states. The collective set of results strongly suggests that KinCon is a versatile tool that can be used to study many kinases of biomedical and fundamental importance. Given that kinases are extensively studied by researchers in academia or industry, KinCon could have a broad impact as well.

Weaknesses:

This manuscript, however, also has several weaknesses that I outline below. These weaknesses decrease the overall level of impact on the manuscript, as is.<br /> • The manuscript is exceedingly long. For instance, the introduction provides background information for each kinase that is further expanded in the results section. I think the background information for each kinase in the Introduction and Results sections can be significantly reduced to highlight the major points. Otherwise, not only does the manuscript become too long, but also the main points get diluted.

• Similarly, the figure legends are very long, providing information that is already in the main text or in Methods. The authors should provide the essential information to understand the figure.

• A major concern throughout the manuscript is the use of the word "dynamics," which is used in the text in various contexts. The authors should clarify what they understand for dynamics of conformation. Are they measuring how the time-dependent process by which the kinase is interconverting between active and inactive states? It seems to me that the assays in this report evaluate a population of kinases that are in an open or close conformation (i.e., a particular state in each experimental condition) but there is not direct information how the kinase goes from one state to the other. In that sense, the use of dynamics is unclear. Also, the use of dynamics in different sentences in ambiguous. Here are a few examples but this should be revised throughout the manuscript:<br /> - Line 27: dynamics of full-length protein kinases. Is this referred to dynamics of conformational interconversion between inactive and active states?<br /> - Line 138: dynamic functioning of kinases. No clear what that means.<br /> - Line 276: ... alters KinCon dynamics. Not clear if they are measuring time-dependent process or a single point.<br /> - Figure legend 4F: dynamics of CDK4/6 reporters. Again, not clear how the assay is measuring dynamics.<br /> Nonetheless, in my opinion the authors use proper terminology that describes their assay in which the term dynamics is not used: Title (... impact of protein and small molecule interactions on kinase conformations) and Line 89 (... reporter can be used to track conformational changes of kinases...)

• The authors use the phrase that KinCon has predictive capabilities (abstract and line 142). What do the authors refer to this?

• The authors indicate that KinCon is a highly sensitive assay. Can the authors elaborate on what high sensitivity means? For example, can they discuss how other fluorescence-based approaches that are less sensitive would not be able to accomplish the same type of results or derive similar conclusions? Can they provide a resolution metric both in space and time? Given that the authors state that this is a technical report, this information is of relevance.

• The authors nicely describe how KinCon works in Figure 1B and part of 1C. I do think that the bottom of panel 1C needs to be revised, as well as the text describing the potential scenarios of potency, efficacy and synergism.<br /> - One issue with this part of Figure 1C is that it is not clear what the x-axis in the 3 plots refer to. Is this time? Is this concentration of a small molecule, inhibitor or binding partner? This was confusing also in the context of the term dynamics used throughout the text. The terms potency, efficacy and synergism should be subtitles or the panels and the x-axis should be better defined, especially for a non-specialized reader.<br /> - Related to this part of Figure 1C is the text. The authors mention potency, effectiveness and synergy (Line 195). Can the authors use more fundamental terminology related to these three scenarios, for example, changes in activation constant, percent of protein activates? Also, why synergy is only related to effectiveness? Can synergy also be associated to potency?<br /> - Lastly, the use of these three cartoons gives the impression that the experimental results to come will follow a similar representation. Instead, the results are presented in bar plots for many different conditions. I think this will lead to confusion for a broad audience.

• For a non-expert reader, can the authors clarify the use of tracking basal conformations vs. transient over-expression of the various KinCon constructs? Moreover, the authors use the term transient over-expression for 10, 16, 24 and 48 h (Line 203). This, to a non-expert reader, seems not transient.

• Regarding Figure 1E and similar graphical representations: Why is the signal (RLU) non-linear with time? If the fluorescence of the KinCon construct is linearly related with its expression or concentration inside the cell, one would expect a linear increase. Have the authors plotted RLU/Expression band intensity to account for changes in protein concentration? For instance, some of the results within Figure 3 are normalized to concentration on the reporter expression level.

• For the results with LKB1, the authors claim that intermediate fold change in fluorescence (Figure 2E) is due to a partially closed intermediate state (Line 262). Can the authors discard the possibility by which there is a change in populations of active and inactive that on average give intermediate values?

• The authors claim in Line 274 that mutations located at the interface of the LKB1/STRADalkpha complex affect interactions and hypothesize that allosteric communication between LKB1 and STRADalpha is essential for function. Given that this mutations are at the interaction interface, why would the authors postulate an allosteric mechanism that evokes an effect distant to the interaction/active site? Could it be that function requires surface contacts alone that are disrupted by the mutations?

• I was unable to find text to explain the following: Figure 2I shows the mutation R74A as n.s., but in the text only W308C is mentioned to not change fluorescence. Could the authors clarify why R74A is not discussed in the text? Maybe this reviewer missed the text in which it was discussed. Similarly, the author states in line 326 that the study included an analysis of RIPK2. However, I was unable to find results, graphs or additional text discussing RIPK2.

• Some figures of RLU use absolute values, percentages and fold change. Is there a reason why the authors use different Y-axis values? These should be explained and justified in Methods. Similarly, bars for wt in Figures 3D, G, or 4D, E,F show no errors. How are the authors normalizing the data and repeats so that there is no error, and are they treating the rest of the data (i.e., mutants and/or treated with small molecules) in the same way?

• Lastly, the section starting in Line 472 reads more like a discussion of results from different type of inhibitors used in this study that results on its own. The authors should consider a new subtitle as results or make this section a discussion.

Reviewer #2 (Public Review):

Summary:

Protein kinases have been very successfully targeted with small molecules for several decades, with many compounds (including clinical drugs) bringing about conformational changes that are also relevant to broader interactions with the cellular signaling networks that they control. The authors set out to develop a targeted biosensor approach to evaluate distinct kinase conformations in cells for multiple kinases in the context of incoming signals, other proteins and small molecule binding, with a broad goal of using the KinCon assay to confirm (and perhaps predict) how drug binding or signal perception changes conformations and outputs in the presence of cellular complexes; this work will likely impact on the field with cellular reporters of kinase conformations a useful addition to the toolbox.

Strengths:

The KinCon reporter platform has previously been validated for well-known kinases; in this study, the team evaluate how to employ a full-length kinase (often containing a known pathological mutation). The sensitive detection method is based on a Renilla luciferase (RLuc)protein fragment complementation assay, where individual RLuc fragments are present at the N and the C terminus of the kinase. This report, which is both technical and practical in nature, co-expresses the kinase with known interactors (at low levels) in a high throughput format and then performs pharmacological evaluation with known small molecule kinase modulators. This is explained nicely in Figure 1, as are the signaling pathways that are being evaluated. Data demonstrate that V600E BRAF iexposed to vemurafenib is converted to the inactive conformation, as expected. In contrast, the more closed STRAD𝛼 and LKB1 KinCon conformations appear to represent the more active state of the complexed kinase, and a W308C mutation (evaluated alongside others) reverses this effect. The authors then evaluated necroptotic signaling in the context of RIPK1/3 under conditions where RIPK1 and RIPK3 are active, confirming that the reporters highlight the active states of both kinases. Exposure to compounds that are known to engage with the RIPK1 arm of the pathway induce bioluminescence changes consistent with the opening (inactivation) of the kinase. Finally, the authors move to an important drug target for which clinical drugs have arrived relatively recently; the CDK4/6 complexes. These are of additional importance because kinase-independent functions also exist for CDK6, and the effects of drugs in cells usually relies on a downstream marker, rather than demonstration of direct protein complex engagement. The data presented are interpreted as the formation of complexes with the CDK inhibitor p16INK4a; reducing the affinity of the interaction through mutations drives an inactive conformation, whilst the application of CDK4/6 inhibitors does not, implying binding to the active conformation.

Weaknesses:

(1) The work is very solid, and uses examples from the literature and also extends into new experimental space. An obvious weakness is mentioned by the authors for the CKDK data, in that measurements with Cyclin D (the activating subunit) are not characterised, although Cyclin D might be assumed to be present?<br /> (2) The work with the trimeric LKB1 complex involves pseudokinase, STRADalpha, whose conformation is also examined as a function of LKB1 status; since STRAD is an activator of LKB1, a future goal should be the evaluation of the complex in the presence of STRAD inhibitory/activating small molecules.

Reviewer #2 (Public Review):

In this study, Zhenbang Ye and colleagues investigate the links between microenvironment signatures, gene expression profiles, and prognosis in diffuse large B-cell lymphoma (DLBCL). They show that increased tumor purity (ie, a higher proportion of tumor cells relative to surrounding stromal components) is associated with worse prognosis. They then show that three genes associated with tumor purity (VCAN, CD3G, and C1QB) correlate with patterns of immune cell infiltration and can be used to create a risk scoring system that predicts prognosis, which can be replicated by immunohistochemistry (IHC), and response to some therapies.

(1) The two strengths of the study are its relatively large sample size (n = 190) and the strong prognostic significance of the risk scoring system. It is worth noting that the validation of this scoring with IHC, a simple technique already routinely used for the diagnosis and classification of DLBCL, increases the potential for clinical translation. However, the correlative nature of the study limits the conclusions that can be drawn in regards to links between the risk scoring system, the tumor microenvironment, and the biology of DLBCL.

(2) The tumor microenvironment has been extensively studied in DLBCL and a prognostic implication has already been established (for instance, Steen et al., Cancer Cell, 2021). In addition, associations have already been established in non-Hodgkin lymphoma between prognosis and expression of C1QB (Rapier-Sharman et al., Journal of Bioinformatics and Systems Biology, 2022), VCAN (S. Hu et al., Blood, 2013), and CD3G (Chen et al., Medical Oncology, 2022). Nevertheless, one of the strengths and novelty aspect of the study is the combination of these 3 genes into a risk score that is also valid by immunohistochemistry (IHC), which substantially facilitates a potential clinical translation.

(3) Figures 1A-B: tumor purity is calculated using the ESTIMATE (Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data) algorithm (Yoshihara et al., Nature Communications, 2013). The ESTIMATE algorithm is based on two gene signatures ("stromal" and "immune"). It is therefore expected that tumor purity measured by the ESTIMATE algorithm will correlate with the expression of multiple genes. Importantly, C1QB is included in the stromal signature of the ESTIMATE algorithm meaning that, by definition, it will be correlated with tumor purity in that setting.

(4) Figure 2A: as established in figure 1C, high tumor purity is associated with worse prognosis. Later in the manuscript, it is also shown that C1QB expression is associated with worse prognosis. However, figure 2A shows that C1QB is associated with decreased tumor purity. It therefore makes it less likely that the prognostic role of C1QB expression is related to its impact on tumor purity. The prognostic impact could be related to different patterns of immune cell infiltration, as shown later. However, the evidence presented in the study is correlative and nature and not sufficient to draw this conclusion.

(5) Figure 3G: although there is a strong prognostic implication of the risk score on prognosis, the correlation between the risk score and tumor purity is significant but not very strong (R = 0.376). It is therefore likely that other important biological factors explain the correlation between the risk score and prognosis, as suggested in the gene set enrichment analysis that is later performed.

(6) Figure 6: the drug sensitivity analysis includes a wide range of established and investigational drugs with varied mechanisms of action. Although the difference in sensitivity between tumors with low and high risk scores show statistical significance for certain drugs, the absolute difference appears small in most cases and is of unclear biological significance. In addition, even though the risk score is statistically related to drug sensitivity, there is no direct evidence that the differences in drug sensitivity are directly related to tumor purity.

Reviewer #1 (Public Review):

The authors describe a framework for working with genotype data and associated metadata, specifically geared towards ancient DNA. The Poseidon framework aims to address long-standing data coordination issues in ancient population genomics research. These issues can usefully be thought of as two primary, separate problems:

(1) The genotype merging problem. Often, genotype calls made by a new study are not made publicly available, or they are only made available in an ad-hoc fashion without consistency in formatting between studies. Other users will typically want to combine genotypes from many previously published studies with their own newly produced genotypes, but a lack of coordination and standards means that this is challenging and time-consuming.

(2) The metadata problem. All genomes need informative metadata to be usable in analyses, and this is even more true for ancient genomes which have temporal and often cultural dimensions to them. In the ancient DNA literature, metadata is often only made available in inconsistently formatted supplementary tables, such that reuse requires painstakingly digging through these to compile, curate and harmonise metadata across many studies.

Poseidon aims to solve both of these problems at the same time, and additionally provide a bit of population genetics analysis functionality. The framework is a quite impressive effort, that clearly has taken a lot of work and thought. It displays a great deal of attention to important aspects of software engineering and reproducibility. How much usage it will receive beyond the authors themselves remains to be seen, as there is always a barrier to entry for any new sophisticated framework. But in any case, it clearly represents a useful contribution to the human ancient genomics community.

The paper is quite straightforward in that it mainly describes the various features of the framework, both the way in which data and metadata are organised, and the various little software tools provided to interact with the data. This is all well-described and should serve as a useful introduction for any users of the framework, and I have no concerns with the presentation of the paper. Perhaps it gets a bit too detailed for my taste at times, but it's up to the authors how they want to write the paper.

I thus have no serious concerns with the paper. I do have some thoughts and comments on the various choices made in the design of the framework, and how these fit into the broader ecosystem of genomics data. I wouldn't necessarily describe much of what follows as criticism of what the authors have done - the authors are of course free to design the framework and software that they want and think will be useful. And the authors clearly have done more than basically anyone else in the field to tackle these issues. But I still put forth the points below to provide some kind of wider discussion within the context of ancient genomics data management and its future.

* * *

The authors state that there is no existing archive for genotype data. This is not quite true. There is the European Variation Archive (EVA, https://www.ebi.ac.uk/eva/), which allows archiving of VCFs and is interlinked to raw data in the ENA/SRA/DDBJ. If appropriately used, the EVA and associated mainstream infrastructure could in principle be put to good use by the ancient genomics community. In practice, it's basically not used at all by the ancient genomics community, and partly this is because EVA doesn't quite provide exactly what's needed (in particular with regards to metadata fields). Poseidon aims to provide a much more custom-tailored solution for the most common use cases within the human ancient DNA field, but it could be argued that such a solution is only needed because the ancient genomics community has largely neglected the mainstream infrastructure. In some sense, by providing such a custom-tailored solution that is largely independent of the mainstream infrastructure, I feel like efforts such as Poseidon (and AADR) - while certainly very useful - might risk contributing to further misaligning the ancient genomics community from the rest of the genomics community, rather than bringing it closer. But the authors cannot really be blamed for that - they are simply providing a resource that will be useful to people given the current state of things.

The BioSamples database (https://www.ebi.ac.uk/biosamples/) is an attempt to provide universal sample IDs across the life sciences and is used by the archives for sequence reads (ENA/SRA/DDBJ). Essentially every published ancient sample already has a BioSample accession, because this is required for the submission of sequence reads to ENA/SRA/DDBJ. It would thus have seemed natural to make BioSamples IDs a central component of Poseidon metadata, so as to anchor Poseidon to the mainstream infrastructure, but this is not really done. There are some links being made to ENA in the .ssf "sequence source" files used by the Poseidon package, including sample accessions, but this seems more ad-hoc.

The package uses PLINK and EIGENSTRAT file formats to represent genotypes, which in my view are not particularly good formats for long-term and rigorous data management in genomics. These file formats cannot appropriately represent multiallelic loci, haplotype phase, or store information on genotype qualities, coverage, etc. The standard in the rest of genomics is VCF, a much more robust and flexible format with better software built around it. Insisting on keeping using these arguably outdated formats is one way in which the ancient genomics community risks disaligning itself from the mainstream.

I could not find any discussion of reference genomes: knowing the reference genome coordinate system is essential to using any genotype file. For comparison, in the EVA archive, every VCF dataset has a "Genome Assembly" metadata field specifying the accession number of the reference genome used. It would seem to me like a reference genome field should be part of a Poseidon package too. In practice, the authors likely use some variant of the hg19 / GRCh37 human reference, which is still widely used in ancient genomics despite being over a decade out of date. Insisting on using an outdated reference genome is one way in which the ancient genomics community is disaligning itself from the mainstream, and it complicates comparisons to data from other sub-fields of genomics.

A fundamental issue contributing to the genome merging problem, not unique to ancient DNA, is that genotype files are typically filtered to remove sites that are not polymorphic within the given study - this means that files from two different studies will often contain different and not fully overlapping sets of sites, greatly complicating systematic merging. I don't see any discussion of how Poseidon deals with this. In practice, it seems the authors are primarily concerned with data on the commonly used 1240k array set, such that the set of SNPs is always well-defined. But does Poseidon deal with the more general problem of non-overlapping sites between studies, or is this issue simply left to the user to worry about? This would be of relevance to whole-genome sequencing data, and there are certainly plenty of whole-genome datasets of great interest to the research community (including archaic human genomes, etc).

In principle, it seems the framework could be species-agnostic and thus be useful more generally beyond humans (perhaps it would be enough to add just one more "species" metadata field?). It is of course up to the authors to decide how broadly they want to cater.

Reviewer #2 (Public Review):

Summary:

Schmid et al. provide details of their new data management tool Poseidon which is intended to standardise archaeogenetic genotype data and combine it with the associated standardised metadata, including bibliographic references, in a way that conforms to FAIR principles. Poseidon also includes tools to perform standard analyses of genotype files, and the authors pitch it as the potential first port of call for researchers who are planning on using archaeogenetic data in their research. In fact, Poseidon is already up and running and being used by researchers working in ancient human population genetics. To some extent, it is already on its way to becoming a fundamental resource.

Strengths:

A similar ancient genomics resource (The Ancient Allen Database) exists, but Poseidon is several steps ahead in terms of integration and standardisation of metadata, its intrinsic analytical tools, its flexibility, and its ambitions towards being independent and entirely community-driven. It is clear that a lot of thought has gone into each aspect of what is a large and dynamic package of tools and overall it is systematic and well thought through.

Weaknesses:

The main weakness of the plans for Poseidon, which admirably the authors openly acknowledge, is in how to guarantee it is maintained and updated over the long term while also shifting to a fully independent model. The software is currently hosted by the MPI, although the authors do set out plans to move it to a more independent venue. However, the core team comprising the authors is funded by the MPI, and so the MPI is also the main funder of Poseidon. The authors do state their ambition to move towards a community-driven independent model, but the details of how this would happen are a bit vague. The authors imagine that authors of archaeogenetic papers would upload data themselves, thereby making all authors of archaeogenetics papers the voluntary community who would take on the responsibility of maintaining Poseidon. Archaeogeneticists generally are committed enough to their field that there is a good chance such a model would work but it feels haphazard to rely on goodwill alone. Given there needs to be a core team involved in maintaining Poseidon beyond just updating the database, from the paper as it stands it is difficult to see how Poseidon might be weaned off MPI funding/primary involvement and what the alternative is. However, the same anxieties always surround these sorts of resources when they are first introduced. The main aim of the paper is to introduce and explain the resource rather than make explicit plans for its future and so this is a minor weakness of the paper overall.

Reviewer #1 (Public Review):

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

Detailed major concerns:

Related to Figure 1.<br /> It is imperative to ensure consistency in the number of animals analyzed across the different graphs. The current resolution of the images appears to be low, resulting in unsharp visuals that hinder the interpretation of data beyond the presence of "white dots". To address this issue, it is recommended to enhance the resolution of the images and consider incorporating zoom-in features to facilitate a clearer visualization of the observed differences. Moreover, it would be beneficial to include a complete WB analysis for the cell death pathways analyzed. These adjustments will significantly improve the clarity and interpretability of Figure 1.

Related to Figure 2.<br /> It is essential to ensure consistency in the number of animals analyzed across the different graphs, as indicated by n=6 in the figure legend (similar to Figure 1). Additionally, it is crucial to distinguish between male and female subjects in the dot plots to assess any potential gender-based differences, which should be consistent throughout the paper. To achieve this, the dots plot should be harmonized to clearly differentiate between males and females and investigate if there are any disparities between the genders. Moreover, it is imperative to correlate hepatic inflammation with the activation of Kupffer cells, infiltrating monocytes, and/or hepatic stellate cells (HSCs). Therefore, conducting flow cytometry would be instrumental in achieving this correlation. Additionally, the staining for Ki67 appears to be non-specific, showing a granular pattern reminiscent of bile crystals rather than the expected nuclear staining of hepatocytes or immune cells. It is crucial to ensure specific staining for Ki67, and conducting in vitro experiments on primary hepatocytes could further elucidate the proliferation process. These experiments are relatively straightforward to implement and would provide valuable insights into the mechanisms underlying hepatic inflammation and proliferation.

Related to Figure 3 & related to Figure 4.<br /> The immunofluorescence data presented are not entirely convincing and are insufficient to conclusively demonstrate the recruitment of monocytes. Previous suggestions for flow cytometry studies remain pertinent and are indeed necessary to bolster the robustness of the data and conclusions. Conducting flow cytometry analyses would provide more accurate and quantitative assessments of monocyte recruitment, ensuring the reliability of the findings and strengthening the overall conclusions of the study. Regarding the single-cell RNA sequencing analysis presented in the manuscript, it's worth questioning its relevance and depth of information provided. While it successfully identifies a quantitative difference in the cellular composition of the liver between control and knockout mice, it may fall short in elucidating the intricate interactions between different cell populations, which are crucial for understanding the underlying mechanisms of hepatic inflammation. Therefore, I propose considering alternative bioinformatic analyses, such as CellPhone-CellChat, which could potentially provide a more comprehensive understanding of the cellular dynamics and interactions within the liver microenvironment. By examining the dialogue between different cell clusters, these analyses could offer deeper insights into the functional consequences of Ripk1 deficiency in hepatocytes and its impact on hepatic inflammation during fasting.

Related to Figure 5.<br /> What additional insights do the data from Figure 5 provide compared to the study published in Nat Comms, which demonstrated that RIPK1 regulates starvation resistance by modulating aspartate catabolism (PMID: 34686667)?

Related to Figure 6.<br /> The data presented in Figure 7 are complementary and do not introduce new mechanistic insights.

Related to Figure 7.<br /> The data from Figure 7 suggest that RIPK1 in hepatocytes is responsible for the observed damage. However, it has been previously demonstrated that inhibition of RIPK1 activity in macrophages protects against the development of MASLD (PMID: 33208891). One possible explanation for these findings could be that the overreaction of macrophages to fasting, coupled with the absence of RIPK1 in hepatocytes (an indirect effect), contributes to the observed damage. Considering this, complementing hepatocytes with a kinase-dead version of RIPK1 could be a valuable approach to further refine the molecular aspect of the study. This would allow for a more precise investigation into the specific role of RIPK1's scaffolding or kinase function in response to starvation in hepatocytes. Such experiments could provide additional insights into the mechanisms underlying the observed effects and help delineate the contributions of RIPK1 in different cell types to metabolic stress responses.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

However, the findings are not novel enough because the pro-survival role of RIPK1 scaffold is well-established and several similar pieces of research already exist. Moreover, the mechanism is not very clear and needs additional experiments.

Reviewer 1 (Public Review):

Multiple sclerosis (MS) is a debilitating autoimmune disease that causes loss of myelin in neurons of the central nervous system. MS is characterized by the presence of inflammatory immune cells in several brain regions as well as the brain barriers (meninges). This study aims to understand the local immune hallmarks in regions of the brain parenchyma that are adjacent to the leptomeninges in a mouse model of MS. The leptomeninges are known to be a foci of inflammation in MS and perhaps "bleed" inflammatory cells and molecules to adjacent brain parenchyma regions. To do so, they use novel technology called spatial transcriptomics so that the spatial relationships between the two regions remain intact. The study identifies canonical inflammatory genes and gene sets such as complement and B cells enriched in the parenchyma in close proximity to the leptomeninges in the mouse model of MS but not control. The manuscript is very well written and easy to follow. The results will become a useful resource to others working in the field and can be followed by time series experiments where the same technology can be applied to the different stages of the disease.

Comments on revised version:

I agree that the authors successfully addressed most of my comments/critiques.<br /> However, the fact that the control mice were not injected with CFA is somewhat concerning, because it will be hard to interpret the cause of the transcriptomic readouts described in this study. Some of the described effects might be due to CFA (which was used in the EAE but not the "naive" group), and not necessarily to the relapsing-remitting EAE immune features recapitulated in this mouse model. Moreover, this caveat associated with the "naive" control group is not being clearly stated throughout the manuscript and might go unnoticed to readers.<br /> The authors should clearly state, in the methods section (in the section "Induction of SJL EAE"), that the naive control group was not injected with CFA.<br /> Additionally, this potential confounder, of not using a control group injected with the same CFA regimen of the EAE group, should be mentioned in paragraph two of the discussion alongside the other limitations of the study already highlighted by the authors (or in another section of the discussion).

Reviewer 2 (Public Review):

Accumulating data suggests that the presence of immune cell infiltrates in the meninges of the multiple sclerosis brain contributes to the tissue damage in the underlying cortical grey matter by the release of inflammatory and cytotoxic factors that diffuse into the brain parenchyma. However, little is known about the identity and direct and indirect effects of these mediators at a molecular level. This study addresses the vital link between an adaptive immune response in the CSF space and the molecular mechanisms of tissue damage that drive clinical progression. In this short report the authors use a spatial transcriptomics approach using Visium Gene Expression technology from 10x Genomics, to identify gene expression signatures in the meninges and the underlying brain parenchyma, and their interrelationship, in the PLP-induced EAE model of MS in the SJL mouse. MRI imaging using a high field strength (11.7T) scanner was used to identify areas of meningeal infiltration for further study. They report, as might be expected, the upregulation of genes associated with the complement cascade, immune cell infiltration, antigen presentation, and astrocyte activation. Pathway analysis revealed the presence of TNF, JAK-STAT and NFkB signaling, amongst others, close to sites of meningeal inflammation in the EAE animals, although the spatial resolution is insufficient to indicate whether this is in the meninges, grey matter, or both.

UMAP clustering illuminated a major distinct cluster of upregulated genes in the meninges and smaller clusters associated with the grey matter parenchyma underlying the infiltrates. The meningeal cluster contained genes associated with immune cell functions and interactions, cytokine production, and action. The parenchymal clusters included genes and pathways related to glial activation, but also adaptive/B-cell mediated immunity and antigen presentation. This again suggests a technical inability to resolve fully between the compartments as immune cells do not penetrate the pial surface in this model or in MS. Finally, a trajectory analysis based on distance from the meningeal gene cluster successfully demonstrated descending and ascending gradients of gene expression, in particular a decline in pathway enrichment for immune processes with distance from the meninges.

Comments on revised version:

The authors have addressed all of my comments regarding the lack of spatial resolution between the grey matter and the overlying meninges and also concerning the difficulties in extrapolating from this mouse model to MS itself.<br /> I am however very concerned about the lack of the correct control group. Immunization of rodents with complete freunds adjuvant (albeit with pertussis toxin) gives rise to widespread microglial activation, some immune cell infiltration and also structural changes to axons, particularly at nodes of Ranvier (https://doi.org/10.1097/NEN.0b013e3181f3a5b1). This will inevitably make it difficult to interpret the transcriptomics results, depending on whether these changes are reversible or not and the time frame of the reversal. In the C57Bl6 EAE models adjuvant induced microglial activation becomes chronic, whereas the axonal changes do reverse by 10 weeks. Whether this is the same in SJL EAE model using CFA alone is not clear.

Reviewer #1 (Public Review):

In the manuscript by Su et al., the authors present a massively parallel reporter assay (MPRA) measuring the stability of in vitro transcribed mRNAs carrying wild-type or mutant 5' or 3' UTRs transfected into two different human cell lines. The goal presented at the beginning of the manuscript was to screen for effects of disease-associated point mutations on the stability of the reporter RNAs carrying partial human 5' or 3' UTRs. However, the majority of the manuscript is dedicated to identifying sequence components underlying the differential stability of reporter constructs. This shows that TA dinucleotides are the most predictive feature of RNA stability in both cell lines and both UTRs.<br /> The effect of AU rich elements (AREs) on RNA stability is well established in multiple systems, and the present study confirms this general trend but points out variability in the consequence of seemingly similar motifs on RNA stability. For example, the authors report that a long stretch of Us has extreme opposite effects on RNA stability depending on whether it is preceded by an A (strongly destabilizing) or followed by an A (strongly stabilizing). While the authors interpretation of a context-dependence of the effect is certainly well-founded, it seems counterintuitive that the preceding or following A would be the (only) determining factor. This points to a generally reductionist approach taken by the authors in the analysis of the data and in their attempt to dissect the contribution of "AU rich sequences" to RNA stability, with a general tendency to reduce the size and complexity of the features (e.g. to dinucleotides). While this certainly increases the statistical power of the analysis due to the number of occurrences of these motifs, it limits the interpretability of the results. How do TA dinucleotides per se contribute to destabilizing the RNA, both in 5' and 3' UTRs, but (according to limited data presented) not in coding sequences? What is the mechanism? RBPs binding to TA dinucleotide containing sequences are suggested to "mask" the destabilizing effect, thereby leading to a more stable RNA. Gain of TA dinucleotides is reported to have a destabilizing effect, but again no hypothesis is provided as to the underlying molecular mechanism. In addition to reducing the motif length to dinucleotides, the notion of "context dependence" is used in a very narrow sense; especially when focusing on simple and short motifs, a more extensive analysis of the interdependence of these features (beyond the existing analysis of the relationship between TA-diNTs and GC content) could potentially reveal more of the context dependence underlying the seemingly opposite behavior of very similar motifs.

The present MPRAs measures the effect of UTR sequences in one specific reporter context and using one experimental approach (following the decay of in vitro transcribed and transfected RNAs). While this approach certainly has its merits compared to other approaches, it also comes with some caveats: RNA is delivered naked, without bound RBPs and no nuclear history, e.g. of splicing (no EJCs), editing and modifications. One way to assess the generalizability of the results as well as the context dependence of the effects is to perform the same analysis on existing datasets of RNA stability measurements obtained through other methods (e.g. transcription inhibition). Are TA dinucleotides universally the most predictive feature of RNA half-lives?

