Reviewer #1 (Public Review):

Summary:<br /> This Research Advance is an extension of this group's prior paper published in 2022 on the conserved roles of the Hippo pathway effector Yorkie in C. owczarzaki (PMID: 35659869). This species is an amoeba that holds an important phylogenetic position as a close relative of multicellular animals. The prior study used genome editing to delete the C. owczarzaki Yki (termed coYki) and found that Yki is not required for proliferation but instead regulates cell contractility and cell aggregation. In the current study, the authors wanted to address whether Hippo pathway kinases - coHippo (coHpo) and coWarts (coWts) - regulate coYki and whether they are dispensable for proliferation but instead regulate cell contractility and cell aggregation. They used genome editing to delete coHpo and coWts singly in C. owczarzaki. Both mutant strains had increased nuclear location of transfected coYki (tagged with Scarlet), suggesting that Hippo kinase pathway regulation of Yki is conserved in this organism. Neither kinase is required for proliferation. Either kinase mutant strain had a significantly increased percentage of cells that were elongated, which was relatively rare in a control population. The incident of elongation could be enhanced in both kinase-mutant and in control cells when myosin inhibitors were added to the media. coHpo and coWts-mutant aggregates were more tightly packed than control cell aggregates, which they hypothesize is due to the increased contractility seen in kinase-mutant cells. They could reduce the density of packing in kinase-mutant aggregates when they treated the cells with myosin or F-actin inhibitors. To test whether the effects observed in kinase-mutant strains were due to increased Yki activation, they generated a coYki with four S to A substitutions (termed coYki4SA), which should produce a dominant-active Yki impervious to phosphorylation and hence inactivation by Hippo kinases. Control C. owczarzaki cells transfected with coYki4SA had increased cell density in aggregates and elongation in adherent cells. These results support their conclusions that coHpo and coWts regulate cell contractility and cell packing through coYki.

Strengths:<br /> The major strengths of the paper include high quality data, robust analyses of the data, and a well-written manuscript. The combination of genome editing in C. owczarzaki, transfection of C. owczarzaki, and time-lapse movies of adherent cells generally support the conclusions (1) that control of cell density is an ancient function of the Hippo pathway; (2) that Hippo pathway control of cytoskeletal properties and contractile behavior underlie its regulation of cell density, and (3) that Hippo kinase control of Yki localization is likely an ancient function of the pathway.

Weaknesses:<br /> There are only minor weaknesses. (1) Fig. 3C needs the "still" for the movie of control C. owczarzaki (in Movie S1). (2) The elongated cell shape is seen infrequently in control cells, and I wonder whether these events are transient inactivation of coHpo or coWts in these cells. Perhaps the authors could comment on this in the discussion. (3) Does C. owczarzaki normally aggregate or this is a lab-specific phenotype? For example, the slime mold Dictyostelium discoideum forms aggregates during its life cycle. Could some additional information about C. owczarzaki be added to the introduction?

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to provide information about the likely function of uncharacterised genes in fission yeast. The authors highlight the bias in the literature to well-studied genes/proteins and the fact that the functions of many proteins that are conserved from yeast to humans remain unknown. Initial functional characterisation could provide the impetus for researchers to dedicate time and resources to detailed investigations of protein function. The authors subject the fission yeast deletion set to a battery of perturbations (drug treatments etc) and measured the resultant colony size. In total, 131 conditions were analysed for nearly 3,500 mutants, representing a rich dataset. Clustering analysis was then used to identify common phenotype patterns and thereby infer protein functions using a "guilt by association approach. To assign potential GO terms to uncharacterised proteins, the authors developed a new computational approach (NET-FF) which combined two previous approaches, which they validated against curated annotations on the S. pombe database Pombase. Finally, the authors chose a group of genes which their analysis predicted to be involved in cellular ageing for experimental validation, cross-validating a priority unstudied novel gene (SPAC23C4.09c) to be involved in this process. Overall, the functional analysis performed in this manuscript is rigorous, thorough and incorporates some novel approaches leading to new insights and predicted protein functions. It will be an important resource for the fission yeast community.

Reviewer #1 (Public Review):

Summary:

In this paper, Jeong et al. investigate the prevalence and cause of TADs that are preserved in eukaryotic cells after cohesin depletion. The authors perform an extensive analysis of previously published Hi-C data, and find that roughly 15% of TADs are preserved in both mouse liver cells and in HCT-116 cells. They confirm previous findings that epigenetic mismatches across the boundaries of TADs can cause TAD preservation. However, the authors also find that not all preserved TADs can be explained this way. Jeong et al. provide an argument based on polymer simulations that "physical boundaries" in 3D structures provide an additional mechanism that can lead to TAD preservation. However, in its current form, we do not find the argumentation and evidence that leads to this claim to be fully compelling.

Strengths:

We appreciate the extensive statistical analysis performed by the authors on the extent to which TAD's are preserved; this does seem like a novel and valuable contribution to the field.

Weaknesses:

1. As the authors briefly note, the fact that compartmentalization due to epigenetic mismatches can cause TAD-like structures upon cohesin depletion has already been discussed in the literature; see for example Extended Data Figure 8 in (Schwarzer et al., 2017) or the simulation study (Nuebler et al., 2018). We are hence left with the impression that the novelty of this finding is somewhat overstated in this manuscript.<br /> 2. It is not quite clear what the authors conceptually mean by "physical boundaries" and how this could offer additional insight into preserved TADs. First, the authors use the CCM model to show that TAD boundaries correlate with peaks in the single cell boundary probability distribution of the model. This finding is consistent with previous reports that TAD-like structures are present in single cells, and that specific TAD boundaries only arise as a population average. The finding based on the CCM simulations hence seems to be that preserved TADs also arise as a population average in cohesin-depleted cells, but we do not follow what the term "physical boundaries" refers to in this context. The authors then use the Hi-C data to infer a maximum-entropy-based HIPPS model. They find that preserved TADs often have boundaries that correspond to peaks in the single cell boundary probabilities of the inferred model. The authors seem to imply that these peaks in the boundary probability correspond to "physical boundaries" that cause the preservation of TADs. This argument seems circular; the model is based on inferring interaction strengths between monomers, such that the model recreates the input Hi-C map. This means that the ensemble average of the model should have a TAD boundary where one is present in the input Hi-C data. A TAD boundary in the Hi-C data would then seem to imply a peak in the model's single-cell boundary probability. (The authors do display two examples where this is not the case in Fig.3h, but looking at these cases by eye, they do not seem to correspond to strong TAD boundaries.) "Physical boundaries" in the model are hence a consequence of the preserved TADs, rather than the other way around, as the authors seem to suggest. At the very least the boundary probability in the HIPPS model is not an independent statistic from the Hi-C map (on which their model is constrained), so we have concerns about using the physical boundaries idea to understand where some of the preserved TADs come from.

References:<br /> Nuebler, J., Fudenberg, G., Imakaev, M., Abdennur, N., & Mirny, L. A. (2018). Chromatin organization by an interplay of loop extrusion and compartmental segregation. Proceedings of the National Academy of Sciences of the United States of America, 115(29), E6697-E6706. https://doi.org/10.1073/PNAS.1717730115/SUPPL_FILE/PNAS.1717730115.SAPP.PDF

Schwarzer, W., Abdennur, N., Goloborodko, A., Pekowska, A., Fudenberg, G., Loe-Mie, Y., Fonseca, N. A., Huber, W., Haering, C. H., Mirny, L., & Spitz, F. (2017). Two independent modes of chromatin organization revealed by cohesin removal. Nature 2017 551:7678, 551(7678), 51-56. https://doi.org/10.1038/nature24281

Joint Public Review:

This paper by Castello-Serrano et al. addresses the role of lipid rafts in trafficking in the secretory pathway. By performing carefully controlled experiments with synthetic membrane proteins derived from the transmembrane region of LAT, the authors describe, model and quantify the importance of transmembrane domains in the kinetics of trafficking of a protein through the cell. Their data suggest affinity for ordered domains influences the kinetics of exit from the Golgi. Additional microscopy data suggest that lipid-driven partitioning might segregate Golgi membranes into domains. However, the relationship between the partitioning of the synthetic membrane proteins into ordered domains visualised ex vivo in GPMVs, and the domains in the TGN, remain at best correlative. Additional experiments that relate to the existence and nature of domains at the TGN are necessary to provide a direct connection between the phase partitioning capability of the transmembrane regions of membrane proteins and the sorting potential of this phenomenon.

The authors have used the RUSH system to study the traffic of model secretory proteins containing single-pass transmembrane domains that confer defined affinities for liquid ordered (lo) phases in Giant Plasma Membrane derived Vesicles (GPMVs), out of the ER and Golgi. A native protein termed LAT partitioned into these lo-domains, unlike a synthetic model protein termed LAT-allL, which had a substituted transmembrane domain. The authors experiments provide support for the idea that ER exit relies on motifs in the cytosolic tails, but that accelerated Golgi exit is correlated with lo domain partitioning.

Additional experiments provided evidence for segregation of Golgi membranes into coexisting lipid-driven domains that potentially concentrate different proteins. Their inference is that lipid rafts play an important role in Golgi exit. While this is an attractive idea, the experiments described in this manuscript do not provide a convincing argument one way or the other. It does however revive the discussion about the relationship between the potential for phase partitioning and its influence on membrane traffic.

Our detailed comments are listed below:

ER exit:<br /> The experiments conducted to identify an ER exit motif in the C-terminal domain of LAT are straightforward and convincing. This is also consistent with available literature. The authors should comment on whether the conservation of the putative COPII association motif (detailed in Fig. 2A) is significantly higher than that of other parts of the C-terminal domain. One cause of concern is that addition of a short cytoplasmic domain from LAT is sufficient to drive ER exit, and in its absence the synthetic constructs are all very slow. However, the argument presented that specific lo phase partitioning behaviour of the TMDs do not have a significant effect on exit from the ER is a little confusing. This is related to the choice of the allL-TMD as the 'non-lo domain' partitioning comparator. Previous data has shown that longer TMDs (23+) promote ER export (eg. Munro 91, Munro 95, Sharpe 2005). The mechanism for this is not, to my knowledge, known. One could postulate that it has something to do with the very subject of this manuscript- lipid phase partitioning. If this is the case, then a TMD length of 22 might be a poor choice of comparison. A TMD 17 Ls' long would be a more appropriate 'non-raft' cargo. It would be interesting to see a couple of experiments with a cargo like this.

Golgi exit:<br /> For the LAT constructs, the kinetics of Golgi exit as shown in Fig. 3B are surprisingly slow. About half of the protein remains in the Golgi at 1 h after biotin addition. Most secretory cargo proteins would have almost completely exited the Golgi by that time, as illustrated by VSVG in Fig. S3. There is a concern that LAT may have some tendency to linger in the Golgi, presumably due to a factor independent of the transmembrane domain, and therefore cannot be viewed as a good model protein. For kinetic modeling in particular, the existence of such an additional factor would be far from ideal. A valuable control would be to examine the Golgi exit kinetics of at least one additional secretory cargo.

Comments about the trafficking model<br /> 1. In Figure 1E, the export of LAT-TMD from the ER is fitted to a single-exponential fit that the authors say is "well described". This is unclear and there is perhaps something more complex going on. It appears that there is an initial lag phase and then similar kinetics after that - perhaps the authors can comment on this?

2. The model for Golgi sorting is also complicated and controversial, and while the authors' intention to not over-interpreting their data in this regard must be respected, this data is in support of the two-phase Golgi export model (Patterson et al PMID:18555781). Furthermore contrary to the statement in lines 200-202, the kinetics of VSVG exit from the Golgi (Fig. S3) are roughly linear and so are NOT consistent with the previous report by Hirschberg et al. Moreover, the kinetics of LAT export from the Golgi (Fig. 3B) appear quite different, more closely approximating exponential decay of the signal. These points should be described accurately and discussed.

Relationship between membrane traffic and domain partitioning:<br /> 1. Phase segregation in the GPMV is dictated by thermodynamics given its composition and the measurement temperature (at low temperatures 4degC). However at physiological temperatures (32-37degC) at which membrane trafficking is taking place these GPMVs are not phase separated. Hence it is difficult to argue that a sorting mechanism based solely on the partitioning of the synthetic LAT-TMD constructs into lo domains detected at low temperatures in GPMVs provide a basis (or its lack) for the differential kinetics of traffic of out of the Golgi (or ER). The mechanism in a living cell to form any lipid based sorting platforms naturally requires further elaboration, and by definition cannot resemble the lo domains generated in GPMVs at low temperatures.

2. The lipid compositions of each of these membranes - PM, ER and Golgi are drastically different. Each is likely to phase separate at different phase transition temperatures (if at all). The transition temperature is probably even lower for Golgi and the ER membranes compared to the PM. Hence, if the reported compositions of these compartments are to be taken at face value, the propensity to form phase separated domains at a physiological temperature will be very low. Are ordered domains even formed at the Golgi at physiological temperatures?

3. The hypothesis of 'lipid rafts' is a very specific idea, related to functional segregation, and the underlying basis for domain formation has been also hotly debated. In this article the authors conflate thermodynamic phase separation mechanisms with the potential formation of functional sorting domains, further adding to the confusion in the literature. To conclude that this segregation is indeed based on lipid environments of varying degrees of lipid order, it would probably be best to look at the heterogeneity of the various membranes directly using probes designed to measure lipid packing, and then look for colocalization of domains of different cargo with these domains.

4. In the super-resolution experiments (by SIM- where the enhancement of resolution is around two fold or less compared to optical), the authors are able to discern a segregation of the two types of Golgi-resident cargo that have different preferences for the lo-domains in GPMVs. It should be noted that TMD-allL and the LATallL end up in the late endosome after exit of the Golgi. Previous work from the Bonafacino laboratory (PMID: 28978644) has shown that proteins (such as M6PR) destined to go to the late endosome bud from a different part of the Golgi in vesicular carriers, while those that are destined for the cell surface first (including TfR) bud with tubular vesicular carriers. Thus at the resolution depicted in Fig 5, the segregation seen by the authors could be due to an alternative explanation, that these molecules are present in different areas of the Golgi for reasons different from phase partitioning. The relatively high colocalization of TfR with the GPI probe in Fig 5E is consistent with this explanation. TfR and GPI prefer different domains in the GPMV assays yet they show a high degree of colocalization and also traffic to the cell surface.

Reviewer #1 (Public Review):

Summary:<br /> This important study from Godneeva et al. establishes a Drosophila model system for understanding how the activity of Tif1 proteins is modified by SUMO. The authors nicely show that Bonus, like homologous mammalian Tif1 proteins, is a repressor, and that it interacts with other co-repressors Mi-2/NuRD and setdb1 in Drosophila ovaries and S2 cells. They also show that Bonus is SUMOylated by Su(var)2-10 on at least one lysine at its N-terminus to promote its interaction with setdb1. By combining nice biochemistry with an elegant reporter gene approach, they show that SUMOylation is important for Bonus interaction with setdb1, and that this SUMO-dependent interaction triggers high levels of H3K9me3 deposition and gene silencing. While there are still major questions of how SUMO molecularly promotes this process, this study is a valuable first step that opens the door for interesting future experimentation.

Major Point:<br /> The RNAseq and ChIPseq data is not available. This is critical for the review of the paper and would help the readers and reviewers interpret the Bonus mutant phenotype and its mechanism of repressing genes.

1) The author's conclusion that Bonus SUMOylation is "essential for its chromatin localization" is not supported by the data. Figure 5F shows less 3KR mutant in the chromatin fraction but there is still significant signal.<br /> 2) The author's conclusion that Bonus is SUMOylated at a single site close to its N-terminus is not necessarily true. In several SUMO and Bonus blots throughout the paper (5B, 6C, S4A), there are >2 differentially migrating species that could represent more than one SUMO added to Bonus. While the single K20R mutation eliminates all of these species in Fig 5C, it is possible that K20R SUMOylation is required for additional SUMOylation events on other residues. One way to determine if Bonus is SUMOylated on multiple sites is to add recombinant SUMO protease to the extract and see if multiple higher molecular weight bands collapse into a single migrating species (implying multiple SUMOs) or multiple migrating species (implying something else is altering gel migration).<br /> 3) The authors state that most upregulated genes in BonusGLKD are not highly enriched in H3K9me3. The heatmap in figure 3D is not an ideal presentation of this argument. The authors should show an example of what the signal on a highly enriched gene looks like for comparison. The authors also argue that because most upregulated genes in BonusGLKD are not highly enriched in H3K9me3, they must be indirectly repressed. Another possibility is that bonus-mediated H3K9me3 is only important (and present) during early nurse cell differentiation and is later lost and dispensable during the rapid endocycles. After bonus establishes repression though H3K9me3, it might be maintained through bonus-Mi2/Nurd, something else, or nothing at all. The authors could discuss this possibility or perform H3K9me3 ChIP during cyst formation and early nurse cell differentiation rather than in whole ovaries, which are enriched for later stages.<br /> 4) The BonusGLKD RNAseq analysis is underwhelming. The conclusion that "Bonus represses tissue-specific genes" has limited value. Every gene that is not expressed in ovaries is "tissue-specific." What subset of tissue-specific genes does Bonus repress? What common features do these genes have and how do they compare to other sets of tissue-specific genes, such as those reportedly repressed by setdb1, Polycomb proteins, small ovary, l(3)mbt, and stonewall (among others in female germ cells). Comparing these available data sets could help the authors understand the mechanism of Bonus repression and how BonusGLKD leads to sterility. The authors could also further analyze the differences between nos-Gal4 and MT-Gal4 to better understand why nos- but not MT-driven knockdown is sterile.

Main Study Limitations:<br /> 1) It is unclear which genes are directly vs indirectly regulated by bonus, which makes it difficult to understand Bonus's repressive mechanism. Several lines of experiments could help resolve this issue. 1) Bonus ChIPseq, which the authors mentioned was difficult. 2) RNAseq of BonusGLKD rescued with KR3 mutation. This would help separate SUMO/setdb1-dependent regulation from Mi-2 dependent regulation. Similarly, comparing differentially expressed genes in Su(var)2-10GLKD, setdb1GLKD, 3KR rescue, and MI-2 GLKD could identify overlapping targets and help refine how bonus represses subsets of genes through these different corepressors.

2) The paper falls short in discussing how SUMO might promote repression. This is important when considering the conservation (of lack thereof) of SUMOylation sites in Tif1 proteins in distantly related animals. One piece of data that was not discussed is the apparent localization of SUMOylated bonus in the cytoplasmic fraction of the blot in Figure 5F. Su(var)2-10 is mostly a nuclear protein, so is bonus SUMOylated in the nucleus and then exported to the cytoplasm? Also, setdb1 is a nuclear protein, so it is unlikely that the SUMOylated bonus directly interacts with setdb1 on target genes. Together with Fig 5E (unSUMOylatable Bonus aggregates in the nucleus), one could make a model where SUMO solubilizes bonus (perhaps by disassembling aggregates) and indirectly allows it to associate with setdb1 and chromatin. It is also important to note that in Figure 5I, the K3R mutation appears to lessen but not eliminate Bonus interaction with setdb1. This data again disfavors a model where SUMO establishes an interaction interface between setdb1 and Bonus. To determine which form of Bonus interacts with setdb1, the authors could perform a setdb1 pulldown and monitor the SUMOylation state of coIPed Bonus through mobility shift. If mostly unSUMOylated bonus interacts with setdb1, and SUMO indirectly promotes Bonus interaction with setdb1 (perhaps by disassembling Bonus aggregates), then the precise locations of Bonus SUMOylation sites could more easily shift during evolution, disfavoring the author's convergent evolution hypothesis.

Reviewer #1 (Public Review):

In the study by Venkat et al. the authors expand the current knowledge of allosteric diversity in the human kinome by c-terminal splicing variants using as a paradigm DCLK1. In this work, the authors provide evolutionary and some mechanistic evidence about how c-terminal isoform specific variants generated by alternative splicing can regulate catalytic activity by means of coupling specific phosphorylation sites to dynamical and conformational changes controlling active site and substrate pocket occupancy, as well as interfering with protein-protein interacting interfaces that altogether provides evidence of c-terminal isoform specific regulation of the catalytic activity in protein kinases.

The paper is overall well written, the rationale and the fundamental questions are clear and well explained, the evolutionary and MD analyses are very detailed and well explained. Overall I think this is a study that brings some new aspects and concepts that are important for the protein kinase field, in particular the allosteric regulation of the catalytic core by c-terminal segments, and how evolutionary cues generate more sophisticated mechanisms of allosteric control in protein kinases.

Current submission: I have read and gone through the revised manuscript and the rebuttal letter and I confirm that the authors did an excellent job answering all the comments satisfactorily.

Communication Is Symbolic/Arbitrary * symbols, which are marks/objects that represent something else by association, are something that are taught and can be highly subjective depending on multiple aspects. words are visual symbols, and thus can have major forms of differential meanings. Meaning can change from contextual changes, cultural changes, physical body language, etc. Symbols are arbitrary; there is no reason why dog means the large quadrapedal canine creature. You can change the symbol and the true nature would not be affected.

Communication Is Shared Meaning * Symbols many be "meaningless" in a conceptual and disconnected sense, but they carry heavy meaning when individuals have an agreed sense and deliberate identity to it. Culture is a major form of how important group consensus ties to meaning both linguistically and socially. Beyond culture a major part of communication deals with experiences from an abstract intimate sense of self. Even members of the same culture will have different perceptions of an experience, which can make sharing difficult. human interpretation lends to answers objectively "correct" in one persons mind, but false in another. These are connotative definitions vs denotative definitions.

Communication Involves Intentionality

• we are in a constant state of communication, whether intended or not, but verbal communication almost always comes with a premediate sense of conviction and intention. Direct intention towards a shared topic makes communication more effective. Body language, is inversely related in that many times it can be an unconscious decision without intention.

Dimensions of Communication

• there are dimensions, or levels of interpretation during communication. Relational dimension describes how different relationships allow for specific tones, like friendship, cordial business, etc, and using specific tones for wrong situations could be seen as inappropriate. without having a clear understanding of the relationship you have with a person could make the reception of a message clouded or confusing. Content dimension refers to explicit information and the wordage used to convey a certain message.

Communication Is a Process

• communication is ongoing and dynamic, and even if a relationship with an individual start at one level of communication can mean it cant grow to be a "deeper" level where you can communicate more freely. Think of inside jokes.

Communication Is Culturally Determined

• Culture is learned and alters our perceptions of the world and what is considered "normal". Religion is a major aspect of culture that defines morality, values, etc. These perceptions are hard to pinpoint until interacting with someone who doesn't share those values.

Communication Occurs in a Context

• external forces are a major influence in how we interact/commun. yelling out "fire!" at a movie theatre vs yelling that as a lyric at a concert carry two very context dependent results.

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

In this nice study, the authors set out to investigate the role of the canonical circadian gene Clock in the rhythmic biology of the basal metazoan Nematostella vectensis, a sea anemone, which might illuminate the evolution of the Clock gene functionality. To achieve their aims the team generated a Clock knockout mutant line (Clock-/- ) by CRISPR/Cas9 gene deletion and subsequent crossing. They then compared wild-type (WT) with Clock-/- animals for locomotor activity and transcriptomic changes over time in constant darkness (DD) and under light/dark cycles to establish these phenotypes under circadian control and those driven by light cycles. In addition, they used Hybridization Chain Reaction-In situ Hybridization (HCR-ISH) to demonstrate the spatial expression of Clock and a putative circadian clocl-controlled gene Myh7 in whole-mounted juvenile anemones.

The authors demonstrate that under LD both WT and Clock-/- animals were behaviourally rhythmic but under DD the mutants lost this rhythmicity, indicating that Clock is necessary for endogenous rhythms in activity. With altered LD regimes (LD6:6) they show also that Clock is light-dependent. RNAseq comparisons of rhythmic gene expression in WT and Clock-/- animals suggest that clock KO has a profound effect on the rhythmic genome, with very little overlap in rhythmic transcripts between the two phenotypes; of the rhythmic genes in both LD and DD in WT animals (220- termed clock-controlled genes, CCGS) 85% were not rhythmic in Clock-/- animals in either light condition. In silico gene ontology (GO) analysis of CCGS reflected process associated with circadian control. Correspondingly, those genes rhythmic in KO animals under DD (here termed neoCCGs) were not rhythmic in WT, lacked upstream E-box motifs associated with circadian regulation, and did not display any GO enrichment terms. 'Core' circadian genes (as identified in previous literature) in WT and Clock-/- animals were only rhythmic under entrainment (LD) conditions whilst Clock-/- displayed altered expression profiles under LD compared to WT. Comparing CCGs with previous studies of cycling genes in Nematostellar, the authors selected a gene from 16 rhythmic transcripts. One of these, Myh7 was detectable by both RNAseq and HCR-ISH and considered a marker of the circadian clock by the authors.

The authors claim that the study reveals insights into the evolutionary origin of circadian timing; Clock is conserved across distant groups of organisms, having a function as a positive regulator of the transcriptional translational feedback loop at the heart of daily timing, but is not a central element of the core feedback loop circadian system in this basal species. Their behavioural and transcriptomic data largely support the claims that Clock is necessary for endogenous daily activity but that the putative molecular circadian system is not self-sustained under constant darkness (this was known already for WT animals)- rather it is responsive to light cycles with altered dynamics in Clock-/- specimens in some core genes under LD. In the main, I think the authors achieved their aims and the manuscript is a solid piece of important work. The Clock-/- animal is a useful resource for examining time-keeping in a basal metazoan.

The work described builds on other transcriptomic-based works on cnidaria, including Nematostellar, and does probe into the molecular underpinnings with a loss-of-function in a gene known to be core in other circadian systems. The field of chronobiology will benefit from the evolutionary aspect of this work and the fact that it highlights the necessity to study a range of non-model species to get a fuller picture of timing systems to better appreciate the development and diversity of clocks.

Strengths:<br /> The generation of a line of Clock mutant Nematostellar is a very useful tool for the chronobiological community and coupled with a growing suite of tools in this species will be an asset. The experiments seem mostly well conceived and executed (NB see 'weaknesses'). The problem tackled is an interesting one and should be an important contribution to the field.

