Reviewer #3 (Public Review):

Summary:<br /> The manuscript from Tariq and Maurici et al. presents important biochemical and biophysical data linking protein phosphorylation to phase separation behavior in the repressive arm of the Neurospora circadian clock. This is an important topic that contributes to what is likely a conceptual shift in the field.

Reviewer #3 (Public Review):

Summary<br /> Pham, Pahuja, Hagenbeek, et al. have conducted a comprehensive range of assays to biochemically and genetically determine TEAD degradation through RNF146 ubiquitination. Additionally, they designed a PROTAC protein degrader system to regulate the Hippo pathway through TEAD degradation. Overall, the data appears robust. However, the manuscript lacks detailed methodological descriptions, which should be addressed and improved. For instance, the methods used to analyze the K48 ubiquitination site on TEAD and the gene expression analysis of Hippo Signaling are unclear. Furthermore, the multiple proteomics, RNA-seq, and ATAC-seq data must be made publicly available upon publication to ensure reproducibility. Most of the main figures are of low resolution, which needs addressing.

Strengths:<br /> - A broad range of assays was used to robustly determine the role of RNF146 in TEAD degradation.<br /> - Development of novel PROTAC for degrading TEAD.

Weaknesses:<br /> - An orthogonal approach is needed (e.g., PARP1 inhibitor) to demonstrate PARP1's dependency in TEAD ubiquitination.

- The data from Table 2 is unclear in illustrating the association of identified K48 ubiquitination with TEAD4, especially since the experiments were presumably to be conducted on whole cell lysates with KGG enrichment. This raises the possibility that the K48 ubiquitination could originate from other proteins. Alternatively, if the authors performed immunoprecipitation on TEAD followed by mass spectrometry, this should be explicitly described in the text and materials and methods section.

- Figure 2D: The methodology for measuring the Hippo signature is unclear, as is the case for Figures 7E and F regarding the analysis of Hippo target genes.

- Figure S3F requires quantification with additional replicates for validation.

- There is a misleading claim in the discussion stating "TEAD PARylation by PAR-family members (Figure 3)"; however, the demonstration is only for PARP1, which should be corrected.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, Curran et al investigate the role of Ntn, Slit1, and Slit 2 in the axon patterning of DRG neurons. The paper uses mouse genetics to perturb each guidance molecule and its corresponding receptor. Cre-based approaches and immunostaining of DRG neurons are used to assess the phenotypes. Overall, the study uses the strength of mouse genetics and imaging to reveal new genetic modifiers of DRG axons. The conclusions of the experiments match the presented results. The paper is an important contribution to the field, as evidence that dorsal funiculus formation is impacted by Ntn and Slit signaling. However, there are some potential areas of the manuscript that should be edited to better match the results with the conclusions of the work.

Strengths:<br /> The manuscript uses the advantage of mouse genetics to investigate the axon patterning of DRG neurons. The work does a great job of assessing individual phenotypes in single and double mutants. This reveals an intriguing cooperative and independent function of Ntn, Slit1, and Slit2 in DRG axon patterning. The sophisticated triple mutant analysis is lauded and provides important insight.

Weaknesses:<br /> Overall, the manuscript is sound in technique and analysis. However, the majority of the manuscript is about the dorsal funiculus and not the bifurcation of the axons, as the title would make a reader believe. Further, the manuscript would provide a more scholarly discussion of the current knowledge of DRG axon patterning and how their work fits into that knowledge.

Reviewer #3 (Public Review):

In this work, the authors shed light onto the structures of Fusarium oxysporum f.sp. lycopersici proteins involved in the infection of tomato. They unravelled several new secreted effector protein structures and additionally used computational approaches to structurally classify the remaining effectors known from this pathogen. Through this they uncovered a new and unique structural class of proteins which they found to be present and widely distributed in fungal plant pathogens and plant symbiotic fungi. The authors further predicted structural models for the full SIX effector set revealing that genome-proximal effector pairs share similar structural classes. Building on their Avr1 structure, the authors also define the C-terminal domain and specific amino acid residues that are essential to Avr1 detection by its cognate immune receptor.

A major strength of this work is a portfolio of several (Avr1, Avr3, SIX6, SIX8) new structurally resolved proteins which led to the discovery that several of them fall into the same structural class. These findings are supported by strong results.

The experiments addressing the structure-function relationship of Avr1's avirulence activity are highly relevant to our understanding of disease resistance mechanisms against Fusarium. Additional controls would allow for better support of the conclusions to be drawn. An example is FonSIX4's cell death activity in N.benthamiana leaves and whether FonSIX4 cdll death is indeed dependent on the tomato I receptor. Complementary work in Fusarium mutants lacking Avr1 and expressing chimeric versions would document that the observations from transient expressions in Nicotiana benthamiana are relevant in the biological context of a Fusarium/tomato interaction.

The discovered solvent-exposed residues conditioning Avr1 recognition by the I receptor seem to be positioned in an area of the protein which had previously been highlighted as being highly variable in FOLD proteins of symbiotic fungi but it is not clear from the work whether this is indeed the case or whether Avr1 differs significantly in its structure from FOLD proteins found in other fungi.<br /> It remains to be tested whether the residues conditioning avirulence activity are also crucial for virulence activity in Fusarium.

This work uncovered a new structural class of proteins with critical roles in plant-pathogen interactions. Structure-based predictions and genome-wide comparisons have emerged as a new approach enabling the identification of similar proteins with divergent sequences. The work undertaken by the authors adds to a growing body of work in plant-microbe research, predominantly from pathogenic organisms, and more recently in symbiotic fungi.

Reviewer #3 (Public Review):

Summary:<br /> In this study, the authors investigated the potential of nifuroxazide to enhance responsiveness to radiotherapy, employing both an in vitro cell culture system and an in vivo syngeneic mouse tumor model.

Strengths:<br /> The researchers conducted a series of experiments to elucidate the role of nifuroxazide in facilitating the radiotherapy-induced reduction of proliferation, migration, and invasion of HepG2 cells.

Weaknesses:<br /> The evidence supporting the claim that nifuroxazide contributes to the degradation of radiotherapy-induced upregulation of PD-L1 via the ubiquitin-proteasome pathway is still relatively weak.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript introduces a new computational framework for choosing 'the best method' according to the case for getting the best possible structural prediction for the CDR-H3 loop. The authors show their strategy improves on average the accuracy of the predictions on datasets of increasingly difficulty in comparison to several state-of-the-art methods. They also show the benefits of improving the structural predictions of the CDR-H3 in the evaluation of different properties that may be relevant for drug discovery and therapeutics design.

Strengths:<br /> Authors introduce a novel framework, which can be easily adapted and improved. Authors use a well defined dataset to test their new method. A modest average accuracy gain is obtained in comparison to other state-of-the art methods for the same task, while avoiding for testing different prediction approaches. Although the accuracy gain is mainly ascribed to easy cases, the accuracy and precision for moderate to challenging cases is comparable to the best PLM methods (see Fig. 4b and Extended Data Fig. 2), reflecting the present methodological limit in the field.

Weaknesses:<br /> The proposed method lacks of a confidence score or a warning to help guiding the users in moderate to challenging cases.

Reviewer #3 (Public Review):

Summary:<br /> This study examines the impact of CTRP10/C1QL2 absence on obesity and metabolic health in mice. Female mice lacking CTRP10 tend to develop obesity, particularly on a high-fat diet. Surprisingly, they do not display the typical metabolic traits associated with obesity, like fatty liver or glucose intolerance. This indicates a disconnection between weight gain and metabolic issues in these female mice. The research underscores the need to understand sex-specific factors in how obesity influences metabolic health.

Strengths:<br /> The study provides compelling evidence regarding Ctrp10's role in female-specific metabolic regulation in mice, shedding light on its potential significance in metabolically healthy obese (MHO) individuals.

Weaknesses:<br /> -The analysis and description of sex-specific human data require more details to highlight the relevance of Ctrp10 mouse data and the analysis of differentially expressed genes in humans.<br /> -There's a lack of analysis regarding secreted Ctrp10 under various dietary conditions.<br /> -The study didn't assess adipose tissue function to evaluate metabolic health.

Reviewer #3 (Public Review):

Summary:

Bartolome et al. present ProteasomeID, a novel method to identify components, interactors, and (potentially) substrates of the proteasome in cell lines and mouse models. As a major protein degradation machine that is highly conserved across eukaryotes, the proteasome has historically been assumed to be relatively homogeneous across biological scales (with few notable exceptions, e.g., immunoproteasomes and thymoproteasomes). However, a growing body of evidence suggests that there is some degree of heterogeneity in the composition of proteasomes across cell tissues, and can be highly dynamic in response to physiologic and pathologic stimuli. This work provides a methodological framework for investigating such sources of variation. The authors start by adapting the increasingly popular biotin ligation strategy for labelling proteins coming into close proximity with one of three different subunits of the proteasome, before proceeding with PSMA4 for further development and analysis based on their preliminary labelling data. In a series of well-constructed and convincing validation experiments, the authors go on to show that the tagged PSMA4 construct can be incorporated into functional proteasomes, and is able to label a broad set of known proteasome components and interacting proteins in HEK293T cells. They also attempt to identify novel proteasomal degradation substrates with ProteasomeID; while this was convincing for known substrates with particularly short half-lives (exemplified by the transcription factor c-myc), follow-up validation experiments with other substrates were less clear. One of the most compelling results was from a similar experiment to confirm proteasomal degradation induced by a BRD-targeting PROTAC, which I think is likely to be of keen interest to the targeted degradation community. Finally, the authors establish a ProteasomeID mouse model, and demonstrate its utility across several tissues.

Strengths:

1) ProteasomeID itself is an important step forward for researchers with an interest in protein turnover across biological scales (e.g., in sub-cellular compartments, in cells, in tissues, and whole organisms). I especially see interest from two communities: those studying fundamental proteostasis in physiological and pathologic processes (e.g., ageing; tissue-specific protein aggregation diseases), and those developing targeted protein degradation modalities (e.g., PROTACs; molecular glues). All the datasets generated and deposited here are likely to provide a rich resource to both. The HEK293T cell line data are a valuable proof-of-concept to allow expansion into more biologically-relevant cell culture settings; however, I envision the greatest innovation here to be the mouse model. For example, in the targeted protein degradation space, two major hurdles in early-stage pre-clinical development are (i) evaluation of degradation efficacy across disease-relevant tissues, and (ii) toxicity and safety implications caused by off-target degradation, e.g., of newly-identified molecular glues and/or in particularly-sensitive tissues. The ProteasomeID mouse allows early in vivo assessment of both these questions. The results of the BRD PROTAC experiment in 293T cells provides an excellent in vitro proof-of-concept for this approach.

2) The mass spectrometry-based proteomics workflows used and presented throughout the manuscript are robust, rigorous, and convincing. For example, the algorithm the authors use for defining enrichment score cut-offs are logical and based on rational models, rather than on arbitrary cut-offs that are common for similar proteomics studies. The construction (and subsequent validation) of both BirA*- and miniTurbo- tagged PSMA4 variants also increases the utility of the method, allowing researchers to choose the variant with the labelling time-scale required for their particular research question.

3) The optimised BioID and TurboID protocol the authors develop (summarised in Fig. S2A) and validate (Fig. S2B-D) is likely to be of broad interest to cell and molecular biologists beyond the protein degradation field, given that proximity labelling is a current gold-standard in global protein:protein interaction profiling.

Limitations:

I think the authors do an excellent job in highlighting the limitations of ProteasomeID throughout the Results and Discussion. I do have some specific comments that might provide additional context for the reader.

1) The authors do a good job in showing that a substantial proportion of PSMA4-BirA* is incorporated into functional proteasome particles; however, it is not immediately clear to me how much background (false-positive IDs) might be contributed by the ~40 % of PSMA4-BirA* that is not incorporated into the mature core particle (based on the BirA* SEC-MS traces in Fig. 2b and S3b, i.e., the large peak ~ fraction 20). Are there any bands lower down in the native gel shown in Fig. 2c, i.e., corresponding to lower molecular weight complexes or monomeric PSMA4-BirA*? The enrichment of proteasome assembly factors in all the ProteasomeID experiments might suggest the presence of assembly intermediates, which might themselves become substrates for proteasomal degradation (as has been shown for other incompletely-assembled protein complexes, e.g., the ribosome, TRiC/CCT).

2) Although the authors attempt to show that BirA* tagging of PSMA4 does not interfere with proteasome activity (Fig. 2e-f), I think the experimental evidence for this is incomplete. They show that the overall chymotrypsin-like activity (attributable to PSMB5) in cells expressing PSMA4-BirA* is not markedly reduced compared with control BirA*-expressing cells. However, they do not show that the activity of the specific proteasome sub-population that contains PSMA4-BirA* is unaffected (e.g., by purifying this sub-population via the Flag tag). The proteasome activity of the sub-population of wild-type proteasome complexes that do not contain the PSMA4-BirA* (~50%, based on the earlier immunoblots) could account for the entire chymotrypsin-like activity-especially in the context of HEK293T cells, where steady-state proteasome levels are unlikely to be limiting. It would also be useful to assess any changes in tryspin- and caspase- like activities, especially as tagging of PSMA4 could conceivably interfere with the activity of some PSMB subunits, but not others.

3) I was left unsure of the general utility of ProteasomeID for identifying novel proteasomal substrates in homeostatic or stressed conditions. The immunoblots for the two candidates the authors follow up in Fig. 4g was not especially clear; the reduction in the bands are modest, at best. Furthermore, classifying candidates based on enrichment following proteasome inhibition with MG-132 have the potential to lead to a high number of false positives. ProteasomeID's utility in identifying potential substrates in more targeted settings (e.g., molecular glues, off-target PROTAC substrates) is far more apparent.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Zhang et al. analyzes the function of the centrosomal protein CEP44 in centriole duplication and in the formation of the mitotic spindle. The first part addresses the role of CEP44 at centrioles. Using mostly RNAi-mediated depletion in cell lines and in some cases KO cells, the authors find increased centriole numbers in depleted cells and, based on quantification of centrioles stained with various centriole markers as well as live imaging, conclude that this is due to premature centriole disengagement and overduplication. The second part, which is largely independent of the first, focuses on the role of CEP44 in the mitotic spindle. The authors find that CEP44 is phosphorylated in mitosis in an Aurora A-dependent manner and identify the phosphorylation site, which controls CEP44 spindle localization and functions in maintaining spindle integrity.

Strength:<br /> The manuscript makes the interesting observation that reduced expression of CEP44 is observed in breast cancer and correlated with poor survival in patients.<br /> The analysis of mitotic phosphorylation including the identification of the modified site and its role in spindle recruitment is interesting and useful.

Weakness:<br /> The authors seem to largely ignore previously published work that contrasts with the findings presented in the current study. The previous work found a role of CEP44 in centriole formation and centrosome conversion and observed reduced centriole numbers in depleted cells, whereas the current study claims the opposite, a role in centriole engagement that leads to overduplication and increased centriole number in depleted cells. However, the supporting evidence is not strong enough, especially in light of the previous work. Considering that CEP44 depletion also disrupts mitosis, which could affect centriole numbers by failed segregation/division, a more careful analysis in synchronized cultures would be needed. Also, cell cycle analysis would be required to rule out cell cycle effects in CEP44-depleted cells, which could also explain altered centriole numbers. Moreover, the quality of the imaging is often not sufficient to support the claims.<br /> The second part is largely disconnected from the first and reads as if it was a separate study. There is no attempt to integrate both parts. For example, the second part seems to largely focus on normal bipolar spindles, even though the first part reveals multipolarity as a phenotype after CEP44 knockdown. It remains unclear if the spindle defects are due to centriole defects, defective spindle microtubule stability/organization, or both, and whether the centriole-localized or spindle-localized CEP44 is involved.

Another weak aspect is that neither for RNAi nor for KO cells the authors show that CEP44 is depleted at centrioles and to what extent. This is only shown in cell extract.

Reviewer #3 (Public Review):

Summary:<br /> The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:<br /> The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1, and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs.

Weaknesses:<br /> Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. In addition, some conclusions about the role of specific DDC proteins in cell cycle arrest at G2/M need further experimental support. The results with Bfa1/Bub2 are surprising and somewhat unexpected. There is no clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block.

Reviewer #3 (Public Review):

Summary:<br /> Prior research on SCC3, a cohesin subunit protein, in yeast and Arabidopsis has underscored its vital role in cell division. This study investigated into the specific functions of SCC3 in rice mitosis and meiosis. In a weakened SCC3 mutant, sister chromatids separating was observed in anaphase I, resulting in 24 univalents and subsequent sterility. The authors meticulously documented SCC3's loading and degradation dynamics on chromosomes, noting its impact on DNA replication. Despite the loss of homologous chromosome pairing and synapsis in the mutant, chromosomes retained double-strand breaks without fragmenting. Consequently, the authors inferred that in the scc3 mutant, DNA repair more frequently relies on sister chromatids as templates compared to the wild type.

Strengths:<br /> The study presents exceptionally well-executed research in the field of rice cytogenetics.

Weaknesses:<br /> While the paper's conclusions are generally well-supported, further substantiation is needed for the claim that SCC3 inhibits template choice for sister chromatids. To bolster this conclusion, I recommend that the authors perform whole-genome sequencing on parental and F1 individuals from two rice variants, subsequently calculating the allele frequencies at heterozygous sites in the F1 individuals. If SCC3 indeed inhibits inter-sister chromatid repair in the wild type, we would anticipate a higher frequency of inter-homologous chromosome repair (i.e., gene conversion). This should be manifested as a bias away from the Mendelian inheritance ratio (50:50) in the offspring of the wild type compared to the offspring of the scc3+/- mutant.

Reviewer #3 (Public Review):

Summary:<br /> This manuscript reports the novel observation of alterations in the nuclear pore (NUP) components and the function of the nuclear envelope in knock-in models of APP and presenilin mutations. The data show that loss of NUP immunoreactivity (IR) and pore density are observed at times prior to plaque deposition in this model. The loss of NUP IR is correlated with an increase in intraneuronal Abeta IR with two monoclonal antibodies that react with the N-terminus of Abeta. Similar results are observed in cultured neurons from APP-KI and Wt mice where further results with cultured neurons indicate that Abeta "drives" this process: incubation of neurons with oligomeric, but not monomeric or fibrillar Abeta causes loss of NUP IR, incubation with conditioned media from KI cells but not wt cells also causes loss of NUP IR and treatment with the gamma secretase inhibitor, NAPT partially blocks the loss of NUP IR. Further data show that nuclear envelope function is altered in KI cells and KI cells are more sensitive to TNFalpha-induced necroptosis. This is potentially an important and significant report, but how this fits within the larger picture of what is known about amyloid aggregation and accumulation and pathogenesis in neurons needs to be clarified. The results from mouse brains are strong, while the results from cultured cells are in some instances are of a lower magnitude, less convincing, ambiguous, and sometimes over-interpreted.

Strengths:<br /> 1. Loss of NUP expression and activity is a novel observation.<br /> 2. Its association with intraneuronal Abeta immunoreactivity suggests an association with Alzheimer's disease.<br /> 3. The experiments generally appear to be well-controlled.<br /> 4. Multiple approaches are sometimes used to increase the robustness of the data.

Weaknesses:<br /> 1. It does not consider the relationship of the findings here to other published work on the intraneuronal perinuclear and nuclear accumulation of amyloid in other transgenic mouse models and in humans.<br /> 2. It appears to presume that soluble, secreted Abeta is responsible for the effect rather than the insoluble amyloid fibrils.<br /> 3. Most of the critical findings on the association with Abeta and the functional consequences are done in cultured neurons and not in mouse models.<br /> 4. There is no evidence from the human brain that would strengthen the significance.<br /> 5. It is not clear when the alteration in NUP expression begins in the KI mice as there is no time at which there is no difference between NUP expression in KI and Wt and the earliest time shown is 2 months. If NUP expression is decreased from the earliest times at birth, then this makes the significance of the observation of the association with amyloid pathology less clear.

Reviewer #3 (Public Review):

This study presents results arguing that the mammalian acetyltransferase Tip60/KAT5 auto-acetylates itself on one specific lysine residue before the MYST domain, which in turn favors not only nuclear localization but also condensate formation on chromatin through LLPS. The authors further argue that this modification is responsible for the bulk of Tip60 autoacetylation and acetyltransferase activity towards histone H4. Finally, they suggest that it is required for association with txn factors and in vivo function in gene regulation and DNA damage response.

These are very wide and important claims and, while some results are interesting and intriguing, there is not really close to enough work performed/data presented to support them. In addition, some results are redundant between them, lack consistency in the mutants analyzed, and show contradiction between them. The most important shortcoming of the study is the fact that every single experiment in cells was done in over-expressed conditions, from transiently transfected cells. It is well known that these conditions can lead to non-specific mass effects, cellular localization not reflecting native conditions, and disruption of native interactome. On that topic, it is quite striking that the authors completely ignore the fact that Tip60 is exclusively found as part of a stable large multi-subunit complex in vivo, with more than 15 different proteins. Thus, arguing for a single residue acetylation regulating condensate formation and most Tip60 functions while ignoring native conditions (and the fact that Tip60 cannot function outside its native complex) does not allow me to support this study.

Specific points:<br /> -It is known that over-expression after transient transfection can lead to non-specific acetylation of lysines on the proteins, likely in part to protect from proteasome-mediated degradation. It is not clear whether the Kac sites targeted in the experiments are based on published/public data. In that sense, it is surprising that the K327R mutant does not behave like a HAT-dead mutant (which is what exactly?) or the K187R mutant as this site needs to be auto-acetylated to free the catalytic pocket, so essential for acetyltransferase activity like in all MYST-family HATs. In addition, the effect of K187R on the total acetyl-lysine signal of Tip60 is very surprising as this site does not seem to be a dominant one in public databases.

-As the physiological relevance of the results is not clear, the mutants need to be analyzed at the native level of expression to study real functional effects on transcription and localization (ChIP/IF). It is not clear the claim that Tip60 forms nuclear foci/punctate signals at physiological levels is based on what. This is certainly debated because in part of the poor choice of antibodies available for IF analysis. In that sense, it is not clear which Ab is used in the Westerns. Endogenous Tip60 is known to be expressed in multiple isoforms from splice variants, the most dominant one being isoform 2 (PLIP) which lacks a big part (aa96-147) of the so-called IDR domain presented in the study. Does this major isoform behave the same?

-It is extremely strange to show that the K187R mutant fails to get in the nuclei by cell imaging but remains chromatin-bound by fractionation... If K187 is auto-acetylated and required to enter the nucleus, why would a HAT-dead mutant not behave the same?

-If K187 acetylation is key to Tip60 function, it would be most logical (and classical) to test a K187Q acetyl-mimic substitution. In that sense, what happens with the R188Q mutant? That all goes back to the fact that this cluster of basic residues looks quite like an NLS.

-The effect of the mutant on the TIP60 complex itself needs to be analyzed, e.g. for associated subunits like p400, ING3, TRRAP, Brd8...

-The discussion is excessively long without addressing the obvious questions mentioned above.

Reviewer #3 (Public Review):

In this study, the authors demonstrated a new mechanism by which the protein stability of EZH2 is regulated. This mechanism is multifaceted and yet the authors provided evidence for every step of regulation. EZH2 is monomethylated at K20 by SET7, which can be removed by LSD1 and recognized by L3MBTL3. L3MBTL3 recruits the ubiquitin E3 ligase CRLDCAF5 to EZH2 via methylation of K20, which results in polyubiquitylation and proteasomal degradation of the histone methyltransferase. Additionally, they found that AKT-mediated phosphorylation of EZH2 at S21 blocks monomethylation at K20 and vice versa. Finally, they demonstrated in the K20R GEMM model that stabilization of EZH2 protein leads to reactive hyperplasia and hematopoiesis. In general, this study reveals an interesting and novel mechanism underlying the regulation of the epigenetic mark H3K27me3 and the oncogenic function of EZH2. The authors have considered every aspect of the signaling pathway that regulates the protein stability of EZH2. The data was comprehensive, rigorous, and supportive of the conclusions they made. Their results may help explain some of the conflicting results that previous studies have reported.

However, there are still some issues with the significance of the work and the quality of the data. The major issues are:<br /> 1. The converged effect of EZH2 methylation and phosphorylation on H3K27me3 is unclear.<br /> 2. How the methylation-phosphorylation switch of EZH2 determines the biological phenotypes they observed is not addressed.<br /> 3. Some of the data in the manuscript is conflicting.

Reviewer #3 (Public Review):

In their manuscript, Pivirotto et al. make an unexpected observation that a set of candidate beneficial alleles according to the Evolutionary Probability method (EP) have estimated ages thousands of years older than control alleles of similar frequency and outside of functional segments. To explain this unexpectedly older ages, the authors propose a number of interesting evolutionary processes related to balancing selection, including staggered sweeps.

It is important to first mention that the authors do find that as expected, deleterious alleles are younger than controls. This provides evidence that the allele age estimates used by the authors are of sufficient quality to detect age differences between groups of genes. I am also convinced by the fact that EP can be used to focus on a set of alleles substantially enriched in deleterious ones, given the very clear frequency patterns related to EP.

I have a number of concerns about the manuscript, including one rather serious one.

My main concern is that many of the observations made by the authors could be caused by mispolarization of alleles, where either (i) mostly low frequency derived alleles are mischaracterized as ancestral and the other, actually ancestral allele is mischaracterized as a high frequency derived allele, or (ii) mostly low frequency ancestral alleles are mischaracterized as derived. Unfortunately, the authors do not even mention the risk of mispolarization in their manuscript. This is a serious problem for this manuscript because ancestral alleles annotated as derived are by definition going to generate older age estimates than if they were truly derived. It would be very useful to be able to have a look at the full distribution of allele ages rather than just confidence intervals as in Figure 1. I happen to have experience with mispolarization of high frequency ancestral alleles as derived by a maximum likelihood method, different from the one used by the authors (Keightley et al Genetics 2018), where the mispolarization became visible as a very suspicious SFS with a visible excess of high frequency variants, especially those expected to be functional (because of the relatively larger corresponding supply of low frequency deleterious functional variants). Even if the ML method used by the authors is not the same, mispolarization is still a serious risk. Glémin et al. Genome Research 2015 also found that mispolarization is far from being a negligible issue.