The authors conclude their study with a meta-analysis of genes with increased TA dinucleotides in 5' and 3'UTRs, showing that specific functional groups are overrepresented among these genes. In addition, they provide evidence for an effect of disease-associated UTR mutations on endogenous RNA stability. While these elements link back to the original motivation of the study (screening for effects of point mutations in 5' and 3' UTRs), they provide only a limited amount of additional insights.

In summary, this manuscript presents an interesting addition to the long-standing attempts at dissecting the sequence basis of RNA stability in human cells. The analysis is in general very comprehensive and sound; however, at times the goal of the authors to find novelty and specificity in the data overshadows some analyses. One example is the case where the authors try to show that TA-dinucleotides and GC content are decoupled and not merely two sides of the same coin. They claim that the effect of TA dinucleotides is different between high- and low-GC content contexts but do not control for the fact that low GC-content regions naturally will contain more TA dinucleotides and therefore the effect sizes and the resulting correlation between TA-diNT rate and stability will be stronger (Fig. 5A). A more thorough analysis and greater caution in some of the claims could further improve the credibility of the conclusions.

Reviewer #2 (Public Review):

Summary of goals:

Untranslated regions are key cis-regulatory elements that control mRNA stability, translation, and translocation. Through interactions with small RNAs and RNA binding proteins, UTRs form complex transcriptional circuitry that allows cells to fine-tune gene expression. Functional annotation of UTR variants has been very limited, and improvements could offer insights into disease relevant regulatory mechanisms. The goals were to advance our understanding of the determinants of UTR regulatory elements and characterize the effects of a set of "disease-relevant" UTR variants.

Strengths:

The use of a massively parallel reporter assay allowed for analysis of a substantial set (6,555 pairs) of 5' and 3' UTR fragments compiled from known disease associated variants. Two cell types were used.

The findings confirm previous work about the importance of AREs, which helps show validity and adds some detailed comparisons of specific AU-rich motif effects in these two cell types.

Using a Lasso regression, TA-dinucleotide content is identified as a strong regulator of RNA stability in a context dependent manner based on GC content and presence of RNA binding protein binding motifs. The findings have potential importance, drawing attention to a UTR feature that is not well characterized.

The use of complementary datasets, including from half-life analyses of RNAs and from random sequence library MRPA's, is a useful addition and supports several important findings. The finding the TA dinucleotides have explanatory power separate from (and in some cases interacting with) GC content is valuable.

The functional enrichment analysis suggests some new ideas about how UTRs may contribute to regulation of certain classes of genes.

Weaknesses:

It is difficult to understand how the calculations for half-life were performed. The sequencing approach measures the relative frequency of each sequence at each time point (less stable sequences become relatively less frequent after time 0, whereas more stable sequences become relatively more frequent after time 0). Since there is no discussion of whether the abundance of the transfected RNA population is referenced to some external standard (e.g., housekeeping RNAs), it is not clear how absolute (rather than relative) half-lives were determined.

Fig. S1A and B are used to assess reproducibility. They show that read counts at a given time point correlate well across replicate experiments. However, this is not a good way to assess reproducibility or accuracy of the measurements of t1/2 are. (The major source of variability in read counts in these plots - especially at early time points - is likely the starting abundance of each RNA sequence, not stability.) This creates concerns about how well the method is measuring t1/2. Also creating concern is the observation that many RNAs are associated with half-lives that are much longer than the time points analyzed in the study. For example, based upon Figure S1 and Table S1 correctly, the median t1/2 for the 5' UTR library in HEK cells appears to be >700 minutes. Given that RNA was collected at 30, 75, and 120 minutes, accurate measurements of RNAs with such long half lives would seem to be very difficult.

There is no direct comparison of t1/2 between the two cell types studied for the full set of sequences studied. This would be helpful in understanding whether the regulatory effects of UTRs are generally similar across cell lines (as has been shown in some previous studies) or whether there are fundamental differences. The distribution of t1/2's is clearly quite different in the two cell lines, but it is important to know if this reflects generally slow RNA turnover in HEK cells or whether there are a large number of sequence-specific effects on stability between cell lines. A related issue is that it is not clear whether the relatively small number of significant variant effects detected in HEK cells versus SH-SY5Y cells is attributable to real biological differences between cell types or to technical issues (many fewer read counts and much longer half lives in HEK cells).

The general assertion is made in many places that TA dinucleotides are the most prominent destabilizing element in UTRs (e.g., in the title, the abstract, Fig. 4 legend, and on p. 12). This appears to be true for only one of the two cell lines tested based on Fig. 3.

Appraisal and impact:

The work adds to existing studies that previously identified sequence features, including AREs and other RNA binding protein motifs, that regulate stability and puts a new emphasis on the role of "TA" (better "UA") dinucleotides. It is not clear how potential problems with the RNA stability measurements discussed above might influence the overall conclusions, which may limit the impact unless these can be addressed.

It is difficult to understand whether the importance of TA dinucleotides is best explained by their occurrence in a related set of longer RBP binding motifs (see Fig 5J, these motifs may be encompassed by the "WWWWWW cluster") or whether some other explanation applies. Further discussion of this would be helpful. Does the LASSO method tend to collapse a more diverse set of longer motifs that are each relatively rare compared to the dinucleotide? It remains unclear whether TA dinucleotides are associated with less stability independent of the presence of the known larger WWWWWWW motif. As noted above, the importance of TA dinucleotides in the HEK experiments appears to be less than is implied in the text.

The inclusion of more than a single cell type is an acknowledgement of the importance of evaluating cell type-specific effects. The work suggests a number of cell type-specific differences, but due to technical issues (especially with the HEK data, as outlined above) and the use of only two cell lines, it is difficult to understand cell type effects from the work.

The inclusion of both 3' and 5' UTR sequences distinguishes this work from most prior studies in the field. Contrasting the effects of these regions on stability is of interest, although the role of these UTRs (especially the 5' UTR) in translational regulation is not assessed here.

Reviewer #3 (Public Review):

Summary:

In their manuscript titled "Multiplexed Assays of Human Disease‐relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability" the authors aim to investigate the effect of sequence variations in 3'UTR and 5'UTRs on the stability of mRNAs in two different human cell lines.

To do so, the authors use a massively parallel reporter assay (MPRA). They transfect cells with a set of mRNA reporters that contain sequence variants in their 3' or 5' UTRs, which were previously reported in human diseases. They follow their clearance from cells over time relative to the matching non-variant sequence. To analyze their results, they define a set of factors (RBP and miRNA binding sites, sequence features, secondary structure etc.) and test their association with differences in mRNA stability. For features with a significant association, they use clustering to select a subset of factors for LASSO regression and identify factors that affect mRNA stability.<br /> They conclude that the TA dinucleotide content of UTRs is the strongest destabilizing sequence feature. Within that context, elevated GC content and protein binding can protect susceptible mRNAs from degradation. They also show that TA dinucleotide content of UTRs affects native mRNA stability, and that it is associated with specific functional groups. Finally, they link disease associated sequence variants with differences in mRNA stability of reporters.

Strengths:

(1) This work introduces a different MPRA approach to analyze the effect of genetic variants. While previous works in tissue culture use DNA transfections that require normalization for transcription efficiency, here the mRNA is directly introduced into cells at fixed amounts, allowing a more direct view of the mRNA regulation.

(2) The authors also introduce a unique analysis approach, which takes into account multiple factors that might affect mRNA stability. This approach allows them to identify general sequence features that affect mRNA stability beyond specific genetic variants, and reach important insights on mRNA stability regulation. Indeed, while the conclusions to genetic variants identified in this work are interesting, the main strength of the work involve general effect of sequence features rather than specific variants.

(3) The authors provide adequate supports for their claims, and validate their analysis using both their reporter data and native genes. For the main feature identified, TA di-nucleotides, they perform follow-up experiments with modified reporters that further strengthen their claims, and also validate the effect on native cellular transcripts (beyond reporters), demonstrating its validity also within native scenarios.

(4) The work provides a broad analysis of mRNA stability, across two mRNA regulatory segments (3'UTR and 5'UTR) and is performed in two separate cell-types. Comparison between two different cell-types is adequate, and the results demonstrate, as expected, the dependence of mRNA stability on the cellular context. Analysis of 3'UTR and 5'UTR regulatory effects also shows interesting differences and similarities between these two regulatory regions.

Weaknesses:

(1) The authors fail to acknowledge several possible confounding factors of their MPRA approach in the discussion.<br /> First, while transfection of mRNA directly into cells allows to avoid the need to normalize for differences in transcription, the introduction of naked mRNA molecules is different than native cellular mRNAs and could introduce biases due to differences in mRNA modifications, protein associations etc. that may occur co-transcriptionally.<br /> Second, along those lines, the authors also use in-vitro polyadenylation. The length of the polyA tail of the transfected transcripts could potentially be very different than that of native mRNAs and also affect stability.

(2) The analysis approach used in this work for identifying regulatory features in UTRs was not previously used. As such, lack of in-depth details of the methodology, and possibly also more general validation of the approach, is a drawback in convincing the reader in the validity of this approach and its results.<br /> In particular, a main point that is not addressed is how the authors decide on the set of "factors" used in their analysis? As choosing different sets of factors might affect the results of the analysis. For example, the choice to use 7-mer sequences within the factors set is not explained, particularly when almost all motifs that are eventually identified (Figure 3B-E) are shorter.<br /> In addition, the authors do not perform validations to demonstrate the validity of their approach on simulated data or well-established control datasets. Such analysis would be helpful to further convince the reader in the usefulness and robustness of the analysis.

(3) The analysis and regression models built in this work are not thoroughly investigated relative to native genes within cells. The effect of sequence "factors" on native cellular transcripts' stability is not investigated beyond TA di-nucleotides, and it is unclear to what degree do other predicted factors also affect native transcripts.

Reviewer #1 (Public Review):

Summary:

This study offers a new perspective. ACTL7A and ACTL7B play roles in epigenetic regulation in spermiogenesis. Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization, and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. It has been reported that ACTL7A is localized on the acrosome in mouse sperms (Boëda et al., 2011). Previous studies have identified ACTL7A mutations (c.1118G>A:p.R373H; c.1204G>A:p.G402S, c.1117C>T:p.R373C), All these variants were located in the actin domain and were predicted to be pathogenic, affecting the number of hydrogen bonds or the arrangement of nearby protein structures (Wang et al., 2023; Xin et al., 2020; Zhao et al., 2023; Zhou et al., 2023). This work used AI to model the role of ACTL7A/B in the nucleosome remodeling complex and proposed a testis-specific conformation of SCRAP complex. This is different from previous studies.

Strengths:

This study provides a new perspective to reveal the additional roles of these proteins.

Weaknesses:

The results section contains a substantial background description. However, the results and discussion sections require streamlining. There is a lack of mutual support for data between the sections, and direct data to support the authors' conclusions are missing.

Reviewer #2 (Public Review):

Summary:

How dynamics of gene expression accompany cell fate and cellular morphological changes is important for our understanding of molecular mechanisms that govern development and diseases. The phenomenon is particularly prominent during spermatogenesis, the process which spermatogonia stem cells develop into sperm through a series of steps of cell division, differentiation, meiosis, and cellular morphogenesis. The intricacy of various aspects of cellular processes and gene expression during spermatogenesis remains to be fully understood. In this study, the authors found that testis-specific actin-related proteins (which usually participate in modifying cells' cytoskeletal systems) ACTL7A and ACTL7B were expressed and localized in the nuclei of mouse spermatocytes and spermatids. Based on this observation, the authors analyzed protein sequence conservations of ACTL7B across dozens of species and identified a putative nuclear localization sequence (NLS) that is often responsible for the nuclear import of proteins that carry them. Using molecular biology experiments in a heterologous cell system, the authors verified the potential role of this internal NLS and found it indeed could facilitate the nuclear localization of marker proteins when expressed in cells. Using gene-deleted mouse models they generated previously, the authors showed that deletion of Actl7b caused changes in gene expression and mis-localization of nucleosomal histone H3 and chromatin regulator histone deacetylase HDAC1 and 2, supporting their proposed roles of ACTL7B in regulating gene expression. The authors further used alpha-Fold 2 to model the potential protein complexes that could be formed between the ARPs (ACTL7A and ACTL7B) and known chromatin modifiers, such as INO80 and SWI/SNF complexes and found that consistent with previous findings, it is likely that ACTL7A and ACTL7B interact with the chromatin-modifying complexes through binding to their alpha-helical HSA domain cooperatively. These results suggest that ACTL7B possesses novel functions in regulating chromatin structure and thus gene expression beyond conventional roles of cytoskeleton regulation, providing alternative pathways for understanding how gene expression is regulated during spermatogenesis and the etiology of relevant infertility diseases.

Strengths:

The authors provided sufficient background to the study and discussions of the results. Based on their previous research, this study utilized numerous methods, including protein complex structural modeling method alpha-fold 2 Multimers, to further investigate the functional roles of ACTL7B. The results presented here are in general of good quality. The identification of a potential internal NLS in ACTL7B is mostly convincing, in line with the phenotypes presented in the gene deletion model.

Weaknesses:

While the study offered an interesting new look at the functions of ARP proteins during spermatogenesis, some of the study is mainly theoretical speculations, including the protein complex formation. Some of the results may need further experimental verifications, for example, differentially expressed genes that were found in potentially spermatogenic cells at different developmental stages, in order to support the conclusions and avoid undermining the significance of the study.

Reviewer #3 (Public Review):

In this manuscript, Pierre Ferrer and colleagues explore the exciting possibility that, in the male germ line, the composition and function of deeply conserved chromatin remodeling complexes is fine-tuned by the addition of testis-specific actin-related proteins (ARPs). In this regard, the Authors aim to extend previously reported non-canonical (transcriptional) roles of ARPs in somatic cells to the unique developmental context of the germ line. The manuscript is focused on the potential regulatory role in post-meiotic transcription of two ARPs: ACTL7A and ACTL7B (particularly the latter). The canonical function of both testis-specific ARPs in spermatogenesis is well established, as they have been previously shown to be required for the extensive cellular morphogenesis program driving post-meiotic development (spermiogenesis). Disentangling the actual functions of ACTL7A and ACTL7B as transcriptional regulators from their canonical role in the profound morphological reshaping of post-meiotic cells (a process that also deeply impacts nuclear architecture and regulation) represents a key challenge in terms of interpreting the reported findings (see below).

The authors begin by documenting, via fluorescence microscopy, the intranuclear localization of ACTL7B. This ARP is convincingly shown to accumulate in the nucleus of spermatocytes and spermatids. Using a series of elegant reporter-based experiments in a somatic cell line, the authors map the driver of this nuclear accumulation to a potential NLS sequence in the ACTL7B actin-like body domain. Ferrer and colleagues then performed a testicular RNA-seq analysis in ACTL7B KO mice to define the putative role of ACTL7B in male germ cell transcription. They report substantial changes to the testicular transcriptome - particularly the upregulation of several classes of genes - in ACTL7B KO mice. However, wild-type testes were used as controls for this experiment, thus introducing a clear confounding effect to the analysis (ACTL7B KO testes have extensive post-meiotic defects due to the canonical role of ACTL7B in spermatid development). Then, the authors employ cutting-edge AI-driven approaches to predict that both ACTL7A and ACTL7B are likely to bind to four key chromatin remodeling complexes. Although these predictions are based on a robust methodology, they would certainly benefit from experimental validation. Finally, the authors associate the loss of ACTL7B with decreased lysine acetylation and lower levels of the HDAC1 and HDAC3 chromatin remodelers in the nucleus of developing spermatids.

Globally, these data may provide important insight into the unique processes male germ cells employ to sustain their extraordinarily complex transcriptional program. Furthermore, the concept that (comparably younger) testis-specific proteins can be incorporated into ancient chromatin remodeling complexes to modulate their function in the germ line is timely and exciting.

It is my opinion that the manuscript would benefit from additional experimental validation to better support the authors' conclusions. In particular, I believe that addressing two critical points would substantially strengthen the message of the manuscript:

(1) The proposed role of ACTL7B in post-meiotic transcriptional regulation temporally overlaps with the protein's previously reported canonical functions in spermiogenesis (PMID: 36617158 and 37800308). Indeed, the canonical functions of ACTL7B have been shown to have a profound effect at the level of spermatid morphology and to impact nuclear organization. This potentially renders the observed transcriptional deregulation in ACTL7B KO testes an indirect consequence of spermatid morphology defects. I acknowledge that it is experimentally difficult to disentangle the proposed intranuclear roles of ACTL7B from the protein's well-documented cytoplasmic function. Perhaps the generation of a NLS-scrambled ACTL7B variant could offer some insight. In light of the substantial investment this approach would represent, I would suggest, as an alternative, that instead of using wild-type testes as controls for the transcriptome and chromatin localization assays, the authors consider the possibility of using testicular tissue from a mutant with similarly abnormal spermiogenesis but due to transcription-independent defects. This would, in my opinion, offer a more suitable baseline to compare ACTL7B KO testes with.

(2) The manuscript would greatly benefit if experimental validation of the AI-driven predictions were to be provided (in terms of the binding capacity of ACTL7A and ACTL7B to key chromatin remodeling complexes). More so it seems that the authors have the technical expertise / available mass spectrometry data required for this purpose (lines 664-665). Still on this topic, given the predicted interactions of ACTL7A and ACTL7B with the SRCAP, EP400, SMARCA2 and SMARCA4 complexes (Figure 7), it is rather counter-intuitive that the Authors chose for their immunofluorescence assays, in ACTL7B KO testes, to determine the chromatin localization of HDAC1 and HDAC3, rather than that of any of above four complexes.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Bimbard et al., a new method to perform stable recordings over long periods of time with neuropixels, as well as the technical details on how the electrodes can be explanted for follow-up reuse, is provided. I think the description of all parts of the method is very clear, and the validation analyses (n of units per day over time, RMS over recording days...) are very convincing. I however missed a stronger emphasis on why this could provide a big impact on the ephys community, by enabling new analyses, new behavior correlation studies, or neurophysiological mechanisms across temporal scales that were previously inaccessible with high temporal resolution (i.e. not with imaging).

Strengths:

Open source method. Validation across laboratories. Across species (mice and rats) demonstration of its use and in different behavioral conditions (head-fixed and freely moving).

Weaknesses:

Weak emphasis on what can be enabled with this new method that didn't exist before.

Reviewer #2 (Public Review):

Summary:

This work by Bimbard et al., introduces a new implant for Neuropixels probes. While Neuropixels probes have critically improved and extended our ability to record the activity of a large number of neurons with high temporal resolution, the use of these expensive devices in chronic experiments has so far been hampered by the difficulty of safely implanting them and, importantly, to explant and reuse them after conclusion of the experiment. The authors present a newly designed two-part implant, consisting of a docking and a payload module, that allows for secure implantation and straightforward recovery of the probes. The implant is lightweight, making it amenable for use in mice and rats, and customizable. The authors provide schematics and files for printing of the implants, which can be easily modified and adapted to custom experiments by researchers with little to no design experience. Importantly, the authors demonstrate the successful use of this implant across multiple use cases, in head-fixed and freely moving experiments, in mice and rats, with different versions of Neuropixels probes, and across 8 different labs. Taken together, the presented implants promise to make chronic Neuropixel recordings and long-term studies of neuronal activity significantly easier and attainable for both current and future Neuropixels users.

Strengths:

- The implants have been successfully tested across 8 different laboratories, in mice and rats, in head-fixed and freely moving conditions, and have been adapted in multiple ways for a number of distinct experiments.

- Implants are easily customizable and the authors provide a straightforward approach for customization across multiple design dimensions even for researchers not experienced in design.

- The authors provide clear and straightforward descriptions of the construction, implantation, and explant of the described implants.

- The split of the implant into a docking and payload module makes reuse even in different experiments (using different docking modules) easy.

- The authors demonstrate that implants can be re-used multiple times and still allow for high-quality recordings.

- The authors show that the chronic implantations allow for the tracking of individual neurons across days and weeks (using additional software tracking solutions), which is critical for a large number of experiments requiring the description of neuronal activity, e.g. throughout learning processes.

- The authors show that implanted animals can even perform complex behavioral tasks, with no apparent reduction in their performance.

Weaknesses:

- While implanted animals can still perform complex behavioral tasks, the authors describe that the implants may reduce the animals' mobility, as measured by prolonged reaction times. However, the presented data does not allow us to judge whether this effect is specifically due to the presented implant or whether any implant or just tethering of the animals per se would have the same effects.

- While the authors make certain comparisons to other, previously published approaches for chronic implantation and re-use of Neuropixels probes, it is hard to make conclusive comparisons and judge the advantages of the current implant. For example, while the authors emphasize that the lower weight of their implant allows them to perform recordings in mice (and is surely advantageous), the previously described, heavier implants they mention (Steinmetz et al., 2021; van Daal et al., 2021), have also been used in mice. Whether the weight difference makes a difference in practice therefore remains somewhat unclear.

- The non-permanent integration of the headstages into the implant, while allowing for the use of the same headstage for multiple animals in parallel, requires repeated connections and does not provide strong protection for the implant. This may especially be an issue for the use in rats, requiring additional protective components as in the presented rat experiments.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Bimbard and colleagues describe a new implant apparatus called "Apollo Implant", which should facilitate recording in freely moving rodents (mice and rats) using Neuropixels probes. The authors collected data from both mice and rats, they used 3 different versions of Neuropixels, multiple labs have already adopted this method, which is impressive. They openly share their CAD designs and surgery protocol to further facilitate the adaptation of their method.

Strengths:

Overall, the "Apollo Implant" is easy to use and adapt, as it has been used in other laboratories successfully and custom modifications are already available. The device is reproducible using common 3D printing services and can be easily modified thanks to its CAD design (the video explaining this is extremely helpful). The weight and price are amazing compared to other systems for rigid silicon probes allowing a wide range of use of the "Apollo Implant".

Weaknesses:

The "Apollo Implant" can only handle Neuropixels probes. It cannot hold other widely used and commercially available silicon probes. Certain angles and distances are not possible in their current form (distance between probes 1.8 to 4mm, implantation depth 2-6.5 mm, or angle of insertion up to 20 degrees).

Reviewer #1 (Public Review):

Summary:

This paper presents a mechanistic study of rDNA origin regulation in yeast by SIR2. Each of the ~180 tandemly repeated rDNA gene copies contains a potential replication origin. Early-efficient initiation of these origins is suppressed by Sir2, reducing competition with origins distributed throughout the genome for rate-limiting initiation factors. Previous studies by these authors showed that SIR2 deletion advances replication timing of rDNA origins by a complex mechanism of transcriptional de-repression of a local PolII promoter causing licensed origin proteins (MCMcomplexes) to re-localize (slide along the DNA) to a different (and altered) chromatin environment. In this study, they identify a chromatin remodeler, FUN30, that suppresses the sir2∆ effect, and remarkably, results in a contraction of the rDNA to about one-quarter it's normal length/number of repeats, implicating replication defects of the rDNA. Through examination of replication timing, MCM occupancy and nucleosome occupancy on the chromatin in sir2, fun30, and double mutants, they propose a model where nucleosome position relative to the licensed origin (MCM complexes) intrinsically determines origin timing/efficiency. While their interpretations of the data are largely reasonable and can be interpreted to support their model, a key weakness is the connection between Mcm ChEC signal disappearance and origin firing. While the cyclical chromatin association-dissociation of MCM proteins with potential origin sequences may be generally interpreted as licensing followed by firing, dissociation may also result from passive replication and as shown here, displacement by transcription and/or chromatin remodeling. Moreover, linking its disappearance from chromatin in the ChEC method with such precise resolution needs to be validated against an independent method to determine the initiation site(s). Differences in rDNA copy number and relative transcription levels also are not directly accounted for, obscuring a clearer interpretation of the results. Nevertheless, this paper makes a valuable advance with the finding of Fun30 involvement, which substantially reduces rDNA repeat number in sir2∆ background. The model they develop is compelling and I am inclined to agree, but I think the evidence on this specific point is purely correlative and a better method is needed to address the initiation site question. The authors deserve credit for their efforts to elucidate our obscure understanding of the intricacies of chromatin regulation. At a minimum, I suggest their conclusions on these points of concern should be softened and caveats discussed. Statistical analysis is lacking for some claims.

Strengths are the identification of FUN30 as suppressor, examination of specific mutants of FUN30 to distinguish likely functional involvement. Use of multiple methods to analyze replication and protein occupancies on chromatin. Development of a coherent model.

Weaknesses are failure to address copy number as a variable; insufficient validation of ChEC method relationship to exact initiation locus; lack of statistical analysis in some cases.

Additional background and discussion for public review:

This paper broadly addresses the mechanism(s) that regulate replication origin firing in different chromatin contexts. The rDNA origin is present in each of ~180 tandem repeats of the rDNA sequence, representing a high potential origin density per length of DNA (9.1kb repeat unit). However, the average origin efficiency of rDNA origins is relatively low (~20% in wild-type cells), which reduces the replication load on the overall genome by reducing competition with origins throughout the genome for limiting replication initiation factors. Deletion of histone deacetylase SIR2, which silences PolII transcription within the rDNA, results in increased early activation or the rDNA origins (and reduced rate of overall genome replication). Previous work by the authors showed that MCM complexes loaded onto the rDNA origins (origin licensing) were laterally displaced (sliding) along the rDNA, away from a well-positioned nucleosome on one side. The authors' major hypothesis throughout this work is that the new MCM location(s) are intrinsically more efficient configurations for origin firing. The authors identify a chromatin remodeling enzyme, FUN30, whose deletion appears to suppress the earlier activation of rDNA origins in sir2∆ cells. Indeed, it appears that the reduction of rDNA origin activity in sir2∆ fun30∆ cells is severe enough to results in a substantial reduction in the rDNA array repeat length (number of repeats); the reduced rDNA length presumably facilitates it's more stable replication and maintenance.

Analysis of replication by 2D gels is marginally convincing, using 2D gels for this purpose is very challenging and tricky to quantify. The more quantitative analysis by EdU incorporation is more convincing of the suppression of the earlier replication caused by SIR2 deletion.

To address the mechanism of suppression, they analyze MCM positioning using ChEC, which in G1 cells shows partial displacement of MCM from normal position A to positions B and C in sir2∆ cells and similar but more complete displacement away from A to positions B and C in sir2fun30 cells. During S-phase in the presence of hydroxyurea, which slows replication progression considerably (and blocks later origin firing) MCM signals redistribute, which is interpreted to represent origin firing and bidirectional movement of MCMs (only one direction is shown), some of which accumulate near the replication fork barrier, consistent with their interpretation. They observe that MCMs displaced (in G1) to sites B or C in sir2∆ cells, disappear more rapidly during S-phase, whereas the similar dynamic is not observed in sir2∆fun30∆. This is the main basis for their conclusion that the B and C sites are more permissive than A. While this may be the simplest interpretation, there are limitations with this assay that undermine a rigorous conclusion (additional points below). The main problem is that we know the MCM complexes are mobile so disappearance may reflect displacement by other means including transcription which is high is the sir2∆ background. Indeed, the double mutant has greater level of transcription per repeat unit which might explain more displaced from A in G1. Thus, displacement might not always represent origin firing. Because the sir2 background profoundly changes transcription, and the double mutant has a much smaller array length associated with higher transcription, how can we rule out greater accessibility at site A, for example in sir2∆, leading to more firing, which is suppressed in sir2 fun30 due to greater MCM displacement away from A?

I think the critical missing data to solidly support their conclusions is a definitive determination of the site(s) of initiation using a more direct method, such as strand specific sequencing of EdU or nascent strand analysis. More direct comparisons of the strains with lower copy number to rule out this facet. As discussed in detail below, copy number reduction is known to suppress at least part of the sir2∆ effect so this looms over the interpretations. I think they are probably correct in their overall model based on the simplest interpretation of the data but I think it remains to be rigorously established. I think they should soften their conclusions in this respect.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors follow up on their previous work showing that in the absence of the Sir2 deacetylase the MCM replicative helicase at the rDNA spacer region is repositioned to a region of low nucleosome occupancy. Here they show that the repositioned displaced MCMs have increased firing propensity relative to non-displaced MCMs. In addition, they show that activation of the repositioned MCMs and low nucleosome occupancy in the adjacent region depend on the chromatin remodeling activity of Fun30.

Strengths:

The paper provides new information on the role of a conserved chromatin remodeling protein in the regulation of origin firing and in addition provides evidence that not all loaded MCMs fire and that origin firing is regulated at a step downstream of MCM loading.

Weaknesses:

The relationship between the author's results and prior work on the role of Sir2 (and Fob1) in regulation of rDNA recombination and copy number maintenance is not explored, making it difficult to place the results in a broader context. Sir2 has previously been shown to be recruited by Fob1, which is also required for DSB formation and recombination-mediated changes in rDNA copy number. Are the changes that the authors observe specifically in fun30 sir2 cells related to this pathway? Is Fob1 required for the reduced rDNA copy number in fun30 sir2 double mutant cells?

Reviewer #3 (Public Review):

Summary:

Heterochromatin is characterized by low transcription activity and late replication timing, both dependent on the NAD-dependent protein deacetylase Sir2, the founding member of the sirtuins. This manuscript addresses the mechanism by which Sir2 delays replication timing at the rDNA in budding yeast. Previous work from the same laboratory (Foss et al. PLoS Genetics 15, e1008138) showed that Sir2 represses transcription-dependent displacement of the Mcm helicase in the rDNA. In this manuscript, the authors show convincingly that the repositioned Mcms fire earlier and that this early firing partly depends on the ATPase activity of the nucleosome remodeler Fun30. Using read-depth analysis of sorted G1/S cells, fun30 was the only chromatin remodeler mutant that somewhat delayed replication timing in sir2 mutants, while nhp10, chd1, isw1, htl1, swr1, isw2, and irc5 had not effect. The conclusion was corroborated with orthogonal assays including two-dimensional gel electrophoresis and analysis of EdU incorporation at early origins. Using an insightful analysis with an Mcm-MNase fusion (Mcm-ChEC), the authors show that the repositioned Mcms in sir2 mutants fire earlier than the Mcm at the normal position in wild type. This early firing at the repositioned Mcms is partially suppressed by Fun30. In addition, the authors show Fun30 affects nucleosome occupancy at the sites of the repositioned Mcm, providing a plausible mechanism for the effect of Fun30 on Mcm firing at that position. However, the results from the MNAse-seq and ChEC-seq assays are not fully congruent for the fun30 single mutant. Overall, the results support the conclusions providing a much better mechanistic understanding how Sir2 affects replication timing at rDNA,

Strengths

(1) The data clearly show that the repositioned Mcm helicase fires earlier than the Mcm in the wild type position.<br /> (2) The study identifies a specific role for Fun30 in replication timing and an effect on nucleosome occupancy around the newly positioned Mcm helicase in sir2 cells.