Weaknesses:<br /> I think the claims about shedding light on the evolutionary origin of circadian time maintenance are a little bold. I agree that the data do point to an alternative role for Clock in this animal in light responsiveness, but this doesn't illuminate the evolution of time-keeping more broadly in my view. In addition, these are transcriptomic data and so should be caveated- they only demonstrate the expression of genes and not physiology beyond that. The time-course analysis is weakened by its low resolution, particularly for the RAIN algorithm when 4-hour intervals constrain the analysis. I accept that only 24h rhythms were selected in the analysis from this but, it might be that detail was lost - I think a preferred option would be 2 or 3-hour resolution or 2 full 24h cycles of analysis.

The authors discount the possibility of the observed 12h rhythmicity in Clock-/- animals by exposing them to LD6:6 cycles before free-running them in DD. I suggest that LD cycles are not a particularly robust way to entrain tidal animals as far as we know. Recent papers show inundation/mechanical agitation are more reliable cues (Kwiatkowski ER, et al. Curr Biol. 2023, 2;33(10):1867-1882.e5. doi: 10.1016/j.cub.2023.03.015; Zhang L., et al Curr Biol. 2013, 23;19, 1863-1873 doi.org/10.1016/j.cub.2013.08.038.) and might be more effective in revealing endogenous 12h rhythms in the absence of 24h cues.

Reviewer #1 (Public Review):

In their manuscript, Laporte et al. analyze the process of formation of the quiescent-cell nuclear microtubule (Q-nMT) bundle, a set of parallel MTs that emanate from the nuclear side of the spindle pole bodies (SPBs) upon the entry of Saccharomyces cerevisiae cells in quiescence. Based on their results, the authors propose that Q-nMT bundle formation is a multistep process that comprises three distinct sequential phases. The authors further evaluate the role of different factors during the growth of the Q-nMT bundle upon quiescence entry, as well as during the disassembly of this structure once cells resume their proliferation.

The Q-nMT is an interesting cellular structure whose physiological function is still widely unknown. Hence, providing new insights into the dynamics of Q-nMT bundle formation and identifying new factors involved in this process is an interesting topic of relevance in the field. The authors made a substantial effort in order to evaluate Q-nMT bundle formation and provide a considerable amount of data, mainly obtained from microscopy analyses. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented.

Despite the interest in the study, there are important issues that could affect the validity of the conclusions drawn in the manuscript. In this way, regarding the analysis of the dynamics of Q-nMT bundle formation, the experimental set up described in some of the experiments raises certain concerns, which mostly derive from the nocodazole treatments and the use of this microtubule-depolymerizing agent as the only approach to evaluate the stability of the Q-nMT bundle. On the other hand, regarding the factors involved in Q-nMT formation, the differences in microtubule length with the wild-type strain, despite being statistically significant, are really subtle for many of the mutants analyzed (e.g., bir1, slk19, etc.). Additionally, there are proteins that are proposed to participate in the process of Q-nMT formation and whose expression during quiescence needs to be demonstrated. Finally, although the cell viability defects observed for some of the mutants in these factors could be certainly associated with the lack of expression or mutation of the specific gene under evaluation, in none of the cases can they be directly attributed to a defect in aberrant Q-nMT bundle formation.

Based on the aforementioned reasons, and despite the considerable effort by the authors, it is my impression that many of the conclusions of the manuscript are not sufficiently justified by the data provided. Additional evidence, including the incorporation of key experimental controls that are currently missing, would be required in order to more strongly support the conclusions of the manuscript.

Reviewer #1 (Public Review):

In their study, Aman et al. utilized single cell transcriptome analysis to investigate wild-type and mutant zebrafish skin tissues during the post-embryonic growth period. They identified new epidermal cell types, such as ameloblasts, and shed light on the effects of TH on skin morphogenesis. Additionally, they revealed the important role of the hypodermis in supporting pigment cells and adult stripe formation. Overall, I find their figures to be of high quality, their analyses to be appropriate and compelling, and their major claims to be well-supported by additional experiments. Therefore, this study will be an important contribution to the field of vertebrate skin research.

Reviewer #1 (Public Review):

Polymorphisms in genes in the human leukocyte antigen (HLA) class II region comprise the most important inherited risk factors for many autoimmune diseases including type 1 diabetes (T1D) and celiac disease (CD). The paper focuses on the novel triad ((SNPs): rs3135394, rs9268645, and rs3129877) finding quite interesting results. The paper suggests further studies at the molecular and structural level to increase our fundamental knowledge of the etiology of autoimmune deceases.

Reviewer #1 (Public Review):

Summary:

Parkinson and colleagues address an interesting and important question, i.e., whether the bumblebee Bombus terrestris can perceive field-realistic concentrations of different pesticides in a sugar solution mimicking nectar. The study directly follows up on a previous study conducted by the same team (Kessler et al. 2015, Nature), which was partly questioned by another more recent study (Arce et al. 2018, Proc. R. Soc. B). The authors apply a combination of electrophysiological measurements and behavioral feeding tests to answer this question. Their results strongly suggest that B. terrestris workers are not able to perceive field-realistic doses of pesticides in a sugar solution. They additionally show that B. terrestris can physiologically differentiate between solutions varying in sugar composition.

Strengths:

Sophisticated methodology, a combination of approaches, clear and precise language

Weaknesses:

Topic and study implications could be discussed more broadly, the statistical approach is not fully clear to me.

Reviewer #1 (Public Review):

This work investigates the function of the PTB domain containing adaptor protein Numb in skeletal muscle structure and function. In particular, the effects of reduced Numb expression in aging muscle is proposed as a mechanism for reduced contractile function associated with sarcopenia. Using ex-vivo analysis of conditional Numb and Numblike knockout muscle the authors demonstrate that loss of Numb but not the related Numblike expression perturbs muscle muscle force generation. In order to explore the molecular mechanisms involved, Numb interacting proteins were identified in C2C12 cell cultured myotubes by immunoprecipitation and LC-MS/MS. The authors identify Septin 7 as a Numb binding protein and demonstrate that loss of Numb/Numblike in myofibers causes changes in Septin 7 subcellular localization. Several questions remain. The authors could provide further clarity on the expression of Numb and Numb-like proteins and the specificity of antibodies used in this study since some Numb antibodies recognize both Numb and Numblike. The authors focus on septin 7 amongst the list of potential Numb interactions identified by AP-MS. Of note, septin 2, 9 and 10 were also identified in the AP-MS experiment. Whether these septins form a complex or are also disrupted by Numb/Numblike loss remains an interesting area for further investigation. Additional investigation of the specificity and mapping of the Numb-Septin 7 (or another Septin) interaction would be of interest and provide an approach for future studies to demonstrate the biological relevance and specificity of the Numb-Septin 7 interaction in skeletal muscle.

Reviewer #1 (Public Review):

Summary:

Pineda et al investigate the association of the hypothesis that Dux4, an embryonic transcription factor, expression in tumor cells is associated with immune evasion and resistance to immunotherapy. They analyze existing cohorts of bulk RNAseq sequenced tumors across cancer types to identify Dux4 expression and association with survival. They find that Dux4 expression is detected in a higher proportion of metastatic tumors compared to primary tumors, is associated with decreased immune infiltrate and a variety of immune metrics and previously nominated immune signatures, and do an in depth evaluation of a cohort of metastatic urothelial cell carcinoma, finding that Dux4 expression is associated with a more immunodeficient tumor microenvironment (desert or excluded microenvironment) and worse survival in this aPDL1 treated cohort. They then find that Dux4 expression is a major independent predictor of survival in this cohort using different types of survival analyses (KM, Cox PH, and random survival forests). With prior existing biological data supporting the hypothesis (in prior work, the senior author has demonstrated Dux4 expression causally suppresses MHC-I expression in interferon-gamma treated cell lines), the current work links Dux4 expression with less immune activity in clinical tumor samples and with survival in ICI treated urothelial carcinomas, and demonstrates that Dux4 expression provides independent information towards survival including other molecular and clinical characteristics (TMB, ECOG PS as the other strongest markers), and provides interesting resolution on landmark analyses with TMB and Dux4 expression providing greater informativeness at later survival landmarks (e.g. 1 year and later), while ECOG PS has strong informativeness already at earlier time points. This work provides impetus towards more mechanistic and functional dissection of the mechanism of Dux4-associated changes with the tumor microenvironment (e.g. in vivo mouse studies) as well as potential interventional studies (e.g. Dux4 as a target in combination therapies). What the work does not provide is additional resolution on the mechanism of how Dux4 may be associated with a more immunodeficient microenvironment.

The conclusions are generally well supported, but there are issues that would benefit from clarification and extension:

- The finding that Dux4 expression is detected in a higher proportion of metastatic tumors and at higher levels compared to primaries (Fig 1BC) is striking. However, at least for one tumor type (melanoma), the "primary" samples are sourced as n=400+ tumors from TCGA, but the TCGA melanoma cohort is comprised of mostly metastatic (n=81 primary and 367 metastatic tumors in the PanCan Atlas), so it is unclear whether this is correctly interpreted. The analysis of tumors with matched FFPE and flash frozen samples with hybrid probe capture and polyA sequencing, respectively is a nice validation to show that the difference in Dux4 expression is not due to differences in preservation of starting material/sequencing in the metastatic samples vs primary samples (S1BC). However, the cited work from which this data arises (D. Robinson et al 2015) is a study of a cohort of prostate cancers with polyA bulk RNAseq sequencing and at least in that work does not seem to have matched FFPE sequencing, making the provenance of this data unclear at a minimum.

- The findings that Dux4 expression in the metastatic urothelial carcinoma setting is associated with a more immunodeficient microenvironment (Figure 2) is clear and unambiguous using multiple lines of data and analyses (bulk RNAseq, DUX4-positive vs DUX4-negative tumors, different immune cell and cytokine signatures; IHC showing an association with immune deserts and immune excluded phenotypes). However, this is an association and does not demonstrate causality.

- The survival analyses (Fig 3,4,5) show fairly convincingly that Dux4 provide independent predictive information beyond clinical variables and TMB towards survival in the aPDL1 treated metastatic urothelial carcinoma cohort, however, there are different choices of Dux4 expression categorization where the rationale is not clearly justified (e.g. Dux4 expression < 0.5 TPM and > 1 TPM in Fig 3, < 0.25 TPM and > 1 TPM in Fig 4 and 5) by either the underlying distribution (e.g. a bimodal distribution) or some type of percentile split.

- The authors demonstrate that adding Dux4 to clinical markers and TMB results in an improved predictive model for survival, but there are a few questions regarding this model as a clinical biomarker<br /> o Is Dux4 expression better than other correlated immune signatures/markers (e.g. interferon gamma, T effector signature, overall immune infiltrate) in providing additional information?<br /> o Since Dux4 expression is categorized to < 0.25 TPM and > 1 TPM, not all patients are included in the model (i.e. between 0.25 TPM and 1 TPM). How many patients this excludes is unclear, and is important to know if this is to be a clinically relevant biomarker.

- The use of random survival forests to quantify the (predictive) marginal effect of Dux4+ vs Dux4- expression on survival in a non-parametric model as well as shed light on association with survival at different landmark times using Shapley values is quite interesting and well conducted.

Reviewer #1 (Public Review):

In this study, the researchers aimed to investigate the cellular landscape and cell-cell interactions in cavernous tissues under diabetic conditions, specifically focusing on erectile dysfunction (ED). They employed single-cell RNA sequencing to analyze gene expression patterns in various cell types within the cavernous tissues of diabetic individuals. The researchers identified decreased expression of genes associated with collagen or extracellular matrix organization and angiogenesis in several cell types, including fibroblasts, chondrocytes, myofibroblasts, valve-related lymphatic endothelial cells, and pericytes. They also discovered a newly identified marker, LBH, that distinguishes pericytes from smooth muscle cells in mouse and human cavernous tissues. Furthermore, the study revealed that pericytes play a role in angiogenesis, adhesion, and migration by communicating with other cell types within the corpus cavernosum. However, these interactions were found to be significantly reduced under diabetic conditions. The study also investigated the role of LBH and its interactions with other proteins (CRYAB and VIM) in maintaining pericyte function and highlighted their potential involvement in regulating neurovascular regeneration. Overall, the manuscript is well-written and the study provides novel insights into the pathogenesis of ED in patients with diabetes and identifies potential therapeutic targets for further investigation.

Joint Public Review:

In this manuscript, the authors proposed an approach to systematically characterise how heterogeneity in a protein signalling network affects its emergent dynamics, with particular emphasis on drug-response signalling dynamics in cancer treatments. They named this approach Meta Dynamic Network (MDN) modelling, as it aims to consider the potential dynamic responses globally, varying both initial conditions (i.e., expression levels) and biophysical parameters (i.e., protein interaction parameters). By characterising the "meta" response of the network, the authors propose that the method can provide insights not only into the possible dynamic behaviours of the system of interest but also into the likelihood and frequency of observing these dynamic behaviours in the natural system.

The authors studied the Early Cell Cycle (ECC) network as a proof of concept, specifically focusing on PI3K, EGFR, and CDK4/6, with particular interest in identifying the mechanisms that cancer could potentially exploit to display drug resistance. The biochemical reaction model consists of 50 equations (state variables) with 94 kinetic parameters, described using SBML and computed in Matlab. Based on the simulations, the authors concluded the following main points: a large number of network states can facilitate resistance, the individual biophysical parameters alone are insufficient to predict resistance, and adaptive resistance is an emergent property of the network. Finally, the authors attempt to validate the model's prediction that differential core sub-networks can drive drug resistance by comparing their observations with the knock-out information available in the literature. The authors identified subnetworks potentially responsible for drug resistance through the inhibition of individual pathways. Importantly, some concerns regarding the methodology are discussed below, putting in doubt the validity of the main claims of this work.

While the authors proposed a potentially useful computational approach to better understand the effect of heterogeneity in a system's dynamic response to a drug treatment (i.e., a perturbation), there are important weaknesses in the manuscript in its current form:

(1) It is unclear how the random parameter sets (i.e., model instances) and initial conditions are generated, and how this choice biases or limits the general conclusions for the case studied. Particularly, it is not evident how the kinetic rates are related to any biological data, nor if the parameter distributions used in this study have any biological relevance.<br /> (2) Related to this problem, it is not clear whether the considered 100,000 random parameter samples sufficiently explore parameter space due to the combinatorial explosion that arises from having 94 free parameters, nor 100,000 random initial conditions for a system with 50 species (variables).<br /> (3) Moreover, the authors filter out all the cases with stiff behaviour. This filtering step appears to select model parameters based on computational convenience, rather than biological plausibility.<br /> (4) Also, it is not clear how exactly the drug effect is incorporated into the model (e.g., molecular inhibition?), nor how it is evaluated in the dynamic simulations (e.g., at the beginning of the simulation?). Moreover, in a complex network, the results may differ depending on whether the inhibition is applied from the start or after the network has reached a stable state.<br /> (5) On the same line, the conclusions need to be discussed in the context of stability, particularly when evaluating the role of initial conditions. As stable steady states are determined by the model parameters, once again, the details of how the perturbation effect is evaluated on the simulation dynamics are critical to interpret the results.<br /> (6) The presented validation of the model results (Fig. 7) is only qualitative, and the interpretation is not carefully discussed in the manuscript, particularly considering the comparison between fold-change responses without specifying the baseline states.

Reviewer #1 (Public Review):

Wang and all present an interesting body of work focused on the effects of high altitude and hypoxia on erythropoiesis, resulting in erythrocytosis. This work is specifically focused on the spleen, targeting splenic macrophages as central cells in this effect. This is logical since these cells are involved in erythrophagocytosis and iron recycling. The results suggest that hypoxia induces splenomegaly with decreased number of splenic macrophages. There is also evidence that ferroptosis is induced in these macrophages, leading to cell destruction. However, additional data demonstrates that RBC clearance is increased, aka shortening the RBC lifespan, calling into question whether splenic function is impaired in hypoxia or whether the spleen enlargement is compensatory, leading to increased erythropoiesis; similarly, increased iron in the spleen provides potential evidence of enhanced erythrophagocytosis with iron release. Many of the reviewers' prior comments are not addressed or only superficially addressed and the additional experimental results and text to the background and discussion sections in the revised manuscript does not increase enthusiasm or clarity. Taken together, there are many issues with the presented results, with somewhat superficial data, with overstated conclusions, decreasing confidence that the hypotheses and observed results are directly causally related to hypoxia in the way that the authors propose.

Reviewer #1 (Public Review):

It is well established that tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a leading cause of mortality and morbidity worldwide. However, the only vaccine licensed against tuberculosis is Bacille Calmette Guerin (BCG), has been around for nearly a century, and has limited efficacy in adults. Herein, the authors sought to investigate the effectiveness of a nanoparticle-based formulation of a subunit vaccine composed of Mtb lipid and protein antigens. The authors found that they were able to load the lipid, mycolic acid, into their nanoparticles without disrupting the architecture and that the loaded particles activated T cells both in vitro and in vivo. Moreover, when they vaccinated with particles loaded with both lipid and protein antigens, they found that the lipid antigen persisted, and mycolic acid-specific T cells were able to be activated 6 weeks post-vaccination, in contrast to peptide-specific T cells. The authors investigated further and found that persistence required the nanoparticle encapsulation, rather than free lipid, and that it was independent of route (intratracheal, intravenous, or subcutaneous) of administration. To address the mechanisms underlying antigen persistence, the authors loaded the nanoparticles with a dye and demonstrated that the nanoparticle encapsulated lipid antigen was primarily stored in lung alveolar macrophages and that CD1b+ dendritic cells presented the antigen to mycolic acid specific T cells. Finally, the authors conducted mixed bone marrow chimera studies to examine the phenotype of the mycolic acid specific T cells and found that the memory T cell population phenotypically resembled T follicular helper, regulatory T cells, and exhausted T cells. Interestingly, while a large percentage of these lipid antigen specific T cells in the lymph nodes, lung and spleen were CXCR5+PD1+, the cells were still proliferating (Ki67+). Overall, this is a comprehensive study that has the potential to significantly enhance the field.

Reviewer #1 (Public Review):

Murphy, Fancy and Skene performed a reanalysis of snRNA-seq data from Alzheimer Disease (AD) patients and healthy controls published previously by Mathys et al. (2019), arriving at the conclusion that many of the transcriptional differences described in the original publication were false positives. This was achieved by revising the strategy for both quality control and differential expression analysis. I believe the authors' intention was to show the results of their reanalysis not as a criticism of the original paper (which can hardly be faulted for their strategy which was state-of-the-art at the time and indeed they took extra measures attempting to ensure the reliability of their results), but primarily to raise awareness and provide recommendations for rigorous analysis of sc/snRNA-seq data for future studies.

STRENGTHS:

The authors demonstrate that the choice of data analysis strategy can have a vast impact on the results of a study, which in itself may not be obvious to many researchers.

The authors apply a pseudobulk-based differential expression analysis strategy (essentially, adding up counts from all cells per individual and comparing those counts with standard RNA-seq differential expression tests), which is (a) in line with latest community recommendations, (b) different from the "default options" in most popular scRNA-seq analysis suites, and (c) explains the vastly different number of DEGs identified by the authors and the original publication. The recommendation of this approach together with a detailed assessment of the DEGs found by both methodologies could be a useful finding for the research community. Unfortunately, it is currently not fully substantiated and is confounded with concurrent changes in QC measures (see weaknesses).

The authors show a correlation between the number of DEGs and the number of cells assessed, which indicates a methodological shortcoming of the original paper's approach (actually, the authors of the original paper already acknowledged that the lesser number of DEGs for rare cell types was a technical artefact). To be educational for the reader it would be important to provide more information about the DEGs that were "found" and those that were "lost". Given vast inter-individual heterogeneity in humans, it is likely that the study was underpowered to find weaker differences using the pseudobulks (Fig. 1B shows that only genes with more than 4-fold change were found "significant").

All code and data used in this study are publicly available to the readers.

WEAKNESSES:

The authors interpret the fact that they found fewer DEGs with their method than the original paper as a good thing by making the assumption that all genes that were not found were false positives. However, they do not prove this, and it is likely that at least some genes were not found due to a lack of statistical power and not because they were actually "incorrect". The original paper also performed independent validations of some genes that were not found here.

I am concerned that the only DEGs found by the authors are in the rare cell types, foremost the rare microglia (see Fig. 1f). It is unclear to me how many cells the pseudo-bulk counts were based on for these cells types, but it seems that (a) there were few and (b) there were quite few reads per cells. If both are the case, the pseudobulk counts for these cell populations might be rather noisy and the DEG results are liable to outliers with extreme fold changes.

The authors claim they improved the quality control of the dataset. While I do not think they did anything wrong per se, the authors offer no objective metric to assess this putative improvement. This is another major weakness of the paper as it confounds the results of the improved (?) differential analysis strategy and dilutes the results. I detail this weakness in the two following points:

Removing low-quality cells: The authors apply a new QC procedure resulting in the removal of some 20k more cells than in the original publication. They state "we believe the authors' quality control (QC) approach did not capture all of these low quality cells" (l. 26). While all the QC metrics used are very sensible, it is unclear whether they are indeed "better". For instance, removal with a mitochondrial count of <5% seems harsh and might account for a large proportion of additional cells filtered out in comparison to the original analysis. There is no blanket "correct cutoff" for this percentage. For instance, the "classic" Seurat tutorial https://satijalab.org/seurat/articles/pbmc3k_tutorial.html uses the 5% threshold chosen by the authors, an MAD-based selection of cutoff arrived at 8% here https://www.sc-best-practices.org/preprocessing_visualization/quality_control.html, another "best practices" guide choses by default 10% https://bioconductor.org/books/3.17/OSCA.basic/quality-control.html#quality-control-discarded, etc. Generally, the % of mitochondrial reads varies a lot between datasets. As far as I can tell, the original paper did not use a fixed threshold but instead used a clustering approach to identify cells with an "abnormally high" mitochondrial read fraction. That also seems reasonable. Overall, I cannot assess whether the new QC is really more appropriate than the original analysis and the authors do not provide any evidence in favor of their strategy.

Batch correction: "Dataset integration has become a standard step in single-cell RNA-Seq protocols" (l. 29). While it is true that many authors now choose to perform an integration step as part of their analysis workflow, this is by no means uncontroversial as there is a risk of "over-integration" and loss of true biological differences. Also, there are many different methods for dataset integration out there, which will all have different results. More importantly, the authors go on "we found different cell type proportions to the authors (Fig. 1a) which could be due to accounting for batch effects" but offer no support for the claim that the batch effects are indeed related to the observed differences. An alternative explanation would be a selective loss/gain of certain cell types during quality control. The original paper stated concerns about losing certain cell types (microglia, which do not seem to be differentially abundant in the original paper / new analysis).

Relevant literature is incompletely cited. Instead of referring to reviews of best practices and benchmarks comparing methods for batch correction and or differential analysis, the authors only refer to their own previous work.

Due to a lack of comparison with other methods and due to the fact that the author's methodology was only applied to a single dataset, the paper presents merely a case study, which could be useful but falls short of providing a general recommendation for a best practice workflow.

APPRAISAL:

The manuscript could help to increase awareness of data analysis choices in the community, but only if the superiority of the methodology was clearly demonstrated. The recommended pseudobulk differential expression approach along with the indication of drastic differences that this might have on the results is the main output of the current manuscript, but it is difficult to assess unequivocally how this influenced the results because the differential analysis comes after QC and cell type annotation, which have also been changed in comparison to the original publication. In my opinion, the purpose of the paper might be better served by focusing on the DE strategy without changing QC and instead detailing where/how DEGs were gained/lost and supporting whether these were false positives.

Reviewer #1 (Public Review):

Summary: Cullinan et al. explore the hypothesis that the cytoplasmic N- and C-termini of ASIC1a, not resolved in x-ray or cryo-EM structures, form a dynamic complex that breaks apart at low pH, exposing a C-terminal binding site for RIPK1, a regulator of necrotic cell death. They expressed channels tagged at their N- and C-termini with the fluorescent, non-canonical amino acid ANAP in CHO cells using amber stop-codon suppression. Interaction between the termini was assessed by FRET between ANAP and colored transition metal ions bound either to a cysteine reactive chelator attached to the channel (TETAC) or metal-chelating lipids (C18-NTA). A key advantage to using metal ions is that they are very poor FRET acceptors, i.e. they must be very close to the donor for FRET to occur. This is ideal for measuring small distances/changes in distance on the scales expected from the initial hypothesis. In order to apply chelated metal ions, CHO cells were mechanically unroofed, providing access to the inner leaflet of the plasma membrane. At high pH, the N- and C- termini are close enough for FRET to be measured, but apparently too far apart to be explained by a direct binding interaction. At low pH, there was an apparent increase in FRET between the termini. FRET between ANAP on the N-and C-termini and metal ions bound to the plasma membrane suggests that both termini move away from the plasma membrane at low pH. The authors propose an alternative hypothesis whereby close association with the plasma membrane precludes RIPK1 binding to the C-terminus of ASIC1a.

Strengths: The findings presented here are certainly valuable for the ion channel/signaling field and the technical approach only increases the significance of the work. The choice of techniques is appropriate for this study and the results are clear and high quality. Sufficient evidence is presented against the starting hypothesis.

Weaknesses: I have a few questions about certain controls and assumptions that I would like to see discussed more explicitly in the manuscript.

--My biggest concern is with the C-terminal citrine tag. Might this prevent the hypothesized interaction between the N- and C-termini? What about the serine to cysteine mutations? The authors might consider a control experiment in channels lacking the C-terminal FP tag.

--Figure 2 supplement 1 shows apparent read-through of the N-terminal stop codons. Given that most of the paper uses N-terminal ANAP tags, this figure should be moved out of the supplement. Do N-terminally truncated subunits form functional channels? Do the authors expect N-terminally truncated subunits to co-assemble in trimers with full-length subunits? The authors should include a more explicit discussion regarding the effect of truncated channels on their FRET signal in the case of such co-assembly.

