Reviewer #1 (Public Review):

Koumoundourou et al., identify a pathway downstream of Bcl11b that controls synapse morphology and plasticity of hippocampal mossy fiber synapses. Using an elegant combination of in vivo, ex vivo, and in vitro approaches, the authors build on their previous work that indicated C1ql2 as a functional target of Bcl11b (De Bruyckere et al., 2018). Here, they examine the functional implications of C1ql2 at MF synapses in Bcl11b cKO mice and following C1ql2 shRNA. The authors find that Bcl11b KO and shRNA against C1ql2 significantly reduces the recruitment of synaptic vesicles and impairs LTP at MF synapses. Importantly, the authors test a role for the previously identified C1ql2 binding partner, exon 25b-containing Nrxn3 (Matsuda et al., 2016), as relevant at MF synapses to maintain synaptic vesicle recruitment. To test this, the authors developed a K262E C1ql2 mutant that disrupts binding to Nrxn3. Curiously, while Bcl11b KO and C1ql2 KD largely phenocopy (reduced vesicle recruitment and impaired LTP), only vesicle recruitment is dependent on C1ql2-Nrxn3 interactions. These findings provide new insight into the functional role of C1ql2 at MF synapses. The authors utilize a multidisciplinary approach to convincingly demonstrate a role for C1ql2-Nrxn3(25b+) interactions for vesicle recruitment and a Nrxn3(25b+)-independent role for C1ql2 in LTP, The authors establish an important signaling pathway that offers insight into how disruptions of Bcl11b contribute to synapse dysfunction and provide a much needed advance toward understanding the functional consequences of neurexin alternative splicing.

Reviewer #1 (Public Review):

Summary:<br /> This is an important work showing that loss of LRRK function causes late-onset dopaminergic neurodegeneration in a cell-autonomous manner. One of the LRRK members, LRRK2, is of significant translational importance as mutations in LRRK2 cause late-onset autosomal dominant Parkinson's disease (PD). While many in the field assume that LRRK2 mutant causes PD via increased LRRK2 activity (i.e., kinase activity), it is not a settled issue as not all disease-causing mutant LRRK2 exhibits increased activity. Further, while LRRK2 inhibitors are under clinical trials for PD, the consequence of chronic, long-term LRRK2 inhibition is unknown. Thus, studies evaluating the long-term impact of LRRK deficit have important translational implications. Moreover, because LRRK proteins, particularly LRRK2, are known to modulate immune response and intracellular membrane trafficking, the study's results and the reagents will be valuable for others interested in LRRK function.

Strengths:<br /> This report describes a mouse model where LRRK1 and LRRK2 genes are conditionally deleted in dopaminergic neurons. Previously, this group showed that while loss of LRRK2 expression does not cause brain phenotype, loss of both LRRK1 and LRRK2 causes a later onset, progressive degeneration of catecholaminergic neurons, Dopaminergic (DAergic) neurons in substantia niga (SN) and Noradrenergic neurons in Locus Coeruleus (LC). However, because LRRK genes are widely expressed with some peripheral phenotypes, it was unknown if the neurodegeneration in LRRK double Knock Out (DKO) was cell autonomous. To rigorously test this question, the authors have generated a double conditional KO allele where both LRRK1 and LRRK2 genes were targeted to contain loxP sites. In my view, this was beyond what is normally required as most investigators might just combine one KO allele with another floxed allele. The authors provide a rigorous validation showing that the Driver (DAT-Cre) is expressed in the majority of DAergic neurons in SN and that LRRK levels are decreased selectively in the ventral midbrain. Using these mice, the authors show that the number of DA neurons is average at 15 but significantly decreased at 20 months of age. Moreover, the authors show that the number of apoptotic neurons is increased by ~2X in aged SN, demonstrating increased ongoing cell death, as well as an increase in activated microglia. The degeneration is limited to DA neurons as LC neurons are not lost as this population does not express DAT. Overall, the mouse genetics and experimental analysis were performed in a rigorous manner and the results were statistically sound and compelling.

Weakness: I only have a few minor comments. First, in PD and other degenerative conditions, axons and terminals loss occurs prior to cell bodies. It might be beneficial to show the status of DAergic markers in the striatum. Second, previous studies indicate that very little, if any, LRRK1 is expressed in SN DAergic neurons. This also seems to be the case with the Allen Brain Atlas profile. Thus, it is preferable that authors discuss the discrepancy as authors seem to imply significant LRRK1 expression in DA neurons.

Revision: I appreciate the authors revising the manuscript with additional data that have clarified my prior comments. They now show that TH levels in the striatum decrease with SNpc neurons. Further, while there is some disagreement regarding the expression LRRK1 in SNpc, the authors provide a convincing case that LRRK1 function is important in SNpc DA neurons.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript by Liu et al explores the role of the UPR and immune regulators in the evaluation of nutritional quality in C. elegans. They identify neuronal UPR activation and the MAPK PMK-1 as key responders to low food quality. In particular, the data suggest that these pathways are activated by low levels of vitamin C synthesis that result from the low sugar levels present in heat-killed E. coli.

Strengths:<br /> The results are intriguing and expand our understanding both of physiological food evaluation systems, and of the known roles of stress response pathways in organismal physiology. The authors use a range of techniques, encompassing imaging, metabolomic analysis, gene expression analysis, and behavioural assays, to support their claims.

Weaknesses:<br /> There is limited mechanistic analysis in the study. In particular, how does low vitamin C trigger UPR activation? This is an intriguing finding that, if followed up, could potentially reveal a novel mechanism of UPR activation. In addition, how is the activation of the PMK-1 pathway driven by/coordinated with UPR activation? The data in some figures is not as convincing as it could be: the magnitude of the effect size is small in the supplementation experiments, and the statistical tests used are not always appropriate to enable multiple comparisons.

Reviewer #1 (Public Review):

Summary:<br /> The authors introduce two preparations for observing large-scale cortical activity in mice during behavior. Alongside this, they present intriguing preliminary findings utilizing these methods. This paper is poised to be an invaluable resource for researchers engaged in extensive cortical recording in behaving mice.

Strengths:<br /> -Comprehensive methodological detailing:<br /> The paper excels in providing an exceptionally detailed description of the methods used. This meticulous documentation includes a step-by-step workflow, complemented by thorough workflow, protocols, and a list of materials in the supplementary materials.

-Minimal movement artifacts:<br /> A notable strength of this study is the remarkably low movement artifacts. To further underscore this achievement, a more robust quantification across all subjects, coupled with benchmarking against established tools (such as those from suite2p), would be beneficial.

Insightful preliminary data and analysis:<br /> The preliminary data unveiled in the study reveal interesting heterogeneity in the relationships between neural activity and detailed behavioral features, particularly notable in the lateral cortex. This aspect of the findings is intriguing and suggests avenues for further exploration.

Weaknesses:<br /> -Clarification about the extent of the method in the title and text:<br /> The title of the paper, using the term "pan-cortical," along with certain phrases in the text, may inadvertently suggest that both the top and lateral view preparations are utilized in the same set of mice. To avoid confusion, it should be explicitly stated that the authors employ either the dorsal view (which offers limited access to the lateral ventral regions) or the lateral view (which restricts access to the opposite side of the cortex). For instance, in line 545, the phrase "lateral cortex with our dorsal and side mount preparations" should be revised to "lateral cortex with our dorsal or side mount preparations" for greater clarity.

-Comparison with existing methods:<br /> A more detailed contrast between this method and other published techniques would add value to the paper. Specifically, the lateral view appears somewhat narrower than that described in Esmaeili et al., 2021; a discussion of this comparison would be useful. Furthermore, the number of neurons analyzed seems modest compared to recent papers (50k) - elaborating on this aspect could provide important context for the readers.

-Discussion of methodological limitations:<br /> The limitations inherent to the method, such as the potential behavioral effects of tilting the mouse's head, are not thoroughly examined. A more comprehensive discussion of these limitations would enhance the paper's balance and depth.

-Preliminary nature of results:<br /> The results are at a preliminary stage; for example, the B-soid analysis is based on a single mouse, and the validation data are derived from the training data set. The discrepancy between the maps in Figures 5e and 6e might indicate that a significant portion of the map represents noise. An analysis of variability across mice and a method to assign significance to these maps would be beneficial.

-Analysis details:<br /> More comprehensive details on the analysis would be beneficial for replicability and deeper understanding. For instance, the statement "Rigid and non-rigid motion correction were performed in Suite2p" could be expanded with a brief explanation of the underlying principles, such as phase correlation, to provide readers with a better grasp of the methodologies employed.

Reviewer #1 (Public Review):

Granados-Aparici et al., investigate somatic-germline interactions in female mice. Mammalian oocytes are nurtured in multi-cellular ovarian follicles and communication with surrounding somatic cells is critical for oocyte development. This study focused on transzonal projections (TZP) extending from granulosa cells to the surface of oocytes and documented the importance of SMAD4, a TGF- β mediator, in regulating the TZPs. They propose a model in which individual TZPs contact the surface of the oocyte and stably attach if there is sufficient N-cadherin. In SMAD4-depleted cells, there is insufficient N-cadherin to stabilize the attachment. The TZP continues to elongate but eventually retracts. Their model is well supported by their experimental evidence and the manuscript is both well-formulated and written.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes the crystal structures of Streptococcus pneumoniae NOXs. Crystals were obtained for the wild-type and mutant dehydrogenase domain, as well as for the full-length protein comprising the membrane domain. The manuscript further carefully studies the enzyme's kinetics and substrate-specificity properties. Streptococcus pneumoniae NOX is a non-regulated enzyme, and therefore, its structure should provide a view of the NOX active conformation. The structural and biochemical data are discussed on this ground.

Strengths:<br /> This is very solid work. The protein chemistry and biochemical analysis are well executed and carefully described. Similarly, the crystallography must be appreciated given the difficulty of obtaining good enzyme preparations and the flexibility of the protein. Even if solved at medium resolution, the crystal structure of the full-length protein conveys relevant information. The manuscript nicely shows that the domain rotations are unlikely to be the main mechanistic element of NOX regulation. It rather appears that the NADPH-binding conformation is pivotal to enzyme activation. The paper extensively refers to the previous literature and analyses the structures comprehensively with a comparison to previously reported structures of eukaryotic and prokaryotic NOXs.

Weaknesses:<br /> The manuscript is not always very clear with regard to the analysis of NADPH binding. The last section describes a "crevice" featured by the NADPH-binding sites in NOXs. It remains unclear whether this element corresponds to the different conformations of the protein C-terminal residues or more extensive structural differences. This point must be clarified.<br /> A second less convincing point concerns the nature of the electron acceptor. The manuscript states that this NOX might not physiologically act as a ROS producer. A question then immediately arises: Is this protein an iron reductase? Can the authors better discuss or provide more data about this point?

Reviewer #1 (Public Review):

This paper performed a functional analysis of the poorly characterized pseudo-phosphatase Styxl2, one of the targets of the Jak/Stat pathway in muscle cells. The authors propose that Styxl2 is essential for de novo sarcomere assembly by regulating autophagic degradation of non-muscle myosin IIs (NM IIs). Although a previous study by Fero et al. (2014) has already reported that Styxl2 is essential for the integrity of sarcomeres, this study provides new mechanistic insights into the phenomenon. In vivo studies in this manuscript are compelling; however, I feel the contribution of autophagy in the degradation of NM IIs is still unclear.

Reviewer #1 (Public Review):

Summary:<br /> This paper presents evidence that a relatively common genetic variant tied to several disease phenotypes affects the interaction between the mRNA of CCL2 and the RNA binding protein HuR. CCL2 is an immune cell chemoattractant protein.

Strengths:<br /> The study is well conducted with relevant controls. The techniques are appropriate, and several approaches provided concordant results that were generally supportive of the conclusions reached. The impact of this work, identifying a genetic variant that works by altering the binding of an RNA-regulatory protein, has important implications given that the HuR protein could be a drug target to improve its function and override this genetic change. This could have important implications for a number of diseases where this genetic variant contributes to disease risk.

Weaknesses:<br /> The authors need to do a better job of citing prior work. Certain details of the experimental protocols need to be further elaborated or clarified to contextualize the significance of the findings, Some of the findings need to be better described.

Reviewer #1 (Public Review):

The authors examine the fascinating question of how T lymphocytes regulate proteome expression during the dramatic cell state change that accompanies the transition from the resting quiescent state to the activated, dividing state. Orthogonal, complementary assays for translation (RPM/RTA, metabolic labeling) are combined with polyribosome profiling and quantitative, biochemical determinations of protein and ribosome content to explore this question, primarily in the OT-I T lymphocyte model system. The authors conclude that the ratio of protein levels to ribosomes/protein synthesis capacity is insufficient to support activation-coupled T cell division and cell size expansion. The authors hint at cellular mechanisms to explain this apparent paradox, focusing on protein acquisition strategies, including emperipolesis and entosis, though these remain topic areas for future study.

The strengths of the paper include the focus on a fundamental biological question - the transcriptional/translational control mechanisms that support the rapid, dramatic cell state change that accompanies lymphocyte activation from the quiescent to activated state, the use of orthogonal approaches to validate the primary findings, and the creative proposal for how this state change is achieved.

The weakness of the work is that several cellular regulatory processes that could explain the apparent paradox are not explored, though they are accessible to experimental analysis. In the accounting narrative that the authors highlight, a thorough accounting of the cellular process inventory that could support the cell state change should be further explored before committing to the proposal, provocative as it is, that protein acquisition provides a principal mechanism for supporting lymphocyte activation cell state change.

Appraisal and Discussion:

1) Relating to the points raised above, two recent review articles explore this topic area and highlight important areas of study in RNA biology and translational control that likely contribute to the paradox noted by the authors: Choi et al. 2022,<br /> doi.org/10.4110/in.2022.22.e39 ("RNA metabolism in T lymphocytes") and Turner 2023, DOI: 10.1002/bies.202200236 ("Regulation and function of poised mRNAs in lymphocytes"). These should be cited, and the broader areas of RNA biology discussed by these authors integrated into the current manuscript.

2) The authors cite the Wolf et al. study from the Geiger lab (doi.org/10.1038/s41590-020-0714-5, ref. 41) though largely to compare determined values for ribosome number. Many other elements of the Wolf paper seem quite relevant, for example, the very high abundance of glycolytic enzymes (and whose mRNAs are quite abundant as well), where (and as others have reported) there is a dramatic activation of glycolytic flux upon T cell activation that is largely independent of transcription and translation, the evidence for "pre-existing, idle ribosomes", the changes in mRNA copy number and protein synthesis rate Spearman correlation that accompanies activation, and that the efficiencies of mRNA translation are heterogeneous. These data suggest that more accounting needs to be done to establish that there is a paradox.

As one example, what if glycolytic enzyme protein levels in the resting cell are in substantial excess of what's need to support glycolysis (likely true) and so translational upregulation can be directed to other mRNAs whose products are necessary for function of the activated cell? In this scenario the dilution of glycolytic enzyme concentration that would come with cell division would not necessarily have a functional consequence. And the idle ribosomes could be recruited to key subsets of mRNAs (transcriptionally or post-transcriptionally upregulated) and with that a substantial remodeling of the proteome (authors ref. 44). The study of Ricciardi et al. 2018 (The translational machinery of human CD4+ T cells is poised for activation and controls the switch from quiescence to metabolic remodeling (doi.org/10.1016/j.cmet.2018.08.009) is consistent with this possibility. That study, and the short reviews noted above, are useful in highlighting the contributions of selective translational remodeling and the signaling pathways that contribute to the cell state change of T cell activation. From this perspective an alternative view can be posited, where the quiescent state is biologically poised to support activation, where subsets of proteins and mRNAs are present in far higher levels than that necessary to support basal function of the quiescent lymphocyte. In such a model, the early stages of lymphocyte activation and cell division are supported by this surplus inventory, with transcriptional activation, including ribosomal genes, primarily contributing at later stages of the activation process. An obvious analogy is the developing Drosophila embryo where maternal inheritance supports early-stage development and zygotic transcriptional contributions subsequently assuming primary control (e.g. DOI 10.1002/1873-3468.13183 , DOI: 10.1126/science.abq4835). To pursue that biological logic would require quantifying individual mRNAs and their ribosome loading states, mRNA-specific elongation rates, existing individual protein levels, turnover rates of both mRNAs and proteins, ribosome levels, mean ribosome occupancy state, and how each of these parameters are altered in response to activation. Such accounting could go far to unveil the paradox. This is a considerable undertaking, though, and outside the scope of the current paper.

Regarding the revised manuscript:

I am largely satisfied with the authors responses to the review and have but a few remaining thoughts, some mirrored in the comments from the other reviewers and some that came to mind upon reading the revision.

1) In the Introduction, it would be (have been) helpful if in paragraph two, it was stated that the current study was designed to test that assumption made in prior reports that the fold-increase in protein synthesis in response to mitogen activation was sufficient to endow the daughter cells with "the same protein content as their progenitor".

2) The primary conclusion, that "...protein synthesis activity or capacity of in vivo activated T cells does not support their doubling times" remains, to my eye, insufficiently supported by the data, though I agree it is a rational interpretation. My concern is that the devil is deeper in the details and without knowing the mRNA transcriptome composition pre- and post-activation, mean CDS length, 5' UTR structural features, perhaps codon optimality, etc., etc., the broader conclusion could be premature. As a first check, it would be useful to determine poly(A) mRNA and ribosome concentrations/cell, pre- and post-activation, and subsequently to compare mRNA transcriptome compositions in greater detail. Do mRNA:ribosome levels and ratios diverge as a consequence of activation? Poly(A) mRNA compositions? Does protein half-life change pre- and post-activation? mRNA half-life? My view is that additional molecular accounting is likely necessary to be confident in the primary conclusion.

3) I did not provide a clear description of the alternative interpretation I was imagining, which is that in the resting, unstimulated state, mRNA:ribosome and/or protein levels may be much higher than that necessary for lymphocyte viability. As in early development, this could be a mechanism to then provide sufficient protein synthesis capacity and/or proteins to daughter cells following activation of cell division and cell growth. In other words, it's a dynamic range question; the daughter cells exploit "unused" protein synthesis capacity to sustain their growth and division. Quantification and analysis of the additional variables noted in point 2) could reconcile the different interpretations.

Reviewer #1 (Public Review):

The association of vitamin D supplementation in reducing Asthma risk is well studied, although the mechanistic basis for this remains unanswered. In the presented study, Kilic and co-authors aim to dissect the pathway of Vitamin D-mediated amelioration of allergic airway inflammation. They use initial leads from bioinformatic approaches, which they then associate with results from a clinical trial (VDAART) and then validate them using experimental approaches in murine models. The authors identify a role of VDR in inducing the expression of the key regulator Ikzf3, which possibly suppresses the IL-2/STAT5 axis, consequently blunting the Th2 response and mitigating allergic airway inflammation.

The major strength of the paper lies in its interdisciplinary approach, right from hypothesis generation, and linkage with clinical data, as well as in the use of extensive ex vivo experiments and in vivo approaches using knock-out mice. The study presents some interesting findings including an inducible baseline absence/minimal expression of VDR in lymphocytes, which could have physiological implications and needs to be explored in future studies.<br /> The study presents a potential for further dissection of relevant pathophysiological pathways to explain certain seemingly associative results, and allow for a more effective translation.