Weaknesses

(1) It is unclear which strains were used in each experiment.<br /> (2) The relevance of the fun30 phospho-site mutant (S20AS28A) is unclear.<br /> (3) For some experiments (Figs. 3, 4, 6) it is unclear whether the data are reproducible and the differences significant. Information about the number of independent experiments and quantitation is lacking. This affects the interpretation, as fun30 seems to affect the +3 nucleosome much more than let on in the description.

Reviewer #2 (Public Review):

The authors examine the impact of optogenetic inhibition of hippocampal axon terminals in the retrosplenial cortex (RSP) during the performance of a working memory T-maze task. Performance on a delayed non-match-to-place task was impaired by such inhibition. The authors also report that inhibition is associated with faster decision-making and that the effects of inhibition can be observed over several subsequent trials. The work seems reasonably well done and the role of hippocampal projections to retrosplenial cortex in memory and decision-making is very relevant to multiple fields. However, the work should be expanded in several ways before one can make firm conclusions on the role of this projection in memory and behavior.

Comments on revised version:

The authors have provided their comments on the concerns voiced in my first review. I remain of the opinion that the experiments do not extend beyond determining whether disruption of hippocampal to retrosplenial cortex connections impacts spatial working memory. Given the restricted level of inquiry and the very moderate effect of the manipulation on memory, the work, in my opinion, does not provide significant insight into the processes of spatial working memory nor the function of the hippocampal to retrosplenial cortex connection.

Reviewer #1 (Public Review):

Summary: In this study, the authors address whether the dorsal nucleus of the inferior colliculus (DCIC) in mice encodes sound source location within the front horizontal plane (i.e., azimuth). They do this using volumetric two-photon Ca2+ imaging and high-density silicon probes (Neuropixels) to collect single-unit data. Such recordings are beneficial because they allow large populations of simultaneous neural data to be collected. Their main results and the claims about those results are the following:

1) DCIC single-unit responses have high trial-to-trial variability (i.e., neural noise);

2) approximately 32% to 40% of DCIC single units have responses that are sensitive to sound source azimuth;

3) single-trial population responses (i.e., the joint response across all sampled single units in an animal) encode sound source azimuth "effectively" (as stated in title) in that localization decoding error matches average mouse discrimination thresholds;

4) DCIC can encode sound source azimuth in a similar format to that in the central nucleus of the inferior colliculus (as stated in Abstract);

5) evidence of noise correlation between pairs of neurons exists;

and 6) noise correlations between responses of neurons help reduce population decoding error.

While simultaneous recordings are not necessary to demonstrate results #1, #2, and #4, they are necessary to demonstrate results #3, #5, and #6.

Strengths:<br /> - Important research question to all researchers interested in sensory coding in the nervous system.<br /> - State-of-the-art data collection: volumetric two-photon Ca2+ imaging and extracellular recording using high-density probes. Large neuronal data sets.<br /> - Confirmation of imaging results (lower temporal resolution) with more traditional microelectrode results (higher temporal resolution).<br /> - Clear and appropriate explanation of surgical and electrophysiological methods. I cannot comment on the appropriateness of the imaging methods.

Strength of evidence for claims of the study:

1) DCIC single-unit responses have high trial-to-trial variability -<br /> The authors' data clearly shows this.

2) Approximately 32% to 40% of DCIC single units have responses that are sensitive to sound source azimuth -<br /> The sensitivity of each neuron's response to sound source azimuth was tested with a Kruskal-Wallis test, which is appropriate since response distributions were not normal. Using this statistical test, only 8% of neurons (median for imaging data) were found to be sensitive to azimuth, and the authors noted this was not significantly different than the false positive rate. The Kruskal-Wallis test was not performed on electrophysiological data. The authors suggested that low numbers of azimuth-sensitive units resulting from the statistical analysis may be due to the combination of high neural noise and relatively low number of trials, which would reduce statistical power of the test. This may be true, but if single-unit responses were moderately or strongly sensitive to azimuth, one would expect them to pass the test even with relatively low statistical power. At best, if their statistical test missed some azimuth-sensitive units, they were likely only weakly sensitive to azimuth. The authors went on to perform a second test of azimuth sensitivity-a chi-squared test-and found 32% (imaging) and 40% (e-phys) of single units to have statistically significant sensitivity. This feels a bit like fishing for a lower p-value. The Kruskal-Wallis test should have been left as the only analysis. Moreover, the use of a chi-squared test is questionable because it is meant to be used between two categorical variables, and neural response had to be binned before applying the test.

3) Single-trial population responses encode sound source azimuth "effectively" in that localization decoding error matches average mouse discrimination thresholds -<br /> If only one neuron in a population had responses that were sensitive to azimuth, we would expect that decoding azimuth from observation of that one neuron's response would perform better than chance. By observing the responses of more than one neuron (if more than one were sensitive to azimuth), we would expect performance to increase. The authors found that decoding from the whole population response was no better than chance. They argue (reasonably) that this is because of overfitting of the decoder model-too few trials used to fit too many parameters-and provide evidence from decoding combined with principal components analysis which suggests that overfitting is occurring. What is troubling is the performance of the decoder when using only a handful of "top-ranked" neurons (in terms of azimuth sensitivity) (Fig. 4F and G). Decoder performance seems to increase when going from one to two neurons, then decreases when going from two to three neurons, and doesn't get much better for more neurons than for one neuron alone. It seems likely there is more information about azimuth in the population response, but decoder performance is not able to capture it because spike count distributions in the decoder model are not being accurately estimated due to too few stimulus trials (14, on average). In other words, it seems likely that decoder performance is underestimating the ability of the DCIC population to encode sound source azimuth.<br /> To get a sense of how effective a neural population is at coding a particular stimulus parameter, it is useful to compare population decoder performance to psychophysical performance. Unfortunately, mouse behavioral localization data do not exist. Therefore, the authors compare decoder error to mouse left-right discrimination thresholds published previously by a different lab. However, this comparison is inappropriate because the decoder and the mice were performing different perceptual tasks. The decoder is classifying sound sources to 1 of 13 locations from left to right, whereas the mice were discriminating between left or right sources centered around zero degrees. The errors in these two tasks represent different things. The two data sets may potentially be more accurately compared by extracting information from the confusion matrices of population decoder performance. For example, when the stimulus was at -30 deg, how often did the decoder classify the stimulus to a lefthand azimuth? Likewise, when the stimulus was +30 deg, how often did the decoder classify the stimulus to a righthand azimuth?

4) DCIC can encode sound source azimuth in a similar format to that in the central nucleus of the inferior colliculus -<br /> It is unclear what exactly the authors mean by this statement in the Abstract. There are major differences in the encoding of azimuth between the two neighboring brain areas: a large majority of neurons in the CNIC are sensitive to azimuth (and strongly so), whereas the present study shows a minority of azimuth-sensitive neurons in the DCIC. Furthermore, CNIC neurons fire reliably to sound stimuli (low neural noise), whereas the present study shows that DCIC neurons fire more erratically (high neural noise).

5) Evidence of noise correlation between pairs of neurons exists -<br /> The authors' data and analyses seem appropriate and sufficient to justify this claim.

6) Noise correlations between responses of neurons help reduce population decoding error -<br /> The authors show convincing analysis that performance of their decoder increased when simultaneously measured responses were tested (which include noise correlation) than when scrambled-trial responses were tested (eliminating noise correlation). This makes it seem likely that noise correlation in the responses improved decoder performance. The authors mention that the naïve Bayesian classifier was used as their decoder for computational efficiency, presumably because it assumes no noise correlation and, therefore, assumes responses of individual neurons are independent of each other across trials to the same stimulus. The use of decoder that assumes independence seems key here in testing the hypothesis that noise correlation contains information about sound source azimuth. The logic of using this decoder could be more clearly spelled out to the reader. For example, if the null hypothesis is that noise correlations do not carry azimuth information, then a decoder that assumes independence should perform the same whether population responses are simultaneous or scrambled. The authors' analysis showing a difference in performance between these two cases provides evidence against this null hypothesis.

Minor weakness:<br /> - Most studies of neural encoding of sound source azimuth are done in a noise-free environment, but the experimental setup in the present study had substantial background noise. This complicates comparison of the azimuth tuning results in this study to those of other studies. One is left wondering if azimuth sensitivity would have been greater in the absence of background noise, particularly for the imaging data where the signal was only about 12 dB above the noise. The description of the noise level and signal + noise level in the Methods should be made clearer. Mice hear from about 2.5 - 80 kHz, so it is important to know the noise level within this band as well as specifically within the band overlapping with the signal.

Reviewer #2 (Public Review):

In the present study, Boffi et al. investigate the manner in which the dorsal cortex of the of the inferior colliculus (DCIC), an auditory midbrain area, encodes sound location azimuth in awake, passively listening mice. By employing volumetric calcium imaging (scanned temporal focusing or s-TeFo), complemented with high-density electrode electrophysiological recordings (neuropixels probes), they show that sound-evoked responses are exquisitely noisy, with only a small portion of neurons (units) exhibiting spatial sensitivity. Nevertheless, a naïve Bayesian classifier was able to predict the presented azimuth based on the responses from small populations of these spatially sensitive units. A portion of the spatial information was provided by correlated trial-to-trial response variability between individual units (noise correlations). The study presents a novel characterization of spatial auditory coding in a non-canonical structure, representing a noteworthy contribution specifically to the auditory field and generally to systems neuroscience, due to its implementation of state-of-the-art techniques in an experimentally challenging brain region. However, nuances in the calcium imaging dataset and the naïve Bayesian classifier warrant caution when interpreting some of the results.

Strengths:<br /> The primary strength of the study lies in its methodological achievements, which allowed the authors to collect a comprehensive and novel dataset. While the DCIC is a dorsal structure, it extends up to a millimetre in depth, making it optically challenging to access in its entirety. It is also more highly myelinated and vascularised compared to e.g., the cerebral cortex, compounding the problem. The authors successfully overcame these challenges and present an impressive volumetric calcium imaging dataset. Furthermore, they corroborated this dataset with electrophysiological recordings, which produced overlapping results. This methodological combination ameliorates the natural concerns that arise from inferring neuronal activity from calcium signals alone, which are in essence an indirect measurement thereof.

Another strength of the study is its interdisciplinary relevance. For the auditory field, it represents a significant contribution to the question of how auditory space is represented in the mammalian brain. "Space" per se is not mapped onto the basilar membrane of the cochlea and must be computed entirely within the brain. For azimuth, this requires the comparison between miniscule differences between the timing and intensity of sounds arriving at each ear. It is now generally thought that azimuth is initially encoded in two, opposing hemispheric channels, but the extent to which this initial arrangement is maintained throughout the auditory system remains an open question. The authors observe only a slight contralateral bias in their data, suggesting that sound source azimuth in the DCIC is encoded in a more nuanced manner compared to earlier processing stages of the auditory hindbrain. This is interesting, because it is also known to be an auditory structure to receive more descending inputs from the cortex.

Systems neuroscience continues to strive for the perfection of imaging novel, less accessible brain regions. Volumetric calcium imaging is a promising emerging technique, allowing the simultaneous measurement of large populations of neurons in three dimensions. But this necessitates corroboration with other methods, such as electrophysiological recordings, which the authors achieve. The dataset moreover highlights the distinctive characteristics of neuronal auditory representations in the brain. Its signals can be exceptionally sparse and noisy, which provide an additional layer of complexity in the processing and analysis of such datasets. This will be undoubtedly useful for future studies of other less accessible structures with sparse responsiveness.

Weaknesses:<br /> Although the primary finding that small populations of neurons carry enough spatial information for a naïve Bayesian classifier to reasonably decode the presented stimulus is not called into question, certain idiosyncrasies, in particular the calcium imaging dataset and model, complicate specific interpretations of the model output, and the readership is urged to interpret these aspects of the study's conclusions with caution.

I remain in favour of volumetric calcium imaging as a suitable technique for the study, but the presently constrained spatial resolution is insufficient to unequivocally identify regions of interest as cell bodies (and are instead referred to as "units" akin to those of electrophysiological recordings). It remains possible that the imaging set is inadvertently influenced by non-somatic structures (including neuropil), which could report neuronal activity differently than cell bodies. Due to the lack of a comprehensive ground-truth comparison in this regard (which to my knowledge is impossible to achieve with current technology), it is difficult to imagine how many informative such units might have been missed because their signals were influenced by spurious, non-somatic signals, which could have subsequently misled the models. The authors reference the original Nature Methods article (Prevedel et al., 2016) throughout the manuscript, presumably in order to avoid having to repeat previously published experimental metrics. But the DCIC is neither the cortex nor hippocampus (for which the method was originally developed) and may not have the same light scattering properties (not to mention neuronal noise levels). Although the corroborative electrophysiology data largely eleviates these concerns for this particular study, the readership should be cognisant of such caveats, in particular those who are interested in implementing the technique for their own research.

A related technical limitation of the calcium imaging dataset is the relatively low number of trials (14) given the inherently high level of noise (both neuronal and imaging). Volumetric calcium imaging, while offering a uniquely expansive field of view, requires relatively high average excitation laser power (in this case nearly 200 mW), a level of exposure the authors may have wanted to minimise by maintaining a low the number of repetitions, but I yield to them to explain. Calcium imaging is also inherently slow, requiring relatively long inter-stimulus intervals (in this case 5 s). This unfortunately renders any model designed to predict a stimulus (in this case sound azimuth) from particularly noisy population neuronal data like these as highly prone to overfitting, to which the authors correctly admit after a model trained on the entire raw dataset failed to perform significantly above chance level. This prompted them to feed the model only with data from neurons with the highest spatial sensitivity. This ultimately produced reasonable performance (and was implemented throughout the rest of the study), but it remains possible that if the model was fed with more repetitions of imaging data, its performance would have been more stable across the number of units used to train it. (All models trained with imaging data eventually failed to converge.) However, I also see these limitations as an opportunity to improve the technology further, which I reiterate will be generally important for volume imaging of other sparse or noisy calcium signals in the brain.

Transitioning to the naïve Bayesian classifier itself, I first openly ask the authors to justify their choice of this specific model. There are countless types of classifiers for these data, each with their own pros and cons. Did they actually try other models (such as support vector machines), which ultimately failed? If so, these negative results (even if mentioned en passant) would be extremely valuable to the community, in my view. I ask this specifically because different methods assume correspondingly different statistical properties of the input data, and to my knowledge naïve Bayesian classifiers assume that predictors (neuronal responses) are assumed to be independent within a class (azimuth). As the authors show that noise correlations are informative in predicting azimuth, I wonder why they chose a model that doesn't take advantage of these statistical regularities. It could be because of technical considerations (they mention computing efficiency), but I am left generally uncertain about the specific logic that was used to guide the authors through their analytical journey.

That aside, there remain other peculiarities in model performance that warrant further investigation. For example, what spurious features (or lack of informative features) in these additional units prevented the models of imaging data from converging? In an orthogonal question, did the most spatially sensitive units share any detectable tuning features? A different model trained with electrophysiology data in contrast did not collapse in the range of top-ranked units plotted. Did this model collapse at some point after adding enough units, and how well did that correlate with the model for the imaging data? How well did the form (and diversity) of the spatial tuning functions as recorded with electrophysiology resemble their calcium imaging counterparts? These fundamental questions could be addressed with more basic, but transparent analyses of the data (e.g., the diversity of spatial tuning functions of their recorded units across the population). Even if the model extracts features that are not obvious to the human eye in traditional visualisations, I would still find this interesting.

Finally, the readership is encouraged to interpret certain statements by the authors in the current version conservatively. How the brain ultimately extracts spatial neuronal data for perception is anyone's guess, but it is important to remember that this study only shows that a naïve Bayesian classifier could decode this information, and it remains entirely unclear whether the brain does this as well. For example, the model is able to achieve a prediction error that corresponds to the psychophysical threshold in mice performing a discrimination task (~30 {degree sign}). Although this is an interesting coincidental observation, it does not mean that the two metrics are necessarily related. The authors correctly do not explicitly claim this, but the manner in which the prose flows may lead a non-expert into drawing that conclusion. Moreover, the concept of redundancy (of spatial information carried by units throughout the DCIC) is difficult for me to disentangle. One interpretation of this formulation could be that there are non-overlapping populations of neurons distributed across the DCIC that each could predict azimuth independently of each other, which is unlikely what the authors meant. If the authors meant generally that multiple neurons in the DCIC carry sufficient spatial information, then a single neuron would have been able to predict sound source azimuth, which was not the case. I have the feeling that they actually mean "complimentary", but I leave it to the authors to clarify my confusion, should they wish.

In summary, the present study represents a significant body of work that contributes substantially to the field of spatial auditory coding and systems neuroscience. However, limitations of the imaging dataset and model as applied in the study muddles concrete conclusions about how the DCIC precisely encodes sound source azimuth and even more so to sound localisation in a behaving animal. Nevertheless, it presents a novel and unique dataset, which, regardless of secondary interpretation, corroborates the general notion that auditory space is encoded in an extraordinarily complex manner in the mammalian brain.

Reviewer #3 (Public Review):

Summary: Boffi and colleagues sought to quantify the single-trial, azimuthal information in the dorsal cortex of the inferior colliculus (DCIC), a relatively understudied subnucleus of the auditory midbrain. They used two complementary recording methods while mice passively listened to sounds at different locations: a large volume but slow sampling calcium-imaging method, and a smaller volume but temporally precise electrophysiology method. They found that neurons in the DCIC were variable in their activity, unreliably responding to sound presentation and responding during inter-sound intervals. Boffi and colleagues used a naïve Bayesian decoder to determine if the DCIC population encoded sound location on a single trial. The decoder failed to classify sound location better than chance when using the raw single-trial population response but performed significantly better than chance when using intermediate principal components of the population response. In line with this, when the most azimuth dependent neurons were used to decode azimuthal position, the decoder performed equivalently to the azimuthal localization abilities of mice. The top azimuthal units were not clustered in the DCIC, possessed a contralateral bias in response, and were correlated in their variability (e.g., positive noise correlations). Interestingly, when these noise correlations were perturbed by inter-trial shuffling decoding performance decreased. Although Boffi and colleagues display that azimuthal information can be extracted from DCIC responses, it remains unclear to what degree this information is used and what role noise correlations play in azimuthal encoding.

Strengths: The authors should be commended for collection of this dataset. When done in isolation (which is typical), calcium imaging and linear array recordings have intrinsic weaknesses. However, those weaknesses are alleviated when done in conjunction with one another - especially when the data largely recapitulates the findings of the other recording methodology. In addition to the video of the head during the calcium imaging, this data set is extremely rich and will be of use to those interested in the information available in the DCIC, an understudied but likely important subnucleus in the auditory midbrain.

The DCIC neural responses are complex; the units unreliably respond to sound onset, and at the very least respond to some unknown input or internal state (e.g., large inter-sound interval responses). The authors do a decent job in wrangling these complex responses: using interpretable decoders to extract information available from population responses.

Weaknesses:<br /> The authors observe that neurons with the most azimuthal sensitivity within the DCIC are positively correlated, but they use a Naïve Bayesian decoder which assume independence between units. Although this is a bit strange given their observation that some of the recorded units are correlated, it is unlikely to be a critical flaw. At one point the authors reduce the dimensionality of their data through PCA and use the loadings onto these components in their decoder. PCA incorporates the correlational structure when finding the principal components and constrains these components to be orthogonal and uncorrelated. This should alleviate some of the concern regarding the use of the naïve Bayesian decoder because the projections onto the different components are independent. Nevertheless, the decoding results are a bit strange, likely because there is not much linearly decodable azimuth information in the DCIC responses. Raw population responses failed to provide sufficient information concerning azimuth for the decoder to perform better than chance. Additionally, it only performed better than chance when certain principal components or top ranked units contributed to the decoder but not as more components or units were added. So, although there does appear to be some azimuthal information in the recoded DCIC populations - it is somewhat difficult to extract and likely not an 'effective' encoding of sound localization as their title suggests.

Although this is quite a worthwhile dataset, the authors present relatively little about the characteristics of the units they've recorded. This may be due to the high variance in responses seen in their population. Nevertheless, the authors note that units do not respond on every trial but do not report what percent of trials that fail to evoke a response. Is it that neurons are noisy because they do not respond on every trial or is it also that when they do respond they have variable response distributions? It would be nice to gain some insight into the heterogeneity of the responses. Additionally, is there any clustering at all in response profiles or is each neuron they recorded in the DCIC unique? They also only report the noise correlations for their top ranked units, but it is possible that the noise correlations in the rest of the population are different. It would also be worth digging into the noise correlations more - are units positively correlated because they respond together (e.g., if unit x responds on trial 1 so does unit y) or are they also modulated around their mean rates on similar trials (e.g., unit x and y respond and both are responding more than their mean response rate). A large portion of trial with no response can occlude noise correlations. More transparency around the response properties of these populations would be welcome.

It is largely unclear what the DCIC is encoding. Although the authors are interested in azimuth, sound location seems to be only a small part of DCIC responses. The authors report responses during inter-sound interval and unreliable sound-evoked responses. Although they have video of the head during recording, we only see a correlation to snout and ear movements (which are peculiar since in the example shown it seems the head movements predict the sound presentation). Additional correlates could be eye movements or pupil size. Eye movement are of particular interest due to their known interaction with IC responses - especially if the DCIC encodes sound location in relation to eye position instead of head position (though much of eye-position-IC work was done in primates and not rodent). Alternatively, much of the population may only encode sound location if an animal is engaged in a localization task. Ideally, the authors could perform more substantive analyses to determine if this population is truly noisy or if the DCIC is integrating un-analyzed signals.

Although this critique is ubiquitous among decoding papers in the absence of behavioral or causal perturbations, it is unclear what - if any - role the decoded information may play in neuronal computations. The interpretation of the decoder means that there is some extractable information concerning sound azimuth - but not if it is functional. This information may just be epiphenomenal, leaking in from inputs, and not used in computation or relayed to downstream structures. This should be kept in mind when the authors suggest their findings implicate the DCIC functionally in sound localization.

It is unclear why positive noise correlations amongst similarly tuned neurons would improve decoding. A toy model exploring how positive noise correlations in conjunction with unreliable units that inconsistently respond may anchor these findings in an interpretable way. It seems plausible that inconsistent responses would benefit from strong noise correlations, simply by units responding together. This would predict that shuffling would impair performance because you would then be sampling from trials in which some units respond, and trials in which some units do not respond - and may predict a bimodal performance distribution in which some trials decode well (when the units respond) and poor performance (when the units do not respond).

Significance: Boffi and colleagues set out to parse the azimuthal information available in the DCIC on a single trial. They largely accomplish this goal and are able to extract this information when allowing the units that contain more information about sound location to contribute to their decoding (e.g., through PCA or decoding on top unit activity specifically). The dataset will be of value to those interested in the DCIC and also to anyone interested in the role of noise correlations in population coding. Although this work is first step into parsing the information available in the DCIC, it remains difficult to interpret if/how this azimuthal information is used in localization behaviors of engaged mice.

Reviewer #1 (Public Review):

Summary:

The authors aim at dissecting the relationship between hair-cell directional mechanosensation and orientation-linked synaptic selectivity, using mice and the zebrafish. They find that Gpr156 mutant animals homogenize the orientation of hair cells without affecting the selectivity of afferent neurons, suggesting that hair-cell orientation is not the feature that determines synaptic selectivity. Therefore, the process of Emx2-dependent synaptic selectivity bifurcates downstream of Gpr156.

Strengths:

This is an interesting and solid paper. It solves an interesting problem and establishes a framework for the following studies. That is, to ask what are the putative targets of Emx2 that affect synaptic selectivity.<br /> The quality of the data is generally excellent.

Weaknesses:

The feeling is that the advance derived from the results is very limited.

Reviewer #2 (Public Review):

Summary:

The authors inquire in particular whether the receptor Gpr156, which is necessary for hair cells to reverse their polarities in the zebrafish lateral line and mammalian otolith organs downstream of the differential expression of the transcription factor Emx2, also controls the mechanosensitive properties of hair cells and ultimately an animal's behavior. This study thoroughly addresses the issue by analyzing the morphology, electrophysiological responses, and afferent connections of hair cells found in different regions of the mammalian utricle and the Ca2+ responses of lateral line neuromasts in both wild-type animals and gpr156 mutants. Although many features of hair cell function are preserved in the mutants-such as development of the mechanosensory organs and the Emx2-dependent, polarity-specific afferent wiring and synaptic pairing-there are a few key changes. In the zebrafish neuromast, the magnitude of responses of all hair cells to water flow resembles that of the wild-type hair cells that respond to flow arriving from the tail. These responses are larger than those observed in hair cells that are sensitive to flow arriving from the head and resemble effects previously observed in Emx2 mutants. The authors note that this behavior suggests that the Emx2-GPR156 signaling axis also impinges on hair cell mechanotransduction. Although mutant mice exhibit normal posture and balance, they display defects in swimming behavior. Moreover, their vestibulo-ocular reflexes are perturbed. The authors note that the gpr156 mutant is a good model to study the role of opposing hair cell polarity in the vestibular system, for the wiring patterns follow the expression patterns of Emx2, even though hair cells are all of the same polarity. This paper excels at describing the effects of gpr156 perturbation in mouse and zebrafish models and will be of interest to those studying the vestibular system, hair cell polarity, and the role of inner-ear organs in animal behavior.

Strengths:

The study is exceptional in including, not only morphological and immunohistochemical indices of cellular identity but also electrophysiological properties. The mutant hair cells of murine maculæ display essentially normal mechanoelectrical transduction and adaptation-with two or even three kinetic components-as well as normal voltage-activated ionic currents.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Napoli et al, the authors study the intracellular function of Cytosolic S100A8/A9 a myeloid cell soluble protein that operates extracellularly as an alarmin, whose intracellular function is not well characterized. Here, the authors utilize state-of-the-art intravital microscopy to demonstrate that adhesion defects observed in cells lacking S100A8/A9 (Mrp14-/-) are not rescued by exogenous S100A8/A9, thus highlighting an intrinsic defect. Based on this result subsequent efforts were employed to characterize the nature of those adhesion defects.

Strengths:

The authors convincingly show that Mrp14-/- neutrophils have normal rolling but defective adhesion caused by impaired CD11b activation (deficient ICAM1 binding). Analysis of cellular spreading (defective in Mrp14-/- cells) is also sound. The manuscript then focuses on selective signaling pathways and calcium measurements. Overall, this is a straightforward study of biologically important proteins and mechanisms.

Weaknesses:

Some suggestions are included below to improve this manuscript.

Reviewer #2 (Public Review):

Summary:

Napoli et al. provide a compelling study showing the importance of cytosolic S100A8/9 in maintaining calcium levels at LFA-1 nanoclusters at the cell membrane, thus allowing the successful crawling and adherence of neutrophils under shear stress. The authors show that cytosolic S100A8/9 is responsible for retaining stable and high concentrations of calcium specifically at LFA-1 nanoclusters upon binding to ICAM-1, and imply that this process aids in facilitating actin polymerisation involved in cell shape and adherence. The authors show early on that S100A8/9 deficient neutrophils fail to extravasate successfully into the tissue, thus suggesting that targeting cytosolic S100A8/9 could be useful in settings of autoimmunity/acute inflammation where neutrophil-induced collateral damage is unwanted.

Strengths:

Using multiple complementary methods from imaging to western blotting and flow cytometry, including extracellular supplementation of S100A8/9 in vivo, the authors conclusively prove a defect in intracellular S100A8/9, rather than extracellular S100A8/9 was responsible for the loss in neutrophil adherence, and pinpointed that S100A8/9 aided in calcium stabilisation and retention at the plasma membrane.

Weaknesses:

(1) Extravasation is shown to be a major defect of Mrp14-/- neutrophils, but the Giemsa staining in Figure 1H seems to be quite unspecific to me, as neutrophils were determined by nuclear shape and granularity. It would have perhaps been more clear to use immunofluorescence staining for neutrophils instead as seen in Supplementary Figure 1A (staining for Ly6G or other markers instead of S100A9).

(2) The representative image for Mrp14-/- neutrophils used in Figure 4K to demonstrate Ripley's K function seems to be very different from that shown above in Figures 4C and 4F.

(3) Although the authors have done well to draw a path linking cytosolic S100A8/9 to actin polymerisation and subsequently the arrest and adherence of neutrophils in vitro, the authors can be more explicit with the analysis - for example, is the F-actin co-localized with the LFA-1 nanoclusters? Does S100A8/9 localise to the membrane with LFA-1 upon stimulation? Lastly, I think it would have been very useful to close the loop on the extravasation observation with some in vitro evidence to show that neutrophils fail to extravasate under shear stress.

Reviewer #1 (Public Review):

I appreciate the normative approach of the PEA model and am eager to examine this model in the future. However, two minor issues remain:

(1) Clarification on the PReMo Model:

The authors state, "The PReMo model proposes that this drift comprises two phases: initial proprioceptive recalibration and subsequent visual recalibration." This description could misinterpret the intent of PReMo. According to PReMo, the time course of the reported hand position is merely a read-out of the *perceived hand position* (x_hat in your paper). Early in adaptation, the perceived hand position is biased by the visual cursor (x_hat in the direction of the cursor); towards the end, due to implicit adaptation, x_hat reduces to zero. This is the same as PEA. I recommend that the authors clarify PReMo's intent to avoid confusion.

Note, however, the observed overshoot of 1 degree in the reported hand position. In the PReMo paper, we hypothesized that this effect is due to the recalibration of the perceived visual target location (inspired by studies showing that vision is also recalibrated by proprioception, but in the opposite direction). If the goal of implicit adaptation is to align the perceived hand position (x_hat) with the perceived target position (t_hat), then there would be an overshoot of x_hat over the actual target position.

PEA posits a different account for the overshoot. It currently suggests that the reported hand position combines x_hat (which takes x_p as input) with x_p itself. What is reasoning underlying the *double occurrence* of x_p?