--As the epitope used for the western blots in Figure 2 and supplements is part of the C-terminal tag, these blots do not provide an estimate of the fraction of C-terminally truncated channels (those that failed to incorporate ANAP at the stop codon). What effect would C-terminally truncated channels have on the FRET signal if incorporated into trimers with full-length subunits?

--Some general discussion of these results in the context of trimeric channels would be helpful. Is the putative interaction of the termini within or between subunits? Are the distances between subunits large enough to preclude FRET between donors on one subunit and acceptor ions bound on multiple subunits?

--The authors conclude that the relatively small amount of FRET between the cytoplasmic termini suggests that the interaction previously modeled in Rosetta is unlikely. Is it possible that the proposed structure is correct, but labile? For example, could it be that the FRET signal is the time average of a state in which the termini directly interact (as in the Rosetta model) and one in which they do not?

Reviewer #1 (Public Review):

In this manuscript, Nagel et al. sought to comprehensively characterize the composition of urinary compounds, some of which are putative chemosignals. They used urines from adult males and females in three different strains, including one wild-derived strain. By performing mass spectrometry of two classes of compounds: volatile organic compounds and proteins, they found that urines from inbred strains are qualitatively similar to those of a wild strain. This finding is significant because there is a high degree of genetic diversity in wild mice, with chemosensory receptor genes harboring many polymorphisms.

In the second part of this work, the authors used calcium imaging to monitor the pattern of vomeronasal neuron responses to these urines. By performing pairwise comparisons, the authors found a large degree of strain-specific response and a relatively minor response to sex-specific urinary stimuli. This is a finding generally in agreement with previous calcium imaging work by Ron Yu and colleagues in 2008. The authors extend the previous work by using urines from wild mice. They further report that the concentration diversity of urinary compounds in different urine batches is largely uncorrelated with the activity profiles of these urines. In addition, the authors found that the patterns of vomeronasal neuron response to urinary cues are not identical when measured using different recipient strains. This fascinating finding, however, requires an additional control to exclude the possibility that this is not due to sampling error.

There are several weaknesses in this manuscript, including the lack of analysis of the compositions of sulfated steroids and other steroids, which have been proposed to be the major constituents of vomeronasal ligands in urines and the indirect (correlational) nature of their mass spectrometry data and activity data.

Overall, the major contribution of this work is the identification of specific molecules in mouse urines. This work is likely to be of significant interest to researchers in chemosensory signaling in mammals and provides a systematic avenue to exhaustively identify vomeronasal ligands in the future.

Reviewer #1 (Public Review):

In this study, the authors build upon previous research that utilized non-invasive EEG and MEG by analyzing intracranial human ECoG data with high spatial resolution. They employed a receptive field mapping task to infer the retinotopic organization of the human visual system. The results present compelling evidence that the spatial distribution of human alpha oscillations is highly specific and functionally relevant, as it provides information about the position of a stimulus within the visual field.

Using state-of-the-art modeling approaches, the authors not only strengthen the existing evidence for the spatial specificity of the human dominant rhythm but also provide new quantification of its functional utility, specifically in terms of the size of the receptive field relative to the one estimated based on broad band activity.

The present manuscript currently omits the complementary view that the retinotopic map of the visual system might be related to eye movement control. Previous research in non-human primates using microelectrode stimulation has clearly shown that neuronal circuits in the visual system possess motor properties (e.g. Schiller and Styker 1972, Schiller and Tehovnik 2001). More recent work utilizing Utah arrays, receptive field mapping, and electrical stimulation further supports this perspective, demonstrating that the retinotopic map functions as a motor map. In other words, neurons within a specific area responding to a particular stimulus location also trigger eye movements towards that location when electrically stimulated (e.g. Chen et al. 2020).

Similarly, recent studies in humans have established a link between the retinotopic variation of human alpha oscillations and eye movements (e.g., Quax et al. 2019, Popov et al. 2021, Celli et al. 2022, Liu et al. 2023, Popov et al. 2023). Therefore, it would be valuable to discuss and acknowledge this complementary perspective on the functional relevance of the presented evidence in the discussion section.

Reviewer #1 (Public Review):

This study investigates the impact of recurrent connections on grid fields generated in networks trained by adjusting the strength of feedforward spatial inputs. The main result is that if the recurrent connections in the network are given a 1D continuous attractor architecture, then aligned grid firing patterns emerge in the network following training. Detailed analyses of the low dimensional dynamics of the resulting networks are then presented. The simulations and analyses appear carefully carried out.

The feedforward model investigated by the authors (previously introduced by Kropff & Treves, 2008) is an interesting and important alternative to models that generate grid firing patterns through 2-dimensional continuous attractor network (CAN) dynamics. However, while both classes of model generate grid fields, in making comparisons the manuscript is insufficiently clear about their differences. In particular, in the CAN models grid firing is a direct result of their 2-D architecture, either a torus structure with a single activity bump (e.g. Guanella et al. 2007, Pastoll et al. 2013), or sheet with multiple local activity bumps (Fuhs & Touretzky, Burak & Fiete, 2009). In these models, spatial input can anchor the grid representations but is not necessary for grid firing. By contrast, in the feedforward models neurons transform existing spatial inputs into a grid representation. Thus, the two classes of model implement different computations; CANs path integrate, while the feedforward models transform spatial representations. A demonstration that a 1D CAN generates coordinated 2D grid fields would be surprising and important, but it's less clear why coordination between grids generated by the feedforward mechanism would be surprising. As written, it's unclear which of these claims the study is trying to make. If the former, then the conclusion doesn't appear well supported by the data as presented, if the latter then the results are perhaps not so unexpected, and the imposed attractor dynamics may still not be relevant.

Whichever claim is being made, it could be helpful to more carefully evaluate the model dynamics given predictions expected for the different classes of model. Key questions that are not answered by the manuscript include:

- At what point is the 1D attractor architecture playing a role in the models presented here? Is it important specifically for training or is it also contributing to computation in the fully trained network?

- Is an attractor architecture required at all for emergence of population alignment and gridness? Key controls missing from Figure 2 include training on networks with other architectures. For example, one might consider various architectures with randomly structured connectivity (e.g. drawing weights from exponential or Gaussian distributions).

- In the trained models do the recurrent connections substantially influence activity in the test conditions? Or after training are the 1D dynamics drowned out by feedforward inputs?

- What is the low dimensional structure of the input to the network? Can the apparent discrepancy between dimensionality of architecture and representation be resolved by considering structure of the inputs, e.g. if the input is a 2 dimensional representation of location then is it surprising that the output is too?

- What happens to representations in the trained networks presented when place cells remap? Is the 1D manifold maintained as expected for CAN models, or does it reorganise?

Reviewer #1 (Public Review):

Summary:<br /> Mitochondria is the power plant of the cells including neurons. Thomas et al. characterized the distribution of mitochondria in dendrites and spines of L2/3 neurons from the ferret visual cortex, for which visually driven calcium responses of individual dendritic spines were examined. The authors analyzed the relationship between the position of mitochondria and the morphology or orientation selectivity of nearby dendrite spines. They found no correlation between mitochondrion location and spine morphological parameters associated with the strength of synapses, but correlation with the spine-somatic difference of orientation preference and local heterogeneity in preferred orientation of nearby spines. Moreover, they reported that the spines that have a mitochondrion in the head or neck are larger in size and have stronger orientation selectivity. Therefore, they proposed that "mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs."

Strengths:<br /> This paper attempted to address a fundamental question: whether the distribution of the mitochondria along the dendrites of visual cortical neurons is associated with the functions of the spines, postsynaptic sites of excitatory synapses. Two state of the art techniques (2 photon Ca imaging of somata and spines and EM reconstructions of cortical pyramidal neurons) had been used, which provides a great opportunity to examine and correlate the function of spine ultrastructure and spatial distribution of dendritic mitochondria.

Weaknesses:<br /> Overall, the findings are interesting. However, the study lacks the data providing insights into either the mechanisms or the functional meaning of the pattern of mitochondrion distribution along the dendrites, which restricts the significance of the study. It also suffers from small correlation coefficients and small sample sizes (60-121 spines in 4 neurons) as well as missing some important analysis.

Reviewer #1 (Public Review):

Summary<br /> In this manuscript, Hagihara et al. characterized the relationship between the changes in lactate and pH and the behavioral phenotypes in different animal models of neuropsychiatric disorders at a large-scale level. The authors have previously reported that increased lactate levels and decreased pH are commonly observed in the brains of five genetic mouse models of schizophrenia (SZ), bipolar disorder (BD), and autism spectrum disorder (ASD). In this study, they expanded the detection range to 109 strains or conditions of animal models, covering neuropsychiatric disorders and neurodegenerative disorders. Through statistical analysis of the first 65 strains/conditions of animal models which were set as exploratory cohort, the authors found that most strains showed decreased pH and increased lactate levels in the brains. There was a significant negative correlation between pH and lactate levels both at the strain/condition level and the individual animal level. Besides, only working memory was negatively correlated with brain lactate levels. These results were successfully duplicated by studying the confirmative cohort, including 44 strains/conditions of animal models. In all strains/conditions, the lactate levels were not correlated with age, sex, or storage duration of brain samples.

Strengths<br /> 1. The manuscript is well-written and structured. In particular, the discussion is really nice, covering many potential mechanisms for the altered lactate levels in these disease models.<br /> 2. Tremendous efforts were made to recruit a huge number of various animal models, giving the conclusions sufficient power.

Weaknesses<br /> 1. The biggest concern of this study is the limited novelty. The point of "altered pH and/or lactate levels in the brains from human and rodent animals of neuropsychiatric disorders" has been reported by the same lab and other groups in many previous papers.<br /> 2. This study is mostly descriptive, lacking functional investigations. Although a larger cohort of animal models were studied which makes the conclusion more solid, limited conceptual advance is contributed to the relevant field, as we are still not clear about what the altered levels of pH and lactate mean for the pathogenesis of neuropsychiatric disorders.<br /> 3. The experiment procedure is also a concern. The brains from animal models were acutely collected without cardiac perfusion in this study, which suggests that resident blood may contaminate the brain samples. The lactate is enriched in the blood, making it a potential confounded factor to affect the lactate levels as well as pH in the brain samples.<br /> 4. The lactate and pH levels may also be affected by other confounded factors, such as circadian period, and locomotor activity before the mice were sacrificed. This should also be discussed in the paper.<br /> 5. Another concern is the animal models. Although previous studies have demonstrated that dysfunctions of these genes could cause related phenotypes for certain disorders, many of them are not acknowledged by the field as reliable disease models. Besides, gene deficiency could also cause many known or unknown unrelated phenotypes, which may contribute to the altered levels of lactate and pH, too. In this circumstance, the conclusion "pH and lactate levels are transdiagnostic endophenotype of neuropsychiatric disorders" is somewhat overstated.<br /> 6. The negative correlationship between pH and lactate is rather convincing. However, how much the contribution of lactate to pH is not tested. In addition, regarding pH and lactate, which factor contributes most to the pathogenesis of neuropsychiatric disorders is also unclear. These questions may need to be addressed in the future study.<br /> 7. The authorship is open to question. Most authors listed in this paper may only provide mice strains or brain samples. Maybe it is better just to acknowledge them in the acknowledgments section.<br /> 8. The last concern is about the significance of this study. Although the majority of strains showed increased lactate, some still showed decreased lactate levels in the brains. These results suggested that lactate or pH is an endophenotype for neuropsychiatric disorders, but it is hard to serve as a good diagnostic index as the change is not unidirectional in different disorders. In other words, the relationship between lactate level and neuropsychiatric disorders is not exclusive.

Reviewer #1 (Public Review):

This study by Hormigo et al. examines the relationship between activity in the zona incerta (ZI) and behavior. The authors aim to assess the hypothesis that the ZI might mediate a general behavioral function, namely the distribution of information about ongoing movement to other brain areas that regulate behavior. Given the heterogeneity of prior literature on the ZI, this topic is important and interesting. The study employs a strong diversity of technical approaches, spanning electrophysiological recordings, calcium imaging, optogenetics, virally-mediated cell-type ablation, and several behavioral assays. The output is a large dataset where each experiment is useful and interesting, and together, the results could be interpreted as consistent with the prospect of the ZI mediating a general function. However, there are notable weaknesses in the current version of this paper. First, it is unclear whether the experiments and analyses were set up to be able to rule out more specific candidate functions of the ZI. Second, many important details of the experiments and their results are hard to decipher given the current descriptions and presentations of the data.

The paper could be significantly strengthened by including more details from each experiment, stronger justifications for the limited behaviors and experimental analyses performed, and, finally, a broader analysis of how the recorded activity in the ZI relates to behavioral parameters.

(1) Anatomical specification: The ZI contains many distinct subdivisions--each with its own topographically organized inputs/outputs and putative functions. The current manuscript doesn't reference these known divisions or their behavioral distinctions, and one cannot tell exactly which portion(s) of the ZI was included in the current study.

Moreover, the elongated structure of the ZI makes it very difficult to specifically or completely infect virally. The data could be better interpreted if the paper included basic information on the locations of recordings, the extent of the AAV spread in the ZI in each viral experiment, and what fraction of infected neurons were inside versus outside ZI.

(2) Electrophysiological recording on the treadmill: The authors are commended for this technically very difficult experiment. The authors do not specify, however, how they knew when they were recording in ZI rather than surrounding structures, particularly given that recording site lesions were only performed during the last recording session. A map of the locations of the different classes of units would be valuable data to relate to the literature.

(3) The rationale of the analysis of activity with respect to "movement peak": It is unclear why the authors did not assess how ZI activity correlates with a broad set of movement parameters, but rather grouped heterogeneous behavioral epochs to analyze firing with respect to "movement peaks".

(4) The display of mean categorical data in various figures is interesting, however, the reader cannot gather a very detailed view of ZI firing responses or potential heterogeneity with so little information about their distributions.

(5) Somatosensory firing responses in ZI: It is unclear why the authors chose the specific stimuli used in the study. How often did they evoke reflexive motor responses? What was the latency of sensory-evoked responses in ZI activity and the latency of the reflexive movement?

(6) It would be valuable to see example traces in Figure 3 to get a better sense of the time course and contexts under which Ca signals in ZI tracks movement. What is the typical latency? What is the typical range of magnitudes of responses? Does the Ca signal track both fast and slow movements? How are the authors sure that there are no movement artifacts contributing to the calcium imaging? It seems there is more information in the dataset that could be valuable.

(7) Figure 4: The rationale for quantifying the F/Fo responses over a 6-second window, rather than with respect to discrete movement parameters, is not well explained. What types of movement are binned in this approach and might this broad binning hinder the ability to detect more specific relationships between activity and movement?

(8) Separation of sensory and motor responses in Figure 5: The current data do not adequately differentiate whether the responses are sensory or motor given the high correlation of the sensory inputs driving motor responses. Because isoflurane can diminish auditory responses early in the auditory pathway, this reviewer is not convinced the isoflurane experiments are interpretable.

(9) Given the broad duration of the mean avoidance response (Fig. 6 C, bottom), it would be useful to know to what extent this plot reflects a prolonged behavior or is the result of averaging different animals/trials with different latencies. Given that the shapes of the F/Fo responses in ZI appear similar across avoids and escapes (Fig. 6D), despite their apparent different speeds and movement durations (Fig 6C), it would be valuable to know how the timing of the F/Fo relates to movement on a trial-by-trial basis.

(10) Lesion quantification: One cannot tell what rostral-caudal extent of ZI was lesioned and quantified in this experiment. It would be easier to interpret if also plotted for each animal, so the reader can tell how reliable the method is. The mean ablation would be better shown as a normalized fraction of cells. Although the authors claim the lesions have little impact on behavior, it appears the incompleteness of the lesions could warrant a more conservative interpretation.

(11) Optogenetics: the location of infected neurons is poorly described, including the rostral-caudal extent and the fraction of neurons inside and outside of ZI. Moreover, it is unclear how strongly the optogenetic manipulations in this study are expected to affect neuronal activity in ZI.

Reviewer #1 (Public Review):

In this paper, the interocular/binocular combination of temporal luminance modulations is studied. Binocular combination is of broad interest because it provides a remarkable case study of how the brain combines information from different sources. In addition, the mechanisms of binocular combination are of interest to vision scientists because they provide insight into when/where/how information from two eyes is combined.

This study focuses on how luminance flicker is combined across two eyes, extending previous work that focused mainly on spatial modulations. The results appear to show that temporal modulations are combined in different ways, with additional differences between subcortical and cortical pathways.

The manuscript has been revised to address prior reviewers' comments. It now provides more justification for the empirical choices made by the authors, and a better illustration of the methods. That said, the paper would still benefit from an expanded rationale for significance beyond this specific area. There were no substantive changes made to the abstract or introduction, and only little to the discussion.

Reviewer #1 (Public Review):

In this manuscript Rubin and Aso provide important new tools for the study of learning and memory in Drosophila. In flies, olfactory learning and memory occurs at the Mushroom Body (MB) and is communicated to the rest of the brain through Mushroom Body Output Neurons (MBONs). Previously, typical MBONs were thoroughly studied. Here, atypical MBONs that have dendritic input both within the MB lobes and in adjacent brain regions are studied. The authors describe new cell-type-specific GAL4 drivers for the majority of atypical MBONs (and other MBONs) and using an optogenetic activation screen they examined their ability to drive behaviors and learning.

The experiments in this manuscript were carefully performed and the results are clear. The tools provided in this manuscript are of great importance to the field.

Reviewer #1 (Public Review):

The goal of the authors is to use whole-exome sequencing to identify genomic factors contributing to asthenoteratozoospermia and male infertility. Using whole-exome sequencing, they discovered homozygous ZMYND12 variants in four unrelated patients. They examined the localization of key sperm tail components in sperm from the patients. To validate the findings, they knocked down the ortholog in Trypanosoma brucei. They further dissected the complex using co-immunoprecipitation and comparative proteomics with samples from Trypanosoma and Ttc29 KO mice. They concluded that ZMYND12 is a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.

The major strengths are that the authors used the cutting-edge technique, whole-exome sequencing, to identify genes associated with male infertility, and used a new model organism, Trypanosoma brucei to validate the findings, together with other high-throughput tools, including comparative proteomics to dissect the protein complex essential for normal sperm formation/function. The major weakness is that limited samples could be collected from the patients for further characterization by other approaches, including western blotting and TEM.

In general, the authors achieved their goal, and the conclusion is supported by their results. The findings not only provide another genetic marker for the diagnosis of asthenoteratozoospermia but also enrich the knowledge of cilia/flagella.

Reviewer #1 (Public Review):

It has been shown previously that maternal aging in mice is associated with an increase in accumulation of damaged mitochondria and activation of parkin-mediated autophagy (see DOI: 10.1080/15548627.2021.1946739). It has also been shown that C-natriuretic peptide (CNP) regulates oocyte meiotic arrest and that its use during in vitro oocyte maturation can improve parameters associated with decreased oocyte quality. Here the authors tested whether use of CNP treatment in vivo could improve oocyte quality and fertility of aged mice, for which they provided convincing evidence. They also attempted to determine how CNP improves oocyte developmental competence. They showed a correlation between CNP use in vivo and the appearance (and some functional qualities) of cytoplasmic organelles more closely approximating those of oocytes from young mice. However, this correlation could not be interpreted to imply causation. Additional experiments performed using CNP during in vitro maturation were not properly controlled and so are not possible to interpret.

A strength of the manuscript is that the authors use an in vivo treatment to improve oocyte quality rather than just using CNP during oocyte maturation in vitro as has been done previously. This strategy provides more potential for improving oocyte quality - over the course of oocyte growth and maturation - rather than just the final few hours of maturation alone. This strategy also has the potential to be translated into a more generally useful clinical therapeutic method that using CNP during in vitro maturation. However, it is difficult to glean information regarding how CNP might have its effects in vivo. A range of models are used in the manuscript with a mix of in vivo studies with in vitro experiments, which results in some disconnect between systemic CNP and its reported intrafollicular action as well as in the short-term versus longer-term actions of CNP on oocyte quality. Specifically, CNP was shown to be reduced in the plasma of aged mice, but this was not shown in the granulosa cells, which are the reported source of CNP that acts on oocytes. Whether the ovarian source of CNP is reduced in aged females was not demonstrated, and CNP is not known to act on oocytes through an endocrine effect. In vivo treatments with CNP by i.p. injection were performed, but the dose (120 ug/kg) and time (14 days) of treatment were not validated by any prior experiments to give them physiological relevance.

Weaknesses:

1. The Results section is not always clear regarding what CNP treatment was done - in vivo injections or in vitro maturation. For example, what is the difference, if any, between Figures 2C-D and Figures S2A-B?

This remains unclear in the revised manuscript.

2. Immature oocytes from aged females (~1 year) were treated with a two-step culture system with a pre-IVM step with CNP. Controls included oocytes from young (6-8 weeks) females or oocytes from aged females treated by conventional IVM. The description of these methods suggests that control oocytes did not receive an equivalent pre-IVM culture, hence the relevance of comparisons of CNP-treated versus control oocyte is questionable. This concern has not been addressed in the revised manuscript. It was observed that aged oocytes pre-cultured in CNP improved polar body extrusion rates and meiotic spindle morphology compared to oocytes in conventional IVM, as has been well established. The description of statistical methods does not make clear whether the PBE rate in CNP-treated old oocytes remained significantly lower than young controls.

This concern has not been addressed in the revised manuscript.

3. The main effect of the CNP 2-week treatment appears to be increasing the number of follicles that grow into secondary and antral stages, but there is no attempt made to discover the mechanism by which this occurs and therefore to understand why there might be an increase in the number of ovulated eggs, quality of the eggs, and litter size. It is also not clear how an intraperitoneal injection can guarantee its effectiveness because the half-life of CNP is very short, only a few minutes.

This concern has not been addressed in the revised manuscript.

4. Meiotic spindle morphology, as well as a number of putative markers of cytoplasmic maturation are also suggested to be improved after pre-culture with CNP. In each case a subjective interpretation of "normal" morphology of these markers is derived from observations of the young controls and the proportions of oocytes with normal or abnormal appearance is evaluated. However, parameters that define abnormal patterns of these markers appear to be subjective judgements, and whether these morphological patterns can be mechanistically attributed to the differences in developmental potential cannot be concluded.

This concern has not been addressed in the revised manuscript.

5. In addition to the localization patterns of mitochondria, the mitochondrial membrane potential, oocyte ATP content and ROS levels were assessed through more objective quantitative methods. These are well known to be defective in oocytes of aged females and CNP treatment improved these measures. Mitochondrial dysfunction is the most obvious link between oocyte apoptosis, autophagy, cytoplasmic organelle miss-localization and aberrant spindle morphology. Among the most intriguing results is the finding that CNP mediated a cAMP-dependent protein kinase (PKA) dependent reduction in mitochondrial autophagy mediators PINK and Parkin and reduced the recruitment of Parkin to mitochondria in oocytes. However, it may not be possible to directly link this observation to the improvements in IVM oocyte quality, since PINK/Parkin assessments were performed in oocytes from cultured follicles treated with CNP for 6 days.

This weakness has not been addressed in the revised manuscript.

6. The gold standard assay for oocyte quality is embryo transfer and live birth. The authors assessed the impact of maturing oocytes in vitro in the presence of CNP on oocyte quality by less robust assays (e.g., preimplantation embryo development in vitro), so the impact on oocyte quality is less certain.

This weakness has not been addressed in the revised manuscript.

7. The numbers of embryos should have been corrected for the number of eggs fertilized as a starting point so that the percentage that developed to each stage could be expressed as a percentage of successfully fertilized eggs rather than overall percentages. As currently shown in the Figures and described in the Legend, there is no information regarding what the percentage on the y-axis means. For example, does Figure 4B show the number of 2C embryos divided by the number of eggs inseminated? Or is it divided by the number of successfully fertilized eggs, and if so, how was that assessed?

There is no additional information provided in the revised manuscript to address these concerns.

8. When fewer eggs are fertilized, the numbers of embryos per group are lower and so the impact of culturing multiple embryos together is lost. As a result, it is possible that culture conditions rather than oocyte quality drove the differences in the numbers of embryos that achieved each stage of development.

This concern has not been addressed in the revised manuscript. Similar numbers of oocytes were cultured together, but not similar numbers of fertilized oocytes, or embryos.

9. Not all claims in the Discussion are supported by the evidence provided. For example, "In addition, the findings demonstrated that CNP improved cytoplasmic maturation events by maintaining normal CG, ER and Golgi apparatus distribution and function in aged oocytes" but it was never demonstrated that the altered distribution had any functional impact.

This concern has not been addressed in the revised manuscript.

10. Incompleteness and errors in the Methods section reduce confidence in many of the results reported.

This concern has not been addressed in the revised manuscript.

11. The methods used for Statistical Analysis are never explained in either the Methods or the Figure legends. It is unclear whether appropriate analyses were done, and it is frequently unclear what was the sample size and how many times a particular experiment was repeated. These weaknesses detract from confidence in the data.

This concern has not been addressed adequately in the revised manuscript.

Reviewer #1 (Public Review):

This manuscript describes a series of experiments documenting trophic egg production in a species of harvester ant, Pogonomyrmex rugosus. In brief, queens are the primary trophic egg producers, there is seasonality and periodicity to trophic egg production, trophic eggs differ in many basic dimensions and contents relative to reproductive eggs, and diets supplemented with trophic eggs had an effect on the queen/worker ratio produced (increasing worker production).

The manuscript is very well prepared and the methods are sufficient. The outcomes are interesting and help fill gaps in knowledge, both on ants as well as insects, more generally. More context could enrich the study and flow could be improved.

Reviewer #1 (Public Review):

The authors report a study, where they have sequenced whole genomes of four individuals of an extinct species of butterfly from western North America (Glaucopsyche xerces), along with seven genomes of a closely related species (Glaucopsyche lygdamus), mainly from museum specimens, several to many decades old. They then compare these fragmented genomes to a high-quality, chromosome-level assembly of a genome of a European species in the same genus (Glaucopsyche alexis). They find that the extinct species shows clear signs of declining population sizes since the last glacial period and an increase in inbreeding, perhaps exacerbating the low viability of the populations and contributing to the extinction of the species.