Several results in the study suggest multiple factors and pathways influencing the phenotype seen, which could be explored in the future. The inferences of this study also need to be read in the context of the different sub-phenotypes and endotypes of Asthma, where the Th2 response may not be predominant. While this does not undermine the importance of this elegant study, it is essential to emphasise a holistic picture while interpreting the results.

Reviewer #1 (Public Review):

The manuscript investigates the role of membrane contact sites (MCSs) and sphingolipid metabolism in regulating vacuolar morphology in the yeast Saccharomyces cerevisiae. The authors show that tricalbin (1-3) deletion leads to vacuolar fragmentation and the accumulation of the sphingolipid phytosphingosine (PHS). They propose that PHS triggers vacuole division through MCSs and the nuclear-vacuolar junction (NVJ). The study presents some solid data and proposes potential mechanisms underlying vacuolar fragmentation driven by this pathway. Although the manuscript is clear in what the data indicates and what is more hypothetical, the story would benefit from providing more conclusive evidence to support these hypothesis. Overall, the study provides valuable insights into the connection between MCSs, lipid metabolism, and vacuole dynamics.

Reviewer #1 (Public Review):

Summary:<br /> This paper addresses the mechanisms positioning microtubule asters in Drosophila explants. Taking advantage of a genetic mutant, blocking the cell cycle in early embryos, the authors generate embryos with centrosomes detached from nuclei and then study the positioning mechanisms of such asters in explants. They conclude that asters interact via pushing forces. While this is an artificial system, understanding the mechanics of asters positioning, in particular, whether forces are pushing or pulling is an important one.

Strengths:<br /> The major strength of this paper is the series of laser cutting experiments supporting that asters position via pushing forces acting both on the boundary (see below for a relevant comment) and between asters. The combination of imaging, data analysis and mathematical modeling is also powerful.

Weaknesses:<br /> This paper has overlap in the conclusions with a previous paper from the same authors, so its impact is reduced. In Figure 2, the tracking of fluid flows is hard to see and better quantifications/analyses would lead to stronger conclusions. In Figure 4, it is not clear that the acceleration is significant and no statistical test is provided or described, as far as I can tell.

Reviewer #1 (Public Review):

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

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

At the top of the results, the authors should say what species they're working with and give some background about the nature of the reduced genome. It is important to know what the changes were and especially how much of the genome was deleted. Some insights into the genes that were deleted would also be useful context for understanding the evolution experiment. This could be included in the introduction or results.

Reviewer #1 (Public Review):

Summary:

The manuscript by Hann et. al examines the role of survival motor neuron protein (SMN) in lateral plate mesoderm-derived cells using the Prrx1Cre to elucidate how changing cell-specific SMN levels coordinate aspects of the spinal muscular atrophy (SMA) pathology. SMN has generally been studied in neuronal cells, and this is one of the first insights into non-neuronal cells that may contribute to SMA disease. The authors generated 3 mouse lines: a Prrx1;Smnf/f conditional null mouse, as well as, single and double copy Prrx1;Smnf/f;SMN2 mice carrying either one or two copies of a human SMN2 transgene. First, the bone development and growth of all three were assessed; the conditional null Smn mutation was lethal shortly after birth, while the SMN2 2-copy mutant did not exhibit bone growth phenotypes. Meanwhile, single-copy SMN2 mutant mice showed reduced size and shorter limbs with shorter proliferative and hypertrophic chondrocyte zones. The authors suggested that this was cell autonomous by assessing the expression of extrinsic factors known to modulate proliferation/differentiation of growth plate chondrocytes. After assessing bone phenotypes, the authors transitioned to the assessments of neuromuscular junction (NMJ) phenotypes, since there are documented neuromuscular impairments in SMA and the Prrx1Cre transgene is expressed in muscle-associated fibro-adipogenic progenitors (FAPs). Neonatal NMJ development was unchanged in mutant mice with two copies of SMN2 , but adult single-copy SMN2 mutant mice had abnormal NMJ morphology, altered presynaptic neurotransmission, and problematic nerve terminal structure. Finally, the authors sought to assess the ability to rescue NMJ phenotypes via FAP cell transplantation and showed wild-type FAPs were able to reduce pre/postsynaptic fragmentation and neurofilament varicosities.

Strengths:

The conditional genetic approaches are novel and interestingly demonstrate the potential for chondrocyte and fibro-adipogenic progenitor-specific contributions to the SMA pathology.

The characterizations of the neuromuscular and NMJ phenotypes are relatively strong.

The data strongly suggest a non-neuronal contribution to SMA, which indicates a need for further mechanistic (cellular and molecular) studies to better understand SMA.

Weaknesses:

The skeletal analyses are not rigorous and likely do not get to the core of how SMN regulates bone development.

The overall work is descriptive and lacks convincing mechanisms.

Additional experimentation is likely needed to fully justify the conclusions.

Reviewer #1 (Public Review):

People can perform a wide variety of different tasks, and a long-standing question in cognitive neuroscience is how the properties of different tasks are represented in the brain. The authors develop an interesting task that mixes two different sources of difficulty, and find that the brain appears to represent this mixture on a continuum, in the prefrontal areas involved in resolving task difficulty. While these results are interesting and in several ways compelling, they overlap with previous findings and rely on novel statistical analyses that may require further validation.

Strengths<br /> 1. The authors present an interesting and novel task for combining the contributions of stimulus-stimulus and stimulus-response conflict. While this mixture has been measured in the multi-source interference task (MSIT), this task provides a more graded mixture between these two sources of difficulty.

2. The authors do a good job triangulating regions that encoding conflict similarity, looking for the conjunction across several different measures of conflict encoding. These conflict measures use several best-practice approaches towards estimating representational similarity.

3. The authors quantify several salient alternative hypothesis, and systematically distinguish their core results from these alternatives.

4. The question that the authors tackle is important to cognitive control, and they make a solid contribution.

Concerns<br /> 1. The framing of 'infinite possible types of conflict' feels like a strawman. While they might be true across stimuli (which may motivate a feature-based account of control), the authors explore the interpolation between two stimuli. Instead, this work provides confirmatory evidence that task difficulty is represented parametrically (e.g., consistent with literatures like n-back, multiple object tracking, and random dot motion). This parametric encoding is standard in feature-based attention, and it's not clear what the cognitive map framing is contributing.

2. The representations within DLPFC appear to treat 100% Stoop and (to a lesser extent) 100% Simon differently than mixed trials. Within mixed trials, the RDM within this region don't strongly match the predictions of the conflict similarity model. It appears that there may be a more complex relationship encoded in this region.

3. To orthogonalized their variables, the authors need to employ a complex linear mixed effects analysis, with a potential influence of implementation details (e.g., high-level interactions and inflated degrees of freedom).

Reviewer #1 (Public Review):

Summary:

Kinase inhibitors represent a highly valuable class of drugs as evidenced by their continued clinical success. The target landscape of kinase targeting small molecules can be leveraged to alter multiple phenotypes with increasing complexity that broadly aligns with increasing target promiscuity. This 'tools and resources' contribution provides a starting point for researchers interested in aligning kinase inhibitor activity with cytokine/chemokine stimulated signal transduction networks.

Strengths:

KinCytE is a forward-thinking database that yields hypothesis-generating options for researchers interested in pharmacologically modulating cytokine/chemokine signaling.

Weaknesses:

As a 'tools and resources' contribution, the primary (potential) weakness will be the authors' willingness to update and improve the tool. KinCytE will require frequent updating to better inform users in terms of contextual cytokine/chemokine stimulated signaling and the target landscape of those agents that are included as options.

Reviewer #1 (Public Review):

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

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

Weaknesses:<br /> The data on blood neutrophil count is really intriguing and seems to provide a very powerful yet easy-to-measure method to differentiate survival vs. death in TBM patients. It would be quite useful in this case to perform predictive analysis to see if neutrophil count alone, or in combination with gene signature, can predict (or better predict) mortality, as it would be far easier for clinical implementation than the RNA-based method. Moreover, genes associated with increased neutrophil activation and decreased T cell activation both have significantly higher enrichment scores in TBM (Figure 9) and in morality (Figure 8). While I understand the basis of selecting hub genes in the significant modules, they often do not represent these biological pathways (at least not directly associated in most cases). If genes were selected based on these biologically relevant pathways, would they have better predictive values?

Reviewer #1 (Public Review):

Summary:<br /> The investigators have performed a state-of-the art systematic review and meta-analysis of studies that may help to answer the research question: if administration of multiple antibiotics simultaneously prevents antibiotic resistance development in individuals. The amount of studies eligible for analysis is very low, and within that low number, there is huge variability in bug-drug combinations studied and most studies had a high risk of bias, further limiting the capability of meta-analysis to answer the research question. In addition, based on I2 values there is also huge statistical heterogeneity between outcomes of studies compared, further limiting the predictive value of meta-analysis. In fact, the only 2 studies meeting all eligibility criteria addressed the treatment of mycobacterium tuberculosis, for which the research question is hardly applicable. The authors, therefore, conclude that "our analysis could not identify any benefit or harm of using a higher or a lower number of antibiotics regarding within-patient resistance development." Apart from articulating this knowledge gap, the findings will not have consequences for patient care, but may stimulate the scientific community to better address this research question in future studies.

Strengths:<br /> The systematic and rigorous approach for the review and meta-analysis.

Weaknesses:<br /> None identified.

Reviewer #1 (Public Review):

Summary:

This manuscript by Vuong and colleagues reports a study that pooled data from 3 separate longitudinal studies that collectively spanned an observation period of over 15 years. The authors examined for correlation between viraemia measured at various days from illness onset with thrombocytopaenia and severe dengue, according to the WHO 2009 classification scheme. The motivation for this study is both to support the use of viraemia measurement as a prognostic indicator of dengue and also when an antiviral drug becomes licensed for use, to guide the selection of patients for antiviral therapy. They found that the four DENVs show differences in peak and duration of viraemia and that viraemia levels before day 5 but not those after from illness onset correlated with platelet count and plasma leakage at day 7 onwards. They concluded that the viraemia kinetics call for early measurement of viraemia levels in the early febrile phase of illness.

Strengths:

This is a unique study due to the large sample size and longitudinal viraemia measurements in the study subjects. The data addresses a gap in information in the literature, where although it has been widely indicated that viraemia levels are useful when collected early in the course of illness, this is the first time anyone has systematically examined this notion.

Weaknesses:

The study only analysed data from dengue patients in Vietnam. Moreover, the majority of these patients had DENV-1 infection; few had DENV-4 infection. The data could thus be skewed by the imbalance in the prevalence of the different types of DENV during the period of observation. The use of patient-reported time of symptom onset as a reference point for viraemia measurement is pragmatic although there is subjectivity and thus noise in the data.

Reviewer #1 (Public Review):

Summary:

The authors presented here a novel 3D fibroblast culture and transdifferentiation approach for potential meat production with GelMA hydrogel.

Strengths:

1. Reduced serum concentration for 3D chicken fibroblast culture and transdifferentiation is optimized.<br /> 2. Efficient myogenic transdifferentiation and lipogenesis as well as controlled fat deposition are achieved in the 3D GelMA.

Weaknesses:

1. While the authors stated the rationale of using fibroblasts instead of myogenic/adipogenic stem cells for meat production, the authors did not comment on the drawbacks/disadvantages of genetic engineering (e.g., forced expression of MyoD) in meat production.<br /> 2. While the authors cited one paper to state the properties and applications of GelMA hydrogel in tissue engineering and food processing, concerns/examples of the food safety with GelMA hydrogel are not discussed thoroughly.<br /> 3. In Fig. 4C, there seems no significant difference in the Vimentin expression between Fibroblast_MyoD and Myofibroblast. The conclusion of "greatly reduced in the myogenic transdifferentiated cells" is overstated.<br /> 4. The presented cell culture platform is only applied to chicken fibroblasts and should be tested in other species such as pigs and fish.

Reviewer #1 (Public Review):

In this manuscript, Manessero and colleagues argue that the prefrontal cortex (PFC), given its exquisite capability to down-regulate down-stream regions central in driving emotional responses to threat, maybe a promising target to stimulate in order to reduce aberrant fear memory responses. They aim to differ from previous studies that tested the strengthening of extinction learning, by merely focusing on the expression of threat memory without extinction learning. Given that other studies have often focused on the dorsolateral prefrontal cortex as promising target to regulate fear responses, they also ran experiments to directly compare effectiveness of targeting the mPFC and dlPFC in reducing fear memory responses. These aims are all focused on what the authors describe as "implicit memory", but they also test the effects of the interventions on "explicit memory" of the presented cues. However, in the introduction, the authors do not explicitly describe what their aim or theoretical rationale to implement these tests was. Likewise, the authors implemented generalisation stimuli (i.e., cues similar to the original CS) in the implicit memory tests, but the aim of these tests is also not explained.

In order to test their hypotheses, the authors adopt a single-cue fear conditioning paradigm where participants learned to associate an auditory cue with the occurrence of short electrical stimulation across 15 repetitions of the CS-US pairing (80% reinforcement rate). One week later, for the second session, this cue was again presented 4 times, along with 2 types of generalization stimuli, that were each also presented four times. This test session took place in another environment. Conditioned skin conductance responses were measured as index of defensive responding. In the critical condition, during 10 minutes prior to these cue presentations, repetitive Transcranial Magnetic Stimulation (rTMS) was applied to specifically target the medial PFC. Another independent group of participants completed a two-alternative forced-choice (2AFC) explicit recognition test, to inquire to what extent they could recognize whether a given tone was presented during the conditioning phase (basically a source memory task). Finally, a two-alternative forced-choice (2AFC) perceptual discrimination test was presented, to ascertain that participants could discern the different tones presented. The second session was repeated yet another week later, but without any rTMS and in the original conditioning context again, to test whether any potential fear dampening effects were retained.

The observations are quite straightforward: compared to sham and an active control group, mPFC stimulation prior to fear memory retrieval resulted in an immediate reduction of conditioned responses, a difference that was consistent across all 4 test trials. Also conditioned responses to the generalization cues were reduced upon mPFC stimulation. These effects seemed to be specific for memories, since responding to novel unconditioned cues (loud female scream) were not affected by prior mPFC stimulation. Likewise, measures of explicit memory were unaffected. In separate experiments, stimulation of the mPFC also outperformed stimulation of the dlPFC. This pattern of results was again observed during the tests a week later.

The authors conclude that, since these outcomes were observed in the absence of extinction training, the rTMS procedure directly modulated the defensive responses activated by the threat memory trace. The fact that defensive responses to novel unconditioned stimuli were not affected are in line with earlier observations that the mPFC seems critical for the expression of conditioned but not innate fear. Given that dlPFC stimulation seemed less effective, the mPFC may be the most suitable candidate for future therapeutic interventions.

Major strengths:<br /> - Earlier work delving into the involvement of the prefrontal cortex in fear regulation has not only revealed a central role for the mPFC, but also for the dlPFC. An important strength of this study is that the authors therefore also directly compare groups that are targeted in either one of these regions, thereby revealing that even though stimulating the dlPFC results in some fear reduction, the effect is much stronger for mPFC. Another nice consequence of this extra group is that the earlier observations when targeting the mPFC are being replicated.

- It is important to test novel avenues to achieve enduring fear dampening effects of interventions. An intervention that only exerts immediate but transient effects does not bring much clinical value. So the fact that this study incorporates a follow-up test and then shows that the acute fear dampening effects are retained in the absence of any TMS stimulation certainly is important.

- It is only natural to show defensive responses to cues that previously have been paired with something aversive, like a shock. For this reason, generalized fear responses to cues that are similar to fear cues but in fact innocuous is considered maladaptive, and at the core of anxiety disorders. A strength of the paper is that the authors have added generalization tests in addition to (adaptive) fear retention, to ascertain that their intervention in fact also targets maladaptive responding.

Major weaknesses<br /> - There are two major weaknesses in this paper, that can have a potentially detrimental consequence for the robustness of the results and conclusions. First of all, even though comparing the effect of mPFC stimulation with other groups that have been stimulated in other brain regions is important, another comparison - perhaps an even more essential one - is lacking: is there a significant reduction in conditioned fear responses after targeting the mPFC as compared to that group's own fear acquisition (or at least the final phase of acquisition)? Instead, the authors compare fear responses with responses PRIOR to conditioning, which is not meaningful. The same goes for the long-term follow-up: also here, a comparison with fear responding prior to the intervention is lacking. Such a reduction in conditioned fear responding should be larger than any reduction (e.g., due to habituation or forgetting) in fear responding in the (sham) control groups (i.e., an overall interaction between group and fear responding should be present). Whether this is the case is unfortunately unknown, since the fear acquisition data (neither raw, nor pre-processed) are not to be found in the manuscript, and are therefore also not included in any of the analyses. Since there is also no safe control stimulus, the crucial comparison is made entirely between-subjects, and for such a comparison groups of n~20 are quite modest.

- Second, against commons practice, the authors commence by square root transforming all SCR data to normalize the data (while this should only be done in the final phase or preprocessing, if the variables entered in the statistical tests require so), only to then again normalize these obtained values by dividing by the unconditioned responses of the participants, that then are used to calculate differences scores with preconditioning. In these descriptions it is unclear which unconditioned stimulus it was (the original one, from conditioning?) and whether it was standardised to the highest response or an average of all the responses. Decisions that are taken in these early pre-processing steps can have a gigantic impact on the outcomes and conclusions, so this is not trivial. One may say that this cannot explain the group effects that have been observed, given the fact that all groups have been pre-processed in the same way. However, the mPFC group of interest seems to display relatively high unconditioned responses - standardising with these measures may result in relatively low conditioned responses in this particular group. This shortcoming is therefore closely related to the point made above: given that conditioning data would be standardised in the same vein, a test that included the within-subject comparison between acquisition and post-intervention is absolutely crucial to ascertain that the effects observed are not merely due to coincidences in pre-processing values and pre-existing group differences.

- In addition to the above-described main analyses, some other potential weaknesses concern the analysis strategy applied to the generalization tests. Several ANOVAS are being run, one to test for the pattern of generalization responses within-subjects (i.e., the CS, NS1 and NS2), and several ones to compare each of these between the three groups. But such analyses are not warranted in the absence of an overall interaction between the within subject factors and group factor. Such overall omnibus tests however are lacking, and the high number of separate anovas risks false positives (i.e., these comparisons should have been made with planned contrasts). The fact that the included factors and levels are not being described, makes it generally hard to gauge what variables exactly have been entered in every analysis.

Further remarks:<br /> - There is a possibility that a re-analysis of the data using properly preprocessed SCR data along with analyses that include comparisons with the conditioned responses during acquisition reveal a different pattern of results. Therefore, whether the authors truly achieved their aims and whether the results support their conclusions is as of yet undecided.

- Even if the pattern of results holds, then the claim that the long-term follow-up reductions of fear were achieved in the absence of any extinction cannot be made with confidence: after all, upon mPFC stimulation during the second session, the CS was presented four times, and so were each of the two generalization stimuli. So perhaps extinction was not complete, but almost certainly some extinction has taken place: it is well-known that the strongest extinction-learning typically takes place in the first trials (e.g., due to higher prediction errors). The authors do not give any alternative theoretical explanation for the enduring reduction of fear reduction, which would be interesting to learn their thoughts on.