There seem to be three alternatives that seem more plausible (and could lead to the same overshooting): 1) increasing x_p's contribution (assuming visual uncertainty increases when the visual cursor is absent during the hand report phase), 2) decreasing sigma_p (assuming that participants pay more attention to the hand during the report phase), 3) it could be that the perceived target position undergoes recalibration in the opposite direction to proprioceptive recalibration. All these options, at least to me, seem equally plausible and testable in the future.

(2) Effect of Visual Uncertainty on Error Size:

I appreciate the authors' response about methodological differences between the cursor cloud used in previous studies and the Gaussian blob used in the current study. However, it is still not clear to me how the authors reconcile previous studies showing that visual uncertainty reduced implicit adaptation for small but not large errors (Tsay et al, 2021; Makino, et al 2023) with the current findings, where visual uncertainty reduced implicit adaptation for large but not small errors.

Could the authors connect the dots here: I could see that the cursor cloud increases potential overlap with the visual target when the visual error is small, resulting in intrinsic reward-like mechanisms (Kim et al, 2019), which could potentially explain attenuated implicit adaptation for small visual errors. However, why would implicit adaptation in response to large visual errors remain unaffected by the cursor cloud? Note that we did verify that sigma_v is increased in (Tsay et al. 2021), so it is unlikely due to the cloud simply failing as a manipulation of visual uncertainty.

In addition, we also reasoned that testing individuals with low vision could offer a different test of visual uncertainty (Tsay et al, 2023). The advantage here is that both control and patients with low vision are provided with the same visual input-a single cursor. Our findings suggest that uncertainty due to low vision also shows reduced implicit adaptation in response to small but not large errors, contrary to the findings in the current paper. Missing in the manuscript is a discussion related to why the authors' current findings contradict those of previous results.

Reviewer #2 (Public Review):

Summary:

The authors present the Perceptual Error Adaptation (PEA) model, a computational approach offering a unified explanation for behavioral results that are inconsistent with standard state-space models. Beginning with the conventional state-space framework, the paper introduces two innovative concepts. Firstly, errors are calculated based on the perceived hand position, determined through Bayesian integration of visual, proprioceptive, and predictive cues. Secondly, the model accounts for the eccentricity of vision, proposing that the uncertainty of cursor position increases with distance from the fixation point. This elegantly simple model, with minimal free parameters, effectively explains the observed plateau in motor adaptation under the implicit motor adaptation paradigm using the error-clamp method. Furthermore, the authors experimentally manipulate visual cursor uncertainty, a method established in visuomotor studies, to provide causal evidence. Their results show that the adaptation rate correlates with perturbation sizes and visual noise, uniquely explained by the PEA model and not by previous models. Therefore, the study convincingly demonstrates that implicit motor adaptation is a process of Bayesian cue integration

Strengths:

In the past decade, numerous perplexing results in visuomotor rotation tasks have questioned their underlying mechanisms. Prior models have individually addressed aspects like aiming strategies, motor adaptation plateaus, and sensory recalibration effects. However, a unified model encapsulating these phenomena with a simple computational principle was lacking. This paper addresses this gap with a robust Bayesian integration-based model. Its strength lies in two fundamental assumptions: motor adaptation's influence by visual eccentricity, a well-established vision science concept, and sensory estimation through Bayesian integration. By merging these well-founded principles, the authors elucidate previously incongruent and diverse results with an error-based update model. The incorporation of cursor feedback noise manipulation provides causal evidence for their model. The use of eye-tracking in their experimental design, and the analysis of adaptation studies based on estimated eccentricity, are particularly elegant. This paper makes a significant contribution to visuomotor learning research.

The authors discussed in the revised version that the proposed model can capture the general implicit motor learning process in addition to the visuomotor rotation task. In the discussion, they emphasize two main principles: the automatic tracking of effector position and the combination of movement cues using Bayesian integration. These principles are suggested as key to understanding and modeling various motor adaptations and skill learning. The proposed model could potentially become a basis for creating new computational models for skill acquisition, especially where current models fall short.

Weaknesses:

The proposed model is described as elegant. In this paper, the authors test the model within a limited example condition, demonstrating its relevance to the sensorimotor adaptation mechanisms of the human brain. However, the scope of the model's applicability remains unclear. It has shown the capacity to explain prior data, thereby surpassing previous models that rely on elementary mathematics. To solidify its credibility in the field, the authors must gather more supporting evidence.

Reviewer #3 (Public Review):

(2.1) Summary

In this paper, the authors model motor adaptation as a Bayesian process that combines visual uncertainty about the error feedback, uncertainty about proprioceptive sense of hand position, and uncertainty of predicted (=planned) hand movement with a learning and retention rate as used in state space models. The model is built with results from several experiments presented in the paper and is compared with the PReMo model (Tsay, Kim et al., 2022) as well as a cue combination model (Wei & Körding, 2009). The model and experiments demonstrate the role of visual uncertainty about error feedback in implicit adaptation.

In the introduction, the authors notice that implicit adaptation (as measured in error-clamp based paradigms) does not saturate at larger perturbations, but decreases again (e.g. Moorehead et al., 2017 shows no adaptation at 135{degree sign} and 175{degree sign} perturbations). They hypothesized that visual uncertainty about cursor position increases with larger perturbations since the cursor is further from the fixated target. This could decrease importance assigned to visual feedback which could explain lower asymptotes.

The authors characterize visual uncertainty for 3 rotation sizes in a first experiment, and while this experiment could be improved, it is probably sufficient for the current purposes. Then the authors present a second experiment where adaptation to 7 clamped errors are tested in different groups of participants. The models' visual uncertainty is set using a linear fit to the results from experiment 1, and the remaining 4 parameters are then fit to this second data set. The 4 parameters are 1) proprioceptive uncertainty, 2) uncertainty about the predicted hand position, 3) a learning rate and 4) a retention rate. The authors' Perceptual Error Adaptation model ("PEA") predicts asymptotic levels of implicit adaptation much better than both the PReMo model (Tsay, Kim et al., 2022), which predicts saturated asymptotes, or a causal inference model (Wei & Körding, 2007) which predicts no adaptation for larger rotations. In a third experiment, the authors test their model's predictions about proprioceptive recalibration, but unfortunately compare their data with an unsuitable other data set (Tsay et al. 2020, instead of Tsay et al. 2021). Finally, the authors conduct a fourth experiment where they put their model to the test. They measure implicit adaptation with increased visual uncertainty, by adding blur to the cursor, and the results are again better in line with their model (predicting overall lower adaptation), than with the PReMo model (predicting equal saturation but at larger perturbations) or a causal inference model (predicting equal peak adaptation, but shifted to larger rotations). In particular the model fits for experiment 2 and the results from experiment 4 show that the core idea of the model has merit: increased visual uncertainty about errors dampens implicit adaptation.

(2.2) Strengths

In this study the authors propose a Perceptual Error Adaptation model ("PEA") and the work combines various ideas from the field of cue combination, Bayesian methods and new data sets, collected in four experiments using various techniques that test very different components of the model. The central component of visual uncertainty is assessed in a first experiment. The model uses 4 other parameters to explain implicit adaptation. These parameters are: 1) a learning and 2) a retention rate, as used in popular state space models and the uncertainty (variance) of 3) predicted and 4) proprioceptive hand position. In particular, the authors observe that asymptotes for implicit learning do not saturate, as claimed before, but decrease again when rotations are very large and that this may have to do with visual uncertainty (e.g. Tsay et al., 2021, J Neurophysiol 125, 12-22). The final experiment confirms predictions of the fitted model about what happens when visual uncertainty is increased (overall decrease of adaptation). By incorporating visual uncertainty depending on retinal eccentricity, the predictions of the PEA model for very large perturbations are notably different from, and better than, the predictions of the two other models it is compared to. That is, the paper provides strong support for the idea that visual uncertainty of errors matters for implicit adaptation.

(2.3) Weaknesses

Although the authors don't say this, the "concave" function that shows that adaptation does not saturate for larger rotations has been shown before, including in papers cited in this manuscript.

The first experiment, measuring visual uncertainty for several rotation sizes in error-clamped paradigms has several shortcomings, but these might not be so large as to invalidate the model or the findings in the rest of the manuscript. There are two main issues we highlight here. First, the data is not presented in units that allow comparison with vision science literature. Second, the 1 second delay between movement endpoint and disappearance of the cursor, and the presentation of the reference marker, may have led to substantial degradation of the visual memory of the cursor endpoint. That is, the experiment could be overestimating the visual uncertainty during implicit adaptation.

The paper's third experiment relies to a large degree on reproducing patterns found in one particular paper, where the reported hand positions - as a measure of proprioceptive sense of hand position - are given and plotted relative to an ever present visual target, rather than relative to the actual hand position. That is, 1) since participants actively move to a visual target, the reported hand positions do not reflect proprioception, but mostly the remembered position of the target participants were trying to move to, and 2) if the reports are converted to a difference between the real and reported hand position (rather than the difference between the target and the report), those would be on the order of ~20{degree sign} which is roughly two times larger than any previously reported proprioceptive recalibration, and an order of magnitude larger than what the authors themselves find (1-2{degree sign}) and what their model predicts. Experiment 3 is perhaps not crucial to the paper, but it nicely provides support for the idea that proprioceptive recalibration can occur with error-clamped feedback.

Perhaps the largest caveat to the study is that it assumes that people do not look at the only error feedback available to them (and can explicitly suppress learning from it). This was probably true in the experiments used in the manuscript, but unlikely to be the case in most of the cited literature. Ignoring errors and suppressing adaptation would also be a disastrous strategy to use in the real world, such that our brains may not be very good at this. So the question remains to what degree - if any - the ideas behind the model generalize to experiments without fixation control, and more importantly, to real life situations.

Reviewer #1 (Public Review):

This is a very interesting paper which addresses how auditory cortex represents sound while an animal is performing an auditory task. The study involves psychometric and neurophysiological measurements from ferrets engaged in a challenging tone in noise discrimination task, and relates these measurements using neurometric analysis. A novel neural decoding technique (decoding-based dimensionality reduction or dDR, introduced in a previous paper by two of the authors) is used to reduce bias so that stimulus parameters can be read out from neuronal responses.

The central finding of the study is that, when an animal is engaged in a task, non-primary auditory cortex represents task-relevant sound features in a categorical way. In primary cortex, task engagement also affects representations, but in a different way - the decoding is improved (suggesting that representations have been enhanced), but is not categorical in nature. The authors argue that these results are compatible with a model where early sensory representations form an overcomplete representation of the world, and downstream neurons flexibly read out behaviourally relevant information from these representations.

I find the concept and execution of the study very interesting and elegant. The paper is also commendably clear and readable. The differences between primary and higher cortex are compelling and I am largely convinced by the authors' claim that they have found evidence that broadly supports a mixed selectivity model of neural disentanglement along the lines of Rigotti et al (2013). I think that the increasing body of evidence for these kinds of representations is a significant development in our understanding of higher sensory representations. I also think that the dDR method is likely to be useful to researchers in a variety of fields who are looking to perform similar types of neural decoding analysis.

Reviewer #2 (Public Review):

This study compares the activity of neural populations in the primary and non-primary auditory cortex of ferrets while the animals actively behaved or passively listened to a sound discrimination task. Using a variety of methods, the authors convincingly show differential effects of task engagement on population neural activity in primary vs non-primary auditory cortex; notably that in the primary auditory cortex, task-engagement (1) improves discriminability for both task-relevant and non-task relevant dimensions, and (2) improves the alignment between covariability and sound discrimination axes; whereas in the non-primary auditory cortex, task-engagement (1) improves discriminability for only task-relevant dimensions, and (2) does not affect the alignment between covariability and sound discrimination axes. They additionally show that task-engagement changes in gain can account for the selectivity noted in the discriminability of non-primary auditory neurons. They also admirably attempt to isolate task-engagement from arousal fluctuations, by using fluctuations in pupil size as a proxy for physiological arousal. This is a well-carried out study with thoughtful analyses which in large part achieves its aims to evaluate how task-engagement changes neural activity across multiple auditory regions . As with all work, there are several caveats or areas for future study/analysis. First, the sounds used here (tones, and narrow-band noise) are relatively simple sounds; previous work suggests that exactly what activity is observed within each region (e.g., sensory only, decision-related, etc) may depend in part upon what stimuli are used. Therefore, while the current study adds importance to the literature, future work may consider the use of more varied stimuli. Second, the animals here were engaged in a behavioral task; but apart from an initial calculation of behavioral d', the task performance (and its effect on neural activity) is largely unaddressed.

Reviewer #1 (Public Review):

The main focus of the current study is to identify the anatomical core of an expiratory oscillator in the medulla using pharmacological disinhibition. Although expiration is passive in normal eupneic conditions, activation of the parafacial (pFL) region is believed to evoke active expiration in conditions of elevated ventilatory demands. The authors and others in the field have previously attempted to map this region using pharmacological, optogenetic and chemogenetic approaches, which present with their own challenges.

In the present study, the authors take a systematic approach to determine the precise anatomical location within the ventral medulla's rostro-caudal axis where the expiratory oscillator is located. The authors used a bicuculline (a GABA-A receptor antagonist) and fluorobeads solution at 5 distinct anatomical locations to study the effects on neuronal excitability and functional circuitry in the pFL. The effects of bicuculline on different phases of the respiratory cycle were characterized using a multidimensional cycle-by-cycle analysis. This analysis involved measuring the differences in airflow, diaphragm electromyography (EMG), and abdominal EMG signals, as well as using a phase-plane analysis to analyze the combined differences of these respiratory signals. Anatomical immunostaining techniques were also used to complement the functional mapping of the pFL.

Major strengths of this work include a robust study design, complementary neurophysiological and immunohistochemical methods and the use of a novel phase-plane analysis. The authors construct a comprehensive functional map revealing functional nuances in respiratory responses to bicuculline along the rostrocaudal axis of the parafacial region. They convincingly show that although bicuculline injections at all coordinates of the pFL generated an expiratory response, the most rostral locations in the lateral parafacial region play the strongest role in generating active expiration. These were characterized by a strong impact on the duration and strength of ABD activation, and a robust change in tidal volume and minute ventilation. The authors also confirmed histologically that none of the injection sites overlapped grossly with PHOX2B+ neurons, thus confirming the specificity of the injections in the pFL and not the neighboring RTN.

Although a caveat of the approach is that bicuculine injections have indiscriminate effects on other neuronal populations in the region (GABAergic, glycinergic, and glutamatergic), the results can largely be interpreted as modulation of neuronal populations in different regions of the pFL have differential effects on expiratory output. This limitation of the pharmacological approach has also been aptly discussed by the authors.

Collectively, these findings advance our understanding of the presumed expiratory oscillator, the pFL, and highlight the functional heterogeneity in the functional response of this anatomical structure.

Reviewer #3 (Public Review):

Summary:<br /> The study conducted by Pisanski et al investigates the role of the lateral parafacial area (pFL) in controlling active expiration. Stereotactic injections of bicuculline were utilized to map various pFL sites and their impact on respiration. The results indicate that injections at more rostral pFL locations induce the most robust changes in tidal volume, minute ventilation, and combined respiratory responses. The study indicates that the rostro-caudal organization of the pFL and its influence on breathing is not simple and uniform.

Strengths:<br /> The data provide novel insights into the importance of rostral locations in controlling active expiration. The authors use innovative analytic methods to characterize the respiratory effects of bicuculline injections into various areas of the pFL.

Weaknesses:<br /> Bicuculline injections increase the excitability of neurons. Aside of blocking GABA receptors, bicuculline also inhibits calcium-activated potassium currents and potentiates NMDA currents, thus insights into the role of GABAergic inhibition are limited.<br /> Increasing the excitability of neurons provides little insights into the activity pattern and function of the activated neurons. Without recording from the activated neurons, it is impossible to know whether an effect on active expiration or any other respiratory phase is caused by bicuculline acting on rhythmogenic neurons or tonic neurons that modulate respiration. While this approach is inappropriate to study the functional extent of the conditional "oscillator" for active expiration, it still provides valuable insights into this region's complex role in controlling breathing .

Reviewer #1 (Public Review):

Summary :

This study presents valuable data on diurnal patterns in aphid (Rhopalosiphum padi) feeding behavior and transcriptome profiles. The authors measured honeydew production by the aphids on plants and artificial diet during the day and night and conducted a comprehensive feeding behavior study using EPG with many biological replicates at 6 time-points in 24 hours. They also conducted transcriptome analyses of three samples of each 30 aphids at these time points. Differentially expressed transcripts were grouped into four clusters with distinct expression patterns. The expression of two genes found to be diurnally rhythmic was knocked down with RNAi and these aphids did less well, especially at night. They also analyzed the differential expression of candidate effector genes and found rhythmic ones to be enriched for more expression in aphid heads versus bodies - this pattern is expected given that effectors are most likely expressed in the salivary glands. Knockdown of a known effector (C002) that is diurnally rhythmic, and a novel effector gene, was found to alter aphid feeding dynamics and performance.

Strengths:

The manuscript was highly accessible, with clear writing, and the figures provided were both comprehensive and of good quality. The datasets generated from this research are valuable to the research field, especially the findings for honeydew secretion, EPG analysis, and transcriptome experiments.

The datasets generated in this study will be useful to scientists working on aphids and aphid-plant interactions and will inform similar studies on other insect species.

Weaknesses:

The weaknesses mainly relate to the (depth of) analyses and interpretation of the data. Also, some methods require more explanation, as follows:

In Figure 1, data show that aphids produce more honeydew at night than during the day. This suggests that the aphids ingest more phloem (E2 phase). However, in Figure 1d the duration of the E2 phase does not show obvious differences among the time points in the 24 hours. The authors contribute the explanation that the aphids may osmoregulate more during the night, leading to more honeydew secretion at night. This may be the case, but there could be other explanations. For example, the physiology, including regulation of water transport, of plants is known to change during night/day. The authors may focus this section more on the differences in the E1 phase, as this involves the delivery of aphid saliva and effectors into the plant phloem.

Transcriptome data shown in Figure 2 (and the experimental procedure of Figure 5b) appears to be based on three biological replicates. However, these replicates appear to have been harvested at the same time in the experiment, and this makes them technical replicates, not biological replicates. The inclusion of true biological replicates that include samples from time series experiments done on different days should be considered.

The authors conducted knockdown experiments targeting aquaporin 1 and gut sucrase 1 in aphids, resulting in reduced nymph production and decreased honeydew secretion. It is concluded that these results indicate significant roles of aquaporin 1 and gut sucrase 1 in diurnal regulation. However, it is essential to consider that these genes likely play crucial roles in aphid physiology beyond diurnal rhythms. Consequently, reduced expression would naturally impair aphid performance. The dsAQP1 and dsSUC1 aphids consistently produced less honeydew, regardless of the time of day, indicating a broader impact of gene knockdown. The observed increase of the phenotype at night may not be attributable to the specific roles of these genes in diurnal regulation but rather due to heightened aphid activity during that time (as evidenced by increased honeydew secretion) that could magnify the impact of the knockdown effect, making it easier to observe. Therefore, the knockdown of aquaporin 1 and gut sucrase 1 may exert a general negative influence on aphid fitness, independently of diurnal factors.

To analyze the roles of genes in diurnal regulation, additional controls should be incorporated. This could involve the knockdown of genes with essential functions that are not influenced by diurnal rhythms, providing a baseline comparison. Furthermore, consider including genes known to be involved in diurnal regulation in other insects, as documented in the existing literature, in the experimental design.

The same arguments as for aquaporin 1 and gut sucrase 1 above may be made for knockdown of effector genes (Figure 4). It has already been shown that knockdown of C002 impacts aphid performance, and the data herein may be explained by a general lower performance of aphids rather than a specific function of these effectors in diurnal regulation. It is also expected that knockdown of the effectors has less impact on aphids feeding from artificial diets. This does not necessarily indicate the role of the effectors in diurnal regulation.

In the abstract and elsewhere, the authors assert priority by stating, "...the first evidence of...". However, it's important to note that priority claims are often challenging to verify across many fields. Instead of relying solely on claims of precedence, the evidence presented in the research could stand on its own merit.

Conclusion:

The study presents intriguing new findings, particularly in the realms of honeydew analysis, EPG, and transcriptome analysis. However, the interpretation of subsequent studies employing gene knockdowns needs further consideration.

Reviewer #2 (Public Review):

Summary:

The authors conducted a time-course of whole-body transcriptional analysis of a pest aphid, Rhopalosiphum padi, and identified four major clusters of the genes that show diurnal rhythmicity in transcription. In addition, they conducted the analysis of aphid feeding behaviour and showed that aphids salivate longer from the end of the day toward the beginning of the night while their phloem feeding time does not change throughout the day. The genes up-regulated at night time were enriched with the genes involved in metabolic activities, collaborating with the results showing a higher number of honeydew excretion at night. The authors identified the list of candidate salivary genes that show diurnal rhythmicity in the transcription and silenced a salivary gene C002 and the candidate salivary gene E8696. Silencing of these genes reduced aphid fecundity and survival rate on the host plant but not on the artificial diet.

Strengths:

The time-course transcription study and its analysis will be of interest to researchers studying diurnal rhythms in insect biology. Also, the analysis of aphid feeding behaviour at different times of day is interesting. This study provides variable resources for those who study insect biology.

Weaknesses:

It is not clear to me which data was used to define the putative salivary effectors for R. padi, but the candidate salivary gene list made by Thorpe et al consists of the aphid genes encoding secreted proteins that are up-regulated in the head samples compared to the body samples. Although some proteins were confirmed to be secreted into the aphid saliva, many genes in the list are not confirmed to be expressed in the aphid salivary glands, and their products are not confirmed to be secreted into the saliva and the plant. Is E8696 expressed in the aphid salivary glands and secreted into its host plant? Without the data confirming the expression of the gene in the salivary glands and its secretion into the saliva and into the host plant, we cannot call the protein a salivary protein. Furthermore, without the observation that E8696 has some effect on plant biology, we cannot call it an aphid effector. Therefore, I cannot agree with the parts of the manuscript that refer to E8686 as an aphid salivary effector.

It is interesting to know that some candidate salivary gene expression showed a diurnal rhythm. However, without the knowledge of the functions of the salivary effectors, especially their targets, it is not possible to conclude that the rhythmical expression is important for the aphid performance. In addition, I wonder whether the increase in gene expression is directly correlated with the increase of protein secretion into the saliva and the plant.

Finally, the authors examined aphid survival, fecundity, and feeding behaviour. Those are important for overall aphid performance, but they do not "shape" aphid colonization. Aphid colonisation is shaped by the mechanisms by which aphids find and select their host plant and start to feed on it. Therefore, I do not agree with the title of this manuscript and some parts of the discussion.

I would like the authors to develop how the knowledge of the diurnal rhythm of aphid feeding can contribute to optimise pest management. I see that there are some differences in aphid metabolism and feeding behaviour between day and night, but I would like to hear how such knowledge can optimise pest management strategies.

Reviewer #1 (Public Review):

The authors perform RNA-seq on FACS isolated neurons from adult worms at days 1 and 8 of adulthood to profile the gene expression changes that occur with cognitive decline. Supporting data are included indicating that by day 7 of adulthood, learning and memory are reduced, indicating that this timepoint or after represents cognitively aged worms. Neuronal identity genes are reduced in expression within the cognitively aged worms, whereas genes involved in proteostasis, transcription/chromatin, and the stress response are elevated. A number of specific examples are provided, representing markers of specific neuronal subtypes, and correlating expression changes to the erosion of particular functions (e.g. motor neurons, chemosensory neurons, aversive learning neurons, etc).

To investigate whether upregulation of genes in neurons with age is compensatory or deleterious, the authors reduced expression of a set of three significantly upregulated genes and performed behavioral assays in young adults. In each case, reduction of expression improved memory, consistent with a model in which age-associated increases impair neuronal function.

The authors then characterize learning and memory in wild type, daf-2, and daf-2/daf-16 worms with age and find that daf-2 worms have an extended ability to learn for approximately 10 days longer that wild types. This was daf-16 dependent. Memory was extended in daf-2 as well, and strikingly, daf-2;daf-16 had no short term memory even at day 1. Transcriptomic analysis of FACS-sorted neurons was performed on the three groups at day 8. The authors focus their analysis on daf-2 vs. daf-2;daf-16 and present evidence that daf-2 neurons express a stress-resistance gene program. They also find small differences between the N2 and daf-2;daf-16 neurons, which correlate with the observed behavioral differences, though these differences are modest.

The authors tested eight candidate genes that were more highly expressed in daf-2 neurons vs. daf-2;daf-16 and showed that reduction of 2 and 5 of these genes impaired learning and memory, respectively, in daf-2 worms. This finding implicates specific neuronal transcriptional targets of IIS in maintaining cognitive ability in daf-2 with age, which, importantly, are distinct from those in young wild type worms.

Overall, this is a strong study with rigorously performed experiments. The authors achieved their aim of identifying transcriptional changes in neurons that underlie loss of learning and memory in C. elegans, and how cognition is maintained in insulin/IGF-1-like signaling mutants.

Reviewer #2 (Public Review):

Weng et al. perform a comprehensive study of gene expression changes in young and old animals, in wild-type and daf-2 insulin receptor mutants, in the whole animal and specifically in the nervous system. Using this data, they identify gene families that are correlated with neuronal ageing, as well as a distinct set of genes that are upregulated in neurons of aged daf-2 mutants. This is particularly interesting as daf-2 mutants show both extended lifespan and healthier neurons in aged animals, reflected by better learning/memory in older animals compared with wild-type controls. Indeed, knockdown of several of these upregulated genes resulted in poorer learning and memory. In addition, the authors showed that several genes upregulated during ageing in wild-type neurons also contribute to learning and memory; specifically, knockdown of these genes in young animals resulted in improved memory. This indicates that (at least in this small number of cases), genes that show increased transcript levels with age in the nervous system somehow suppress memory, potentially by having damaging effects on neuronal health.

Finally, from a resource perspective, the neuronal transcriptome provided here will be very useful for C. elegans researchers as it adds to other existing datasets by providing the transcriptome of older animals (animals at day 8 of adulthood) and demonstrating the benefits of performing tissue-specific RNAseq instead of whole-animal sequencing.

The work presented here is of high quality and the authors present convincing evidence supporting their conclusions.

Reviewer #3 (Public Review):

Summary

In this manuscript, Weng et al. identify the neuron specific transcriptome that impacts age dependent cognitive decline. The authors design a pipeline to profile neurons from wild type and long-lived insulin receptor/IGF-1 mutants using timepoints when memory functions are declining. They discover signatures unique to neurons which validates their approach. The authors identify that genes related to neuronal identity are lost with age in wild type worms. For example, old neurons reduce the expression of genes linked to synaptic function and neuropeptide signaling and increase the expression of chromatin regulators, insulin peptides and glycoproteins. Depletion of selected genes which are upregulated in old neurons (utx-1, ins-19 and nmgp-1) leads to improved short memory function. This indicates that some genes that increase with age have detrimental effects on learning and memory. The pipeline is then used to test neuronal profiles of long-lived insulin/IGF-1 daf-2 mutants. Genes related to stress response pathways are upregulated in long lived daf-2 mutants (e.g. dod-24, F08H9.4) and those genes are required for improved neuron function.

Strengths

The manuscript is well written, and the experiments are well described. The authors take great care to explain their reasoning for performing experiments in a specific way and guide the reader through the interpretation of the results, which makes this manuscript an enjoyable and interesting read. The authors discover novel regulators of learning and memory using neuron-specific transcriptomic analysis in aged animals, which underlines the importance of cell specific deep sequencing. The timepoints of the transcriptomic profiling are elegantly chosen, as they coincide with the loss of memory and can be used to specifically reveal gene expression profiles related to neuron function. The authors discuss on the dod-24 example how powerful this approach is. In daf-2 mutants whole-body dod-24 expression differs from neuron specific profiles, which underlines the importance of precise cell specific approaches. This dataset provides a very useful resource for the C. elegans and aging community as it complements existing datasets with additional time points and neuron specific deep profiling.

Reviewer #1 (Public Review):

Summary:

This computational modeling study builds on multiple previous lines of experimental and theoretical research to investigate how a single neuron can solve a nonlinear pattern classification task. The authors construct a detailed biophysical and morphological model of a single striatal medium spiny neuron, and endow excitatory and inhibitory synapses with dynamic synaptic plasticity mechanisms that are sensitive to (1) the presence or absence of a dopamine reward signal, and (2) spatiotemporal coincidence of synaptic activity in single dendritic branches. The latter coincidence is detected by voltage-dependent NMDA-type glutamate receptors, which can generate a type of dendritic spike referred to as a "plateau potential." The proposed mechanisms result in moderate performance on a nonlinear classification task when specific input features are segregated and clustered onto individual branches, but reduced performance when input features are randomly distributed across branches. Given the high level of complexity of all components of the model, it is not clear which features of which components are most important for its performance. There is also room for improvement in the narrative structure of the manuscript and the organization of concepts and data.

Strengths:

The integrative aspect of this study is its major strength. It is challenging to relate low-level details such as electrical spine compartmentalization, extrasynaptic neurotransmitter concentrations, dendritic nonlinearities, spatial clustering of correlated inputs, and plasticity of excitatory and inhibitory synapses to high-level computations such as nonlinear feature classification. Due to high simulation costs, it is rare to see highly biophysical and morphological models used for learning studies that require repeated stimulus presentations over the course of a training procedure. The study aspires to prove the principle that experimentally-supported biological mechanisms can explain complex learning.

Weaknesses:

The high level of complexity of each component of the model makes it difficult to gain an intuition for which aspects of the model are essential for its performance, or responsible for its poor performance under certain conditions. Stripping down some of the biophysical detail and comparing it to a simpler model may help better understand each component in isolation. That said, the fundamental concepts behind nonlinear feature binding in neurons with compartmentalized dendrites have been explored in previous work, so it is not clear how this study represents a significant conceptual advance. Finally, the presentation of the model, the motivation and justification of each design choice, and the interpretation of each result could be restructured for clarity to be better received by a wider audience.