The study really highlights how museum specimens can be used to understand the genetic variability of populations and species in the past, up to a century or more ago. This is an incredibly valuable tool, and can potentially help us to quickly identify whether current populations of rare and declining species are in danger due to inbreeding, or whether at least their genetic integrity is in good condition and other factors need to be prioritised in their conservation. In the case of extinct species, sequencing museum specimens is really our only window into the dynamics of genomic variability prior to extinction, and such information can help us understand how genetic variation is related to extinction.

I think the authors have achieved their goal admirably, they have used a careful approach to mapping their genomic reads to a related species with a high-quality genome assembly. They might miss out on some interesting genetic information in the unmapped reads, but by and large, they have captured the essential information on genetic variability within their mapped reads. Their conclusions on the lower genetic variability in the extinct species are sound, and they convincingly show that Glaucopyche xerces is a separate species to Glaucopsyche lygdamus (this has been debated in the past).

Reviewer #1 (Public Review):

First, I agree with the authors of this manuscript that conformational changes in the XFEL structures with 2.8 A resolution are not reliable enough for demonstrating the subtle changes in the electron transfer events in this bacterial photosynthesis system. Actually, the data statistics in the paper by Dods et al. showed that the high-resolution range of some of the XFEL datasets may include pretty high noise (low CC1/2 and high Rsplit) so the comparison of the subtle conformational changes of the structures is problematic.

The manuscript by Gai Nishikawa investigated time-dependent changes in the energetics of the electron transfer pathway based on the structures by Dods et al. by calculating redox potential of the active and inactive branches in the structures and found no clear link between the time-dependent structural changes and the electron transfer events in the XFEL structures published by Dods, R.et al. (2021). This study provided validation for the interpretation of the structures of those electron-transferring proteins.

The paper was well prepared.

Comments on latest version:

The revisions the authors have made have improved the manuscript.

Reviewer #1 (Public Review):

Summary: This paper performs fine-mapping of the silkworm mutants bd and its fertile allelic version, bdf, narrowing down the causal intervals to a small interval of a handful of genes. In this region, the gene orthologous to mamo is impaired by a large indel, and its function is later confirmed using expression profiling, RNAi, and CRISPR KO. All these experiments are convincingly showing that mamo is necessary for the suppression of melanic pigmentation in the silkworm larval integument.

The authors also use in silico and in vitro assays to probe the potential effector genes that mamo may regulate.

Strengths: The genotype-to-phenotype workflow, combining forward (mapping) and reverse genetics (RNAi and CRISPR loss-of-function assays) linking mamo to pigmentation are extremely convincing.

Weaknesses:

1) The last section of the results, entitled "Downstream target gene analysis" is primarily based on in silico genome-wide binding motif predictions.<br /> While the authors identify a potential binding site using EMSA, it is unclear how much this general approach over-predicted potential targets. While I think this work is interesting, its potential caveats are not mentioned. In fact the Discussion section seems to trust the high number of target genes as a reliable result. Specifically, the authors correctly say: "even if there are some transcription factor-binding sites in a gene, the gene is not necessarily regulated by these factors in a specific tissue and period", but then propose a biological explanation that not all binding sites are relevant to expression control. This makes a radical short-cut that predicted binding sites are actual in vivo binding sites. This may not be true, as I'd expect that only a subset of binding motifs predicted by Positional Weight Matrices (PWM) are real in vivo binding sites with a ChIP-seq or Cut-and-Run signal. This is particularly problematic for PWM that feature only 5-nt signature motifs, as inferred here for mamo-S and mamo-L, simply because we can expect many predicted sites by chance.

2) The last part of the current discussion ("Notably, the industrial melanism event, in a short period of several decades ... a more advanced self-regulation program") is flawed with important logical shortcuts that assign "agency" to the evolutionary process. For instance, this section conveys the idea that phenotypically relevant mutations may not be random. I believe some of this is due to translation issues in English, as I understand that the authors want to express the idea that some parts of the genome are paths of least resistance for evolutionary change (e.g. the regulatory regions of developmental regulators are likely to articulate morphological change). But the language and tone is made worst by the mention that in another system, a mechanism involving photoreception drives adaptive plasticity, making it sound like the authors want to make a Lamarckian argument here (inheritance of acquired characteristics), or a point about orthogenesis (e.g. the idea that the environment may guide non-random mutations).<br /> Because this last part of the current discussion suffers from confused statements on modes and tempo of regulatory evolution and is rather out of topic, I would suggest removing it.

In any case, it is important to highlight here that while this manuscript is an excellent genotype-to-phenotype study, it has very few comparative insights on the evolutionary process. The finding that mamo is a pattern or pigment regulatory factor is interesting and will deserve many more studies to decipher the full evolutionary study behind this Gene Regulatory Network.

Minor Comment :

The gene models presented in Figure 1 are obsolete, as there are more recent annotations of the Bm-mamo gene that feature more complete intron-exon structures, including for the neighboring genes in the bd/bdf intervals. It remains true that the mamo locus encodes two protein isoforms.<br /> An example of the Bm-mamo locus annotation, can be found at : https://www.ncbi.nlm.nih.gov/gene/101738295<br /> RNAseq expression tracks (including from larval epidermis) can be displayed in the embedded genome browser from the link above using the "Configure Tracks" tool.

Based on these more recent annotations, I would say that most of the work on the two isoforms remains valid, but FigS2, and particularly Fig.S2C, need to be revised.

Reviewer #1 (Public Review):

This manuscript presents an important study that contributes to our understanding of the reliability of ancient environmental DNA (aeDNA) extracted from sediment cores. The authors address the potential biases and challenges associated with using aeDNA to infer past ecosystems, specifically focusing on the case of mammoths and woolly rhinoceroses in the Yamal peninsula, West Siberia.

The introduction provides an overview of the significance of sedimentary deposits as archives of past ecosystem changes and illustrates the remarkable insights gained from previous studies using aeDNA, highlighting its potential for reconstructing paleoecology, phylogeography, and understanding extirpation and extinction events of keystone taxa. The authors then report the detection of DNA and near complete mitochondrial genomes of multiple mammoth and woolly rhinoceros individuals in the sampled sediment cores (which are dated to the last few centuries). The authors then employed additional methods to confirm the presence of ancient DNA from mammoths in these sediment cores. Conventional PCR and Sanger sequencing of a mammoth COI fragment confirmed the amplification of mammoth DNA. Mammal metabarcoding and droplet digital PCR (ddPCR) further supported the detection of mammoth DNA in both cores.

The hybridisation enrichment experiment results showed high read counts assigned to Mammuthus, ranging from 2,852 to 72,919 reads per library in core LK-001. Negative controls did not produce any reads assigned to mammals, indicating the absence of contamination. The study also revealed the presence of woolly rhinoceros sequences in the sediment cores, with 12 out of 23 libraries producing more than 100 reads assigned to woolly rhinoceros. The total number of woolly rhinoceros reads was 2,737, and the cumulative mitogenome coverage reached 44%.

The authors carefully addressed the incongruity between the temporal occurrence of these extinct species and the presence of their DNA in recent sediments. They proposed several mechanisms that could explain the recovery of Pleistocene megafaunal DNA in the sediment cores. The minor amount of ancient DNA post mortem damage observed in the mammoth sequences indicates exceptional preservation, consistent with an origin from (recent) permafrost. The dynamics of permafrost thawing and redeposition in the study area provide a plausible explanation for the presence of ancient DNA in the sediments. The authors discuss potential mechanisms for the redistribution of Late Pleistocene material in the sediments, including thermo-denudation processes, methane emissions from degrading permafrost, and the formation of taliks and methane seepage. These processes can disturb the stratigraphy of lake sediments and potentially mix ancient material within the modern sediments. I believe the conclusions are supported by the data and the manuscript is well-written and clear to follow for the reader.

Reviewer #1 (Public Review):

This manuscript by Neininger-Castro and colleagues presents a novel automatic image analysis method for assessing sarcomeres, the basic units of myofibrils and validates this tool in a couple of experimental approaches that interfere with sarcomere assembly in iPSC-cardiomyocytes (iPSC-CM).

Automatic quantification of sarcomeres is definitely something that is useful to the field. I am surprised that there is no reference in the manuscript to SarcTrack, published by Toepfer and colleagues in 2019 (PMID 30700234), which has exactly the same purpose. The advantage of the image analysis software presented in the current manuscript appears to me to be that it can cover both mature sarcomeres and nascent sarcomeres in premyofibrils effectively.

Reviewer #1 (Public Review):

The authors use electrophysiological and behavioral measurements to examine how animals could reliably determine odor intensity/concentration across repeated experiences. Because stimulus repetition leads to short-term adaptation evidenced by reduced overall firing rates in the antennal lobe and firing rates are otherwise concentration-dependent, there could be an ambiguity in sensory coding between reduced concentration or more recent experience. This would have a negative impact on the animal's ability to generate adaptive behavioral responses that depend on odor intensities. The authors conclude that changes in concentration alter the constituent neurons contributing to the neural population response, whereas adaptation maintains the 'activated ensemble' but with scaled firing rates. This provides a neural coding account of the ability to distinguish odor concentrations even after extended experience. Additional analyses attempt to distinguish hypothesized circuit mechanisms for adaptation but are inconclusive. A larger point that runs through the manuscript is that overall spiking activity has an inconsistent relationship with behavior and that the structure of population activity may be the more appropriate feature to consider.

To my knowledge, the dissociation of effects of odor concentration and adaptation on olfactory system population codes was not previously demonstrated. This is a significant contribution that improves on any simple model based on overall spiking activity. The primary result is most strikingly supported by visualization of a principal components analysis in Figure 4. However, there are some weaknesses in the data and analyses that limit confidence in the overall conclusions.

1) Behavioral work interpreted to demonstrate discrimination of different odor concentrations yields inconsistent results. Only two of the four odorants follow the pattern that is emphasized in the text (Figure 1F). Though it's a priori unlikely that animals are incapable of distinguishing odor concentrations at any stage in adaptation, the evidence presented is not sufficient to reach this conclusion.<br /> 2) While conclusions center on concepts related to the combination of activated neurons or the "active ensemble", this specific level of description is not directly demonstrated in any part of the results. We see individual neural responses and dimensional reduction analyses, but we are unable to assess to what extent the activated ensemble is maintained across experience.<br /> 3) There is little information about the variance or statistical strength of results described at the population level. While the PCA presents a compelling picture, the central point that concentration changes and adaptation alter population responses across separable dimensions is not demonstrated quantitatively. The correlation analysis that might partially address this question is presented to be visually interpreted with no additional testing.<br /> 4) Results are often presented separately for each odor stimulus or for separate datasets including two odor stimuli. An effort should be made to characterize patterns of results across all odor stimuli and their statistical reliability. This concern arises throughout all data presentations.<br /> 5) The relevance of the inconclusive analysis of inferred adaptation mechanisms in Figure 2d-f and the single experiment including a complex mixture in Figure 7 to the motivating questions for this study are unclear.<br /> 6) Throughout the description of the results, typical standards for statistical reporting (sample size, error bars, etc.) are not followed. This prevents readers from assessing effect sizes and undermines the ability to assign a confidence to any particular conclusion.

DOI: 10.1371/journal.pbio.3002223

Resource: RRID:ZFIN_ZDB-ALT-160830-1

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-160830-1

What is this?

(~14:00) The way to gain massive results is to have massive irrational goals complemented by small reasonable steps or milestones.

Big goals motivate. Big goals give focus and clarity, they are a filter (see Dr. Benjamin Hardy's content); they allow for easy application of the power law.

(~13:00) Koe argues for making information relevant (Dr. Sung always says you must make info relevant) through the learning for the solving of a particular problem, either for a client, your business, or your personal life. Your problem becomes the lense through which you learn.

For self-education this is ideal.

Dr. Sung's approach differs in that he advocates for the creation of relevancy through inquiry (the asking of relational questions) which is also incredibly powerful, however this is more suited to gaining more motivation for forced learning, i.e., in the formal education system.

In addition, Koe's lense is, I think, more of a high-level filter, whereas Sung's questioning is applicable on the content level. Therefore, both approaches could be, and should be, combined into the same overall (self-)educational system.

(~10:20) Koe makes a very, very, very valid point about education:

I quote: "There is one thing that the school system did get right which is consistent, daily education in hopes for a better future. But, schools don't prioritize curiosity, so most people hate learning by the time they graduate." (emphasis added by me)

The larger point that Koe is making is that if we own anything in life, it is our mind; for everything else can be taken away from us; as such, we must spend a significant amount of effort to cultivate it, grow it, care for it, and make it unique.

(~6:07) Koe argues that specializing, or focusing on one aspect only, limits your potential in every conceivable way.

I think I agree, yet I do also think there is a place for that... It depends on the person and what they enjoy. However, I might still be mistaken.

Koe argues for the following trait of a modern renaissance man (or woman):

• Self-Educated
• Pursue Interest
• Leverage the Digital World
• Exercise (physical training)
• Conscious about their health
• Social
• Doing meaningful work
• Acknowledgement of the Spiritual

Dan Koe seems to argue against a specialistic education based on the argument that it is nigh-impossible for a teenager to decide what they want (to be) for the rest of their lives. He also gives the argument that it results in a lack of creativity and underlying knowledge (that which connects the dots, instead of compartmentalization) which would result in abnormal performance.

I can bypass the limitation of the first point by giving the counter-point that when one has an insane amount of metacognition, which can be trained, it does not matter if one changes path later; why? Because one can easily learn the new subject matter and skills.

However, the second point is interesting and I think I agree with it. That said, I think there is a continuum, instead of only two points, between super-specialists and super-generalists. I myself enjoy specializing. And I believe a team of specialists (that can also work together) can accomplish much more than one (or even multiple) generalist.

Ten minutes before sleep, do the following: PRAY

It's a combination of visualization, commitment, and meditation

Request the subconscious through this act of prayer.

Also visualize the outcome and process of that which you aspire to do the following day, and even that which you want to achieve the following month(s). Thus, visualize the following: Big Picture, Milestones, and yourself the next day.

In the morning, process your subconscious state by instead of immediately inputting, you start outputting!

This can be done through journaling.

Put the phone on airplane mode (in addition to blocking blue light) before sleep, for quite some time before sleep, in order to avoid (over)stimulation and the creation of dopamine which negatively impacts (falling a)sleep

What is done right before and right after sleep sets the stage for literally everything.

How you do anything is how you do everything.

Reviewer #1 (Public Review):

The authors put forth the hypothesis that hepatocyte and/or non-parenchymal liver MCT1 may be responsible for physiologic effects (lower body weight gain and less hepatic steatosis) in MCT1 global heterozygote mice. They generate multiple tools to test this hypothesis, which they combine with mouse diets that induce fatty liver, steatohepatitis and fibrosis. Novel findings include that deletion of hepatocyte MCT1 does not change liver lipid content, but increases liver fibrosis. Deletion of hepatic stellate cell (HSC) MCT1 does not substantially affect any liver parameter, but concomitant HSC MCT1 deletion does reverse fibrosis seen with hepatocyte MCT1 knockout or knockdown. In both models, plasma lactate levels do not change, suggesting that liver MCT1 does not substantially affect systemic lactate. In general, the data match the conclusions of the manuscript, and the studies are well-conducted and well-described. Further work would be necessary to dissect mechanism of fibrosis with hepatocyte MCT1, and whether this is due to changes in local lactate (as speculated by the authors) or another MCT1 substrate. This would be important to understand this novel potential cross-talk between hepatocytes and HSCs.

A parallel and perhaps more important advance is the generation of new methodology to target HSC in mice, using modified siRNA and by transduction of AAV9-Lrat-Cre. Both methods would reduce the need to cross floxed mice with the Lrat-Cre allele, saving time and resources. These tools were validated to an extent by the authors, but not sufficiently to ensure that there is no cross-reactivity with other liver cell types. For example, AAV9-Lrat-Cre-transduced MCT1 floxed mice show compelling HSC but not hepatocyte Mct1 knockdown, but other liver cell types should be assessed to ensure specificity. This is particularly important as overall liver Mct1 decreased by ~30% in AAV9-Lrat-Cre-transduced mice, which may exceed HSC content of these mice, especially when considering a 60-70% knockdown efficiency. This same issue also affects Chol-MCT1-siRNA, which the authors demonstrate to affect hepatocytes and HSC, but likely affects other cell types not tested. As this is a new and potentially valuable tool, it would be important to assess Mct1 expression across more non-parenchymal cells (i.e. endothelial, cholangiocytes, immune cells) to determine penetration and efficacy.

Reviewer #1 (Public Review):

The cerebral cortex, or surface of the brain, is where humans do most of their conscious thinking. In humans, the grooves (sulci) and bumps (convolutions) have a particular pattern in a region of the frontal lobe called Broca's area, which is important for language. Specialists study features imprinted on the internal surfaces of braincases in early hominins by casting their interiors, which produces so-called endocasts. A major question about hominin brain evolution concerns when, where, and in which fossils a humanlike Broca's area first emerged, the answer to which may have implications for the emergence of language. The researchers used advanced imaging technology to study the endocast of a hominin (KNM-ER 3732) that lived about 1.9 million years ago (Ma) in Kenya to test a recently published hypothesis that Broca's remained primitive (apelike) prior to around 1.5 Ma. The results are consistent with the hypothesis and raise new questions about whether endocasts can be used to identify the genus and/or species of fossils.

Reviewer #1 (Public Review):

This work describes a structural analysis of the tripartite HipBST toxin-antitoxin (TA) system, which is related to the canonical two-component HipBA system composed of the HipA serine-threonine kinase toxin and the HipB antitoxin. The crystal structure of the kinase-inactive HipBST complex of the Enteropathogenic E. coli O127:H6 was solved and revealed that HipBST forms a hetero-hexameric complex composed of a dimer of HipBST heterotrimers that interact via the HipB subunit. The HipS antitoxin shows a structural resemblance to HipA N-terminal region and the HipT toxin represents to the core kinase domain of HipA, indicating that in HipBST the hipA toxin gene was likely split in two genes, namely hipS and hipT.

The structure also reveals a conserved and essential Trp residue within the HipS antitoxin, which likely prevents the conserved "Gly-rich loop" of HipT from adopting an inward conformation needed for ATP binding. This work also shows that the regulating Gly-rich loop of the HipT toxin contains conserved phosphoserine residues essential for HipT toxicity that are key players within the HipT active site interacting network and which likely control antitoxin binding and/or activity.

Strengths:

The manuscript is well written and the experimental work well executed. It shows that major features of the classical two-component HipAB TA system have somehow been rerouted in the case of the tripartite HipBST. This includes the N-terminal domain of the HipA toxin, which now functions as bona fide antitoxin, and the partly relegated HipB antitoxin, which could only function as a transcription regulator. In addition, this work shows a new mode of inhibition of a kinase toxin and highlights the impact of the phosphorylation state of key toxin residues in controlling the activity of the antitoxin.

Weaknesses:

A major weakness of this work is the lack of data concerning the role of HipB, which likely does not act as an antitoxin. Does it act as a transcriptional regulator of the hipBST operon and to what extent both HipS and HipT contribute to such regulation? These are still open questions.

In addition, there is no in-depth structural comparison between the structure of the HipBST solved in the work and the two recent structures of HipBST from Legionella. This is also a major weakness of this work.

Reviewer #1 (Public Review):

In this manuscript, Knecht, Sirias et al describe toxin-immunity pair from Proteus mirabilis. Their observations suggest that the immunity protein could protect against non-cognate effectors from the same family. They analyze these proteins by dissecting them into domains and constructing chimeras which leads them to the conclusion that the immunity can be promiscuous and that the binding of immunity is insufficient for protective activity.

Strengths:

The manuscript is well written and the data are very well presented and could be potentially interesting. The phylogenetic analysis is well done, and provides some general insights.

Weaknesses:

1) Conclusions are mostly supported by harsh deletions and double hybrid assays. The later assays might show binding, but this method is not resolutive enough to report the binding strength. Proteins could still bind, but the binding might be weaker, transient, and out-competed by the target binding.

2) While the authors have modeled the structure of toxin and immunity, the toxin-immunity complex model is missing. Such a model allows alternative, more realistic interpretation of the presented data. Firstly, the immunity protein is predicted to bind contributing to the surface all over the sequence, except the last two alpha helices (very high confidence model, iPTM>0.8). The N terminus described by the authors contributes one of the toxin-binding surfaces, but this is not the sole binding site. Most importantly, other parts of the immunity protein are predicted to interact closer to the active site (D-E-K residues). Thus, based on the AlphaFold model, the predicted mechanism of immunization remains physically blocking the active site. However, removing the N terminal part, which contributes large interaction surface will directly impact the binding strength. Hence, the toxin-immunity co-folding model suggests that proper binding of immunity, contributed by different parts of the protein, is required to stabilize the toxin-immunity complex and to achieve complete neutralization. Alternative mechanisms of neutralization might not be necessary in this case and are difficult to imagine for a DNAse.

3) Dissection of a toxin into two domains is also not justified from a structural point of view, it is probably based on initial sequence analyses. The N terminus (actually previously reported as Pone domain in ref 21) is actually not a separate domain, but an integral part of the protein that is encased from both sides by the C terminal part. These parts might indeed evolve faster since they are located further from the active site and the central core of the protein. I am happy to see that the chimeric toxins are active, but regarding the conservation and neutralization, I am not surprised, that the central core of the protein fold is highly conserved. However, "deletion 2" is quite irrelevant - it deletes the central core of the protein, which is simply too drastic to draw any conclusions from such a construct - it will not fold into anything similar to an original protein, if it will fold properly at all.

4) Regarding the "promiscuity" there is always a limit to how similar proteins are, hence when cross-neutralization is claimed authors should always provide sequence similarities. This similarity could also be further compared in terms of the predicted interaction surface between toxin and immunity.

Overall, it looks more like a regular toxin-immunity couple, where some cross-reactions with homologues will, of course, be possible, depending on how far the sequences have deviated. Nevertheless, taking all of the above into account, these results do not challenge toxin-immunity specificity dogma.

Joint Public Review

Introduction - Well written and placed within the current trends of unprecedented biodiversity loss, with emphasis in freshwater ecosystems. The authors identify three important points as to why biodiversity action plans have failed. Namely, community changes occur over large spatio-temporal scales and monitoring programs capture a fraction of these long-term dynamics (e.g. few decades) which although good at capturing trends in biodiversity change, they often fail at identifying the drivers of these changes. Additionally, most of these rely on manual sorting of samples, overlooking cryptic diversity, or state-of-the-art techniques such as sedimentary DNA (sedaDNA) which allow studying decade-long dynamics, usually focus on specific taxonomic groups unable to represent community-level changes. Secondly, the authors identify that biodiversity is threatened by multiple factors and are rarely studied in tandem. Finally, the authors stress the need for high-throughput approaches to study biodiversity changes since historically, most conservation efforts rely on highly specialized skills for biodiversity monitoring, and even well studied species have relatively short time series data. The authors identify a model freshwater lake (Lake Ring, Denmark) - suitable due to its well documented history over the last 100 years - to present a comprehensive framework using metabarcoding, chemical analysis and climatic records for identifying past and current impacts on this ecosystem arising from multiple abiotic environmental stressors.

Results - They are brief and should expand some more. Particularly, there are no results regarding metabarcoding data (number of reads, filtering etc.). These details are important to know the quality of the data which represents the bulk of the analyses. Even the supplementary material gives little information on the metabarcoding results (e.g. number of ASVs - whether every ASV of each family were pooled etc.). The drivers of biodiversity change section could be restructured and include main text tables showing the families positively or negatively correlated with the different variables (akin to table S2 but simplified).

Discussion<br /> The discussion is well written identifying first, some of the possible caveats of this study, particularly regarding classification of metabarcoding data, its biases and the possible DNA degradation of ancient sediment DNA. The authors discuss how their results fit to general trends showing how agricultural runoff and temperature drive changes in freshwater functional biodiversity primarily due to their synergistic effects on bioavailability, adsorption, etc. The authors highlight the advantage of using a system-level approach rather than focusing on taxa-specific studies due to their indicator status. Similarly, the authors justify the importance of studying community composition as far back as possible since it reveals unexpected patterns of ecosystem resilience. Lake Ring, despite its partially recovered status, has not returned to its semi-pristine levels of biodiversity and community assemblage. Additionally, including enzyme activity allows to assess functional diversity of the studied environment although reference databases of these pathways are still lacking. Finally, the authors discuss the implications of their findings under a conservation and land management framework suggesting that by combining these different approaches, drivers of biodiversity stressors can be derived with high accuracy allowing for better informed mitigation and conservation efforts.

Reviewer #1 (Public Review):

In the best genetically and biochemically understood model of eukaryotic DNA replication, the budding yeast, Saccharomyces cerevisiae, the genomic locations at which DNA replication initiates are determined by a specific sequence motif. These motifs, or ARS elements, are bound by the origin recognition complex (ORC). ORC is required for loading of the initially inactive MCM helicase during origin licensing in G1. In human cells, ORC does not have a specific sequence binding domain and origin specification is not specified by a defined motif. There have thus been great efforts over many years to try to understand the determinants of DNA replication initiation in human cells using a variety of approaches, which have gradually become more refined over time.

In this manuscript Tian et al. combine data from multiple previous studies using a range of techniques for identifying sites of replication initiation to identify conserved features of replication origins and to examine the relationship between origins and sites of ORC binding in the human genome. The authors identify a) conserved features of replication origins e.g. association with GC-rich sequences, open chromatin, promoters and CTCF binding sites. These associations have already been described in multiple earlier studies. They also examine the relationship of their determined origins and ORC binding sites and conclude that there is no relationship between sites of ORC binding and DNA replication initiation. While the conclusions concerning genomic features of origins are not novel, if true, a clear lack of colocalization of ORC and origins would be a striking finding. However, the majority of the datasets used do not report replication origins, but rather broad zones in which replication origins fire. Rather than refining the localisation of origins, the approach of combining diverse methods that monitor different objects related to DNA replication leads to a base dataset that is highly flawed and cannot support the conclusions that are drawn, as explained in more detail below.