Mispolarization of low frequency alleles may be especially prominent in the case of mispolarized deleterious alleles associated with a very negative delta-EP, that then appear as alleles with a very positive delta-EP. Focusing on high delta-EP alleles may then in fact enrich the dataset in mispolarized alleles that then result in older age estimates. Looking at Figure 1B especially, I am worried by the fact that very high delta-EP values seem to go back to the frequencies observed for very negative delta-EP. This is what mispolarization of low frequency alleles might cause as a pattern, in this case especially low frequency ancestral alleles being misidentified as derived?

The authors can address the possible issue of mispolarization in multiple ways. First, they can use simulations of sequences to estimate amounts of mispolarization based on their polarization approach, using substitutions/mutation rates as realistic as possible.<br /> Second, the authors could check if there is suspicious symmetry in the distribution of delta-EP between alleles at frequency f and alleles at frequency 1-f. This pattern could be generated by mispolarization.

My second less serious concern has to do with the use of high delta-EP as evidence that alleles are beneficial. The validation set from the Patel & Kumar 2019 paper is arguably small with 24 known selected variants. It does not follow from the fact that a small set of known selected variants have higher delta-EP, that all variants with high delta-EP tend to be beneficial. This is especially true in the case where beneficial variants tend to be rare, and there are then far more variants expected with high delta-EP than there are beneficial variants. I am willing to change my mind on this if the overall results can be shown to be robust after accounting for allele mispolarization.

Third, I like the idea of staggered sweeps to explain the results, but I am wondering if there is any evidence in the literature of interference between deleterious and advantageous variants that the authors could base their proposed explanation on.

Finally, and I realize that it is a bit of a stretch, I am wondering if the authors could better justify their choices of methods to estimate the age of alleles. What about ARGweaver, Relate or tsdate? How do these methods compare with GEVA? From looking at the literature I could not find a direct comparison of the precision of GEVA compared to these other tools, but it may be worth at least discussing that the results could be further put to the test with other available ARG-based tools to estimate allele ages. Wilder Wohns et al. Science 2022 compare the performance of these different ARG methods with ancient DNA data, and in fact find that GEVA does not perform as well as for example Relate or tsdate.

Reviewer #3 (Public Review):

Summary: In this manuscript, Blaeser et al. explore the link between CSD and headache pain. How does an electrochemical wave in the brain parenchyma, which lacks nociceptors, result in pain and allodynia in the V1-3 distribution? Prior work had established that CSD increased the firing rate of trigeminal neurons, measured electrophysiologically at the level of the peripheral ganglion. Here, Blaeser et al. focus on the fine afferent processes of the trigeminal neurons, resolving Ca2+ activity of individual fibers within the meninges. To accomplish these experiments, the authors injected AAV encoding the Ca2+ sensitive fluorophore GCamp6s into the trigeminal ganglion, and 8 weeks later imaged fluorescence signals from the afferent terminals within the meninges through a closed cranial window. They captured activity patterns at rest, with locomotion, and in response to CSD. They found that mechanical forces due to meningeal deformations during locomotion (shearing, scaling, and Z-shifts) drove non-spreading Ca2+ signals throughout the imaging field, whereas CSD caused propagating Ca2+ signals in the trigeminal afferent fibers, moving at the expected speed of CSD (3.8 mm/min). Following CSD, there were variable changes in basal GCamp6s signals: these signals were unchanged in the majority of fibers, signals increased (after a ~20 min delay) in 10% of fibers, and signals decreased in 20% of fibers. Bouts of locomotion were less frequent following CSD, but when they did occur, they elicited more robust GCamp6s signals than pre-CSD. These findings advance the field, suggesting that headache pain following CSD can be explained on the basis of peripheral cranial nerve activity, without invoking central sensitization at the brain stem/thalamic level. This insight could open new pathways for targeting the parenchymal-meningeal interface to develop novel abortive or preventive migraine treatments.

Strengths: The manuscript is well-written. The studies are broadly relevant to neuroscientists and physiologists, as well as neurologists, pain clinicians, and patients with migraine with aura and acephalgic migraine. The studies are well-conceived and appear to be technically well-executed.

Weaknesses: In the present study, conclusions are based entirely on fluorescence signals from GCamp6s. Fluorescence experiments should be interpreted cautiously in the context of CSD. GCamp6 fluorophores are strongly pH dependent, with decreased signal at acidic pH values (at matched Ca2+ concentration). CSD induces an impressive acidosis transient in the brain parenchyma, so one wonders whether the suppression of activity reported in the wake of CSD (Figure 2) in fact reflects decreased sensitivity of the GCamp6 reporter, rather than decreased activity in the fibers. If intracellular pH in trigeminal afferent fibers acidifies in the wake of CSD, GCamp6s fluorescence may underestimate the actual neuronal activity.

Reviewer #3 (Public Review):

The potential for sexual selection and the extent of sexual dimorphism in gene expression have been studied in great detail in animals, but hardly examined in plants so far. In this context, the study by Zhao, Zhou et al. al represents a welcome addition to the literature.

Relative to the previous studies in Angiosperms, the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers).

DOI: 10.1016/j.xpro.2023.102810

Resource: ZFIN_ZDB-GENO-040723-3

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-040723-3

What is this?

Reviewer #3 (Public Review):

Summary:<br /> The authors consider various statistical testing frameworks for model selection in the context of neuronal tuning. They consider cross-validation as a baseline scheme, and show various corrections and modifications to existing cross-validation schemes together with the underlying data/sign shuffling procedures for finding null distributions. Through careful simulations, they show that some of these tests are expectedly too conservative or too optimistic, and show that a log-likelihood-based test statistic with a cyclic shift permutation test for obtaining null distribution and Bonferroni correction strikes the right balance between hits and false detection. They further apply these tests to calcium imaging data from the mouse entorhinal cortex to identify grid cells (i.e., cells for which position is selected as a relevant variable).

Strengths:<br /> The paper is very well written, easy to follow, and enjoyable to read. It addresses an important issue in modern neuroscience, which is drawing conclusions based on data with missing or (unaccounted for) auto-correlated covariates.

Weaknesses:<br /> The paper would benefit from including more rigorous theoretical justification on why some of the procedures examined here outperform the others. This could be done in a stylized example with a Gaussian linear model, for which some of the used statistics have well-known distributions.

Comparisons with false discovery rate (FDR) control, as a more appropriate measure of performance when dealing with many comparisons, would benefit the existing comparisons merely based on Bonferroni correction.

Including spiking history in the generalized linear models (GLMs) used in analyzing the mouse data could be beneficial, as existing literature points to the importance of spiking history as a relevant covariate.

Reviewer #3 (Public Review):

Summary: Drosophila larvae exhibit characteristic escape behavior in response to a noxious stimulus. The underlying nociceptive circuit that regulates the temporal dynamics of escape behavior - bending, rolling, and crawling remains unclear. Using behavioral prototypes with optical stimulation and imaging, the authors show the function of descending neurons (SeIN128) in the termination of the rolling and subsequent initiation of the crawling behavior. The study further establishes the functional connectome of SeIN128, Basin-2, and A00c neurons, forming an inhibitory feedback circuit that regulates the rolling-escape sequences.

Strength: The study provides anatomical and functional evidence for temporal dynamics of escape behaviors in Drosophila larvae. Authors convincingly show the function of bilaterally descending neurons (previously identified SeIN128 neurons) in the transition of escape sequences. Based on the previous studies and functional connectome analysis, the study shows that SeIN128 neurons form a GABAergic feedback circuit with Basin-2, a second-order interneuron, and A00c, an ascending neuron downstream of Basin-2. Activation of SeIN128 neurons terminates the rolling by suppressing Basin-2 activity, facilitating subsequent rapid escape crawling. Thus, it establishes the function of feedback inhibition in temporal dynamics of escape behavior and contributes to a mechanistic understanding of the nociceptive circuits.

Weakness: The manuscript is written clearly; however, the presentation of the data needs to be improved for readability. The data and discussion establish the function of SeIN128 and Basin-2 in escape behavior, but the role of A00c neurons needs to be clarified.

Reviewer #3 (Public Review):

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

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

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

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

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

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

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

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

Specific comments:<br /> A small part of the manuscript relies on replicating or modeling the proprioceptive recalibration in a study we think does NOT measure proprioceptive recalibration (Tsay, Parvin & Ivry, JNP, 2020). In this study, participants reached for a visual target with a clamped cursor, and at the end of the reach were asked to indicate where they thought their hand was. The responses fell very close to the visual target both before and after the perturbation was introduced. This means that the difference between the actual hand position, and the reported/felt hand position gets very large as soon as the perturbation is introduced. That is, proprioceptive recalibration would necessarily have roughly the same magnitude as the adaptation displayed by participants. That would be several times larger than those found in studies where proprioceptive recalibration is measured without a visual anchor. The data is plotted in a way that makes it seem like the proprioceptive recalibration is very small, as they plot the responses relative to the visual target, and not the discrepancy between the actual and reported hand position. It seems to us that this study mostly measures short-term visual memory (of the target location). What is astounding about this study is that the responses change over time to begin with, even if only by a tiny amount. Perhaps this indicates some malleability of the visual system, but it is hard to say for sure.

Regardless, the results of that study do not form a solid basis for the current work and they should be removed. We would recommend making use of the dataset from the same authors, who improved their methods for measuring proprioception shifts just a year later (Tsay, Kim, Parvin, Stover, and Ivry, JNP, 2021). Although here the proprioceptive shifts during error-clamp adaptation (Exp 2) were tiny, and not quite significant (p<0.08), the reports are relative to the actual location of the passively placed unseen hand, measured in trials separate from those with reach adaptation and therefore there is no visual target to anchor their estimates to.

Experiment 1 measures visual uncertainty with increased rotation size. The authors cite relevant work on this topic (Levi & Klein etc) which has found a linear increase in uncertainty of the position of more and more eccentrically displayed stimuli.

First, this is a question where the reported stimuli and effects could greatly benefit from comparisons with the literature in vision science, and the results might even inform it. In order for that to happen, the units for the reported stimuli and effects should (also) be degrees of visual angle (dva).

As far as we know, all previous work has investigated static stimuli, where with moving stimuli, position information from several parts of the visual field are likely integrated over time in a final estimate of position at the end of the trajectory (a Kalman filter type process perhaps). As far as we know, there are no studies in vision science on the uncertainty of the endpoint of moving stimuli. So we think that the experiment is necessary for this study, but there are some areas where it could be improved.

Then, the linear fit is done in the space of the rotation size, but not in the space of eccentricity relative to fixation, and these do not necessarily map onto each other linearly. If we assume that the eye-tracker and the screen were at the closest distance the manufacturer reports it to work accurately at (45 cm), we would get the largest distances the endpoints are away from fixation in dva. Based on that assumed distance between the participant and monitor, we converted the rotation angles to distances between fixation and the cursor endpoint in degrees visual angle: 0.88, 3.5, and 13.25 dva (ignoring screen curvature, or the absence of it). The ratio between the perturbation angle and retinal distance to the endpoint is roughly 0.221, 0.221, and 0.207 if the minimum distance is indeed used - which is probably fine in this case. But still, it would be better to do fit in the relevant perceptual coordinate system.

The first distance (4 deg rotation; 0.88 dva offset between fixation and stimulus) is so close to fixation (even at the assumed shortest distance between eye and screen) that it can be considered foveal and falls within the range of noise of eye-trackers + that of the eye for fixating. There should be no uncertainty on or that close to the fovea. The variability in the data is likely just measurement noise. This also means that a linear fit will almost always go through this point, somewhat skewing the results toward linearity. The advantage is that the estimate of the intercept (measurement noise) is going to be very good. Unfortunately, there are only 2 other points measured, which (if used without the closest point) will always support a linear fit. Therefore, the experiment does not seem suitable to test linearity, only to characterize it, which might be sufficient for the current purposes. We'd understand if the effort to do a test of linearity using many more rotations requires too much effort. But then it should be made much clearer that the experiment assumes linearity and only serves to characterize the assumed linearity.

Final comment after the consultation session:<br /> There were a lot of discussions about the actual interpretation of the behavioral data from this paper with regards to past papers (Tsay et al. 2020 or 2021), and how it matches the different variables of the model. The data from Tsay 2020 combined both proprioceptive information (Xp) and prediction about hand position (Xu) because it involves active movements. On the other hand, Tsay et al. 2021 is based on passive movements and could provide a better measure of Xp alone. We would encourage you to clarify how each of the variables used in the model is mapped onto the outcomes of the cited behavioral experiments.

The reviewers discussed this point extensively during the consultation process. The results reported in the Tsay 2020 study reflect both proprioception and prediction. However, having a visual target contributes more than just prediction, it is likely an anchor in the workspace that draws the response to it. Such that the report is dominated by short-term visual memory of the target (which is not part of the model). However, in the current Exp 3, as in most other work investigating proprioception, this is calculated relative to the actual direction.

The solution is fairly simple. In Experiment 3 in the current study, Xp is measured relative to the hand without any visual anchors drawing responses, and this is also consistent with the reference used in the Tsay et al 2021 study and from many studies in the lab of D. Henriques (none of which also have any visual reach target when measuring proprioceptive estimates). So we suggest using a different data set that also measures Xp without any other influences, such as the data from Tsay et al 2021 instead.

These issues with the data are not superficial and can not be solved within the model. Data with correctly measured biases (relative to the hand) that are not dominated by irrelevant visual attractors would actually be informative about the validity of the PEA model. Dr. Tsay has so much other that we recommend using a more to-the-point data set that could actually validate the PEA model.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Davidsen and collaborators introduce jAspSnFR3, a new version of aspartate biosensor derived from iGluSnFR3, that allows to monitor in real-time aspartate levels in cultured cells. A selective amino acids substitution was applied in a key region of the template to switch its specificity from glutamate to aspartate. The jAspSnFR3 does not respond to other tested metabolites and performs well, is not toxic for cultured cells, and is not affected by temperature ensuring the possibility of using this tool in tissues physiologically more relevant. The high affinity for aspartate (KD=50 uM) allowed the authors to measure fluctuations of this amino acid in the physiological range. Different strategies were used to bring aspartate to the minimal level. Finally, the authors used jAspSnFR3 to estimate the intracellular aspartate concentration.

Strengths:<br /> One of the highlights of the manuscript was a treatment with asparagine during glutamine starvation. Although didn`t corroborate the essentiality of asparagine in glutamine depletion, the measurement of aspartate during this supplementation is a glimpse of how useful this sensor can be.

Weaknesses:<br /> Although this is a well-performed study, I have some comments for the authors to address:<br /> 1-A red tag version of the sensor (jAspSnFR3-mRuby3) was generated for normalization purposes, with this the authors plan to correct GFP signal from expression and movement artifacts. I naturally interpret "movement artifacts" as those generated by variations in cell volume and focal plane during time-lapse experiments. However, it was mentioned that jAspSnFR3-mRuby3 included a histidine tag that may induce a non-specific effect (responses to the treatment with some amino acids). This suggests that a version without the tag needs to be generated and that an alternative design needs to be set for normalization purposes. A nuclear-localized RFP was expressed in a second attempt to incorporate RFP as a normalization signal. Here the cell lines that express both signals (sensor and RFP) were generated by independent lentiviral transductions (insertions). Unless the number of insertions for each construct is known, this approach will not ensure an equimolar expression of both proteins (sensor and RFP). In this scenario is not clear how the nuclear expression of RFP will help the correction by expression or monitor changes in cell volume. The authors may be interested in attempting a bicistronic system to express both the sensor and RFP.<br /> 2-The authors were interested in establishing the temporal dynamics of aspartate depletion by genetics and pharmaceutical means. For the inhibition of mitochondrial complex I rotenone and metformin were used. Although the assays are clearly showing aspartate depletion the report of cell viability is missing. Considering that glutamine deprivation induces arrest in cell proliferation, I think will be important to know the conditions of the cell cultures after 60 hours of treatment with such inhibitors.<br /> 3-The pH sensitivity was checked in vitro with jAspSnFR3-mRuby3 and the sensor reported suitable for measurements at physiological pH. It would be an opportunity to revisit the analysis for pH sensitivity in cultured cells using an untagged version of jAspSnFR3 coupled, for example, to a sensor for pH.<br /> 4-While the authors take an interesting approach to measuring intracellular aspartate concentration, it will be highly desirable if a calibration protocol can be designed for this sensor. Clearly, glutamine depletion grants a minimal ("zero") aspartate concentration. However, having a more dynamic way for calibration will facilitate the introduction of this tool for metabolism studies. This may be achieved by incorporating a cultured cell that already expresses the transporter or by ectopic expression in the cells that have already been used.

Reviewer #3 (Public Review):

The first part of the review was prepared after the first submission of the paper. After this, the authors made several changes in the manuscript. These changes are assessed at the end of the review.

First part:

The paper by Deng, Kumar, Cavalli, Klyachko describe that, unlike in other cell types, loss of Fmr1 decreases the excitability of hippocampal mossy cells due to up-regulation of Kv7 currents. They also show evidence that while muting mossy cells appears to be a compensatory mechanism, it contribute to the higher activity of the dentate gyrus, because the removal of mossy cell output alleviate the inhibition of dentate principal cells. This may be important for the patho-mechanism in Fragile X syndrome caused by the loss of Fmr1.

These experiments were carefully designed, and the results are presented ‎in a very logical, insightful and self-explanatory way. Therefore, this paper represents strong evidences for the claims of the authors. In the current state of the manuscript there are only a few points that need additional explanation.

One of the results, that is shown in the supplementary dataset, does not fit to the main conclusions. Changes in the mEPSC frequency suggest that in addition to the proposed network effects, there are additional changes in the synaptic machinery or synapse number that are independent of the actual activity of the neurons. Since the differences of the mEPSC and sEPSC frequencies are similar and because only the latter can signal network effects, while the former is typically interpreted as a presynaptic change, it cannot be claimed that sEPSC frequency changes are due to the hypo-excitability of mossy cells.

An apparent technical issue may imply a second weak point in the interpretation of the results. Because the IPSCs in the PP stimulation experiments (Fig8) start within a few milliseconds, it is unlikely that its first ‎components originate from the PP-GC-MC-IN feedforward inhibitory circuit. The involvement of this circuit and MCs in the Kv7-dependent excitability changes is the main implication of the results of this paper. But this feedforward inhibition requires three consecutive synaptic steps and EPSP-AP couplings, each of them lasting for at least 1ms + 2-5ms. Therefore, the inhibition via the PP-GC-MC-IN circuit can be only seen from 10-20ms after PP stimulation. The earlier components of the cPSCs should originate from other circuit elements that are not related to the rest of the paper. Therefore, more isolated measurements on the cPSC recordings are needed ‎which consider only the later phase of the IPSCs. This can be either a measurement on the decay phase or a pharmacological manipulation that selectively enhance/inhibit a specific component of the proposed circuit.

I suggest refraining from the conclusions saying "‎MCs provide at least ~51% of the excitatory drive onto interneurons in WT and ~41% in KO mice", because too many factors (eg. IN celll types, slice condition, synaptic reliability) are not accounted for these actual numbers, and these values are not necessary for the general observation of the paper.

There are additional minor issues about the presentation of the results that are explained in the private recommendation for the Authors.

Review after the revision:

The authors accepted my suggestions and made changes in the manuscript to address my point about the interpretation of the mEPSC changes.<br /> The second point was related to the interpretation of the stimulation evoked multisynaptic compound responses. Specifically, the IPSC components in the PP stimulation experiments start within a few milliseconds, and I pointed out that it is unlikely that its first ‎components originate from the PP-GC-MC-IN feedforward inhibitory circuit. The authors provided strong arguments for the interpretation of these compound responses in their reply and the conclusions are consistent with these complex results.

Additional minor issues were fully addressed.

I still think that this is a strong paper that provides new insights into the mechanisms of Fragile X syndrome at the level of single neurons and local network. The extensive series of experiments convincingly support the main findings that in addition to contributing to the underlying mechanisms of this disease also highlight how delicately neuronal activity is balanced even in constrained conditions.

Reviewer #3 (Public Review):

The goal of the current manuscript is to investigate how changes in transporter substrate specificity emerge through experimental evolution. The authors investigate the APC family of amino acid transporters, a large family with many related transporters that together cover the spectrum of amino acid uptake in yeast.

The authors use a clever approach for their experimental evolutions. By deleting 10 amino acid uptake transporters in yeast, they develop a strain that relies on amino acid import by introducing APC transporters under nitrogen-limiting conditions. They can thus evolve transporters towards the transport of new substrates if no other nitrogen source is available. The main takeaway from the paper is that it is relatively easy for the spectrum of substrates in a particular transporter of this family to shift, as a number of single mutants are identified that modulate substrate specificity. In general, transporters evolved towards gain-of-function mutations (better or new activities) and also confer transport promiscuity, expanding the range of amino acids transported.

The data in the paper support the conclusions, in general, and the outcomes (evolution towards promiscuity) agree with the literature available for soluble enzymes. However, it is also a possibility that the design of these experiments selects for promiscuity among amino acids. The selections were designed such that yeast had access to amino acids that were already transported, with a greater abundance of the amino acid that was the target of selection. Under these conditions, it seems probable that the fittest variants will provide the yeast access to all amino acid substrates in the media, and unlikely that a specificity swap would occur, limiting the yeast to only the new amino acid.

The authors also examine the fitness costs of mutants, but only in the narrow context of growth on a single (original) amino acid under conditions of nitrogen limitation. Amino acid uptake is typically tightly controlled because some amino acids (or their carbon degradation products) are toxic in excess. This paper does not address or discuss whether there might be a fitness cost to promiscuous mutants in conditions where nitrogen is not limiting.

Reviewer #3 (Public Review):

Summary:

In this study, Bomba-Warczak et al focused on reproductive aging, and they presented a map for long-lived proteins that were stable during reproductive lifespan. The authors used MIMS to examine and show distinct molecules in different cell types in the ovary and tissue regions in a 6 month mice group, and they also used proteomic analysis to present different LLPs in ovaries between these two timepoints in 6-month and 10-month mice. The authors also examined the LLPs in oocytes in the 6-months mice group and indicated that these were nuclear, cytoskeleton, and mitochondria proteins.

Strengths:

Overall, this study provided basic information or a 'map' of the pattern of long-lived proteins during aging, which will contribute to the understanding of the defects caused by reproductive aging.

Weaknesses:

The 6-month mice were used as an aged model; no validation experiments were performed with proteomics analysis only.

Reviewer #3 (Public Review):

Summary:

The manuscript authored by Lan Guan and colleagues reveals the structure of the cytosol-facing conformation of the MelB sodium/Li coupled permease using the nab-Fab approach and cryoEM for structure determination. The study reveals the conformational transitions in the melB transport cycle and allows understanding the role of sugar and ion specificities within this transporter.

Strengths:

The study employs a very exciting strategy of transferring the CDRS of a conformation specific nano body to the nab-fab system to determine the inward-open structure of MelB. The resolution of the structure is reasonable enough to support the major conclusions of the study. This is overall a well-executed study.

Weaknesses:

The authors seem to have mixed up the exothermic and endothermic aspects of ITC binding in their description. Positive heats correspond to endothermic heat changes in ITC and negative heat changes correspond to exothermic heats. The authors seem to suggest the opposite. This is consistently observed throughout the manuscript.

Reviewer #3 (Public Review):

Hung et al provide a well-written manuscript focused on understanding how Eklf mutation confers anticancer and longevity advantages in vivo.

The authors were responsive to the reviewers comments in some aspects. However, the manuscript continues to suffer from significantly overstated claims that are not mitigated in the revision. While additional data has been added, it is unclear how this new data provides clarity to the overall premise of this observational study. Importantly, the authors have added a second model of hepatocellular carcinoma with findings that are consistent with the melanoma model previously reported. In addition, they make more clear that the previously published manuscript on this subject was use of older donors for BMT while now they use younger donors. This is at best incremental. It remains unclear whether Eklf exerts its effect on resistance to malignant progression / metastasis by modulating Pd1 or Pdl1 vs. increasing NK cells as the authors provide evidence of both and do not resolve which mechanism is primarily involved. Finally, there is no evidence that Eklf mutation confers "an anti-disease and anti-aging" effect as at best the data provides evidence of resistance to malignant progression / metastasis in melanoma and hepatoma models.

The work is impactful as it provides evidence of anticancer effect of a specific hematological mutation but the mechanism by which this occurs is not completely elucidated by this work.

Reviewer #3 (Public Review):

In multiple cancers, the key roles of B cells are emerging in the tumor microenvironment (TME). The authors of this study appropriately introduce that B cells are relatively under-characterised in the TME and argue correctly that it is not known how the B cell receptor (BCR) repertoires across tumors, lymph nodes, and peripheral blood relate. The authors therefore supply a potentially useful study evaluating the tumor, lymph node, and peripheral blood BCR repertoires and site-to-site as well as intra-site relationships. The authors employ sophisticated analysis techniques, although the description of the methods is incomplete. Among other interesting observations, the authors argue that the tumor BCR repertoire is more closely related to that of draining lymph node (dLN) than the peripheral blood in terms of clonal and isotype composition. Furthermore, the author's findings suggest that tumor-infiltrating B cells (TIL-B) exhibit a less mature and less specific BCR repertoire compared with circulating B cells. Overall, this is a potentially useful work that would be of interest to both medical and computational biologists working across cancer. However, there are aspects of the work that would have benefitted from further analysis and areas of the manuscript that could be written more clearly and proofread in further detail.