Reviewer #2 (Public Review):

Summary:

The study explores how single striatal projection neurons (SPNs) utilize dendritic nonlinearities to solve complex integration tasks. It introduces a calcium-based synaptic learning rule that incorporates local calcium dynamics and dopaminergic signals, along with metaplasticity to ensure stability for synaptic weights. Results show SPNs can solve the nonlinear feature binding problem and enhance computational efficiency through inhibitory plasticity in dendrites, emphasizing the significant computational potential of individual neurons. In summary, the study provides a more biologically plausible solution to single-neuron learning and gives further mechanical insights into complex computations at the single-neuron level.

Strengths:

The paper introduces a novel learning rule for training a single multicompartmental neuron model to perform nonlinear feature binding tasks (NFBP), highlighting two main strengths: the learning rule is local, calcium-based, and requires only sparse reward signals, making it highly biologically plausible, and it applies to detailed neuron models that effectively preserve dendritic nonlinearities, contrasting with many previous studies that use simplified models.

Weaknesses:

I am concerned that the manuscript was submitted too hastily, as evidenced by the quality and logic of the writing and the presentation of the figures. These issues may compromise the integrity of the work. I would recommend a substantial revision of the manuscript to improve the clarity of the writing, incorporate more experiments, and better define the goals of the study.

Major Points:

(1) Quality of Scientific Writing: The current draft does not meet the expected standards. Key issues include:

i. Mathematical and Implementation Details: The manuscript lacks comprehensive mathematical descriptions and implementation details for the plasticity models (LTP/LTD/Meta) and the SPN model. Given the complexity of the biophysically detailed multicompartment model and the associated learning rules, the inclusion of only nine abstract equations (Eq. 1-9) in the Methods section is insufficient. I was surprised to find no supplementary material providing these crucial details. What parameters were used for the SPN model? What are the mathematical specifics for the extra-synaptic NMDA receptors utilized in this study? For instance, Eq. 3 references [Ca2+]-does this refer to calcium ions influenced by extra-synaptic NMDARs, or does it apply to other standard NMDARs? I also suggest the authors provide pseudocodes for the entire learning process to further clarify the learning rules.

ii. Figure quality. The authors seem not to carefully typeset the images, resulting in overcrowding and varying font sizes in the figures. Some of the fonts are too small and hard to read. The text in many of the diagrams is confusing. For example, in Panel A of Figure 3, two flattened images are combined, leading to small, distorted font sizes. In Panels C and D of Figure 7, the inconsistent use of terminology such as "kernels" further complicates the clarity of the presentation. I recommend that the authors thoroughly review all figures and accompanying text to ensure they meet the expected standards of clarity and quality.

iii. Writing clarity. The manuscript often includes excessive and irrelevant details, particularly in the mathematical discussions. On page 24, within the "Metaplasticity" section, the authors introduce the biological background to support the proposed metaplasticity equation (Eq. 5). However, much of this biological detail is hypothesized rather than experimentally verified. For instance, the claim that "a pause in dopamine triggers a shift towards higher calcium concentrations while a peak in dopamine pushes the LTP kernel in the opposite direction" lacks cited experimental evidence. If evidence exists, it should be clearly referenced; otherwise, these assertions should be presented as theoretical hypotheses. Generally, Eq. 5 and related discussions should be described more concisely, with only a loose connection to dopamine effects until more experimental findings are available.

(2) Goals of the Study: The authors need to clearly define the primary objective of their research. Is it to showcase the computational advantages of the local learning rule, or to elucidate biological functions?

i. Computational Advantage: If the intent is to demonstrate computational advantages, the current experimental results appear inadequate. The learning rule introduced in this work can only solve for four features, whereas previous research (e.g., Bicknell and Hausser, 2021) has shown capability with over 100 features. It is crucial for the authors to extend their demonstrations to prove that their learning rule can handle more than just three features. Furthermore, the requirement to fine-tune the midpoint of the synapse function indicates that the rule modifies the "activation function" of the synapses, as opposed to merely adjusting synaptic weights. In machine learning, modifying weights directly is typically more efficient than altering activation functions during learning tasks. This might account for why the current learning rule is restricted to a limited number of tasks. The authors should critically evaluate whether the proposed local learning rule, including meta-plasticity, actually offers any computational advantage. This evaluation is essential to understand the practical implications and effectiveness of the proposed learning rule.

ii. Biological Significance: If the goal is to interpret biological functions, the authors should dig deeper into the model behaviors to uncover their biological significance. This exploration should aim to link the observed computational features of the model more directly with biological mechanisms and outcomes.

Reviewer #1 (Public Review):

Summary:

Schweibenz et al are investigating how cells with lower levels of Tai are out-competed by neighboring wild-type (WT) cells. They show that clones homozygous for a tai hypomorphic mutation are disadvantaged and are killed by apoptosis. But tai-low clones are partially rescued when generated in a background that is heterozygous for mutations in apoptotic genes, in the Hippo pathway component warts, or for the Wg/Wnt pathway negative regulator Apc. They then follow up in the link between tai LOF and Wg. The story then shifts away from clones and into experiments that have Tai RNAi depletion or Tai over-expression in the posterior compartment of the wing disc, using the anterior compartment as a control. These non-clonal experiments show that depletion of Tai in the posterior compartment of wing discs results in less Wg in this compartment. This is shown to be due to a reduction in the glypican Dally-like protein (Dlp). The fact that long-range Wg is reduced in tai-depleted discs that also show a reduction in Dlp, suggests that Tai somehow positively promotes Wg distribution. There is some data in the supplementary materials suggesting that Tai promotes dlp mRNA expression but this was not compelling. In fact, the compelling data was that Dlp protein in tai mutant clones is not abundantly on the cell surface, but instead somehow retained in the mutant cell. The authors don't further examine Dlp protein in tai clones. The final figure (Figure 8) shows that there is less Wg at the DV margin in wing discs when tai is depleted from wg-producing cells. In sum, the authors have uncovered some interesting results, but the story has some unresolved issues that, if addressed, could boost its impact. Additionally, the preprint seems to have 2 stories, one about tai and cell competition and the other about tai and Wg distribution. It would be helpful to reorder the figures and improve the narrative so that these are better integrated with each other.

Strengths:

The authors are studying competition between tai-low clones and their fitter WT neighbors, and have uncovered an interesting connection to Wg.

Weaknesses:

(1) It would be good to know whether the authors can rescue tai-low clones by over-expression UAS-Dlp.

(2) The data about tai-promoting dlp (Figure S4) is not compelling as there are no biological replicates and no statistical analyses.

(3) The data on Wg distribution seems disjointed from the data about cell competition. The authors could refocus the paper to emphasize the cell competition story. The role of Dlp in Wg distribution is well established, so the authors could remove or condense these results. The story really could be Figsured 1, 2, 3 and 7 and keep the paper focused on cell competition. The authors could then discuss Dlp as needed for Wg signaling transduction, which is already established in the literature.

(4) The model of tai controlling dlp mRNA and Dlp protein distribution is confusing. In fact, the data for the former is weak, while the data for the latter is strong. I suggest that the authors focus on the altered Dlp protein distribution on tai-low clones. It would also be helpful to prove the Wg signaling is impeded in tai clones (see #5 below).

(5) I don't know if the Fz3-RFP reported for Wg signaling works in imaginal discs, but if it does then the authors could make clones in this background to prove that cell-autonomous Wg signaling is reduced in tai-low clones.

Reviewer #2 (Public Review):

The authors investigate the properties of the transcriptional co-activator Taiman in regulating tissue growth. In previously published work they had shown that cells that overexpress Tiaman in the pupal wing can cause the death of thoracic cells adjacent to the wing tip to die and thus allow the wing to invade the thorax. This was mediated by the secretion of Spz ligands. Here, they investigate the properties of cells that are homozygous for a hypomorphic allele of taiman (tai). They show that homozygous mutant clones are much smaller than their wild-type twin spots and that cells in the clones are dying by apoptosis which is inferred from elevated levels of anti-Dcp1 staining (Figure 1).

By generating clones during eye development, the authors screen for dominant modifiers that increase the representation of homozygous tai tissue in the adult eye (Figure 2). They find that reducing the levels of hid, the entire rpr/hid/grim locus and Apc (and/or Apc2) each increase the representation of tai clones. They then show that the survival of tissue to the adult stage correlates with the size of lones in the third-instar larval wing disc (Figure 3). The rest of the study derives from the modification of the phenotype by Apc and investigates the interaction between Wnt signaling and tai clone survival.

The authors then investigate interactions between tai and the wingless (wg) pathway. First, they show that increasing tai expression increases the expression of a wg reporter (nkd-lacZ) while reducing tai levels decreases its expression (Figure 4) indicating that wg signaling is likely reduced when tai levels are decreased. This finding is strengthened by examining wg-lacZ expression since the expression of this reporter is normally restricted to the D/V boundary in the wing disc by feedback inhibition via Wg signaling. Expression of the reporter is increased when tai expression is reduced and decreased when tai expression is increased (Figure 5).

The authors then look at Wg protein away from the DV boundary. They find increased levels when tai expression is increased and decreased levels when tai is decreased. They conclude that tai activity increased Wg protein in cells (Figure 6). They suggest that this could be the result of the regulation of expression of Dally-like protein (Dlp). Consistent with this idea, increasing tai expression increases Dlp levels, and decreasing tai decreases Dlp levels (Figure 7). They then show that increasing Dlp levels when tai is reduced increases Wg levels which presumably means that Dlp is epistatic to tai. Puzzlingly, increasing both tai and Dlp decreases Wg.

The authors then examine the effect of reducing Dlp in the cells that secrete Wg. They find that increasing tai results in the diffusion of Wg further from its source while reducing tai reduces its spread (Figure 8). They then show that in clones with reduced tai, there is increased cytoplasmic Dlp (Figure 9). They therefore propose that tai clones fail to survive because they do not secrete enough Dlp which results in reduced capture of the Wg for those cells and hence decreased Wg signaling.

Evaluation

While the authors present good evidence in support of most of their conclusions, there are alternative explanations in many cases that have not been excluded.

From the results in Figure 1 (and Figure 3), the authors conclude that "The data indicate the existence of an extracellular competition mechanism that allows normal tai[wt] cells to kill tai[k15101] neighbors" (line 127). However, the experiments have been done with a single allele, and these experiments do not exclude the possibility that there is another mutation on the same chromosome arm that is responsible for the observed phenotype. Since the authors have a UAS-tai stock, they could strengthen their results using a MARCM experiment where they could test whether the expression of UAS-tai rescues the elimination of tai mutant clones. Alternatively, they could use a second (independent) allele to demonstrate that the phenotype can be attributed to a reduction in tai activity.

By screening for dominant modifiers of a phenotype one would not expect to identify all interacting genes - only those that are haploinsufficient in this situation. The authors have screened a total of 21 chromosomes for modification and have not really explained which alleles are nulls and which are hypomorphs. The nature of each of the alleles screened needs to be explained better. Also, the absence of a dominant modification does not necessarily exclude a function of that gene or pathway in the process. This is especially relevant for the Spz/Toll pathway which the authors have previously implicated in the ability of tai-overexpressing cells to kill wild-type cells. The most important discovery from this screen is the modification by the Apc alleles. This part of the paper would be strengthened by testing for modification by other components of the Wingless pathway. The authors show modification by Apc[MI01007] and the double mutant Apc[Q8] Apc2[N175A]. Without showing the Apc[Q8] and Apc2[N175A] alleles separately, it is hard to know if the effect of the double mutant is due to Apc, Apc2,` or the combination.

RNAi of tai seems to block the formation of the Wg gradient. If so, one might expect a reduction in wing size. Indeed, this could explain why the wings of tai/Df flies are smaller. The authors mention briefly that the posterior compartment size is reduced when tai-RNAi is expressed in that compartment. However, this observation merits more emphasis since it could explain why tai/Df flies are smaller (Are their wings smaller?).

In Figure 7, the authors show the effect of manipulating Tai levels alone or in combination with increasing Dlp levels. However, they do not include images of Wg protein distribution upon increasing Dlp levels alone.

In Figure 8, there is more Wg protein both at the DV boundary and spreading when tai is overexpressed in the source cells using bbg-Gal4. However, in an earlier experiment (Figure 5C) they show that the wg-lacZ reporter is downregulated at the DV boundary when tai is overexpressed using en-Gal4. They therefore conclude that wg is not transcriptionally upregulated but is, instead secreted at higher levels when tai is expressed in the source cells. Wg protein is reduced in the DV stripe with tai is overexpressed using the en-Gal4 driver (Figure 6B') and is increased at the same location when tai is overexpressed with the bbg-Gal4 driver. (Figure 8) I don't know how to reconcile these observations.

In Figure 9, the tai-low clones have elevated levels of Dlp. How can this be reconciled with the tai-RNAi knockdown shown in Figure 7C' where reducing tai levels causes a strong reduction in Dlp levels?

Reviewer #3 (Public Review):

Summary:

In this study, Schweibenz et al., identify the transcriptional coactivator, Taiman (Tai), as a factor that determines the fitness level of epithelial cells by regulating Wingless (Wg), which is an important determinant of cellular fitness. Taiman determines cellular fitness level by regulating levels of cell-surface glypican Dally-like protein (Dlp), which regulates extracellular Wingless (Wg) distribution. Thus, by affecting levels of Wg via glypican regulation, Tai participates in determining cellular fitness, and cells with low Tai levels are eliminated as they are deprived of adequate Wg levels.

Strengths:

(1) The authors make a strong case for the effect of tai on Dlp and Wg levels in experiments where a relatively large group of cells have reduced tai levels.<br /> (2) The claim that tai-low clones are competitively eliminated is supported by experiments that show cell death in them, and their elimination at different time points.<br /> (3) The manuscript is well written.

Weaknesses:

(1) The study has relatively weak evidence for the mechanism of cell competition mediated by Dlp and Wg.

(2) More evidence is required to support the claim that dlp transcription or endocytosis is affected in tai clones.

Other comments:

(1) The authors put the study in the context of cell competition, and the first figure indeed is convincing in this regard. However, most of the rest of the study is not in the clonal context, and mainly relies on RNAi KD of tai in the posterior compartment, which is a relatively large group of cells. I understand why the authors chose a different approach to investigate the role of tai in cell competition. However because ubiquitous loss of tai results in smaller organs, it is important to determine to what extent reducing levels of tai in the entire posterior compartment compares with clonal elimination i.e. cell competition. This is important in order to determine to what extent the paradigm of Tai-mediated regulation of Dlp levels and by extension, Wg availability, can be extended as a general mechanism underlying competitive elimination of tai-low clones. If the authors want to make a case for mechanisms involved in the competitive elimination of tai clones, then they need to show that the KD of tai in the posterior compartment shows hallmarks of cell competition. Is there cell death along the A/P boundary? Or is the compartment smaller because those cells are growing slower? Are the levels of Myc/DIAP1, proteins required for fitness, affected in en>tai RNAi cells?

2) The authors do not have direct/strong evidence of changes in dlp mRNA levels or intracellular trafficking. To back these claims, the authors should look for dlp mRNA levels and provide more evidence for Dlp endocytosis like an antibody uptake assay or at the very least, a higher resolution image analysis showing a change in the number of intracellular Dlp positive punctae. Also, do the authors think that loss of tai increases Dlp endocytosis, making it less available on the cell surface for maintaining adequate extracellular Wg levels?

3) The data shown in the last figure is at odds with the model (I think) the authors are trying to establish: When cells have lower Tai levels, this reduces Dlp levels (S2) presumably either by reducing dlp transcription and/or increasing (?) Dlp endocytosis. This in turn reduces Wg (availability) in cells away from source cells (Figure 6). The reduced Wg availability makes them less fit, targeting them for competitive elimination. But in tai clones, I do not see any change in cell-surface Dlp (9B) (I would have expected them to be down based on the proposed model). The authors also see more total Dlp (9A) (which is at odds with S2 assuming data in S2 were done under permeabilizing conditions.).

As a side note, because Dlp is GPI-anchored, the authors should consider the possibility that the 'total' Dlp staining observed in 9A may not be actually total Dlp (and possibly mostly intracellular Dlp, since the permeabilizing membranes with detergent will cause some (most?) Dlp molecules to be lost, and how this might be affecting the interpretation of the data. I think one way to address this would be to process the permeabilized and non-permeabilized samples simultaneously and then image them at the same settings and compare what membrane staining in these two conditions looks like. If membrane staining in the permeabilized condition is decreased compared to non-permeabilized conditions, and the signal intensity of Dlp in permeabilized conditions remains high, then the authors will have evidence to support increased endocytosis in tai clones. Of course, these data will still need to be reconciled with what is shown in S2.

Reviewer #1 (Public Review):

In this revised manuscript, authors have conducted epigenetic and transcriptomic profiling to understand how environmental chemicals such as BPS can cause epimutations that can propagate to future generations. They used isolated somatic cells from mice (Sertoli, granulosa), pluripotent cells to model preimplantation embryos (iPSCs) and cells to model the germline (PGCLCs). This enabled them to model sequential steps in germline development, and when/how epimutations occur. The major findings were that BPS induced unique epimutations in each cell type, albeit with qualitative and quantitative cell-specific differences; that these epimutations are prevalent in regions associated with estrogen-response elements (EREs); and that epimutations induced in iPSCs are corrected as they differentiate into PGCLCs, concomitant with the emergence of de novo epimutations. This study will be useful in understanding multigenerational effects of EDCs, and underlying mechanisms.

Strengths include:

(1) Using different cell types representing life stages of epigenetic programming and during which exposures to EDCs have different effects. This progression revealed information both about correction of epimutations and the emergence of new ones in PGCLCs.<br /> (2) Work conducted by exposing iPSCs to BPS or vehicle, then differentiating to PGCLCs, revealed that novel epimutations emerged.<br /> (3) Relating epimutations to promoter and enhancer regions

A few weaknesses remain: Authors need to discuss the limitations of the small sample size. The supplemental data, while extremely helpful, requires better organization.

Reviewer #2 (Public Review):

Summary:

This manuscript uses cell lines representative of germ line cells, somatic cells and pluripotent cells to address the question of how the endocrine disrupting compound BPS affects these various cells with respect to gene expression and DNA methylation. They find a relationship between the presence of estrogen receptor gene expression and the number of DNA methylation and gene expression changes. Notably, PGCLCs do not express estrogen receptors and although they do have fewer changes, changes are nevertheless detected, suggesting a nonconical pathway for BPS-induced perturbations. Additionally, there was a significant increase in the occurrence of BPS-induced epimutations near EREs in somatic and pluripotent cell types compared to germ cells. Epimutations in the somatic and pluripotent cell types were predominantly in enhancer regions whereas that in the germ cell type was predominantly in gene promoters.

Strengths:

The strengths of the paper include the use of various cell types to address sensitivity of the lineages to BPS as well as the observed relationship between the presence of estrogen receptors and changes in gene expression and DNA methylation.

Weaknesses:

The weakness includes the fact that exposures are more complicated in a whole organism than in an isolated cell line.

Reviewer #1 (Public Review):

This manuscript by Bai et al concerns the expression of Scleraxis (Scx) by muscle satellite cells (SCs) and the role of that gene in regenerative myogenesis. The authors report the expression of this gene associated with tendon development in satellite cells. Genetic deletion of Scx in SCs impairs muscle regeneration, and the authors provide evidence that SCs deficient in Scx are impaired in terms of population growth and cellular differentiation. Overall, this report provides evidence of the role of this gene, unexpectedly, in SC function and adult regenerative myogenesis.

There are a few minor points of concern.

(1) From the data in Figure 1, it appears that all of the SCs, assessed both in vitro and in vivo, express Scx. The authors refer to a scRNA-seq dataset from their lab and one report from mdx mouse muscle that also reveals this unexpected gene expression pattern. Has this been observed in many other scRNA-seq datasets? If not, it would be important to discuss potential explanations as to why this has not been reported previously.

(2) A major point of the paper, as illustrated in Fig. 3, is that Scx-neg SCs fail to produce normal myofibers and renewed SCs following injury/regeneration. They mention in the text that there was no increased PCD by Caspase staining at 5 DPI. A failure of cell survival during the process of SC activation, proliferation, and cell fate determination (differentiation versus self-renewal) would explain most of the in vivo data. As such, this conclusion would seem to warrant a more detailed analysis in terms of at least one or two other time points and an independent method for detecting dead/dying cells (the in vitro data in Fig. 4F is also based on an assessment of activated Caspase to assess cell death). The in vitro data presented later in Fig. S4G, H do suggest an increase in cell loss during proliferative expansion of Scx-neg SCs. To what extent does cell loss (by whatever mechanism of cell death) explain both the in vivo findings of impaired regeneration and even the in vitro studies showing slower population expansion in the absence of Scx?

(3) I'm not sure I understand the description of the data or the conclusions in the section titled "Basement membrane-myofiber interaction in control and Scx cKO mice". Is there something specific to the regeneration from Scx-neg myogenic progenitors, or would these findings be expected in any experimental condition in which myogenesis was significantly delayed, with much smaller fibers in the experimental group at 5 DPI?

(4) The data presented in Fig. 4B showing differences in the purity of SC populations isolated by FACS depending on the reporter used are interesting and important for the field. The authors offer the explanation of exosomal transfer of Tdt from SCs to non-SCs. The data are consistent with this explanation, but no data are presented to support this. Are there any other explanations that the authors have considered and that could be readily tested?

(5) The Cut&Run data of Fig. 6 certainly provide evidence of direct Scx targets, especially since the authors used a novel knock-in strain for analyses. The enrichment of E-box motifs provides support for the 207 intersecting genes (scRNA-seq and Cut&Run) being direct targets. However, the rationale elaborated in the final paragraph of the Results section proposing how 4 of these genes account for the phenotypes on the Scx-neg cells and tissues is just speculation, however reasonable. These are not data, and these considerations would be more appropriate in the Discussion in the absence of any validation studies.

Reviewer #2 (Public Review):

Summary:

Scx is a well-established marker for tenocytes, but the expression in myogenic-lineage cells was unexplored. In this study, the authors performed lineage-trace and scRNA-seq analyses and demonstrated that Scx is expressed in activated SCs. Further, the authors showed that Scx is essential for muscle regeneration using conditional KO mice and identified the target genes of Scx in myogenic cells, which differ from those of tendons.

Strengths:

Sometimes, lineage-trace experiments cause mis-expression and do not reflect the endogenous expression of the target gene. In this study, the authors carefully analyzed the unexpected expression of Scx in myogenic cells using some mouse lines and scRNA-seq data.

Weaknesses:

Scx protein expression has not been verified.

Reviewer #1 (Public Review):

Tleiss et al. demonstrate that while commensal Lactiplantibacillus plantarum freely circulate within the intestinal lumen, pathogenic strains such as Erwinia carotovora or Bacillus thuringiensis are blocked in the anterior midgut where they are rapidly eliminated by antimicrobial peptides. This sequestration of pathogenic bacteria in the anterior midgut requires the Duox enzyme in enterocytes, and both TrpA1 and Dh31 in enteroendocrine cells. This effect induces muscular muscle contraction, which is marked by the formation of TARM structures (thoracic ary-related muscles). This muscle contraction-related blocking happens early after infection (15mins). On the other side, the clearance of bacteria is done by the IMD pathway possibly through antimicrobial peptide production while it is dispensable for the blockage. Genetic manipulations impairing bacterial compartmentalization result in abnormal colonization of posterior midgut regions by pathogenic bacteria. Despite a functional IMD pathway, this ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense.

This important work substantially advances our understanding of the process of pathogen clearance by identifying a new mode of pathogen eradication from the insect gut. The evidence supporting the authors' claims is solid and would benefit from more rigorous experiments.

(1) The authors performed the experiments on Drosophila larvae. I wonder whether this model could extend to adult flies since they have shown that the ROS/TRPA1/Dh31 axis is important for gut muscle contraction in adult flies. If not, how would the authors explain the discrepancy between larvae and adults?

(2) The authors performed their experiments and proposed the models based on two pathogenic bacteria and one commensal bacterial at a relatively high bacterial dose. They showed that feeding Bt at 2X1010 or Ecc15 at 4X108 did not induce a blockage phenotype. I wonder whether larvae die under conditions of enteric infection with low concentrations of pathogenic bacteria. If larvae do not show mortality, what is the mechanism for resisting low concentrations of pathogenic bacteria? Why is this model only applied to high-dose infections?

(3) The authors claim that the lock of bacteria happens at 15 minutes while killing by AMPs happens 6-8 hours later. What happened during this period? More importantly, is IMD activity induced in the anterior region of the larval gut in both Ecc15 and Bt infection at 6 hours after infection? Are they mostly expressed in the anterior midgut in both bacterial infections? Several papers have shown quite different IMD activity patterns in the Drosophila gut. Zhai et al. have shown that in adult Drosophila, IMD activity was mostly absent in the R2 region as indicated by dpt-lacZ. Vodovar et al. have shown that the expression of dpt-lacZ is observable in proventriculus while Pe is not in the same region. Tzou et al. showed that Ecc15 infection induced IMD activity in the anterior midgut 24 hours after infection. Using TrpA1 and Dh31 mutant, the authors found both Ecc15 and Bt in the posterior midgut. Why are they not evenly distributed along the gut? Last but not least, does the ROS/TrpA1/Dh31 axis affect AMP expression?

(4) The TARM structure part is quite interesting. However, the authors did not show its relevance in their model. Is this structure the key-driven force for the blocking phenotype and killing phenotype? Is the ROS/TrpA1/Dh31 axis required to form this structure?

Reviewer #2 (Public Review):

This article describes a novel mechanism of host defense in the gut of Drosophila larvae. Pathogenic bacteria trigger the activation of a valve that blocks them in the anterior midgut where they are subjected to the action of antimicrobial peptides. In contrast, beneficial symbiotic bacteria do not activate the contraction of this sphincter, and can access the posterior midgut, a compartment more favorable to bacterial growth.

Strengths:

The authors decipher the underlying mechanism of sphincter contraction, revealing that ROS production by Duox activates the release of DH31 by enteroendocrine cells that stimulate visceral muscle contractions. The use of mutations affecting the Imd pathway or lacking antimicrobial peptides reveals their contribution to pathogen elimination in the anterior midgut.

Weaknesses:

- The mechanism allowing the discrimination between commensal and pathogenic bacteria remains unclear.

- The use of only two pathogens and one symbiotic species may not be sufficient to draw a conclusion on the difference in treatment between pathogenic and symbiotic species.

- We can also wonder how the process of sphincter contraction is affected by the procedure used in this study, where larvae are starved. Does the sphincter contraction occur in continuous feeding conditions? Since larvae are continuously feeding, is this process physiologically relevant?

Reviewer #1 (Public Review):

Summary:

Sammons, Masserini et al. examine the connectivity of different types of CA3 pyramidal cells ("thorny" and "athorny"), and how their connectivity putatively contributes to their relative timing in sharp-wave-like activity. First, using patch-clamp recordings, they characterize the degree of connectivity within and between athorny and thorny cells. Based upon these experimental results, they compute a synaptic product matrix, and use this to inform a computational model of CA3 activity. This model finds that this differential connectivity between these populations, augmented by two different types of inhibitory neurons, can account for the relative timing of activity observed in sharp waves in vivo.

Strengths:

The patch-clamp experiments are exceptionally thorough and well done. These are very challenging experiments and the authors should be commended for their in-depth characterization of CA3 connectivity.

Weaknesses:

(1) The computational elements of this study feel underdeveloped. Whereas the authors do a thorough job experimentally characterizing connections between excitatory neurons, the inhibitory neurons used in the model seem to be effectivity "fit neurons" and appear to have been tuned to produce the emergent properties of CA3 sharp wave-like activity. Although I appreciate the goal was to implicate CA3 connectivity contributions to activity timing, a stronger relationship seems like it could be examined. For example, did the authors try to "break" their model? It would be informative if they attempted different synaptic product matrices (say, the juxtaposition of their experimental product matrix) and see whether experimentally-derived sequential activity could not be elicited. It seems as though this spirit of analysis was examined in Figure 4C, but only insofar as individual connectivity parameters were changed in isolation.

(2) Additional explanations of how parameters for interneurons were incorporated in the model would be very helpful. As it stands, it is difficult to understand the degree to which the parameters of these neurons are biologically constrained versus used as fit parameters to produce different time windows of activity in types of CA3 pyramidal cells.

Reviewer #2 (Public Review):

Sharp wave ripples are transient oscillations occurring in the hippocampus that are thought to play an important role in organising temporal sequences during the reactivation of neuronal activity. This study addresses the mechanism by which these temporal sequences are generated in the CA3 region focusing on two different subtypes of pyramidal neurons, thorny and athorny. Using high-quality electrophysiological recordings from up to 8 pyramidal neurons at a time the authors measure the connectivity rates between these pyramidal cell subtypes in a large dataset of 348 cells. This is a significant achievement and provides important data. The most striking finding is how similar connection characteristics are between cell types. There are no differences in synaptic strength or failure rates and some small differences in connectivity rates and short-term plasticity. Using model simulations, the authors explore the implications of the differences in connectivity rates for the temporal specificity of pyramidal cell firing within sharp-wave ripple events. The simulations show that the experimentally observed connectivity rates may contribute to the previously observed temporal sequence of pyramidal cell firing during sharp wave ripples.

The conclusions drawn from the simulations are not experimentally tested so remain theoretical. In the simple network model, the authors include basket cell and anti-SWR interneurons but the connectivity of these cell types is not measured experimentally and variations in interneuron parameters may also influence temporal specificity of firing. In addition, the influence of short-term plasticity measured in their experiments is not tested in the model. Interestingly, the experimental data reveal a large variability in many of the measured parameters. This may strongly influence the firing of pyramidal cells during SWRs but it is not represented within the model which uses the averaged data.

Reviewer #3 (Public Review):

Summary:

The hippocampal CA3 region is generally considered to be the primary site of initiation of sharp wave ripples-highly synchronous population events involved in learning and memory although the precise mechanism remains elusive. A recent study revealed that CA3 comprises two distinct pyramidal cell populations: thorny cells that receive mossy fiber input from the dentate gyrus, and athorny cells that do not. That study also showed that it is athorny cells in particular that play a key role in sharp wave initiation. In the present work, Sammons, Masserini, and colleagues expand on this by examining the connectivity probabilities among and between thorny and athorny cells. First, using whole-cell patch clamp recordings, they find an asymmetrical connectivity pattern, with athorny cells receiving the most synaptic connections from both athorny and thorny cells, and thorny cells receiving fewer. They then demonstrate in spiking neural network simulations how this asymmetrical connectivity may underlie the preferential role of athorny cells in sharp wave initiation.