- If the results hold and satisfiable reasons are provided as to why the effects remain visible in the follow-up, this study could be a valuable contribution to the field: it may refocus future studies to the mPFC as major target to not only promote acute fear regulation, but perhaps even more importantly form a clinical perspective, a route for enduring fear reductions.

Reviewer #1 (Public Review):

In this manuscript, the Authors implement a delayed feedback control method and use it for the first time in biological neuronal networks. They extend a well-established computational theory and expand it into the biological realm. With this, they obtain novel evidence, never considered before, that showcases the difference between simulated neuronal networks and biological ones. Furthermore, they optimize the DFC method to achieve optimal results in the control of cell excitability in the content of biological neuronal networks, taking advantage of a closed-loop stimulation setup that, by itself, is not trivial to build and operate and that will certainly have a positive impact the fields of cellular and network electrophysiology.

Regarding the results, it would be very constructive if the Authors could share the code for the quasi-real-time interface with the Multichannel Systems software (current and older hardware versions), as this represents likely a bottleneck preventing more researchers to implement such an experimental paradigm.

On the data focusing on the effects of the DFC algorithms on neuronal behavior, the evidence is very compelling, although more care should be devoted to the statistical analyses, since some of the applied statistical tests are not appropriate. In a more biological sense, further discussion and clarification of the experimental details would improve this manuscript, making it more accessible and clearer for researchers across disciplines (i.e., ranging from computational to experimental Neuroscience) and increasing the impact of this research.

In summary, this work represents a necessary bridge between recent advances in computational neuroscience and the biological implementation of neuronal control mechanisms.

Reviewer #1 (Public Review):

The manuscript by Grove and colleagues analyzes the role of TEAD1 transcription factors in all events regulating PNS myelin formation and maintenance and regeneration. Throughout the manuscript, the authors compare the results obtained to those they previously described in YAP/TAZ double knockout mice. Strengths of the manuscript are combined in vivo analyses by generating mutants constitutively lacking TEAD1 expression in myelinating Schwann cells (P0Cre//TEAD1f/f mice: cKO) and mutants in which TEAD1 expression can be ablated after tamoxifen-mediated recombination is myelinating Schwann cells (PlpCreER//TEAD1f/f mice: iKO). Using this approach the authors were able to assess the role of TEAD1 in all aspects related to PNS myelin: formation as well as maintenance and remyelination after injury. By exploiting these models, they were able to define the role of TEAD1 in regulating Schwann cell proliferation as well as in the cholesterol biosynthetic pathway.

Collectively, their data indicate that TEAD 1 has a composite role in PNS myelination being required for developmental myelination, but dispensable for myelin maintenance. Further, they also describe a role for TEAD1 in promoting PNS remyelination after an injury event.

Despite these strengths, there are some weaknesses that should be addressed by the authors:

1. The manuscript would benefit from better and more detailed analysis of the role of the other TEAD transcription factors, as they are likely redundant in function to TEAD1. For example, since in cKO mice some fibers can escape the sorting defect and eventually myelinate, albeit at a lower level, could they determine whether TEAD2-4 transcription factors might compensate for TEAD1 absence in this setting?

2. A striking result of the study is the morphological defects observed in the process of axonal sorting and in the Remak fibers formation of TEAD1 cKO mice. To explain the sorting defect, the authors correctly analyze Schwann cell proliferation. However, since axonal sorting is mediated by the interaction between the extracellular matrix and intracellular cytoskeleton rearrangement they should address also these two aspects. As per the Remak bundles and the poly-axonal myelination they observe, it is difficult to reconcile this "abnormal" myelination with the fact that TEAD 1 cKO mice have a very severe myelinating phenotype, which is persistent in adulthood.

3. In the analyses of the cholesterol biosynthetic pathway, TEAD1 seems to be only partly involved. Again, which is the role of any of the other TEADs?

4. Why do cKO mice die before P60?

Reviewer #2 (Public Review):

In this paper, the authors discover that postsynaptic mitochondria in C. elegans govern glutamate receptor trafficking dynamics. The core results are two-fold. For one, they find that loss or inhibition of mcu-1 - the C. elegans mitochondrial calcium uniporter - increases GLR-1 glutamate receptor accumulation at the postsynaptic dendritic sites and enhances its trafficking dynamics. The authors hypothesize that this effect on glutamate receptors may have something to do with mitochondrial ROS production. This is because ROS is a by-product of normal oxidative phosphorylation, downstream of calcium import. Indeed, the generation of artificially high amounts of mitochondrial ROS has the opposite effect of mcu-1 loss: decreased glutamate receptor subunit accumulation. Collectively, the results support the idea that mitochondrial function can control receptor dynamics at synaptic sites. This is interesting because tight control of synaptic function likely combines several mitochondrial functions: energy production, calcium buffering, and (here) ROS signaling.

STRENGTHS

• The C. elegans genetic model is a strength because the authors are able to make refined conclusions by classical loss-of-function mutants (e.g., mcu-1) along with an impressive cytological toolkit to examine GLR-1 dynamics.

• The use of pharmacology as a second means to test those genetic conclusions is a strength.

• The authors' careful reagent verification of reporters (Ca2+, ROS, etc.) is a strength.

• The ability to link fundamental mitochondrial processes to GLR-1 exocytosis will expand how the field thinks about mitochondrial synapse function.

WEAKNESSES

For the most part, the data in the paper support the conclusions, and the authors were careful to try experiments in multiple ways. But please see below:

• (Main Point) The data are good, but they fall short of mechanism (e.g., Line 322). Figure 6 is accurate as drawn. But calcium and ROS are not abstract signals. They are likely exerting affirmative actions on specific targets. The Discussion does acknowledge this in terms of ROS and it speculates on possible targets.

The general idea seems to be that mitochondria import calcium through MCU-1 (and interacting factors). As a result, oxidative phosphorylation successfully occurs and mitochondrial ROS is a signaling by-product that signals glutamate receptors not to undergo exocytosis. But there are other interpretations of what might happen in between. In fact, if OXPHOS is disrupted, it is known that this can generate a lot more mitochondrial ROS than the normal by-product levels.

This reviewer wonders if excess ROS would cause an extreme response. Or alternatively, if scavenging ROS via pharmacological scavengers or SOD expression would reverse the effects.

Small Points

• 33.3 mHz - just making sure, do the authors mean once every 30 seconds? That would be more straightforward.

• Figure 2 is confusing. The text says that the mcu-1 mutants have a GLR-1::GFP FRAP rate that is comparable to controls (Lines 165-167). But Figure 2E suggests that it is markedly less, which is the opposite result of the slight increase in rate resulting from Ru360 treatment. And is the explanation why the GLR-1::GFP results differ from the SEP::GLR-1 results a difference between total GFP vs. surface GFP?

• I could not watch Video 2 (not sure if it is the file or just the copy I downloaded).

• It is good that the authors tried both optical stimulation and mechanical stimulation (dropping culture plates to stimulate the worms, Figure 3). Why was the mechanical stimulation set aside for further tests in the paper?

• Does this process affect all kinds of transport, or is it just the glutamate receptors? Was anything else examined?

Reviewer #1 (Public Review):

Payne et al. have investigated the neural basis of VOR adaptation with the goal of constraining sites and mechanisms of plasticity supporting cerebellar learning. This has been an area of intense debate for decades; previous competing models have argued extensively about the sites of plasticity and the strength of eye velocity feedback/ efference copy signals to Purkinje cells has been central to the debate. This paper nicely explores the consequences of varying the strength of this feedback and in so doing, provides a potential explanation for why Purkinje cell responses during VOR cancellation could exhibit stronger responses following learning, despite net depression of the strength of their vestibular inputs. In that sense it provides some reconciliation of existing models. The work appears to be well done and the paper is well written. The manuscript could be improved and the significance of the work clarified and enhanced by contextualizing the work more appropriately within the existing literature in this area.

Reviewer #1 (Public Review):

This interesting study by Miyano combines slice electrophysiology and superresolution microscopy to address the role of RBP2 in Ca2+ channel clustering and neurotransmitter release at hippocampal mossy fiber terminals. While a number of studies demonstrated a critical role for RBPs in clustering Ca2+ channels at other synapses and some provided evidence for a role of the protein in molecular coupling of Ca2+ channels and release sites, the present study targets another key synapse that is an important model for presynaptic studies and offers access to a microdomain controlled synaptic vesicle (SV) release mechanism with low initial release probability.

Summarizing a large body of high quality work, the authors demonstrate reduced Ca2+ currents and a reduced release probability. They attribute the latter to the reduced Ca2+ influx and can restore release by increasing Ca2+ influx. Moreover they propose an altered fusion competence of the SVs, which is not so strongly supported by the data in my view.

The effects are relatively small, but I think the careful analysis of the RBP role at the mossy fiber synapse is an important contribution.

Share an example of a technology you frequently use and share some of its advantages and disadvantages.

Joint Public Review:

In this manuscript, the authors examined the role of transcription readout and intron retention in increasing transcription of transposable elements during aging in mammals. It is assumed that most transposable elements have lost the regulatory elements necessary for transcription activation. Using available RNA-seq datasets, the authors showed that an increase in intron retention and readthrough transcription during aging contributes to an increase in the number of transcripts containing transposable elements.

Previously, it was assumed that the activation of transposable elements during aging is a consequence of a gradual imbalance of transcriptional repression and a decrease in the functionality of heterochromatin (de repression of transcription in heterochromatin). Therefore, this is an interesting study with important novel conclusion.

The authors revised the manuscript in accordance with the comments. Overall, the manuscript is useful because it shows that there is no direct connection between increased levels of transposon RNA and aging, and further demonstrates the disorganization of the transcriptional apparatus during aging.

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #1 (Public Review):

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

Strengths:

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

Novelty: The development of two novel active learning batch selection methods is a commendable contribution. The study also adds value by curating new affinity datasets that provide chronological information on state-of-the-art experimental strategies.<br /> Comprehensive Evaluation: Testing the proposed methods on multiple public datasets with varying optimization goals and sizes enhances the credibility and generalizability of the findings. The focus on comparing the performance of the new methods against existing batch selection methods further strengthens the evaluation.

Weaknesses:

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

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

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

Suggestions for Improvement:

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

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

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

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

Reviewer #2 (Public Review):

Summary:

This paper tests the idea that schooling can provide an energetic advantage over solitary swimming. The present study measures oxygen consumption over a wide range of speeds, to determine the differences in aerobic and anaerobic cost of swimming, providing a potentially valuable addition to the literature related to the advantages of group living.

Strengths:<br /> The strength of this paper is related to providing direct measurements of the energetics (oxygen consumption) of fish while swimming in a group vs solitary. The energetic advantages of schooling has been claimed to be one of the major advantages of schooling and therefore a direct energetic assessment is a useful result.

Weaknesses:

1) Regarding the fish to water volume ratio, the arguments raised by the authors are valid. However, the ratio used is still quite high (as high as >2000 in solitary fish), much higher than that recommended by Svendsen et al (2006). Hence this point needs to be discussed in the ms (summarising the points raised in the authors' response)

2) Wall effects: Fish in a school may have been swimming closer to the wall. The fact that the convex hull volume of the fish school did not change as speed increased is not a demonstration that fish were not closer to the wall, nor is it a demonstration that wall effect were not present. Therefore the issue of potential wall effects is a weakness of this paper.

3) The authors stated "Because we took high-speed videos simultaneously with the respirometry measurements, we can state unequivocally that individual fish within the school did not swim closer to the walls than solitary fish over the testing period". This is however not quantified.

4) Statistical analysis. The authors have dealt satisfactorily with most of the comments.<br /> However :<br /> (a) the following comment has not been dealt with directly in the ms "One can see from the graphs that schooling MO2 tends to have a smaller SD than solitary data. This may well be due to the fact that schooling data are based on 5 points (five schools) and each point is the result of the MO2 of five fish, thereby reducing the variability compared to solitary fish."<br /> (b) Different sizes were used for solitary and schooling fishes. The authors justify using larger fish as solitary to provide a better ratio of respirometer volume to fish volume in the tests on individual fish. However, mass scaling for tail beat frequency was not provided. Although (1) this is because of lack of data for this species and (2) using scaling exponent of distant species would introduce errors of unknown magnitude, this is still a weakness of the paper that needs to be acknowledged here and in the ms.

Reviewer #1 (Public Review):

Summary:<br /> The authors describe their work on finding optimal ways of infusing organoids into mice. They describe five delivery methods and compare organoid survival two weeks after delivery. This work is concluded with the use of a vascularized chamber being the most optimal for organoid viability.

Strengths:<br /> The aim is to have a preclinical, translational model to test methods of organoid infusion. This is important and timely to the field.

Weaknesses:<br /> - A clear aim seems to be missing, although I can extract this from the manuscript. The approach is described a bit cryptically. The manuscript could use a bit more explanation here and there.<br /> - Although the authors themselves argue in the introduction that the use of mice is not optimal, they show a mouse study in which human-derived iPSC organoids are infused in mice.<br /> - As far as I can extract from the Methods section, only one iPSC line was used. Given the huge donor variance, it is essential to repeat the work with multiple iPSC lines.<br /> - I am missing the right control groups, especially for the surgical groups. And the group size is very variable (3 to 7 mice per group). Three per group is then somewhat small in size.

Reviewer #1 (Public Review):

This is a very well-written and performed study describing a TOPBP1 separation of function mutation, resulting in defective MSCI maintenance but normal sex body formation. The phenotype differs from that of a previous TOPBP1 null allele, in which both MSCI and sex body formation were defective. Additional defects in CHK phosphorylation and SETX localization are also described.

Strengths:

The study is very rigorous, with a remarkably large number spectrum of techniques deployed to support the conclusions

Weaknesses

The study claims that MSCI is initiated but not maintained in the mutant. I think alternative hypotheses are possible.

Reviewer #1 (Public Review):

Summary:

In this study, the authors developed a mathematical model to predict human biological ages using physiological traits. This model provides a way to identify environmental and genetic factors that impact aging and lifespan.

Strengths:

1. The topic addressed by the authors - human age predication using physiological traits - is an extremely interesting, important, and challenging question in the aging field. One of the biggest challenges is the lack of well-controlled data from a large number of humans. However, the authors took this challenge and tried their best to extract useful information from available data.

2. Some of the findings can provide valuable guidelines for future experimental design for human and animal studies. For example, it was found that this mathematical model can best predict age when all different organ and physiological systems are sampled. This finding makes sense in general but can be, and has been, neglected when people use molecular markers to predict age. Most of those studies have used only one molecular trait or different traits from one tissue.

Weaknesses:

1. As I mentioned above, the Biobank data used here are not designed for this current study, so there are many limitations for model development using these data, e.g., missing data points and irrelevant measurements for aging. This is a common caveat for human studies and has been discussed by the authors.

2. There is no validation dataset to verify the proposed model. The authors suggested that human biological age can be predicted with high accuracy using 12 simple physiological measurements. It will be super useful and convincing if another biobank dataset containing those 12 traits can be applied to the current model.

Reviewer #1 (Public Review):

The manuscript by Kadkova et al. describes an electrophysiological analysis of 3 neurodevelopmental disease-causing SNAP-25 mutations in hippocampal neuron autaptic cultures. The work expands on a prior study of these 3 mutations, along with several others in SNAP-25, that was performed in acutely dissociated hippocampal cultures by another group (Alten et al, 2021). Most of the physiology defects found are pretty similar for the 3 mutations the two research groups characterized, with differences largely found in the effects on the size of the readily releasable pool (RRP) of SVs. These differences could be due to technical differences in the approach but are also likely to reflect in part differences in autapses as a model that have been previously described. In addition to the physiological analysis in cultured neurons, the current work extends the analysis beyond the prior study by analyzing the effects of these SNAP-25 mutations in in vitro liposome fusion assays with purified proteins, and some modeling of the effects on energy landscapes during priming and fusion.

The authors use lentiviral expression of wildtype or one of the 3 mutants in SNAP-25 autaptic neurons and assay neuronal survival and synaptic output. The authors also combine wildtype with each of the 3 mutants as well, given these diseases manifest as spontaneous mutations in only 1 of the SNAP-25 alleles, suggesting a dominant effect. The authors observe that the V48F and D166Y alleles (that are suggested to disrupt the Syt1-SNAP-25/SNARE interface) result in a very large increase in spontaneous release that exceeds the Syt1 null mutant alone, suggesting an effect on spontaneous SV release beyond a lack of Syt1 regulation of SNARE-mediated fusion. In contrast, Syt1 nulls have a much more severe loss of evoked release, through both V48F and D166Y also have modest decreases in release. They find both mutants also cause a decrease in the RRP. Applying some modeling for these results, the authors suggest V48F and D166Y lowers the energy barrier for fusion, creating the enhanced spontaneous release rates and causing a decrease of the RRP. They also find evidence for reduced SV priming. In contrast, a SNAP-25 I167N disease mutation in the SNARE assembly interaction layer causes dramatic decreases in both evoked and spontaneous release, consistent with a disruption to SNARE assembly/stability. In vitro fusion assays with these mutant SNAP-25 alleles was also done and provided supportive evidence for these interpretations for all 3 alleles. The ability to control calcium, Syt1, PIP2 and Complexin levels in the in vitro assays provided additional information on defining the precise steps of the fusion process these mutations disrupt. Together, the study indicates the I167N mutation acts as a dominant-negative allele to block fusion, while the other two alleles have both loss- and gain-of function properties that cause more complex disruptions that decrease evoked release while dramatically enhancing spontaneous fusion.

Overall, these results build on prior work and shed light on how disruptions to the SNAP-25 t-SNARE alter the process of SV priming and fusion.

Reviewer #1 (Public Review):

Summary:<br /> Moon et al analyse ECoG data obtained during speech listening and focus on the relationship of two aspects: 1) delays between voltage signals at individual electrodes to other electrodes in the vicinity and 2) the power of those signals in a range of spectral bands. They find that high power in frequencies below 30 Hz is correlated with longer delays. They further look for this pattern of results in an oscillator model.

Strengths:<br /> The manuscript examines whether a finding made in cats in the late 90s generalises to intracranial recordings from humans. Specifically, the amplitude of low-frequency oscillations should be related to the delay of cross-correlation between areas. The authors find evidence for such a relationship and show this in individual participants. After inspecting this phenomenon from many different angles, they also added an oscillator model and claimed that they found a similar pattern there. As such, the manuscript reports an extensive body of work carried out on high-quality data.

Weaknesses:<br /> The manuscript's readability and flow could be optimised: terms are used that aren't explained, and the structure seems somewhat convoluted. Showing single-subject results is laudable, however, the authors could consider adding group results that integrate across participants, and perhaps relaying single-participant plots to the supplemental material. The manuscript would benefit if analyses were motivated more clearly. Sometimes, I am unsure why a given analysis was carried out, why it was carried out in a specific way, and what question it was intended to answer.