Major Strengths:

1. The authors provide a unique analysis of BCR repertoires across tumor, dLN, and peripheral blood. The work provides useful insights into inter- and intra-site BCR repertoire heterogeneity. While patient-to-patient variation is expected, the findings with regard to intra-tumor and intra-dLN heterogeneity with the use of fragments from the same tissue are of importance, contribute to the understanding of the TME, and will inform future study design.

2. A particular strength of the study is the detailed CDR3 physicochemical properties analysis which leads the authors to observations that suggest a less-specific BCR repertoire of TIL-B compared to circulating B cells.

Major Weaknesses:

1. The study would have benefitted from a deeper biological interpretation of the data. While given the low number of patients one can plausibly understand a reluctance to speculate about clinical details, there is limited discussion about what may contribute to observed heterogeneity. For example, for the analysis of three lymph nodes taken per patient which were examined for inter-LN heterogeneity, there is a lack of information regarding these lymph nodes. 'LN3' is deemed as exhibiting the most repertoire overlap with the tumor but there is no discussion as to why this may be the case.

2. At times the manuscript is difficult to follow. In particular, the 'Intra-LN heterogeneity' section follows the 'LN-LN heterogeneity in colorectal cancer' section and compares the overlap of LN fragments (LN11, LN21, LN31) with the tumor in two separate patients (Fig 6A). In the previous section (LN-LN), LN11, LN21, LN31 are names given to separate lymph nodes from the same patient. The fragments are referred to as 'LN2' and the nodes in the previous section are referred to similarly. This conflation of naming for nodes and fragments is confusing.

3. There is a duplicated paragraph in 'Short vs long trees' and the following section 'Productive involvement in hypermutation lineages depends on CDR3 characteristics.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript reports new molecular characterization of the Haemophilus influenza tripartite ATP-independent periplasmic (TRAP) transporter of N-acetylneuraminate (Neu5Ac). This membrane transporter is important for the virulence of the pathogen. H. influenza lacks Neu5Ac biosynthetic pathway, and utilizes the TRAP transporter to import it. Neu5Ac is used as a nutrient source but also as a protection from human immune response. The transporter is composed of two fused membrane subunits, HiSiaQM, and one soluble, periplasmic subunit HiSiaP. HiSiaP, by binding to the substrate Neu5Ac, changes its conformation, allowing its binding to HiSiaQM, followed by Neu5Ac and Na+ transport to the cytoplasm. The combination of structural, biophysical and biochemical approaches provides a solid basis for describing the functioning of the Haemophilus influenza Neu5Ac TRAP transporter, which is essential for the pathogen virulence.

Strengths:<br /> The paper describes the electron microscopy structure of HiSiaQM, thanks to its solubilization in L-MNG followed by exchange to amphipol or nanodisc. In these conditions, HiSiaQM consists in a mixture of monomers and dimers, as characterized by analytical ultracentrifugation. The cryo-EM analysis shows two types of dimers: one in an antiparallel configuration, which is artifactual, and a parallel one, which may be physiologically relevant. Cryo-EM on the dimers allows high resolution (≈ 3 Å) structure determination. The structure is the first one of a fused SiaQM, and is the first obtained without megabody. The work highlights structural elements (fusion helix, lipids) that could modulate transport. The authors checked the functionality of the purified HiSiaQM, which, after reconstitution in liposome, displays a significantly larger Neu5Ac transport activity compared to the non-fused PpSiaQM homolog. The work identifies Na+ binding sites, and the putative Neu5Ac binding site. From analytical ultracentrifugation using fluorescently labelled HiSiaP, the authors show that HiSiaP is able to interact with HiSiaQM monomer and dimer, with a low but physiologically relevant affinity. HiSiaP interaction with HiSiaQM was modelled using AlphaFold2, and discussed in view of published activity on mutants, and new transport activity assays using SiaQM and SiaP from different organisms. In conclusion, the combination of structural, biophysical and biochemical approaches provides a solid basis for describing the functioning of this TRAP fused transporter.

Weakness: This work evidences in vitro a HiSiaQM dimer, whose in vivo relevance is not ascertained. However, the authors are very careful, they do not to over-interpret their data, and their conclusions regarding the transporter structure and function are valid irrespective of its state of association.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors are trying to delineate the mechanism underlying the osteonecrosis of the femoral head.

Strengths:

The authors provided compelling in vivo and in vitro data to demonstrate Col2+ cells and Osx+ cells were differentially expressed in the<br /> the femoral head. Moreover, inducible knockout of β-catenin in Col2+ cells but not<br /> Osx+ cells lead to a GONFH-like phenotype including fat accumulation, subchondral<br /> bone destruction and femoral head collapse, indicating that imbalance of osteogenic/adipogenic differentiation of Col2+ cells play an important role in GONFH pathogenesis. Therefore, this manuscript provided the mechanistic insights of osteonecrosis as well as potential therapeutic target for disease treatment.

Weaknesses: Additional in depth discussion regarding the phenotype observed in mice is highly encouraged.

Reviewer #3 (Public Review):

Nguyen, Zhao, et al. used bioinformatic analysis of mutational variants of SARS-CoV-2 Nucleocapsid (N) protein from the large genomic database of SARS-CoV-2 sequences to identify domains and regions of N where mutations are more highly represented and computationally determined the effects of these mutations on the physicochemical properties of the protein. They found that the intrinsically disordered regions (IDRs) of N protein are more highly mutated than structured regions and that these mutations can lead to higher variability in the physical properties of these domains. These computational predictions are compared to in vitro biophysical experiments to assess the effects of identified mutations on the thermodynamic stability, oligomeric state, particle formation, and liquid-liquid phase separation of a few exemplary mutants.

The paper is well-written and easy to follow, and the conclusions drawn are supported by the evidence presented. The analyses and conclusions are interesting and will be of value to virologists, cell biologists, and biophysicists studying SARS-CoV-2 function and assembly. It would be nice if some further extrapolation or comments could be made regarding the effects of the observed mutations on the in vivo behavior and properties of the virus, but I appreciate that this is much higher-order than could be addressed with the approaches employed here.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors investigated the interaction between the pattern recognition receptor MDA5 and 5'ppp-RNA in a teleost fish called Miiuy croaker. They claimed that MDA5 can replace RIG-I in sensing 5'ppp-RNA of Siniperca cheats rhabdovirus (SCRV) in the absence of RIG-I in Miiuy croaker. The recognition of MDA5 to 5'ppp-RNA was also observed in the chicken (Gallus gallus), a bird species that lacks RIG-I. Additionally, they reported that the function of MDA5 can be impaired through m6A-mediated methylation and degradation of MDA5 mRNA by the METTL3/14-YTHDF2/3 regulatory network in Miiuy croaker under SCRV infection. This impairment weakens the innate antiviral immunity of fish and promotes the immune evasion of SCRV.

Strengths:<br /> These findings provide insights into the adaptation and functional diversity of innate antiviral activity in vertebrates.

Weaknesses:<br /> However, there are some major and minor concerns that need to be further addressed. Addressing these concerns will help the authors improve the quality of their manuscript.

One significant issue with the manuscript is that the authors claim to be investigating the role of MDA5 as a substitute for RIG-I in recognizing 5'ppp-RNA, but their study extends beyond this specific scenario. Based on my understanding, it appears that sections 2.2, 2.3, 2.5, 2.6, and 2.7 do not strictly adhere to this particular scenario. Instead, these sections tend to investigate the functional involvement of Miiuy croaker MDA5 in the innate immune response to viral infection. Furthermore, the majority of the data is focused on Miiuy croaker MDA5, with only a limited and insufficient study on chicken MDA5. Consequently, the authors cannot make broad claims that their research represents events in all RIG-I deficient species, considering the limited scope of the species studied.

The current title of the article does not align well with its actual content. It is recommended that the focus of the research be redirected to the recognition function and molecular mechanism of MDA5 in the absence of RIG-I concerning 5'ppp-RNA. This can be achieved through bolstering experimental analysis in the fields of biochemistry and molecular biology, as well as enhancing theoretical research on the molecular evolution of MDA5. It is advisable to decrease or eliminate content related to m6A modification.

Additionally, the main body of the writing contains several aspects that lack rigor and tend to exaggerate, necessitating significant improvement.

Reviewer #3 (Public Review):

Summary:<br /> This study considers how to model distinct host cell states that correspond to different stages of a viral infection: from naïve and susceptible cells to infected cells and a minority of important interferon-secreting cells that are the first line of defense against viral spread. The study first considers the distinct host cell states by analyzing previously published single-cell RNAseq data. Then an agent-based model on a square lattice is used to probe the dependence of the system on various parameters. Finally, a simplified version of the model is explored, and shown to have some similarity with the more complex model, yet lacks the dependence on the interferon range. By exploring these models one gains an intuitive understanding of the system, and the model may be used to generate hypotheses that could be tested experimentally, telling us "when to be surprised" if the biological system deviates from the model predictions.

Strengths:<br /> - Clear presentation of the experimental findings and a clear logical progression from these experimental findings to the modeling.<br /> - The modeling results are easy to understand, revealing interesting behavior and percolation-like features.<br /> - The scaling results presented span several decades and are therefore compelling.<br /> - The results presented suggest several interesting directions for theoretical follow-up work, as well as possible experiments to probe the system (e.g. by stimulating or blocking IFN secretion).

Weaknesses:<br /> - Since the "range" of IFN is an important parameter, it makes sense to consider lattice geometries other than the square lattice, which is somewhat pathological. Perhaps a hexagonal lattice would generalize better.

- Tissues are typically three-dimensional, not two-dimensional. (Epithelium is an exception). It would be interesting to see how the modeling translates to the three-dimensional case. Percolation transitions are known to be very sensitive to the dimensionality of the system.

- The fixed time-step of the agent-based modeling may introduce biases. I would consider simulating the system with Gillespie dynamics where the reaction rates depend on the ambient system parameters.

- Single-cell RNAseq data typically involves data imputation due to the high sparsity of the measured gene expression. More information could be provided on this crucial data processing step since it may significantly alter the experimental findings.

Justification of claims and conclusions:<br /> The claims and conclusions are well justified.

Reviewer #3 (Public Review):

Summary: Bidirectional transsynaptic signaling via cell adhesion molecules and cell surface receptors contributes to the remarkable specificity of synaptic connectivity in the brain. Zaman et al., investigates how the receptor tyrosine kinase Kit and its trans-cellular kit ligand regulate molecular layer interneuron (MLI)- Purkinje cell (PC) connectivity in the cerebellum. Presynaptic Kit is specific for MLIs, and forms a trans-synaptic complex with Kit ligand in postsynaptic PC cells. The authors begin by generating Kit cKOs via an EUCOMM allele to enable cell-type specific Kit deletion. They cross this Kit cKO to the MLI-specific driver Pax2-Cre and conduct validation via Kit IHC and immunoblotting. Using this system to examine the functional consequences of presynaptic MLI Kit deletion onto postsynaptic PC cells, they record spontaneous and miniature synaptic currents from PC cells and find a selective reduction in IPSC frequency. Deletion of Kit ligand from postsynaptic PC cells also results in reduced IPSC frequency, together supporting that this trans-synaptic complex regulates GABAergic synaptic formation or maturation. The authors then show that sparse Kit ligand overexpression in PCs decreases neighboring uninfected control sIPSCs in a potential competitive manner.

Strengths: Overall, the study addresses an important open question, the data largely supports the authors conclusions, the experiments appear well-performed, and the manuscript is well-written. I just have a few suggestions to help shore up the author's interpretations and improve the study.

Weaknesses:

The strong decrease in sIPSC frequency and amplitude in control uninfected cells in Figure 4 is surprising and puzzling. The competition model proposed is one possibility, and I think the authors need to do additional experiments to help support or refute this model. The authors can conduct similar synaptic staining experiments as in Fig S4 but in their sparse infection paradigm, comparing synapses on infected and uninfected cells. Additional electrophysiological parameters in the sparse injection paradigm, such as mIPSCs or evoked IPSCs, would also help support their conclusions.

The authors should validate KL overexpression and increased cell surface levels using their virus to support their overexpression conclusions.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors found that FBXO24, a testis-enriched F-box protein, is indispensable for male fertility. Fbxo24 KO mice exhibited malformed sperm flagellar and compromised sperm motility.

Strengths:

The phenotype of Fbxo24 KO spermatozoa was well analyzed.

Weaknesses:

The authors observed numerous membraneless electron-dense granules in the Fbxo24 KO spermatozoa. They also showed abnormal accumulation of two importins, IPO5 and KPNB1, in the Fbxo24 KO spermatozoa. However, the data presented in the manuscript do not support the conclusion that FBXO24 ensures male fertility by preventing the abnormal accumulation of membraneless granules in sperm flagella, as indicated in the manuscript title.

Reviewer #3 (Public Review):

Overall, this is a strong manuscript that uses multiple current techniques to provide specific mechanistic insight into prior discoveries of the contributions of the Bcl11b transcription factor to mossy fiber synapses of dentate gyrus granule cells. The authors employ an adult deletion of Bcl11b via Tamoxifen-inducible Cre and use immunohistochemical, electron microscopy, and electrophysiological studies of synaptic plasticity, together with viral rescue of C1ql2, a direct transcriptional target of Bcl11b or Nrxn3, to construct a molecular cascade downstream of Bcl11b for DG mossy fiber synapse development. They find that C1ql2 re-expression in Bcl11b cKOs can rescue the synaptic vesicle docking phenotype and the impairments in MF-LTP of these mutants. They also show that C1ql2 knockdown in DG neurons can phenocopy the vesicle docking and plasticity phenotypes of the Bcl11b cKO. They also use artificial synapse formation assays to suggest that C1ql2 functions together with a specific Nrxn3 splice isoform in mediating MF axon development, extending these data with a C1ql2-K262E mutant that purports to specifically disrupt interactions with Nrxn3. All of the molecules involved in this cascade are disease-associated and this study provides an excellent blueprint for uncovering downstream mediators of transcription factor disruption. Together this makes this work of great interest to the field. Strengths are the sophisticated use of viral replacement and multi-level phenotypic analysis while weaknesses include the linkage of C1ql2 with a specific Nrxn3 splice variant in mediating these effects.

Here is an appraisal of the main claims and conclusions:

1. C1ql2 is a downstream target of Bcl11b which mediates the synaptic vesicle recruitment and synaptic plasticity phenotypes seen in these cKOs. This is supported by the clear rescue phenotypes of synapse anatomy (Fig.2) and MF synaptic plasticity (Fig.3). One weakness here is the absence of a control assessing over-expression phenotypes of C1ql2. It's clear from Fig.1D that viral rescue is often greater than WT expression (totally expected). In the case where you are trying to suppress a LoF phenotype, it is important to make sure that enhanced expression of C1ql2 in a WT background does not cause your rescue phenotype. A strong overexpression phenotype in WT would weaken the claim that C1ql2 is the main mediator of the Bcl11b phenotype for MF synapse phenotypes.

2. Knockdown of C1ql2 via 4 shRNAs is sufficient to produce the synaptic vesicle recruitment and MF-LTP phenotypes. This is supported by clear effects in the shRNA-C1ql2 groups as compared to nonsense-EGFP controls. One concern (particularly given the use of 4 distinct shRNAs) is the potential for off-target effects, which is best controlled for by a rescue experiment with RNA-insensitive C1ql2 cDNA as opposed to nonsense sequences, which may not elicit the same off-target effects.

3. C1ql2 interacts with Nrxn3(25b+) to facilitate MF terminal SV clustering. This claim is theoretically supported by the HEK cell artificial synapse formation assay (Fig.5), the inability of the K262-C1ql2 mutation to rescue the Bcl11b phenotype (Fig.6) and the altered localization of C1ql2 in the Nrxn1-3 deletion mice (Fig.7). Each of these lines of experimental evidence has caveats that should be acknowledged and addressed. Given the hypothesis that C1ql2 and Nrxn3b(25b) are expressed in DG neurons and work together, the heterologous co-culture experiment seems weird. Up till now, the authors are looking at pre-synaptic function of C1ql2 since they are re-expressing it in DGNs. The phenotypes they are seeing are also pre-synaptic and/or consistent with pre-synaptic dysfunction. In Fig.5, they are testing whether C1ql2 can induce pre-synaptic differentiation in trans, i.e. theoretically being released from the 293 cells "post-synaptically". But the post-synaptic ligands (Nlgn1 and and GluKs) are not present in the 293 cells, so a heterologous synapse assay doesn't really make sense here. The effect that the authors are seeing likely reflects the fact that C1ql2 and Nrxn3 do bind to each other, so C1ql2 is acting as an artificial post-synaptic ligand, in that it can cluster Nrxn3 which in turn clusters synaptic vesicles. But this does not test the model that the authors propose (i.e. C1ql2 and Nrxn3 are both expressed in MF terminals). Perhaps a heterologous assay where GluK2 is put into HEK cells and the C1ql2 and Nrxn3 are simultaneously or individually manipulated in DG neurons?

4. K262-C1ql2 mutation blocks the normal rescue through a Nrxn3(25b) mechanism (Fig.6). The strength of this experiment rests upon the specificity of this mutation for disrupting Nrxn3b binding (presynaptic) as opposed to any of the known postsynaptic C1ql2 ligands such as GluK2. While this is not relevant for interpreting the heterologous assay (Fig.5), it is relevant for the in vivo phenotypes in Fig.6. Similar approaches as employed in this paper can test whether binding to other known postsynaptic targets is altered by this point mutation.

5. Altered localization of C1ql2 in Nrxn1-3 cKOs. These data are presented to suggest that Nrx3(25b) is important for localizing C1ql2 to the SL of CA3. Weaknesses of this data include both the lack of Nrxn specificity in the triple a/b KOs as well as the profound effects of Nrxn LoF on the total levels of C1ql2 protein. Some measure that isn't biased by this large difference in C1ql2 levels should be attempted (something like in Fig.1F).

Reviewer #3 (Public Review):

Kang, Huang, and colleagues have provided new data to address concerns regarding confirmation of LRRK1 and LRRK2 deletion in their mouse model and the functional impact of the modest loss of TH+ neurons observed in the substantia nigra of their double KO mice. In the revised manuscript, the new data around the characterization of the germline-deleted LRRK1 and LRRK2 mice add confidence that LRRK1 and LRRK2 can be deleted using the genetic approach. They have also added new text to the discussion to try and address some of the comments and questions raised regarding how LRRK1/2 loss may impact cell survival and the implications of this work for PD-linked variants in LRRK2 and therapeutic approaches targeting LRRK2.

The new data provides additional support for the author's claims. I have provided below some suggestions for clarification/additions to the text that can be addressed without additional experiments.

1) The authors added additional text highlighting that more studies are warranted in mice where LRRK1/2 are deleted in other CNS cell types (microglia/astrocytes) to understand cell extrinsic drivers of the autophagy deficits observed in their previous work. It still remains unclear how loss of LRRK1/2 leads to increased apoptosis and gliosis in dopaminergic neurons in a cell-intrinsic manner, and, as suggested in the original review, it would be helpful to add some text to the discussion speculating on potential mechanisms by which this might occur.

2) Revisions have been made to the discussion to clarify their rationale around how variants in LRRK2 associated with PD may be loss-of-function to support the relevance of this mouse model to phenotypes observed in PD. However, as written, the argument that PD-linked variants are loss-of-function is based on the fact that the double KO mice have a mild loss of TH+ neurons while the transgenic mice overexpressing PD-linked LRRK2 variants often do not and that early characterization of kinase activity was done in vitro are relatively weak. Given that the majority of evidence generated by many labs in the field supports a gain-of-function mechanism, the discussion should be further tempered to better highlight the uncertainty around this (rather than strongly arguing for a loss-of-function effect). This could include the mention of increased Rab phosphorylation observed in cellular and animal models and opposing consequences on lysosomal function observed in cellular studies in KO and pathogenic variant expressing cells. Further, a reference to the Whiffen et al. 2020 paper mentioned by another reviewer should be included in the discussion for completeness.

Reviewer #3 (Public Review):

Summary:<br /> Animals can evaluate food quality in many ways. In contrast to the rapid sensory evaluation with smell and taste, the mechanism of slow nutrient sensation and its impact on food choice is unexplored. The authors utilize C. elegans larvae and their bacterial food as an elegant model to tackle this question and reveal the detailed molecular mechanism to avoid nutrient-poor foods.

Strengths:<br /> The strength of this study is that they identified the molecular identities of the critical players in bacterial food and C. elegans using unbiased approaches, namely metabolome analysis, E. coli mutant screening, and RNA sequencing. Furthermore, they strengthen their findings by thorough experiments combining multiple methods such as genetics, fluorescent reporter analysis, and Western blot.

Weaknesses:<br /> The major caveat of this study is the reporter genes. The transcriptional reporters were used to monitor the UPRER and immune responses in the intestine of C. elegans. However, their tissue-specific rescue experiments suggest that the genes in the UPRER and immune response function in the neurons. Thus, we should carefully interpret the results of the reporter genes.

Overall, this work provides convincing data to support their model. In the C. elegans field, the behaviors of larvae are not well studied compared to adults. This work will pose an interesting question about the difference between larvae and adults in nutrition sensing in C. elegans and provide a framework and candidate molecules to be studied in other organisms.

Reviewer #3 (Public Review):

Summary<br /> In their manuscript, Vickers and McCormick have demonstrated the potential of leveraging mesoscale two-photon calcium imaging data to unravel complex behavioural motifs in mice. Particularly commendable is their dedication to providing detailed surgical preparations and corresponding design files, a contribution that will greatly benefit the broader neuroscience community as a whole. The quality of the data is high, but it is not clear whether this is available to the community, some datasets should be deposited. More importantly, the authors have acquired activity-clustered neural ensembles at an unprecedented spatial scale to further correlate with high-level behaviour motifs identified by B-SOiD. Such an advancement marks a significant contribution to the field. While the manuscript is comprehensive and the analytical strategy proposed is promising, some technical aspects warrant further clarification. Overall, the authors have presented an invaluable and innovative approach, effectively laying a solid foundation for future research in correlating large-scale neural ensembles with behavioural. The implementation of a custom sound insulator for the scanner is a great idea and should be something implemented by others.

This is a methods paper, but there is no large diagram that shows how all the parts are connected, communicating, and triggering each other. This is described in the methods, but a visual representation would greatly benefit the readers looking to implement something similar. The authors should cite sources for the claims stated in lines 449-453 and cite the claim of the mouse's hearing threshold mentioned in lines 463. No stats for the results shown in Figure 6e, it would be useful to know which of these neural densities for all areas show a clear statistical significance across all the behaviors. While I understand that this is a methods paper, it seems like the authors are aware of the literature surrounding large neuronal recordings during mouse behavior. Indeed, in lines 178-179, the authors mention how a significant portion of the variance in neural activity can be attributed to changes in "arousal or self-directed movement even during spontaneous behavior.". Why then did the authors not make an attempt at a simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc). These models are straightforward to implement, and indeed it would benefit this work if the model extracts information on par with what is known from the literature.

Specific strengths and weaknesses with areas to improve:

The paper should include an overall cartoon diagram that indicates how the various modules are linked together for the sampling of both behaviour and mesoscale GCAMP. This is a methods paper, but there is no large diagram that shows how all the parts are connected, communicating, and triggering each other.

The paper contains many important results regarding correlations between behaviour and activity motifs on both the cellular and regional scales. There is a lot of data and it is difficult to draw out new concepts. It might be useful for readers to have an overall figure discussing various results and how they are linked to pupil movement and brain activity. A simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc) may help in this regard.

Previously, widefield imaging methods have been employed to describe regional activity motifs that correlate with known intracortical projections. Within the authors' data it would be interesting to perhaps describe how these two different methods are interrelated, they do collect both datasets. Surprisingly, such macroscale patterns are not immediately obvious from the authors' data. Some of this may be related to the scaling of correlation patterns or other factors. Perhaps there still isn't enough data to readily see these and it is too sparse.

In lines 71-71, the authors described some disadvantages of one-photon widefield imaging including the inability to achieve single-cell resolution. However, this is not true. In recent years, the combination of better surgical preparations, camera sensors, and genetically encoded calcium indicators has enabled the acquisition of single-cell data even using one-photon widefield imaging methods. These methods include miniscopes (Cai et al., 2016), multi-camera arrays (Hope et al., 2023), and spinning disks (Xie et al., 2023).

Cai, Denise J., et al. "A shared neural ensemble links distinct contextual memories encoded close in time." Nature 534.7605 (2016): 115-118.<br /> Hope, James, et al. "Brain-wide neural recordings in mice navigating physical spaces enabled by a cranial exoskeleton." bioRxiv (2023).<br /> Xie, Hao, et al. "Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution." Nature Biomedical Engineering (2023): 1-14.

The authors' claim of achieving optical clarity for up to 150 days post-surgery with their modified crystal skull approach is significantly longer than the 8 weeks (approximately 56 days) reported in the original study by Kim et al. (2016). Since surgical preparations are an integral part of the manuscript, it may be helpful to provide more details to address the feasibility and reliability of the preparation in chronic studies. A series of images documenting the progression optical quality of the window would offer valuable insight.

Reviewer #3 (Public Review):

Wu and colleagues are characterising the function of Styxl2 during muscle development, a pseudo-phosphatase that was already described to have some function in sarcomere morphogenesis or maintenance (Fero et al. 2014). The authors verify a role for Styxl2 in sarcomere assembly/maintenance using zebrafish embryonic muscles by morpholino knock-down and by a conditional Styxl2 allele in mice (knocked-out in satellite cells with Pax7 Cre).

Experiments using a tamoxifen inducible Cre suggest that Styxl2 is dispensable for sarcomere maintenance and only needed for sarcomere assembly.

BioID experiments with Styxl2 in C2C 12 myoblasts suggest binding of nonmuscle myosins (NMs) to Styxl2. Interestingly, both NMs are downregulated when muscles differentiate after birth or during regeneration in mice. This down-regulation is reduced in the Styxl2 mutant mice, demonstrating that Styxl2 is required for the degradation of these NMs.