Strengths:

The authors provide independent validation of some of the findings by Hunt et al. (2018) concerning the distinction between thorny and athorny pyramidal cells in CA3 and advance our understanding of their differential integration in CA3 microcircuits. The properties of excitatory connections among and between thorny and athorny cells described by the authors will be key in understanding CA3 functions including, but not limited to, sharp wave initiation.

As stated in the paper, the modeling results lend support to the idea that the increased excitatory connectivity towards athorny cells plays a key role in causing them to fire before thorny cells in sharp waves. More generally, the model adds to an expanding pool of models of sharp wave ripples which should prove useful in guiding and interpreting experimental research.

Weaknesses:

The mechanism by which athorny cells initiate sharp waves in the model is somewhat confusingly described. As far as I understood, random fluctuations in the activities of A and B neurons provide windows of opportunity for pyramidal cells to fire if they have additionally recovered from adaptive currents. Thorny and athorny pyramidal cells are then set in a winner-takes-all competition which is quickly won by the athorny cells. The main thesis of the paper seems to be that athorny cells win this competition because they receive more inputs both from themselves and from thorny cells, hence, the connectivity "underlies the sequential activation". However, it is also stated that athorny cells activate first due to their lower rheobase and steeper f-I curve, and it is also indicated in the methods that athorny (but not thorny) cells fire in bursts. It seems that it is primarily these features that make them fire first, something which apparently happens even when the A to A connectivity is set to 0-albeit with a very small lag. Perhaps the authors could further clarify the differential role of single cell and network parameters in determining the sequential activation of athorny and thorny cells. Is the role of asymmetric excitatory connectivity only to enhance the initial intrinsic advantage of athorny cells? If so, could this advantage also be enhanced in other ways?

Although a clear effort has been made to constrain the model with biological data, too many degrees of freedom remain that allow the modeler to make arbitrary decisions. This is not a problem in itself, but perhaps the authors could explain more of their reasoning and expand upon the differences between their modeling choices and those of others. For example, what are the conceptual or practical advantages of using adaptation in pyramidal neurons as opposed to short-term synaptic plasticity as in the model by Hunt et al.? Relatedly, what experimental observations could validate or falsify the proposed mechanisms?

In the data by Hunt et al., thorny cells have a higher baseline (non-SPW) firing rate, and it is claimed that it is actually stochastic correlations in their firing that are amplified by athorny cells to initiate sharp waves. However, in the current model, the firing of both types of pyramidal cells outside of ripples appears to be essentially zero. Can the model handle more realistic firing rates as described by Hunt et al., or as produced by e.g., walking around an environment tiled with place cells, or would that trigger SPWs continuously?

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 is 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.<br /> (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 others 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.<br /> (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 suggests 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.<br /> (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 limitation of their method.<br /> (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 show 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 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 (clear from Figure 8) but as the authors comment they have not identified this residue(s).

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 (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<br /> D342 significantly decrease 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 has 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:

There are inherent limitations with the methodology used such as the MEMENTO and constant pH MD that have been briefly noted in the manuscript.

Reviewer #1 (Public Review):

Summary:

TMC7 knockout mice were generated by the authors and the phenotype was analyzed. They found that Tmc7 is localized to Golgi and is needed for acrosome biogenesis.

Strengths:

The phenotype of infertility is clear, and the results of TMC7 localization and the failed acrosome formation are highly reliable. In this respect, they made a significant discovery regarding spermatogenesis.

In the original version, I pointed out the gap between their pH/calcium imaging data and the hypothesis of ion channel function of TMC7 in the Golgi. Now the author agrees and has changed the description to be reasonable. Additional experiments were also performed, and I can say that they have answered my concern adequately.

I would say it is good to add any presumed mechanism for the observed changes in pH and calcium concentration in the cytoplasm this time.

Reviewer #2 (Public Review):

Summary:

This study presents a significant finding that enhances our understanding of spermatogenesis. TMC7 belongs to a family of transmembrane channel-like proteins (TMC1-8), primarily known for their role in the ear. Mutations to TMC1/2 are linked to deafness in humans and mice and were originally characterized as auditory mechanosensitive ion channels. However, the function of the other TMC family members remains poorly characterized. In this study, the authors begin to elucidate the function of TMC7 in acrosome biogenesis during spermatogenesis. Through analysis of transcriptomics datasets, they elevated levels of TMC7 in round spermatids in both mouse and human testis. They then generate Tmc7-/- mice and find that male mice exhibit smaller testes and complete infertility. Examination of different developmental stages reveals spermatogenesis defects, including with reduced sperm count, elongated spermatids and large vacuoles. Additionally, abnormal acrosome morphology are observed beginning at the early-stage Golgi phase, indicating TMC7's involvement in proacrosomal vesicle trafficking and fusion. They observed localization of TMC7 in the cis-Golgi and suggest that its presence is required for maintaining Golgi integrity, with Tmc7-/- leading to reduced intracellular Ca2+, elevated pH and increased ROS levels, likely resulting in spermatid apoptosis. Overall, the work delineates a new function of TMC7 in spermatogenesis and the authors propose that that its ion channel and/or scramblase activity is likely important for Golgi homeostasis. This work is of significant interest to the community and is of high quality.

Strengths:

The biggest strength of the paper is the phenotypic characterization of the TMC7-/- mouse model, which has clear acrosome biogenesis/spermatogenesis defects. This is the main claim of the paper and it is supported with the data that are presented.

Weaknesses:

It isn't clear whether TMC7 functions as an ion channel from the current data presented in this paper, but the authors are careful in their interpretation and present this merely as a hypothesis supporting this idea.

Reviewer #3 (Public Review):

Summary:

In this study, Wang et al. have demonstrated that TMC7, a testis-enriched multipass transmembrane protein, is essential for male reproduction in mice. Tmc7 KO male mice are sterile due to reduced sperm count and abnormal sperm morphology. TMC7 co-localizes with GM130, a cis-Golgi marker, in round spermatids. The absence of TMC7 results in reduced levels of Golgi proteins, elevated abundance of ER stress markers, as well as changes of Ca2+ and pH levels in the KO testis. However, further confirmation is required because the analyses were performed with whole testis samples in spite of the differences in the germ cell composition in WT and KO testis. In addition, the causal relationships between the reported anomalies await thorough interrogation

Strengths:

By using PD21 testes, the revised assays have consolidated that depletion of TMC7 leads to a reduced level of Ca2+ and an elevated level of ROS in the male germ cells. The immunohistochemistry analyses have clearly indicated the reduced abundance of GM130, P115, and GRASP65 in the knockout testis.

Weaknesses:

The Discussion section contains sentences reiterating the Introduction and Results of this manuscript (e.g., Lines 79-85 and 231-236; Lines 175-179 and 259-263). Those read repetitive and can be removed.

Future studies are required to decipher how TMC7 stabilizes Golgi structure, coordinates vesicle transport, and maintains the germ cell homeostasis.

Reviewer #1 (Public Review):

Overall, the data presented in this manuscript is of good quality. Understanding how cells control RPA loading on ssDNA is crucial to understanding DNA damage responses and genome maintenance mechanisms. The authors used genetic approaches to show that disrupting PCNA binding and SUMOylation of Srs2 can rescue the CPT sensitivity of rfa1 mutants with reduced affinity for ssDNA. In addition, the authors find that SUMOylation of Srs2 depends on binding to PCNA and the presence of Mec1.

Noted weaknesses include the lack of evidence supporting that Srs2 binding to PCNA and its SUMOylation occur at ssDNA gaps, as proposed by the authors. Also, the mutants of Srs2 with impaired binding to PCNA or impaired SUMOylation showed no clear defects in checkpoint dampening, and in some contexts, even resulted in decreased Rad53 activation. Therefore, key parts of the paper would benefit from further experimentation and/or clarification.

Major Comments

(1) The central model proposed by the authors relies on the loading of PCNA at the 3' junction of an ssDNA gap, which then mediates Srs2 recruitment and RPA removal. While several aspects of the model are consistent with the data, the evidence that it is occurring at ssDNA gaps is not strong. The experiments mainly used CPT, which generates mostly DSBs. The few experiments using MMS, which mostly generates ssDNA gaps, show that Srs2 mutants lead to weaker rescue in this context (Figure S1). How do the authors explain this discrepancy? In the context of DSBs, are the authors proposing that Srs2 is engaging at later steps of HR-driven DSB repair where PCNA gets loaded to promote fill-in synthesis? If so, is RPA removal at that step important for checkpoint dampening? These issues need to be addressed and the final model adjusted.

(2) The data in Figure 3 showing that Srs2 mutants reduce Rad53 activation in the rfa1-zm2 mutant are confusing, especially given the claim of an anti-checkpoint function for Srs2 (in which case Srs2 mutants should result in increased Rad53 activation). The authors propose that Rad53 is hyperactivated in rfa1-zm2 mutant because of compromised ssDNA protection and consequential DNA lesions, however, the effects sharply contrast with the central model. Are the authors proposing that in the rfa1-zm2 mutant, the compromised protection of ssDNA supersedes the checkpoint-dampening effect? Perhaps a schematic should be included in Figure 3 to depict these complexities and help the reader. The schematic could also include the compensatory dampening mechanisms like Slx4 (on that note, why not move Figure S2 to a main figure?... and even expand experiments to better characterize the compensatory mechanisms, which seem important to help understand the lack of checkpoint dampening effect in the Srs2 mutants)

(3) The authors should demarcate the region used for quantifying the G1 population in Figure 3B and explain the following discrepancy: By inspection of the cell cycle graph, all mutants have lower G1 peak height compared to WT (CPT 2h). However, in the quantification bar graph at the bottom, ΔPIM has higher G1 population than the WT.

Reviewer #2 (Public Review):

Summary:

This is an interesting paper that delves into the post-translational modifications of the yeast Srs2 helicase and proteins with which it interacts in coping with DNA damage. The authors use mutants in some interaction domains with RPA and Srs2 to argue for a model in which there is a balance between RPA binding to ssDNA and Srs2's removal of RPA. The idea that a checkpoint is being regulated is based on observing Rad53 and Rad9 phosphorylation (so there are the attributes of a checkpoint), but evidence of cell cycle arrest is lacking. The only apparent delay in the cell cycle is the re-entry into the second S phase (but it could be an exit from G2/M); but in any case, the wild-type cells enter the next cell cycle most rapidly. No direct measurement of RPA residence is presented.

Strengths:

Data concern viability assays in the presence of camptothecin and in the post-translational modifications of Srs2 and other proteins.

Weaknesses:

There are a couple of overriding questions about the results, which appear technically excellent. Clearly, there is an Srs2-dependent repair process here, in the presence of camptothecin, but is it a consequence of replication fork stalling or chromosome breakage? Is repair Rad51-dependent, and if so, is Srs2 displacing RPA or removing Rad51 or both? If RPA is removed quickly what takes its place, and will the removal of RPA result in lower DDC1-MEC1 signaling?

Moreover, It is worth noting that in single-strand annealing, which is ostensibly Rad51 independent, a defect in completing repair and assuring viability is Srs2-dependent, but this defect is suppressed by deleting Rad51. Does deleting Rad51 have an effect here?

Neither this paper nor the preceding one makes clear what really is the consequence of having a weaker-binding Rfa1 mutant. Is DSB repair altered? Neither CPT nor MMS are necessarily good substitutes for some true DSB assay.

With camptothecin, in the absence of site-specific damage, it is difficult to test these questions directly. (Perhaps there is a way to assess the total amount of RPA bound, but ongoing replication may obscure such a measurement). It should be possible to assess how CPT treatment in various genetic backgrounds affects the duration of Mec1/Rad53-dependent checkpoint arrest, but more than a FACS profile would be required.

It is also notable that MMS treatment does not seem to yield similar results (Fig. S1).

Reviewer #3 (Public Review):

The superfamily I 3'-5' DNA helicase Srs2 is well known for its role as an anti-recombinase, stripping Rad51 from ssDNA, as well as an anti-crossover factor, dissociating extended D-loops and favoring non-crossover outcome during recombination. In addition, Srs2 plays a key role in ribonucleotide excision repair. Besides DNA repair defects, srs2 mutants also show a reduced recovery after DNA damage that is related to its role in downregulating the DNA damage signaling or checkpoint response. Recent work from the Zhao laboratory (PMID: 33602817) identified a role of Srs2 in downregulating the DNA damage signaling response by removing RPA from ssDNA. This manuscript reports further mechanistic insights into the signaling downregulation function of Srs2.

Using the genetic interaction with mutations in RPA1, mainly rfa1-zm2, the authors test a panel of mutations in Srs2 that affect CDK sites (srs2-7AV), potential Mec1 sites (srs2-2SA), known sumoylation sites (srs2-3KR), Rad51 binding (delta 875-902), PCNA interaction (delta 1159-1163), and SUMO interaction (srs2-SIMmut). All mutants were generated by genomic replacement and the expression level of the mutant proteins was found to be unchanged. This alleviates some concern about the use of deletion mutants compared to point mutations. The double mutant analysis identified that PCNA interaction and SUMO sites were required for the Srs2 checkpoint dampening function, at least in the context of the rfa1-zm2 mutant. There was no effect of these mutants in a RFA1 wild-type background. This latter result is likely explained by the activity of the parallel pathway of checkpoint dampening mediated by Slx4, and genetic data with an Slx4 point mutation affecting Rtt107 interaction and checkpoint downregulation support this notion. Further analysis of Srs2 sumoylation showed that Srs2 sumoylation depended on PCNA interaction, suggesting sequential events of Srs2 recruitment by PCNA and subsequent sumoylation. Kinetic analysis showed that sumoylation peaks after maximal Mec1 induction by DNA damage (using the Top1 poison camptothecin (CPT)) and depended on Mec1. These data are consistent with a model that Mec1 hyperactivation is ultimately leading to signaling downregulation by Srs2 through Srs2 sumoylation. Mec1-S1964 phosphorylation, a marker for Mec1 hyperactivation and a site found to be needed for checkpoint downregulation after DSB induction did not appear to be involved in checkpoint downregulation after CPT damage. The data are in support of the model that Mec1 hyperactivation when targeted to RPA-covered ssDNA by its Ddc2 (human ATRIP) targeting factor, favors Srs2 sumoylation after Srs2 recruitment to PCNA to disrupt the RPA-Ddc2-Mec1 signaling complex. Presumably, this allows gap filling and disappearance of long-lived ssDNA as the initiator of checkpoint signaling, although the study does not extend to this step.

Strengths

(1) The manuscript focuses on the novel function of Srs2 to downregulate the DNA damage signaling response and provide new mechanistic insights.

(2) The conclusions that PCNA interaction and ensuing Srs2-sumoylation are involved in checkpoint downregulation are well supported by the data.

Weaknesses

(1) Additional mutants of interest could have been tested, such as the recently reported Pin mutant, srs2-Y775A (PMID: 38065943), and the Rad51 interaction point mutant, srs2-F891A (PMID: 31142613).

(2) The use of deletion mutants for PCNA and RAD51 interaction is inferior to using specific point mutants, as done for the SUMO interaction and the sites for post-translational modifications.

(3) Figure 4D and Figure 5A report data with standard deviations, which is unusual for n=2. Maybe the individual data points could be plotted with a color for each independent experiment to allow the reader to evaluate the reproducibility of the results.

Reviewer #2 (Public Review):

Summary:

Tian et al. aimed to assess differences in biological motion (BM) perception between children with and without ADHD, as well as relationships to indices of social functioning and possible predictors of BM perception (including demographics, reasoning ability and inattention). In their study, children with ADHD showed poorer performance relative to typically developing children in three tasks measuring local, global, and general BM perception. The authors further observed that across the whole sample, performance in all three BM tasks was negatively correlated with scores on the social responsiveness scale (SRS), whereas within groups a significant relationship to SRS scores was only observed in the ADHD group and for the local BM task. Local and global BM perception showed a dissociation in that global BM processing was predicted by age, while local BM perception was not. Finally, general (local & global combined) BM processing was predicted by age and global BM processing, while reasoning ability mediated the effect of inattention on BM processing.

Strengths:

Overall, the manuscript is presented in a clear fashion and methods and materials are presented with sufficient detail so the study could be reproduced by independent researchers. The study uses an innovative, albeit not novel, paradigm to investigate two independent processes underlying BM perception. The results are novel and have the potential to have wide-reaching impact on multiple fields.

Weaknesses:

The manuscript has improved in clarity and conceptual and methodological considerations in response to the last review. However, the reported results still provide incomplete support for the claims the authors make in the paper, due to differences between correlations not passing significance thresholds.

Reviewer #3 (Public Review):

The authors presented point light displays of human walkers to children (mean = 9 years) with and without ADHD to compare their biological motion perception abilities, and relate them to IQ, social responsiveness scale (SRS) scores and age. They report that children with ADHD were worse at all three biological motion tasks, but that those loading more heavily on local processing related to social interaction skills and global processing to age. The valuable and solid findings are informative for understanding this complex condition, as well as biological motion processing mechanisms in general. However, the correlations present a pattern that needs further examination in future studies because many of the differences between correlations are not significant.

Strengths:

The authors present differences between ADHD and TD children in biological motion processing, and this question has not received as much attention as equivalent processing capabilities in autism. They use a task that appears well controlled. They raise some interesting mechanistic possibilities for differences in local and global motion processing, which are distinctions worth exploring. The group differences will therefore be of interest to those studying ADHD, as well as other developmental conditions, and those examining biological motion processing mechanisms in general.

Weaknesses:

The data are not strong enough to support claims about differences between global and lobal processing wrt social communication skills and age. The mechanistic possibilities for why these abilities may dissociate in such a way are interesting, but the crucial tests of differences between correlations do not present a clear picture. Further empirical work would be needed to test this further. Specifics:

The authors state frequently that it was the local BM task that related to social communication skills (SRS) and not the global tasks. However, the results section shows a correlation between SRS and all three tasks. The only difference is that when looking specifically within the ADHD group, the correlation is only significant for the local task. The supplementary materials demonstrate that tests of differences between correlations present an incomplete picture. Currently they have small samples for correlations, so this is unsurprising.

Theoretical assumptions. The authors make some statements about local vs global biological motion processing that may have been made in previous studies, but would appear controversial and not definitive. E.g., that local BM processing does not improve with age.

Reviewer #1 (Public Review):

This study seeks to understand how selective mRNA translation informs cellular identity using the Drosophila brain as a model. Using drivers specific for either neurons or glia, the authors express a tagged large ribosomal subunit protein, which they then use as a handle for isolating total mRNA and ribosome footprints. Throughout the study, they compare these data sets to transcriptional and ribosome profiles from the whole fly head, which contains multiple cell types including fat tissue, pigment cells and others, in addition to neurons and glia. Using GO term analyses, they demonstrate the specificity of their cell-type-based ribosome profiling: known glial mRNAs are efficiently translated in glia and likewise in neurons as well. In further examining their RNAseq data set, they find that "neuronal" mRNAs, such as ion channels, are expressed in both neurons and glia, but are translated at higher rates in neurons. Based on this, they hypothesize that neuronal mRNAs are actively suppressed in glia, and next seek to determine the underlying mechanism. By meta-analysis of all mapped ribosome footprints, they find that glia have higher ribosome occupancies in the 5' leader of neuronal mRNAs. This is corroborated by individual ribosome occupancy profiles for several neuronal mRNAs. In 5'leaders containing upstream AUG codons, they find that the glial data sets show an enrichment of ribosomes at these upstream start sites. They thus conclude that that 5' leaders containing upstream AUGs confer translational suppression in glia.

Overall, the sequencing data sets generated in this study and their subsequent bioinformatic analyses seem robust and reliable. Their data echo the trends of cell-type specific translational profiles seen in previous studies (e.g. 27380875, 30650354), and making their data sets and analyses accessible to the broader scientific community would be quite helpful. The findings are presented in a logical and methodical manner, and the data are depicted clearly. The authors' results that 5' leaders facilitate translation suppression is well-supported in literature. However, they overinterpret their data by claiming that such suppression is key for maintaining glial/neuronal identity (it is even featured in their title), but do not present any evidence that loss of such regulation has any impact on cellular identity. In many places, the authors do not acknowledge possible biases in their analytical methods, or consider alternate explanations for their data. These weaken the manuscript in its current form, but many of these issues which I describe below, are rectifiable with modest effort.

(1) The authors' data in Fig. 2-S1A-B shows substantial cell-to-cell variation in RpL3::FLAG expression. The authors do not consider that this variation may cause certain neuronal/glial types to be overrepresented in their datasets. In related, the authors do not discuss whether RpL3::FLAG only present in the cell body or if it is also trafficked to the neuronal/glial processes where localized translation is known to occur (reviewed in 31270476).

(2) The RNA-seq data set that they use to calculate translation efficiency (TE) only represents mRNAs associated with RpL3::FLAG, which is part of the large ribosome subunit. As the authors are likely aware, there are mRNAs on which the full ribosome moiety does not assemble and these are effectively excluded from this data set. Ideally, a more complete picture of the mRNA landscape can be obtained by 40S subunit profiling but I appreciate that this is technically very challenging. At minimum, this caveat needs to be acknowledged.

How does the TPM of differentially regulated transcripts (such as those in Fig. 2H) compare between whole heads, neurons and glia? Since the whole head RNA-seq data was not from an enriched sample, this might serve as a decent proxy for showing that the neuron/glia RNA-seq data sets are representative of RNA abundance.

(3) The analysis in Fig. 2F shows that low abundance mRNAs in glia are further translationally suppressed, which the authors point out in lines 151-152. However, this data also shows that mRNAs with a 1:1 ration in neuron:glia (which fall in the 0.5-1 and 1-2 bin) have a TE-1; this suggests that on average, mRNAs that are equally abundant are translated equally efficiently. This is the opposite of the thesis presented in Fig. 2G-H where many mRNAs of equal abundance in neurons and glia are actually poorly translated in glia. How do the authors reconcile these observations?

It is also unclear from the manuscript whether all mRNAs were considered for the analysis in Fig. 2F or if some cutoff was employed.

(4) Throughout the manuscript the authors favor a "translation suppression" model wherein glia (for example) actively suppress neuronal mRNAs, and this is substantiated in Fig. 3C showing higher ribosome occupancy on 5' leaders than in coding regions. However, they show no evidence that glial mRNAs (such as those indicated in Fig. 2B and 2-S2B) present a different pattern, say that of higher ribosome occupancy in CDS vs. 5' leaders. This type of a positive control is a glaring omission from many of their analyses, including ribosome occupancy at upstream AUG codons (Fig. 4).

In related, to make a broad case (as they do in the title) that differential translation regulation specifies multiple cell types, it is necessary to show the corollary: that glial mRNAs (repo, bnb, pnt, etc) are suppressed in neurons. There is an inkling of this evidence in Fig. 3-S1 where fat body mRNAs in neurons are shown to have low ribosome occupancy in the CDS regions and enhanced occupancy in the 5' leader region. This data is not quantified, nor is a control neuron mRNA shown as a reference for what the ribosome occupancy profile of an actively translated mRNA looks like in a neuron.

(5) The cell-type specific ribosome profiling data sets in the manuscript are from mRNAs associated with 80s subunits that have been treated with cycloheximide during sample preparation. Cycloheximide, and many other translation inhibitors, are known to non-uniformly bias reads towards start codons (PMID: 22056041,22927429). This important caveat and its implications on the start-codon occupancy analysis in Fig. 4 are not acknowledged in the manuscript.<br /> Again, the ideal resolution would be ribosome profiling data set from 40S footprinting or harringtonine-treated samples (PMIDs: 32589966, 27487212, 32589964) to show true accumulation of ribosomes at AUG codons. In the absence of such a data set, a comparative meta-analysis of the ribosome distribution around upstream and initiation AUG codons of differentially translated transcripts from neurons would be a useful control.

(6) The authors chose Rhodopsin 1 (Rh1) as a model mRNA which is translated efficiently in neurons but suppressed in glia. Though the data in Fig. 2-S3B shows higher TE for Rh1 in neurons, the data in 5A show lower ribosome occupancy in the Rh1 CDS in neuron samples (at least in the fragment of the CDS visible). These data are somewhat contradictory.<br /> Further, given that the neuron data are from all nsyb-positive cells but that Rh1 is expressed only in R1-R6 photoreceptors, it is unclear what motivated them to chose Rh1 as opposed to an mRNA that is more broadly expressed in neurons.

(7) Similar to the heterogeneity in nsyb- and repo-GAL4 expression in Fig. 2-S1A-B, Fig. 5C shows substantial variation in the expression of the UAS-GFP reporter driven by tub-GAL4. This variable GAL4 activity makes the mRNA abundance data difficult to interpret. Also, since the authors presume that Rh1 mRNA is expressed in glia (it is not annotated in the RNA-seq analysis in Fig. 2-S2B), would Rh1-GAL4 not be a more apt driver?<br /> These issues are further compounded by the lack of a cellular compartment marker (repo marks glial nuclei) which makes it impossible to determine which cell the mRNA signal is in. There are also no negative controls are presented for the mRNA probes.

Most confoundingly though, the control reporter itself seems to show variable translation efficiencies from one cell to another, with high-GFP protein cells showing lower GFP mRNA and vice versa.<br /> The mRNA:protein ratio may be easier to examine by using repo-GAL4 to specifically drive the Rh1-reporter expression in glia (such as in Fig. 5-S1A) rather than simultaneous expression in both neurons and glia using tub-GAL4.

Comments post revision: The authors have satisfactorily addressed most of my concerns with the study. I appreciate their patient clarification of many of my points, and the revision to text+figures appending more controls. My only minor gripe remains that while their data beautifully show that there is differential regulation of transcripts across neurons and glia, they do not provide evidence that such regulation is required for cell identity. However, I appreciate this is a large experimental ask worthy of another study in and of itself. Overall, I peg this an excellent study that adds substantially to the field of cell-type specific mRNA translation regulation.

Reviewer #3 (Public Review):

It is well established that there is extensive post-transcriptional gene regulation in nervous systems, including the fly brain. For example, dynamic regulation of hundreds of genes during photoreceptor development could only be observed at the level of translated mRNAs, but not the entire transcriptomes. The present study instead addresses the role of differential translational regulation between cell types (or rather classes: neurons and glia, as both are still highly heterogenous groups) in the adult fly brain. By performing bulk RNA-seq and Ribo-seq on the same lysates, the authors are able to compare translation efficiency (TE) of all transcripts between neurons and glia. Many genes display differential TE, but interestingly, they tend to be the genes that already show strong differences at their mRNA level. The most striking observation is the finding that neuronal transcripts in glia display increased ribosome stalling at their 5' UTR, and in particular at the start codons of short "upstream ORFs". This could suggest that glia specifically employ a mechanism to upregulate upstream ORF translation, enabling them to better suppress the expression of the genes that have them. And neuronal genes tend to have longer 5' UTRs, perhaps to facilitate this type of regulation.

However, it is difficult to evaluate the functional significance of these differences because the authors provide only one follow-up experiment to their RNA-seq analysis. Venus expressed with the Rh1 UTR sequences may be displaying differential levels between glia and neurons, but I find this image (Fig. 5C) rather unconvincing to support that conclusion. There are no quantifications of colocalization, or even sample size information provided for this experiment. And if there is indeed a difference, it would still be difficult argue this is because of the 5' stalling phenomenon authors observe with Rh1, because they switched both the 5' and 3' UTRs.

I also find it puzzling that the TE differences between the groups are mostly among the transcripts that are already strongly differentially expressed at the transcriptional level. The authors would like to frame this as a mechanism of 'contrast sharpening'; but it is unclear why that would be needed. Rh1, for instance, is not just differentially expressed between neurons and glia, but it is actually only expressed by a very specific neuronal type (photoreceptors). Thus it's not clear to me why the glia would need this 5' stalling mechanism to fully suppress Rh1 expression, while all the other neurons can apparently do so without it.

Response to authors' revisions:

The authors have addressed most of the technical points in their revised manuscript. However, it is still rather unclear whether this mechanism would have any significant impact on differential gene expression between cell types in vivo. Considering that it's mostly occurring on genes that are already strongly differentially transcribed, that doesn't appear very likely.

Reviewer #3 (Public Review):

Summary:

The authors elucidated 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. 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. They finally propose that USP8 dissociates Rabx-5 from early endosomes facilitating endosome maturation.

Strengths:

The authors have created significant, multifaceted tools for investigating systems involved in endosome dynamics control in both worm and human cells, which will help many members of the cell biology community. The study discovered an intriguing relationship between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, expanding USP8's targets and modes of action. The results provide significant contributions to our knowledge of how endosomal maturation works.

Weaknesses:

The rationales could have been stated clearer to help the readers.

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 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 localization of USP8 to early endosomes are 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 that Rabex5 are 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 evidences 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 in 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 that whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion.<br /> - The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient of both factors displayed similar defects in late endosomes/lysosomes. But the authors didn't confirm whether and/or to which extent that USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model.

Reviewer #1 (Public Review):

This study makes an interesting finding: a polyunsaturated fatty acid, Lin-Glycine, increases the conductance of KCNQ1/KCNE1 channels by stabilizing a state of the selectivity filter that allows K+ conduction. The stabilization of a conducting state appears well supported by single channel analysis, though some technical details are missing and presentations confusing. The linkage to PUFA action through the selectivity filter is supported by disruption of PUFA effects by mutation of residues which change conformation in two KCNQ1 structures from the literature. A definitive functional experiment is conducted by single channel recordings with selectivity filter domain mutation Y315F which ablates the Lin-Glycine effect on Gmax. The computational exploration of two selectivity filter structures proposed to interact distinctly with Lin-Glycine is informative, however the relation of the closed selectivity filter structures to the [K+] concentration in which it was obtained and inactivation in other channels is ignored. Overall, the major claim of the abstract is well-supported: "... that the selectivity filter in KCNQ1 is normally unstable ... and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state."