Reviewer #1 (Public Review):

Summary:<br /> In this important work, the authors propose and test a model for the control of murine ultrasonic vocalizations (USV) in which two independent mechanisms, involving changes in laryngeal opening or airflow, control vocal tone. They present compelling experimental evidence for this dual control model by demonstrating the ability of freely behaving adult mice to generate vocalizations with various intonations by modulating both the breathing pattern and the laryngeal muscles. They also present novel evidence that these mechanisms are encoded in the brainstem vocalization central neural pattern generator, particularly in the component in the medulla called the intermediate reticular oscillator (iRO). The results presented clearly advance understanding of the developmental nature of the iRO, its ability to intrinsically generate and control many of the dynamic features of USV including those related to intonation, and its coordination with/control of expiratory airflow patterns. This work will interest neuroscientists investigating the neural generation and control of vocalization, breathing, and more generally, neuromotor control mechanisms.

Strengths:<br /> Important features and novelty of this work include:

1) The study employs an effective combination of anatomical, molecular, and functional/ behavioral approaches to examine the hypothesis and provide novel data indicating that variations in expiratory airflow can change the pitch patterns of adult murine USV.

2) The results significantly extend the authors' previous work that identified the iRO in neonatal mice by now presenting data that functionally demonstrates the existence of the critical Penk+Vglut2+ iRO neurons in adult mice, indicating that the iRO neurons maintain their function in generating vocalization throughout development.

3) The results convincingly demonstrate that the iRO neurons encode and can generate vocalizations by modulating both breathing and the laryngeal muscles.

4) The anatomical mapping and tracing results establish an important set of input and output circuit connections to the iRO, including input from the vocalization-promoting subregions of the midbrain periaqueductal gray (PAG), as well as output axonal projections to laryngeal motoneurons, and to the respiratory rhythm generator in the preBötzinger complex.

5) These studies advance the important concept that the brainstem vocalization pattern generator integrates with the medullary respiratory pattern generator to control expiratory airflow as a key mechanism to produce various USV types characterized by different pitch patterns.

Weaknesses:<br /> A limitation is that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow have not been fully worked out, requiring future studies.

Reviewer #1 (Public Review):

Summary:<br /> This work studies spatiotemporal patterns of structure-function coupling in developing brains, using a large set of imaging data acquired from children and young adults aged 5-22. Magnetic resonance imaging data of brain structure and function were obtained from a publicly available database, from which structural and functional features and measures were derived. The authors examined the spatial patterns of structure-function coupling and how they evolve with brain development. This work further examined correlations between brain structure-function coupling and behaviour, and explored evolutionary, microarchitectural and genetic bases that could potentially account for the observed patterns.

Strengths:<br /> The strength of this work is the use of currently available state-of-the-art analysis methods, along with a large set of high-quality imaging data, and comprehensive examination of structure-function coupling in developing brains. The results are comprehensive and illuminative.

Weakness:<br /> As in most other studies, transcriptomic and cellular architectures of structure-function coupling were characterized only on the basis of a common atlas in this work.

The authors have achieved their aims in this study, and the findings provide mechanistic insights into brain development, which could inspire further basic and clinical studies along this line.

Reviewer #1 (Public Review):

He et al. investigate the requirement and function of Blimp1 (encoded by Prdm1) in murine NK cells and ILC1. Employing a conditional knockout mouse model (Prdm1flox x Ncr1cre), the authors describe impaired abundance and maturation of Prdm1-deficient NK cells and ILC1 in different tissues. Blimp1-deficient NK cells have reduced expression of cytotoxic molecules (Gzmb, Prf1) and, in some instances, Ifng production, and Prdm1flox x Ncr1cre mice show impaired tumor control in experimental metastasis models. Using single-cell RNA sequencing analysis, the authors propose that Prdm1 regulates JunB expression and NK cell maturation. Based on in silico analyses, the authors suggest manifold intercellular communication between NK/ILC1 and macrophages. Without following up on any of these potentially interesting suggestions, the authors conclude their study reiterating that Prdm1 regulates IFNg-production of tumor-infiltrating NK cells and ILC1.

Many of the reported functions of Blimp1 in NK cells have previously been identified using a mixed-chimera strategy comparing Prdm1 WT and KO NK cells (Kallies et al., Blood 2011). Here, the authors expand on these findings using a conditional model to delete Prdm1 in NK/ILC1 and single-cell sequencing and provide a more refined analysis of the functions of Blimp1 in these cells. Cell-chat analysis suggests close interactions of Blimp-dependent NK/ILC1 subsets with hepatic macrophages, but these suggestions are not followed up by experiments. Potentially interesting differences in the macrophage compartment of Ncr1-Cre x Prdm1-fl/fl mice are suggested by the scc-RNA-Seq data but are not validated e.g. by FACS. The study falls short in providing new mechanistic insights. Nevertheless, it is an interesting confirmation of "old" suggestions in a more refined setting, and the provided single-cell mRNA-Seq data represents a potentially valuable resource for the community. There are some control analyses that are required to support the conclusions of the authors, and I have a few suggestions that would help to improve the manuscript.

Major comments:

- The authors do not control for the potential effects of Cre expression. Expression of Cre from within the Ncr1 locus (using the mouse model established by Narni-Mancinelli et al.) has significant effects on NK cells and especially ILC1s (reducing their frequency and absolute numbers and altering their functionality. The authors should characterize the Ncr1cre mice used here (developed by Shanghai Model Organism Center) in this regard and should use proper controls (Ncr1Cre+ Prdm1wt/wt as control for Ncr1Cre+ Prdm1fl/fl, instead of WT littermates) for all of their key data, e.g. those depicted in Fig 1FG, 2ADFH, 7D, S2,3,4.

- Several of the phenotypic findings on NK cells have been described before by Kallies et al. in 2011 (Ref 29), although using a different genetic Prdm1-ablation model (Prdm1-GFP/GFP knockin/knockout model). This study reported impaired NK cell maturation, reduced Gzmb expression, impaired in vivo cytotoxicity against subcutaneous RMA-S cells, impaired in vitro proliferation, comparable in vitro killing, increase in BM NK cell numbers. The authors should discuss/mention this more prominently in their manuscript, and highlight where they confirm or refine these previous findings, and where they actually provide new information.

- What is the reason to refer to the enriched cluster in Blimp1-deficient NK cells as "Junbhi"? There is no follow-up for a function of Junb, and there are many other genes upregulated in these cells. Most critically, these cells seem to represent exactly the c-Kithi cells that Kallies et al. already showed and discussed in their paper. The authors should stain for Kit, and also refer to this. Also, MacKay et al. performed Blimp1-Chip-Seq (in T cells), maybe it would be interesting to check to which of the identified DEGs Blimp1 can bind.

- cNK cells are considered circulating cells, that transiently pass through the liver. Previous studies have suggested almost identical gene expression patterns in hepatic and splenic NK cells. In functional tests, they often "perform" identically. I am therefore a bit surprised that the authors find a differential dependency of Blimp1 for the IFNg production of splenic (no role of Blimp1) versus hepatic (Blimp1 regulating IFNg production) NK cells (Fig S3). Do the authors have any suggestions on that? The analyses are performed by 12+4h stimulations with IL12/18, which could involve the effects of altered bystander cells (as suggested by Figure 6). Therefore, these analyses should be provided upon standard 4h stimulations with IL12/18 and also with PMA/I under BFA. Note: liver and splenic cNK cells look quite different in the chosen histograms in Figures 7 A, B, C, yet there is massive variability in these analyses - is there any systematic/technical problem?

- Figure 4 H/I - In contrast to NK cells in Fig 4E, F, the KO and WT ILC1s seem to co-cluster largely. Authors should validate differentially expressed genes. How strong is the effect of Blimp1 in ILC1s? Or is Blimp1 a critical TF driving effector differentiation in NK cells, while it has only subtle effects in ILC1 (these may be regulated by Hobit?)? This seems an interesting finding that should at least be discussed. For these types of small differences in ILC1, FACS confirmation analyses should be performed and findings be reevaluated using Cre-expressing controls (see above).

The authors describe and discuss some of Figure 1 and 2 data as if Blimp1 would be involved in alternative NK versus ILC1 fates, but there is no evidence for this.

- There are several recent studies suggesting a role for Hobit, homologue of Blimp1, in NK cells and in ILC1, and in the control of liver metastases. The authors should discuss similar and unique functions of Hobit and Blimp1, also in the regulation of gene expression patterns, and should refer to these studies.

- Figure 4: The authors should discuss (and cross-validate) their liver gene expression analyses in the context of published datasets of NK and ILC1, such as the ones by Lopez et al, Friedrich et al, Ducimetiere et al and Yomogida et al.

Reviewer #1 (Public Review):

Summary:<br /> Functionally important alternative isoforms are gold nuggets found in a swamp of errors produced by the splicing machinery.

The architecture of eukaryotic genomes, when compared with prokaryotes, is characterised by a preponderance of introns. These elements, which are still present within transcripts, are rapidly removed during the splicing of messenger RNA (mRNA), thus not contributing to the final protein. The extreme rarity of introns in prokaryotes, and the elimination of these introns from mRNAs before translation into protein, raises questions about the function of introns in genomes. One explanation comes from functional biology: introns are thought to be involved in post-transcriptional regulation and in the production of translational variants. The latter function is possible when the positions of the edges of the spliced intron vary. While some light has been shed on specific examples of the functional role of alternative splicing, to what extent are they representative of all introns in metazoans?

In this study, the hypothesis of a functional role for alternative splicing, and therefore to a certain extent for introns, is evaluated against another explanation coming from evolutionary biology: isoforms are above all errors of imprecision by the molecular machinery at work during splicing. This hypothesis is based on a principle established by Motoo Mikura, which has become central to population genetics, explaining that the evolutionary trajectory of a mutation with a given effect is intimately linked to the effective population size (Ne) where this mutation emerges. Thus, the probability of fixation of a weakly deleterious mutation increases when Ne decreases, and the probability of fixation of a weakly advantageous mutation increases when Ne increases. The genomes of populations with low Ne are therefore expected to accumulate more weakly deleterious mutations and fewer weakly advantageous mutations than populations with high Ne. In this framework, if splicing errors have only small effects on the fitness of individuals, then natural selection cannot increase the precision of the splicing machinery, allowing tolerance for the production of alternative isoforms.

In the past, the debate opposed one-off observations of effectively functional isoforms on the one hand, to global genomic quantities describing patterns without the possibility of interpreting them in detail. The authors here propose an elegant quantitative approach in line with the expected continuous variation in the effectiveness of selection, both between species and within genomes. The result describing the inter-specific pattern on a large scale confirms what was already known (there is a negative relationship between effective size and average alternative splicing rate). The essential novelty of this study lies in 1) the quantification, for each intron studied, of the relative abundance of each isoform, and 2) the analysis of a relationship between this abundance and the evolutionary constraints acting on these isoforms.

What is striking is the light shed on the general very low abundance of alternative isoforms. Depending on the species, 60% to 96% of cases of alternatively spliced introns lead to an isoform whose abundance is less than 5% of the total variants for a given intron.

In addition to the fact that 60%-96% of the total isoforms are more than 20 times less abundant than their majority form, this large proportion of alternative isoforms exhibit coding-phase shift at rates similar to what would be expected by chance, i.e. for a third of them, which reinforces the idea that there is no particular constraint on these isoforms.

The remaining 4%-40% of isoforms see their coding-phase shift rate decrease as their relative abundance increases. This result represents a major step forward in our understanding of alternative splicing and makes it possible to establish a quantitative model directly linking the relative abundance of an isoform with a putative functional role concerning only those isoforms produced in abundance. Only the (rare) isoforms which are abundantly produced are thought to be involved in a biological function.

Within the same genome, the authors show that only highly expressed genes, i.e. those that tend to be more constrained on average, are also the genes with the lowest alternative splicing rates on average.

The comparison between species in this study reveals that the smaller the effective size of a species, the more its genome produces isoforms that are low in abundance and low in constraint. Conversely, species with a large effective size relatively reduce rare isoforms, and increase stress on abundant isoforms.

To sum up:<br /> • the higher the effective size of a species, the fewer introns are spliced.<br /> • highly expressed genes are spliced less.<br /> • when splicing occurs, it is mainly to produce low-abundance isoforms.<br /> • low-abundance isoforms are also less constrained.

Taken together, these results reinforce a quantitative view of the evolution of alternative splicing as being mainly the product of imprecision in the splicing machinery, generating a great deal of molecular noise. Then, out of all this noise, a few functional gold nuggets can sometimes emerge. From the point of view of the reviewer, the evolutionary dynamics of genomes are depressing. The small effective population sizes are responsible for the accumulation of multiple slightly deleterious introns. Admittedly, metazoan genomes try to get rid of these introns during RNA maturation, but this mechanism is itself rendered imprecise by population sizes.

Strengths:<br /> • The authors simultaneously study the effects of effective population size, isoform abundance, and gene expression levels on the evolutionary constraints acting on isoforms. Within this framework, they clearly show that an isoform becomes functionally important only under certain rare conditions.<br /> • The authors rule out an effect putatively linked to variations in expression between different organs which could have biased comparisons between different species.

Weaknesses:<br /> • While the longevity of organisms as a measure of effective size seems to work overall, it may not be relevant for discriminating within a clade. For example, within Hymenoptera, we might expect them to have the same overall longevity, but that effective size would be influenced more by the degree of sociality: solitary bees/ants/wasps versus eusocial. I am therefore certain that the relationship shown in Figure 4D is currently not significant because the measure of effective size is not relevant for Hymenoptera. The article would have been even more convincing by contrasting the rates of alternative splicing between solitary versus social hymenopterans.<br /> • When functionalist biologists emphasise the role of the complexity of living things, I'm not sure they're thinking of the comparison between "drosophila" and "homo sapiens", but rather of a broader evolutionary scale. Which gives the impression of an exaggeration of the debate in the introduction.

Reviewer #1 (Public Review):

Summary:

This important work advances our understanding of sperm motility regulation during fertilization by uncovering the midpiece/mitochondria contraction associated with motility cessation and structural changes in the midpiece actin network as its mode of action. The evidence supporting the conclusion is solid, with rigorous live cell imaging using state-of-the-art microscopy, although more functional analysis of the midpiece/mitochondria contraction would have further strengthened the study. The work will be of broad interest to cell biologists working on the cytoskeleton, mitochondria, cell fusion, and fertilization.

Strengths:

The authors demonstrate that structural changes in the flagellar midpiece F-actin network are concomitant to midpiece/mitochondrial contraction and motility arrest during sperm-egg fusion by rigorous live cell imaging using state-of-art microscopy.

Weaknesses:

Many interesting observations are listed as correlated or in time series but do not necessarily demonstrate the causality and it remains to be further tested whether the sperm undergoing midpiece contraction are those that fertilize or those that are not selected. Further elaboration of the function of the midpiece contraction associated with motility cessation (a major key discovery of the manuscript) would benefit from a more mechanistic study.

Reviewer #1 (Public Review):

Summary:

Numerous pathways have been proposed to elucidate the nongenomic actions of progesterone within both male and female reproductive tissues. The authors employed the Xenopus oocyte system to investigate the PLA2 activity of ABHD2 and the downstream lipid mediators in conjunction with mPRb and P4, on their significance in meiosis. The research has been conducted extensively and is presented clearly.

Strengths:

While the interaction between membranous PR and ABHD2 is not a novel concept, this present study exhibits several strengths:

1. mPRbeta, a member of the PAQR family, has been elusive in terms of detailed signal transduction. Through mutation studies involving the Zn binding domain, the authors discovered that the hydrolase activity of mPRbeta is not essential for meiosis and oocyte maturation. Instead, they suggest that ABHD2, acting as a coreceptor of mPRbeta, demonstrates phospholipase activity, indicating that downstream lipid mediators may play a dominant role when stimulated by progesterone.

2. Extensive exploration of downstream signaling pathways and the identification of several potential meiotic activity-related lipid mediators make this aspect of the study novel and potentially significant.

Weaknesses:

However, there are some weaknesses and areas that need further clarification:

1. The mechanism governing the molecular assembly of mPRbeta and ABHD2 remains unclear. Are they constitutively associated or is their association ligand-dependent? Does P4 bind not only to mPRbeta but also to ABHD2, as indicated in Figure 6J? In the latter case, the reviewer suggests that the authors conduct a binding experiment using labeled P4 with ABHD2 to confirm this interaction and assess any potential positive or negative cooperativity with a partner receptor.

2. The authors have diligently determined the metabolite profile using numerous egg cells. However, the interpretation of the results appears incomplete, and inconsistencies were noted between Figure 2B and Supplementary Figure 2C. Furthermore, PGE2 and D2 serve distinct roles and have different elution patterns by LC-MS/MS, thus requiring separate measurements. In addition, the extremely short half-life of PGI2 necessitates the measurement of its stable metabolite, 6-keto-PGF1a, instead. The authors also need to clarify why they measured PGF1a but not PGF2a.

3. Although they propose PGs, LPA, and S1P are important downstream mediators, the exact roles of the identified lipid mediators have not been clearly demonstrated, as receptor expression and activation were not demonstrated. While the authors showed S1PR3 expression and its importance by genetic manipulation, there was no observed change in S1P levels following P4 treatment (Supplementary Figure 2D). It is essential to identify which receptors (subtypes) are expressed and how downstream signaling pathways (PKA, Ca, MAPK, etc.) relate to oocyte phenotypes.

These clarifications and further experiments would enhance the overall impact and comprehensiveness of the study.

Reviewer #1 (Public Review):

Summary:

In this study, Yue et al. re-processed publicly available DNA methylation data (published in 2012 and 2017 from the Meissner lab) from pre- and post-implantation mouse embryos. Against the global wave of genome-wide reduction of DNA methylation occurring during pre-implantation development, they detected a slight increase (~1% on average) of DNA methylation at gene promoter regions during the transition from 8-cell to blastocyst stage. They claim that many such promoters are located in the X chromosome. Subsequently, they knocked down Dnmt3b (presumably because of its upregulation during the transition from the 8-cell to blastocyst stage) and detected the aberrant patterning of H3K27me3 in the mutant female embryos. Based on this observation, they claim that imprinted X-chromosome inactivation is impaired in the Dnmt3b-Kd pre-implantation embryos. Finally, they propose a model where such an increase of DNA methylation together with H3K27me3 regulates imprinted X-chromosome inactivation in the pre-implantation embryos. While their observation is of potential interest, the current version of the work fails to provide enough evidence to support their conclusions. Below are suggestions and comments on the manuscript.

Major issues:

1. Sex of the embryos of the genome-wide bisulfite-sequencing data<br /> The authors re-analyzed publicly available genome-wide DNA methylation data from the Meissner lab published in 2012 and 2017. The former used reduced representation bisulfite sequencing (RRBS) and the latter used whole-genome bisulfite sequencing (WGBS). Based mainly on the RRBS data, Yue et al. detected de novo DNA methylated promoters during the transition from 8-cell to blastocyst against the global wave of genome-wide DNA demethylation. They claim that such promoter regions are enriched at the "inactive" X chromosome. However, it would be difficult to discuss DNA methylation at inactive X-chromosomes as the RRBS data were derived from a mixture of male and female embryos. It would also be notable that the increase of DNA methylation at these promoter regions is ~1% on average. Such a slight increase in DNA methylation during pre-implantation development could also be due to the developmental variations between the embryos or between the sexes of embryos.