Methods to determine sites at which DNA replication is initiated can be divided into two groups based on the genomic resolution at which they operate. Techniques such as bubble-seq, ok-seq can localise zones of replication initiation in the range ~50kb. Such zones may contain many replication origins. Conversely, techniques such as SNS-seq and ini-seq can localise replication origins down to less than 1kb. Indeed, the application of these different approaches has led to a degree of controversy in the field about whether human replication does indeed initiate at discrete sites (origins), or whether it initiates randomly in large zones with no recurrent sites being used. However, more recent work has shown that elements of both models are correct i.e. there are recurrent and efficient sites of replication initiation in the human genome, but these tend to be clustered and correspond to the demonstrated initiation zones (Guilbaud et al., 2022).

These different scales and methodologies are important when considering the approach of Tian et al. The premise that combining all available data from five techniques will increase accuracy and confidence in identifying the most important origins is flawed for two principal reasons. First, as noted above, of the different techniques combined in this manuscript, only SNS-seq can actually identify origins rather than initiation zones. It is the former that matters when comparing sites of ORC binding with replication origin sites if a conclusion is to be drawn that the two do not co-localise.

Second, the authors give equal weight to all datasets. Certainly, in the case of SNS-seq, this is not appropriate. The technique has evolved over the years and some earlier versions have significantly different technical designs that may impact the reliability and/or resolution of the results e.g. in Foulk et al. (Foulk et al., 2015), lambda exonuclease was added to single stranded DNA from a total genomic preparation rather than purified nascent strands), which may lead to significantly different digestion patterns (ie underdigestion). Curiously, the authors do not make the best use of the largest SNS-seq dataset (Akerman et al., 2020) by ignoring these authors separation of core and stochastic origins. By blending all data together any separation of signal and noise is lost. Further, I am surprised that the authors have chosen not to use data and analysis from a recent study that provides subsets of the most highly used and efficient origins in the human genome, at high resolution (Guilbaud et al., 2022).

References:

Akerman I, Kasaai B, Bazarova A, Sang PB, Peiffer I, Artufel M, Derelle R, Smith G, Rodriguez-Martinez M, Romano M, Kinet S, Tino P, Theillet C, Taylor N, Ballester B, Méchali M (2020) A predictable conserved DNA base composition signature defines human core DNA replication origins. Nat Commun, 11: 4826

Foulk MS, Urban JM, Casella C, Gerbi SA (2015) Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res, 25: 725-735

Guilbaud G, Murat P, Wilkes HS, Lerner LK, Sale JE, Krude T (2022) Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation. Nucleic Acids Res, 50: 7436-7450

Reviewer #1 (Public Review):

The authors sought to resolve the coordinated functions of the two muscles that primarily power flight in birds (supracoracoideus and pectoralis), with particular focus on the pectoralis. Technology has limited the ability to resolve some details of pectoralis function, so the authors developed a model that can make accurate predictions about this muscle's function during flight. The authors first measured aerodynamic forces, wing shape changes, and pectoralis muscle activity in flying doves. They used cutting-edge techniques for the aerodynamic and wing shape measurements and they used well-established methods to measure activity and length of the pectoralis muscle. The authors then developed two mathematical models to estimate the instantaneous force vector produced by the pectoralis throughout the wing stroke. Finally, the authors applied their mathematical models to other-sized birds in order to compare muscle physiology across species.

The strength of the methods is that they smoothly incorporate techniques from many complementary fields to generate a comprehensive model of pectoralis muscle function during flight. The high-speed structured-light technique for quantifying surface area during flight is novel and cutting-edge, as is the aerodynamic force platform used. These methods push the boundaries of what has historically been used to quantify their respective aspects of bird flight and their use here is exciting. The methods used for measuring muscle activation and length are standard in the field. Together, these provide both a strong conceptual foundation for the model and highlight its novelty. This model allows for estimations of muscle function that are not feasible to measure in live birds during flight at present. The weakness of this approach is that it relies heavily on a series of assumptions. While the research presented in this paper makes use of powerful methods from multiple fields, those methods each have assumptions inherent to them that simplify the biological system of study. This reduction in the complexity of phenomena allows the specific measurements to be made. In joining the techniques of multiple fields to study the greater complexity of the phenomenon of interest, the assumptions are all incorporated also. Furthermore, assumptions are inherent to mathematical modeling of biological phenomena. That being said, the authors acknowledge and justify their assumptions at each step and their model seems to be quite good at predicting muscle function.

Indeed, the authors achieve their aims. They effectively integrate methods from multiple disciplines to explore the coordination and function of the pectoralis and supracoracoideus muscles during flight. The conclusions that the authors derive from their model address the intended research aim.

The authors demonstrate the value of such interdisciplinary research, especially in studying complex behaviors that are difficult or infeasible to measure in living animals. Additionally, this work provides predictions for muscle function that can be tested empirically. These methods are certainly valuable for understanding flight but also have implications for biologists studying movement and muscle function more generally.

Reviewer #1 (Public Review):

In this paper, Schalcher et al. examined how barn owls' landing force affects their hunting success during two hunting strategies: strike hunting and sit-and-wait hunting. They tracked tens of barn owls that raised their nestlings in nest boxes and utilized high-resolution GPS and acceleration loggers to monitor their movements. In addition, camcorders were placed near their nest boxes and used to record the prey they brought to the nest, thus measuring their foraging success.

This study generated a unique dataset and provided new insights into the foraging behavior of barn owls. The researchers discovered that the landing force during hunting strikes was significantly higher compared to the sit-and-wait strategy. Additionally, they found a positive relationship between landing force and foraging success during hunting strikes, whereas, during the sit-and-wait strategy, there was a negative relationship between the two. This suggests that barn owls avoid detection by generating a lower landing force and producing less noise. Furthermore, the researchers observed that environmental characteristics affect barn owls' landing force during sit-and-wait hunting. They found a greater landing force when landing on buildings, a lower landing force when landing on trees, and the lowest landing force when landing on poles. The landing force also decreased as the time to the next hunting attempt decreased. These findings collectively suggest that barn owls reduce their landing force as an acoustic camouflage to avoid detection by their prey.

The main strength of this work is the researchers' comprehensive approach, examining different aspects of foraging behavior, including high-resolution movement, foraging success, and the influence of the environment on this behavior, supported by impressive data collection. The weakness of this study is that the results only present a partial biological story contained within the data. The focus is on acoustic camouflage without addressing other aspects of barn owls' foraging strategy, leaving the reader with many unanswered questions. These include individual differences, direct measurements of owls' fitness, a detailed analysis of the foraging strategy of males and females, and the collective effort per nest box. However, it is possible that these data will be published in a separate paper.

The results presented support the authors' conclusion that lower landing force during sit-and-wait hunting increases hunting success, likely due to a decreased probability of detection by their prey, resulting in acoustic camouflage. The authors also argue that hunting success is crucial for survival, and thus, acoustic camouflage has a direct link to fitness. While this statement is reasonable, it should be presented as a hypothesis, as no direct evidence has been provided here. However, since information about nestling survival is typically monitored when studying behavior during the breeding period, the authors' knowledge of the effect of acoustic camouflage on owls' fitness can probably be provided. Furthermore, it will be interesting to further examine the foraging strategies used by different individuals during foraging, the joint foraging success of both males and females within each nest box, and the link between landing force and foraging success if the data are available. However, even without this additional analysis on survival, this paper provides an unprecedented dataset and the first measurement of landing force during hunting in the wild. It is likely to inspire many other researchers currently studying animal foraging behavior to explore how animals' movements affect foraging success.

Reviewer #1 (Public Review):

After revision, the manuscript is clearly improved and I thank the authors for their efforts. Yet, two contentious issues remain.

Firstly, I am skeptical whether the circularity issue has been resolved.

The authors equate uncertainty in the outcome of interactions with social complexity and they then diagnose for these three species that higher social complexity correlates with higher communicative complexity. Yet, there is still an inherent link between the occurrence of signals and other behaviours that allow the authors to determine the outcome of an interaction.

I do agree with the authors' conclusion that the three species vary in terms of the predictability of their signaling behaviour and the outcome of interactions. I just think the observed link between the two is not very surprising or informative, but rather inevitable.

Secondly, I am still not convinced that visual communication is more prevalent in situations with higher predation pressure. There are two reasons: relying on visual communication requires that the recipients, typically one's group members, are actually looking at the signaler when they produce the signal. The vocal-auditory channel in contrast, has a much higher potential to reach all recipients, even when visual communication in impaired. In addition, the idea that predators use acoustic signals to single out individuals and preferentially attack them, is poorly corroborated by data, especially for terrestrial predators. In contrast, there is ample evidence that prey species direct their calls at terrestrial predators (mobbing calls against snakes, antelope vigorously snorting against lions and leopards). See also this paper by Griesser (PMID 23941356).

Reviewer #1 (Public Review):

There are a number of outstanding questions concerning how cohesin turnover on DNA is controlled by various accessory factors and how such turnover is controlled by post-translational modification. In this paper, Nasmyth et al. perform a series of AlphaFold structure predictions that aim to address several of these outstanding questions. Their structure predictions suggest that the release factor WAPL forms a ternary complex with PDS5 and SA/SCC3. This ternary complex appears to be able to bind the N-terminal end of SCC1, suggesting how formation of such a complex could stabilize an open state of the cohesin ring. Additional calculations suggest how the Eco/ESCO acetyltransferases and Sororin engage the SMC3 head domain presumably to protect against WAPL-mediated release.

This work thus demonstrates the power of AF prediction methods and how they can lead to a number of interesting and testable hypotheses that can transform our understanding of cohesin regulation. These findings require orthogonal experimental validation, but authors argue convincingly that such validation should not be a pre-requisite to publication.

In their revised version, the authors did not systematically include model confidence scores, and it therefore remains difficult for the reader to evaluate the reliability of the models obtained. The authors correctly point out that such metrics are available on Figshare. It is therefore possible to obtain such information. The caveat is that it remains to the user to identify and extract the relevant information. While they claim that they have labeled N- and C-termini in their figures, no such labeling can be seen in the revised version. Addition of such labels, at least for some of the figures, would help the user to navigate the models.

Also, PAE plots still contain chain labels (A,B,C etc.) and it is not always clear which protein is being referred to. Which segment does the reader need to focus on? The authors claim that PAE plots are now amended but no such changes can be seen. At least for the key models proposed, the authors should have facilitated access and help readers interpret the different plots/models. While it is possible to load the different PDB files from Figshare, the current version still requires that the reader then works out what segments are used and how they correspond to chain labels shown.

It is exciting to see AF-multimer predictions being applied to cohesin. As some of the reported interactions are not universally conserved and some involve relatively small interfaces the possibility arises that these interfaces show poor or borderline confidence scores. As some of these interfaces map to mutants that have previously been obtained by hypothesis-free genetic screens and mutational analyses, they appear nevertheless valid. Thus, an important point to make is that even interfaces that show modest confidence scores may turn out to be valid while others may be not.

Reviewer #1 (Public Review):

Cell death plays a critical role on regulating organogenesis. During tooth morphogenesis, apoptosis of embryonic dental tissue plays critical roles on regulating tooth germ development. The current study focused on ferroptosis, another way of cell death which has rarely been investigated in tooth development, and showed it may also play an important role on regulating the tooth dimension. The topic is novel and interesting, but the experimental design has many flaws which significantly compromised the study.

1. The entire study was based on ex vivo tooth germ explant culture. Mandibular tooth germs of E15.5 (bell stage) were isolated for ex vivo culture. Most tooth germ explant culture experiments were actually using tooth germ of much earlier stages (E11.5-E13.5) for organ culture. After E16.5, both the large size and initially formed enamel/dentin could prevent nutrition from penetrating inside. Also, using tooth germ of earlier stage will help identify impact of ferroptosis upon early tooth development.<br /> 2. Due to limited penetration, the ex vivo culture in the study lasted for no more than 5 days. I would recommend the authors to perform kidney capsule transplantation as an alternative approach, which can support tooth germ development much longer even into root formation.<br /> 3. The major justification of using tooth germ ex vivo culture as the model in the study was to "conduct high-throughput analysis". However, the study could hardly be qualified as a high-throughput analysis. I would recommend the authors perform RNA sequencing for comparing tooth germs before/after erastin treatment. Such experiments won't take too much time or resource.<br /> 4. Although the study mostly used molars as the model, the in vivo iron concentration was only demonstrated on incisors, but not molars (Figure 1).<br /> 5. Phenotype analysis in Figure 2 is too superficial. Only dimensional information was provided. Cusps number, cusps distribution pattern and rooth/furcation formation were not evaluated. Differentiation of amelobast/odontoblast was not evaluated. The proliferation rate in the dental epithelium/mesenchyme was not analyzed.<br /> 6. Low magnification images should be included in Figure 3 to display the entire tooth germs.<br /> 7. In Figure 4, does ferroptotic inhibitor eliminate the iron accumulation in the tooth germ? How about the expression level of several target genes shown in Figure 3?<br /> 8. The manuscript has many typos and grammar mistakes. All "submandibular" should be simply "mandibular". "eastin" should be "erastin" (line 92). "partly" should be "partially" (line 611).

Reviewer #1 (Public Review):

Comments on the original submission:

Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) has shown that PIP5pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulate the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch".

Comments on revised manuscript:

In this revised manuscript, Touray et al. have responded to reviewers' comments with some revisions satisfactorily. However, the authors still haven't addressed some key scientific rigor issues, which are listed below:

1. It is critical to clearly state whether the observations are made for the endogenous WT protein or the tagged protein. It is good that the authors currently clearly indicate the results observed in vivo are for the RAP1-HA protein. However, this is not as clearly stated for in vitro EMSA analyses. In addition, in discussion, the authors simply assumed that the c-terminally tagged RAP1 behaves the same as WT RAP1 and all conclusions were made about WT RAP1.

There are two choices here. The authors can validate that RAP1-HA still retains RAP1's essential function as a sole allele in T. brucei cells (as was recommended previously). Indeed, HA-tagged RAP1 has been studied before, but it is the N-terminally HA-tagged RAP1 that has been shown to retain its essential functions. Adding the HA tag to the C-terminus of RAP1 may well cause certain defects to RAP1. For example, N-terminally HA-tagged TERT does not complement the telomere shortening phenotype in TERT null T. brucei cells, while C-terminally GFP-tagged TERT does, indicating that HA-TERT is not fully functional while TERT-GFP likely has its essential functions (Dreesen, RU thesis). Although RAP1-HA behaves similar to WT RAP1 in many ways, it is still not fully validated that this protein retains essential functions of RAP1. By the way, it has been published that cells lacking one allele of RAP1 behave as WT cells for cell growth and VSG silencing (Yang et al. 2009, Cell; Afrin et al. 2020, mSphere). In addition, although RAP1 may interact with TRF weakly, the interaction is direct, as shown in yeast 2-hybrid analysis in (Yang et al. 2009, Cell).

Alternatively, if the authors do not wish to validate the functionality of RAP1-HA, they need to add one paragraph at the beginning of the discussion to clearly state that RAP1-HA may not behave exactly as WT RAP1. This is important for readers to better interpret the results. In addition, the authors need to tune down the current conclusions dramatically, as all described observations are made on RAP1-HA but not the WT RAP1.

For a similar reason, the current EMSA results truly reflect how C-terminally His6-tagged RAP1 and RAP1 fragments behave. If the authors choose not to remove the His6 tag, it is essential that they use "RAP1-His6" to refer to these recombinant proteins. It is also essential for the authors to clearly state in the discussion that the tagged RAP1 fragments bind DNA, but the current data do not reveal whether WT RAP1 binds DNA. In addition, the authors incorrectly stated that "disruption of the MybL domain sequence did not eliminate RAP1-telomere binding in vivo" (lines 165-166). In ref 29, deletion of Myb domain did not abolish RAP1-telomere association. However, point mutations in MybL domain that abolish RAP1's DNA binding activities clearly disrupted RAP1's association with the telomere chromatin. Therefore, the current observation is not completely consistent with that published in ref 29.

2. There is no evidence, in vitro or in vivo, that binding PI(3,4,5)P3 to RAP1 causes conformational change in RAP1. The BRCT domain of RAP1 is known for its ability to homodimerize (Afrin et al. 2020, mSphere). It is therefore possible that binding of PI(3,4,5)P3 to RAP1 simply disrupts its homodimerization function. The authors clearly have extrapolated their conclusions based on available data. It is therefore important to revise the discussion and make appropriate statements.

Reviewer #1 (Public Review):

This paper describes the role of WRNIP1 AAA+ ATPase, particularly its UBZ domain for ubiquitin-binding, but not ATPase, to prevent the formation of the R-loop when DNA replication is mildly perturbated. By combining cytological analysis for DNA damage, R-loop, and chromosome aberration with the proximity ligation assay for colocalization of various proteins involved in DNA replication and transcription, the authors provide solid evidence to support the claim. The authors also revealed a distinct role of WRNIP1 in the prevention of R-loop-induced DNA damage from FANCD2, which is inconsistent with the known relationship between WRNIP1 and FANCD2 in the repair of crosslinks.

Reviewer #1 (Public Review):

Summary:<br /> This research offers an in-depth exploration and quantification of social vocalization within three families of Mongolian gerbils. In an enlarged, semi-natural environment, the study continuously monitored two parent gerbils and their four pups from P14 to P34. Through dimensionality reduction and clustering, a diverse range of gerbil call types was identified. Interestingly, distinct sets of vocalizations were used by different families in their daily interactions, with unique transition structures exhibited across these families. The primary results of this study are compelling, although some elements could benefit from clarification

Strengths:<br /> Three elements of this study warrant emphasis. Firstly, it bridges the gap between laboratory and natural environments. This approach offers the opportunity to examine natural social behavior within a controlled setting (such as specified family composition, diet, and life stages), maintaining the social relevance of the behavior. Secondly, it seeks to understand short-timescale behaviors, like vocalizations, within the broader context of daily and life-stage timescales. Lastly, the use of unsupervised learning precludes the injection of human bias, such as pre-defined call categories, allowing the discovery of the diversity of vocal outputs.

Weaknesses:<br /> 1. While the notable differences in vocal clusters across families are convincing, the drivers of these differences remain unclear. Are they attributable to "dialect," call usage, or specific vocalizing individuals (e.g., adults vs. pups)? Further investigation, via a literature review or additional observation, into acoustic differences between adult and pup calls is recommended. Moreover, a consistent post-weaning decrease in the bottom-left cluster (Fig. S3) invites interpretation: could this reflect drops in pup vocalization?

2. Developmental progression, particularly during pre-weaning periods when pup vocal output remains unstable, might be another factor influencing cross-family vocal differences. Representing data from this non-stationary process as an overall density map could result in the loss of time-dependent information. For instance, were dominating call types consistently present throughout the recording period, or were they prominent only at specific times? Displaying the evolution of the density map would enhance understanding of this aspect.

3. Family-specific vocalizations were credited to the transition structure, a finding that may seem obvious if the 1-gram (i.e., the proportion of call types) already differs. This result lacks depth unless it can be demonstrated that, firstly, the transition matrix provides a robust description of the data, and secondly, different families arrange the same set of syllables into unique sequences.

Reviewer #1 (Public Review):

In this manuscript, Davidsen and coworkers describe the development of a novel aspartate biosensor jAspSNFR3. This collaborative work supports and complements what was reported in a recent preprint by Hellweg et al., (bioRxiv; doi: 10.1101/2023.05.04.537313). In both studies, the newly engineered aspartate sensor was developed from the same glutamate biosensor previously developed by the authors of this manuscript. This coincidence is not casual but is the result of the need to find tools capable of measuring aspartate levels in vivo. Therefore, it is undoubtedly a relevant and timely work carried out by groups experienced in aspartate metabolism and in the generation of metabolite biosensors.

Reviewer #1 (Public Review):

The authors have developed an open-source high-resolution microscope that is easily accessible to scientists, students, and the general public. The microscope is specifically designed to work with incubators and can image cells in culture over long periods. The authors provide detailed instructions for building the microscope and the necessary software to run it using off-the-shelf components. The system has great potential for studying cell biology and various biological processes.

The authors' work will make scientific instruments more accessible and remove obstacles to the free diffusion of capabilities and know-how in science. This important contribution will enable more people to conduct scientific research.

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 measurement 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 #1 (Public Review):

Summary:<br /> Tian et al. investigated the effects of emotional signals in biological motion on pupil responses. In this study, subjects were presented with point-light biological motion stimuli with happy, neutral, and sad emotions. Their pupil responses were recorded with an eye tracker. Throughout the study, emotion type (i.e., happy/sad/neutral) and BM stimulus type (intact/inverted/non-BM/local) were systematically manipulated. For intact BM stimuli, happy BM induced a larger pupil diameter than neutral BM, and neutral BM also induced a larger pupil diameter than sad BM. Importantly, the diameter difference between happy and sad BM correlated with the autistic trait of individuals. These effects disappeared for the inverted BM and non-BM stimuli. Interestingly, both happy and sad emotions show superiority in pupil diameter.

Strengths:<br /> 1. The experimental conditions and results are very easy to understand.<br /> 2. The writing and data presentation are clear.<br /> 3. The methods are sound. I have no problems with the experimental design and results.

Weaknesses:<br /> 1. My main concern is the interpretation of the intact and local condition results. The processing advantage of happy emotion is not surprising given a number of existing studies. However, the only difference here seems to be the smaller (or larger) pupil diameter for sad compared to neutral in the intact (or local, respectively) condition. The current form only reports this effect but lacks in-depth discussions and explanations as to why this is the case.

2. I also found no systematic discussion and theoretical contributions regarding the correlation with the autistric trait. If the main point of this paper is to highlight an implicit and objective behavioral marker of the autistric trait, more interpretation and discussion of the links between the results and existing findings in ASD are needed.

Reviewer #1 (Public Review):

The manuscript by Zhu and colleagues aimed to clarify the importance of isoform diversity of PCDHg in establishing cortical synapse specificity. The authors optimized 5' single-cell sequencing to detect cPCDHg isoforms and showed that the pyramidal cells express distinct combinations of PCDHg isoforms. Then, the authors conducted patch-clamp recordings from cortical neurons whose PCDHg diversity was disrupted. In the elegant experiment in Figure 3, the authors demonstrated that the neurons expressing the same sets of cPCDHg isoforms are less likely to form synapses with each other, suggesting that identical cPCDHg isoforms may have a repulsive effect on synapse formation. Importantly, this phenomenon was dependent on the similarity of the isoforms present in neurons but not on the amount of proteins expressed.

One of the major concerns in an earlier version was whether PCDHg isoforms, which are expressed at a much lower level than C-type isoforms, have true physiological significance. The authors conducted additional experiments to address this point by using PCDHg cKO and provided convincing data supporting their conclusion. The results from PCDHg C4 overexpression, showing no impact on synaptic connectivity, further clarified the importance of isoforms. I have no further concerns, however, I would like to point out that the evidence for the necessity of the PCDHg isoform is still lacking because most experiments were done by overexpression. It would be helpful for the readers if the authors could add this point to the discussion.

Reviewer #1 (Public Review):

Spikol et al. investigate the roles of two distinct populations of neurons in the nucleus incertus (NI). The authors established two new transgenic lines that label gsc2- and rln3a-expressing neurons. They show that the gsc2+ and rln3a+ NI neurons show divergent projection patterns and project to different parts of the interpeduncular nucleus (IPN), which receive inputs from habenula (Hb). Furthermore, calcium imaging shows that gsc2 neurons are activated by the optogenetic activation of the dorsal Hb-IPN and respond to aversive electric shock stimuli, while rln3a neurons are highly spontaneously active. The ablation of rln3a neurons, but not gsc2 neurons, alters locomotor activity of zebrafish larvae.

The strength of the paper is their genetic approach that enabled the authors to characterize many different features of the two genetically targeted populations in the NI. These two neuronal populations are anatomically closely apposed and would have been indistinguishable without their genetic tools. Their analyses provide valuable information on the diverse anatomical, physiological and behavioral functions of the different NI subtypes. On the other hand, these pieces of evidence are only loosely linked with each other to reach a mechanistic understanding of how the NI works in a circuit. For example, the anatomical study revealed the connections from the NI to the IPN, while the optogenetic mapping experiments investigate the other way around, i.e. the connection from the IPN to the NI.

Reviewer #1 (Public Review):

This study examines whether the human brain uses a hexagonal grid-like representation to navigate in a non-spatial space constructed by competence and trustworthiness. To test this, the authors asked human participants to learn the levels of competence and trustworthiness for six faces by associating them with specific lengths of bar graphs that indicate their levels in each trait. After learning, participants were asked to extrapolate the location from the partially observed morphing bar graphs. Using fMRI, the authors identified brain areas where activity is modulated by the angles of morphing trajectories in six-fold symmetry. The strength of this paper lies in the question it attempts to address. Specifically, the question of whether and how the human brain uses grid-like representations not only for spatial navigation but also for navigating abstract concepts, such as social space, and guiding everyday decision-making. This question is of emerging importance.

The weak points of this paper are that its findings are not sufficiently supporting their arguments, and there are several reasons for this:

1. Does the grid-like activity reflect 'navigation over the social space' or 'navigation in sensory feature space'? The grid-like representation in this study could simply reflect the transition between stimuli (the length of bar graphs). Participants in this study associated each face with a specific length of two bars, and the 'navigation' was only guided by the morphing of a bar graph image. Moreover, any social cognition was not required to perform the task where they estimate the grid-like activity. To make social decision-making that was conducted separately, we do not know if participants needed to navigate between faces in a social space. Instead, they can recall bar graphs associated with faces and compute the decision values by comparing the length of bars. Notably, in the trust game in this study, competence and trustworthiness are not equally important to make a decision (Equation 1). The expected value is more sensitive to one over the other. This also suggests that the space might not reflect social values but perceptual differences.

2. Does the brain have a common representation of faces in a social space? In this study, participants don't need to have a map-like representation of six faces according to their levels of social traits. Instead, they can remember the values of each trait. The evidence of neural representations of the faces in a 2-dimensional social space is lacking. The authors argued that the relationship between the reaction times and the distances between faces provides evidence of the formation of internal representations. However, this can be found without the internal representation of the relationships between faces. If the authors seek internal representations of the faces in the brain, it would be important to show that this representation is not simply driven by perceptual differences between bar graphs that participants may recall in association with each face.