Reviewer #2 (Public Review):

Summary:

Golluscio et al. addresses one of the mechanisms of IKs (KCNQ1/KCNE1) channel upregulation by a polyunsaturated fatty acid (PUFA). PUFAs are known to upregulate KCNQ1 and KCNQ1/KCNE1 channels by two mechanisms: one shifts the voltage dependence to the negative direction, and the other increases the maximum conductance (Gmax). While the first mechanism is known to affect the voltage sensor equilibrium by charge effect, the second mechanism is less known. By applying the single-channel recordings and mutagenesis on the putative binding sites (most of them related to the selectivity filter), they concluded that the selectivity filter is stabilized to a conductive state by PUFA binding.

Strengths:

The manuscript employed single-channel recordings and directly assessed the behavior of the selectivity filter. The method is straightforward and convincing enough to support the claims.

Weaknesses:

Although the analysis using selectivity filter mutants supports the hypothesis that PUFA binding stabilizes the conducting state of the filter, it may be somewhat speculative how PUFAs bind to the KCNQ1 channel in the presence of KCNE1.

Reviewer #3 (Public Review):

Summary:

This manuscript reveals an important mechanism of KCNQ1/IKs channel gating such that the open state of the pore is unstable and undergoes intermittent closed and open conformations. PUFA enhances the maximum open probability of IKs by binding to a crevice adjacent to the pore and stabilize the open conformation. This mechanism is supported by convincing single channel recordings that show empty and open channel traces and the ratio of such traces is affected by PUFA. In addition, mutations of the pore residues alter PUFA effects, convincingly supporting that PUFA alters the interactions among these pore residues.

Strengths:

The data are of high quality and the description is clear.

Reviewer #1 (Public Review):

Summary:

The current manuscript provides strong evidence that the molecular function of SLC35G1, an orphan human SLC transporter, is citrate export at the basolateral membrane of intestinal epithelial cells. Multiple lines of evidence, including radioactive transport experiments, immunohistochemical staining, gene expression analysis, and siRNA knockdown are combined to deduce a model of the physiological role of this transporter.

Strengths:

The experimental approaches are comprehensive, and together establish a strong model for the role of SLC35G1 in citrate uptake. The observation that chloride inhibits uptake suggests an interesting mechanism that exploits the difference in chloride concentration across the basolateral membrane.

Weaknesses:

Some aspects of the results would benefit from a more thorough discussion of the conclusions and/or model.

For example, the authors find that SLC35G1 prefers the dianionic (singly protonated) form of citrate, and rationalize this finding by comparison with the substrate selectivity of the citrate importer NaDC1. However, this comparison has weaknesses when considering the physiological pH for SLC35G1 and NaDC1. NaDC1 binds citrate at a pH of ~5.4 (the pKa of citrate is 5.4, so there is a lot of dianionic citrate present under physiological circumstances). SLC35G1 binds citrate under pH conditions of ~7.5, where a very small amount of dianionic citrate is present. The data clearly show a pH dependence of transport, and the authors rule out proton coupling, but the discrepancy between the pH dependence and the physiological expectations should be addressed/commented on.

The rationale for the series of compounds tested in Figure 1F, which includes metabolites with carboxylate groups, a selection of drugs including anion channel inhibitors and statins, and bile acids, is not described. Moreover, the lessons drawn from this experiment are vague and should be expanded upon. It is not clear what, if anything, the compounds that reduce citrate uptake have in common.

The transporter is described as a facilitative transporter, but this is not established definitively. For example, another possibility could involve coupling citrate transport to another substrate, possibly even chloride ion.

Reviewer #2 (Public Review):

Summary:

The primary goal of this study was to identify the transport pathway that is responsible for the release of dietary citrate from enterocytes into blood across the basolateral membrane.

Strengths:

The transport pathway responsible for the entry of dietary citrate into enterocytes was already known, but the transporter responsible for the second step remained unidentified. The studies presented in this manuscript identify SLC35G1 as the most likely transporter that mediates the release of absorbed citrate from intestinal cells into the serosal side. This fills an important gap in our current knowledge of the transcellular absorption of dietary citrate. The exclusive localization of the transporter in the basolateral membrane of human intestinal cells and the human intestinal cell line Caco-2 and the inhibition of the transporter function by chloride support this conclusion.

Weaknesses:

(i) The substrate specificity experiments have been done with relatively low concentrations of potential competing substrates, considering the relatively low affinity of the transporter for citrate. Given that NaDC1 brings in not only citrate as a divalent anion but also other divalent anions such as succinate, it is possible that SLC35G1 is responsible for the release of not only citrate but also other dicarboxylates. But the substrate specificity studies show that the dicarboxylates tested did not compete with citrate, meaning that SLc35G1 is selective for the citrate (2-), but this conclusion might be flawed because of the low concentration of the competing substrates used in the experiment.

(ii) The authors have used MDCK cells for assessment of the transcellular transfer of citrate via SLC35G1, but it is not clear whether this cell line expresses NaDC1 in the apical membrane as the enterocytes do. Even though the authors expressed SLC35G1 ectopically in MDCK cells and showed that the transporter localizes to the basolateral membrane, the question as to how citrate actually enters the apical membrane for SLC35G1 in the other membrane to work remains unanswered.

(iii) There is one other transporter that has already been identified for the efflux of citrate in some cell types in the literature (SLC62A1, PLoS Genetics; 10.1371/journal.pgen.1008884), but no mention of this transporter has been made in the current manuscript.

Reviewer #3 (Public Review):

Summary:

Mimura et al describe the discovery of the orphan transporter SLC35G1 as a citrate transporter in the small intestine. Using a combination of cellular transport assays, they show that SLC35G1 can mediate citrate transport in small intestinal cell lines. Furthermore, they investigate its expression and localization in both human tissue and cell lines. Limited evidence exists to date on both SLC35G1 and citrate uptake in the small intestine, therefore this study is an important contribution to both fields. However, the main claims by the authors are only partially supported by experimental evidence.

Strengths:

The authors convincingly show that SLC35G1 mediates uptake of citrate which is dependent on pH and chloride concentration. Putting their initial findings in a physiological context, they present human tissue expression data of SLC35G. Their Transwell assay indicates that SLC35G1 is a citrate exporter at the basolateral membrane.

Weaknesses:

Further confirmation and clarification are required to claim that the SLC indeed exports citrate at the basolateral membrane as concluded by the authors. Most experiments measure citrate uptake, but the authors state that SLC35G1 is an exporter, mostly based on the lack of uptake at physiological conditions faced at the basolateral side. The Transwell assay in Figure 1L is the only evidence that it indeed is an exporter. However, in this experiment, the applied chloride concentration was not according to the proposed model (120mM at the basolateral side). The Transwell assay, or a similar assay measuring export instead of import, should be carried out in knockdown cells to prove that the export indeed occurs through SLC35G1 and not through an indirect effect. Related to the mentioned chloride sensitivity, it is unclear how the proposed model works if the SLC faces high chloride conditions under physiological conditions though it is inhibited by chloride.

Reviewer #1 (Public Review):

The study identifies the epigenetic reader SntB as a crucial transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus, although the precise molecular mechanisms remain elusive. Using homologous recombination, researchers constructed sntB gene deletion (ΔsntB), complementary (Com-sntB), and HA tag-fused sntB (sntB-HA) strains. Results indicated that deletion of the sntB gene impaired mycelial growth, conidial production, sclerotia formation, aflatoxin synthesis, and host colonization compared to the wild type (WT). The defects in the ΔsntB strain were reversible in the Com-sntB strain.

Further experiments involving ChIP-seq and RNA-seq analyses of sntB-HA and WT, as well as ΔsntB and WT strains, highlighted SntB's significant role in the oxidative stress response. Analysis of the catalase-encoding catC gene, which was upregulated in the ΔsntB strain, and a secretory lipase gene, which was downregulated, underpinned the functional disruptions observed. Under oxidative stress induced by menadione sodium bisulfite (MSB), the deletion of sntB reduced catC expression significantly. Additionally, deleting the catC gene curtailed mycelial growth, conidial production, and sclerotia formation, but elevated reactive oxygen species (ROS) levels and aflatoxin production. The ΔcatC strain also showed reduced susceptibility to MSB and decreased aflatoxin production compared to the WT.

This study outlines a pathway by which SntB regulates fungal morphogenesis, mycotoxin synthesis, and virulence through a sequence of H3K36me3 modification to peroxisomes and lipid hydrolysis, impacting fungal virulence and mycotoxin biosynthesis.

The authors have achieved majority of their aims at the beginning of the study, finding target genes, which led to catC mediated regulation of development, growth and aflatoxin metabolism. Overall most parts of the study is solid and clear.

Reviewer #2 (Public Review):

Summary:

This work is of great significance in revealing the regulatory mechanisms of pathogenic fungi in toxin production, pathogenicity, and in its prevention and pollution control. Overall, this is generally an excellent manuscript.

Strengths:

The data in this manuscript is robust and the experiments conducted are appropriate.

Weaknesses:

(1) The authors found that SntB played key roles in oxidative stress response of A. flavus by ChIP-seq and RNA sequencing. To confirm the role of SntB in oxidative stress, authors have better to measure the ROS levels in the ΔsntB and WT strains, besides the ΔcatC strain.<br /> (2) Why the authors only studied the function of catC among the 7 genes related to oxidative response listed in Table S14.

Reviewer #1 (Public Review):

By using a series of biochemical methods based on proteomic and metabolomic approaches, this study aims at: (1) validating the specific targeting of a biologically active molecule (MIPS2673) towards a defined (and unique?) protein target within a parasite, and (2) exploring whether it is possible to extrapolate which metabolic pathway has been disrupted.

Strength/Weaknesses

-The chemoproteomic approach, convincingly shows that MIPS2673 more significantly "protects" the putative target (PfA-M1) against thermal degradation or against enzymatic attack (by proteinase K). Proteomic studies are carried using parasite extracts enriched in late trophozoites (30-38 h pi), and are restricted to the soluble proteins fraction.<br /> -The metabolomic approach, documents the ability of MIPS2673 to selectively increase the number of non-hydrolyzed dipeptides in treated versus untreated parasites, further arguing for selective targeting of PfA-M1 and impairment of hemoglobin breakdown by the parasite.<br /> -The revised version now also considers and further studies the additional putative targets identified by one proteomic approach (but not the other one), which is both more critical of the results obtained and more realistic.<br /> The work as a whole is highly interesting, both for the specific topic of PfA-M1's role in parasite biology and for the method, applicable to other malarial drug contexts.

Reviewer #2 (Public Review):

In this manuscript, the authors first developed a new small molecular inhibitor that could target specifically the M1 metalloproteases of both important malaria parasite species Plasmodium falciparum and P. vivax. This was done by a chemical modification of a previously developed molecule that targets PfM1 as well as PfM17 and possibly other Plasmodial metalloproteases. After the successful chemical synthesis, the authors showed that the derived inhibitor, named MIPS2673, has a strong antiparasitic activity with IC50 342 nM and it is highly specific for M1. With this in mind, the authors first carried out two large-scale proteomics to confirm the MIPS2673 interaction with PfM1 in the context of the total P. falciparum protein lysate. This was done first by using thermal shift profiling and subsequently limited proteolysis. While the first demonstrated overall interaction, the latter (limited proteolysis) could map more specifically the site of MIPS2673-PfM1 interaction, presumably the active site. Subsequent metabolomics analysis showed that MIPS2673 cytotoxic inhibitory effect leads to the accumulation of short peptides many of which originate from hemoglobin. Based on that the authors argue that the MIPS2673 mode of action (MOA) involves inhibition of hemoglobin digestion that in turn inhibits the parasite growth and development.

Comments on the revised version:

The authors addressed all my comments from the previous round of reviews.

Reviewer #3 (Public Review):

Summary:

Overall, this is an interesting series of experiments which have identified a putative inhibitor of the Plasmodium M1 alanyl aminopeptidases, PfA-M1 and PvA-M1. The weaknesses include the lack of additional analysis of additional targets identified in the chemoproteomic approaches.

Strengths:

The main strengths include the synthesis of MIPS2673 which is selectively active against the enzyme and in whole cell assay.

Weaknesses:

The authors have addressed the previously identified weaknesses and have now provided additional data and explanations. They have modified their conclusions to indicate the limitations of their work.

Reviewer #1 (Public Review):

Summary:

Khan et. al., investigated the functional redundancy of the non-canonical L-cysteine synthases of M. tuberculosis, CysM and CysK2, focussing on their role in mitigating the effects of host-derived stress. They found that while deletion mutants of the two synthases (Rv∆cysM, Rv∆cysK2) have similar transcriptomes under standard conditions, their transcriptional response to oxidative stress is distinct. The impact of deleting the synthases also differentially affected the pools of L-cysteine-derived metabolites. They show that the mutants (Rv∆cysM, Rv∆cysK2) have impaired survival in peritoneal macrophages and in a mouse model of infection. Importantly, they show that the survival of the mutants increases when the host are defective in producing reactive oxygen and nitrogen species, linking the phenotype to a defect in combating host-derived stress. Finally, they show that compounds inhibiting L-cysteine synthases reduces intracellular survival of M. tuberculosis.

Strengths:

(1) The distinct transcriptome of the Rv∆cysM and Rv∆cysK2 mutants in the presence of oxidative stress provides solid evidence that these mutants are distinct in their response to oxidative stress, and suggests that they are not functionally redundant.<br /> (2) The use of macrophages from phox-/- and INF-/- mice and an iNOS inhibitor for the intracellular survival assays provides solid evidence that the survival defect seen for the Rv∆cysM and Rv∆cysK2 mutants is related to their reduced ability to combat host-derive oxidative and nitrosative stress. This is further supported by the infection studies in phox-/- and INF-/- mice.

Weaknesses:

Inclusion of the complemented strains in the metabolite study would strengthen the data. Furthermore, using an alternate method to quantify the MSH:MSSM ratio would provide insight into the redox homoeostasis in mutants in the presence and absence of CHP to support the statement that "deletion or inhibition of CysM or CysK2 perturbs redox homeostasis of Mtb".

The authors sought to investigate the functional redundancy of the non-canonical L-cysteine synthases CysM and CysK2. While their distinct transcriptional response to oxidative stress suggests distinct physiological roles, the study did not explore these differences, and therefore provides only preliminary insight into the underlying reasons for this observation. In the context of drug development, this work suggests that while L-cysteine synthases inhibitors do not have high potency for killing intracellular M. tuberculosis, they have potential for decreasing the pathogen's survival in the presence of host-derive stress.

Reviewer #3 (Public Review):

The manuscript by Bimai et al describes a structural and functional characterization of an anaerobic ribonucleotide reductase (RNR) enzyme from the human microbe, P. copri. More specifically, the authors aimed to characterize the mechanism by how (d)ATP modulates nucleotide reduction in this anaerobic RNR, using a combination of enzyme kinetics, binding thermodynamics, and cryo-EM structural determination, complemented by hydrogen-deuterium exchange (HDX). One of the principal findings of this paper is the ordering of a NxN 'flap' in the presence of ATP that promotes RNR catalysis and the disordering (or increased protein dynamics) of both this flap and the glycyl radical domain (GRD) when the inhibitory effector, dATP, binds. The latter is correlated with a loss of substrate binding, which is the likely mechanism for dATP inhibition. It is important to note that the GRD is remote (>30 Ang) from the binding site of the dATP molecule, suggesting long-range communication of the structural (dis)ordering. The authors also present evidence for a shift in oligomerization in the presence of dATP. The work does provide evidence for new insights/views into the subtle differences of nucleotide modulation (allostery) of RNR, in a class III system, through long-range interactions.

The strengths of the work are the impressive, in-depth structural analysis of the various regulated forms of PcRNR by (d)ATP using cryo-EM. The authors present seven different models in total, with striking differences in oligomerization and (dis)ordering of select structural features, including the GRD that is integral to catalysis. The authors present several, complementary biochemical experiments (ITC, MST, EPR, kinetics) aimed at resolving the binding and regulatory mechanism of the enzyme by various nucleotides. The authors present a good breadth of the literature in which the focus of allosteric regulation of RNRs has been on the aerobic orthologues.

The addition of hydrogen-deuterium exchange mass spectrometry (HDX-MS) complements the results originating from cryo-EM data. Most notable, is the observation of the enhanced exchange (albeit quite subtle) of the GRD domain in the presence of dATP that matches the loss of structural information in this region in the cryo-EM data. The most pronounced and compelling HDX results are seen in the form of dATP-induced protection of peptides immediately adjacent to the b-hairpin at the s-site, where dATP is expected to bind based on cryo-EM. It is clear that the presence of dATP increases the rigidity of this region.

Reviewer #1 (Public Review):

Valk and Engert et al. examined the potential relations between three different mental training modules, hippocampal structure and functional connectivity, and cortisol levels (stress) over a 9-month period. They found that among the three types of mental training: Presence (attention and introspective awareness), Affect (socio-emotional - compassion and prosocial motivation), and Perspective (socio-cognitive - metacognition and perspective taking) modules; Affect training most robustly related to changes in hippocampal structure and function - specifically, CA1-3 subfields of the hippocampus. Moreover, change in intrinsic functional connectivity related to changes in diurnal cortisol release and long-term cortisol exposure. These changes are proposed to result from a combination of factors, which is supported by multivariate analyses showing changes across subfields and training content relate to cortisol changes.

The authors demonstrate that mindfulness training programs are a potential avenue for stress interventions that impact hippocampal structure and cortisol, providing a promising approach to improve health. The data contribute to the literature on plasticity of hippocampal subfields during adulthood, the impact of mental training interventions on the brain, and the link between CA1-3 and both short- and long-term stress changes.

The authors thoughtfully approached the study of hippocampal subfields, utilizing a method designed for T1w images that outperformed Freesurfer 5.3 and that produced comparable results to an earlier version of ASHS. The authors note the limitations of their approaches and provide detailed information on the data used and analyses conducted. The results provide a strong basis from which future studies can expand using computational approaches or more fine-grained investigations of the impact of mindfulness training on cortisol levels and the hippocampus.

Reviewer #3 (Public Review):

Summary:

This study profiled the single-cell transcriptome of human spermatogenesis and provided many potential molecular markers for developing testicular puncture-specific marker kits for NOA patients.

Strengths:

Perform single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on testicular tissues from two OA patients and three NOA patients.

Weaknesses:

Most results are analytical and lack specific experiments to support these analytical results and hypotheses.

Reviewer #1 (Public Review):

Summary:

The manuscript is dedicated heavily to cell type mapping and identification of sub-type markers in the human testis but does not present enough results from cross-investigation between NOA cases versus control. Their findings are mostly based on transcriptome and the authors do not make enough use of the scATAC-seq data in their analyses as they put forward in the title. Overall, the authors should do more to include the differential profile of NOA cases at the molecular level - specific gene expression, chromatin accessibility, TF binding, pathway, and signaling that are perturbed in NOA patients that may be associated with azoospermia.

Strengths:

(1) The establishment of single-cell data (both RNA and ATAC) from the human testicular tissues is noteworthy.

(2) The manuscript includes extensive mapping of sub-cell populations with some claimed as novel, and reports marker gene expression.

(3) The authors present inter-cellular cross-talks in human testicular tissues that may be important in adequate sperm cell differentiation.

Weaknesses:

(1) A low sample size (2 OA and 3 NOA cases). There are no control samples from healthy individuals.

(2) Their argument about interactions between germ and Sertoli cells is not based on statistical testing.

(3) Rationale/logic of the study. This study, in its present form, seems to be more about the role of sub-Sertoli population interactions in sperm cell development and does not provide enough insights about NOA.

(4) The authors do not make full use of the scATAC-seq data.

Reviewer #2 (Public Review):

Summary:

Shimin Wang et al. investigated the role of Sertoli cells in mediating spermatogenesis disorders in non-obstructive azoospermia (NOA) through stage-specific communications. The authors utilized scRNA-seq and scATAC-seq to analyze the molecular and epigenetic profiles of germ cells and Sertoli cells at different stages of spermatogenesis.

Strengths:

By understanding the gene expression patterns and chromatin accessibility changes in Sertoli cells, the authors sought to uncover key regulatory mechanisms underlying male infertility and identify potential targets for therapeutic interventions. They emphasized that the absence of the SC3 subtype would be a major factor contributing to NOA.

Weaknesses:

Although the authors used cutting-edge techniques to support their arguments, it is difficult to find conceptual and scientific advances compared to Zeng S et al.'s paper (Zeng S, Chen L, Liu X, Tang H, Wu H, and Liu C (2023) Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia. Front. Endocrinol. 14:1138386.). Overall, the authors need to improve their manuscript to demonstrate the novelty of their findings in a more logical way.

Reviewer #1 (Public Review):

In this manuscript, Leikina et al. investigate the role of redox changes in the ubiquitous protein La in the promotion of osteoclast fusion. In a recently published manuscript, the investigators found that osteoclast multinucleation and resorptive activity are regulated by a de-phosphorylated and proteolytically cleaved form of the La protein that is present on the cell surface of differentiating osteoclasts. In the present work, the authors build upon these findings to determine the physiologic signals that regulate La trafficking to the cell membrane and ultimately, the ability of this protein to promote fusion. Building upon other published studies that show (1) that intracellular redox signaling can elicit changes in the confirmation and localization of La, and (2) that osteoclast formation is dependent on ROS signaling, the authors hypothesize that oxidation of La in response to intracellular ROS underlies the re-localization of La to the cell membrane and that this is necessary for its pro-fusion activity. The authors test this hypothesis in a rigorous manner using antioxidant treatments, recombinant La protein, and modification of cysteine residues predicted to be key sites of oxidation. Osteoclast fusion is then monitored in each condition using fluorescence microscopy. These data strongly support the conclusion that oxidized La is de-phosphorylated, increases in abundance at the cell surface of differentiating osteoclasts, and promotes cell-cell fusion. A strength of this manuscript is the use of multiple complementary approaches to test the hypothesis, especially the use of Cys mutant forms of La to directly tie the observed phenotypes to changes in residues that are key targets for oxidation. The manuscript is also well-written and describes a clearly articulated hypothesis based on a precise summation of the existing literature. The findings of this manuscript will be of interest to researchers in the field of bone biology, but also more generally to cell biologists. The data in this manuscript may also lead to future studies that target La for bone diseases in which there is increased osteoclast activity. The weaknesses of the manuscript are minor and predominantly relate to data presentation choices. These weaknesses do not detract from the overall conclusions of the study.

Reviewer #2 (Public Review):

Summary:

Bone resorption by osteoclasts plays an important role in bone modeling and homeostasis. The multinucleated mature osteoclasts have higher bone-resorbing capacity than their mononuclear precursors. The previous work by authors has identified that increased cell-surface level of La protein promotes the fusion of mononuclear osteoclast precursor cells to form fully active multinucleated osteoclasts. In the present study, the authors further provided convincing data obtained from cellular and biochemical experiments to demonstrate that the nuclear-localized La protein where it regulates RNA metabolism was oxidized by redox signaling during osteoclast differentiation and the modified La protein was translocated to the osteoclast surface where it associated with other proteins and phospholipids to trigger cell-cell fusion process. The work provides novel mechanistic insights into osteoclast biology and provides a potential therapeutic target to suppress excessive bone resorption in metabolic bone diseases such as osteoporosis and arthritis.

Strengths:

Increased intracellular ROS induced by osteoclast differentiation cytokine RANKL has been widely studied in enhancing RANKL signaling during osteoclast differentiation. The work provides novel evidence that redox signaling can post-translationally modify proteins to alter the translocation and functions of critical regulators in the late stage of osteoclastogenesis. The results and conclusions are mostly supported by the convincing cellular and biochemical assays,

Weaknesses:

The lack of in vivo studies in animal models of bone diseases such as postmenopausal osteoporosis, inflammatory arthritis, and osteoarthritis reduces the translational potential of this work.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Beardslee and Schmitz, the authors undertook a screen for potential degrons - short peptide sequences at the C-terminus that would target the toxin VapC for degradation. The authors randomly mutagenized 5 amino acids appended to the C-terminus of VapC and transformed this library into E. coli to look for surviving cells when the VapC gene was expressed. The authors found an enrichment for tags ending Ala-Ala, and found that this enrichment was dependent on the presence of the ClpXP protease, since this sequence was not similarly enriched in a mutant lacking this protease. Moreover, the authors identify the sequence FKLVA as the tag with the highest fold enrichment in the screen and confirm that GFP tagged with this sequence is degraded by ClpXP with similar kinetics to GFP tagged with the ssrA-derived tag.

Strengths:

This study has two major implications for understanding the nature of degrons in E. coli. First, peptides ending Ala-Ala, and especially degrons resembling the ssrA degron are likely the most degradation-promoting sequences in E. coli. Second, these findings suggest that ClpXP is the most central protease, at least for this particular protein with a randomized C-terminus under the particular conditions of this screen. It is also notable that the ribosome quality control protein RqcH tags truncated proteins with an alanine tag in a template-free manner when the large ribosomal subunit is obstructed. Although E. coli doesn't encode RqcH, the utility of alanine-tagging for protein degradation likely extends to other organisms.

Weaknesses:

The authors remark and show that mutations that inactivate the VapC protein are enriched potentially more than the proteolysis tags. This is a limitation of the study and the authors have done well to describe this as it will inform future screens. Perhaps using a protein with more intermediate toxicity in future screens would help to prioritize C-terminal mutations instead of toxin-inactivating mutations.

For clarity, the authors should explain why the NNK structure of the random codons was used. Why is it important that the codon end with a G or T?

Authors state on page 7 that by determining enrichment of individual tags they can rank the relative Km for proteolysis of the individual tags. This statement is not accurate since the tag could variously impact its association with any of the proteases in the cell. Since Km is specific to each particular protease, these can't be ranked in vivo when all proteases are present.

Reviewer #2 (Public Review):

Summary:

The authors studied the sequence determinants of C-terminal tags that govern protein degradation in bacteria. They introduce a new strategy to determine degron sequences: Detox (Degron Enrichment by toxin). This unbiased approach links degron efficiency to cell growth as degrons are C-terminally fused to the toxin VapC, which inhibits protein translation. Selecting for bacterial growth and thus toxin degradation enabled the identification of potent degron derived from a randomized library of pentapeptides. Remarkably, most degrons show sequence similarity to the SsrA-tag, which is fused to incomplete polypeptides at stalled ribosomes by the tmRNA-tagging system. These findings underline the extraordinary efficiency of the SsrA-tag and the ClpXP protease in removing incomplete polypeptides and demonstrate that most proteins are spared from degradation by harboring different C-termini. The introduced method will be highly useful to determine degron sequences in other positions and other bacterial species.

Strengths:

The work introduces an innovative and powerful strategy to identify degron sequences in bacteria. The study is well-controlled and results have been thoroughly analyzed. It will now become important to broaden the technology, making it also accessible for more complex degrons.

Weaknesses:

The approach is efficient in identifying strong degron sequences that are predominantly recognized by the ClpXP protease. The sequence specificity of other proteolytic systems, however, is not efficiently addressed, pointing to a potential limitation of this technology. The GS-rich linker sequence connecting the degron and the toxin might also impact proteolysis and thus outcome.

Reviewer #3 (Public Review):

Summary:

This manuscript by Beardslee and Schmitz reports discoveries made from a genetic screen to identify C-terminal degrons that cause the efficient depletion of a potent toxin, which allowed for a deep assessment of amino acid patterns that promote protein turnover.

Strengths:

The key findings are that SsrA-like C termini are a dominant class of efficient degrons and that ClpP (X/A) mediates the turnover in most cases. Moreover, the data provides insight into the importance of residues situated farther into the degron and reveals aspects of the ClpX engagement and processivity process. The manuscript is clearly written and there is ample supporting data for the conclusions drawn. The figures are also informative.

Weaknesses:

There are only a few minor suggestions on data interpretation.

(1) Page 6: It is stated that "We plated cells on media containing 0 - 1% arabinose inducer, and observed that stronger induction of untagged VapC indeed correlates with smaller colony size; ... We conclude that VapC levels have a titratable effect on growth rate."

In E. coli with intact arabinose import/response systems, sub-saturating levels of arabinose do not generally lower the induction level of the PBAD promoter in each cell; rather, a sub-population of cells becomes induced [PMID: 9223333]. The bulk observation is a reduced expression level, and, in this case, slowed growth, but it seems more likely that the slow growth observed is from the induced cohort dying off as the cultures and colonies develop.

(2) Page 8: "At 6-hours post-induction,..."

Because these experiments were enrichments from initial pools of clones, the number of cell divisions is more informative than the hours of outgrowth or culture densities at harvest. It would be helpful if the authors could indicate, or at least estimate, the number of cell divisions. this could then be included in the results or methods section.

(3) Page 12: "It is possible that these sequences compromise VapC folding and solubility, or mimic inhibitory interactions made by hydrophobic segments of the VapB antitoxin that block VapC activity (43, 59)."

Later in the manuscript, Lon is presented as a minor player in the overall story, but Lon prefers hydrophobic degrons. Could that hydrophobic class be Lon substrates? (Possibly presented as an additional mechanism here or in the discussion of this class of tags.)

(4) Page 13: "Arg in the 2nd position was also associated with proteolysis, yet Arg is virtually absent from proteobacterial ssrA sequences."

The nucleic acid changes required for evolutionary drift from the predominant amino acid codons at this position in proteobacteria to Arg may require moving through several codons that notably impair the performance of the degron. Such a constraint may also be responsible, in part, for the observed conservation.

Reviewer #1 (Public Review):

Summary:

This manuscript addresses the temporal patterns in how cholinergic signaling to the gut affects the lifespan of the worm C. elegans, which should make the manuscript of wide interest to those who study aging, as well as those who study the brain-gut axis in health and disease. The authors show that early acetylcholine (ACh) signaling to the intestine via the ACR-6 receptor shortens worm lifespan, which depends on the DAF-16/FOXO transcription factor. However, later ACh signaling to the intestine via the GAR-3 receptor extends lifespan, which in turn depends on the heat shock factor HSF-1. The authors also show a potential mechanism through which these two temporal patterns of ACh signaling might be coordinated to influence longevity in the worm, and possibly in other animals.