2. Imprinted X-chromosome inactivation and evaluation of H3K27me3 (related to Figures 2C, D; 3F; Figure2-supplement 2 F, G; Figure3-supplement 3G)<br /> Based on the slight change in the H3K27me3 signals in the Dnmt3b-Kd blastocysts, the authors claim that imprinted X-chromosome inactivation is impaired in the mutant embryo. It would be not easy to reach this conclusion from such a rough analysis of H3K27me3 presented in Figure 2C, D. Rigorous quantification/evaluation of the H3K27me3 signals in the Dnmt3b-Kd embryos should be considered. Additional evidence for the impairment of H3K27me3 in the mutant embryos should also be provided (expression of a subset of X-linked genes by RNA-FISH or RT-PCR etc.). Though technically challenging, high-resolution genome-wide approach such as ChIP-seq of H3K27me3 in the Dnmt3b-kd female embryos (with traceable SNPs between maternal and paternal X chromosome to distinguish inactive and active X-chromosome) could more precisely evaluate regions that lose H3K27me3 in the X-chromosome (de novo DNA methylated promoters from 8-cell to blastocyst, for example).

3. Analysis of the developmental potential of Dnmt3b-kd embryos<br /> While the authors claim that Dnmt3b-mediated de novo DNA methylation plays an important role in imprinted X-chromosome inactivation, it remains unclear whether the analysis presented in Figure 4 is derived from "female" embryos. This analysis seemed confusing as the authors claim that de novo DNA methylation in the promoter regions during the transition from 8-cell to blastocyst regulates imprinted X-chromosome inactivation, but this should not happen in the male embryos. Was the impairment of embryonic proliferation and differentiation observed in both male and female embryos? Or is this specific to the female embryos? We think that the sex of the embryos would be critical for the analysis presented in Figure 4.

Reviewer #1 (Public Review):

Summary:

This manuscript reports experiments designed to dissect the function of N-cadherin during mammalian folliculogenesis, using the mouse as a model system. Prior studies have shown that this is the principal cadherin expressed by the follicular granulosa cells. Two main strategies are used - small-molecule inhibitors that target N-cadherin and a conditional knockout where the gene encoding N-cad is deleted in granulosa cells. The authors also take advantage of the ability to reproduce key events of folliculogenesis, such as oocyte meiotic maturation, in vitro. Four main conclusions are drawn from the studies: (i) cadherin-based cell contact is required to maintain cadherin (N-cad in the granulosa cells; E-cad in the oocyte) at the plasma membrane; (ii) N-cad is required for cumulus layer expansion; (iii) N-cad is required for meiotic maturation of the oocyte; (iv) N-cad is required for ovulation.

Strengths:

The experiments are logically conceived, clearly described and presented, and carefully interpreted. A key strength of the paper is that multiple approaches have been used (drugs, knockouts, immunofluorescence, PLA, in vitro and in vivo studies). Taken together, they clearly establish essential roles for N-cadherin during folliculogenesis.

It is intriguing that, when cadherin activity is impaired, the cadherins are lost from the plasma membrane. This suggests that, in a multicellular context, interactions with other cadherins, either in cis within the same cell or in trans with a neighboring cell, are required to maintain cadherins at the membrane. Hence, beyond their significance for understanding female reproductive biology, these experiments have broader implications for cell biology.

Weaknesses:

A few points could be considered or clarified by the authors:

The YAP experiments were confusing to the reviewer. CRS-066 increased YAP activity, as indicated by increased expression of target genes. Since CRS-066 prevents expansion, this result suggests that YAP antagonizes expansion. Therefore, blocking YAP should favor expansion. Yet, the YAP inhibitor impaired expansion. In the reviewer's eyes, these results seem to be contradictory.

It is intriguing that the inhibitors were able to efficiently block oocyte maturation. Oocytes from which the cumulus granulosa cells have been removed (denuded) will mature in vitro in the absence of LH or EGF. Since the effect of the inhibitors is to break the contact between the cumulus cells and oocyte, one might have predicted that this would not impair the ability of the oocytes to mature. Perhaps the authors could comment on this.

Regarding the experiments where the inhibitors were administered intra-peritoneally, the authors might comment on the rationale for choosing the doses that were used. An additional point to consider is that, since N-cadherin is expressed in a variety of tissues, an effect of interfering with N-cadherin at these non-ovarian sites could indirectly influence ovarian function.

Reviewer #1 (Public Review):

Weinberger et al. use different fate-mapping models, the FIRE model and PLX-diet to follow and target different macrophage populations and combine them with single-cell data to understand their contribution to heart regeneration after I/R injury. This question has already been addressed by other groups in the field using different models. However, the major strength of this manuscript is the usage of the FIRE mouse model that, for the first time, allows specific targeting of only fetal-derived macrophages.

The data show that the absence of resident macrophages is not influencing infarct size but instead is altering the immune cell crosstalk in response to injury, which is in line with the current idea in the field that macrophages of different origins have distinct functions in tissues, especially after an injury.

To fully support the claims of the study, specific targeting of monocyte-derived macrophages or the inhibition of their influx at different stages after injury would be of high interest.

In summary, the study is well done and important for the field of cardiac injury. But it also provides a novel model (FIRE mice + RANK-Cre fate-mapping) for other tissues to study the function of fetal-derived macrophages while monocyte-derived macrophages remain intact.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Xiong L et al., the authors have uncovered an important link between innate immune signaling and hair regeneration. The authors provide convincing evidence supporting the critical roles of TLR2 in sensing CEP levels in hair follicles, counteracting the action of BMP signaling, and facilitating the activation of HFSCs during the hair cycle and wound repair. Importantly, the authors also propose that decreased CEP production and TLR2 expression might be factors contributing to the decreased hair regeneration associated with aging.

Strengths:

The experiments in this manuscript are well-designed and presented. The authors provided extensive evidence supporting the roles of TLR2 signaling in regulating hair follicle stem cell functions. Importantly, the findings from this paper could have sustained impacts on our understanding of the roles of innate immunity in regulating tissue regeneration in the absence of inflammation.

Weaknesses:

1. The central conclusion of this study is that the activation of TLR2 can suppress BMP signaling. However, the molecular link between TLR2 and BMP signaling is still missing. Given the importance of this finding, it would be intriguing to further investigate how TLR2 activation suppresses BMP signaling. A better characterization of the molecular-level interaction between TLR2 and BMP signaling can further enhance the impact of this study.

2. The authors imply that the decreased CEP level in aged mice could lead to deficient TLR2 signaling, which could further cause aging-associated hair regeneration defects. But this has not been demonstrated. What are the BMPs and pSmad1/5 levels in aged skin? Another important experiment to confirm the importance of this link during aging would be to inject CEP into the aged skin and examine whether this could restore hair regeneration in aged mice.

3. The impacts of CEP/TLR2 on proliferation of keratinocytes is still weak. How much of this effect is a result of NFkB activation, and how much is simply due to inhibiting BMP signaling?

Updated comments on the revised manuscript:<br /> The authors have addressed my previous questions.

Reviewer #1 (Public Review):

Summary:

In the article titled "Hammerhead-type FXR agonists induce an eRNA FincoR that ameliorates nonalcoholic steatohepatitis in mice," the authors explore the role of the Farnesoid X Receptor (FXR) in treating metabolic disorders like NASH. They identify a new liver-specific long non-coding RNA (lncRNA), FincoR, regulated by FXR, notably induced by agonists such as tropifexor. The study shows that FincoR plays a significant role in enhancing the efficacy of tropifexor in mitigating liver fibrosis and inflammation associated with NASH, suggesting its potential as a novel therapeutic target. The study makes a promising contribution to understanding the role of FincoR in alleviating liver fibrosis in NASH, providing initial insights into the mechanisms involved. While it offers a valuable starting point, there is potential for further exploration into the functional roles of FincoR and their specific actions in human NASH cases. Building upon the current findings to elucidate more detailed mechanistic pathways through which FincoR exerts its therapeutic effects in liver disease would elevate the research's significance and potential impact in the field.

Strengths:

This study stands out for its comprehensive and unbiased approach to investigating the role of FincoR, a liver-specific lncRNA, in the treatment of NASH. Key strengths include: 1) The application of advanced sequencing methods like GRO-seq and RNA-seq offered a comprehensive and unbiased view of the transcriptional changes induced by tropifexor, particularly highlighting the role of FincoR. 2) Utilizing a genetic mouse model of FXR KO and a FincoR liver-specific knockdown (FincoR-LKD) mouse model provided a controlled and relevant environment for studying NASH, allowing for precise assessment of tropifexor's therapeutic effects. 3) The inclusion of tropifexor, an FDA-approved FXR agonist, adds significant clinical relevance to the study. It bridges the gap between experimental research and potential therapeutic application, providing a direct pathway for translating these findings into real-world clinical benefits for NASH patients. 4) The study's rigorous experimental design, incorporating both negative and positive controls, ensured that the results were specifically attributable to the action of FincoR and tropifexor.

Weaknesses:

The study presents several notable weaknesses that could be addressed to strengthen its findings and conclusions: 1) The authors focus on FincoR, but do not extensively test other lncRNAs identified in Figure 1A. A more comprehensive approach, such as rescue experiments with these lncRNAs, would provide a better understanding of whether similar roles are played by other lncRNAs in mitigating NASH. 2) FincoR was chosen for further study primarily because it is the most upregulated lncRNA induced by GW4064. Including another GW4064-induced lncRNA as a control in functional studies would strengthen the argument for FincoR's unique role in NASH. 3) The study does not conclusively demonstrate whether FincoR is specifically expressed in hepatocytes or other liver cell types. Conducting FincoR RNA-FISH with immunofluorescent experiments or RT-PCR, using markers for different liver cell types, would clarify its expression profile. 4) Understanding the absolute copy number of FincoR is crucial. Determining whether there are sufficient copies of FincoR to function as proposed would lend more credibility to its suggested role. 5) The manuscript, although technically proficient, does not thoroughly address the relevance of these findings to human NASH. Questions like the conservation of FincoR in humans and its potential role in human NASH should be discussed.

Reviewer #1 (Public Review):

Glaucoma is the leading cause of irreversible blindness worldwide, affecting more than 80 million people. Primary open angle glaucoma (POAG) is the prevalent form of glaucoma, while prevalence of primary angle closure glaucoma (PACG) is highest in Asia compared to over the world. Early detection of glaucoma and severity prediction is mandatory, and therefore the main aim of this study focused on characterizing the metabolite profile associated with PACG, identify potential blood diagnostic markers, assess their specificity for PACG and verify their applicability to predict progression of visual field loss. To this end, Li et al. implemented a 5-phases multicenter prospective study to identify novel candidate biomarkers of PACG. A total of 616 individuals were recruited, identifying 1464 distinct metabolites in the serum by metabolomics and chemiluminescence immunoassays. By applying different machine learning algorithms the metabolite androstenedione showed good discrimination between PACG and control subjects, both the discovery and validation phases. This metabolite also showed alterations in the aqueous humor and higher levels of androstenedione seemed to be associated with faster loss of visual field. Overall, the authors claimed that serum androstenedione levels may provide a new biomarker for early detection and monitoring/predicting PACG severity/progression.

Strengths:

• Omics research on glaucoma is constrained by inadequate sample sizes, a dearth of validation sets to corroborate findings and absence of specificity analyses. The 5-phases study designed try overcoming these limitations. The proposed study design is very robust, with well described discovery set (1 and 2), validation phase (1 and 2), supplemental phase and cohort phase. Large and well-characterized patients with adequate control subjects contributed to the robustness of the results.<br /> • Combining untargeted and targeted metabolomics using mass spectrometry instruments (high resolution and low resolution) with an additional chemiluminiscence immunoassay determining androstenedione levels<br /> • Androstenedione achieved better diagnostic accuracy across the discovery and validation sets, with AUC varying between 0.85 and 1.0. Interestingly, baseline androstenedione levels can predict glaucoma progression via visual field loss results.<br /> • Positive correlation was observed between levels of androstenedione in serum and aqueous humor of PACG patients.<br /> • A level higher of 1.66 ng/mL of the metabolite androstenedione seems to imply high risk of visual field loss. Androstenedione may serve as predictor of glaucomatous visual field progression.

Weaknesses:

• A single biomarker seems very unlikely to be of much help in the detection of glaucoma due to the etiological heterogeneity of the disease, the existence of different subtypes, and the genetic variability among patients. Rather, a panel of biomarkers may provide more useful information for clinical prediction, including better sensitivity and specificity. The inclusion of additional metabolites already identifying in the study, in combination, may provide more reliable and correct assignment results.<br /> • The number of samples in the supplementary phase is low, larger samples sizes are mandatory to confirm the diagnostic accuracy.<br /> • Cohorts from different populations are needed to verify the applicability of this candidate biomarker.<br /> • Sex hormones seem to be associated also with other types of glaucoma, such as primary open-angle glaucoma (POAG), although the molecular mechanisms are unclear (see doi:10.1167/iovs.17-22708). The inclusion of patients diagnosed with other subtypes of glaucoma, like POAG, may contribute to determine the sensitivity and specificity of the proposed biomarker. Androstenedione levels should be determined in POAG, NTG or PEXG patients.<br /> • In addition, the levels of androstenedione were found significantly altered during other diseases as described by the authors or by conditions like polycystic ovary syndrome, limiting the utility of the proposed biomarker.<br /> • Uncertainty of the androstenedione levels compromises its usefulness in clinical practice.

Reviewer #1 (Public Review):

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

The paper addresses some interesting points, however some questions arise regarding some of the methods employed. Firstly, I am wondering why VOCs (as measured by GC-MS) were not quantified. While I understand that quantification is time consuming and requires more work, it allows for comparisons to be made between lines of the same species, as well as across other literature on the subject. Simply relying on the area under the curve and presenting results using arbitrary units is not enough for analyses like these. AU values do not allow for conclusions regarding total quantities, and while I understand that this is not the main focus of this paper, it raises a lot of uncertainty for readers (for example, the references cited show that TMTT has been found to accumulate at similar levels of caryophyllene, however the AU values reported are an order of magnitude higher for TMTT. Again, without actual quantification this is meaningless, but for readers it is confusing).

With regards to the correlation analyses shown in figure 6, the results presented in many of the correlation plots are not actually informative. While there is a trend, I do not think that this is an appropriate way to show the data, as there are clearly other relationships at play. The comparison between plants under continuous light and normal light:dark conditions is interesting.

This paper addresses a very interesting idea and I look forward to seeing further work that builds on these ideas.

Reviewer #1 (Public Review):

Summary:<br /> Overall, this study provides a meticulous comparison of developmental transcriptomes between two sub-species of the annelid Streblospio benedicti. Different lineages of S. benedicti maintain one of two genetically programmed alternative life histories, the ancestral planktotrophic or derived lecithotrophic forms of development. This contrast is also seen at the inter-species level in many marine invertebrate taxa, such as echinoderms and molluscs. The authors report relatively (surprisingly?) modest differences in transcriptomes overall but also find some genes whose expression is essentially morph-specific (which they term "exclusive").

Strengths:<br /> The study is based on a dense and appropriately replicated sampling of early development. The tight clustering of each stage/morph combination in PCA space suggests the specimens were accurately categorized. The similar overall trajectories of the two morphs were surprising to me for two stages: 1) the earliest stage (16-cell), at which we might expect maternal differences due to the several-fold difference in zygote size, and 2) the latest stage (1-week), where there appears to be the most obvious morphological difference. This is why we need to do experiments!

The examination of F1 hybrids was another major strength of the study. It also produced one of the most surprising results: though intermediate in phenotype, F1 embryos have the most distinct transcriptomes, and reveal a range of fixed, compensatory differences in the parental lines.

Weaknesses:<br /> Overall I really enjoyed this paper, but I see a few places where it can be tightened and made more insightful. These relate to better defining the basis for "exclusive" expression (regulation or gene presence/absence?), providing more examples of how specific genes related to trophic mode behave, and placing the study in the context of similar work in other phyla.

Reviewer #2 (Public Review):

The authors investigate the transcriptional regulation of cysteine dioxygenase (CDO-1) in C. elegans and its role in maintaining cysteine homeostasis. They show that high cysteine levels activate cdo-1 transcription through the hypoxia-inducible transcription factor HIF-1. Using transcriptional and translational reporters for CDO-1, the authors propose that a negative feedback pathway involving RHY-1, CYSL-1, EGL-9 and HIF-1 in regulating cysteine homeostasis.

Genetics is a notable strength of this study. The forward genetic screen, gene interaction and epistasis analyses are beautifully designed and rigorously conducted, yielding solid and unambiguous conclusions on the genetic pathway regulating CDO-1. The writing is clear and accessible, contributing to the overall high quality of the manuscript.<br /> Addressing the specifics of cysteine supplementation and interpretation regarding the cysteine homeostasis pathway would further clarify the paper and strengthen the study's conclusions.

First, the authors show that the supplementation of exogenous cysteine activates cdo-1p::GFP. Rather than showing data for one dose, the author may consider presenting dose-dependency results and whether cysteine activation of cdo-1 also requires HIF-1 or CYSL-1, which would be important data given the focus and major novelty of the paper in cysteine homeostasis, not the cdo-1 regulatory gene pathway. While the genetic manipulation of cdo-1 regulators yields much more striking results, the effect size of exogenous cysteine is rather small. Does this reflect a lack of extensive condition optimization or robust buffering of exogenous/dietary cysteine? Would genetic manipulation to alter intracellular cysteine or its precursors yield similar or stronger effect sizes?

Second, there remain several major questions regarding the interpretation of the cysteine homeostasis pathway. How much specificity is involved for the RHY-1/CYSL-1/EGL-9/HIF-1 pathway to control cysteine homeostasis? Is the pathway able to sense cysteine directly or indirectly through its metabolites or redox status in general? Given the very low and high physiological concentrations of intracellular cysteine and glutathione (GSH, a major reserve for cysteine), respectively, there is a surprising lack of mention and testing of GSH metabolism. In addition, what are the major similarities and differences of cysteine homeostasis pathways between C. elegans and other systems (HIF dependency, transcription vs post-transcriptional control)? These questions could be better discussed and noted with novel findings of the current study that are likely C. elegans specific or broadly conserved.

All of my comments and questions above have been satisfactorily addressed in the revised manuscript.

Reviewer #1 (Public Review):

Summary:<br /> This study reports that IT neurons have biased representations toward low spatial frequency (SF) and faster decoding of low SFs than high SFs. High SF-preferred neurons, and low SF-preferred neurons to a lesser degree, perform better category decoding than neurons with other profiles (U and inverted U shaped). SF coding also shows more sparseness than category coding in the earlier phase of the response and less sparseness in the later phase. The results are also contrasted with predictions of various DNN models.

Strengths:<br /> The study addressed an important issue on the representations of SF information in a high-level visual area. Data are analyzed with LDA which can effectively reduce the dimensionality of neuronal responses and retain category information.

Weaknesses:<br /> The results are likely compromised by improper stimulus timing and unmatched spatial frequency spectrums of stimuli in different categories.