Impressively, reducing one NM (zMyh10) by double morpholino injection in a Styxl2 morphant zebrafish, does improve zebrafish mobility and sarcomere structure. Degradation of Mhy9 is also stimulated in cell culture if Styxl2 is co-expressed. Surprisingly, the phosphatase domain is not needed for these degradation and sarcomere structure rescue effects. Inhibitor experiments suggest that Styxl2 does promote the degradation of NMs by promoting the selective autophagy pathway.

Strengths:<br /> A major strength of the paper is the combination of various systems, mouse and fish muscles in vivo to test Styxl2 function, and cell culture including a C2C12 muscle cell line to assay protein binding or protein degradation as well as inhibitor studies that can suggest biochemical pathways.<br /> A second strength is that this manuscript sheds new light on the still ill-characterised mechanism of sarcomere assembly in skeletal muscles.

Weakness:<br /> The weaknesses of this manuscript have been largely eliminated during revision.

Reviewer #3 (Public Review):

Perhaps not unexpectedly, the proposed revisions consist of textual revisions only. Yewdell added a touch of levity with his H.G. Wells foundation as a source of $$ for a time machine. The paper does not establish striking new facts, in my opinion, but will stimulate discussion.

One point to consider: the relevance of the human T cell activation experiments is now downplayed even further, by the authors themselves, no less. I would suggest leaving the actual data out altogether and conclude with a statement: "Similar experiments conducted on activated human T cells showed significantly worse activation and may therefore not allow a head-to-head comparison with the results of our experimentst performed on mouse T cells. Not only might one consider the mode of activation (PMA/ionomycin) non-physiological, the activation status achievedwas less than that seen for the OT-1 model. " or something similar to that effect. In the present weakened form, I do not believe that the human data add anything of substance to the paper and are more of a distraction. The authors would increase the impact and readability of their paper if they omitted the human data.

Reviewer #3 (Public Review):

In this manuscript, the authors investigated the effects of deletion of the ER-plasma membrane/Golgi tethering proteins tricalbins (Tcb1-3) on vacuolar morphology to demonstrate the role of membrane contact sites (MCSs) in regulating vacuolar morphology in Saccharomyces cerevisiae. Their data show that tricalbin deletion causes vacuolar fragmentation possibly in parallel with TORC1 pathway. In addition, their data reveal that levels of various lipids including ceramides, long-chain base (LCB)-1P, and phytosphingosine (PHS) are increased in tricalbin-deleted cells. The authors find that exogenously added PHS can induce vacuole fragmentation and by performing analyses of genes involved in sphingolipid metabolism, they conclude that vacuolar fragmentation in tricalbin-deleted cells is due to the accumulated PHS in these cells. Importantly, exogenous PHS- or tricalbin deletion-induced vacuole fragmentation was suppressed by loss of the nucleus vacuole junction (NVJ), suggesting the possibility that PHS transported from the ER to vacuoles via the NVJ triggers vacuole fission. Of note, the authors find that hyperosmotic shock increases intracellular PHS levels, suggesting a general role of PHS in vacuole fission in response to physiological vacuolar division-inducing stimuli.

This work provides valuable insights into the relationship between MCS-mediated sphingolipid metabolism and vacuole morphology. The conclusions of this paper are mostly supported by their results, but inclusion of direct evidence indicating increased transport of PHS from the ER to vacuoles via NVJ in response to vacuolar division-inducing stimuli would have strengthened this study.

There is another weakness in their claim that the transmembrane domain of Tcb3 contributes to the formation of the tricalbin complex which is sufficient for tethering ER to the plasma membrane and the Golgi complex. Their claim is based only on the structural simulation, but not on by biochemical experiments such as co-immunoprecipitation and pull-down.

Reviewer #3 (Public Review):

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

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

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

Weaknesses:<br /> My major concern is the current form of statistical analysis leads to the conclusion that the dissimilarity is not very strong. Adding some more statistical analysis should substantially improve the strength of the manuscript. As mentioned in the Discussion, I understand that there are more available methods to test for generality in experimental evolution but less for diversity. When it is improper to use a canonical statistical test, a test with some simulation and resampling can be useful. For example, I particularly appreciate the analysis done in Figure 2B. An analysis like that should be done more throughout the entire manuscript.

Reviewer #3 (Public Review):

Summary:

SMN expression in non-neuronal cells, particularly in limb mesenchymal progenitors is essential for the proper growth of chondrocytes and the formation of adult NMJ junctions.

Strengths:

The authors show copy numbers of smndelta7 in MPC influence NMJ structure.

Weaknesses:

Functional recovery by FAP transplantation is not complete. Mesenchymal progenitors are heterogeneous, and how heterogeneity influences this study is not clear. Part of the main findings to show the importance of SMN expression in non-neuronal cells is partly published by the same group (Kim et al., JCI Insight 2022). In the study, the authors used Dpp4(+) cells. The difference between the current study and the previous study is not so clear.

What to listen for in jazz Kernfeld 1995

Reviewer #3 (Public Review):

Summary: In this study, the authors attempt to determine what is the role (and strength) of feedback in a closed-loop (cerebellar) system.

Strengths:

1. By combining extensive data fitting of cerebellar experimental observations this study provides deep insights into existing questions and more broadly on the role of feedback and what are the limitations when inferring feedback in (plastic) neural circuits.

2. Another strength of this study is the gradual build-up of evidence by using models of different complexities to help build the argument that weak feedback is sufficient to explain experimental observations.

3. The paper is well-written and structured.

Weaknesses:

1. In principle feedback can (i) drive dynamics or/and (ii) drive learning directly. Throughout the paper, the authors refer to only the first case (i.e. dynamics). However, the role of feedback in learning is already implicitly assumed by the authors when jointly fitting the model before and after learning. Note that the general conclusion that feedback (in general) is weak may be to the first view (i.e. dynamics), but not the second. Given that a key conclusion of the paper is that no feedback is sufficient to explain the data, this suggests that feedback may instead be used for learning/plasticity.

2. There are some potential limitations of the conclusions drawn due to the model inference methods used. The methods used (fmincon) can easily get stuck in local minima and more importantly they do not provide an overview of the likelihood of parameters given the data. A few studies have now shown that it is important to apply more powerful inference techniques both to infer plasticity (Bykowska et al. Frontiers 2019) and neural dynamics (Gonçalves et al. eLife 2020). As highlighted by Costa et al. Frontiers 2013 using more standard fitting methods can lead to misleading interpretations. Given the large range of experimental data used to constrain the model, this may not be an issue, but it is not explicitly shown.

3. There is some lack of clarity on how the feedback pathways as currently presented should be interpreted in the brain.

4. The functional benefits of having (or not) feedback could be better discussed (related to point 1 above).

5. Some of the key conclusions of the work are not described in the abstract, namely that feedback is weak in the cerebellar system.

Claims:

The argument is well-built throughout the paper, but there are some potential caveats with the general interpretation (see weaknesses).

Impact:

This work has the potential to bring important messages on how best to interpret and infer the role of feedback in neural systems. For the field of the cerebellum, it also proposes solutions to long-standing problems.

Reviewer #3 (Public Review):

The Hippo signalling pathway has been implicated in organ growth through the regulation of cell proliferation and apoptosis. The main transcriptional effectors of this pathway, the Yap and Taz proteins, associate with members of the TEAD family of transcription factors to drive diverse transcriptional programs of proliferation, growth, and differentiation. It has previously been shown that YAP/TAZ are essentially required in Schwann cells for developmental myelination, homeostasis, and regenerative myelination in the peripheral nervous system. All four members of the TEAD family are expressed in the Schwann cell lineage, raising the possibility that different aspects of YAP/TAZ role in the Schwann cell lineage are underpinned by differential associations with TEAD transcription factors. In this study, Grove and colleagues provide convincing evidence that TEAD1 is the main transcription factor through which YAP/TAZ affects myelination in development and following nerve injury. A careful comparison between Schwann cell-specific and inducible Yap/Taz and TEAD1 knock out animals reveal unique and redundant roles for TEAD1 in myelination by regulating Schwann cell proliferation, Krox20-dependent myelin gene expression, and cholesterol biosynthesis. Interestingly, their study appears to reveal a YAP/TAZ independent role for TEAD1 in non-myelinating Schwann cell ensheathment of low calibre axons and Remak bundle formation. The conclusions of this study are based on rigorous biochemical, immune-histochemical, electron microscopic, and functional analysis of mutant and wild-type nerves at different stages of postnatal development and following crush nerve injury.

Perhaps the most surprising finding of this study is that TEAD1 can function independently from YAP/TAZ in one branch of the Schwann cell lineage (the authors had reported earlier that non-myelinating Schwann cells do not express YAP/TAZ).<br /> How TEAD1 transcriptional activity is modulated in these Remak Schwann cells is an interesting avenue of future research.

Reviewer #4 (Public Review):

Using optogenetic stimulation, the authors presented compelling evidence that neuronal activity increases mitochondrial calcium levels, facilitated by the mitochondrial uniporter MCU-1. Through ratiometric measurements, they showed that mitochondrial ROS levels also increase due to neuronal activity via MCU-1. Subsequent FRAP studies were employed to investigate the trafficking of the AMPA receptor, GLR-1. By integrating genetic and pharmacological methodologies, the recovery rate of GLR-1 was assessed. The authors concluded that increased mitochondrial ROS due to neuronal activity reduces the trafficking and exocytosis of AMPA receptors. They proposed that mitochondrial ROS serves as a homeostatic mechanism regulating AMPA receptor trafficking and abundance, thus maintaining synaptic strength. This research is crucial as it provides a direct link between mitochondrial signaling and AMPA receptor trafficking.

However, there are several significant concerns regarding the methodologies and quantifications employed in this manuscript. The authors utilized GLR-SEP to label surface AMPA receptors and relied on the "FRAP rate" as an indicator of the exocytosis rate. The absence of direct visualization of exocytosis using GLR-SEP, and the lack of direct measurements of exocytosis events, casts doubt on the conclusions about ROS's impact on AMPA receptor exocytosis. Furthermore, the "FRAP rate" determined in this study is a combination of recovery rates (incorporating both endosomal trafficking and diffusion) with the mobile fractions of AMPA receptors, potentially weakened interpretations of the findings. A more comprehensive discussion addressing the conflicting effects of MCU-1 and ROS on GLR-GFP FRAP recovery and dendritic trafficking would enable readers to grasp the intricate roles of mitochondrial calcium and ROS in modulating synaptic receptors.

Reviewer #3 (Public Review):

The work presented by Ascencao and coworkers aims to deepen into the process of sex chromosome inactivation during meiosis (MSCI) as a critical factor in the regulation of meiosis progression in male mammals. For this purpose, they have generated a transgenic mouse model in which a specific domain of TOPBP1 protein has been mutated, hampering the binding of a number of protein partners and interfering with the regulatory cascade initiated by ATR. Through the use of immunolocalization of an impressive number of markers of MSCI, phosphoproteomics and single cell RNA sequencing (scRNAseq), the authors are able to show that despite a proper morphological formation of the sex body and the incorporation of most canonical MSCI makers, sex chromosome-liked genes are reactivated at some point during pachytene and this triggers meiosis progression breakdown, likely due to a defective phosphorylation of the helicase SETX.<br /> The manuscript presents a clear advance in the understanding of MSCI and meiosis progression with two main strengths. First, the generation of a mouse model with a very uncommon phenotype. Second, the use of a vast methodological approach. The results are well presented and illustrated. Nevertheless, the discussion could be still a bit tuned by the inclusion of some ideas, and perhaps speculations, that have not been considered.

Reviewer #3 (Public Review):

Summary:<br /> This is my assessment of the manuscript entitled "Inter-regional delays fluctuate in the human cerebral cortex" submitted by Moon et al. to eleventh article deals with an interesting question, namely: how do different areas in the brain synchronize with each other. As the title indicates, the article shows that interregional activity can be more or less out of sync, and that the degree of synchronicity depends on the global power of low and high-frequency oscillations.

Overall, I found the paper interesting, although, as written, it is sometimes not clear why studying these inter-regional delays is important. For a broader audience, it is necessary to better emphasize the relevance of inter-regional delays, and what we learn from studies like this beyond the mechanistic aspect of how waves spread in the human brain. Also, it is important to explain why the task (listening to audio) was chosen, and what this task offers in comparison to, for example, studying spontaneous activity. I understand that intra-cranial data from humans is precious and difficult to obtain, so I am not asking for more data, just for a clear honest explanation of why this task was chosen.

Beyond these minor formatting issues, I have two main concerns on the data analysis and interpretation. In a nutshell, they deal with:

- Cross-correlating alpha power with inter-electrode lags computed from raw signals where alpha itself is included. IMO this could lead to obvious high correlation values simply because low-frequency signals spread passively (with some delays) across electrodes. High-frequency signals spread less and are thus less correlated in neighboring electrodes.

- Possible influence of the referencing scheme on the data. I could not find any information about where reference and ground electrodes were located but I fear that epochs of zero-lag coherence could be simply due to common referencing. Non-zero lag synchrony could be explained by generators becoming more or less active close to the recording electrodes. This is probably the most parsimonious explanation of the activity observed and explaining it does not require any coupled oscillators.

Strengths:<br /> The paper relies on a strong dataset from intracranial recordings in humans. Conceptually the paper has strong value as it seeks to explore global and local activity dynamics within the human brain.

Weaknesses:<br /> There are a number of methodological issues that need to be clarified, which could potentially influence the results obtained and their interpretation (i.e. corr-correlating alpha with itself, the influence of the referencing scheme on inter-electrode lags).

Reviewer #3 (Public Review):

While progressive and also hyperactivated motility are required for sperm to reach the site of fertilization and to penetrate the oocyte's outer vestments, during fusion with the oocyte's plasma membrane it has been observed that sperm motility ceases. Identifying the underlying molecular mechanisms would provide novel insights into a crucial but mostly overlooked physiological change during the sperm's life cycle. In this publication, the authors aim to provide evidence that the helical actin structure surrounding the sperm mitochondria in the midpiece plays a role in regulating sperm motility, specifically the motility arrest during sperm fusion but also during earlier cessation of motility in a subpopulation of sperm post acrosomal exocytosis.

The main observation the authors make is that in a subpopulation of sperm undergoing acrosomal exocytosis and sperm that fuse with the plasma membrane of the oocyte display a decrease in midpiece parameter due to a 200 nm shift of the plasma membrane towards the actin helix. The authors show the decrease in midpiece diameter via various microscopy techniques all based on membrane dyes, bright-field images and other orthogonal approaches like electron microscopy would confirm those observations if true but are missing. The lack of additional experimental evidence and the fact that the authors simultaneously observe an increase in membrane dye fluorescence suggests that the membrane dyes instead might be internalized and are now staining intracellular membranes, creating a false-positive result. The authors also propose that the midpiece diameter decrease is driven by changes in sperm intracellular Ca2+ and structural changes of the actin helix network. Important controls and additional experiments are needed to prove that the events observed by the authors are causally dependent and not simply a result of sperm cells dying.

Reviewer #3 (Public Review):

Summary:

The authors report two P4 receptors, ABHD2 and mPRβ that function as co-receptors to induce PLA2 activity and thus drive meiosis. In their experimental studies, the authors knock down ABHD2 and demonstrated inhibition of oocyte maturation and inactivation of Plk1, MAPK, and MPF, which indicated that ABHD2 is required for P4-induced oocyte maturation. Next, they showed three residues (S207, D345, H376) in the lipase domain that are crucial for ABHD2 P4-mediated oocyte maturation in functional assays. They performed global lipidomics analysis on mPRβ or ABHD2 knockdown oocytes, among which the downregulation of GPL and sphingolipid species were observed, and enrichment in LPA was also detected using their metabolomics method. Furthermore, they investigated pharmacological profiles of enzymes predicted to be important for maturation based on their metabolomic analyses and ascertained the central role of PLA2 in inducing oocyte maturation downstream of P4. They showed the modulation of S1P/S1PR3 pathway on oocyte maturation and the potential role for Gαs signaling and potentially Gβγ downstream of P4.

Strengths:

The authors make a very interesting finding that ABHD2 has PLA2 catalytic activity but only in the presence of mPRβ and P4. Finally, they provided supporting data for a relationship between ABHD2/PLA2 activity and mPRβ endocytosis and further downstream signaling. Collectively, this research report defines early steps in nongenomic P4 signaling, which has broad physiological implications.

Weaknesses:

There were concerns with the pharmacological studies presented. Many of these inhibitors are used at high (double-digit micromolar) concentrations that could result in non-specific pharmacological effects and the authors have provided very little data in support of target engagement and selectivity under the multiple experimental paradigms. In addition, the use of an available ABHD2 small molecule inhibitor was lacking in these studies.

Reviewer #3 (Public Review):

Summary:

The manuscript of Zhou et al. reports a genome wide study of in vitro translation initiation using a novel version of ribosome profiling. Here they probe the role of the key RNA helicase, Ded1 in yeast translation initiation using a reconstituted biochemical system and all polyA+ mRNAs in the cell. The authors use ribosome profiling to identify mRNAs that assemble a preinitiation complex at the AUG start codon (48S PIC). They confirm that Ded1 is required for efficient initiation in highly structured RNAs, leading to an increase in PIC formation at the start codon, and nicely correlate their results with prior in vivo investigations using mutant Ded1s.

Strengths:

Rigorous in vitro biochemistry, careful correlation with in vivo results, genome wide analysis. Novel sequencing-based assay.

Weaknesses:

The slow nature of the biochemical experiments could bias results.

Reviewer #4 (Public Review):

Summary:<br /> This is a revised manuscript that describes a role for cdo-1 in regulating cellular cysteine levels. The authors show that expression of cdo-1, predicted to encode a cysteine dioxygenase, is regulated by HIF-1, the conserved hypoxia-induced transcription factor. The expression of cdo-1 is controlled by the RHY-1/CYSL-1/EGL-9/HIF-1 pathway that has been demonstrated to be involved in the response to H2S.

Strengths:<br /> The new finding of this study is that cdo-1, predicted to encode a cysteine dioxygenase, is expressed in the hypodermis and that hypodermal expression rescues at least one phenotype of the cdo-1(mg622) mutant (ability to survive toxic sulfite accumulation in Moco-deficient conditions). Using sulfite toxicity is an interesting reporter for cellular cysteine abundance.

Weaknesses:<br /> The authors claim more than once that the H2S/Cys responsive pathway is RHY-1 - CYSL-1 - EGL-9 - HIF-1. Their data don't seem to support this claim, as they show that Pcdo-1::GFP is induced in rhy-1 mutants incubated with cysteine. It is therefore not appropriate to claim that "HIF-1-induced cysteine catabolism requires the activity of rhy-1" that they include in the description of the model in Fig 6. There is simply no evidence at all that RHY-1 has any role in modulating the activity of CDO-1 other than through transcriptional activation via HIF-1.

I don't find the arguments that this pathway is required for cysteine homeostasis per se (as claimed in the last sentence of the introduction). The authors expose worms to excess cysteine for 48 hours in liquid culture with bacteria. It is well known in these conditions that the bacteria will produce H2S from the cysteine in the culture. All of the cysteine exposure data shown can be explained by the effect of H2S exposure. This would explain why hif-1 and cysl-1 mutants die but cdo-1 mutants do not, for example. The authors don't provide any data to rule out the possibility that bacterial H2S production underlies these results. This explains why the pathway described in this work is the same as has been previously described. Similarly, there is no evidence at all to support their assertion that there are "other pathways" induced by HIF-1 to deal with sulfite produced by cysteine catabolism. However, if the main problem is H2S production (perhaps by bacteria) then cdo-1 would not be relevant and the mutants would be viable as observed.

In a couple of places, the authors seem to argue that H2S-induced expression is limited to the hypodermis and hypoxia-induced gene expression is mostly in the intestine. This is consistent with the expression of cdo-1 (this work) and nhr-57 (Budde and Roth) but it is not appropriate to generalize this. Previous work from the Ruvkun lab (Ma et al) show that the CYSL-1 regulates expression of HIF-1 targets in neurons. Moreover, HIF-1 protein accumulates in the nucleus of nearly all cells, and there is no reason to believe that there are changes in the expression of other genes in different tissues.

Reviewer #3 (Public Review):

Summary:<br /> The authors attempted to unravel the role of the Ca2+-binding proteins CaBP1 and CaBP2 for the hitherto enigmatic lack of Ca2+-dependent inactivation of Ca2+ currents in sensory inner hair cells (IHCs). As Ca2+ currents through Cav1.3 channels are crucial for exocytosis, the lack of inactivation of those Ca2+ currents is essential for the indefatigable sound encoding by IHCs. Using a deaf mouse model lacking both CaBP1 and CaBP2, the authors convincingly demonstrate that both CaBP1 and CaBP2 together confer a lack of inactivation, with CaBP2 being far more effective. This is surprising given the mild phenotype of the single knockouts, which has been published by the authors before. Re-admission of CaBP2 through viral gene transfer into the inner ear of double-knockout mice largely restored hearing function, normal Ca2+ current properties, and exocytosis.

Strengths:<br /> 1. In vitro electrophysiology: perforated patch-clamp recordings of Ca2+/Ba2+ currents of inner hair cells (IHCs) from 3-4 week-old mice - very difficult recordings - necessary to not interfere with intracellular Ca2+ buffers, including CaBP1 and CaBP2.<br /> 2. Capacitance (exocytosis) recordings from IHCs in perforated patch mode.<br /> 3. The insight that a negative holding potential might underestimate the impact of lack of CaBP1/2 on the inactivation of ICa in IHCs. As the physiological holding potential is much more positive than a preferred holding potential in patch clamp experiments it has a strong impact on inactivation in the pauses between depolarization mimicking receptor potentials.<br /> This truly advances our thinking about the stimulation of IHCs and accumulating inactivation of the Cav1.3 channels.<br /> 4. Insight that the voltage sine method with usual voltage excursions (35 mV) to determine the membrane capacitance (for exocytosis measurements) also favors the inactivated state of Cav1.3 channels<br /> 5. Use of double ko mice (for both CaBP1 and CaBP2, DKO) and use of DKO with virally injected CaBP2-eGFP into the inner ear.<br /> 6. Use of DKO animals/IHCs/SGNs after virus-mediated CaBP2 gene transfer shows a great amount of rescue of the normal ICa inactivation phenotype.<br /> 7. In vivo measurements of SGN AP responses to sound, which is highly demanding.<br /> 8. In vivo measurements of hearing thresholds, DPOAE characteristics, and ABR wave I amplitudes/latencies of DKO mice and DKO+injected mice compared to WT mice.

Very thorough analysis and presentation of the data, excellent statistical analysis.

The authors achieved their aims. Their results fully support their conclusions. The methods used by the authors are state-of-the-art.

The impacts on the field are the following:<br /> Regulation of inactivation of Cav1.3 currents is crucial for the persistent functioning of Cav1.3 channels in sensory transduction.<br /> The findings of the authors better explain the phenotype of the human autosomal recessive DFNB93, which is based on the malfunction of CaBP2.<br /> Future work - by the authors or others - should address the molecular mechanisms of the interaction of CaBP1 and 2 in regulating Cav1.3 inactivation.

Weaknesses:<br /> I do not see weaknesses.<br /> What is not explained (but was not the aim of the authors) is how the CaBPs 1 and 2 interact with the Cav1.3 channels and with each other to reduce CDI. Also, why DFNB93, which is based on mutation of the CaBP2 gene, lead to a severe phenotype in humans in contrast to the phenotype of the CaBP2 ko mouse.

Reviewer #3 (Public Review):

Summary:<br /> The authors continue their study of the experimental model of small cell lung cancer (SCLC) they created from human embryonic stem cells (hESCs) using a protocol for differentiating the hESCs into pulmonary lineages followed by NOTCH signaling inactivation with DAPT, and then knockdown of TP53 and RB1 (RP models) with DOX inducible shRNAs. To this published model, they now add DOX-controlled activation of expression of a MYC or T58A MYC transgenes (RPM and RPMT58A models) and study the impact of this on xenograft tumor growth and metastases. Their major findings are that the addition of MYC increased dramatically subcutaneous tumor growth and also the growth of tumors implanted into the renal capsule. In addition, they only found liver and occasional lung metastases with renal capsule implantation. Molecular studies including scRNAseq showed that tumor lines with MYC or T58A MYC led surprisingly to more neuroendocrine differentiation, and (not surprisingly) that MYC expression was most highly correlated with NEUROD1 expression. Of interest, many of the hESCs with RPM/RPMT58A expressed ASCL1. Of note, even in the renal capsule RPM/RPMT58A models only 6/12 and 4/9 mice developed metastases (mainly liver with one lung metastasis) and a few mice of each type did not even develop a renal sub capsule tumor. The authors start their Discussion by concluding: " In this report, we show that the addition of an efficiently expressed transgene encoding normal or mutant human cMYC can convert weakly tumorigenic human PNEC cells, derived from a human ESC line and depleted of tumor suppressors RB1 and TP53, into highly malignant, metastatic SCLC-like cancers after implantation into the renal capsule of immunodeficient mice.".

Strengths:<br /> The in vivo study of a human preclinical model of SCLC demonstrates the important role of c-Myc in the development of a malignant phenotype and metastases. Also the role of c-Myc in selecting for expression of NEUROD1 lineage oncogene expression.

Weaknesses:<br /> There are no data on results from an orthotopic (pulmonary) implantation on generation of metastases; no comparative study of other myc family members (MYCL, MYCN); no indication of analyses of other common metastatic sites found in SCLC (e.g. brain, adrenal gland, lymph nodes, bone marrow); no studies of response to standard platin-etoposide doublet chemotherapy; no data on the status of NEUROD1 and ASCL1 expression in the individual metastatic lesions they identified.