Considering these caveats, it is hard for me to agree if the authors provide evidence to support their claims.

Reviewer #1 (Public Review):

Summary:

In this study, the authors show that a long-non coding RNA lncDACH1 inhibits sodium currents in cardiomyocytes by binding to and altering the localization of dystrophin. The authors use a number of methodologies to demonstrate that lncDACH1 binds to dystrophin and disrupts its localization to the membrane, which in turn downregulates NaV1.5 currents. Knockdown of lncDACH1 upregulates NaV1.5 currents. Furthermore, in heart failure, lncDACH1 is shown to be upregulated which suggests that this mechanism may have pathophysiolgoical relevance.

Strengths:

1. This study presents a novel mechanism of Na channel regulation which may be pathophysiologically important.

2. The experiments are comprehensive and systematically evaluate the physiological importance of lncDACH1.

Weaknesses:

1. What is indicated by the cytoplasmic level of NaV1.5, a transmembrane protein? The methods do not provide details regarding how this was determined. Do you authors means NaV1.5 retained in various intracellular organelles?

2. What is the negative control in Fig. 2b, Fig. 4b, Fig. 6e, Fig. 7c? The maximum current amplitude in these seem quite different. -40 pA/pF in some, -30 pA/pF in others and this value seems to be different than in CMs from WT mice (<-20 pA/pF). Is there an explanation for what causes this variability between experiments and/or increase with transfection of the negative control? This is important since the effect of lncDACH1 is less than 50% reduction and these could fall in the range depending on the amplitude of the negative control.

3. NaV1.5 staining in Fig. 1E is difficult to visualize and to separate from lncDACH1. Is it possible to pseudocolor differently so that all three channels can be visualized/distinguished more robustly?

4. The authors use shRNA to knockdown lncDACH1 levels. It would be helpful to have a scrambled ShRNA control.

5. Is there any measurement on the baseline levels of LncDACH1 in wild-type mice? It seems quite low and yet is a substantial increase in NaV1.5 currents upon knocking down LncDACH1. By comparison, the level of LncDACH1 seems to be massively upregulated in TAC models. Have the authors measured NaV1.5 currents in these cells? Furthermore, does LncDACH1 knockdown evoke a larger increase in NaV1.5 currents?

6. What do error bars denote in all bar graphs, and also in the current voltage relationships?

Reviewer #1 (Public Review):

Sukumar et al build on a body of work from the Palmer lab that seeks to unravel the transcriptional targets of Alk signaling (a receptor tyrosine kinase). Having uncovered its targets in the mesoderm in an earlier study, they seek to determine its targets in the central nervous system. To do this, they use Targeted DamID (TaDa) in the wild-type and Alk dominant negative background and identify about 1700 genes that might be under the control of Alk signalling. Using their earlier data and applying a set of criteria - upregulated in gain-of-Alk, downregulated in loss-of-Alk, and co-expressed with Alk positive cells in single cell datasets - they arrive upon a single gene, Sparkly, which is predicted to be a neuropeptide precursor.

They generate antibodies and mutants for Sparkly and determine that it is responsive to Alk signalling and is expressed in many neuroendocrine cells, as well as in clock neurons. Though the mutants survive, they have reduced lifespans and are hyperactive. In summary, the authors identify a previously unidentified transcriptional target of Alk signalling, which is likely cleaved into a neuropeptide and is involved in regulating circadian activity.

The data support claims made, are generally well presented and the manuscript clearly written. The link between circadian control of Alk signalling in Clock neurons > Spar expression > ultimately controlling circadian activity, however, was not clear.

Reviewer #1 (Public Review):

This study was designed to examine the bypass of Ras/Erk signaling defects that enable limited regeneration in a mouse model of hepatic regeneration. The authors show that this hepatocyte proliferation is marked by expression of CD133 by groups of cells. The CD133 appears to be located on intracellular vesicles associated with microtubules. These vesicles are loaded with mRNA. The authors conclude that the CD133 vesicles mediate an intercellular signaling pathway that supports cell proliferation. These are new observations that have broad significance to the fields of regeneration and cancer.

The primary observation is that the limited regeneration observed in livers with Ras/Erk signaling defects is associated with CD133 expression by groups of cells. The functional significance of CD133 was tested using Prom1 KO mice - the data presented are convincing.

The major weakness of the study is that some molecular mechanistic details are unclear - this is, in part, due to the extensive new biology that is described. Nevertheless, the data used to support some key points in this study are unclear:

a) What is the evidence that the observed CD133 groups of cells are not due to clonal growth. Is this conclusion based on the time course (the groups appear more rapidly than proliferation) or is this based on the GFP clonal analysis?

b) What is the evidence that the CD133 vesicles mediate intercellular communication. This is an exciting hypothesis, but what is the evidence that this happens? Is this inferred from IEG mRNA diversity? or some other data. Is there direct evidence of transfer - for example, the does the GFP clonal analysis show transfer of GFP that is not mediated by clonal proliferation? Moreover, since the hepatocytes are isogenic, what distinguishes the donor and recipient cells?

Increased clarity concerning what is hypothesis and what is directly supported by data - would improve the presentation of this study.

Reviewer #1 (Public Review):

Summary:<br /> The authors use a combination of ChIP-seq, RNA-seq and ATAC-seq on FACS-purified germ cells to understand the changes in transcription and chromatin landscape of germline stem cells (GSCs) and their progeny during adult oogenesis.

Strengths:<br /> The major strengths of the paper include high quality -omics data, robust analyses of the data, and a well-written manuscript. The data strongly support the conclusions (1) that GSCs have more open chromatin than its differentiating daughter cells, (2) that H3K9me3 heterochromatin forms in 16-cell cyst stage and silences GSC-enriched genes, transposons, and testis-biased and somatic genes; (3) that GSC-enriched genes encoding cell cycle control, protein synthesis and signal transduction reside in clusters in autosomal pericentric regions; and (4) that there is a transcriptionally-driven metabolic reprogramming of nurse (germline) cells to aerobic glycolysis.

These data sets and analyses will have a high impact of the field of germline lifecycle (from GSC to primordial germ cells to GSCs again). The authors will make these data sets available through NCBI GEO and on Github. However, these are not incredibly user friendly and these data sets are extremely useful. I wonder if it is possible to incorporate these valuable data set into existing websites?

Weaknesses:<br /> There are no obvious weaknesses.

Reviewer #1 (Public Review):

The current manuscript builds on a previous publication from the same group(s) (https://doi.org/10.7554/eLife.77779) that identified several new interacting partners of the ESCRT pathway. The authors show via fluorescence polarization anisotropy (FPA) and NMR spectroscopy that the microtubule-interacting and trafficking (MIT) domains of CALPAIN7 bind to the IST1 subunit of the ESCRT-III complex. The authors used a powerful combination of biochemical, structural, and cell biological tools. The experiments are designed well and are performed to a high standard. The vast majority of the conclusions are supported by the data.

The authors report the X-ray crystal structure of the MIT-IST complex show the exact residues involved in the interaction and the mode of binding. They validate their findings and put them in a biological context by introducing mutations into the key residues in the MIT domain of CALPAIN7 and IST domain of ESCRT-III that can disrupt and restore the molecular interactions using in vitro biochemical assays and in vivo immunofluorescence microscopy imaging.

The writing is exceptional. I could not spot a single typographical or grammatical error. The manuscript is easy to read. It is concise and is nicely supported by high-quality figures. It was a pleasure to read.

The authors provide a great amount of detailed information about the experiments performed and have deposited the majority (if not all) of the plasmids used in the study in a public depository. They should be commended for this decision.

Reviewer #1 (Public Review):

Iskusnykh et al. present an elegant and thorough analysis of the role of transcription factor Lmx1a as a master regulator of the cortical hem, which is a secondary organizer in the brain. The authors report that loss of Lmx1a in the hem alters expression levels of Wnts, that Lmx1a is critical for hem progenitors to exit the cell cycle properly, and that Lmx1a loss leads to defects in CR cell differentiation and migration. Furthermore, the authors show that hem-like fate can be induced by overexpressing Lmx1a. This is a fundamental role for a transcription factor that was long used as a hem marker but was never examined for its function in the hem. This study has broader implications for how secondary organizers are created in the embryo and would be of great interest to a wide readership. The conclusions are broadly well supported by the data, though there are a few points of interpretation that need to be addressed.

(1) Figure 3A shows staining intensity in WT and Lmx1a-/- whereas the quantification has Lmx1a+/-. Both genotypes are relevant, -/- and +/-, to test whether the loss of 1 copy of Lmx1a results in a partial diminution of Wnt3a levels. Likewise, it is necessary to examine Wnt3a expression levels in the Wnt3a+/- embryo. Together, these could explain why the Lmx1a+/-; Wnt3a+/- double heterozygote has a DG phenotype, otherwise, it remains an unexplained though interesting observation.

(2) Line 309: "to test Wnt3a as a downstream mediator of Lmx1a function in CH/DG development, we performed an analysis of Lmx1a/Wnt3a double heterozygotes rather than Wnt3a overexpression rescue experiments in Lmx1a -/- mice."

The authors' reasoning is unclear. The double het experiments do not go on to show that one gene acts via the other. It's entirely possible the two act via parallel pathways.<br /> However, since Lmx1a does indeed regulate Wnt3a levels, this is a good argument for suggesting it acts via Wnt3a, even without the overexpression rescue. The authors could reorganize the data and rephrase the definitive "acts via" statement (also in the heading of this section, line 289, and discussion, line 553) to better fit the data.

(3) In the discussion section, the authors should include that trans-hilar and supragranular scaffold is disrupted in Lrp6 and Lef1 single as well as double mutants, which indicates Wnt signaling has a role to play in the morphogenesis of this scaffold. In this context, the author may discuss how Lmx1a could regulate this process via modulating Wnt signaling.

(4) Reduction in Tbr2 levels (Fig4B): E 13.5, not all Tbr2+ cells in the hem show a visible decrease in Tbr2 levels. The CR cells in the marginal zone show faint Tbr2. It would be useful if the staining intensity within the hem was quantified by dividing the section into three bins along the radial axis: Ventricular Zone, "Intermediate" zone, and Marginal zone to get a sense of the intensity profile. Co-labeling with p73 would identify CR cells and distinguish them from hem progenitors.

(5) Are the total number of Prox1+ cells at E14.5 similar between control and Lmx1a-/- ? Might the decrease in Prox1+ cells in the DG of P21 Lmx1a-/- animals occur due to granule cell death or because fewer cells were specified due to lower Wnts from the compromised Lmx1a-/- hem? The authors should examine cell death, labeling with CC3 and Prox1 together to test the cell death angle and discuss if the specification angle applies.

(6) In figure 6, the authors show that Lmx1a OE is sufficient to induce hem-like features, and identify p73+ cells (CR cell lineage). Is the choroid lineage not induced or was it not examined? A line to this effect would be useful. Also, the validation that it is indeed ectopic hem could be stronger with a few additional markers, since this is a striking finding.

Reviewer #1 (Public Review):

Huang, Kevin Y. et al. perform a meta-analysis of single-cell RNA-seq (scRNA-seq) data derived from 11 studies and across six tissues (liver, lung, heart, skin, kidney, endometrium) to address a focused hypothesis: pro-fibrotic SPP1+ macrophages that have been found in liver and lung tissue of idiopathic pulmonary fibrosis patients exist in other human tissues which can result in broader fibrotic disease states. The authors use existing, state-of-the-art single-cell analysis tools to perform the meta-analysis. They convincingly show that the SPP1+ macrophage population can be identified in lung, liver, heart, skin, uterus (endometrium), and kidney clusters derived from each tissues' scRNA-seq data. They further identify three subpopulations of the SPP1+ macrophages: a matrisome-associated macrophages (MAMs) defined as SPP1+MAM+ and two others enriched for inflammatory and ribosomal processes which they group together and define as SPP1+MAM-. Pathway analysis of genes unregulated in SPP1+MAM+ vs SPP1+MAM- cells yields significant enrichment of extracellular matrix remodeling and metabolism-related pathways and genes. This allows them to arrive at SPP1+MAM+ and SPP1+MAM- gene expression signature scores to further highlight the upregulation of these pathways in SPP1+MAM+ macrophages and their role in fibrosis. They explicitly show enrichment for SPP1+MAM+ macrophages in disease compared to healthy control subjects in a variety of tissues and their associated fibrosis-related diseases. Cell differentiation trajectory analysis identified 2 main trajectories: both starting from FCN1+ infiltrating monocytes/macrophages with one moving toward a homeostatic state and another toward SPP1+MAM+. They verified this using an alternative trajectory analysis approach. Importantly, for all tissues and fibrotic diseases, they found SPP1+MAM+ were at the end of the trajectory preceded by the SPP1+MAM- state, suggesting SPP1+MAM+ represents a common polarization state of SPP1+ macrophages. They develop a probability-based score that estimates the propensity of SPP1+MAM- macrophages to differentiate into SPP1+MAM+ and show that this was significantly higher in fibrotic disease subjects compared to healthy controls. They go on to identify the transcription factor networks (regulons) associated with SPP1+MAM+ differentiation and activation. They find a number of enriched regulons/transcription factors and through a linear-modeling trajectory analysis highlight the regulons that are associated specifically with the SPP1+MAM- to SPP1+MAM+ transition. In this way, they prioritize the NFATC1 and HIVEP3 regulations as driving the differentiation of SPP1+MAM- macrophages toward the SPP1+MAM+ polarization state. Finally, given that age is a risk factor for fibrotic disease, they assessed the association of SPP1+MAM+ and SPP1+MAM- gene signatures in healthy control old and young human subjects as well as old and young mice and found SPP1+MAM+ was either exclusively (human) or more significantly (mice) elevated in old versus young compared to SPP1+MAM-.

The strengths of this paper are the authors gathered a number of relevant single-cell RNA-seq data sets from fibrosis-focused studies to address a highly focused hypothesis (stated above). They gained the power to detect the population of SPP1+MAM+ cells by integrating these datasets. The analysis is carried out well using existing state-of-the-art tools. With whatever metric or single cell analysis-based discovery they make about the SPP1+MAM+ subpopulations (e.g., gene signatures, endpoint of trajectory analysis, associated regulons, etc), they compare the relevant scoring metrics in fibrosis and control subjects at every stage of the meta-analysis and find the SPP11+MAM+ is consistently higher across tissues and fibrosis-related diseases.

There are only minor weaknesses in this paper. One is that some of the most highly significant or simply significant results are not shown in main figures but are summarized in supplementary tables (e.g., MYC TARGETS V1 would have appeared as the most significant, highest enriched, and among the largest in terms of set size). Another is analysis criteria that may not yield the most biologically relevant or impactful conclusion (e.g., while the regulon THRA does not display a shift in slopes it shows the strongest, progressive increase going toward the SPP1+MAM+ state).

Reviewer #1 (Public Review):

The manuscript by Salloum and colleagues examines the role of statin-mediated regulation of mitochondrial cholesterol as a determinant of epigenetic programming via JMJD3 in macrophages.

Key strengths of the work include:

1. Mechanistic analysis of how statin treatments can remodel the mitochondrial membrane content via cholesterol depletion which in turn affects JMJD3 levels is a novel concept.

2. Use of RNA-seq and ATAC-seq data provides an avenue for unbiased analysis of the statin effects.

3. Use of methyl-cyclodextrin (MCD) alongside statins increases the robustness of the findings and the use of NFKB inhibitors suggests a mechanistic role for NFKB.

The conclusions are only partially supported by the presented data:

1. There is a lack of any in vivo studies that are required to demonstrate that the concentrations of statins used to induce epigenetic programming of macrophages are physiologically relevant. There have been numerous studies that have examined the anti-inflammatory effects of statins but there is significant debate and controversy regarding the in vivo relevance. Much of the in vivo effects of statins are achieved via changes in systemic cholesterol levels but the direct effects on macrophages are not clear.

2. "Statins" is used globally and it is unclear which statins were used, which doses of statins, and the treatment durations

3. The RNA-seq, ATAC-seq, and selected H3K27 ChIP only show a snapshot of the results without leveraging the power of unbiased analysis. Such an unbiased analysis could show whether the examined genes are indeed the most relevant targets of statins.

4. CCCP depletion can have broad toxic effects and it is difficult to interpret specific roles of ATP synthase from potentially toxic mitochondrial uncoupling.

Reviewer #1 (Public Review):

In this manuscript, Drs. Miura, Mori, and colleagues, first present lineage tracing data using PDGFRa-CreERT2 and Foxa2-Cre drivers to show that PDGFRa+ cells, when lineage-labeled early in development go on to form the lung mesenchyme (but little to none of the epithelium), whereas FOXA2 expressing cells go on to contribute to both the lung epithelium and lung mesenchyme. However, it is already well known that FOXA2 is expressed in the mesendoderm around the time of gastrulation, and that this population generates both endoderm and mesodermal derivatives. As a result, it is not surprising that lineage labeling this population would contribute to both the lung epithelium and lung mesenchyme. The authors use the term bona fide lung (BFL) generative lineage. However, since the mesendoderm contributes to both the endoderm and mesoderm, but is by no means specific to the lung, and as shown in this paper (Figure 2G) the FOXA2 population only generates 30-40% of the mesenchyme, the term BFL is both confusing and misleading.

In the second portion of the manuscript, the authors conditionally delete Fgfr2 using a Foxa2-Cre driver. Although loss of Fgf10 or Fgfr2 is known to result in lung agenesis, deletion of Fgfr2 within the FOXA2+ expressing cells is novel. However, since FOXA2 is broadly expressed within the nascent lung epithelium and Fgfr2 is known to be expressed within the lung epithelium, it isn't entirely clear how much information this adds beyond what already known from other Fgfr2 knockout studies. Perhaps the most interesting aspect of the reported phenotype is that the other organs (e.g. intestine) in these knockout animals appears to be relatively spared. This should be better characterized by the authors, as currently only a few H&E images are shown.

The authors then used conditional blastocyst complementation with nGFP+iPSCs from wild-type mice to rescue the phenotype of the Fgfr2 conditional knockout mice, showing that an embryonic lung is formed. However, blastocyst complementation has previously been performed with other knockout mouse models with severe lung hypoplasia/aplasia, including Dr. Mori's previous Nature Medicine paper. Although most of the previous mouse models target the endoderm/early epithelial cells (e.g. conditional deletion of Ctnnb1, Fgfr2, or global knockout of Nkx2.1; see Li E, et al. Dev Dyn 2021 Jul;250(7):1001-1020; Wen B, Am J Resp Crit Care Med. 2020; in addition to Mori M, Nature Medicine, 2019), Kitahara A, et al (Cell Rep. May 12 2020;31(6):107626) previously reported blastocyst complementation in in Fgf10 null mouse model, so it is not clear what the current study significantly adds contributes to this existing body of literature. The lungs of the mice undergoing blastocyst complementation are also incompletely characterized in the current version of this study. For example, it is unclear how functional these lungs are and whether they are capable of gas exchange after birth.

Reviewer #1 (Public Review):

In this manuscript, the authors examine the role of Sirt2 on cardiac hypertrophy by using 2 in-vivo models- systemic KO of Sirt2 and cardiac-specific KO of Sirt 2. The authors show that Sirt2 is important for the development of heart failure and cardiac hypertrophy. Mechanistically, the authors show that Sirt2 regulates NRF2 and that deletion of Sirt2 is protective through stabilization and increased nuclear translocation of NRF2. The paper is clinically relevant and the data is of high quality.

Reviewer #1 (Public Review):

Sangar and co-workers have shown the role of Syntaxin 6 on prion (PrP) protein aggregation and have claimed a possible molecular mechanism behind its role in pathogenesis of Prion diseases like CJD. Authors have elaborately shown the aggregation kinetics of PrP in absence and presence of Stx6 by their in-house developed NAA assay which allowed them to catch the interaction of PrP with other cellular proteins like Stx6 or Hspa1a. By in vitro aggregation kinetics assays, authors show that Stx6 delays the aggregation kinetics of PrP and leads to amorphous aggregates rather than well-formed fibrils. By a neurite length detection assay with treatment of preformed PrP-fibrils (with or without Stx6 interaction), they show enhanced toxicity of Prp-Stx6 aggregates compared to PrP fibrils. However, there are a number of concerns related to physiological relevance that need to be addressed by the authors.

Reviewer #1 (Public Review):

In this manuscript, the authors compare the behavior of the intracellular ion channel TPC1 in two species of plants. TPC1 channels are cation permeable pseudo tetramers which in plants are expressed in the intracellular vacuole and whose function is not well understood.

It was previously known that calcium ions are modulators of the function of these channels and the authors identify a cluster of negative charges facing the lumen of the vacuole that can regulate the action of calcium and modulate the voltage dependence of the channels. Interestingly, this cluster is not entirely present in the faba bean (Vicia faba) vfTPC1 channel.

Through electrophysiological recordings of transfected channels into TPC1-lacking vacuoles, the authors show that the vfTPC1 channels activate at more negative voltages, even in the presence of high concentrations of calcium, leading to cation entrance into the vacuole and increased excitability, as assessed by action potential firing.

Though the findings are interesting and the methods are of high quality, the authors fail to convey the importance of the problem they are tackling and do not frame their findings in the broad physiology of the plant species they study.

These findings should be of interest to plant physiologists and channel biophysicists interested in TPC1 channels.

(~10:00) "The context determines the meaning of the content."

Thus reframing is very powerful as you recontextualize the past, and therefore see it in a whole new light; the meaning of the past changed.

By asking what you have learned from the past, you become anti-fragile and flexible, as you turn the past into something useful; an asset.

"The past happens for us, not to us."

"How you frame the past influences your expectations for the future."

"You can't disconnect your view of the future from your experience in the present."

"You can't have meaning in the present without hope & purpose in the future."

One of the powerful things about journaling is that you can control the past; reframe it. What is the meaning of the past gets determined by both the present and the future.

Hardy recommends to often (even daily) reflect on the past and notice how different you are now compared to then. What you have achieved, what is possible now that was not possible then, etc.

What did I learn today?

• for: W2W, inequality, 1%, oligarch, elites, kleptocrats, civil society - exploitation, TUSN, Transnational Uncivil Society Network
• title: How the kleptocrats and oligarchs hunt civil society groups to the ends of the Earth
• author: Peter Rinko
• comment
  • This article explores the research work of Alexander Cooley, John Heathershaw and Ricard Soares de Oliverira who coauthored the paper

Transnational Uncivil Society Networks: kleptocracy’s global fightback against liberal activism - reference - https://ora.ox.ac.uk/objects/uuid:5e5a3052-c693-4991-a7cc-bc2b47134467/download_file?file_format=application%2Fpdf&safe_filename=Cooley_et_al_2023_transnational_uncivil_society.pdf&type_of_work=Journal+article

Reviewer #1 (Public Review):

Guan et al. explored the mechanisms responsible for the development, maintenance, and functional properties of a specific subset of unconventional T cells expressing a Va3.2 T cell receptor that recognizes a peptide, QFL, presented by the class Ib protein Qa-1. Prior studies from this group showed that cells from mice deficient in the ER protease ERAAP elicit responses in wild-type animals enriched for Qa-1-restricted CD8 T cells. They further showed that a significant proportion of these responses were directed against the QFL peptide derived from a conserved protein with incompletely understood functions. Many of these so-called QFL T cells expressed Va3.2-Ja21, were present in the spleen of wild-type mice, and exhibited a memory-like phenotype. Due to their relatively low frequency and weak staining with Qa-1 tetramers, analyzing QFL T cells has been challenging. Therefore, the authors generated dextramers, which permitted them to more rigorously identify these cells. They confirmed some of their previous findings and further showed that Va3.2+ and Va3.2- QFL T cells were present in the intestinal epithelium, where they also express CD8alpha homodimers, a characteristic of most small intestinal intraepithelial lymphocytes (siIELs), and most similar to the so-called natural siIELs that acquire their innate functions in the thymus. The authors show that TAP but not Qa-1 or ERAAP expression are required for the development of these cells, and both Qa-1 and ERAAP are required for the natural siIEL phenotype. Some of these findings were confirmed using a new TCR transgenic mouse expressing the QFL TCR. They further show that retention but not homing of QFL T cells to the intestinal epithelium involves commensal microorganisms, and using in silico approaches, they identify a commensal that contains a peptide similar to QFL that can activate QFL T cells. Finally, they show that this organism, P. pentosaceus, can promote gut retention of QFL T cells when it is introduced into germ-free mice. From these findings, the authors conclude that the microbiota influences the maintenance of Qa-1-restricted T cells.

Comments:<br /> 1. Overall, the authors employ a number of new reagents and elegant approaches to explore the development, maintenance, and functional properties of QFL T cells.<br /> 2. Generally, conclusions made are well supported by the data presented.<br /> 3. One limitation of the work is that the immunological functions of QFL T cells remain unclear.<br /> 4. The work covers a lot of ground (intestinal IELs, unconventional T cells, innate/virtual memory T cells, Qa-1/HLA-E, etc) that may not be familiar to many readers.<br /> 5. A few questions remain:<br /> a) Regarding the results for TAP knockout animals, since Qa-1 does not appear to be required for QFL T cell development, the absence of these cells in TAP KO mice cannot be easily explained.<br /> b) The Va3.2 T cells display similarities with previously identified innate/virtual memory T cells, some of which require IL-4 production by CD1d-restricted NKT cells for their intrathymic development, which is not fully discussed.<br /> c) Qa-1/peptide complexes may also be recognized by CD94/NKG2 (both inhibitory and activating) receptors on NK cells and subsets of CD8 T cells, which may complicate data interpretation, but is not noted in the text.<br /> d) Are these conclusions relevant to the human homolog of Qa-1, HLA-E?

Reviewer #1 (Public Review):

By identifying a loss of function mutant of IQCH in an infertile patient, Ruan et al. show that IQCH is essential for spermiogenesis by generating a knockout mouse model of IQCH. Similar to infertile patients with a mutant of IQCH, IQCH knockout mice are characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. Mechanistically, IQCH regulates the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis.