The authors used a very brief stimulus duration (35ms), which would degrade the visual system's contrast sensitivity to medium and high SF information disproportionately (see Nachmias, JOSAA, 1967). Therefore, IT neurons in the study could have received more degraded medium and high SF inputs compared to low SF inputs, which may be at least partially responsible for higher firing rates to low Sf R1 stimuli (Figure 1c) and poorer recall performance with median and high SF R3-R5 stimuli in LDA decoding. The issue may also to some degree explain the delayed onset of recall to higher SF stimuli (Figure 2a), preferred low SF with an earlier T1 onset (Figure 2b), lower firing rate to high SF during T1 (Figure 2c), somewhat increased firing rate to high SF during T2 (because weaker high SF inputs would lead to later onset, Figure 2d).

Figure 3b shows greater face coding than object coding by high SF and to a lesser degree by low SF neurons. Only the inverted-U-shaped neurons displayed slightly better object coding than face coding. Overall the results give an impression that IT neurons are significantly more capable of coding faces than coding objects, which is inconsistent with the general understanding of the functions of IT neurons. The problem may lie with the selection of stimulus images (Figure 1b). To study SF-related category coding, the images in two categories need to have similar SF spectrums in the Fourier domain. Such efforts are not mentioned in the manuscript, and a look at the images in Figure 1b suggests that such efforts are likely not properly made. The ResNet18 decoding results in Figure 6C, in that IT neurons of different profiles show similar face and object coding, might be closer to reality.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript dissects the contribution of the CaBP 1 and 2 on the calcium current in the cochlear inner hair cells. The authors measured the calcium current inactivation from the double knock-out CaBP1 and 2 and showed that both proteins contribute to voltage-dependent and calcium-dependent inactivation. Synaptic release was reduced in the double KO. As a consequence, the authors observed a depressed activity within the auditory nerve. Taken together, this study identifies a new player that regulates the stimulation-secretion coupling in the auditory sensory cells.

Strengths:<br /> In this study, the authors bring compelling evidence that CaBP 1 and 2 are both involved in the inactivation of the calcium current, from cellular up to system level, and by taking care to probe different experimental conditions such as different holding potentials and by rescuing the phenotype with the re-expression of CaBP2. Indeed, while changing the holding potential worsens the secretion, it completely changes the kinetics of the inactivation recovery. It alerts the reader that probing different experimental conditions that may be closer to physiology is better suited to uncovering any deleterious phenotype. This gave pretty solid results.

Weaknesses:<br /> Although this study clearly points out that CaBP1 is involved in the calcium current inactivation, it is not clear how CaBP1 and CaBP2 act together (but this is probably beyond the scope of the study). Another point is that the authors re-express CaBP2 to largely rescue the phenotype in the double KO but no data are available to know whether the re-expression of both CaBP1 and CaBP2 would achieve a full recovery and what would be the effect of the sole re-expression of CaBP1 in the double KO.

Reviewer #1 (Public Review):

Summary:<br /> The authors introduced their previous paper with the concise statement that "the relationships between lineage-specific attributes and genotypic differences of tumors are not understood" (Chen et al., JEM 2019, PMID: 30737256). For example, it is not clear why combined loss of RB1 and TP53 is required for tumorigenesis in SCLC or other aggressive neuroendocrine (NE) cancers, or why the oncogenic mutations in KRAS or EGFR that drive NSCLC tumorigenesis are found so infrequently in SCLC. This is the main question addressed by the previous and current papers.

One approach to this question is to identify a discrete set of genetic/biochemical manipulations that are sufficient to transform non-malignant human cells into SCLC-like tumors. One group reported the transformation of primary human bronchial epithelial cells into NE tumors through a complex lentiviral cocktail involving the inactivation of pRB and p53 and activation of AKT, cMYC, and BCL2 (PARCB) (Park et al., Science 2018, PMID: 30287662). The cocktail previously reported by Chen and colleagues to transform human pluripotent stem-cell (hPSC)-derived lung progenitors (LPs) into NE xenografts was more concise: DAPT to inactivate NOTCH signaling combined with shRNAs against RB1 and TP53. However, the resulting RP xenografts lacked important characteristics of SCLC. Unlike SCLC, these tumors proliferated slowly and did not metastasize, and although small subpopulations expressed MYC or MYCL, none expressed NEUROD1.

MYC is frequently amplified or expressed at high levels in SCLC, and here, the authors have tested whether inducible expression of MYC could increase the resemblance of their hPSC-derived NE tumors to SCLC. These RPM cells (or RPM T58A with stabilized cMYC) engrafted more consistently and grew more rapidly than RP cells, and unlike RP cells, formed liver metastases when injected into the renal capsule. Gene expression analyses revealed that RPM tumor subpopulations expressed NEUROD1, ASCL1, and/or YAP1.

The hPSC-derived RPM model is a major advance over the previous RP model. This may become a powerful tool for understanding SCLC tumorigenesis and progression and for discovering gene dependencies and molecular targets for novel therapies. However, the specific role of cMYC in this model needs to be clarified.

cMYC can drive proliferation, tumorigenesis, or apoptosis in a variety of lineages depending on concurrent mutations. For example, in the Park et al., study, normal human prostate cells could be reprogrammed to form adenocarcinoma-like tumors by activation of cMYC and AKT alone, without manipulation of TP53 or RB1. In their previous manuscript, the authors carefully showed the role of each molecular manipulation in NE tumorigenesis. DAPT was required for NE differentiation of LPs to PNECs, shRB1 was required for expansion of the PNECs, and shTP53 was required for xenograft formation. cMYC expression could influence each of these steps, and importantly, could render some steps dispensable. For example, shRB1 was previously necessary to expand the DAPT-induced PNECs, as neither shTP53 nor activation of KRAS or EGFR had no effect on this population, but perhaps cMYC overexpression could expand PNECs even in the presence of pRB, or even induce LPs to become PNECs without DAPT. Similarly, both shRB1 and shTP53 were necessary for xenograft formation, but maybe not if cMYC is overexpressed. If a molecular hallmark of SCLC, such as loss of RB1 or TP53, has become dispensable with the addition of cMYC, this information is critically important in interpreting this as a model of SCLC tumorigenesis.

To interpret the role of cMYC expression in hPSC-derived RPM tumors, we need to know what this manipulation does without manipulation of pRB, p53, or NOTCH, alone or in combination. Seven relevant combinations should be presented in this manuscript: (1) cMYC alone in LPs, (2) cMYC + DAPT, (3) cMYC + shRB1, (4) cMYC + DAPT + shRB1, (5) cMYC + shTP53, (6) cMYC + DAPT + shTP53, and (7) cMYC + shRB1 + shTP53. Wild-type cMYC is sufficient; further exploration with the T58A mutant would not be necessary.

This reviewer considers that there should be a presentation of the effects of these combinations on LP differentiation to PNECs, expansion of PNECs as well as other lung cells, xenograft formation and histology, and xenograft growth rate and capacity for metastasis. If this could be clarified experimentally, and the results discussed in the context of other similar approaches such as the Park et al., paper, this study would be a major addition to the field.

Reviewer #1 (Public Review):

Summary:<br /> The authors were trying to achieve that Tgif1 expression is regulated by EAK1/2 and PTH in a time-dependent manner, and its roles in suppressing Pak3 for facilitating osteoblast adhesion. The authors further tried to show that the Tgif1-Pak3 signaling plays a significant role in osteoblast migration to the site of bone repair and bone remodeling.

Strengths:<br /> - In a previous study, it was demonstrated that Tgif1 is a target gene of PTH, and the absence of Tgif1 failed to increase bone mass by PTH treatment (Saito et al., Nat Commun., 2019). In this study, the authors found that Tgif1-Pak3 signaling prompts osteoblast migration through osteoblast adhesion to prompt bone regeneration. This novel finding provides a better understanding of how Tgif1 expression in osteoblasts regulates adherence, spreading, and migration during bone healing and bone remodeling.

- The authors demonstrated that ERK1/2 and PTH regulate Tgif1 expression in a time-dependent manner and its role in suppressing Pak3 through various experimental approaches such as luciferase assay, ChIP assay, and gene silencing. These results contribute to the overall strength of the article.

Weaknesses:<br /> -The authors need to further justify why they focused on Pak3 in the introduction by mentioning its known function for cell adhesion.

-Some results indicated statistically significant but small changes. The authors need to explain in the discussion part why they believe this is the major mechanism or why there may be some other possible mechanisms.

-The study does not include enough in vivo data to claim that this mechanism is crucial for bone healing and bone remodeling in vivo.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript entitled "Association with TFIIIC limits MYCN accumulation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II" the authors examine how the cohesin complex component (and RNA pol III associated factor) TFIIIC interacts with MYCN and controls transcription. They confirm that TFIIIC co-purifies with MYCN, dependent on its amino terminus, as shown in previous work. The authors also find that TFIIIC and MYCN are both found in promoter hubs and suggest that TFIIIC inhibits MYCN's association with these hubs. Finally, the authors indicate that TFIIIC/MYCN alters exosome function, and BRCA1-dependent effects, at MYCN-regulated loci.

Strengths:<br /> The authors utilize multiple experimental approaches to investigate the potential biological and genomic impacts of MYCN association with TFIIIC - the findings are interesting in suggesting that this interaction may limit or otherwise regulate MYC activity.

Weaknesses:<br /> (1) In Figure 1, the authors show that TF3C binds to the amino terminus of MYCN (Myc box I region), as shown previously. The data in Figure 1 B-D support, but do not rigorously confirm a 'direct' interaction because it has not been ruled out that accessory proteins mediating the association may be present in the mixture.

(2) The authors indicate in Figure 2 that TF3C has essentially no effect on MYCN-dependent gene expression and/or transcription elongation. Yet a previous study (PMID: 29262328) associated with several of the same authors concluded that TF3C positively affects transcription elongation. The authors make no attempt to reconcile these disparate results and need to clarify this point.

(3) Figures 2B and C show that unphosphorylated pol2 is TSS-centered, and Ser2-P pol2 occupation is centered beyond the TES. From this data, however, the reader can't tell how much of the phospho-Ser2- pol2 is centered on the TSS. The authors should include overall plots over TSS and TES, and also perhaps the gene-body to allow a better comparison for TSS and TES plotted for both antibodies over the collected gene sets.

(4) The authors see more TF3C at promoters in cells with MYCN (Figure 2F). What are the levels of TF3C in the absence and presence of MYCN?

(5) The finding that TF3C is increased at TSS (Figure 2F) doesn't necessarily indicate that 1) MYCN is recruiting TF3C there, and 2) that this is due to the phosphorylation status of pol2. It could mean many other things. The logic of conflating these 3 points based on the data shown is questionable.

(6) Figure 3A doesn't add much to the paper, as it is overplotted and no relationship is clear, except that Pol2 and MYCN occupy many of the same sites. Perhaps a less complex or different type of plot would allow the interactions to be better visible.

(7) That depletion of TF3C leads to increased promoter hubs may or may not have anything to do with its association with MYCN (Figure 4E). This could be a direct consequence of its known structural function in cohesin complexes, and the MYCN changes as a secondary consequence of this (also see point 4, above).

(8) Depletion of TF3C5 results in a loss of EXOSC5 (exosome) at TSS in the presence and absence of MYCN (Figure 5B). As TF3C5 is a cohesin, could this simply be a consequence of genomic structure changes?

(9) The authors suggest that RNA dynamics are affected by changes in exosome function (RNA degradation, etc). What effect, if any does TF3C depletion have on the overall gene expression profile?

Reviewer #1 (Public Review):

Interactions known to be important for melanosome transport include exon F and the globular tail domain (GTD) of MyoVa with Mlph. Motivated by a discrepancy between in vitro and cell culture results regarding necessary interactions for MyoVa to be recruited to the melanosome, the authors used a series of pull-down and pelleting assays experiments to identify an additional interaction that occurs between exon G of MyoVa and Mlph. This interaction is independent of and synergistic with the interaction of Mlph with exon F. However, the interaction of the actin-binding domain of Mlph can occur either with exon G or with the actin filament, but not both simultaneously. These data lead to a modified recruitment model where both exon F and exon G enhance the binding of Mlph to auto-inhibited MyoVa, and then via an unidentified switch (PKA?) the actin-binding domain of Mlph dissociates from MyoVa and interacts with the actin filament to enhance MyoVa processivity.

The only weakness noted is that the authors could have had a more complete story if they pursued whether PKA phosphorylation/dephosphorylation of Mlph is indeed the switch for the actin-binding domain of Mlph to interact with exon G versus the actin filament.

Reviewer #1 (Public Review):

Summary:<br /> The authors establish a recombinant insect cell expression and purification scheme for the antiviral Dicer complex of C. elegans. In addition to Dicer-1, the complex harbors two additional proteins, the RIG-I-like helicase DRH-1, and the dsRNA-binding protein RDE-4. The authors show that the complex prefers blunt-end dsRNA over dsRNAs that contain overhangs. Furthermore, whereas ATP-dependent dsRNA cleavage only exacerbates regular dsRNA cleavage activity, the presence of RDE-4 is essential to ATP-dependent and ATP-independent dsRNA cleavage. Single-particle cryo-EM studies of the ternary C. elegans Dicer complex reveal that the N-terminal domain of DRH-1 interacts with the helicase domain of DCR-1, thereby relieving its autoinhibitory state. Lastly, the authors show that the ternary complex is able to processively cleave long dsRNA, an activity primarily relying on the helicase activity of DRH-1.

Strengths:<br /> • First thorough biochemical characterization of the antiviral activity of C. elegans Dicer in complex with the RIG-I-like helicase DRH-1 and the dsRNA-binding protein RDE-4.<br /> • Discovery that RDE-4 is essential to dsRNA processing, whereas ATP hydrolysis is not.<br /> • Discovery of an autoinhibitory role of DRH-1's N-terminal domain (in analogy to the CARD domains of RIG-I).<br /> • First structural insights into the ternary complex DCR-1:DRH-1:RDE-4 by cryo-EM to medium resolution.<br /> • Trap experiments reveal that the ternary DCR-1 complex cleaves blunt-ended dsRNA processively. Likely, the helicase domain of DRH-1 is responsible for this processive cleavage.

Weaknesses:<br /> • Cryo-EM Structure of the ternary Dicer-1:DRH-1:RED-4 complex to only medium resolution.<br /> • High-resolution structure of the C-terminal domain of DRH-1 bound to dsRNA does not reveal the mechanism of how blunt-end dsRNA and overhang-containing one are being discriminated.<br /> • The cryo-EM structure of DCR1:DRH-1:RDE-4 in the presence of ATP only reveals the helicase and CTD domains of DRH-1 bound to dsRNA. No information on dsRNA termini recognition is presented. The paragraph seems detached from the general flow of the manuscript.<br /> • The antiviral DCR-1:DRH-1:RDE-4 complex shows largely homologous activities and regulation than Drosophila Dicer-2.

Reviewer #1 (Public Review):

Summary:

The authors have developed and optimized a footprinting assay to monitor the recruitment of mRNAs to a reconstituted translation initiation system. This assay is named Recruitment-Sequencing (Rec-Seq) and enables the analysis of many purified mRNAs in the reconstituted system.

This system possesses the ability to determine how competition occurs between mRNAs for the initiation machinery. This is the first approach using a reconstituted system that enables this important feature, and this is an important advance for the field.

Strengths:

Using purified mRNAs in a fully reconstituted system and being able to monitor start site selection is an important advance. The method enables one to observe changes in mRNA recruitment and start site selection in response to the absence or presence of different initiation components or accessory proteins.

Weaknesses:

Start site fidelity in purified reconstituted systems can be dramatically altered in different buffer conditions. Interpretation of the observed changes to start site selection in mRNAs in the absence or presence of Ded1 using only the one buffer condition used is therefore limited.

Reviewer #1 (Public Review):

Summary:<br /> In their manuscript, Kong Fang et al describe a robust pipeline for the isolation of small extracellular vesicles through a combination of size exclusion chromatography and miniaturized density gradient separation. Subsequently, they prove that the method is reproducible and suitable for small-volume operations while at the same time not compromising the quality of vesicles.

Strengths:<br /> The paper narrates a robust method for purifying high-quality sEVs from small amounts of blood plasma. They also demonstrate that through this approach, they can derive sEVs without compromising the protein composition, integrity of the vesicles, or contamination with other proteins or lipids.

Weaknesses:<br /> The paper is a nice summary of how to enrich sEVs from blood samples. Although well performed and substantiated with data, the paper primarily deals with method development and optimisation.

Reviewer #1 (Public Review):

In recent years, these investigators have been engaged in a debate regarding the classification of the sacral parasympathetic system as "sympathetic" rather than "parasympathetic," based on shared developmental ontogeny of spinal preganglionic neurons. In this current study, these investigators conducted single-cell RNAseq analyses of four groups of autonomic neurons: paravertebral sympathetic neurons (stellate and lumbar train ganglia), prevertebral sympathetic neurons (coeliac-mesenteric ganglia), rostral parasympathetic ganglia (sphenopalatine ganglia), and the caudal pelvic ganglia (containing traditionally recognized sacral "parasympathetic cholinergic neurons," which the investigators sought to challenge in terms of nomenclature). The authors argued that the pelvic ganglionic neurons shared the expression of more genes with sympathetic ganglia, as opposed to parasympathetic ganglia. Additionally, the pelvic neurons did not express a set of genes observed in the rostral parasympathetic sphenopalatine ganglia. Based on these findings, they claimed that the sacral autonomic system should be considered sympathetic rather than parasympathetic.

However, noradrenergic sympathetic neurons and cholinergic neurons, by the virtue of expressing different neurotransmitters, could have distinct roles. It is true that some cholinergic neurons reside in the sympathetic train ganglia as well, such as those innervating the sweat gland and some vascular systems; in this sense, the pelvic ganglia share some features with sympathetic ganglia, except that the pelvic ganglia contain a much higher percentage of cholinergic neurons compared with sympathetic ganglia. It is much simpler and easier to divide the autonomic nervous system into sympathetic neurons that relieve noradrenaline versus parasympathetic neurons that relieve acetylcholine, and these two systems often act in antagonistic manners, even though in some cases, these two systems can work synergistically. As such, it is not justified to claim that "pelvic organs receive no parasympathetic innervation".

Reviewer #1 (Public Review):

This paper aims to address the establishment and maintenance of neural circuitry in the case of a massive loss of neurons. The authors used genetic manipulations to ablate the principal projection neurons, the mitral/tufted cells, in the mouse olfactory bulb. Using diphtheria toxin (Tbx21-Cre:: loxP-DTA line) the authors ablated progressively large numbers of M/T cells postnatally. By injecting diphtheria toxin (DT) into the Tbx21-Cre:: loxP-iDTR line, the authors were able to control the timing of the ablation in the adult stage. Both methods led to the successful elimination of a majority of M/TCs by 4 months of age. The authors made a few interesting observations. First, they found that the initial pruning of the remaining M/T cell primary dendrite was unaffected. However, in adulthood, a significant portion of these cells extended primary dendrites to innervate multiple glomeruli. Moreover, the incoming olfactory sensory neuron (OSN) axons, as examined for those expressing the M72 receptor, showed a divergent innervation pattern as well. The authors conclude that M/T cell density is required to maintain the dendritic structures and the olfactory map. To address the functional consequences of eliminating a large portion of principal neurons, the authors conducted a series of behavioral assays. They found that learned odor discrimination was largely intact. On the other hand, mating and aggression were reduced. The authors concluded that learned behaviors are more resilient than innate ones.