Reviewer #3 (Public Review):

Summary:<br /> Vidal et al. investigated how TFIIIC may mediate MYCN effects on transcription. The work builds upon previous reports from the same group where they describe MYCN interactors in neuroblastoma cells (Buchel et al, 2017), which include TFIIIC, and their different roles in MYCN-dependent control of RNA polymerase II function (Herold et al, 2019) (Roeschert et al, 2021) (Papadopoulus et al, 2022). Using baculovirus expression systems, they confirm that MYCN-TFIIIC interaction is direct and likely relevant for neuroblastoma cell proliferation. However, transcriptomics analyses led them to conclude that TFIIC is largely dispensable for MYCN-dependent gene expression. Instead, they propose that TFIIC limits MYCN-mediated promoter-promoter 3D chromatin contacts, which would in turn facilitate the recruitment of the nascent RNA degradation machinery and restrict the accumulation of non-phosphorylated RNA polymerase II at promoters. How this mechanism may impact on MYCN-driven neuroblastoma cell biology remains to be elucidated.

Strengths:<br /> This study presents a nice variety of genomic datasets addressing the specific role of TFIIIC in MYCN-dependent functions. In particular, the technically challenging HiChIP sequencing experiments performed under various conditions provide very useful information about the interplay between MYCN and TFIIIC in the regulation of 3D chromatin contacts. The authors show that MYCN and TFIIIC participate both in unique and overlapping long-range chromatin contacts and that the expression of each of these proteins limits the function of the other. Together, their results suggest a dynamic and interconnected relationship between MYCN and TFIIIC in regulating 3D chromatin contacts.

Weaknesses:<br /> The connection between the three major findings presented in this study regarding the role of TFIIIC in the regulation of MYCN function remains unclear. Specifically, how the TFIIIC-dependent restriction of MYCN localization to promoter hubs enhances the association of factors involved in nascent RNA degradation to prevent the accumulation of inactive RNA polymerase II at promoters is not apparent. As they are currently presented, these findings appear as independent observations. Cross-comparison of the different datasets obtained may provide some insight into addressing this question.

Another concern involves the disparities in RNA polymerase II ChIP-seq results between this study and earlier ones conducted by the same group. In Figure 2, the authors demonstrate that activation of MYCN results in a reduction of non-phosphorylated RNA polymerase II across all expressed genes. This discovery contradicts prior findings obtained using the same methodology, where it was concluded that the expression of MYCN had no significant effect on the chromatin association of hypo-phosphorylated RNA polymerase II (Buchel et al, 2017). In this regard, the choice of the 8WG16 antibody raises concern, as fluctuations in the signal may be attributed to changes in the phosphorylation levels of the C-terminal domain. It remains unclear why the authors decided against using antibodies targeting the N-terminal domain of RNA polymerase II, which are unaffected by phosphorylation and consistently demonstrated a significant signal reduction upon MYCN activation in their previous studies (Buchel et al, 2017) (Herold et al, 2019). Similarly, the authors previously proposed that depletion of TFIIIC5 abrogates the MYCN-dependent increase of Ser2-phosphorylated RNA polymerase II (Buchel et al, 2017), whereas they now show that it has no obvious impact. These aspects need clarification.

Finally, the varied techniques employed to explore the role of TFIIIC in MYCN-dependent recruitment of nascent RNA degradation factors make it challenging to draw definitive conclusions about which factor is affected and which one is not. While conducting ChIPseq experiments for all factors may be beyond the scope of this manuscript, incorporating proximity ligation assays (PLA) or ChIP-qPCR assays with each factor would have enabled a more direct and comprehensive comparison.

Reviewer #3 (Public Review):

Summary:

This study investigates how various behavioral features are represented in the medial prefrontal cortex (mPFC) of rats engaged in a naturalistic foraging task. The authors recorded electrophysiological responses of individual neurons as animals transitioned between navigation, reward consumption, avoidance, and escape behaviors. Employing a range of computational and statistical methods, including artificial neural networks, dimensionality reduction, hierarchical clustering, and Bayesian classifiers, the authors sought to predict from neural activity distinct task variables (such as distance from the reward zone and the success or failure of avoidance behavior). The findings suggest that mPFC neurons alternate between at least two distinct functional modes, namely spatial encoding and threat evaluation, contingent on the specific location.

Strengths:

This study attempts to address an important question: understanding the role of mPFC across multiple dynamic behaviors. The authors highlight the diverse roles attributed to mPFC in previous literature and seek to explain this apparent heterogeneity. They designed an ethologically relevant foraging task that facilitated the examination of complex dynamic behavior, collecting comprehensive behavioral and neural data. The analyses conducted are both sound and rigorous.

Weaknesses:

The primary concern with this study is the absence of direct evidence regarding the role of the mPFC in the foraging behavior of the rats. The ability to predict heterogeneous variables from the population activity of a specific brain area does not necessarily imply that this brain area is computing or using this information. In light of recent reports revealing the distributed nature of neural coding, conducting direct causal experiments would be essential to draw conclusions about the role of the mPFC in spatial encoding and/or threat evaluation. Alternatively, a comparison with the activity from a different brain region could provide valuable insights (or at the very least, a comparison between PL and IL within the mPFC). Moreover, given that high-dimensional movement has been shown to be reflected in the neural activity across the entire dorsal cortex, more thorough comparisons between the neural encoding of task variables and movement would help rule out the possibility that the heterogeneous encoding observed in the mPFC is merely a reflection of the rats' movements in different behavioral modes. Lastly, the main claim of the paper is that the mPFC population switches between different functional modes depending on the context. However, no dynamic analysis or switching model has been employed to directly support this hypothesis.

Conclusion:

To strengthen the argument and offer novel insights into the functions of the mPFC, it would be important to conduct a more comprehensive analysis if additional data cannot be provided.

Reviewer #3 (Public Review):

Summary:<br /> Raudales et al. aimed at providing an insight into the brain-wide distribution and synaptic connectivity of bona fide GABAergic inhibitory interneuron subtypes focusing on the axo-axonic cell (AAC), one of the most distinctive interneuron subtypes, which innervates the axon initial segments of glutamatergic projection neurons. They establish intersectional genetic strategies that enable them to specifically and comprehensively capture AACs based on their lineage (Nkx2.1) and marker expression (Unc5b, Pthlh). They find that AACs are deployed across essentially all the pallium-derived brain structures as well as the anterior olfactory nucleus, taenia tecta, and lateral septum. They show that AACs in distinct areas and layers of the neocortex as well as different subregions of the hippocampal formation display unique soma and synaptic density and morphological variations. Rabies virus-based retrograde monosynaptic input tracing reveals that AACs in the neocortex, the hippocampus, and the basolateral amygdala receive synaptic inputs from common as well as specific brain regions and supports the utility of this novel genetic approach. This study elucidates brain-wide neuroanatomical features and morphological variations of AACs with solid techniques and analysis. Their novel AAC-targeting strategies will facilitate the study of their development and function in different brain regions. The conclusions in this paper are well supported by the data. However, there are a few comments to strengthen this study.

1) The definition of putative AAC (pAAC) is unclear and Table 1 may not be accurate. Although the authors find synaptic cartridges of RFP-labeled cells in the claustro-insular complex and the dorsal endopiriform nuclei, they still consider these cells as pAACs (not validated). The authors claim that without examining the presence of synaptic cartridges, RFP-labeled cells in the hypothalamus and the bed nuclei of the stria terminalis (BNST) are pAACs while those in the L4 of the somatosensory cortex in Pthlh;Nkx2.1;Ai65 mice are non-AACs. In Table 1, the BNST is supposed to contain AACs (validated), but in the text, the authors claim that RFP-labeled cells in the BNST are pAACs. Could the authors clarify how AACs, pAACs, and non-AACs are defined?

2) The intersectional strategies presented in this study could also specifically capture developing AACs. If so, how early are AACs labeled in the brain? It would also be nice if the authors could add a simple schematic like Fig. 1a showing the time course of Pthlh expression.

Reviewer #3 (Public Review):

Summary:<br /> Zhao et al. address the question of whether intermediate states of the epithelial-to-mesenchymal transition (EMT) exist in a natural developmental context as well as in cancer cells. This is important not only for our understanding of these developmental systems but also for their development as resources for new anti-cancer approaches. Guided by single-cell RNA sequencing analysis of delaminating mouse cranial neural crest cells, they identify two distinct populations with transcriptional signatures intermediate between neuroepithelial progenitors and migrating crest. Both clusters are intermediate spatially and actively cycling, with one in S-phase and one in G2/M. They show that blocking progression through S phase prior to the onset of delamination and knockdown of intermediate state marker Dlc1 both reduce the number of migratory cells that have completed EMT. Overall, the work provides a modern take and new insights into the classical developmental process of neural crest delamination.

Strengths:<br /> • Deep analysis of the scRNAseq dataset revealed previously unappreciated cell populations intermediate between premigratory and migratory crest.<br /> • The observation that delaminating/intermediate neural crest cells appear to be in S or G2/M phase is interesting and worth reporting, though the ultimate significance remains unclear, given that they do not make distinct derivatives depending on their cycle state.<br /> • The authors employ new methods for multiplex spatial imaging to more accurately define their populations of interest and their relative positions.<br /> • The authors present evidence that intermediate state gene Dlc1 (a Rho GAP) is not just a marker but functionally required for neural crest delamination in mice, as previously shown in chicken.

Weaknesses:<br /> • Similar experiments involving blockade of cell cycle progression and Dlc1 dose manipulation were previously performed in chick models, as noted in the discussion. The newly-defined intermediate states give added context to the results, but they are not entirely novel.<br /> • The putative intermediate cells differentially express mRNAs for genes involved in cell adhesion, polarity, and protrusion relative to bona fide premigratory cells (Fig. 2E). This is persuasive evidence, but only differentially expressed genes are shown. Discussing those markers that have not yet changed, e.g. Cdh1 or Zo1 (?), would be instructive and help to clarify the order of events.<br /> • It is unclear whether the two putative intermediate state clusters differ other than their stage of the cell cycle. Based on the trajectory analysis in Fig. 3C-D, the authors state that these two populations form simultaneously and independently but then merge into a single population. However, without further differential expression, it seems more plausible that they represent a single population that is temporarily bifurcated due to cell cycle asynchrony.<br /> • The authors do not present an in-depth comparison of these neural crest intermediate states to previously reported cancer intermediate states. This analysis would reveal how similar the signatures are and thus how extrapolatable these and future findings in delaminating neural crest are to different types of cancer.<br /> • Lines 265-289 (Fig. 4): The aphidicolin treatments appear to have been started before NC delamination begins in earnest, so the fact that there are any migratory SOX10+ neural crest in the treated embryos at all indicates that progression through S-phase is not explicitly required for delamination. The authors surmise that the successfully delaminated cells may instead have been in G2/M phase (perhaps representing cluster 10') already at the start of treatment and thus able to progress through EMT, while S phase intermediate and true premigratory cells were not. This is plausible. However, the reduction in SOX10+ cells may be in part or wholly attributable to inhibition of proliferation AFTER delamination. Showing that there are premigratory NCCs in G2/M at ~E8.0 would bolster the argument that this population is present from the earliest stages.

Reviewer #3 (Public Review):

Seba et al. investigate whether chromosomal recruitment of the E. coli SMC complex MukBEF is initiated at a single site, how MukBEF activity is excluded from the replication terminus region, and whether its recruitment and activity depend on DNA replication. Upon induction of MukBEF, the authors find that chromosomal long-range contacts increase globally rather than from a single site. Using large-scale chromosome rearrangements, they show that matS sites can insulate separate areas of high MukBEF activity from each other. This suggests that MukBEF loads at multiple sites in the genome. Finally, the authors propose that MukBEF associates preferentially with newly replicated DNA, based on ChIP-seq experiments after DNA replication arrest.

The conclusions of the paper are well supported by the data. The ratiometric contact analyses and range-of-contact analyses are compelling and nicely show the interplay between MukBEF and its proposed unloader MatP/matS. I particularly enjoyed the chromosome re-arrangement experiments, which lend strong support to the idea that MukBEF activity is independent of a centralized loading site.<br /> The enrichment of MukBEF in newly replicated regions is convincing, despite somewhat small effect sizes. The suggestion that matS density controls MukBEF activity is appealing, but will need additional support from more systematic studies. It is based on a comparison of only two strains (looking at different combinations of three matS sites), and the effect size is small. As it is, differences in matS sequence composition and genomic context cannot be factored out.

Overall, the work is an important advance in our understanding of bacterial chromosome organization. It will be of broad interest to chromosome biologists and bacterial cell biologists.

Reviewer #3 (Public Review):

In this study, Sun et al examine the role of the splicing factor SRSF1 in spermatogenesis in mice. Alternative splicing is important for spermatogenic development, but its regulation and major developmental roles during spermatogenesis are not well understood. The authors set out to better define both SRSF1 function in testes and the contribution of alternative splicing. They generate several large 'omics datasets to define SRSF1 targets in testis, including RNA interactions by CLIP-seq in whole testis, protein interactions by IP-mass spec in whole testis, and RNA sequencing to detect expression levels and splice variants. They also examine the phenotype of germline conditional knockouts (cKO) for Srsf1, using the early-acting Vasa-Cre, and find a severe depletion of germ cells starting at 7 days post partum (dpp) and culminating with a lack of germ cells (Sertoli Cell Only Syndrome) by adulthood. They detect differences in gene expression as well as differences in splicing between control and knockout, including 9 genes that are downregulated, experience alternative splicing, and whose transcripts are also bound by SRSF1, and identify the Tial1/Tiar transcript as one of these targets. They conclude that SRSF1 is required for homing and self-renewal of precursor spermatogonial stem cells, and suggest that this role may be mediated in part though its regulation of Tial1/Tiar splicing.

Strengths of the paper include detailed phenotyping of the Srsf1 cKO, which convincingly supports the Sertoli Cell Only phenotype, establishes the timing of the first appearance of the spermatogonial defect, and provides new insight into the role of splicing factors and SRSF1 specifically in spermatogenesis. Another strength is the generation of CLIP-seq, IP-MS, and RNA-seq datasets which will be a useful resource for the field of germ cell development. Overall, the results support the claims made. While the study does not provide a full mechanistic understanding of how alternative splicing mediated by SRSF1 affects SSC precursors, the contributions are novel and useful, and will be of interest to the fields of alternative splicing and male reproductive biology.

Reviewer #3 (Public Review):

Summary:

The authors have undertaken a study to rigorously characterize the possible role of eIF2A in regulating translation in yeast. The authors test for a role of eIF2A in the absence or presence of cellular stress and conclude that eIF2A does not play any significant role in regulating translation initiation in yeast.

The authors have used rigorous experimental approaches, including genome wide ribosome profiling analysis in the absence or presence of stress, to show that eIF2A does not function in translation initiation on most mRNAs in yeast. Interestingly, the authors do identify a small number of mRNAs that possess some eIF2A dependency, so they constructed reporters to rigorously test them. One mRNA, HKR1, appears to possess a degree of eIF2A-dependent translation regulation.

No role of eIF2A in translation initiation is apparent and one limitation of the study is that the authors do not determine what function eIF2A plays in yeast.

Reviewer #3 (Public Review):

Summary:<br /> The work in this manuscript builds on prior efforts by this team to understand how sterols are biosynthesized and utilized in bacteria. The study reports a new function for three genes encoded near sterol biosynthesis enzymes, suggesting the resulting proteins function as a sterol transport system. Biochemical and structural characterization of the two soluble components of the pathway establishes that both proteins can bind sterols, with a preference for 4-methylated derivatives. High-resolution x-ray structures of the apoproteins reveal hydrophobic cavities of the appropriate size to accommodate these substrates. Docking and molecular dynamics simulations confirm this observation and provide specific insights into residues involved in substrate binding.

Strengths:<br /> The manuscript is comprehensive and well-written. The annotation of a new function in a set of proteins related to bacterial sterol usage is exciting and likely to enable further study of this phenomenon - which is currently not well understood. The work also has implications for improving our understanding of lipid usage in general among bacterial organisms.

Reviewer #3 (Public Review):

Summary: The study conducted by Ouasti et al. is an elegant investigation of fission yeast CAF-1, employing a diverse array of technologies and genetic alterations to dissect its functions and their interdependence. These functions play a critical role in specifying interactions vital for DNA replication, heterochromatin maintenance, and DNA damage repair, and their dynamics involve multiple interactions. The authors have extensively utilized various in vitro and in vivo tools to validate their model and emphasize the dynamic nature of this complex.

Strengths: Their work is supported by robust experimental data from multiple techniques, including NMR and SAXS, which validate their molecular model. They conducted in vitro interactions using EMSA and isothermal microcalorimetry, in vitro histone deposition using Xenopus high-speed egg extract, and systematically generated and tested various genetic mutants for functionality in in vivo assays. They successfully delineated domain-specific functions using in vitro assays and could validate their roles to large extent using genetic mutants. One significant revelation from this study is the unfolded nature of the acidic domain, observed to fold when binding to histones. Additionally, the authors also elucidated the role of the long KER helix in mediating DNA binding and enhancing the association of CAF-1 with PCNA. The paper effectively addresses its primary objective.

Weaknesses: A few relatively minor unresolved aspects persist, which, if clarified or experimentally addressed by the authors, could further bolster the study.<br /> 1. The precise function of the WHD domain remains elusive. Its deletion does not result in DNA damage accumulation or defects in heterochromatin maintenance. This raises questions about the biological significance of this domain and whether it is dispensable. While in vitro assays revealed defects in chromatin assembly using this mutant (Figure 5), confirming these phenotypes through in vivo assays would provide additional assurance that the lack of function is not simply due to the in vitro system lacking PTMs or other regulatory factors.<br /> 2. The observation of increased Pcf2-gfp foci in pcf1-ED* cells, particularly in mono-nucleated (G2-phase) and bi-nucleated cells with septum marks (S-phase), might suggest the presence of replication stress. This could imply incomplete replication in specific regions, leading to the persistence of Caf1-ED*-PCNA factories throughout the cell cycle. To further confirm this, detecting accumulated single-stranded DNA (ssDNA) regions outside of S-phase using RPA as an ssDNA marker could be informative.<br /> 3. Moreover, considering the authors' strong assertion of histone binding defects in ED* through in vitro assays (Figure 2d and S2a), these claims could be further substantiated, especially considering that some degree of histone deposition might still persist in vivo in the ED* mutant (Figure 7d, viable though growth defective double ED*+hip1D mutants). For example, the approach, akin to the one employed in Fig. 6a (FLAG-IPs of various Pcf1-FLAG-tagged mutants), could also enable a comparison of the association of different mutants with histones and PCNA, providing a more thorough validation of their findings.<br /> 4. It would be valuable for the authors to speculate on the necessity of having disordered regions in CAF1. Specifically, exploring the overall distribution of these domains within disordered/unfolded structures could provide insightful perspectives. Additionally, it's intriguing to note that the significant disparities observed among mutants (ED*, PIP*, and KER*) in in vitro assays seem to become more generic in vivo, except for the indispensability of the WHD-domain. Could these disordered regions potentially play a crucial role in the phase separation of replication factories? Considering these questions could offer valuable insights into the underlying mechanisms at play.

Reviewer #3 (Public Review):

The dogma in the Trypanosome field is that transmission by Tsetse flies is ensured by stumpy forms. This has been recently challenged by the Engstler lab (Schuster et al. ), who showed that slender forms can also be transmitted by teneral flies. In this work, the authors aimed to test whether transmission by slender forms is possible and frequent. The authors observed that most stumpy forms infections with teneral and adult flies were successful while only 1 out of 24 slender form infections were successful.

In this revised version of the manuscript, the authors made some text changes and included statistical testing as a new section of the Materials and Methods. It seems the comparison of midgut infection in adult vs teneral flies was significant in most of the conditions. However, the critical comparison is still missing: within each type of fly (adult or teneral), was the MG infection significantly different between slender and stumpy forms?

Given no additional experiments were performed, it remains unknown why this work and Schuster et al. reached different conclusions. As a result it remains unclear in which conditions slender forms could be important for transmission. Several variables could explain differences between the two groups: the strain used, the presence or absence of N-acetylglucosamine and/or glutathione, how Tsetse colonies were maintained, thorough molecular and cellular characterisation of slender and stumpy forms (to avoid using intermediate forms as slender forms), comparison to recent field parasite strains.

Reviewer #3 (Public Review):

Summary<br /> The authors of this work aim to address the challenge of accurately and efficiently identifying protein binding sites from sequences. They recognize that the limitations of current methods, including reliance on multiple sequence alignments or experimental protein structure, and the under-explored geometry of the structure, which limit the performance and genome-scale applications. The authors have developed a multi-task network called GPSite that predicts binding residues for a range of biologically relevant molecules, including DNA, RNA, peptides, proteins, ATP, HEM, and metal ions, using a combination of sequence embeddings from protein language models and ESMFold-predicted structures. Their approach attempts to extract residual and relational geometric contexts in an end-to-end manner, surpassing current sequence-based and structure-based methods.

Strengths<br /> 1. The GPSite model's ability to predict binding sites for a wide variety of molecules, including DNA, RNA, peptides, and various metal ions.<br /> 2. Based on the presented results, GPSite outperforms state-of-the-art methods in several benchmark datasets.<br /> 3. GPSite adopts predicted structures instead of native structures as input, enabling the model to be applied to a wider range of scenarios where native structures are rare.<br /> 4. The authors emphasize the low computational cost of GPSite, which enables rapid genome-scale binding residue annotations, indicating the model's potential for large-scale applications.

Weaknesses<br /> 1. One major advantage of GPSite, as claimed by the authors, is its efficiency. Although the manuscript mentioned that the inference takes about 5 hours for all datasets, it remains unclear how much improvement GPSite can offer compared with existing methods. A more detailed benchmark comparison of running time against other methods is recommended (including the running time of different components, since some methods like GPSite use predicted structures while some use native structures).<br /> 2. Since the model uses predicted protein structure, the authors have conducted some studies on the effect of the predicted structure's quality. However, only the 0.7 threshold was used. A more comprehensive analysis with several different thresholds is recommended.<br /> 3. To demonstrate the robustness of GPSite, the authors performed a case study on human GR containing two zinc fingers, where the predicted structure is not perfect. The analysis could benefit from more a detailed explanation of why the model can still infer the binding site correctly even though the input structural information is slightly off.<br /> 4. To analyze the relatively low AUC value for protein-protein interactions, the authors claimed that it is "due to the fact that protein-protein interactions are ubiquitous in living organisms while the Swiss-Prot function annotations are incomplete", which is unjustified. It is highly recommended to support this claim by showing at least one example where GPSite's prediction is a valid binding site that is not present in the current Swiss-Prot database or via other approaches.<br /> 5. The authors reported that many GPSite-predicted binding sites are associated with known biological functions. Notably, for RNA-binding sites, there is a significantly higher proportion of translation-related binding sites. The analysis could benefit from a further investigation into this observation, such as the analyzing the percentage of such interactions in the training site. In addition, if there is sufficient data, it would also be interesting to see the cross-interaction-type performance of the proposed model, e.g., train the model on a dataset excluding specific binding sites and test its performance on that class of interactions.

Reviewer #3 (Public Review):

Summary:<br /> The primary aim of this manuscript was to investigate how context, defined from visual object information in multimodal movies, impacts the neural representation of concrete and abstract conceptual knowledge. The authors first conduct a series of analyses to identify context-independent regional responses to concrete and abstract concepts in order to compare these results with the networks observed in prior research using non-naturalistic paradigms. The authors then conduct analyses to investigate whether the regional response to abstract and concrete concepts changes when the concepts are either contextually situated or displaced. A concept is considered displaced if the visual information immediately preceding the word is weakly associated with the word whereas a concept is situated if the association is high. The results suggest that, when ignoring context, abstract and concrete concepts engage different brain regions with overlap in core language areas. When context is accounted for, however, similar brain regions are activated for processing concrete and situated abstract concepts and for processing abstract and displaced concrete concepts. The authors suggest that contextual information dynamically changes the brain regions that support the representation of abstract and concrete conceptual knowledge.

Strengths:<br /> There is significant interest in understanding both the acquisition and neural representation of abstract and concrete concepts, and most of the work in this area has used highly constrained, decontextualized experimental stimuli and paradigms to do so. This manuscript addresses this limitation by using multimodal narratives which allows for an investigation of how context-sensitive the regional response to abstract and concrete concepts is. The authors characterize the regional response in a comprehensive way.

Weaknesses:<br /> The context measure is interesting, but I'm not convinced that it's capturing what the authors intended. In analysing the neural response to a single word, the authors are presuming that they have isolated the window in which that concept is processed and the observed activation corresponds to the neural representation of that word given the prior context. I question to what extent this assumption holds true in a narrative when co-articulation blurs the boundaries between words and when rapid context integration is occurring. Further, the authors define context based on the preceding visual information. I'm not sure that this is a strong manipulation of the narrative context, although I agree that it captures some of the local context. It is maybe not surprising that if a word, abstract or concrete, has a strong association with the preceding visual information then activation in the occipital cortex is observed. I also wonder if the effects being captured have less to do with concrete and abstract concepts and more to do with the specific context the displaced condition captures during a multimodal viewing paradigm. If the visual information is less related to the verbal content, the viewer might process those narrative moments differently regardless of whether the subsequent word is concrete or abstract. I think the claims could be tailored to focus less generally on context and more specifically on how visually presented objects, which contribute to the ongoing context of a multimodal narrative, influence the subsequent processing of abstract and concrete concepts.

Reviewer #3 (Public Review):

Yuan et al., set out to examine the role of functional and structural interaction between Slack and NaVs on the Slack sensitivity to quinidine. Through pharmacological and genetic means they identify NaV1.6 as the privileged NaV isoform in sensitizing Slack to quinidine. Through biochemical assays, they then determine that the C-terminus of Slack physically interacts with the N- and C-termini of NaV1.6. Using the information gleaned from the in vitro experiments the authors then show that virally-mediated transduction of Slack's C-terminus lessens the extent of SlackG269S-induced seizures. These data uncover a previously unrecognized interaction between a sodium and a potassium channel, which contributes to the latter's sensitivity to quinidine.