Although this manuscript contains a potentially interesting piece of work that delineates a mechanism of IQCH that associates with spermatogenesis, this reviewer feels that a number of issues require clarification and re-evaluation for a better understanding of the role of IQCH in spermatogenesis. With the shortage of logic and supporting data, causal relationships are still not clear among IQCH, CaM, and HNRPAB. The most serious point in this manuscript could be that the authors try to generalize their interpretations with a model that is too simplified from limited pieces of their data. The way the data and the logic are presented needs to be largely revised, and several interpretations should be supported by direct evidence.

Reviewer #1 (Public Review):

Recent studies in plants and human cell lines argued for a central role of 1,5-InsP8 as the central nutrient messenger in eukaryotic cells, but previous studies concluded that this function is performed by 1-InsP7 in baker's yeast. Chabert et al now performed an elegant set of capillary electrophoresis coupled to mass spectrometry time course experiments to define the cellular concentrations of different inositol pyrophosphosphates (PP-InsPs) in wild-type yeast cells under normal and phosphate (Pi) starvation growth conditions. These experiments, in my opinion, form the center of the present study and clearly highlight that the levels of all major PP-InsPs drop under Pi starvation, with the 1,5-InsP8 isomer showing the most rapid changes.

The analysis of known mutants in the PP-InsP biosynthetic pathways furthermore demonstrate that loss-of-function of the PPIP5K enzymes Kcs1 and Vip1 result in a loss of 1,5-InsP8 and a hyperaccumulation of 5-InsP7, respectively. In line with this, loss-of-function of known PP-InsP phosphatases Ddp1 and Swi14 result in hyperaccumulation of either 1- or 5-InsP7, as anticipated from their in vitro substrate specificities. These experiments are of high technical quality and add to our understanding of the kinetics of PP-InsP metabolism/catabolism in yeast.

Next, the authors use changes in subcellular localisation of the central transcription factor Pho4 to assay at which time point after onset of Pi starvation the PHO pathway becomes activated. The early onset of the response, the behavior of the kcs1D mutant and of the ksc1D/vip1D all strongly argue for 1,5-InsP8 as the central nutrient messenger. I find this part of the manuscript well argued, nicely correlating PP-InsP levels, dynamics and the different mutant phenotypes.

The third part of the manuscript is a structure-function study of the CDK inhibitor Pho81, basically using a reverse genetics approach. This analysis demonstrates at the genetic level that the Pho81 SPX domain is required for activation of the PHO pathway. Next, the authors design point mutations that should block either interaction of Pho81-SPX with 1,5-InsP8 or interaction of Pho81 with the Pho80/Pho85 complex. In my opinion, these data can only provide limited insight into the molecular mechanism, as no complementary in vitro binding assays / in vivo co-IP experiments with the wild-type and mutant forms of Pho81 are presented. This seems to be due to technical limitations in recombinantly expressing and purifying the respective Pho81 protein for in vitro PP-InsP binding and protein - protein interaction assays.

Taken together, the work by Chabert et al, reinvestigates and clarifies the activation of the yeast PHO pathway by PP-InsP nutrient messengers and their cellular SPX receptors. From this work, a more unified eukaryotic mechanism emerges, in which 1,5-InsP8 represents the central signaling molecule in different species, with conserved SPX receptors sensing this signaling molecule.

Reviewer #1 (Public Review):

The authors of the manuscript cultivated a Planctomycetes strain affiliated with Phycisphaerae. The strain was one of the few Planctomycetes from deep-sea environments and demonstrated several unique characteristics, such as being the only known Phycisphaerae using a budding mode of division, extensive involvement in nitrate assimilation, and being able to release phage particles without cell death. The manuscript is generally well-written. However, a few issues need to be more clearly addressed, especially regarding the identification and characterization of the phage.

Reviewer #1 (Public Review):

The authors investigate how the viscoelasticity of the fingertip skin can affect the firing of mechanoreceptive afferents and they find a clear effect of recent physical skin state (memory), which is different between afferents. The manuscript is extremely well-written and well-presented. It uses a large dataset of low threshold mechanoreceptive afferents in the fingertip, where it is particularly noteworthy that the SA-2s have been thoroughly analyzed and play an important role here. They point out in the introduction the importance of the non-linear dynamics of the event when an external stimulus contacts the skin, to the point at which this information is picked up by receptors. Although clearly correlated, these are different processes, and it has been very well-explained throughout. I have some comments and ideas that the authors could think about that could further improve their already very interesting paper. Overall, the authors have more than achieved their aims, where their results very much support the conclusions and provoke many further questions. This impact of the previous dynamics of the skin affecting the current state can be explored further in so many ways and may help us to better understand skin aging and the effects of anatomical changes of the skin.

At the beginning of the Results, it states that FA-2s were not considered as stimuli and did not contain mechanical events with frequency components high enough to reliably excite them. Was this really the case, did the authors test any of the FA-2s from the larger dataset? If FA-2s were not at all activated, this is also relevant information for the brain to signal that it is not a relevant Pacinian stimulus (as they respond to everything). Further, afferent receptive fields that were more distant to the stimulus were included, which likely fired very little, like the FA-2s, so why not consider them even if their contribution was low?

One question that I wondered throughout was whether you have looked at further past history in stimulation, i.e. not just the preceding stimulus, but 2 or 3 stimuli back? It would be interesting to know if there is any ongoing change that can be related back further. I do not think you would see anything as such here, but it would be interesting to test and/or explore in future work (e.g. especially with sticky, forceful, or sharp indentation touch). However, even here, it could be that certain directions gave more effects.

Did the authors analyze or take into account the difference between receptive field locations? For example, did afferents more on the sides have lower responses and a lesser effect of history?

Was there anything different in the firing patterns between the spontaneous and non-spontaneously active SA-2s? For example, did the non-spontaneous show more dynamic responses?

Were the spontaneously active SA-2 afferents firing all the time or did they have periods of rest - and did this relate to recent stimulation? Were the spontaneously active SA-2s located in a certain part of the finger (e.g. nail) or were they randomly spread throughout the fingertip? Any distribution differences could indicate a more complicated role in skin sensing.

Did the authors look to see if the spontaneous firing in SA-2s between trials could predict the extent to which the type 1 afferents encode the proceeding stimulus? Basically, does the SA-2 state relate to how the type 1 units fire?

In the discussion, it is stated that "the viscoelastic memory of the preceding loading would have modulated the pattern of strain changes in the fingertip differently depending on where their receptor organs are situated in the fingertip". Can the authors expand on this or make any predictions about the size of the memory effect and the distance from the point of stimulation?

In the discussion, it would be good if the authors could briefly comment more on the diversity of the mechanoreceptive afferent firing and why this may be useful to the system.

Also, the authors could briefly discuss why this memory (or recency) effect occurs - is it useful, does it serve a purpose, or it is just a by-product of our skin structure? There are examples of memory in the other senses where comparisons could be drawn. Is it like stimulus adaptation effects in the other senses (e.g. aftereffects of visual motion)?

One point that would be nice to add to the discussion is the implications of the work for skin sensing. What would you predict for the time constant of relaxation of fingertip skin, how long could these skin memory effects last? Two main points to address here may be how the hydration of the skin and anatomical skin changes related to aging affect the results. If the skin is less viscoelastic, what would be the implications for the firing of mechanoreceptors?

How long does it take for the effect to end? Again, this will likely depend on the skin's viscoelasticity. However, could the authors use it in a psychophysical paradigm to predict whether participants would be more or less sensitive to future stimuli? In this way, it would be possible to test whether the direction modifies touch perception.

Reviewer #1 (Public Review):

This paper describes the development and initial validation of an approach-avoidance task and its relationship to anxiety. The task is a two-armed bandit where one choice is 'safer' - has no probability of punishment, delivered as an aversive sound, but also lower probability of reward - and the other choice involves a reward-punishment conflict. The authors fit a computational model of reinforcement learning to this task and found that self-reported state anxiety during the task was related to a greater likelihood of choosing the safe stimulus when the other (conflict) stimulus had a higher likelihood of punishment. Computationally, this was represented by a smaller value for the ratio of reward to punishment sensitivity in people with higher task-induced anxiety. They replicated this finding, but not another finding that this behavior was related to a measure of psychopathology (experiential avoidance), in a second sample. They also tested test-retest reliability in a sub-sample tested twice, one week apart and found that some aspects of task behavior had acceptable levels of reliability. The introduction makes a strong appeal to back-translation and computational validity. The task design is clever and most methods are solid - it is encouraging to see attempts to validate tasks as they are developed. The lack of replicated effects with psychopathology may mean that this task is better suited to assess state anxiety, or to serve as a foundation for additional task development.

Reviewer #1 (Public Review):

This manuscript describes identification of influential organisms on rice growth and an attempt of validation. The analysis of eDNA on rice pot and mimic field provides rice growth promoting organisms. This approach is novel for plant ecology field. However current results did not fully support whether eDNA analysis-based detection of influencing organism.

The strength of this manuscript is to attempt application of eDNA analysis-based plant growth differentiation. The weakness is too preliminary data and experimental set-up to make any conclusion. The trials of authors experiments are ideal. However, the process of data analysis did not meet certain level. For example, eDNA analysis of different time points on rice growth stages resulted two influential organisms for rice growth. Then they cultivate two species and applied rice seedlings. Without understanding of fitness and robustness, how we can know the effect of the two species on rice growth.

The authors did not check the fate of two species after introducing into rice. If this is true, it is difficult to link between the rice gene expression after treatments and the effectiveness of two species. I think the validation experiment in 2019 needs to be re-conducted.

As authorized gave answered, no strong rationale to select the two species was found. However, I insist that the method has enough novelty to present to general audiences.

Reviewer #1 (Public Review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with a lipid composition similar to the native virion. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. Additional contacts and conformational restraints imposed by ectodomain regions of the envelope glycoprotein, however, remain unaddressed-the size of such simulations likely runs into technical limitations including sampling and compute time.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive. However, given the large amount of data presented within the manuscript, the text would benefit from clearer subsections that delineate discrete mechanistic discoveries, particularly for experimentalists interested in antibody discovery and design. One area the paper does not address involves the polyreactivity associated with membrane binding antibodies-MD simulations and/or pulling velocity experiments with model membranes of different compositions, with and without model antigens, would be needed. Finally, given the challenges in initializing these simulations and their limitations, the text regarding their generalized use for discovery, rather than mechanism, could be toned down.

Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #1 (Public Review):

The authors of the manuscript "High-resolution kinetics of herbivore-induced plant volatile transfer reveal tightly clocked responses in neighboring plants" assessed the effects of herbivory-induced maize volatiles on receiver plants over a period of time in order to assess the dynamics of the responses of receiver plants. Different volatile compound classes were measured over a period of time using PTR-ToF-MS and GC-MS, under both natural light:dark conditions, and continuous light. They also measured gene expression of related genes as well as defense-related phytohormones. The effects of a secondary exposure to GLVs on primed receiver plants were also measured.

The paper addresses some interesting points, however, some questions arise regarding some of the methods employed. Firstly, I am wondering why VOCs (as measured by GC-MS) were not quantified. While I understand that quantification is time-consuming and requires more work, it allows for comparisons to be made between lines of the same species, as well as across other literature on the subject. As experiments with VOC dispensers were also used in this experiment, I find it even more baffling that the authors didn't confirm the concentration of the emission from the plants they used to make sure they matched. The references cited justifying the concentration used (saying it was within the range of GLVs emitted by their plants) to prepare the dispenser were for either a different variety of maize (delprim versus B73) or arabidopsis. Simply relying on the area under the curve and presenting results using arbitrary units is not enough for analyses like these.

With regards to the correlation analyses shown in Figure 6, the results presented in many of the correlation plots are not actually informative. By blindly reporting the correlation coefficient important trends are being ignored, as there are clearly either bimodal relationships (e.g. upper left panel, HAC/TMTT, HAC/MNT) or even stranger relationships (e.g. upper left panel, IND/SQT, IND/MNT) that are not being well explained by a correlation plot. It is not appropriate to discuss the correlation factors presented here and to draw such strong conclusions on emission kinetics. The comparison between plants under continuous light and normal light:dark conditions is interesting, but I think there are better ways to examine these relationships, for example, multivariate analysis might reveal some patterns.

In Figure 2, the elevated concentrations of beta-caryophyllene found in the control plants at 8h and 16.75h measurement timepoints are curious. Is this something that is commonly seen in B73? While there can be discrepancies between emissions and compounds actually present within leaf tissue, it is a little bit odd that such high levels of b-caryophyllene were found at these timepoints, however, this is not reflected in the PTR-ToF-MS measurements of sesquiterpenes. It would be beneficial to include an overview of the mechanism of production and storage of sesquiterpenes in maize leaves, which would clarify why high amounts were found only in the GC-MS analysis and not the PTR-ToF-MS analysis, which is a more sensitive analytical tool. It is possible that the amounts of b-caryophyllene present in the leaf are actually extremely low, however as the values are not given as a concentration but rather arbitrary units, it is not possible to tell. I would include a line explaining what is seen with b-caryophyllene. Additionally, it seems like the amounts of TMTT within the leaf are extraordinarily high (judging only by the au values given for scale), far higher than one would expect from maize.

Reviewer #1 (Public Review):

In this paper, the authors show that disruption of calcineurin, which is encoded by tax-6 in C. elegans, results in increased susceptibility to P. aeruginosa, but extends lifespan. In exploring the mechanisms involved, the authors show that disruption of tax-6 decreases the rate of defecation leading to intestinal accumulation of bacteria and distension of the intestinal lumen. The authors further show that the lifespan extension is dependent on hlh-30, which may be involved in breaking down lipids following deficits in defecation, and nhr-8, whose levels are increased by deficits in defecation. The authors propose a model in which disruption of the defecation motor program is responsible for the effect of calcineurin on pathogen susceptibility and lifespan, but do not exclude the possibility that calcineurin affects these phenotypes independently of defecation.

Reviewer #1 (Public Review):

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

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. Although the authors provide evidence that CRB3 promotes ciliogenesis via (indirect) physical association with Rab11 and gamma-TuRC, the precise mechanism by which CRB3 promotes ciliogenesis remains to be clarified.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript provides some valuable findings concerning the hippocampal circuitry and the potential role of adult-born granule cells in an interesting long-term social memory retrieval. The behavior experiments and strategy employed to understand how adult-born granule cells contribute to long-term social discrimination memory are interesting.

I have a few concerns, however with the strength of the evidence presented for some of the experiments. The data presented and the method described is incomplete in describing the connection between cell types in CA2 and the projections from abGCs. Likewise, I worry about the interpretation of the data in Figures 1 and 2 given the employed methodology. I think that the interpretation should be broadened. This second concern does not impact the interest and significance of the findings.

Strengths:<br /> The behavior experiments are beautifully designed and executed. The experimental strategy is interesting.

Weaknesses:<br /> The interpretation of the results may not be justified given the methods and details provided.

Reviewer #1 (Public Review):

The evolution of dioecy in angiosperms has significant implications for plant reproductive efficiency, adaptation, evolutionary potential, and resilience to environmental changes. Dioecy allows for the specialization and division of labor between male and female plants, where each sex can focus on specific aspects of reproduction and allocate resources accordingly. This division of labor creates an opportunity for sexual selection to act and can drive the evolution of sexual dimorphism.

In the present study, the authors investigate sex-biased gene expression patterns in juvenile and mature dioecious flowers to gain insights into the molecular basis of sexual dimorphism. They find that a large proportion of the plant transcriptome is differentially regulated between males and females with the number of sex-biased genes in floral buds being approximately 15 times higher than in mature flowers. The functional analysis of sex-biased genes reveals that chemical defense pathways against herbivores are up-regulated in the female buds along with genes involved in the acquisition of resources such as carbon for fruit and seed production, whereas male buds are enriched in genes related to signaling, inflorescence development and senescence of male flowers. Furthermore, the authors implement sophisticated maximum likelihood methods to understand the forces driving the evolution of sex-biased genes. They highlight the influence of positive and relaxed purifying selection on the evolution of male-biased genes, which show significantly higher rates of non-synonymous to synonymous substitutions than female or unbiased genes. This is the first report (to my knowledge) highlighting the occurrence of this pattern in plants. Overall, this study provides important insights into the genetic basis of sexual dimorphism and the evolution of reproductive genes in Cucurbitaceae.

There are, however, parts of the manuscript that are not clearly described or could be otherwise improved.

- The number of denovo-assembled unigenes seems large and I would like to know how it compares to the number of genes in other Cucurbitaceae species. The presence of alternatively assembled isoforms or assembly artifacts may be still high in the final assembly and inflate the numbers of identified sex-biased genes.

- It is interesting that the majority of sex-biased genes are present in the floral buds but not in the mature flowers. I think this pattern could be explored in more detail, by investigating the expression of male and female sex-biased genes throughout the flower development in the opposite sex. It is also not clear how the expression of the sex-biased genes found in the buds changes when buds and mature flowers are compared within each sex.

- The statistical analysis of evolutionary rates between male-biased, female-biased, and unbiased genes is performed on samples with very different numbers of observations, therefore, a permutation test seems more appropriate here.

- The impact of pleiotropy on the evolutionary rates of male-biased genes is speculative since only two tissue samples (buds and mature flowers) are used. More tissue types need to be included to draw any meaningful conclusions here.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript of Zhao et al. aimed at investigating the relationships between type 2 diabetes, bone mineral density (BMD) and fracture risk using Mendelian Randomization (MR) approach.<br /> The authors found that genetically predicted T2D was associated with higher BMD and lower risk of fracture, and suggested a mediated effect of RSPO3 level. Moreover, when stratified by the risk factors secondary to T2D, they observed that the effect of T2D on the risk of fracture decreased when the number of risk factors secondary to T2D decreased.

Strengths:<br /> - Important question<br /> - Manuscript is overall clear and well-written<br /> - MR analyses have been conducted properly, which include the usage of various MR methods and sensitivity analyses, and likely meet the criteria of the MR-strobe checklist to report MR results.

Weaknesses:<br /> - Previous MR studies on that topic have not been discussed<br /> - Multivariable MR could have been used to better assessed the mediative effect of BMI or RSPO3 on the relationships between T2D and fracture risk.

Reviewer #1 (Public Review):

In this manuscript, the authors investigate differences between Tibetans and Han Chinese at altitude in terms of placental transcriptomes during full-term pregnancy. Most importantly, they found that the inter-population differentiation is mostly male-specific and the observed direction of transcriptional differentiation seems to be adaptive at high altitude. In general, it is of great importance and provides new insights into the functional basis of Tibetan high-altitude adaptations, which so far have been mostly studied via population genetic measures only. More specifically, I firmly believe that we need more phenotype data (including molecular phenotypes such as gene expression data) to fully understand Tibetan adaptations to high altitude, and this manuscript is a rare example of such a study. I have a few suggestions and/or questions with which I hope to improve the manuscript further, especially in terms of 1) testing if the observed DEG patterns are truly adaptive, and 2) how and whether the findings in this study can be linked to EPAS1 and EGLN1, the signature adaptation genes in Tibetans.

Major Comments:

1. The DEG analysis is the most central result in this manuscript, but the discrepancy between sex-combined and sex-specific DEGs is quite mind-boggling. For those that were differentially expressed in the sex-specific sets but not in the sex-combined one, the authors suggest an opposite direction of DE as an explanation (page 11, Figure S5). But Figure S5A does not show such a trend, showing that down-regulated genes in males are mostly not at all differentially expressed in females. Figure S6B does show such a trend, but it doesn't seem to be a dominant explanation. I would like to recommend the authors test alternative ways of analysis to boost statistical power for DEG detection other than simply splitting data into males and females and performing analysis in each subset. For example, the authors may consider utilizing gene-by-environment interaction analysis schemes here biological sex as an environmental factor.

2. Please clarify how the authors handled multiple testing correction of p-values.

3. The "natural selection acts on the placental DEGs ..." section is potentially misleading readers to assume that the manuscript reports evidence for positive selection on the observed DEG pattern between Tibetans and Han, which is not.<br /> a) Currently the section simply describes an overlap between DEGs and a set of 192 genes likely under positive selection in Tibetans (TSNGs). The overlap is quite small, leading to only 13 genes in total (Figure 6). The authors are currently not providing any statistical measure of whether this overlap is significantly enriched or at the level expected for random sampling.<br /> b) The authors are describing sets of DEGs that seem to affect important phenotypic changes in a consistent and adaptive direction. A relevant form of natural selection for this situation may be polygenic adaptation while the authors only consider strong positive selection at a single variant/gene level.<br /> c) The manuscript is currently providing no eQTL information that can explain the differential expression of key genes. The authors can actually do this based on the genotype and expression data of the individuals in this study. Combining eQTL info, they can set up a test for polygenic adaptation (e.g. Berg and Coop; https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1004412). This will provide a powerful and direct test for the adaptiveness of the observed DEG pattern.

4. The manuscript is currently only minimally discussing how findings are linked to EPAS1 and EGLN1 genes, which show the hallmark signature of positive selection in Tibetans. In fact, the authors' group previously reported male-specific association between EPAS1 SNPs and blood hemoglobin level. Many readers will be intrigued to see a discussion about this point.

Reviewer #1 (Public Review):

The authors report a new bioinformatics pipeline ("SPICE") to predict pairwise cooperative binding-sites based on input ChIP-seq data for transcription factor (TF)-of-interest, analyzed against DNA-binding sites (DNA motifs) in a database (HOCOMOCO). The pipeline also predicts the optimal distance between the paired binding sites. The pipeline correctly predicted known/reported transcription factor cooperations, and also predicted new cooperations, not yet reported in literature. The authors choose to follow up on the predicted interaction between Ikaros and Jun. Using ChIP-seq in mouse B cells, they show extensive overlap in binding regions between Ikaros and Jun in LPS+IL21 stimulated cells. In a human B-lineage cell line (MINO) they show that anti-Ikaros Ab can co-immunoprecipitate Jun protein, and that the MINO cell extracts contain protein(s) that can bind to the CNS9 probe (conserved region upstream of IL10 gene), and that binding is lost upon mutation of two basepairs in the AP1 binding motif, and reduced upon mutation of two basepairs in the non-canonical Ikaros binding motif. Part of this protein complex is super-shifted with an anti-Jun antibody, and more DNA is shifted with addition of an anti-Ikaros antibody.

The authors perform EMSA showing that recombinant Jun can bind to the tested DNA-region (IL10 CNS9) and that addition of recombinant Ikaros (or anti-Ikaros antibody in Fig 3E) can enhance binding (increase amount of DNA shifted). The authors lastly show that the IL10 CNS9 DNA region can enhance transcription in B- and T-cells with a luciferase reporter assay, and that 2 bp mutation of the Ikaros or Jun DNA motifs greatly reduce or abolish this activity.

This is interesting work, with two main contributions: The SPICE pipeline (if made available to the scientific community), and the report of interaction between Ikaros and Jun. However, the distinction between DNA motifs, and the proteins actually binding and having a biological function, should be made clear consistently throughout the manuscript. The same DNA motifs can be bound by multiple factors, for instance within transcription factor families with highly homology in the DNA-binding regions of the proteins.

Some specific points:

SPICE: It is unclear if this is uploaded somewhere to be available to the scientific community.

It was unclear if Ikaros-Jun interaction was initially found from primary Jun ChIP-seq (and secondary Ikaros motif from HOCOMOCO) or from primary Ikaros CHIP-seq (and secondary Jun motif from HOCOMOCO). And - what were the two DNA motifs (primary and secondary, and their distance) from the SPICE analysis?

Authors have mostly careful considerations and statements. One additional comment is that binding does not equal function (Fig 2D), and that opening of chromatin (by any other factor(s)) can give DNA-binding factors (like Ikaros and Jun) the opportunity to bind, without functional consequence for the biological process studied.

Figure 2E: Ikaros is reported to be expressed at baseline in murine B cells, yet the Ikaros ChIP-seq in unstimulated cells had what looks to be no significant or low peaks. LPS stimulation induced strong Ikaros ChIP-seq signal. A western blot showing the Ikaros protein levels in the 3 conditions could help understand if the binding pattern is due to protein expression level induction. Similar for Jun (western in the 3 conditions), which seemed to mainly bind in the LPS+IL21 condition. Furthermore, as also suggested below, tracks showing Ikaros and Jun binding from all conditions (unstimulated, LPS only and LPS+IL21 stimulated cells), at select genomic loci, would be helpful in illustrating this difference in signal between the different cell conditions. This is relevant in regards to the point of cooperativity of binding.

ChIP-seq in mouse B cells showed that Ikaros bound strongly in LPS stimulated cells, in the (relative) absence of Jun binding (Fig. 2C). However, in EMSA (Fig 3C), there is no binding when the AP1 site is mutated, and the authors describe this as Ikaros binding site. What does the Ikaros binding look like at this genomic location in LPS (only) stimulated cells? The authors could show the same figure as in Fig 2F but show Ikaros and Jun ChIP-seq tracks at IL10 CNS9 locus from all conditions to compare binding in unstimulated, LPS and LPS+IL21 cells.

Also: How does this reconcile with the luciferase assay in Fig 4E, where LPS (only) stimulation is used, which in Fig 2E only/mainly induced Ikaros, and not Jun ChIP-seq signal (while EMSA indicate Ikaros cannot bind the site alone, but can enhance Jun-dependent binding).

Comment on statements in results section: The luciferase assays in B and T cells do not demonstrate the role of the proteins Ikaros or Jun directly (page 10, lines 208 and surrounding text). The assay measures an effect of the DNA sequences (implying binding of some transcription factor(s)), but does not identify which protein factors bind there.

Lastly, the authors only discuss Ikaros (using the term IKZF1 which is the gene symbol for the Ikaros protein). There are other Ikaros family members that have high homology and that are reported to bind similar DNA sequences (for instance Aiolos and Helios), which are expressed in B-cells and T-cells. A discussion of this is of relevance, as these are different proteins, although belonging to the same family (the Ikaros-family) of transcription factors. For instance, western for Aiolos and Helios will likely detect Aiolos in the B cells used, and Helios in the T cells used.