The study is technically sound, and the results are clear-cut. The most striking result is the contrast between the normal dendritic pruning during early development and the expanded dendritic innervation in adulthood. It is a novel discovery that can lead to further investigation of how the single-glomerulus dendritic innervation is maintained. The authors conducted a few experiments to address potential mechanisms, but it is inconclusive, as detailed below. It is also interesting to see that the massive neuronal loss did not severely impact learned odor discrimination. This result, together with previous studies showing nearly normal odor discrimination in the absence of large portions of the olfactory bulb or scrambled innervation patterns, attests to the redundancy and robustness of the sensory system. The discussion should take into account these other studies in a historical context.

Main comments:

1. In previous studies, it has been concluded that dendritic pruning unfolds independently, regardless of the innervation pattern or activity of the OSNs. The new observation bolsters this conclusion by showing that a loss of neighboring M/T cells does not affect the developmental process. A more nuanced discussion comparing the results of these studies would strengthen the paper.

2. The authors propose that a certain density of M/T is required to prevent the divergent innervation of primary dendrites, but the evidence is not sufficient to support this proposal. The experiment with low-dose DT injection to ablate a smaller portion of M/T cells did not change the percentage of cells innervating two or more glomeruli. The authors suggest that a threshold must be met, but this threshold is not determined. It would be possible to adjust the DT injection dose to find this threshold.

3. The authors suggest that neural activity is not required for this plasticity. The evidence was derived primarily from naris occlusion and neuronal silencing using Kir2.1. While the results are consistent with the notion, it is a rather narrow interpretation of how neural activity affects circuit configuration. Perturbation of neural activity also entails an increase in firing. Inducing the activity of the neurons may alter this plasticity. Silencing per se may induce a homeostatic response that expands the neurite innervation pattern to increase synaptic input to compensate for the loss of activity. Thus, further silencing the cells may not reduce multi-glomerular innervation, but an increased activity may.

4. There is a discrepancy between this study and the one by Fujimoto et al. (Developmental Cell; 2023), which shows that not only glutamatergic inputs to the primary dendrite can facilitate pruning of remaining dendrites but also Kir2.1 overexpression can significantly perturb dendritic pruning. This discrepancy is not discussed by the authors.

5. An alternative interpretation of the discrepancy between the apparent normal pruning by p10 and expanded dendritic innervation in adulthood is that there are more cells before P10, when ~25% of M/T cells are present, but at a later date only 1-3% are present. The relationship between the number of M/T cells and single glomerulus innervation has not been explored during postnatal development. It would be important to test this hypothesis.

6. The authors attribute the change in the olfactory map to the loss of M/T cells. Another obvious possibility is that the diffused projection is a response to the change in the olfactory bulb size. With less space to occupy, the axons may be forced to innervate neighboring glomeruli. It is not known how the total number of glomeruli is affected. This question could be addressed by tracking developmental changes in bulb volume and glomerular numbers.

7. The retained ability to discriminate odors upon reinforced training is not surprising in light of a number of earlier studies. For example, Slotnick and colleagues have shown that rats losing ~90% of the OB can retain odor discrimination. Weiss et al have shown that humans without an olfactory bulb can perform normal olfactory tasks. Gronowitz et al have used theoretical prediction and experimental results to demonstrate that perturbing the olfactory map does not have a major impact on olfactory discrimination.<br /> Fleischmann et al have shown that mice with a monoclonal nose can discriminate odors. The authors should discuss their results in these contexts.

8. It should be noted that odor discrimination resulting from reinforcement training does not mean normal olfactory function. It is a highly artificial situation as the animals are overtrained. It should not be used as a measure of the robustness of the olfactory sense. Natural odor discrimination (without training), detection threshold, and innate appetitive/aversive response to certain odors may be affected. These experiments were not conducted.

9. The social behaviors were conducted using relatively coarse measures (vaginal plug and display of aggression). Moreover, these behaviors are most likely affected by the disruption of the AOB mitral cells and have little to do with the dendritic pruning process described in the paper. It is misleading to lump social behaviors with innate responses to odors.

Reviewer #1 (Public Review):

The authors use a combination of crop modeling and field experiments to argue that drought during seedling establishment likely severely impacts the yield of pearl millet, an important but understudied cereal crop and that rapid seedling root elongation could play a major role in mitigating this. They further argue that this trait has a strong genetic basis and that major polymorphisms in candidate genes can be identified using standard methods from modern genetics and genomics. Finally, they use homology with the model plant Arabidopsis thaliana to argue that the function of one putatively causal gene is to regulate root cell elongation.

The major strength of this paper is that it convincingly demonstrates how modern methods from plant breeding and model organisms can be combined to address questions of great practical importance in important but poorly understood crops. The notion that it is possible to connect single-locus polymorphism and cellular biology to drought tolerance and crop yield in pearl millet is not a trivial one.

The weakness is obvious: while the argument made is convincing, it must be recognized that the strength of the evidence is by no means of the level expected in a model organism. Conclusions could easily be wrong, and there is no direct evidence that regulatory variation in PgGRXC9 leads to higher crop yield via cell elongation and seedling drought tolerance. However, generating such evidence in a poorly studied crop would be a monumental undertaking, and should probably not be the priority of people working on pearl millet!

The utility of this work is that it suggests that it is practicable to gain valuable insight into crop adaptation by clever use of modern methods from a variety of sources.

Reviewer #1 (Public Review):

Trenker et al. report cryo-EM structures of HER4/HER2 heterodimers and HER4 homodimers bound to Neuregulin-1β (Nrg1β) and Betacellulin (BTC). As observed for prior cryo-EM structures of full-length or near full-length HER-family receptors only the extracellular regions are visualized, presumably owing to flexibility in the relative orientation of extra- and intra-cellular regions. The authors observe no appreciable differences between Nrg1β and BTC bound heterodimers, both ligands, in this case being high-affinity ligands, and modest "scissor-like" differences in the subunit relationships in HER4 homodimers with Nrg1β and BTC bound.

The authors also show that, as they showed for HER3, the HER4 dimerization arm is not indispensable for forming heterodimers with HER2 despite the HER4 dimerization arm forming a more canonical interaction with HER2. Perhaps most interestingly, the authors observe glycan interactions that appear to stabilize intra- and inter-subunit interactions in HER4 homodimers but that inter-subunit glycans are not present in HER2/HER4 heterodimers. The authors speculate that these glycan interactions may contribute to the apparent propensity of HER4 to homodimerize vs. heterodimerize with HER2.

I realize that an important role of reviewers is to provide authors with informed and critical comments, but I found this manuscript a well-written, thoughtful, and important contribution. My only note is that I am not an electron microscopist so have assumed the microscopy has been carried out expertly and rely on other reviewers to vet structure determinations.

Reviewer #1 (Public Review):

Summary:<br /> The paper is an attempt to explain a geographic paradox between infection prevalence and antimalarial resistance emergence. The authors developed a compartmental model that importantly contains antigenic strain diversity and in turn antigen-specific immunity. They find a negative correlation between parasite prevalence and the frequency of resistance emergence and validate this result using empirical data of chloroquine-resistance. Overall, the authors conclude that strain diversity is a key player in explaining observed patterns of resistance evolution across different geographic regions.

The authors pose and address the following specific questions:<br /> 1. Does strain diversity modulate the equilibrium resistance frequency given different transmission intensities?<br /> 2. Does strain diversity modulate the equilibrium resistance frequency and its changes following drug withdrawal?<br /> 3. Does the model explain biogeographic patterns of drug resistance evolution?

Strengths:<br /> The model built by the authors is novel. As emphasized in the manuscript, many factors (e.g., drug usage, vectorial capacity, population immunity) have been explored in models attempting to explain resistance emergence, but strain diversity (and strain specific immunity) has not been explicitly included and thus explored. This is an interesting oversight in previous models, given the vast antigenic diversity of Plasmodium falciparum (the most common human malaria parasite) and its potential to "drive key differences in epidemiological features".

The model also accounts for multiple infections, which is a key feature of malarial infections, with individuals often infected with either multiple Plasmodium species or multiple strains of the same species. Accounting for multiple infections is critical when considering resistance emergence, as with multiple infections there is within-host competition which will mediate the fitness of resistant genotypes. Overall, the model is an interesting combination of a classic epidemiological model (e.g., SIR) and a population genetics model.

In terms of major model innovations, the model also directly links selection pressure via drug administration with local transmission dynamics. This is accomplished by the interaction between strain-specific immunity, generalized immunity and host immune response.

Weaknesses:<br /> The authors emphasize several model limitations, including the specification of resistance by a single locus (thus not addressing the importance of recombination should resistance be specified by more than one locus); the assumption that parasites are independently and randomly distributed among hosts (contrary to empirical evidence); and the assumption of a random association between the resistant genotype and antigenic diversity. However, each of these limitations are addressed in the discussion.

Did the authors achieve their goals? Did the results support their conclusion?<br /> Returning to the questions posed by the authors:<br /> 1. Does strain diversity modulate the equilibrium resistance frequency given different transmission intensities? Yes. The authors demonstrate a negative relationship between prevalence/strain diversity and resistance frequency (Figure 2).

2. Does strain diversity modulate the equilibrium resistance frequency and its changes following drug withdrawal? Yes. The authors find that, under resistance invasion and some level of drug treatment, resistance frequency decreased with the number of strains (Figure 4). The authors also find that lower strain diversity results in a slower decline in resistant genotypes after drug withdrawal and higher equilibrium resistance frequency (Figure 6).

3. Does the model explain biogeographic patterns of drug resistance evolution? Yes. The authors find that their full model (which includes strain-specific immunity) produces the empirically observed negative relationship between resistance and prevalence/strain diversity, while a model only incorporating generalised immunity does not (Figure 8).

Utility of work to others and relevance within and beyond the field?<br /> This work is important because antimalarial drug resistance has been an ongoing issue of concern for much of the 20th century and now 21st century. Further, this resistance emergence is not equitably distributed across biogeographic regions, with South America and Southeast Asia experiencing much of the burden of this resistance emergence. Not only can widespread resistant strains be traced back to these two relatively low-transmission regions, but these strains remain at high frequency even after drug treatment ceases.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, Janssens et al. addressed the challenge of mapping the location of transcriptionally unique cell types identified by single nuclei sequencing (snRNA-seq) data available through the Fly Cell Atlas. They identified 100 transcripts for head samples and 50 transcripts for fly body samples allowing the identification of every unique cell type discovered through the Fly Cell Atlas. To map all of these cell types, the authors divided the fly body into head and body samples and used the Molecular Cartography (Resolve Biosciences) method to visualize these transcripts. This approach allowed them to build spatial tissue atlases of the fly head and body, to identify the location of previously unknown cell types and the subcellular localization of different transcripts. By combining snRNA-seq data from the Fly Cell Atlas with their spatially resolved transcriptomics (SRT) data, they demonstrated an automated cell type annotation strategy to identify unknown clusters and infer their location in the fly body. This manuscript constitutes a proof-of-principle study to map the location of the cells identified by ever-growing single-cell transcriptomic datasets generated by others.

Strengths:<br /> The authors used the Molecular Cartography (Resolve Biosciences) method to visualize 100 transcripts for head samples and 50 transcripts for fly body samples in high resolution. This method achieves high resolution by multiplexing a large number of transcript visualization steps and allows the authors to map the location of unique cell types identified by the Fly Cell Atlas.

Weaknesses:<br /> Combining single-nuclei sequencing (snRNA-seq) data with spatially resolved transcriptomics (SRT) data is challenging, and the methods used by the authors in this study cannot reliably distinguish between cells, especially in brain regions where the processes of different neurons are clustered, such as in neuropils. This means that a grid that the authors mark as a unique cell may actually be composed of processes from multiple cells.

Certainly! Let's break down the provided code snippets:

1. What is it and why is it used?

In Mongoose, a schema is a blueprint for defining the structure of documents within a collection. When you define a schema, you can also attach methods to it. These methods become instance methods, meaning they are available on the individual documents (instances) created from that schema.

Instance methods are useful for encapsulating functionality related to a specific document or model instance. They allow you to define custom behavior that can be executed on a specific document. In the given example, the findSimilarTypes method is added to instances of the Animal model, making it easy to find other animals of the same type.

2. Syntax:

Using methods object directly in the schema options:

javascript const animalSchema = new Schema( { name: String, type: String }, { methods: { findSimilarTypes(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); } } } );

Using methods object directly in the schema:

javascript animalSchema.methods.findSimilarTypes = function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); };

Using Schema.method() helper:

javascript animalSchema.method('findSimilarTypes', function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); });

3. Explanation in Simple Words with Examples:

Why it's Used:

Imagine you have a collection of animals in your database, and you want to find other animals of the same type. Instead of writing the same logic repeatedly, you can define a method that can be called on each animal instance to find similar types. This helps in keeping your code DRY (Don't Repeat Yourself) and makes it easier to maintain.