Reviewer #3 (Public Review):

The manuscript by Zhang and colleagues attempts to combine genetically barcoded rabies viruses with spatial transcriptomics in order to genetically identify connected pairs. The major shortcoming with the application of a barcoded rabies virus, as reported by 2 groups prior, is that with the high dropout rate inherent in single cell procedures, it is difficult to definitively identify connected pairs. By combining the two methods, they are able to establish a platform for doing that, and provide insight into connectivity, as well as pros and cons of their method, which is well thought out and balanced.

The authors did a nice job of addressing my comments which mainly centered around the presentation of data, specificity, and wording.

Reviewer #3 (Public Review):

Summary:<br /> The authors report a scene-selective area in the posterior intraparietal gyrus (PIGS). This area lies outside the classical three scene-selective regions (PPA/TPA, RSC/MPA, TOS/OPA), and is selective for ego-motion.

Strengths:<br /> The authors firmly establish the location and selectivity of the new area through a series of well-crafted controlled experiments. They show that the area can be missed with too much smoothing, thus providing a case for why it has not been previously described. They show that it appears in much the same location in different subjects, with different magnetic field strengths, and with different stimulus sets. Finally, they show that it is selective for ego-motion - defined as a series of sequential photographs of an egocentric trajectory along a path. They further clarify that the area is not generically motion-selective by showing that it does not respond to biological motion without an ego-motion component to it. All statistics are standard and sound; the evidence presented is strong.

Weaknesses:<br /> There are few weaknesses in this work. If pressed, I might say that the stimuli depicting ego-motion do not, strictly speaking, depict motion, but only apparent motion between 2m apart photographs. However, this choice was made to equate frame rates and motion contrast between the 'ego-motion' and a control condition, which is a useful and valid approach to the problem. Some choices for visualization of the results might be made differently; for example, outlines of the regions might be shown in more plots for easier comparison of activation locations, but this is a minor issue.

This is a very strong paper.

Reviewer #3 (Public Review):

Summary:<br /> This study aimed to investigate whether the differences observed in the organization of visual brain networks between blind and sighted adults result from a reorganization of an early functional architecture due to blindness, or whether the early architecture is immature at birth and requires visual experience to develop functional connections. This question was investigated through the comparison of 3 groups of subjects with resting-state functional MRI (rs-fMRI). Based on convincing analyses, the study suggests that: 1) secondary visual cortices showed higher connectivity to prefrontal cortical regions (PFC) than to non-visual sensory areas (S1/M1 and A1) in sighted infants like in blind adults, in contrast to sighted adults; 2) the V1 connectivity pattern of sighted infants lies between that of sighted adults (stronger functional connectivity with non-visual sensory areas than with PFC) and that of blind adults (stronger functional connectivity with PFC than with non-visual sensory areas); 3) the laterality of the connectivity patterns of sighted infants resembled those of sighted adults more than those of blind adults, but sighted infants showed a less differentiated fronto-occipital connectivity pattern than adults.

Strengths:<br /> - The question investigated in this article is important for understanding the mechanisms of plasticity during typical and impaired development, and the approach considered, which compares different groups of subjects including, neonates/infants and blind adults, is highly original.

- Overall, the analyses considered are solid and well-detailed. The results are quite convincing, even if the interpretation might need to be revised downwards, as factors other than visual experience may play a role in the development of functional connections with the visual system.

Weaknesses:<br /> - While it is informative to compare the "initial" state (close to birth) and the "final" states in blind and sighted adults to study the impact of post-natal and visual experience, this study does not analyze the chronology of this development and when the specialization of functional connections is completed. This would require investigating when experience-dependent mechanisms are important for the setting- establishment of multiple functional connections within the visual system. This could be achieved by analyzing different developmental periods in the same way, using open databases such as the Baby Connectome Project. Given the early, "condensed" maturation of the visual system after birth, we might expect sighted infants to show connectivity patterns similar to those of adults a few months after birth.

- The rationale for mixing full-term neonates and preterm infants (scanned at term-equivalent age) from the dHCP 3rd release is not understandable since preterms might have a very different development related to prematurity and to post-natal (including visual) experience. Although the authors show that the difference between the connectivity of visual and other sensory regions, and the one of visual and PFC regions, do not depend on age at birth, they do not show that each connectivity pattern is not influenced by prematurity. Simply not considering the preterm infants would have made the analysis much more robust, and the full-term group in itself is already quite large compared with the two adult groups. The current study setting and the analyses performed do not seem to be an adequate and sufficient model to ascertain that "a few weeks of vision after birth is ... insufficient to influence connectivity".

In a similar way, excluding the few infants with detected brain anomalies (radiological scores higher or equal to 4) would strengthen the group homogeneity by focusing on infants supposed to have a rather typical neurodevelopment. The authors quote all infants as "sighted" but this is not guaranteed as no follow-up is provided.

The post-menstrual age (PMA) at scan of the infants is also not described. The methods indicate that all were scanned at "term-equivalent age" but does this mean that there is some PMA variability between 37 and 41 weeks? Connectivity measures might be influenced by such inter-individual variability in PMA, and this could be evaluated.

- The rationale for presenting results on the connectivity of secondary visual cortices before one of the primary cortices (V1) was not clear to understand. Also, it might be relevant to better justify why only the connectivity of visual regions to non-visual sensory regions (S1-M1, A1) and prefrontal cortex (PFC) was considered in the analyses, and not the ones to other brain regions.

- In relation to the question explored, it might be informative to reposition the study in relation to what others have shown about the developmental chronology of structural and functional long-distance and short-distance connections during pregnancy and the first postnatal months.

- The authors acknowledge the methodological difficulties in defining regions of interest (ROIs) in infants in a similar way as adults. The reliability and the comparability of the ROIs positioning in infants is definitely an issue. Given that brain development is not homogeneous and synchronous across brain regions (in particular with the frontal and parietal lobes showing delayed growth), the newborn brain is not homothetic to the adult brain, which poses major problems for registration. The functional specialization of cortical regions is incomplete at birth. This raises the question of whether the findings of this study would be stable/robust if slightly larger or displaced regions had been considered, to cover with greater certainty the same areas as those considered in adults. And have other cortical parcellation approaches been considered to assess the ROIs robustness (e.g. MCRIB-S for full-terms)?

Reviewer #3 (Public Review):

Summary:<br /> This paper presents evidence from three behavioral experiments that causal impressions of "launching events", in which one object is perceived to cause another object to move, depending on motion direction-selective processing. Specifically, the work uses an adaptation paradigm (Rolfs et al., 2013), presenting repetitive patterns of events matching certain features to a single retinal location, then measuring subsequent perceptual reports of a test display in which the degree of overlap between two discs was varied, and participants could respond "launch" or "pass". The three experiments report results of adapting to motion direction, motion speed, and "object identity", and examine how the psychometric curves for causal reports shift in these conditions depending on the similarity of the adapter and test. While causality reports in the test display were selective for motion direction (Experiment 1), they were not selective for adapter-test speed differences (Experiment 2) nor for changes in object identity induced via color swap (Experiment 3). These results support the notion that causal perception is computed (in part) at relatively early stages of sensory processing, possibly even independently of or prior to computations of object identity.

Strengths:<br /> The setup of the research question and hypotheses is exceptional. The experiments are carefully performed (appropriate equipment, and careful control of eye movements). The slip adaptor is a really nice control condition and effectively mitigates the need to control motion direction with a drifting grating or similar. Participants were measured with sufficient precision, and a power curve analysis was conducted to determine the sample size. Data analysis and statistical quantification are appropriate. Data and analysis code are shared on publication, in keeping with open science principles. The paper is concise and well-written.

Weaknesses:<br /> The biggest uncertainty I have in interpreting the results is the relationship between the task and the assumption that the results tell us about causality impressions. The experimental logic assumes that "pass" reports are always non-causal impressions and "launch" reports are always causal impressions. This logic is inherited from Rolfs et al (2013) and Kominsky & Scholl (2020), who assert rather than measure this. However, other evidence suggests that this assumption might not be solid (Bechlivanidis et al., 2019). Specifically, "[our experiments] reveal strong causal impressions upon first encounter with collision-like sequences that the literature typically labels "non-causal"" (Bechlivanidis et al., 2019) -- including a condition that is similar to the current "pass". It is therefore possible that participants' "pass" reports could also involve causal experiences.

Furthermore, since the only report options are "launch" or "pass", it is also possible that "launch" reports are not indications of "I experienced a causal event" but rather "I did not experience a pass event". It seems possible to me that different adaptation transfer effects (e.g. selectivity to motion direction, speed, or color-swapping) change the way that participants interpret the task, or the uncertainty of their impression. For example, it could be that adaptation increases the likelihood of experiencing a "pass" event in a direction-selective manner, without changing causal impressions. Increases of "pass" impressions (or at least, uncertainty around what was experienced) would produce a leftward shift in the PSE as reported in Experiment 1, but this does not necessarily mean that experiences of causal events changed. Thus, changes in the PSEs between the conditions in the different experiments may not directly reflect changes in causal impressions. I would like the authors to clarify the extent to which these concerns call their conclusions into question.

Leaving these concerns aside, I am also left wondering about the functional significance of these specialised mechanisms. Why would direction matter but speed and object identity not? Surely object identity, in particular, should be relevant to real-world interpretations and inputs of these visual routines? Is color simply too weak an identity?

References:

Bechlivanidis, C., Schlottmann, A., & Lagnado, D. A. (2019). Causation without realism. Journal of Experimental Psychology: General, 148(5), 785-804. https://doi.org/10.1037/xge0000602

Kominsky, J. F., & Scholl, B. J. (2020). Retinotopic adaptation reveals distinct categories of causal perception. Cognition, 203, 104339.

Rolfs, M., Dambacher, M., & Cavanagh, P. (2013). Visual Adaptation of the Perception of Causality. Current Biology, 23(3), 250-254. https://doi.org/10.1016/j.cub.2012.12.017

Reviewer #3 (Public Review):

Summary:<br /> In their study, Zhao et al. investigated the population activity of mirror neurons (MNs) in the premotor cortex of monkeys either executing or observing a task consisting of reaching to, grasping, and manipulating various objects. The authors proposed an innovative method for analyzing the population activity of MNs during both execution and observation trials. This method enabled to isolate the condition-dependent variance in neural data and to study its temporal evolution over the course of single trials. The method proposed by the authors consists of building a time series of "instantaneous" subspaces with single time step resolution, rather than a single subspace spanning the entire task duration. As these subspaces are computed on an instant time basis, projecting neural activity from a given task time into them results in latent trajectories that capture condition-dependent variance while minimizing the condition-independent one. The authors then analyzed the time evolution of these instantaneous subspaces and revealed that a progressive shift is present in subspaces of both execution and observation trials, with slower shifts during the grasping and manipulating phases compared to the initial preparation phase. Finally, they compared the instantaneous subspaces between execution and observation trials and observed that neural population activity did not traverse the same subspaces in these two conditions. However, they showed that these distinct neural representations can be aligned with Canonical Correlation Analysis, indicating dynamic similarities of neural data when executing and observing the task. The authors speculated that such similarities might facilitate the nervous system's ability to recognize actions performed by oneself or another individual.

Strengths:<br /> Unlike other areas of the brain, the analysis of neural population dynamics of premotor cortex MNs is not well established. Furthermore, analyzing population activity recorded during non-trivial motor actions, distinct from the commonly used reaching tasks, serves as a valuable contribution to computational neuroscience. This study holds particular significance as it bridges both domains, shedding light on the temporal evolution of the shift in neural states when executing and observing actions. The results are moderately robust, and the proposed analytical method could potentially be used in other neuroscience contexts.

Weaknesses:<br /> While the overall clarity is satisfactory, the paper falls short in providing a clear description of the mathematical formulas for the different methods used in the study. Moreover, it was not immediately clear why the authors did not consider a (relatively) straightforward metric to quantity the progressive shift of the instantaneous subspaces, such as computing the angle between consecutive subspaces, rather than choosing a (in my opinion) more cumbersome metric based on classification of trajectory segments representing different movements.

Specific comments:<br /> In the methods, it is stated that instantaneous subspaces are found with 3 PCs. Why does it say 2 here? Another doubt on how instantaneous subspaces are computed: in the methods you state that you apply PCA on trial-averaged activity at each 50ms time step. From the next sentence, I gather that you apply PCA on an Nx4 data matrix (N being the number of neurons, and 4 being the trial-averaged activity of the four objects) every 50 ms. Is this right? It would help to explicitly specify the dimensions of the data matrix that goes into PCA computation.

It would help to include some equations in the methods section related to the LSTM decoding. Just to make sure I understood correctly: after having identified the instantaneous subspaces (every 50 ms), you projected the Instruction, Go, Movement, and Holding segments from individual trials (each containing 100 samples, since they are sampled from a 100ms window) onto each instantaneous subspace. So you have four trajectories for each subspace. In the methods, it is stated that a single LSTM classifier is trained for each subspace. Do you also have a separate classifier for each trajectory segment? What is used as input to the classifier? Each trajectory segment should be a 100x3 matrix once projected in an instantaneous subspace. Is that what (each of) the LSTMs take as input? And lastly, what is the LSTM trained to predict exactly? Just a label indicating the type of object that was manipulated in that trial? I apologize if I overlooked any detail, but I believe a clearer explanation of the LSTM, preferably with mathematical formulas, would greatly help readers understand this section.

Reviewer #3 (Public Review):

Summary:<br /> This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts, and heterozygotes) are examined here and whole-cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in the current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition, immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that the removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

1. From previous work it is known that GK,L in type I hair cells have unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+, and modulated by cyclic GMP. The question arises, if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells and immunostaining for Kv1.4 channels in rat utricle presented here in Fig. 6. However, hair cell-specific partner interactions with Kv1.8 that result in GK,L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

2. Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

3. Strong immunosignal appears in the cuticle plates of hair cells in addition to signal in basal regions of hair cells and supporting cells. Please provide a possible explanation for this.

4. A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be very interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Reviewer #4 (Public Review):

The manuscript brings convincing results regarding genes involved in the radio-resistance of tardigrades. It is nicely written and the authors used different techniques to study these genes. There are sometimes problems with the structure of the manuscript but these could be easily solved. According to me, there are also some points which should be clarified in the result sections. The discussion section is clear but could be more detailed, although some results were actually discussed in the results section. I wish that the authors would go deeper in the comparison with other IR-resistant eucaryotes. Overall, this is a very nice study and of interest to researchers studying molecular mechanisms of ionizing radiation resistance.

I have two small suggestions regarding the content of the study itself.

1) I think the study would benefit from the analyses of a gene tree (if feasible) in order to verify if TDR1 is indeed tardigrade-specific.<br /> 2) It would be appreciated to indicate the expression level of the different genes discussed in the study, using, for example, transcript per millions (TPMs).

Reviewer #3 (Public Review):

Cells can oxidize diverse substrates in the mitochondria to sustain cellular energy metabolism. However, all of these substrates require covalent thioester linkage to coenzyme A (CoA). Thus, multiple energy metabolism substrates could potentially compete for a limited pool of mitochondrial CoA. Cells encode a set of mitochondrial acyl-CoA thioesterases (ACOTs) that free CoA up by removing attached substrates. The authors hypothesized that ACOT2, a mitochondrial ACOT with a preference for long-chain acyl-CoA substrates that arise during the oxidation of lipids as a fuel source, could regulate the balance of substrates used in the mitochondria by reducing the oxidation of lipids by removing them from CoA and freeing the mitochondrial pool of CoA for use by other substrates.

To test this hypothesis, the authors generated mice with loss of ACOT2 in the skeletal muscle, where this is most expressed, and assayed the CoA composition of muscle and their glucose/fatty acid catabolism in mice that were challenged with different diets, fasting or exercise to expose the muscle to different substrates conditions. These experiments were complemented with biochemical analysis of mitochondria isolated from the muscle of control and ACOT2 animals exposed to a variety of substrates and challenged with different simulated energy demands.

On the basis of these convincing experiments, the authors argue that loss of ACOT2 both in vivo and in vitro interestingly increases glucose oxidation, while not increasing oxidation of lipids. This is particularly surprising as the CoA competition model would predict that ACOT2 loss would increase lipid oxidation while hindering glucose oxidation. The authors argue that ACOT2 facilitates lipid oxidation due to ACOT2 reversal of lipid ligation to CoA preventing feedback inhibition of the lipid oxidation pathway that occurs when lipid supply outstrips the ability of the lipid oxidation pathway to metabolize the lipids. These findings will be valuable for the field of metabolism providing insight into how ACOTs regulate substrate catabolism in cells and tissues.

Reviewer #3 (Public Review):

Summary:<br /> The authors aimed to investigate how genetic and environmental factors influence the muscle insulin signaling network and its impact on metabolism. They utilized mass spectrometry-based phosphoproteomics to quantify phosphosites in skeletal muscle of genetically distinct mouse strains in different dietary environments, with and without insulin stimulation. The results showed that genetic background and diet both affected insulin signaling, with almost half of the insulin-regulated phosphoproteome being modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affecting insulin signaling in a strain-dependent manner.

Strengths:<br /> Study uses state-of-the-art phosphoproteomics workflow allowing quantification of a large number of phosphosites in skeletal muscle, providing a comprehensive view of the muscle insulin signaling network. The study examined five genetically distinct mouse strains in two dietary environments, allowing for the investigation of the impact of genetic and environmental factors on insulin signaling. The identification of coregulated subnetworks within the insulin signaling pathway expanded our understanding of its organization and provided insights into potential regulatory mechanisms. The study associated diverse signaling responses with insulin-stimulated glucose uptake, uncovering regulators of muscle insulin responsiveness.

Weaknesses:<br /> The limitations acknowledged by the authors, such as the need for larger cohorts and the inclusion of female mice. Moreover as acknowledged by authors, they are unable to dissect to what extent the obesity and different life span cycle for different strain affects insulin signaling. This suggest that further research is needed to validate and expand upon the findings.

Reviewer #3 (Public Review):

Summary:<br /> The authors use docking and molecular dynamics (MD) simulations to investigate transient conformations that are otherwise difficult to resolve experimentally. The docking and simulations suggest an interesting series of events whereby agonists initially bind to the low-affinity site and then flip 180 degrees as the site contracts to its high-affinity conformation. This work will be of interest to the ion channel community and to biophysical studies of pentameric ligand-gated channels.

Strengths:<br /> I find the premise for the simulations to be good, starting with an antagonist-bound structure as an estimate of the low affinity binding site conformation, then docking agonists into the site and using MD to allow the site to relax to a higher affinity conformation that is similar to structures in complex with agonists. I cannot speak to the details of the simulation methods, but the predictions are interesting and provide a view into what a transient conformation that is difficult to observe experimentally might be like.

Weaknesses:<br /> Although the match in simulated vs experimental energies for two ligands was very good, the calculated energies for two other ligands were significantly different than the experiment. It is unclear to what extent the choice of method for the energy calculations influenced the results.

A control simulation, such as for an apo site, is lacking.

Reviewer #3 (Public Review):

In this article by Bastidas et al. the authors examine the functions of the Chlamydia deubiquitinating enzyme 1 (Cdu1) during infections of human cells. First, a mutant lacking Cdu1 but not Cdu2 was constructed using targetron and quantitative proteomics was used to identify differences in ubiquitinated proteins (both host and bacterial) during infection. While they found minimal changes in host protein ubiquitination, they identified three Chlamydia effector proteins, IpaM, InaC and CTL0480 were all ubiquitinated in the absence of Cdu1. Microscopy and immunoprecipitations found Cdu1 directly interacts with these Chlamydia effectors and confirmed that Cdu1 mediates the stabilization of these effectors at the inclusion membrane during late infection time points. Surprisingly rather than deubiquitination driving this stabilization, the acetylation function of Cdu1 was required, and acetylation on lysine residues prevented degradative ubiquitination of Cdu1, IpaM, InaC and CTL0480. In line with this observation the authors show that loss of Cdu1 phenocopies the loss of single effector mutants of InaC, IpaM and CTL0480, including golgi stack formation and the recruitment of MYPT1 to the inclusion. The aggregation of changes to the Chlamydia inclusion does not alter growth but controls extrusion of chlamydia from cells with reduced extrusion in Cdu1 mutant Chlamydia infections. The strengths of the manuscript are the range of assays used to convincingly examine the biochemical and cellular biology underlying Cdu1 functions. The finding that acetylation of lysine residues is a mechanisms for bacterial effectors to block degradative ubiqutination is impactful and will open new investigations into this mechanism for many intracellular pathogens. The authors revisions to the manuscript have addressed my primary concerns and the authors present compelling arguments for remaining questions that are outside the scope of this study. Altogether this is an important series of findings that help to understand the mechanisms underpinning Chlamydia pathogenesis using orthologous methods and is an impactful study.

Reviewer #3 (Public Review):

Summary:<br /> Navratna et al. have solved the first structure of a transmembrane N-acetyltransferase (TNAT), resolving the architecture of human heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) in the acetyl-CoA bound state using single particle cryo-electron microscopy (cryoEM). They show that the protein is a dimer, and define the architecture of the alpha- and beta- GSNAT fragments, as well as convincingly characterizing the binding site of acetyl-CoA.

Strengths:<br /> This is the first structure of any member of the transmembrane acyl transferase superfamily, and as such it provides important insights into the architecture and acetyl-CoA binding site of this class of enzymes.

The structural data is of a high quality, with an isotropic cryoEM density map at 3.3Å facilitating the building of a high-confidence atomic model. Importantly, the density of the acetyl-CoA ligand is particularly well-defined, as are the contacting residues within the transmembrane domain.

The open-to-lumen structure of HSGNAT presented here will undoubtedly lay the groundwork for future structural and functional characterization of the reaction cycle of this class of enzymes.

Weaknesses:<br /> While the structural data for the open-to-lumen state presented in this work is very convincing, and clearly defines the binding site of acetyl-CoA, to get a complete picture of the enzymatic mechanism of this family, additional structures of other states will be required.

A potentially significant weakness of the study is the lack of functional validation. The enzymatic activity of the enzyme characterized was not measured, and the enzyme lacks native proteolytic processing, so it is a little unclear whether the structure represents an active enzyme.

Reviewer #3 (Public Review):

Summary:<br /> The study attempts to shed light on the mechanisms underlying information-seeking in infants by investigating whether infants distinguish between informative and uninformative stimuli to resourcefully allocate their attention. The authors show that 8-month-old infants can learn whether a visual stimulus is informative or uninformative about the location of a later appearing rewarding stimulus by employing statistical regularities from the input. Specifically, infants showed decreased pupil dilation for informative over uninformative cues, which developed over the course of trials as more and more information was gathered from the input. The pattern of learning was in line with a reinforcement learning model which employed a steep learning curve in the beginning followed by a more shallow but steady learning growth over trials. After 17 trials, the authors presented novel cues that shared certain visual features with the previous stimuli and showed that pupil dilation was reduced for novel cues that shared features with the previous informative stimuli, suggesting that infants were able to generalize their acquired knowledge about the informativeness of certain features to novel stimuli. The present study adds to the existing literature about the underlying mechanisms of learning by showing that infants cannot only predict an upcoming stimulus based on statistical regularities of a preceding cue but also the informativeness of the cue itself.

Strengths:<br /> The authors use a suitable method to test the highly relevant question of whether and how infants infer the informativeness of stimuli from experience and whether they can generalize this knowledge to new stimuli. Their experiment is carefully designed and well controlled with conditions closely matched (e.g., the shape and color of objects and the structure of each trial). Their measure of interest (i.e., pupil dilation) is also examined at a time point in each trial when the conditions are the most similar, which further points to a thought-through and careful design. This empirical data is backed up with a computational approach (using a Bayesian model and training a reinforcement learning algorithm) to elucidate the learning mechanisms at play. This approach is explained concisely to readers not familiar with the models.

The results are convincing showing a clear difference between informative and uninformative condition and development over trials. Specifically, this difference is not apparent in the first trial (Fig. 2c) but develops over time which supports a learning trajectory. The data support the authors' conclusion that infants learn about the informativeness of the object cue from the input, and the employed learning algorithms give further insights into the learning trajectory of the infants. Overall, the statistical analyses seem solid and the priors for the Bayesian models are well reported.

Data and scripts are openly available fostering transparency.

Overall, the manuscript is very well and concisely written.

Weaknesses:<br /> The authors' conclusion that infants can generalize the acquired knowledge to similar but novel stimuli is weakened by methodological concerns regarding the analysis. It is not fully clear which trials the authors excluded and analyzed as they do not consistently report the trials in the manuscript (e.g., it is stated that after trial 17 the first generalization trial started, but also that trial 17 was excluded as the first trial of the generalization phase). As there are only a few novel trials and novel and familiar trials alternated, the inclusion or exclusion of trial analyses might have a significant impact on the results. Thus, this needs further clarification. The authors also mentioned that the novel stimuli shared relevant as well as irrelevant features, but it was not clear to me whether the authors could establish that only the relevant features contributed to the observed generalization effect.

Some methodological decisions were not explained and need justification, in particular, as the study is not preregistered. This includes, for example, the exclusion criteria and the choice not to analyze all generalization trials. Further, the authors did not perform model comparison (e.g., their model against a null model) and therefore do not report the strength of evidence for a difference in conditions.

Another weakness is that the sample sizes of 30 infants for the initial part and 19 infants for the generalization part of the experiment are rather small (especially with regard to the chosen weakly informative priors).

Reviewer #3 (Public Review):

Summary:<br /> The publication presents unique in-vivo images of the upper layer of the epidermis of the glabrous skin when a flat object compresses or slides on the fingertip. The images are captured using OCT, and are the process of recovering the strain that fingerprints experience during the mechanical stimulation.

The most important finding is, in my opinion, that fingerprints undergo pure compression/tension without horizontal shear, hinting at the fact that the shear stress caused by the tangential load is transferred to the deeper tissues and ultimately to the mechanoreceptors (SA-I / RA-I).