Strengths:

The authors observed that the functional ablation of acr-2-expressing cholinergic neurons in C. elegans (Pacr-2::TeTx) produced a lifespan curve that intersects the lifespan curve of a wild-type population. The first quartile of Pacr-2::TeTx worms shows a longer lifespan than the first quartile of wild-type worms, whereas the last quartile of Pacr-2::TeTx worms exhibits a shorter lifespan than wild-type. These observations raised the hypothesis that cholinergic neurons have two opposing effects on longevity: an early longevity-inhibiting effect and a later longevity-promoting effect. Much of the data supports the authors' conclusions.

Weaknesses:

While the authors have proved their hypothesis by temporally increasing the activity of cholinergic neurons at different life stages through the auxin-inducible degron system, their work raises two major concerns. First, they might want to discuss the conflicting data from Zullo et al (Nature 2019, vol 574, pp 359-364). For example, the authors show that increasing the activity of acr-2-expressing neurons after the 7th day of adulthood increases lifespan. However, Zullo et al (2019) show that the reciprocal experiment, inhibiting cholinergic neuron activity on the 1st day or the 8th day of adulthood, also increases lifespan. Is this because the two studies are using different promoters, that of the acr-2 ACh receptor (this work) versus that of the unc-17 vesicular ACh transporter (Zullo et al., 2019)? The two genes are expressed in different subsets of cells that do not completely overlap. CeNGEN shows that acr-2 is expressed in motor and non-motor neurons, but some of these neurons are also different from those that express unc-17. Is it possible that different cholinergic neurons also have opposite lifespan effects during adulthood? Or is it because both lack of signaling and hypersignaling can lead to a long-life phenotype? Leinwand et al (eLife 2015, vol 4, e10181) previously suggested that disturbing the balance in neurotransmission alone can extend lifespan. A simple discussion of these possibilities in the Discussion section is likely sufficient. Or can the auxin treatment and removal be confounding factors? Loose and Ghazi (Biol Open 2021, vol 10, bio058703) show that auxin IAA alone can affect lifespan and that this effect can depend on the time the animal is exposed to the auxin.

Second, the daf-16-dependence of the early longevity-inhibiting effect of ACh signaling needs clarification and further experimentation. The authors present a model in Figure 6D, where DAF-16 inhibits longevity. This contradicts published literature. Libina et al (Cell 2003, vol 115, pp 489-502) have shown that intestinal DAF-16 increases lifespan. From the authors' data, it is possible that ACh signaling inhibits DAF-16, not promotes it as they have drawn in Figure 6D. In Figure 3F, the authors used Pacr-2::TeTx, which inhibits cholinergic neuron activity, to show an increase in the expression of DAF-16 targets. Why did the authors not use the worms that express the transgene Pacr-2::syntaxin(T254I), which increases cholinergic neuron activity? What happens to the expression of DAF-16 targets in these animals? Do their expression go down? What happens if intestinal daf-16 is knocked down in animals with increased cholinergic neuron activity, instead of reduced cholinergic neuron activity?

Reviewer #2 (Public Review):

Summary:

In the manuscript "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans", Xu and colleagues examine the role of cholinergic signaling by C. elegans motor neurons in modulating lifespan. The authors show that manipulating motor neuronal activity using genetic techniques can be beneficial or detrimental to lifespan, depending on when motor neuron activity is modulated.

Strengths:

A large body of data showing the effects of knockdown of cholinergic receptors and neurotransmitters on lifespan is presented. This would be of value to the community.

Weaknesses:

However, the studies are incomplete. More rigorous approaches would be needed to support the key conclusions, and substantiate the main findings and pathway components.

Reviewer #3 (Public Review):

I very much enjoyed reading Lingxiu Xu et al.'s paper "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans," where they investigate the mechanisms by which motor neurons regulate lifespan in C. elegans worms. In this paper, they first discover that interfering with synaptic release in cholinergic motor neurons affects lifespan. Using mutants and gene knockdowns they show that these effects are due to the neurotransmitter acetylcholine. They show that the effects of these motor neurons on lifespan are opposite, depending on timed genetic interventions promoting synaptic release. If these interventions occur during development, the lifespan is shortened, but if they occur starting on day 7 of adulthood, then lifespan is lengthened. They then show that the transcription factor daf-16 is required for the former effect, while the transcription factor hsf-1 is required for the latter one. In addition, these early and late effects, they find, required the acetylcholine receptors acr-6 and gar-3, respectively, and intestinal expression of these genes rescues the respective phenotypes. Interestingly, tagging the endogenous acr-6 and gar-3 genes with mCherry, they find that the ACR-6 and GAR-3 proteins are expressed in the intestine, ACR-6 during development, and GAR-3 during adulthood. Based on these findings they propose a model where acetylcholine from motor neurons regulates lifespan by modulating different receptors expressed at different times. These receptors, in turn, affect lifespan in opposing ways via different transcription factors.

Reviewer #4 (Public Review):

This is a very interesting study, where the authors discovered two neuroendocrine signaling circuits with opposite effects on organismal longevity elicited by motor neurons at different ages.

Interestingly, both systems employ the same neurotransmitter (that is, acetylcholine) and signal the intestine. However, one has effects on early life to shorten lifespan whereas the other system is activated in mid-life to extend lifespan. At the mechanistic level, this bidirectional regulation is possible through the recruitment of two different ACh receptors in the gut: ACR-6 and GAR-3. The authors found that ACR-6 expression in the intestine is restricted to early life, whereas GAR-3 expression in the gut is confined to mid-late life. Interestingly, ACR-6 modulates the transcription factor DAF-16, but GAR-3 regulates HSF-1.

The study combines different approaches, including inducible systems (AID) which are critical for the conclusions of the paper. The conclusions are well supported by the experiments and results. The data provide a potential mechanism for the temporal control of lifespan and shed light on the complex role of the nervous system in organismal aging. These results can have important implications for understanding how organismal aging is regulated in a temporal manner by cell non-autonomous mechanisms. I didn't observe significant weaknesses in the study, but I have several comments that I hope the authors will address.

Reviewer #1 (Public Review):

Summary:

The work provides more evidence of the importance of data quality and representation for ligand-based virtual screening approaches. The authors have applied different machine learning (ML) algorithms and data representation using a new dataset of BRAF ligands. First, the authors evaluate the ML algorithms and demonstrate that independently of the ML algorithm, predictive and robust models can be obtained in this BRAF dataset. Second, the authors investigate how the molecular representations can modify the prediction of the ML algorithm. They found that in this highly curated dataset the different molecule representations are adequate for the ML algorithms since almost all of them obtain high accuracy values, with Estate fingerprints obtaining the worst-performing predictive models and ECFP6 fingerprints producing the best classificatory models. Third, the authors evaluate the performance of the models on subsets of different composition and size of the BRAF dataset. They found that given a finite number of active compounds, increasing the number of inactive compounds worsens the recall and accuracy. Finally, the authors analyze if the use of "less active" molecules affect the model's predictive performance using "less active" molecules taken from ChEMBl Database or using decoys from DUD-E. As results, they found that the accuracy of the model falls as the number of "less active" examples in the training dataset increases while the implementation of decoys in the training set generates results as good as the original models or even better in some cases. However, the use of decoys in the training set worsens the predictive power in the test sets that contain active and inactive molecules.

Strengths:

It is a very interesting topic in medicinal chemistry and drug discovery. This work is very well written and contains up-to-date references. The general structure of the work is adequate, allowing easy reading. The hypotheses are clear and were explored correctly. This work provides new evidence about the importance of inferring models from high-quality data and that, if such a condition is met, it is not necessary to use complex computational methods to obtain predictive models. The generated BRAF dataset is also a valuable benchmark dataset for medicinal chemists working in ligand based virtual screening.

Weaknesses:

Leaving aside the new curated BRAF dataset, the work lacks novelty since it is a topic widely studied in chemoinformatics and medicinal chemistry. Furthermore, the conclusions drawn here correspond to the analysis of only one high-quality dataset where the similarity between the molecules is not quantitatively assessed (maybe active and inactive molecules are very dissimilar and any ML algorithm and fingerprint could obtain good results). To generalize the conclusions, it would be fundamental to repeat the analysis with other high-quality datasets.

Some key tasks are not clearly described, for example, there is no information about the new BRAF dataset (e.g., where the molecules were obtained from or why the inactive molecules provide better results than the "less active" from ChEMBL... what differentiates them?). The defintion of an "inactive" compound is not clear. It is not described if global or balanced accuracy was used in the imbalanced datasets. When using decoys to evaluate the models it is important to consider that decoys were generated to be topologically different from active compounds by the comparison of the ECFP4 fingerprints using the Tanimoto coefficient. Therefore, it is quite obvious that when fingerprints are used to characterize molecules, the models will be able to easily discriminate them. It is important to note that this is not necessarily true for models based on other molecular descriptors, since they are not used in the generation of the decoys. In some cases, the differences between accuracies are very small and there are no statistical analyzes to demonstrate whether they are statistically different or not.

Reviewer #2 (Public Review):

Summary:

The authors explored the importance of data quality and representation for ligand-based virtual screening approaches. I believe the results could be of potential benefit to the drug discovery community, especially to those scientists working in the field of machine learning applied to drug research. The in silico design is comprehensive and adequate for the proposed comparisons.

This manuscript by Chong A. et al describes that it is not necessary to resort to the use of sophisticated deep learning algorithms for virtual screening, since based on their results considering conventional ML may perform exceptionally well if fed by the right data and molecular representations.

The article is interesting and well-written. The overview of the field and the warning about dataset composition are very well thought-out and should be of interest to a broad segment of the AI ​​in drug discovery readership. This article further highlights some of the considerations that need to be taken into consideration for the implementation of data-centric AI for computer-aided drug design methods.

Strengths:

This study contributes significantly to the field of machine learning and data curation in drug discovery. The paper is, in general, well-written and structured. However, in my opinion, there are some suggestions regarding certain aspects of the data analyses.

Weaknesses:

The conclusions drawn in the study are based on the analysis of a single dataset, and I am not sure they can be generalized. Therefore, in my opinion, the conclusions are only partially supported by the data. To generalize the conclusions, it is imperative to conduct a benchmark with diverse datasets, for different molecular targets.<br /> The conclusion cannot be immediately extended to molecular descriptors or features different from the ones used in this study<br /> It is advisable to present statistical analyses to ascertain whether the observed differences in metrics hold statistical significance.

Reviewer #3 (Public Review):

Summary:

The authors presented a data-centric ML approach for virtual ligand screening. They used BRAF as an example to demonstrate the predictive power of their approach.

Strengths:

The performance of predictive models in this study is superior (nearly perfect) with respect to exiting methods.

Weaknesses:

I feel the training and testing datasets may not be rigorously constructed. If that is the case, the results would be significantly affected.

I have 3 major comments:

(1) The authors identified ~4100 BRAF actives, then randomly selected 3600 BRAF actives to be part of the training dataset with the remaining 500 actives becoming a part of the hold-out test set. The problem is that, the authors did not evaluate the chemical similarity between the 3600 actives in the training, and the 500 actives in the testing set. If some of them were similar, the testing results would be very good but partially due to information leakage. The authors should carefully examine the chemical similarity between any pairs of their training and testing datasets, before any conclusion is made.

(2) The authors tried to explore the role of dataset size in the performance, in particular, what would happen when the number of actives are reduced. However the minimal number of actives used is 500 while the number of inactives ranges from 500 to 3600. This is quite different from real applications where the number of expected actives in the screening library would be at most 1-2% of the whole database. The authors should further reduced the number of actives (e.g. 125, 25, 5, 1), and evaluate their model's performance.

(3) The authors chose BRAF as example in this study. BRAF is a well studied drug target with thousands of known actives. In real applications, the target may only have a handful of known actives. The authors should try to apply their approach, to a couple other targets that have less known actives than BRAF, to evaluate their method's transferability.

Reviewer #1 (Public Review):

Using a knock-out mutant strain, the authors tried to decipher the role of the last gene in the mycofactocin operon, mftG. They found that MftG was essential for growth in the presence of ethanol as the sole carbon source, but not for the metabolism of ethanol, evidenced by the equal production of acetaldehyde in the mutant and wild type strains when grown with ethanol (Fig 3). The phenotypic characterization of ΔmftG cells revealed a growth-arrest phenotype in ethanol, reminiscent of starvation conditions (Fig 4). Investigation of cofactor metabolism revealed that MftG was not required to maintain redox balance via NADH/NAD+, but was important for energy production (ATP) in ethanol. Since mycobacteria cannot grow via substrate-level phosphorylation alone, this pointed to a role of MftG in respiration during ethanol metabolism. The accumulation of reduced mycofactocin points to impaired cofactor cycling in the absence of MftG, which would impact the availability of reducing equivalents to feed into the electron transport chain for respiration (Fig 5). This was confirmed when looking at oxygen consumption in membrane preparations from the mutant and would type strains with reduced mycofactocin electron donors (Fig 7). The transcriptional analysis supported the starvation phenotype, as well as perturbations in energy metabolism, and may be beneficial if described prior to respiratory activity data.

While the data and conclusions do support the role of MftG in ethanol metabolism, the title of the publication may be misleading as the mutant was able to grow in the presence of other alcohols (Supp Fig S2). Furthermore, the authors propose that MftG could not be involved in acetate assimilation based on the detection of acetate in the supernatant and the ability to grow in the presence of acetate. The minimal amount of acetate detected in the supernatant but a comparative amount of acetaldehyde could point to disruption of an aldehyde dehydrogenase.

The link between mycofactocin oxidation and respiration is shown, however the mutant has an intact respiratory chain in the presence of ethanol (oxygen consumption with NADH and succinate in Fig 7C) and the NADH/NAD+ ratios are comparable to growth in glucose. Could the lack of growth of the mutant in ethanol be linked to factors other than respiration? To this end, bioinformatic investigation or other evidence to identify the membrane-bound respiratory partner would strengthen the conclusions.

Reviewer #2 (Public Review):

Summary

Patrícia Graça et al., examined the role of the putative oxidoreductase MftG in regeneration of redox cofactors from the mycofactocin family in Mycolicibacerium smegmatis. The authors show that the mftG is often co-encoded with genes from the mycofactocin synthesis pathway in M. smegmatis genomes. Using a mftG deletion mutant, the authors show that mftG is critical for growth when ethanol is the only available carbon source, and this phenotype can be complemented in trans. The authors demonstrate the ethanol associated growth defect is not due to ethanol induced cell death, but is likely a result of carbon starvation, which was supported by multiple lines of evidence (imaging, transcriptomics, ATP/ADP measurement and respirometry using whole cells and cell membranes). The authors next used LC-MS to show that the mftG deletion mutant has much lower oxidised mycofactocin (MFFT-8 vs MMFT-8H2) compared to WT, suggesting an impaired ability to regenerate myofactocin redox cofactors during ethanol metabolism. These striking results were further supported by mycofactocin oxidation assays after over-expression of MftG in the native host, but also with recombinantly produced partially purified MftG from E. coli. The results showed that MftG is able to partially oxidise mycofactocin species, finally respirometry measurements with M. smegmatis membrane preparations from WT and mftG mutant cells show that the activity of MftG is indispensable for coupling of mycofactocin electron transfer to the respiratory chain. Overall, I find this study to be comprehensive and the conclusions of the paper are well supported by multiple complementary lines of evidence that are clearly presented.

Strengths

The major strengths of the paper are that it is clearly written and presented and contains multiple, complementary lines of experimental evidence that support the hypothesis that MftG is involved in the regeneration of mycofactocin cofactors, and assists with coupling of electrons derived from ethanol metabolism to the aerobic respiratory chain. The data appear to support the the authors hypotheses.

Weaknesses

No major weaknesses were identified, only minor weaknesses mostly surrounding presentation of data in some figures.

Reviewer #1 (Public Review):

Summary:

The authors set out to measure the diffusion of small drug molecules inside live cells. To do this, they selected a range of flourescent drugs, as well as some commonly used dyes, and used FRAP to quantify their diffusion. The authors find that drugs diffuse and localize within the cell in a way that is weakly correalted with their charge, with positively charged molecules displaying dramatically slower diffusion and a high degree of subcellular localization.<br /> The study is important because it points at an important issue related to the way drugs behave inside cells beyond the simple "IC50" metric (a decidedly mesoscopic/systemic value). The authors conclude, and I agree, that their results point to nuanced effects that are governed by drug chemistry that could be optimized to make them more effective.

Strengths:

The work examines an understudied aspect of drug delivery.<br /> The work uses well-established methodologies to measure diffusion in cells<br /> The work provides an extensive dataset, covering a range of chemistries that are common in small molecule drug design<br /> The authors consider several explanations as to the origin of changes in cellular diffusion

Weaknesses:

The results are described qualitatively, despite quantitative data that can be used to infer the strength of the proposed correlations.<br /> The statistical treatment of the data is not rigorous and not visualized according to best practices, making it difficult for readers to assess the significance of the findings.<br /> Some important aspects of drug behavior are not discussed quantitatively, such as the cell-to-cell or subcellular variability in concentration.<br /> It is unclear if the observed behavior of each drug in the cell actually relates to its efficacy - though this is clearly beyond the scope of this specific work.

Reviewer #2 (Public Review):

Summary: Blocking a weak base compound's protonation increased intracellular diffusion and fractional recovery in the cytoplasm, which may improve the intracellular availability and distribution of weakly basic, small molecule drugs and be impactful in future drug development.

Strengths:

1) The intracellular distribution of drugs and the chemical properties that drive their distribution are much needed in the literature. Thus, the idea behind this paper is of relevance.

2) The study used common compounds that were relevant to others.

3) Altering a compound's pKa value and measuring cytosolic diffusion rates certainly is inciteful on how weak base drugs and their relatively high pKa values affect distribution and pharmacokinetics. This particular experiment demonstrated relevance to drug targeting and drug development.

4) The manuscript was fairly well written.

Weaknesses:

1) Small sample sizes. 2 acids and 1 neutral compound vs 6 weak bases (Figure 1).

2) A comparison between the percentage of neutral and weak base drug accumulation in lysosomes would have helped indicate weak base ion trapping. Such a comparison would have strengthened this study.

3) When cytosolic diffusion rates of compounds were measured, were the lysosomes extracted from the image using Imaris to determine a realistic cytosolic value? In real-time, lysosomes move through the cytosol at different rates. Because weak base drugs get trapped, it is likely the movement of a weak base in the lysosome being measured rather than the movement of a weak base itself throughout the cytosol. This was unclear in the methods. Please explain.

4) Because weak base drugs can be protonated in the cytoplasm, the authors need to elaborate on why they thought that inhibiting lysosome accumulation of weak bases would increase cytosolic diffusion rates. Ion trapping is different than "micrometers per second" in the cytosol. Moreover, treating cells with sodium azide de-acidifies lysosomes and acidifies the cytosol; thus, more protons in the cytosol means more protonation of weak base drugs. The diffusion rates were slowed down in the presence of lysosome inhibition (Figure 7), which is more fitting of the story about blocking protonation increases diffusion rates, but in this case, increasing cytosolic protonation via lysosome de-acidification agents decreases diffusion rates. Please elaborate.

A discussion of the likely impact:<br /> The manuscript certainly adds another dimension to the field of intracellular drug distribution, but the manuscript needs to be strengthened in its current form. Additional experiments need to be included, and there are clarifications in the manuscript that need to be addressed. Once these issues are resolved, then the manuscript, if the conclusions are further strengthened, is much needed and would be inciteful to drug development.

Reviewer #2 (Public Review):

In the manuscript by Maio et al, the authors examined the bioenergetic mechanisms involved in the delayed migration of DC's during Mtb infection. The authors performed a series of in vitro infection experiments including bioenergetic experiments using the Agilent Seahorse XF, and glucose uptake and lactate production experiments. Also, data from SCENITH is included in the revised manuscript as well as some clinical data. This is a well written manuscript and addresses an important question in the TB field.

Reviewer #1 (Public Review):

Summary

The authors use an elegant but somewhat artificial heterodimerisation approach to activate the isolated cytoplasmic domains of different receptor kinases (RKs) including the receptor kinase BRI1 and EFR. The developmental RK BRI1 is known to be activated by the co-receptor BAK1. Active BRI1 is then able to phosphorylate downstream substrates. The immune receptor EFR is also an active protein kinase also activated by the co-receptor BAK1. EFR however appears to have little or no kinase activity but seems to use an allosteric mechanism to in turn enable BAK1 to phosphorylate the substrate kinase BIK1. EFR tyrosine phosphorylation by BAK1 appears to trigger a conformational change in EFR, activating the receptor. Likewise, kinase activating mutations can cause similar conformational transitions in EFR and also in BAK1 in vitro and in planta.

Strengths:

I particularly liked The HDX experiments coupled with mutational analysis (Fig. 2) and the design and testing of the kinase activating mutations (Fig. 3), as they provide novel mechanistic insights into the activation mechanisms of EFR and of BAK1. These findings are nicely extended by the large-scale identification of EFR-related RKs from different species with potentially similar activation mechanisms (Fig. 5).

Overall this is an interesting study that aims to advance our understanding of the activation mechanisms of different plant receptor kinases with important functions in plant immunity.

Reviewer #2 (Public Review):

Summary:

Transmembrane signaling in plants is crucial for homeostasis. In this study, the authors set out to understand to what extent catalytic activity in the EFR tyrosine kinase is required in order to transmit a signal. This work was driven by mounting data that suggest many eukaryotic kinases do not rely on catalysis for signal transduction, relying instead on conformational switching to relay information. The crucial findings reported here involve the realisation that a kinase-inactive EFR can still activate (ie lead to downstream phosphorylation) of its partner protein BAK1. Using a convincing set of biochemical, mass spectrometric (HD-exchange) and in vivo assays, the team suggests a model in which EFR is likely phosphorylated in the canonical activation segment (where two Ser residues are present), which is sufficient to generate a conformation that can activate BAK1 through dimersation. A model is put forward involving C-helix positioning in BAK1, and the model is extended to other 'non-RD' kinases in Arabidopsis kinases that likely do not require kinase activity for signaling.

Strengths:

The work uses logical and well-controlled approaches throughout, and is clear and convincing in most areas, linking data from IPs, kinase assays (including clear 32P-based biochemistry), HD-MX data (from non-phosphorylated EFR) structural biology, oxidative burst data and infectivity assays. Repetitions and statistical analysis all appear appropriate.

Overall, the work builds a convincing story and the discussion does a clear job of explaining the potential impact of these findings (and perhaps an explanation of why so many Arabidopsis kinases are 'pseudokinases', including XPS1 and XIIa6, where this is shown explicitly).

Impact:

The work is an important new step in the huge amount of follow-up work needed to examine how kinases and pseudokinases 'talk' to each other in (especially) the plant kingdom, where significant genetic expansions have occurred. The broader impact is that we might understand better how to manipulate signaling for the benefit of plants and mankind; as the authors suggest, their study is a natural progression both of their own work and the kingdom-wide study of the Kannan group.

Reviewer #3 (Public Review):

The study presents strong evidence for allosteric activation of plant receptor kinases, which enhances our understanding of the non-catalytic mechanisms employed by this large family of receptors.

Plant receptor kinases (RKs) play a critical role in transducing extracellular signals. The activation of RKs involves homo- or heterodimerization of the RKs, and it is believed that mutual phosphorylation of their intracellular kinase domains initiates downstream signaling. However, this model faces a challenge in cases where the kinase domain exhibits pseudokinase characteristics. In their recent study, Mühlenbeck et al. reveal the non-catalytic activation mechanisms of the EFR-BAK1 complex in plant receptor kinase signaling. Specifically, they aimed to determine that the EFR kinase domain activates BAK1 not through its kinase activity, but rather by utilizing a "conformational toggle" mechanism to enter an active-like state, enabling allosteric trans-activation of BAK1. The study sought to elucidate the structural elements and mutations of EFR that affect this conformational switch, as well as explore the implications for immune signaling in plants. To investigate the activation mechanisms of the EFR-BAK1 complex, the research team employed a combination of mutational analysis, structural studies, and hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis. For instance, through HDX-MS analysis, Mühlenbeck et al. discovered that the EFR (Y836F) mutation impairs the accessibility of the active-like conformation. On the other hand, they identified the EFR (F761H) mutation as a potent intragenic suppressor capable of stabilizing the active-like conformation, highlighting the pivotal role of allosteric regulation in BAK1 kinase activation. The data obtained from this methodology strengthens their major conclusion. Moreover, the researchers propose that the allosteric activation mechanism may extend beyond the EFR-BAK1 complex, as it may also be partially conserved in the Arabidopsis LRR-RK XIIa kinases. This suggests a broader role for non-catalytic mechanisms in plant RK signaling.

The allosteric activation mechanism was demonstrated for receptor tyrosine kinases (RTKs) many years ago. A similar mechanism has been suggested for the activation of plant RKs, but experimental evidence for this conclusion is lacking. Data in this study represent a significant advancement in our understanding of non-catalytic mechanisms in plant RK signaling. By shedding light on the allosteric regulation of BAK1, the study provides a new paradigm for future research in this area.

Reviewer #1 (Public Review):

Summary:

The manuscript by Jiayun Li and colleagues aims to provide insight into adipokinetic hormone signaling that mediates the fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Using FISH, qRT-PCR, WB, RNAi, and miRNA-related methods, authors found that knockdown of DcAKH and DcAKHR not only resulted in triacylglycerol accumulation and a decline of glycogen but also significantly decreased fecundity and CLas titer in ovaries. miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes.

Reviewer #2 (Public Review):

Diaphorina citri is the primary vector of Candidatus Liberibacter asiaticus (CLas), but the mechanism of how D. citri maintains a balance between lipid metabolism and increased fecundity after infection with CLas remains unknown. In their study, Li et al. presented convincing methodology and data to demonstrate that CLas exploits AKH/AKHR-miR-34-JH signaling to enhance D. citri lipid metabolism and fecundity, while simultaneously promoting CLas replication. These findings are both novel and valuable, not only have theoretical implications for expanding our understanding of the interaction between insect vectors and pathogenic microorganisms but also provide new targets for controlling D. citri and HLB in practical implications. The conclusions of this paper are well supported by data.

Reviewer #1 (Public Review):

Here, using an organoid system, Wong et al aimed to establish new models of hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), with which they wanted to understand how CSF1R-mutaions affect the phenotypes of microglia/macrophages. They found metabolic changes in microglia/macrophages with mutations, which were associated with a proinflammatory phenotype. In general, the authors tackle important issues and provide valuable tools to investigate the underlying mechanisms for HDLS.

Strength:

The authors establish two HDLS patient-derived iPS cells with their isogeneic controls and provide possible mechanistic insights into the disease mechanisms.

Weakness:

It is unclear how nicely the organoid system in this study can recapitulate the condition in patients with HDLS (e.g. reduced microglia density, downregulated expression of P2YR12, pathological alterations).

The authors generated two different models with distinct mutations that produce different readouts in CSF1R-mediated cellular responses. It is unclear if the different outcomes between HD1 and HD2 are generated simply through different mutations or due to different differentiation efficiency from iMacs.

Suggestions:

(1) This paper would benefit from additional histological analyses to characterize iMac & iMicro at least histologically, which would be helpful for readers to know how nicely the organoid system recapitulates the condition in patients with HDLS.

(2) In addition, in Fig.5E-J the authors could highlight microglia core genes that would be upregulated if iMacs are successfully differentiated into iMicro.

(3) Since there are no direct evidence to confirm that "microglial dysregulation and IL1b signalling contribute to the degenerative neuro-environment in HDLS", the authors should tone down their argument and rephrase the Abstract.

Reviewer #2 (Public Review):

Summary:

This paper investigates a rare and severe brain disease called Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids (HDLS). The authors aimed to understand how mutations in the gene CSF-1R affect microglia, the resident immune cells in the brain, and which alterations and factors lead to the specific pathophysiology. To model the human brain with the pathophysiology of HDLS, they used the human-specific model system of induced pluripotent stem cell (iPSC)-derived forebrain organoids with integrated iPSC-derived microglia (iMicro) from patients with the HDLS-causing mutation and an isogenic cell line with the corrected genome. They found that iPSC-derived macrophages (iMac) with HDLS mutations showed changes in their response, including increased inflammation and altered metabolism. Additionally, they studied these iMacs in forebrain organoids, where they differentiate into iMicro, and showed transcriptional differences in isolated iMicro when carrying the HDLS mutation. In addition, the authors described the influence of the mutation within iMicro on the transcriptional level of neurons and neural progenitor cells (NPCs) in the organoid. They observed that the one mutation showed implications for impaired development of neurons, possibly contributing to the progression of the disease. Overall, this study provides valuable insights into the mechanisms underlying HDLS and emphasizes the importance of studying diseases like these with a suitable model system. These findings, while promising, represent only an initial step towards understanding HDLS and similar neurodegenerative diseases, and thus, their direct translation into new treatment options remains uncertain.

Strengths:

The strength of the work lies in the successful reprogramming of two HDLS patient-derived induced pluripotent stem cells (iPSCs) with different mutations, which is crucial for the study of HDLS using human forebrain organoid models. The use of corrected isogenic iPSC lines as controls increases the validity of the mutation-specific observations. In addition, the model effectively mimics HDLS, particularly concerning deficits in the frontal lobe, mirroring observations in the human brain. Obtaining iPSCs from patients with different CSF1R mutations is particularly valuable given the limitations of rodent and zebrafish models when studying adult-onset neurodegenerative diseases. The study also highlights significant metabolic changes associated with the CSF1R mutation, particularly in the HD2 mutant line, which is confirmed by the HD1 line. In addition, the work shows transcriptional upregulation of the proinflammatory cytokine IL-1beta in cells carrying the mutation, particularly when they phagocytose apoptotic cells, providing further insight into disease mechanisms.

Weaknesses:

Most of the points have been addressed in the revision, but some points remain (see below) and are well within the scope of the current manuscript in this reviewer's opinion.

(1) The characterization of iMicros is incomplete, with limited protein-level analysis (e.g. validate RNA-seq data via flow cytometry, ELISA etc.).

(2) Additionally, the claim of microglial-like morphology lacks adequate evidence, as the provided image is insufficient for such an assessment (also the newly provided Supp. Fig. 3C is insufficient and looks rather like background). Show single channels for each staining. Show examples for both cell lines.

(3) RNA-seq experiments are still difficult to read. A combination of data from both lines into one big analysis would be advantageous. E.g. showing overlapping GO terms for both lines. What is common, what is different in both lines?

(4) Statistical test information is missing in the legends.