Reviewer #1 (Public Review):

In this manuscript, the authors explored the benefits of intermittent fasting on the cardiac physiology through a multi-omics approach and compared different fasting times (IF12; IF 16 and EOD) for a duration of 6 months. Combining the RNA-sequencing, proteomics and phosphor-proteomics analysis, the authors have made an interesting observation that different fasting times would lead to different changes that could be important for the cardiac physiology. Moreover, the changes observed at transcriptional level are different from protein level, suggesting a post-transcriptional regulation mechanism. Using western blot, the authors have confirmed the key signaling pathways, including AMPK, IRS pathway to be significantly altered upon intermittent fasting for 16hrs. Lastly, as a proof of concept for better cardiac function, the animals were challenged with dobutamine and echocardiography was performed to show the mice subjected to intermittent fasting have better cardiac systolic function.

The impact of intermittent fasting on cardiovascular health has been well characterized in several studies. This report appears to be the first one utilizing a multi-omics approach and provided an interesting dataset at transcriptome, proteome and phosphor-proteome levels, and would serve as a valuable data resource for the field. I have the following concerns:

Major concerns:

1) The rationale for choosing the intermittent fasting pattern and timing<br /> While the 16:8 intermittent fasting is relatively standard, what is the rationale to test IF 12 hours? As a 4 hour fasting difference might not cause dramatic changes in transcriptome and proteome. Also, what is the rationale to perform 6 months study? The dobutamine stress test is not a terminal procedure, have the authors examined the cardiac function prior to 6 months to see whether there is a difference?

2) Lack of validation study<br /> One interesting observation from this study is the changes of transcriptome does not reflect all the changes at protein level and there is a differential gene expression pattern in IF12, IF16 and EOD. If this is the case, the authors should select a few important targets and provide both mRNA and protein level analysis, as a proof of concept for the bioinformatics analysis accuracy.

3) Poor western blot image quality<br /> The quality of the western blot has several issues: a. the change of pAMPK/AMPK appears to be a decrease of total AMPK instead of change at p-AMPK level. Same with GSK3a/b. There appears to be an increase of total GSK3a/b. The AKT should also be blotted and quantified at phosphorylation level. The western blot should be clearly labeled, for the ones with double bands, including GSK3a/b, the author should clearly label which is GSK3a and which is GSK3b. For the IRS with non-specific band, the author should point out IRS-1 band itself.

Reviewer #1 (Public Review):

The manuscript focused on roles of a key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1), in the metabolism, gene regulation and homeostasis of invariant natural killer T (NKT_ cells and impact on these T cells' roles during asthma pathogenesis. The authors presented data showing that the acetyl-coA-carboxylase 1 enzyme regulates the expression of PPARg then the function of NKT cells including the secretion of Th2-type cytokines to impact on asthma pathogenesis. The results are clearcut and data were logically presented.

Reviewer #1 (Public Review):

Summary: A well-executed series of experiments that will likely be of immense interest to (a) vector-borne disease researchers and (b) gram-negative sepsis/bacteremia researchers. The study uses comparative transcriptomics to begin probing what makes Peromyscus leucopus a unique host for numerous pathogens. Most issues with the paper are trivial, relating to descriptions of statistical cutoffs. While the paper does not provide mechanistic insight into how P. leucopus restrains its immune response to LPS or other microbial invaders, it is likely that this paper will be frequently consulted by researchers trying to understand that phenomenon.

Strengths:

o Use of outbred M. musculus is a commendable choice for the studies here.<br /> o Excellent decision by the authors to use their published dataset (with appropriate statistical normalization) to improve their statistical power to examine sex-biased gene expression. Is it possible to go one step further and briefly incorporate their prior BALB/c data to see how the BALB/c compare to the outbred mice. This could perhaps be just a PCA plot to see if they cluster with the outbred mice and/or Peromyscus, or are separate.<br /> o The correlations and ratios used to try to understand immune cell dynamics are clever and likely reflect interesting biology, but caution should be used when interpreting these indirect measures. As there are no tools for cell separation in P. leucopus, the authors should continue to include these data to stimulate ideas in the field, but readers should understand the "conclusions" are hypotheses due to the nature of the bulk RNAseq.

Weaknesses:

o Supplemental Table 1 only lists genes that passed the authors statistical thresholds. The full list of genes detected in their analysis should be included with read counts, statistics, etc. as supplemental information<br /> o While P. leucopus is a critical reservoir for B. burgdorferi, caution should be taken in directly connecting the data presented here and the Lyme disease spirochete. While it's possible that P. leucopus have a universal mechanism for limiting inflammation in response to PAMPs, B. burgdorferi lack LPS and so it is also possible the mechanisms that enable LPS tolerance and B. burgdorferi tolerance may be highly divergent.<br /> o Statistical significance is binary and p-values should not be used as the primary comparator of groups (e.g. once a p-value crosses the deigned threshold for significance, the magnitude of that p-value no longer provides biological information). For instance, in comparing GO-terms, the reason for using of high p-value cutoffs ("None of these were up-regulated gene GO terms with p values < 1011 for M. musculus.") to compare species is unclear. If the authors wish to compare effect sizes, comparing enrichment between terms that pass a cutoff would likely be the better choice. Similarly, comparing DEG expression by p-value cutoff and effect size is more meaningful than analyses based on exclusively on p-value: "Of the top 100 DEGs for each species by ascending FDR p value." Description in later figures (e.g. Figure 4) is favored.<br /> o The ability to use of CD45 to normalize data is unclear. Authors should elaborate both on the use of the method and provide some data how the data change when they are normalized. For instance, do correlations between untreated Mus and Peromyscus gene expression improve? The authors seem to imply this should be a standard for interspecies comparison and so it would be helpful to either provide data to support that or, if applicable, use of the technique in literature should be referenced.<br /> o Regarding the ISG data-is a possible conclusion not that Peromyscus don't upregulate the antiviral response because it's already so high in untreated rodents? It seems untreated Peromyscus have ISG expression roughly equivalent to the LPS mice for some of the genes. This could be compared more clearly if genes were displayed as bar plots/box and whisker plots rather than in scatter plots. It is unclear why the linear regression is the key point here rather than normalized differences in expression.<br /> o Some sections of the discussion are under supported:<br />  The claim that low inflammation contributes to increased lifespan is stated both in the introduction and discussion. Is there justification to support this? Do aged pathogen-free mice show more inflammation than aged Peromyscus?<br />  The claim that reduced Peromyscus responsiveness could lead to increased susceptibility to infection is prominently proposed but not supported by any of the literature cited.<br />  References to B. burgdorferi, which do not have LPS, in the discussion need to ensure that the reader understands this and the potential that responses could be very different.

Reviewer #1 (Public Review):

The article by Reversade and colleagues reports new mutations in the PYCR1 in a progeroid disease associated with premature skin aging. Using human cell culture and a newly generated mouse model of PYCR1deficiency they identify a role for this factor in maintaining dermal homeostasis and ECM production. I have some minor concerns about the role of PYCR1 in fibroblast survival vs function and the quantification of western blots.

Reviewer #1 (Public Review):

Summary:

In this excellent manuscript by Egan et al., the authors very carefully dissect the roles of inflammasome components in restricting Salmonella Typhimurium (STm) replication in human macrophages. They show that caspase-1 is essential to mediating inflammasome responses and that caspase-4 contributes to bacterial restriction at later time points. The authors show very clear roles for the host proteins that mediate terminal lysis, gasdermin D and ninjurin-1. The unique finding in this study is that in the absence of inflammasome responses, Salmonella hypereplicates within the cytosol of macrophages. These findings suggest that caspase-1 and possibly caspase-4 play roles in restricting the replication of Salmonella in the cytosol as well as in the Salmonella containing vacuole.

Strengths:

1) The genetic and biochemical approaches have shown for the first time in human macrophages that the caspase-1-GSDMD-NINJ1 axis is very important for restricting intracellular STm replication. In addition, they demonstrate a later role for Casp4 in control of intracellular bacterial replication.

2) In addition, they show that in macrophages deficient in the caspase-1-GSDMD-NINJ1 axis that STm are found replicating in the cytosol, which is a novel finding. The electron microscopy is convincing that STm are in the cytosol.

3) The authors go on to use a chloroquine resistance assay to show that inflammasome signaling also restricts STm within SCVs in human macrophages.

4) Finally, they show that the Type 3 Secretion System encoded on Salmonella Pathogenicity Island 1 contributes to STm's cytosolic access in human macrophages.

Weaknesses:

1) Their results with human macrophages suggest that there are differences between murine and human macrophages in inflammasome-mediated restriction of STm growth. For example, Thurston et al. showed that in murine macrophages that inflammasome activation controls the replication of mutant STm that aberrantly invades the cytosol, but only slightly limits replication of WT STm. In contrast, here the authors found that primed human macrophages rely on caspase-1, gasdermin D and ninjurin-1 to restrict WT STm. I wonder if the priming of the human macrophages in this study could account for the differences in these studies. Along those lines, do the authors see the same results presented in this study in the absence of priming the macrophages with Pam3CSK4. I think that determining whether the control of intracellular STm replication is dependent on priming is very important. Another difference with the Thurston et al. paper is the way that the STm inoculum was prepared - stationary phase bacteria that were opsonized. Could this also account for differences between the two studies rather than differences between murine and human macrophages in inflammasome-dependent control of STm?<br /> 2) The authors show that the pore-forming proteins GSDMD and Ninj1 contribute to control of STm replication in human macrophages. Is it possible that leakage of gentamicin from the media contributes to this control?

3) One major question that remains to be answered is whether casp-1 plays a direct role in the intracellular localization of STm. If the authors quantify the percentage of vacuolar vs. cytosolic bacteria at early time points in WT and casp-1 KO macrophages, would that be the same in the presence and absence of casp-1? If so, then this would suggest that there is a basal level of bacterial-dependent lysis of the SCV and in WT macrophages the presence of cytosolic PAMPS trigger cell death and bacteria can't replicate in the cytosol. However, in the inflammasome KO macrophages, the host cell remains alive and bacteria can replicate in the cytosol.

Reviewer #1 (Public Review):

The manuscript by Sun and colleagues followed their previous findings on the tumor-suppressive role of PDLIM2 in lung cancer. They further investigated various mechanisms, including epigenetic modification, copy number variation, and LOH, that led to the decreased expression of PDLIM2 in human lung cancer. Next, they used a nanoparticle-based approach to specifically restore the expression in mouse lung tumors. They showed that over-expression of PDLIM2 in lung cancer repressed its progression in vivo. Also, this treatment could synergize with chemotherapy and checkpoint inhibitor anti-PD-1. Overall, the results were quite promising and convincing, using a treatment combination that would appear to have the potential for clinical implementation.

Reviewer #1 (Public Review):

Xie and Colleagues propose here to investigate the mechanism by which exercise inhibits bone metastasis progression. The authors describe that osteocyte, sensing mechanical stimulation generated by exercise, inhibit NSCLC cell proliferation and sustain the dormancy thereof by releasing sEVs with tumor suppressor microRNAs. Furthermore, mechanical loading of the tibia inhibited the bone metastasis progression of NSCLC. Interestingly, exercise preconditioning effectively suppressed bone metastasis progression.

Reviewer #1 (Public Review):

In this manuscript, the authors demonstrated that YAP/TAZ promotes P-body formation in a series of cancer cell lines. YAP/TAZ modulates the transcription of multiple P-body-related genes, especially repressing the transcription of the tumor suppressor proline-rich nuclear receptor coactivator 1 (PNRC1) through cooperation with the NuRD complex. PNRC1 functions as a critical repressor in YAP-induced biogenesis of P-bodies and tumorigenesis in colorectal cancer (CRC). Reexpression of PNRC1 or disruption of P-bodies attenuated the protumorigenic effects of YAP. Overall, these findings are interesting and the study was well conducted.

Major concerns:

1. RNAseq data indicated that Yap has the capacity to suppress the expression of numerous genes. In addition to PNRC1, could there be additional Yap targeting factors involved in Yap-mediated the formation of P-bodies?<br /> 2. It is still not clear how PNRC1 regulates P-bodies. Knockdown of PNRC1 prevented the reduction of P-bodies caused by Yap knockdown. How do the genes related to P-bodies that are positively regulated by Yap, such as SAMD4A, AJUBA, and WTIP, change in this scenario? Given that the expression of Yap can differ considerably among various cell types, is it possible for P-bodies to be present in tumor cells lacking Yap expression?<br /> 3. The authors demonstrated that CHD4 can bind to Yap target genes, such as CTGF, AJUBA, SAMD4A (Figure 4 - Figure Supplement 1D). Does the NuRD complex repress the expression of these genes? the NuRD complex could prevent the formation of P-bodies?<br /> 4. YAP/TAZ promotes the formation of P-bodies which contradicts the previous study's conclusion (PMID: 34516278). Please address these inconsistent findings.

Reviewer #1 (Public Review):

This is a technically sound paper focused on a useful resource around the DRGP phenotypes which the authors have curated, pooled, and provided a user-friendly website. This is aimed to be a crowd-sourced resource for this in the future.

The authors should make sure they coordinate as well as possible with the NC datasets and community and broader fly community. It looks reasonable to me but I am not from that community.

I have only one major concern which in a more traditional review setting I would be flagging to the editor to insist the authors did on resubmission. I also have some scene setting and coordination suggestions and some minor textual / analysis considerations.

The major concern is that the authors do not comment on the distribution of the phenotypes; it is assumed it is a continuous metric and well-behaved - broad gaussian. This is likely to be more true of means and medians per line than individual measurements, but not guaranteed, and there could easily be categorical data in the future. The application of ANOVA tests (of the "covariates") is for example fragile for this.

The simplest recommendation is in the interface to ensure there is an inverse normalisation (rank and then project on a gaussian) function, and also to comment on this for the existing phenotypes in the analysis (presumably the authors are happy). An alternative is to offer a kruskal test (almost the same thing) on covariates, but note PLINK will also work most robustly on a normalised dataset.

Minor points:<br /> On the introduction, I think the authors would find the extensive set of human GWAS/PheWAS resources useful; widespread examples include the GWAS Catalog, Open Targets PheWAS, MR-base, and the FinnGen portal. The GWAS Catalog also has summary statistics submission guidelines, and I think where possible meta-data harmonisation should be similar (not a big thing). Of course, DRGP has a very different structure (line and individuals) and of course, raw data can be freely shown, so this is not a one-to-one mapping.

For some authors coming from a human genetics background, they will be interpreting correlations of phenotypes more in the genetic variant space (eg LD score regression), rather than a more straightforward correlation between DRGP lines of different individuals. I would encourage explaining this difference somewhere.

This leads to an interesting point that the inbred nature of the DRGP allows for both traditional genetic approaches and leveraging the inbred replication; there is something about looking at phenotype correlations through both these lenses, but this is for another paper I suspect that this harmonised pool of data can help.

I was surprised the authors did not crunch the number of transcript/gene expression phenotypes and have them in. Is this because this was better done in other datasets? Or too big and annoying on normalisation? I'd explain the rationale to leave these out.

I think 25% FDR is dangerously close to "random chance of being wrong". I'd just redo this section at a higher FDR, even if it makes the results less 'exciting'. This is not the point of the paper anyway.

I didn't buy the extreme line piece as being informative. Something has to be on the top and bottom of the ranks; the phenotypes are an opportunity for collection and probably have known (as you show) and cryptic correlations. I think you don't need this section at all for the paper and worry it gives an idea of "super normals" or "true wild types" which ... I just don't think is helpful.

I'd say "well-established inversion genotypes and symbiot levels" rather than generic covariates. Covariates could mean anything. You have specific "covariates" which might actually be the causal thing.

I wouldn't use the adjective tedious about curation. It's a bit of a value judgement and probably places the role of curation in the wrong way. Time-consuming due to lack of standards and best practice?

Reviewer #1 (Public Review):

General comments:<br /> This paper investigates the pH-specific enzymatic activity of mouse acidic mammalian chitinase (AMCase) and aims to elucidate its function's underlying mechanisms. The authors employ a comprehensive approach, including hydrolysis assays, X-ray crystallography, theoretical calculations of pKa values, and molecular dynamics simulations to observe the behavior of mouse AMCase and explore the structural features influencing its pH-dependent activity.

The study's key findings include determining kinetic parameters (Kcat and Km) under a broad range of pH conditions, spanning from strong acid to neutral. The results reveal pH-dependent changes in enzymatic activity, suggesting that mouse AMCase employs different mechanisms for protonation of the catalytic glutamic acid residue and the neighboring two aspartic acids at the catalytic motif under distinct pH conditions.

The novelty of this research lies in the observation of structural rearrangements and the identification of pH-dependent mechanisms in mouse AMCase, offering a unique perspective on its enzymatic activity compared to other enzymes. By investigating the distinct protonation mechanisms and their relationship to pH, the authors reveal the adaptive nature of mouse AMCase, highlighting its ability to adjust its catalytic behavior in response to varying pH conditions. These insights contribute to our understanding of the pH-specific enzymatic activity of mouse AMCase and provide valuable information about its adaptation to different physiological conditions.

Overall, the study enhances our understanding of the pH-dependent activity and catalytic properties of mouse AMCase and sheds light on its adaptation to different physiological pH environments.

Reviewer #1 (Public Review):

Summary:<br /> This study focuses on the defining cellular pathways critical for tRNA export from the nucleus. While a number of these pathways have been identified, the observation that the primary transport receptors identified thus far (Los1 and Msn5) are not essential and that cells are viable even when both the genes are deleted supports the idea that there are as yet unidentified mediators of tRNA export from the nucleus. This study implicates the helicase Dbp5 in one of these parallel pathways arguing that Dbp5 works in a pathway that is independent of Los1 and/or Msn5. The authors present genetic data to support this conclusion. At least one result suggests that the idea of these pathways in parallel may be too simplistic as deletion of the LOS1 gene, which is not essential decreases the interaction of tRNA export substrate with Dbp5 (Figure 2A). If the two pathways were working in parallel, one might have expected removing one pathway to lead to an increase in the use of the other pathway and hence the interaction with a receptor in that pathway. The authors provide solid evidence that Dbp5 interacts with tRNA directly and that the addition of the factor Gle1 together with the previously identified co-factor InsP6 can trigger helicase activity and release of tRNA. The combination of in vivo studies and biochemistry provides evidence to consider how Dbp5 contributes to the export of tRNA and more broadly adds to the conversation about how coding and non-coding RNA export from the nucleus might be coordinated to control cell physiology.

Strengths and weaknesses:<br /> A major strength of this manuscript is the multi-pronged approach to explore a potential role for the helicase Dbp5 in one of the multiple export pathways for tRNA from the nucleus.

The obvious missing experiment here with respect to genetics is the test of whether deletion of the MSN5 gene in the cells, which combines deletion of LOS1 and the dbp5_R423A allele, shown in Figure 1D would be lethal. This key experiment would lend substance to the argument that Dbp5 functions in a tRNA export pathway that is parallel to the Los1 and Msn5 pathways.

While some of the binding assays show rather modest band shifts (Figure 4B for example), the data in Figure 4A showing that there is no binding detected unless a non-hydrolyzable ATP analogue is employed, argues for specificity in nucleic acid binding. The question that does arise is whether the binding is specific for tRNA.

With the exception of the binding studies, which also employ a mixture of yeast tRNAs, this study relies primarily on a single tRNA species to come to the conclusions drawn. Many other studies have used multiple tRNAs to explore whether pathways characterized are generalizable to other tRNAs.

The authors provide evidence of a model where the helicase Dbp5 plays a role in tRNA export from the nucleus. Further evidence is required to determine whether Dbp5 could function in the same pathway as the previously defined tRNA export receptors, Los1 and Msn5. There are genetic tests that could be performed to explore this question. Some of the biochemistry presented would show when Los1 is absent that the interaction of Dbp5 with tRNA decreases, which could support a model where Dbp5 plays a role in coordination with Los1.

This work allows insight into key questions which still remain about the multiple pathways that are required for tRNA trafficking as well as how transport pathways for coding and non-coding RNAs might be coordinated. These questions are important as many of these pathways may be regulated in response to cellular conditions or during development and defining the fundamental pathways will be critical to understanding these dynamic processes.

Reviewer #1 (Public Review):

The authors present a study focused on addressing the key challenge in drug discovery, which is the optimization of absorption and affinity properties of small molecules through in silico methods. They propose active learning as a strategy for optimizing these properties and describe the development of two novel active learning batch selection methods. The methods are tested on various public datasets with different optimization goals and sizes, and new affinity datasets are curated to provide up-to-date experimental information. The authors claim that their active learning methods outperform existing batch selection methods, potentially reducing the number of experiments required to achieve the same model performance. They also emphasize the general applicability of their methods, including compatibility with popular packages like DeepChem.

Strengths:

Relevance and Importance: The study addresses a significant challenge in the field of drug discovery, highlighting the importance of optimizing the absorption and affinity properties of small molecules through in silico methods. This topic is of great interest to researchers and pharmaceutical industries.

Novelty: The development of two novel active learning batch selection methods is a commendable contribution. The study also adds value by curating new affinity datasets that provide chronological information on state-of-the-art experimental strategies.

Comprehensive Evaluation: Testing the proposed methods on multiple public datasets with varying optimization goals and sizes enhances the credibility and generalizability of the findings. The focus on comparing the performance of the new methods against existing batch selection methods further strengthens the evaluation.

Weaknesses:

Lack of Technical Details: The feedback lacks specific technical details regarding the developed active learning batch selection methods. Information such as the underlying algorithms, implementation specifics, and key design choices should be provided to enable readers to understand and evaluate the methods thoroughly.

Evaluation Metrics: The feedback does not mention the specific evaluation metrics used to assess the performance of the proposed methods. The authors should clarify the criteria employed to compare their methods against existing batch selection methods and demonstrate the statistical significance of the observed improvements.

Reproducibility: While the authors claim that their methods can be used with any package, including DeepChem, no mention is made of providing the necessary code or resources to reproduce the experiments. Including code repositories or detailed instructions would enhance the reproducibility and practical utility of the study.

Suggestions for Improvement:

Elaborate on the Methodology: Provide an in-depth explanation of the two active learning batch selection methods, including algorithmic details, implementation considerations, and any specific assumptions made. This will enable readers to better comprehend and evaluate the proposed techniques.

Clarify Evaluation Metrics: Clearly specify the evaluation metrics employed in the study to measure the performance of the active learning methods. Additionally, conduct statistical tests to establish the significance of the improvements observed over existing batch selection methods.

Enhance Reproducibility: To facilitate the reproducibility of the study, consider sharing the code, data, and resources necessary for readers to replicate the experiments. This will allow researchers in the field to validate and build upon your work more effectively.

Conclusion:

The authors' study on active learning methods for optimizing drug discovery presents an important and relevant contribution to the field. The proposed batch selection methods and curated affinity datasets hold promise for improving the efficiency of drug discovery processes. However, to strengthen the study, it is crucial to provide more technical details, clarify evaluation metrics, and enhance reproducibility by sharing code and resources. Addressing these limitations will further enhance the value and impact of the research.

Reviewer #1 (Public Review):

The authors developed an extension to the pairwise sequentially Markov coalecent model that allows to simultaneously analyze multiple types of polymorphism data. In this paper, they focus on SNPs and DNA methylation data. Since methylation markers mutate at a much faster rate than SNPs, this potentially gives the method better power to infer size history in the recent past. Additionally, they explored a model where there are both local and regional epimutational processes.

Integrating additional types of heritable markers into SMC is a nice idea which I like in principle. However, a major caveat to this approach seems to be a strong dependence on knowing the epimutation rate. In Fig. 6 it is seen that, when the epimutation rate is known, inferences do indeed look better; but this is not necessarily true when the rate is not known. A roughly similar pattern emerges in Supp. Figs. 4-7; in general, results when the rates have to be estimated don't seem that much better than when focusing on SNPs alone. This carries over to the real data analysis too: the interpretation in Fig. 7 appears to hinge on whether the rates are known or estimated, and the estimated rates differ by a large amount from earlier published ones.

Overall, this is an interesting research direction, and I think the method may hold more promise as we get more and better epigenetic data, and in particular better knowledge of the epigenetic mutational process. At the same time, I would be careful about placing too much emphasis on new findings that emerge solely by switching to SNP+SMP analysis.

Reviewer #1 (Public Review):

Summary:<br /> The precise mechanism of how tetraspanin proteins engage in the generation of discs is still an open question in the field of photoreceptor biology. This question is of significance as the lack of photoreceptor discs or defects in disc morphogenesis due to mutations in tetraspanin proteins is a known cause of vision loss in humans. The authors of this study combine TEM and mouse models to tease out the role of tetraspanin proteins, peripherin, and Rom1 in the genesis of the photoreceptor discs. They show that the absence of Rom1 leads to an increase in peripherin and changes in disc morphology. Further rise in peripherin alleviates some of the defects observed in Rom1 knockout animals leading to the conclusion that peripherin can substitute for the absence of Rom1.

Strengths:<br /> A mouse model of Rom1 generated by the McInnes group in 2000 predicted a role for Rom1 in rim closure. They also showed enlarged discs in the absence of Rom1. This study confirmed this finding and showed the compensatory changes in peripherin, maintaining the total levels of tetraspanin proteins. Lack of Rom1 leads to excessive open disks demonstrated by darkly stained tannic acid-accessible areas in TEM. Interestingly, increased peripherin expression can rescue some morphological defects, including maintaining normal disc diameters and incisures. Overall, these observations lead authors to propose a model that ROM1 can be replaced by peripherin.

Weaknesses:<br /> The compensatory increase in peripherin and morphological rescue in the absence of ROM1 is expected, given the previous work from authors showing i) absence of peripherin showing increased ROM1 and ii) "Eliminating Rom1 also increased levels of Prph2/RRCT: mean Prph2/RRCT levels in P30 Prph2+/R retinas were 34% of WT, while levels in Prph2+/R/Rom1−/− retinas were 59% of WT" from Conley, 2019. The current study provides a comprehensive quantitative analysis. However, the mechanism behind the mechanism is unclear and warrants discussion.

Photoreceptor morphology appears better when peripherin is overexpressed. Is there a rescue of rod function (assessed by ERG or equivalent measures) in peripherin OE/Rom1-/- mice? Given the extensive work in this area and the implications the authors allude to at the end, it is important to investigate this aspect.