Strengths:<br /> - Fascinating new insights into the mechanics of glabrous skin. To the best of my knowledge, this is the first experimental evidence of the mechanical deformation of fingerprints when subjected to dynamic mechanical stimulation. The OCT measurement allows an unprecedented measurement of the depth of the skin whereas previous works were limited to tracking the surface deformation.<br /> - The robust data analysis reveals the continuum mechanics underlying the deformation of the fingerprint ridges.

Weaknesses:<br /> I do not see any major weaknesses. The work is mainly experimental and is rigorously executed. Two points pique my curiosity, however:

1. How do the results presented in this study compare with previous finite element analysis? I am curious to know if the claim that the horizontal shear strain is transferred to the previous layer is also captured by these models. The reason is that the FEA models typically use homogeneous materials and whether or not the behavior in-silico and in-vivo matches would offer an idea of the nature of the stratum corneum.<br /> 2. Was there a specific reason why the authors chose to track only one fingerprint? From the method section, it seems that nothing would have prevented tracking a denser point cloud and reconstructing the stain on a section of the skin rather than just one ridge. With such data, the author could extend their analysis to multiple ridges interaction and get a better sense of the behavior of the entire strip of skin.

Reviewer #3 (Public Review):

Rovira, et al., aim to characterize immune cells in the brain parenchyma and identify a novel macrophage population referred to as "dendritic-like cells". They use a combination of single-cell transcriptomics, immunohistochemistry, and genetic mutants to conclude the presence of this "dendritic-like cell" population in the brain. The strength of this manuscript is the identification of dendritic cells in the brain, which are typically found in the meningeal layers and choroid plexus. A weakness is the lack of specific reporters or labeling of this dendritic cell population using specific genes found in their single-cell dataset. Additionally, it is difficult to remove the meningeal layers from the brain samples and thus can lead to confounding conclusions. Overall, I believe this study should be accepted contingent on sufficient labeling of this population and addressing comments.

Reviewer #3 (Public Review):

Summary:<br /> The present manuscript by Shore et al. entitled Reduced GABAergic Neuron Excitability, Altered Synaptic Connectivity, and Seizures in a KCNT1 Gain-of-Function Mouse Model of Childhood Epilepsy" describes in vitro and in silico results obtained in cortical neurons from mice carrying the KCNT1-Y777H gain-of-function (GOF) variant in the KCNT1 gene encoding for a subunit of the Na+-activated K+ (KNa) channel. This variant corresponds to the human Y796H variant found in a family with Autosomal Dominant Nocturnal Frontal lobe epilepsy. The occurrence of GOF variants in potassium channel encoding genes is well known, and among potential pathophysiological mechanisms, impaired inhibition has been documented as responsible for KCNT1-related DEEs. Therefore, building on a previous study by the same group performed in homozygous KI animals, and considering that the largest majority of pathogenic KCNT1 variants in humans occur in heterozygosis, the Authors have investigated the effects of heterozygous Kcnt1-Y777H expression on KNa currents and neuronal physiology among cortical glutamatergic and the 3 main classes of GABAergic neurons, namely those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), crossing KCNT1-Y777H mice with PV-, SST- and PV-cre mouse lines, and recording from GABAergic neurons identified by their expression of mCherry (but negative for GFP used to mark excitatory neurons).

The results obtained revealed heterogeneous effects of the variant on KNa and action potential firing rates in distinct neuronal subpopulations, ranging from no change (glutamatergic and VIP GABAergic) to decreased excitability (SST GABAergic) to increased excitability (PV GABAergic). In particular, modelling and in vitro data revealed that an increase in persistent Na current occurring in PV neurons was sufficient to overcome the effects of KCNT1 GOF and cause an overall increase in AP generation.

Strengths:<br /> The paper is very well written, the results clearly presented and interpreted, and the discussion focuses on the most relevant points.

The recordings performed in distinct neuronal subpopulations are a clear strength of the paper. The finding that the same variant can cause opposite effects and trigger specific homeostatic mechanisms in distinct neuronal populations is very relevant for the field, as it narrows the existing gap between experimental models and clinical evidence.

Weaknesses:<br /> My main concern is in the epileptic phenotype of the heterozygous mice investigated. In fact, in their previous paper the Authors state that "...Kcnt1-Y777H heterozygous mice did not exhibit any detectable epileptiform activity" (first sentence on page 4). However, in the present manuscript, they indicate twice in the discussion section that these mice exhibit "infrequent seizures". This relevant difference needs to be clarified to correctly attribute to the novel pathophysiological mechanism a role in seizure occurrence. Were such infrequent seizures clearly identified on the EEG, or were behavioral seizures? Could the authors quantify this "infrequent" value? This is crucial also to place in the proper perspective the Discussion statement regarding "... the increased INaP contribution to ... network hyperexcitability and seizures".

Also, some statistical analysis seems to be missing. For example, I could not find any for the data shown in Fig. 6. Thus, the following statement: "the model PV neurons responded to KCNT1 GOF with decreased AP firing and an increased rheobase" requires proper statistical evaluation.

Reviewer #3 (Public Review):

Summary:

The fruit fly visual system has provided a powerful context in which to investigate fundamental questions in neural development, phototransduction, and systems neuroscience. Of recent interest is motion processing, particularly how visual motion cues are estimated locally, and then pooled to derive behaviorally meaningful signals. Many of these pooling operations have been shown to take place in the wide-field neurons in the lobula plate, cell types that have been explored using electrophysiological recordings for more than 50 years in a variety of Diptera. However, our understanding of the diversity and connectivity of these cells remains incompletely understood, and is of interest to many.

In this context, Reiser and colleagues describe the anatomy and connectivity of the complete set of Lobula Plate Tangential neurons in Drosophila, using a careful and systematic reconstruction of the FAFB dataset. Leveraging a previous study of retinal geometry, combined with their characterization of the anatomical inputs to the elementary motion detectors, T4 and T5, they then predict the motion sensitivities of each cell, their neurotransmitter identities, and map the connections of many of these cells into the central brain and contralateral optic lobe.

Strengths:

The quality of the connectomic analysis is exceptional, and the quantitative analysis that links connectivity to function is rigorous and impressive. This paper will be an important resource for the community.

Weaknesses:

Some of the findings could be better linked to previously published work in this field, and there may be a minor limitation to the predicted optimal motion axes, given one of the simplifying assumptions made.

Reviewer #3 (Public Review):

Summary:<br /> The work by Masala and colleagues highlights a striking artifact that can result from a particular viral method for expressing genetically encoded calcium indicators (GECIs) in neurons. In a cross-institutional collaboration, the authors find that viral transduction of GECIs in the hippocampus can result in aberrant slow-traveling calcium (Ca2+) micro-waves. These Ca2+ micro-waves are distinct from previously described ictal activity but nevertheless are likely a pathological consequence of overexpression of virally transduced proteins. Ca2+ micro-waves will most likely obscure the physiology that most researchers are interested in studying with GECIs, and their presence indicates that the neural circuit is in an unintended pathological state. Interestingly this pathology was not observed using the same viral transduction methods in the visual cortex. The authors recommend several approaches that may help other experimenters avoid this confound in their own data such as reducing the titer of viral injections or using recombinase-dependent expression. The intent of this manuscript is to raise awareness of the potential unintended consequences of viral overexpression, particularly for GECIs. A rigorous investigation into the exact causes of Ca2+ micro-waves or the mech

Strengths:

The authors clearly demonstrate that Ca2+ micro-waves occur in the CA1 and CA3 regions of the hippocampus following large volume, high titer injections of adeno-associated viruses (AAV1 and AAV9) encoding GECIs. The supplementary videos provide undeniable proof of their existence.

By forming an inter-institutional collaboration, the authors demonstrate that this phenomenon is robust to changes in surgical techniques or imaging conditions.

Weaknesses:

I believe that the weaknesses of the manuscript are appropriately highlighted by the authors themselves in the discussion. I would, however, like to emphasize several additional points.

As the authors state, the exact conditions that lead to Ca2+ micro-waves are unclear from this manuscript. It is also unclear if Ca2+ micro-waves are specific to GECI expression or if high-titer viral transduction of other proteins such as genetically encoded voltage indicators, static fluorescent proteins, recombinases, etc could also cause Ca2+ micro-waves.

The authors almost exclusively tested high titer (>5x10^12 vg/mL) large volume (500-1000 nL) injections using the synapsin promoter and AAV1 serotypes. It is possible that Ca2+ micro-waves are dramatically less frequent when titers are lowered further but still kept high enough to be useful for in vivo imaging (e.g. 1x10^12 vg/mL) or smaller injection volumes are used. It is also possible that Ca2+ micro-waves occur with high titer injections using other viral promoter sequences such as EF1α or CaMKIIα. There may additionally be effects of viral serotype on micro-wave occurrence.

The number of animals in any particular condition are fairly low (Table 1) with the exception of V1 imaging and thy1-GCaMP6 imaging. This prohibits rigorous comparison of the frequency of pathological calcium activity across conditions.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors explore the roles of dact1 and dact2 during zebrafish gastrulation and craniofacial development. Previous studies used morpholino (MO) knockdowns to show that these scaffolding proteins, which interact with disheveled (Dsh), are expressed during zebrafish gastrulation and suggested that dact1 promotes canonical Wnt/B-catenin signaling, while dact2 promotes non-canonical Wnt/PCP-dependent convergent-extension (Waxman et al 2004). This study goes beyond this work by creating loss-of-function mutant alleles for each gene and unlike the MO studies finds little (dact2) to no (dact1) phenotypic defects in the homozygous mutants. Interestingly, dact1/2 double mutants have a more severe phenotype, which resembles those reported with MOs as well as homozygous wnt11/silberblick (wnt11/slb) mutants that disrupt non-canonical Wnt signaling (Heisenberg et al., 1997; 2000). Further analyses in this paper try to connect gastrulation and craniofacial defects in dact1/2 mutants with wnt11/slb and other wnt-pathway mutants. scRNAseq conducted in mutants identifies calpain 8 as a potential new target of dact1/2 and Wnt signaling.

Strengths:<br /> When considered separately the new mutants are an improvement over the MOs and the paper contains a lot of new data.

Weaknesses:<br /> The hypotheses are very poorly defined and misinterpret key previous findings surrounding the roles of wnt11 and gpc4, which results in a very confusing manuscript. Many of the results are not novel and focus on secondary defects. The most novel result of overexpressing calpain8 in dact1/2 mutants is preliminary and not convincing.

Major Comments:<br /> 1) One major problem throughout the paper is that the authors misrepresent the fact that wnt11f2 and gpc4 act in different cell populations at different times. Gastrulation defects in these mutants are not similar: wnt11 is required for anterior mesoderm CE during gastrulation but not during subsequent craniofacial development while gpc4 is required for posterior mesoderm CE and later craniofacial cartilage morphogenesis (LeClair et al., 2009). Overall, the non-overlapping functions of wnt11 and gpc4, both temporally and spatially, suggest that they are not part of the same pathway.

2) There are also serious problems surrounding attempts to relate single-cell data with the other data in the manuscript and many claims that lack validation. For example, in Fig 1 it is entirely unclear how the Daniocell scRNA-seq data have been used to compare dact1/2 with wnt11f2 or gpc4. With no labeling in panel 1E of this figure these comparisons are impossible to follow. Similarly, the comparisons between dact1/2 and gpc4 in scRNA-seq data in Fig. 6 as well as the choices of DEGs in dact1/2 or gpc4 mutants in Fig. 7 seem arbitrary and do not make a convincing case for any specific developmental hypothesis. Are dact1 and gpc4 or dact2 and wnt11 co-expressed in individual cells? Eyeballing similarity is not acceptable.

3) Many of the results in the paper are not novel and either confirm previous findings, particularly Waxman et al (2004), or even contradict them without good evidence. The authors should make sure that dact2 loss-of-function is not compensated for by an increase in dact1 transcription or vice versa. Testing genetic interactions, including investigating the expression of wnt11f2 in dact1/2 mutants, dact1/2 expression in wnt11f2 mutants, or the ability of dact1/2 to rescue wnt11f2 loss of function would give this work a more novel, mechanistic angle.

4) The identification of calpain 8 overexpression in Dact1/2 mutants is interesting, but getting 1/142 phenotypes from mRNA injections does not meet reproducibility standards.

Reviewer #3 (Public Review):

Summary:

This is a really interesting study, looking at the efficacy of AAV-mediated delivery of wt HSPE2 gene into mouse mutants with the goal of rescuing lower urinary tract defects.

Strengths: Nice analysis of muscle physiology ex vivo, interesting approach.

Weaknesses: lack of rigor (see below). This is an awesome opportunity to learn much more about the disease, its affects on neurons, muscle, etc.

* Single-cell analysis of mutants versus control bladder, urethra including sphincter. This would be great also for the community.

* Detailed tables showing data from each mouse examined.

* Survival curves.

* Use of measurements that are done in vivo (spot assay for example). This sounds relatively simple.

* Assessment of viral integration in tissues besides the liver (could be done by QPCR).

* Discuss subtypes of neurons that are present and targeted in the context of mutants and controls.

Reviewer #3 (Public Review):

Summary of the findings:

The authors explore an important question concerning the underlying mechanism of representational drift, which despite intense recent interest remains obscure. The paper explores the intriguing hypothesis that drift may reflect changes in the intrinsic excitability of neurons. The authors set out to provide theoretical insight into this potential mechanism.

They construct a rate model with all-to-all recurrent connectivity, in which recurrent synapses are governed by a standard Hebbian plasticity rule. This network receives a global input, constant across all neurons, which can be varied with time. Each neuron also is driven by an "intrinsic excitability" bias term, which does vary across cells. The authors study how activity in the network evolves as this intrinsic excitability term is changed.

They find that after initial stimulation of the network, those neurons where the excitability term is set high become more strongly connected and are in turn more responsive to the input. Each day the subset of neurons with high intrinsic excitability is changed, and the network's recurrent synaptic connectivity and responsiveness gradually shift, such that the new high intrinsic excitability subset becomes both more strongly activated by the global input and also more strongly recurrently connected. These changes result in drift, reflected by a gradual decrease across time in the correlation of the neuronal population vector response to the stimulus.

The authors are able to build a classifier that decodes the "day" (i.e. which subset of neurons had high intrinsic excitability) with perfect accuracy. This is despite the fact that the excitability bias during decoding is set to 0 for all neurons, and so the decoder is really detecting those neurons with strong recurrent connectivity, and in turn strong responses to the input. The authors show that it is also possible to decode the order in which different subsets of neurons were given high intrinsic excitability on previous "days". This second result depends on the extent by which intrinsic excitability was increased: if the increase in intrinsic excitability was either too high or too low, it was not possible to read out any information about the past ordering of excitability changes.

Finally, using another Hebbian learning rule, the authors show that an output neuron, whose activity is a weighted sum of the activity of all neurons in the network, is able to read out the activity of the network. What this means specifically, is that although the set of neurons most active in the network changes, the output neuron always maintains a higher firing rate than a neuron with randomly shuffled synaptic weights, because the output neuron continuously updates its weights to sample from the highly active population at any given moment. Thus, the output neuron can read out a stable memory despite drift.

Strengths:

The authors are clear in their description of the network they construct and in their results. They convincingly show that when they change their "intrinsic excitability term", upon stimulation, the Hebbian synapses in their network gradually evolve, and the combined synaptic connectivity and altered excitability result in drifting patterns of activity in response to an unchanging input (Fig. 1, Fig. 2a). Furthermore, their classification analyses (Fig. 2) show that information is preserved in the network, and their readout neuron successfully tracks the active cells (Fig. 3). Finally, the observation that only a specific range of excitability bias values permits decoding of the temporal structure of the history of intrinsic excitability (Fig. 2f and Figure S1) is interesting, and as the authors point out, not trivial.

Weaknesses:

1) The way the network is constructed, there is no formal difference between what the authors call "input", Δ(t), and what they call "intrinsic excitability" Ɛ_i(t) (see Equation 3). These are two separate terms that are summed (Eq. 3) to define the rate dynamics of the network. The authors could have switched the names of these terms: Δ(t) could have been considered a global "intrinsic excitability term" that varied with time and Ɛ_i(t) could have been the external input received by each neuron in the network. In that case, the paper would have considered the consequence of "slow fluctuations of external input" rather than "slow fluctuations of intrinsic excitability", but the results would have been the same. The difference is therefore semantic. The consequence is that this paper is not necessarily about "intrinsic excitability", rather it considers how a Hebbian network responds to changes in excitatory drive, regardless of whether those drives are labeled "input" or "intrinsic excitability".

A revised version of the manuscript models "slope-based" excitability changes in addition to "threshold-based" changes. This serves to address the above concern that as constructed here changes in excitability threshold are not distinguishable from changes in input. However, it remains unclear what the model would do should only a subset of neurons receive a given, fixed input. In that case, are excitability changes sufficient to induce drift? This remains an important question that is not addressed by the paper in its current form.

2) Given how the learning rule that defines the input to the readout neuron is constructed, it is trivial that this unit responds to the most active neurons in the network, more so than a neuron assigned random weights. What would happen if the network included more than one "memory"? Would it be possible to construct a readout neuron that could classify two distinct patterns? Along these lines, what if there were multiple, distinct stimuli used to drive this network, rather than the global input the authors employ here? Does the system, as constructed, have the capacity to provide two distinct patterns of activity in response to two distinct inputs?

A revised version of the manuscript addresses this question, demonstrating that the network is capable of maintaining two distinct memories.

Impact:

Defining the potential role of changes in intrinsic excitability in drift is fundamental. Thus, this paper represents an important contribution. What we see here is that changes in intrinsic excitability are sufficient to induce drift. This raises the question for future work of the specific contributions of changing excitability from changing input to representational drift.

Reviewer #3 (Public Review):

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

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

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

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

Additionally, the study assumes that whoever disappeared from the dataset is attrition in academia. While in reality, those attritions could be researchers who moved to another country or another institution that is not included in the AARC (Academic Analytics Research Centre) dataset.

Reviewer #3 (Public Review):

Summary:<br /> This paper focuses on the roles of a toxoplasma protein (SPARKEL) with homology to an elongin C and the kinase SPARK that it interacts with. They demonstrate that the two proteins regulate the abundance of PKA and PKG, and that depletion of SPARKEL reduces invasion and egress (previously shown with SPARK), and that their loss also triggers spontaneous bradyzoite differentiation. The data are overall very convincing and will be of high interest to those who study Toxoplasma and related apicomplexan parasites.

Strengths:<br /> The study is very well executed with appropriate controls. The manuscript is also very well and clearly written. Overall, the work clearly demonstrates that SPARK/SPARKEL regulate invasion and egress and that their loss triggers differentiation.

Weaknesses:<br /> 1. The authors fail to discriminate between SPARK/SPARKEL acting as negative regulators of differentiation as a result of an active role in regulating stage-specific transcription/translation or as a consequence of a stress response activated when either is depleted.

2. The function of SPARKEL has not been addressed. In mammalian cells, Elongin C is part of an E3 ubiquitin ligase complex that regulates transcription and other processes. From what I can tell from the proteomic data, homologs of the Elongin B/C complex were not identified. This is an important issue as the authors find that PKG and PKA protein levels are reduced in the knockdown strains

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors conducted a comprehensive study of the X-linked miR-506 family miRNAs in mice on its origin, evolution, expression, and function. They demonstrate that the X-linked miR-506 family, predominantly expressed in the testis, may be derived from MER91C DNA transposons and further expanded by retrotransposition. By genetic deletion of different combinations of 5 major clusters of this miRNA family in mice, they found these miRNAs are not required for spermatogenesis. However, by further examination, the mutant mice show mild fertility problem and inferior sperm competitiveness. The authors conclude that the X-linked miR-506 miRNAs finetune spermatogenesis to enhance sperm competition.

Strengths:

This is a comprehensive study with extensive computational and genetic dissection of the X-linked miR-506 family providing a holistic view of its evolution and function in mice. The finding that this family miRNAs could enhance sperm competition is interesting and could explain their roles in finetuning germ cell gene expression to regulate reproductive fitness.

Weaknesses:

The authors specifically addressed the function of 5 clusters of X-link miR-506 family containing 19 miRNAs. There is another small cluster containing 3 miRNAs close to the Fmr1 locus. Would this small cluster act in concert with the 5 clusters to regulate spermatogenesis? In addition, any autosomal miR-506 like miRNAs may compensate for the loss of X-linked miR-506 family. These possibilities should be discussed.

Direct molecular link to sperm competitiveness defect remains unclear but is difficult to address.

Reviewer #3 (Public Review):

Summary:

The manuscript by Zhou and colleagues describes the potential of a two-compound combination (2C), CHIR99021 and A-485, which can generate regenerative cardiac cells (RCCs) from human embryonic stem cell-derived TNNT2+ cardiomyocytes. The authors have also demonstrated this phenomenon in neonatal rats CMs in vitro. Further, the administration of 2C can generate RCCs in adult mouse hearts and significantly improve survival and cardiac function in mice subjected to myocardial infarction. Interestingly, 2C treatment induces global changes in transcription and epigenetic modifications.

Strengths of the study:

1. This study describes the potential of 2C in improving the regeneration of the heart post-MI. The findings may have a translation potential. The idea of promoting the regenerative capacity of the heart by reprogramming CMs into RCCs is interesting.

2. The authors have validated the effect of 2C independently in hESCs, rat CMs, and a model of MI.

3. The authors explored the mechanism by Single-cell RNA-seq and Chip-Seq, which points to the transcriptional and epigenetic activation of genes essential for RCC.

Weaknesses of the study:

1. The mechanism involved in the 2C-mediated generation of RCCs is still unclear. The leads found in the RNA-seq and ChIP-seq were not validated experimentally.

2. Considering the very low number of RCCs (0.6%-1.5% of cells) generated, I cannot comprehend how the heart is protected from MI. Did the author believe 2C would affect the survival or metabolism of existing CM under hypoxia? What percentage of cells were regenerated by 2C treatment post-MI?

3. I would like to know about administering 2C in mice, which could have generated RCCs- dedifferentiated CMs in the heart. Does 2C affect the cardiac functions in mice under basal conditions? Also, does 2C administration affect any physiology in mice? The cardiac structural and functional parameters are required post-2C administration.

4. It is also not tested whether 2C would affect other cell types of the heart, including fibroblasts and endothelial cells, in vitro and in vivo. Assuming the level of protection by 2C in mice, it would affect other cell types.

5. It is still being determined how the authors chose the dose of 2C for in vivo and in vitro studies, although the concentration used for screening is different. Assessing the effect of 2C in a dose-dependent manner is essential.

6. A-485 affects H3K27Ac but not H3K9Ac. However, data show that both H3K27Ac and H3K9Ac are affected. An explanation is required.

7. The authors use "regeneration" even at the screening stage. I am wondering if regeneration could be assessed by the experimental approach they adopted.

Reviewer #3 (Public Review):

Summary:<br /> Günther and colleagues leverage ancient DNA data to track the genomic history of one of the most important farm animals (cattle) in Iberia, a region showing peculiarities both in terms of cultural practices as well as a climatic refugium during the LGM, the latter of which could have allowed the survival of endemic lineages. They document interesting trends of hybridisation with wild aurochs over the last 8-9 millennia, including a stabilisation of auroch ancestry ~4000 years ago, at ~20%, a time coincidental with the arrival of domestic horses from the Pontic steppe. Modern breeds such as the iconic Lidia used in bullfighting or bull running retain a comparable level of auroch ancestry.

Strengths:<br /> The generation of ancient DNA data has been proven crucial to unravel the domestication history of traditional livestock, and this is challenging due to the environmental conditions of the Iberian peninsula, less favourable to DNA preservation. The authors leverage samples unearthed from key archaeological sites in Spain, including the karstic system of Atapuerca. Their results provide fresher insights into past management practices, and permit characterisation of significant shifts in hybridization with wild aurochs.

Weaknesses:<br /> - Treatment of post-mortem damage: the base quality of nucleotide transitions was recalibrated down to a quality score of 2, but for 5bp from the read termini only. In some specimens (e.g. moo022), the damage seems to extend further. Why not use dedicated tools (e.g. mapDamage), or check the robustness by conditioning on nucleotide transversions?

- Their more solid analyses are based on qpAdm, but rely on two single-sample donor populations. As the authors openly discuss, it is unclear whether CPC98 is a good proxy for Iberian aurochs despite possibly forming a monophyletic clade (the number of analysed sites is simply too low to assess this monophyly; Supplementary Table S2). Additionally, it is also unclear whether Sub1 was a fully unadmixed domestic specimen, depleted of auroch ancestry. The authors seem to suggest themselves that sex-biased introgression may have already taken place in Anatolia ("suggesting that sex-biased processes already took place prior to the arrival of cattle to Iberia").

Alternatively, I recommend using Struct-f4 as it can model the ancestry of all individuals together based on their f4 permutations, including outgroups and modern data, and without the need to define pure "right" and "left" populations such as CPC98 and Sub1. It should work with low-coverage data, and allows us to do f4-based MDS plots as well as to estimate ancestry proportions (including from ghost populations).

- In the admixture graph analyses (supplementary results), the authors use population groups based on a single sample. If these samples are pseudohaploidised (or if coverage is insufficient to estimate heterozygosity - and it is at least for moo004 and moo014), f3 values are biased, implying that the fitted graph may be wrong. The graph shown in Fig S7 is in fact hard to interpret. For example, the auroch Gyu2 from Anatolia but not the auroch CPC98 also from Anatolia received 62% of ancestry from North Africa? The Neolithic samples moo004 and moo014 also show the same shocking disparity. I would consider re-doing this analysis with more than a sample per population group.

Reviewer #3 (Public Review):

Summary:

The researchers aim add to the literature on faculty career pathways with particular attention to how gender disparities persist in the career and funding opportunities of researchers. The researchers also examine aspects of institutional prestige that can further amplify funding and career disparities. While some factors about individuals' pathways to faculty lines are known, including the prospects of certain K award recipients, the current study provides the only known examination of the K99/R00 awardees and their pathways.

Strengths:

The authors establish a clear overview of the institutional locations of K99 and R00 awardees and the pathways for K99-to-R00 researchers and the gendered and institutional patterns of such pathways. For example, there's a clear institutional hierarchy of hiring for K99/R00 researchers that echo previous research on the rigid faculty hiring networks across fields, and a pivotal difference in the time between awards that can impact faculty careers. Moreover, there's regional clusters of hiring in certain parts of the US where multiple research universities are located. Moreover, documenting the pathways of HBCU faculty is an important extension of the study by Wapman et al. (2022: https://www.nature.com/articles/s41586-022-05222-x), and provides a more nuanced look at the pathways of faculty beyond the oft-discussed high status institutions. (However, there is a need for more refinement in this segment of the analyses). Also, the authors provide important caveats throughout the manuscript about the study's findings that show careful attention to the complexity of these patterns and attempting to limit misinterpretations of readers.

Weaknesses:

The authors have addressed my recommendations in the previous review round in a satisfactory way.

Reviewer #3 (Public Review):

Summary<br /> CLC-2 channels play an important role in cellular homeostasis and electrical excitability, and dysfunctions are associated with aldosteronism and leukodystrophy. Structural insights into the functioning of CLC-2 are just emerging. CLC-2 channels are distinct among the members of the CLC family in that they are activated by hyperpolarization. Earlier studies have implicated channel regulation by a "ball-and-chain" type of channel block mechanism which underlies its strong rectification and use-dependent "run-up" properties. Structural insights into these mechanisms are currently lacking. In this manuscript, Xu et al present CryoEM structures of CLC-2 in the apo and inhibitor-bound conformations in the 2.5-2.7 A resolution range. Several novel structural features are presented that lend support to the "ball-and chain" model, identify an interesting role for the c-terminal domain in gating, and establish the interaction pocket for AK-42. Electrophysiology and simulations nicely support the structural work. Overall, an elegant study, with high-quality data, and a well-presented manuscript.

Strengths<br /> 1. The cryoEM data presented reveals that the channel is in a closed conformation at depolarizing potential (0 mv). Structures for the closed state of CLCs were not previously available. A strong density for Glu205, which constitutes the Egate, allows for an unambiguous assignment of its position at the Scen Cl-binding site, thereby establishing the basis for the block in the closed channel.<br /> 2. The apo state particles were sorted into two classes that differ in the conformation of the CTD. A previously unobserved rearrangement of the CBS region in the CTD is reported wherein the CTD is positioned closer to the TM region in one of the subunits, breaking the C2 symmetry. The data implicates a role for the conformational flexibility of CTD in gating.<br /> 3. The most interesting finding of this work, is perhaps, the presence of an additional density, corresponding to a hairpin-like structure, that is seen only at the subunit where the CTD is positioned away from the TMD. The authors propose that the additional density corresponds to a 13 aa stretch in the N-terminal region. The position of the hairpin at the intracellular mouth of the CL-permeation pathway is likely to impede ion conduction, by a mechanism analogous to the "ball-and-chain" proposed in other voltage-gated channels.<br /> 4. The structure of CLC-2 in complex with a selective inhibitor AK-42 is in a conformation very similar to that of the apo state, with a clear additional density for the AK-42 molecule. Binding site interaction provides insights into AK-42 selectivity for CLC-2 vs CLC-1.

Reviewer #3 (Public Review):

Summary: The authors present a thought-provoking and comprehensive re-analysis of previously published human cell genomics data that seeks to understand the relationship between the sites where the Origin Recognition Complex (ORC) binds chromatin, where the replicative helicase (Mcm2-7) is loaded, and where DNA replication actually begins (origins). The view that these should coincide is influenced by studies in yeast where ORC binds site-specifically to dedicated nucleosome-free origins where Mcm2-7 can be loaded and remains stably positioned for subsequent replication initiation. However, this is most certainly not the case in metazoans where it has already been reported that chromatin bindings sites of ORC and Mcm2-7 do not necessarily overlap, nor do they always overlap with origins. This is likely due to Mcm2-7 possessing linear mobility on DNA (i.e., it can slide) such that other chromatin-contextualized processes can displace it from the site in which it was originally loaded. Additionally, Mcm2-7 is loaded in excess and thus only a fraction of Mcm2-7 would be predicted to coincide with replication start sites. This study reaches a very similar conclusion of these previous studies: they find a high degree of discordance between ORC, Mcm2-7, and origin positions in human cells.

Strengths: The strength of this work is its comprehensive and unbiased analysis of all relevant genomics datasets. To my knowledge, this is the first attempt to integrate these observations. It also is an important cautionary tale to not confuse replication factor binding sites with the genomic loci where replication actually begins, although this point is already widely appreciated in the field.

Weaknesses: The major weakness of this paper is the lack of novel biological insight and that the comprehensive approach taken failed to provide any additional mechanistic insight regarding how and why ORC, Mcm2-7, and origin sites are selected or why they may not coincide.

Reviewer #3 (Public Review):

Sasani et al. develop and implement a new method for mutator allele discovery in the BXD mouse population. This new method, termed "aggregate mutation spectrum distance" or AMSD, carries several notable strengths, including the ability to aggregate de novo mutations across individuals to reduce data sparsity and to combine mutation rate frequencies across multiple nucleotide contexts into a single estimate. As demonstrated by simulations, this method is better suited to mutator discovery under certain scenarios, as compared to conventional QTL or association mapping. Overall, the theoretical premise of the AMSD method is judged to be both strong and innovative, and the methodology could be extended to other species and populations to enable discovery of additional mutator alleles.

The authors then apply their method to the BXD mouse recombinant inbred mapping population. As proof-of-principle, they first successfully re-identify a known mutator locus in this population on chr4. Next, to assess possible genetic interactions involving this known mutator, Sasani et al. condition on the chr4 mutator genotype and reimplement the AMSD scan. This strategy led them to identify a second locus on chr6 that interacts epistatically with the chr4 locus; mice with "D" alleles at both loci exhibit a significantly increased burden of C>A de novo mutations, even though mice with the D allele at the chr6 locus alone show no appreciable increase in the C>A mutation fraction. This exciting discovery not only adds to the catalog of known mutator alleles, but also reveals key aspects of mutator biology and reinforces the hypothesis that segregating variants in genes associated with DNA repair influence germline mutation spectra.

Despite a high level of overall enthusiasm for this work, there are some limitations to the AMSD method. However, it is my judgement that the authors present a balanced summary of the strengths and weaknesses of their method in the revised manuscript. I also think that the authors' conclusions may actually somewhat undersell the scientific impact of their findings. As the authors note, few mutation rate modifiers have been identified in mammals. This is potentially because large- and moderate-effect modifiers are rapidly selected against due to their deleterious effects, but could also be due to pervasive epistasis wherein modifiers are only expressed on certain "permissive" genetic backgrounds, such as the chr6 locus the authors discover in this paper. The potential background dependence of mutator expression could partially shelter it from the action of selection, allowing the allele persist in populations. This discovery has significant implications for our understanding of mutation rate evolution, but only earns a cursory mention in the paper.

Reviewer #3 (Public Review):

Summary:

In the submitted manuscript by Go et al, the authors evaluated the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and made a number of interesting observations, including the following: 1) CCL5 expression within the tumor microenvironment negatively correlated with clinical outcomes in human patients with PDAC; 2) there were both positive and negative correlations between CCL5 expression and the expression of specific genes (e.g. those encoding CD56 and CD16, respectively) included among gene signature lists for Treg, MDSC, TAM, and NK cells; 3) CCR5 inhibition with the inhibitor, maraviroc, reduced Treg infiltration but not that of other immune cell types in an orthotopic murine model of PDAC; 4) CCR5 inhibition augmented anti-PD1 immunotherapy when combined with ionizing radiation (IR) therapy in the murine model; 5) the above therapy resulted in increased infiltration of CD8+ cytotoxic T cells as well as of a subset of NKG2D-negative, tissue-residency (tr) marker expressing NK cells (deemed Cluster 1 NK in their data sets) that inversely correlated with the number of E-cadherin+ cells (i.e. tumor cells) and showed predicted interactions with cDC1 dendritic cells (including XCL1/XCL2 expressed by the NK and XCR1 expressed by the cDC1); 6) the authors identified a number of putative signals stemming from the trNK (e.g. IL-16, TNFSF14, FASLG, CSF, MIF) as well as incoming from cDC1s to NK (e.g. BAG6-NKp30); 7) these trNK cells positively correlated with good outcomes and with CD8+ T cell infiltrations in human PDAC as well as in many other solid tumor types; and 8) importantly, the benefit of IR therapy was specific to the subset of PDAC patients (represented in the TCGA dataset) that were predicted to have low amounts of trNK cells. The authors used murine experimental models, multiplexed imaging analyses, and a number of publicly available sequencing data sets from human tumor samples to perform their investigations. Based on their findings, the authors proposed that combining IR with CCR5 inhibition and anti-PD1 immunotherapy is a promising strategy to treat solid cancers.

Strengths:

Overall, the collective analyses and conclusions appear to be novel and could be of high and rapid impact on the field, particularly in terms of directing clinical trials to incorporate IR with CCR5 inhibition and immunotherapy. The manuscript is well written; the figures are for the most part clear; and the Discussion is very thoughtful.

Weaknesses:

There were a number of minor typographical errors, missing references, or minor issues with the figures. In general, while many of the observations provided strong suggestive evidence of relationships, phenotypes, and functions, the authors often used language to indicate that such things were confirmed, validated, or proven. In fact, there was a paucity of such functional/confirmatory experiments. This does not necessarily detract from the overall significance, excitement for, and potential impact of the study; but the language could likely be adjusted to be more in keeping with the true nature of the findings. The main title and running title are a bit different; consider making them more similar.

Reviewer #3 (Public Review):

This work by Fleck et al. and colleagues documented the auxin feeding-induced effects in adult flies, since auxin could be used in temporally control gene expression using a modified Gal4/Gal80 system. Overall, the experiments were well designed and carefully executed. The results were quantified with appropriate statistical analyses. The paper was also well written and the results were presented logically. Their findings demonstrate that auxin-fed flies have significantly lower triglyceride levels than the control flies using Ultra High-pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS)-based metabolomics assays. Further transcriptome analyses using the whole flies show changes of genes involved in fatty acid metabolism. However, female oogenesis and fecundity do not seem to be affected, at least using the current assays. These results indicate that auxin may not be used in experiments involving lipid-related metabolism, but could be appropriate to be applied for other biological processes. Researchers need to be careful when applying this strategy in their own experimental design and should perform proper controls.

Reviewer #3 (Public Review):

Summary of Work<br /> This paper conducts the largest GWAS study of A. thaliana in response to a viral infection. The paper identifies a 1.5 MB region in the chromosome associated with disease, including SNPs, structural variation, and transposon insertions. Studies further validate the association experimentally with a separate experimental infection procedure with several lines and specific T-DNA mutants. Finally, the paper presents a geographic analysis of the minor disease allele and the major association. The major take-home message of the paper is that structural variants and not only SNPs are important changes associated with disease susceptibility. The manuscript also makes a strong case for negative frequency-dependent selection maintaining a disease susceptibility locus at low frequency.

Strengths and Weaknesses<br /> A major strength of this manuscript is the large sample sizes, careful experimental design, and rigor in the follow-up experiments. For instance, mentioning non-infected controls and using methods to determine if geographic locus associations were due to chance. The strong result of a GWAS-detected locus is impressive given the complex interaction between plant genotypes and strains noted in the results. In addition to the follow-up experiments, the geographic analysis added important context and broadened the scope of the study beyond typical lab-based GWAS studies. I find very few weaknesses in this manuscript.

Support of Conclusions<br /> The support for the conclusions is exceptional. This is due to the massive amount of evidence for each statement and also due to the careful consideration of alternative explanations for the data.

Significance of Work<br /> This manuscript will be of great significance in plant disease research, both for its findings and its experimental approach. The study has very important implications for genetic associations with disease beyond plants.

Reviewer #3 (Public Review):

Summary:<br /> In this interesting study, the authors characterize the mechanisms whereby a C. elegans TGF-beta DAF-7 responds to various forms of food cues to regulate foraging.<br /> Building on their previous findings that characterized the functional role of daf-7 in the ASJ sensory neurons in response to a bacterial pathogen and in regulating searching behaviors, the authors of this manuscript show that ingestion of E. coli OP50, a common laboratory food for the worms, suppresses ASJ expression of daf-7 and secreted water-soluble cues of OP50 increase it. They further show that the level of daf-7 expression in ASJ is positively associated with a higher level of roaming/exploration. The authors identify that the function of a C. elegans ortholog of Anaplastic Lymphoma Kinase in the interneurons AIA regulates ASJ expression of daf-7 in response to food information and the related searching behavior.

Strengths:<br /> The study addresses an important question that appeals to a wide readership. The findings are demonstrated by strong results produced from well designed experiments.

Reviewer #3 (Public Review):

This study is well designed and executed and provides new and important insights into the role of two TFs during the maturation of female gametocytes and fertilization in the mosquito midgut. However, it but would benefit from a more thorough characterization of the phenotype to understand at which step of development these factors are required.

Overall the authors have shown only limited willingness to comprehensively address reviewer concerns and incorporate their suggestions.

Reviewer #3 (Public Review):

Summary & Strengths:

This study is useful in revealing the neural correlates of goal-directed navigation in the rodent parahippocampal regions, including the medial entorhinal cortex, presubiculum, and parasubiculum. It shows that task-relevant information represented by the parahippocampus is strongly related to task performance. It also reports the relationships of navigational factors (e.g., head direction signal) recorded during foraging in an open field with task variables.

Gonzalez and Giocomo investigated the neural activities in the parahippocampal cortex modulated by visual cues and error signals while the animal performed a goal-directed navigation task on the tree maze. They confirmed that the firing rates and spatial firing patterns in the parahippocampus were significantly correlated with the animal's task performance and the general navigational coding in the open field arena. The authors have concluded that the parahippocampal neurons encode mismatch-like signals, suggesting the functional role of the parahippocampus as a feedback system in a goal-directed task. However, a few major concerns should be addressed more closely to support the conclusion.

1) Due to the limitations of histological verification, the neural responses in the medial entorhinal cortex, presubiculum, and parasubiculum are analyzed together, and this limits the study from understanding the differential information processing across these regions. Because the medial entorhinal cortex and the pre/parasubiculum are believed to be located in very different positions in the information flow within the rodent medial temporal lobe with different anatomical connections, it would have been more convincing if the distinctive functions between the regions could be identified.

2) The authors should carefully differentiate rate remapping and global remapping in their analysis. Rate remapping generally indicates firing rate modulation with little or no shift of spatial firing fields (Leutgeb et al., 2005; Colgin et al., 2008). Therefore, the neurons exhibiting global remapping should not be included in the analysis suited for rate remapping (e.g., the encoding model that considers the cue-dependent rate-remapping effect).

3) One of the major findings in this study is that the parahippocampal neural responses to a visual cue or reward were correlated with task performance. One can expect that cue representation before the decision point is likely to have a greater impact on task performance. Although the Uz score between the left cue and right cue seemed not significantly different from zero on the stem, it would be beneficial if the authors verify whether the remapping score based on the firing rate maps will still be correlated with the task performance when examined only before the decision point, not for the entire maze.

4) There is a need to set the analytic epoch in more detail. The boundary between outbound and inbound journeys was set as 'last goal well visit.' However, even in a correct trial, if the reward was not received in the first goal well, an error signal could occur before the animal triggered the second goal well which was rewarding. This might have caused the rate remapping between two cue conditions, specifically on the arms. To eliminate this possibility, it is recommended to set the outbound journey from the home well trigger to the first goal well approach or to select only trials where the animal received rewards from the first goal well triggering.

Weaknesses:

Incomplete results could limit support for the arguments of the study and may require more rigorous analytical methods.

Reviewer #3 (Public Review):

The paper by Li et al. describes the role of the TOR pathway in Aspergillus flavus. The authors tested the effect of rapamycin in WT and different deletion strains. This paper is based on a lot of experiments and work but remains rather descriptive and confirms the results obtained in other fungi. It shows that the TOR pathway is involved in conidiation, aflatoxin production, pathogenicity, and hyphal growth. This is inferred from rapamycin treatment and TOR1/2 deletions. Rapamycin treatment also causes lipid accumulation in hyphae. The phenotypes are not surprising as they have been shown already for several fungi. In addition, one caveat is in my opinion that the strains grow very slowly and this could cause many downstream effects. Several kinases and phosphatases are involved in the TOR pathway. They were known from S. cerevisiae or filamentous fungi. The authors characterized them as well with knock-out approaches.

Reviewer #3 (Public Review):

In this manuscript, the authors explored the transcriptional heterogeneity of the periosteum with single nuclei RNA sequencing. Without prior enrichment of specific populations, this dataset serves as an unbiased representation of the cellular components potentially relevant to bone regeneration. By describing single-cell cluster profiles, the authors characterized over 10 different populations in combined steady state and post-fracture periosteum, including stem cells (SSPC), fibroblast, osteoblast, chondrocyte, immune cells, and so on. Specifically, a developmental trajectory was computationally inferred using the continuum of gene expression to connect SSPC, injury-induced fibrogenic cells (IIFC), chondrocyte, and osteoblast, showcasing the bipotentials of periosteal SSPCs during injury repair. Additional computational pipelines were performed to describe the possible gene regulatory network and the expected pathways involved in bone regeneration. Overall, the authors provided valuable insights into the cell state transitions during bone repair and proposed sets of genes with possible involvements in injury response.

While the highlights of the manuscript are the unbiased characterization of periosteal composition, and the trajectory of SSPC response in bone fracture response, many of the conclusions can be more strongly supported with additional clarifications or extensions of the analysis.

1. As described in the method section, both the steady-state data and full dataset underwent integration before dimensional reduction and clustering. It would be appreciated if the authors could compare the post-integration landscapes of uninjured cells between steady state and full dataset analysis. Specifically, fibroblasts were shown in Figure 1C and 1E, and such annotations did not exist in Figure 2B. Will it be possible that the original 'fibroblasts' were part of the IIFC population?

2. According to Figure 2, immune cells were taking a significant abundance within the dataset, specifically during days 3 & 5 post-fracture. It will be interesting to see the potential roles that immune cells play during bone repair. For example, what are the biological annotations of the immune clusters (B, T, NK, myeloid cells)? Are there any inflammatory genes or related signals unregulated in these immune cells? Do they interact with SSPC or IIFC during the transition?

3. The conclusion of Notch and Wnt signaling in IIFC transition was not sufficiently supported by the analysis presented in the manuscript, which was based on computational inferences. It will be great to add in references supporting these claims or provide experimental validations examining selected members of these pathways.

Reviewer #3 (Public Review):

Summary:<br /> Canelo et al. used a combination of mathematical modeling and behavioral experiments to ask whether flies use an all-or-none EC model or a graded EC model (in which the turn amplitude is modulated by wide-field optic flow). Particularly, the authors focus on the bar-ground discrimination problem, which has received significant attention in flies over the last 50-60 years. First, they use a model by Poggio and Reichardt to model flight response to moving small-field bars and spots and wide-field gratings. They then simulate this model and compare simulation results to flight responses in a yaw-free tether and find generally good agreement. They then ask how flies may do bar-background discrimination (i.e. complex visual environment) and invoke different EC models and an additive model (balancing torque production due to background and bar movement). Using behavioral experiments and simulation supports the notion that flies use an all-or-none EC since flight turns are not influenced by the background optic flow. While the study is interesting, there are major issues with the conceptual framework.

Strengths:<br /> - They ask a significant question related to efference copies during volitional movement.<br /> - The methods are well detailed and the data (and statistics) are presented clearly.<br /> - The integration of behavioral experiments and mathematical modeling of flight behavior.<br /> - The figures are overall very clear and salient.

Weaknesses:<br /> - Omission of saccades: While the authors ask a significant question related to the mechanism of bar-ground discrimination, they fail to integrate an essential component of the Drosophila visuomotor responses: saccades. Indeed, the Poggio and Reichardt model, which was developed almost 50 years ago, while appropriate to study body-fixed flight, has a severe limitation: it does not consider saccades. The authors identify this major issue in the Discussion by citing a recent switched, integrate-and-fire model (Mongeau & Frye, 2017). The authors admit that they "approximated" this model as a smooth pursuit movement. However, I disagree that it is an approximation; rather it is an omission of a motor program that is critical for volitional visuomotor behavior. Indeed, saccades are the main strategy by which Drosophila turn in free flight and prior to landing on an object (i.e. akin to a bar), as reported by the Dickinson group (Censi et al., van Breugel & Dickinson [not cited]). Flies appear to solve the bar-ground discrimination problem by switching between smooth movement and saccades (Mongeau & Frye, 2017; Mongeau et al., 2019 [not cited]). Thus, ignoring saccades is a major issue with the current study as it makes their model disconnected from flight behavior, which has been studied in a more natural context since the work of Poggio.

Critically, recent work showed that a group of columnar neurons (T3) appear specialized for saccadic bar tracking through integrate-and-fire computations, supporting the notion of parallel visual circuits for saccades and smooth movement (Frighetto & Frye, 2023 [not cited]).

A major theme of this work is bar fixation, yet recent work showed that in the presence of proprioceptive feedback, flies do not actually center a bar (Rimniceanu & Frye, 2023). Furthermore, the same study found that yaw-free flies do not smoothly track bars but instead generate saccades. Thus prior work is in direct conflict with the work here. This is a major issue that requires more engagement by the authors.

- Relevance of the EC model: EC-related studies by the authors linked cancellation signals to saccades (Kim et al, 2014 & 2017). Puzzlingly, the authors applied an EC model to smooth movement, when the authors' own work showed that smooth course stabilizing flight turns do not receive cancellation signals (Fenk et al., 2021). Thus, in Fig. 4C, based on the state of the field, the efference copy signal should originate from the torque commands to initiate saccades, and not from torque to generate smooth movement. As this group previously showed, cancellation signals are quantitatively tuned to that of the expected visual input during saccades. Importantly, this tuning would be to the anticipated saccadic turn optic flow. Thus the authors' results supporting an all-or-none model appear in direct conflict with the author's previous work. Further, the addition-only model is not particularly helpful as it has been already refuted by behavioral experiments (Rimneceanu & Frye, Mongeau & Frye).

- Behavioral evidence for all-or-none EC model: The authors state "unless the stability reflex is suppressed during the flies' object evoked turns, the turns should slow down more strongly with the dense background than the sparse one". This hypothesis is based on the fact that the optomotor response magnitude is larger with a denser background, as would be predicted by an EMD model (because there are more pixels projected onto the eye). However, based on the authors' previous work, the EC should be tuned to optic flow and thus the turning velocity (or amplitude). Thus the EC need not be directly tied to the background statistics, as they claim. For instance, I think it would be important to distinguish whether a mismatch in reafferent velocity (optic flow) links to distinct turn velocities (and thus position). This would require moving the background at different velocities (co- and anti-directionally) at the onset of bar motion. Overall, there are alternative hypotheses here that need to be discussed and more fully explored (as presented by Bender & Dickinson and in work by the Maimon group).

Reviewer #3 (Public Review):

In this study, the authors developed and tested a novel framework for extracting muscle synergies. The approach aims at removing some limitations and constrains typical of previous approaches used in the field. In particular, the authors propose a mathematical formulation that removes constrains of linearity and couple the synergies to their motor outcome, supporting the concept of functional synergies and distinguishing the task-related performance related to each synergy. While some concepts behind this work were already introduced in recent work in the field, the methodology provided here encapsulates all these features in an original formulation providing a step forward with respect to the currently available algorithms. The authors also successfully demonstrated the applicability of their method to previously available datasets of multi-joint movements.

Preliminary results positively support the scientific soundness of the presented approach and its potential. The added values of the method should be documented more in future work to understand how the presented formulation relates to previous approaches and what novel insights can be achieved in practical scenarios and confirm/exploit the potential of the theoretical findings.

Reviewer #3 (Public Review):

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

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

Weaknesses:<br /> The presented maps merely reinforce their previous findings, wherein wildtype Cx26 favored a closed conformation in the presence of high PCO2. While the structure of the TM2-TM3 loop may suggest a mechanism for stabilizing the closed conformation, no experimental data was provided to support this mechanism. Additionally, the cryo-EM maps were not effectively presented, making it difficult for readers to grasp the message.

Reviewer #3 (Public Review):

Summary:<br /> Unlike most eukaryotes, Blastocystis has a branched glycolysis pathway, which is split between the cytoplasm and the mitochondrial matrix. An outstanding question was how the glycolytic intermediates generated in the 'preparatory' phase' are transported into the mitochondrial matrix for the 'pay off' phase. Here, the authors use bioinformatic analysis to identify two candidate solute carrier genes, bGIC-1, and bGIC-2, and use biochemical and biophysical methods to characterise their substrate specificity and transport properties. The authors demonstrate that bGIC-2 can transport dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate, establishing this protein as the 'missing link' connecting the two split branches of glycolysis in this branch of single-celled eukaryotes. The authors also present their data on bGIC-1, which suggests a role in anion transport and bOGC, which is a close functional homologue of the human oxoglutarate carrier (hOGC, SLC25A11) and human dicarboxylate carrier (hDIC, SLC25A10).

Strengths:<br /> The results are presented in a clear and logical arrangement, which nicely leads the reader through the process of gene identification and subsequent ligand screening and functional reconstitution. The results are compelling and well supported - the thermal stabilisation data is supported by the exchange studies. Caveats, where apparent, are discussed and rational explanations are given.

Weaknesses:<br /> The study does not contain any significant weaknesses in my view. I would like to see the authors include the initial rate plots used in the main figures (possibly as insets), so we can observe the data points used for these calculations. It would also have been interesting to include the AlphaFold models for bGIC-1 and bGIC-2 and a discussion/rationalisation for the substrate specificity discussed in the study.