Example:

```javascript const mongoose = require('mongoose'); const { Schema } = mongoose;

// Define a schema with a custom instance method const animalSchema = new Schema({ name: String, type: String });

// Add a custom instance method to find similar types animalSchema.methods.findSimilarTypes = function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); };

// Create the Animal model using the schema const Animal = mongoose.model('Animal', animalSchema);

// Create an instance of Animal const dog = new Animal({ type: 'dog', name: 'Buddy' });

// Use the custom method to find similar types dog.findSimilarTypes((err, similarAnimals) => { console.log(similarAnimals); }); ```

In this example, findSimilarTypes is a custom instance method added to the Animal schema. When you create an instance of the Animal model (e.g., a dog), you can then call findSimilarTypes on that instance to find other animals with the same type. The method uses the this.type property, which refers to the type of the current animal instance. This allows you to easily reuse the logic for finding similar types across different instances of the Animal model.

Reviewer #1 (Public Review):

Summary:

In this study, Jeong and Choi examine neural correlates of behavior during a naturalistic foraging task in which rats must dynamically balance resource acquisition with the risk of threat. Rats first learn to forage for sucrose reward from a spout, and when a threat is introduced (an attack-like movement from a "LobsterBot"), they adjust their behavior to continue foraging while balancing exposure to the threat, adopting anticipatory withdrawal behaviors to avoid encounter with the LobsterBot. Using electrode recordings targeting the medial prefrontal cortex (PFC), they identify heterogenous encoding of task variables across prelimbic and infralimbic cortex neurons, including correlates of distance to the reward/threat zone, and correlates of avoidance behavior. Based on analysis of population responses, they suggest that the prefrontal cortex switches between coding schemes to process spatial information or behavioral responses in a context-dependent manner. Characterization of the heterogenous coding scheme by which the frontal cortex represents information in different goal states is an important contribution to our understanding of brain mechanisms underlying flexible behavior in ecological settings.

Strengths:

As many behavioral neuroscience studies employ highly controlled task designs, relatively less is known about how the brain organizes navigation and behavioral selection in naturalistic settings, where environment states and goals are more fluid. Here, the authors take advantage of a natural challenge faced by many animals - how to forage for resources in an unpredictable environment - to investigate neural correlates of behavior when goal states are dynamic. Related to this, they also investigate how prefrontal cortex (PFC) activity can reorganize to support different functional "modes" (here, between a navigational mode and an action-selection mode) for flexible behavior. Overall, an important strength and real value of this study is the design of the behavioral experiment, which is trial-structured, permitting the use of standard methods to analyze neural data, yet rich enough to encourage and permit more natural behavior. The experiment is also phased to measure behavioral changes as animals first encounter a threat, and then learn to adapt their foraging strategy to its presence. Characterization of this adaptation process is itself quite interesting and sets a foundation for further study of threat learning and risk management in the foraging context. Finally, the characterization of single-neuron activity from the prefrontal cortex in this naturalistic setting is an important contribution to the field - previous studies have identified the neural correlates of spatial and behavioral variables in the frontal cortex, but the nature of how these representations co-exist or are dynamically adjusted when animals shift their goals is less clear.

Weaknesses:

While the task design in this study is intentionally stimulus-rich and places a minimal constraint on the animal to preserve naturalistic behavior, this is, unfortunately, a double-edged sword, as it also introduces additional variables that confound some of the neural analysis. Because of this, a general weakness of the study is a lack of clear interpretability of the task variable neural correlates. This is a limitation of the task, which includes many naturally correlated variables - however, I think with some additional analyses, the authors could strengthen some of their core arguments and significantly improve clarity.

For example, the authors argue, based on an ANN decoding analysis (Figure 2b), that PFC neurons encode spatial information - but the spatial coordinate that they decode (the distance to the active foraging zone) is itself confounded by the fact that animals exhibit different behavior in different sections of the arena. From the way the data are presented, it is difficult to tell whether the decoder performance reflects a true neural correlate of distance, or whether it is driven by behavior-associated activity that is evoked by different behaviors in different parts of the arena. The author's claim that PFC neurons encode spatial information could be substantiated with a more careful analysis of single-neuron responses to supplement the decoder analysis. For example, 1) They could show examples of single neurons that are active at some constant distance away from the foraging site, regardless of animal behavior, and 2) They could quantify how many neurons are significantly spatially modulated, controlling for correlates of behavior events. One possible approach to disambiguate this confound could be to use regression-based models of neuron spiking to quantify variance in neuron activity that is explained by spatial features, behavioral features, or both.

The authors also claim that the heterogenous encoding of spatial and behavioral variables in PFC neurons is structured in a particular way that depends on the animal's goal state and/or context (a navigational mode and an action-selection mode). The main evidence supporting this interpretation is a population vector analysis based on principal component projections of neural data (Figure 4), which shows that the population response is different, on average, in the encounter zone compared to the foraging and nesting zones. But again, the different "zones" are obligately correlated with different types of behavior/stimuli. Since some neurons are modulated by events unique to the encounter zone (e.g., licking sucrose water, withdrawing from the LobsterBot, etc.), differences in population activity patterns may simply reflect this behavior/event coding. To substantiate the claim that PFC neurons really switch between different coding "modes," the authors could include a version of this analysis where they have regressed out, or otherwise controlled for, these confounds. Otherwise, the claim that the authors have identified "distinctively different states of ensemble activity," as opposed to simple coding of salient task features, seems premature.

Reviewer #1 (Public Review):

Summary:<br /> The authors aimed to infer the trajectories of long range and local neuronal synchrony across the Alzheimer's disease continuum, relative to neurodegeneration and cognitive decline. The trajectories are inferred using event-based models, which infer a set of data-driven disease stages from a given dataset. The authors develop an adapted event-based modelling approach, in which they characterise each stage as a particular biomarker increasing by a particular z-score deviation from controls. Fitting infers the optimal set of z-scores to use for each biomarker and the order in which each biomarker reaches each z-score. The authors apply this approach to data from 148 individuals (70 cognitively unimpaired older adults and 78 individual with mild cognitive impairment or Alzheimer's disease), identifying trajectories in which long-range (amplitude-envolope correlation) and local (regional spectral power) neuronal synchrony in the alpha and beta bands becomes abnormal prior to neurodegeneration (measured as the volume of the parahippocampal gyrus) and cognitive decline (measured using the mini-mental state examination).

Strengths:<br /> - The main strength is that the authors assess two models. In the first they derive a staging system based only on the volume of the parahippocampal gyrus and mini-mental state examination score. They then investigate how neuronal synchrony metrics change compared to this staging system. In the second they derive a staging system that also includes an average (combined long-range and local) neuronal synchrony metric and investigate how long-range and local synchrony metrics change relative to this staging system. This is a strength as the first model provides confidence that there is not overfitting to the neuronal synchrony data, and the second provides more detailed insights into the dynamics of the early neuronal synchrony changes.<br /> - Another strength is that the authors automatically infer the optimal z-scores to choose, rather than having to pre-select them manually, as in previous approaches.

Weaknesses:<br /> - The authors do not have a dataset for external validation.

Reviewer #1 (Public Review):

In this study, Lin et al developed a protocol termed MOCAT, to perform tissue clearing and labelling on large-scale FFPE mouse brain specimens. They have optimised protocols for dewaxing and adequate delipidation of FFPE tissues to enable deep immunolabelling, even for whole mouse brains. This was useful for the study of disease models such as in an astrocytoma model to evaluate spatial architecture of the tumour and its surrounding microenvironment. It was also used in a traumatic brain injury model to quantify changes in vasculature density and differences in monoaminergic innervation. They have also demonstrated the potential of multi-round immunolabelling using photobleaching, as well as expansion microscopy with FFPE samples using MOCAT.

Strengths:<br /> This paper has demonstrated, with some good imaging examples, that it is possible to perform deep immunostaining with detailed analysis on FFPE samples using MOCAT. The figures provided appeared to be largely convincing with good amount of details.

They have showcased different ways to perform analysis on cleared tissue. For example, the use of lectin-labelled blood vessels as a structural reference for multi-round immunolabelling was very useful. They have also demonstrated how to generate comparable quantitative data on various mouse disease models which will be important for future tissue-clearing studies.

Weaknesses:<br /> Although the authors have proven the feasibility of their techniques on FFPE samples, it is questionable whether this will translate well for human brain tissues. The vast majority of the study data was generated using rodent brain tissues and it appears the technique was only performed on human FFPE tissues no larger than 1 mm in thickness. The PFA/formalin fixation time for the tissue was also limited to 24 hours in this study. Whilst this may be true for most surgical specimens, whole brain specimens in brain banks will often have formalin fixation time exceeding 3 weeks. The issue of prolonged formalin fixation prior to embedding in paraffin wax was not addressed in this study.

Inherent differences in human and rodent brain tissues may affect the effectiveness of immunostaining. In this study, results on human brain specimens appeared to show a reduction in clarity and staining quality at greater imaging depth at 900 µm, particularly for MAP2 and GFAP (Figure 5).

In addition, there are inadequate details in the materials and methods section which may limit the readers' ability to successfully replicate the study or proposed method for tissue clearing. Further details on the optimisation of this protocol and brief details from previously published protocols were not described in the methods section.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, the authors set out to develop genetic tools that can specifically and comprehensively label Axo-Axonic Cells (AACs), also known as Chandelier cells. These AACs possess unique morphological and connectivity features, making them an ideal subject for studying various aspects of cell types across different experimental methods. To achieve both specificity and comprehensiveness in AAC labeling, the authors employ an intersectional strategy that combines lineage origin and molecular markers. This approach successfully targets AACs across the mouse brain and reveals their widespread distribution in various brain structures beyond the previously known regions. Additionally, the authors utilize rabies transneuronal labeling to provide a comprehensive overview of AACs, their variations, and input sources throughout the brain. This experimental approach offers a powerful model system for investigating the role of AACs in circuit development and function across diverse brain regions.

Strengths:<br /> Genetic Tools and Specificity: The authors' genetic tools show qualitative evidence of specificity for AACs, opening new avenues for targeted research on these cells. The use of intersectional strategies enhances the precision of AAC labeling.

Widespread Distribution: The study significantly broadens our understanding of AAC distribution, revealing their presence in brain regions beyond what was previously documented. This expanded knowledge is a valuable contribution to the field.

Transneuronal Labeling: The inclusion of rabies transneuronal labeling provides a comprehensive view of AACs, their variations, and input sources, allowing for a more holistic understanding of their role in neural circuits.

Weaknesses:<br /> Quantitative Analysis: While the claim of specificity appears qualitatively convincing, the manuscript could be improved with more quantitative analysis.

Comprehensiveness Claim: The assertion of comprehensiveness, implying labeling "almost all" AACs in all brain regions, is challenging to substantiate conclusively. Acknowledging the limitations of proving complete comprehensiveness and discussing them in the discussion section would be more appropriate than asserting it in the results section.

Local Inputs: While the manuscript focuses on inter-areal inputs to AACs, it would benefit from exploring local inputs as well. Identifying the local neurons that target AACs and analyzing their patterns could provide valuable insights into AAC function within specific brain regions.

Discussion Focus: The discussion section should delve deeper into the biological implications of the findings, moving beyond technical significance. Exploring similarities and differences in input patterns between AACs and other cell types, and linking them to the locations of starter cells or specific connectivity patterns in the brain, would enrich the discussion. For instance, investigating whether input patterns can be predicted based on the locations of starter cells or connectivity specificity could provide valuable insights.

Reviewer #1 (Public Review):

Summary:<br /> This describes the molecular identity of the intermediate status of cranial neural crest cells (NCCs) during the initial delamination process. Taking advantage of single-cell RNA seq, the authors identify new populations of cells during EMT characterized by a specific set of gene expressions, including Dlc1. Promigratory cranial NCCs differentiate through different trajectories depending on their cell cycle phases but converge into a common progenitor, then differentiate into mesenchymal cells expressing region-specific genes.

Strengths:<br /> Single-cell RNA seq data convincingly support what the authors claim. This is the first time to identify intermediate states between premigratory and migratory cranial NCCs. Silencing one of the marker genes, Dlc1, reduces the migratory activity of cranial NCCs. These findings deepen our understanding of the mechanism of EMT in general.

Weaknesses:<br /> Common and specific features between cranial and trunk NCCs could be described/discussed in-depth. Phenotypic relations between the reduction of delamination and defects found in Dlc1 mutant mice can be discussed.

Reviewer #1 (Public Review):

In this manuscript, Seba et al., investigate the mechanism of chromosome organization by the MukBEF complex in E. coli. They use a combination of Hi-C and ChIP analysis to understand the steps of MukBEF regulation: its unloading from DNA (how MukBEF activity is prevented in the terminus regions of the chromosome by MatP), and its loading onto DNA (how DNA replication influences MukBEF association with the chromosome). Seba et al., induce chromosomal rearrangements to flip the sections of the ter region, thus perturbing matS site numbers and position. They find that MukBEF activity is prevented around matS sites and that higher matS density has greater effect on MukBEF. Separately, using replication mutants and inducible MukBEF expression, they find that MukBEF can associate with the chromosome even in the absence of replication (as seen by the emergence of long-range contacts). However, ChIP data suggests that MukBEF binding to DNA is enriched on newly replicated DNA.

Altogether, this work provides a valuable and comprehensive view of MukBEF-mediated chromosome organization, with insights on the mechanism of the exclusion of MukBEF from the terminus region of the chromosome. The use of the programmed genetic rearrangements is powerful and allows the authors to provide clear and convincing evidence for MukBEF exclusion from ter by matS sites. It is particularly striking to see that MukBEF can promote long-range contacts even in chromosomal regions between two matS, but the complex is excluded from the matS 'zones'. Experiments using cells blocked for replication show that MukBEF can influence chromosome organization in the absence of replication as well. While previous studies have reported some evidences in support of both of the above conclusions, the experiments described here offer a clear and direct demonstration of the same.

Reviewer #1 (Public Review):

In this study, the authors seek to characterize the role of splicing factor SRSF1 during spermatogenesis using Vasa-Cre;Srsf1Fl/del mice model. The authors first revealed that spermatogonia-related genes (e.g., Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes by CLIP-seq. The authors convincingly demonstrated that specific deletion of SRSF1 in mouse gem cells with vasa-cre lead to NOA by impairing homing and failure survival of spermatogonia. To investigate the molecular mechanisms of SRSF1 in spermatogonia, further multiomics analysis including CLIP-seq, IP-MS, and RNA-seq were conducted. The results showed that SRSF1 coordinated with other RNA splicing-related proteins to directly bind and regulate the expression of nine spermatogonia-related genes especially Tial1/Tiar via alternative splicing. The authors revealed the critical role of SRSF1-mediated AS in precursor SSCs homing and survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying the formation of SSC pools and the establishment of niches. This work will be of interest to stem cell and reproductive biologists. The experiments are well-designed and conducted, and the overall methods and results are convincing except for the claim that altered splicing of the Tial1 transcript mediates the effect of SRSF1 loss.

Reviewer #1 (Public Review):

The manuscript aims to provide mechanistic insight into the activation of PI3Kbeta by its known regulators tyrosine phosphorylated peptides, GTP-loaded Rac1 and G-protein beta-gamma subunits. To achieve this the authors have used supported lipid bilayers, engineered recombinant peptides and proteins (often tagged with fluorophores) and TIRF microscopy to enable bulk (averages of many molecules) and single molecule quantitation. The great strength of this approach is the precision and clarity of mechanistic insight. Although the study does not use "in transfecto" or in vivo models the experiments are performed using "physiologically-based" conditions and provide a powerful insight into core regulatory principles that will be relevant in vivo.<br /> The results are beautiful, high quality, well controlled and internally consistent (and with other published work that overlaps on some points) and as a result are compelling. The primary conclusion is that the primary regulator of PI3Kbeta are tyrosine phosphorylated peptides (and by inference tyrosine phsophorylated receptors/adaptors) and that the other activators can synergise with that input but have relatively weak impacts on their own.

Although the methodology is not easily imported, for reasons of both cost and the experience needed to execute them well, the results have broad importance for the field and reverse an impression that had built in large parts of the broader signalling and PI3K communities that all of the inputs to PI3Kbeta were relatively equivalent, however, these conclusions were based on "in cell" or in vivo studies that were very difficult to interpret clearly.

Reviewer #1 (Public Review):

In this revised preprint the authors investigate whether a presumably allosteric P2RX7 activating compound that they previously discovered reduces fibrosis in a bleomycin mouse model. They chose this particular model as publicly available mRNA data indicate that the P2RX7 pathway is downregulated in idiopathic pulmonary fibrosis patients compared to control individuals. In their revised manuscript, the authors use three proxies of lung damage, Ashcroft score, collagen fibers, and CD140a+ cells, to assess lung damage following the administration of bleomycin. These metrics are significantly reduced on HEI3090 treatment. Additional data implicate specific immune cell infiltrates and cytokines, namely inflammatory macrophages and damped release of IL-17A, as potential mechanistic links between their compound and reduced fibrosis. Finally, the researchers transplant splenocytes from WT, NLRP3-KO, and IL-18-KO mice into animals lacking the P2RX7 receptor to specifically ascertain how the transplanted splenocytes, which are WT for P2RX7 receptor, respond to HEI3090 (a P2RX7 agonist). Based on these results, the authors conclude that HEI3090 enhanced IL-18 production through the P2RX7-NLRP3 inflammasome axis to dampen fibrosis.

These findings could be interesting to the field, as there are conflicting results as to whether NLRP3 activation contributes to fibrosis and if so, at what stage(s) (e.g., acute damage phase versus progression). The revised manuscript is more convincing in that three orthogonal metrics for lung damage were quantified.

However, deletion of the P2RX7 receptor itself reduces the extent of fibrosis, suggesting that P2RX7 signaling can be pro-fibrotic. In the absence of P2RX7, the effects of HEI3900 are also abolished, suggesting that HEI3900 acts in part via P2RX7 signaling. This suggests a paradox that P2RX7 signaling can be both detrimental and beneficial in fibrosis and there is need for a better understanding of when P2RX7 signaling is beneficial and when it is detrimental in lung fibrosis. HEI3900-induced activation of P2RX7 seems to be beneficial but this primarily is shown for when fibrosis is already established. As the P2RX7 genetic deletion mouse model has less fibrosis, P2RX7 signaling and inflammasome activation may be deleterious during the formation of disease but it is also possible that HEI3900 has other beneficial effects that are not directly related to P2RX7.

Molecularly, additional evidence on specificity, such as thermal proteome profiling and direct biophysical binding experiments, would also enhance the authors' argument that the compound indeed binds P2RX7 directly and specifically. Since all small molecules have some degree of promiscuity, the absence of an additional P2RX7 modulator, or direct recombinant IL-18 administration, is needed to orthogonally validate the functional importance of this pathway. Another way the authors could probe pathway specificity would involve co-administering α-IL-18 with HEI3090 in several key experiments (similar to Figure 4L).

Reviewer #1 (Public Review):

Summary:<br /> The study follows the role of yeast eIF2A protein as potential translation initiation factor engaged in the non-canonical translation initiation under stress conditions and as a substitute for eIF2. Using ribosome profiling, RNA-Seq and reporter based assays authors evaluated the role of eIF2A protein under regular or stress conditions (cells starved for branched amino acids). Authors found that yeast cells depleted of eIF2A protein do not change significantly their translation initiation, or translation in general. In the contrast to previously reported data for human homolog yeast eIF2A does not significantly contribute to regulation of the uORFs, regardless if they start with canonical AUG or near cognate start codons. eIF2A is not involved in the repression of IRES element in URE2 gene or has a role in purine biosynthesis. It appears that in yeast eIF2A contributes to regulation of very limited number of mRNAs (32 with significant changes in translation efficiency), where only 17 of such messages indeed are consistent with eIF2A deletion and single mRNA (HKR1) could be validated in reporter assay.

Strengths:<br /> The strength of the manuscript is complete analysis and unbiased approach using genomic analysis methods (ribosome profiling and RNA-seq) as well as reporter validation studies. Additional strength of the manuscript is scientific rigor and statistics associated with data analyses, clear data presentation and discussion of the results in the context of the previous studies and results.

Weaknesses:<br /> none noted

Reviewer #1 (Public Review):

In their study, Osorio-Valeriano and colleagues seek to understand how bacterial-specific polymerizing proteins called bactofilins contribute to morphogenesis. They do this primarily in the stalked budding bacterium Hyphomonas neptunium, with supporting work in a spiral-shaped bacterium, Rhodospirillum rubrum. Overall the study incorporates bacterial genetics and physiology, imaging, and biochemistry to explore the function of bactofilins and cell wall hydrolases that are frequently encoded together within an operon. They demonstrate an important, but not essential, function for BacA in morphogenesis of H. neptunium. Using biochemistry and imaging, they show that BacA can polymerize and that its localization in cells is dynamic and cell-cycle regulated. They further demonstrate that BacA likely limits movement of the elongasome into the stalk, spatially confining its activity. The authors then focus on lmdC, which encodes a putative M23 endopeptidase upstream of bacA in H. neptunium, and find that is essential for viability. The purified LmdC C-terminal domain could cleave E. coli peptidoglycan in vitro suggesting that it is a DD-endopeptidase. LmdC interacts directly with BacA in vitro and co-localizes with BacA in cells. To expand their observations, the authors then explore a related endopeptidase/bactofilin pair in R. rubrum; those observations support a function for LmdC and BacA in R. rubrum morphogenesis as well.

An overall strength of this study is the breadth and completeness of approaches used to assess bactofilin and endopeptidase function in cells and in vitro. The authors establish a clear function for BacA in morphogenesis in two bacterial systems, and demonstrate a physical relationship between BacA and the cell wall hydrolase LmdC that may be broadly conserved. The eventual model the authors favor for BacA regulation of morphogenesis in H. neptunium is that it serves as a diffusion barrier and limits movement of morphogenetic machinery like the elongasome into the elongating stalk and/or bud.

The data presented illuminate aspects of bacterial morphogenesis and the physical and functional relationship between polymerizing proteins and cell wall enzymes in bacteria, a recurring theme in bacterial cell biology with a variety of underlying mechanisms. Bactofilins in particular are relatively recently discovered and any new insights into their functions and mechanisms of action are valuable. The findings presented here are likely to interest those studying bacterial morphogenesis, peptidoglycan, and cytoskeletal function.

Joint Public Review:

Summary:

This paper reports how mycobacterial cAMP level is increased under stressful conditions and that the increase is important in the survival of the bacterium in animal hosts.

Strengths:

The authors show that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of a PDE specific to cAMP is significant progress in understanding Mtb pathogenesis. An increase in cAMP apparently increases bacterial survival upon infection. On the practical side, the reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP level. The results here are straightforward, internally consistent, and of both theoretical and applied interests.

Weaknesses:

Repression of PDE promoter by binding of phosphorylated PhoP could have been shown at higher precision. The binding is now somewhere along a roughly 500 bp region. Although the regulation of PDE is shown to be by transcriptional repression only, it has been described as a homeostatic mechanism. The latter would have required a demonstration of both repression and activation by negative feedback.

Reviewer #1 (Public Review):

The paper aims to determine the impact of forest cover and fragmentation on the prevalence of malaria in non-human primates. The paper uses existing spatial datasets, as well as data obtained through published studies on zoonotic malaria. The findings of this study are important, as forest loss is still occurring in the tropics which will impact human infections of zoonotic malaria.

Reviewer #1 (Public Review):

The authors succeeded in establishing experimental and mathematical models for the formation of new blood vessels. The experimental model relies on temporal imaging of multilcellular projections and lumen formation from a single blood vessel embedded in an engineered extracellular matrix. The mathematical model combines both discrete and continuum elements. It would be helpful to understand how the authors came up with phenotypic classes for analyzing their live imaging data. On the modeling side, it would be useful to see whether the claims about Turing patterns could be supported by either a mean-field model or a more thorough parametric analysis of the discreet continuum model. The authors did a good job in comparing their VEGF/Notch mechanism to the EGF/Notch vulval patterning mechanism in C. elegans. The authors might want to look into the literature from studies of the tracheal patterning system in Drosophila when the combined actions of the FGF and Notch signaling specify tip and stalk cells. The similarities are quite striking and are worth noting.

Reviewer #1 (Public Review):

Summary

This article by Zhai et al, investigates sterol transport in bacteria. Synthesis of sterols is rare in bacteria but occurs in some, such as, M capsulatus where the sterols are found primarily in the outer membrane. In a previous paper the authors discovered an operon consisting of five genes, with two of these genes encoding demethylases involved in sterol demethylation. In this manuscript the authors set out to investigate the functions of the other three genes in the operon. Interestingly, through a bioinformatic analysis they show that they are an inner membrane transporter of the RND family, a periplasmic binding protein and an outer membrane associated protein, all potentially involved with lipid transport, so providing a means of transporting the lipids to the outer membrane. These proteins are then extensively investigated through lipid pulldowns, binding analysis on all three, and X-ray crystallography and docking of the latter two.

Strengths<br /> The lipid pulldowns and associated MST binding analysis are convincing, clearly showing that sterols are able to bind to these proteins. The structures of BstB and BstC are high resolution with excellent maps that allow docking studies to be carried out. These structures are distinct from sterol binding proteins in eukaryotes.

Weaknesses<br /> While the docking and molecular dynamics studies are consistent with the binding of sterols to BstB and BstC, this is not backed up particularly well. Their discussion, however, is measured and clearly provides a strong case for further investigation.

Reviewer #1 (Public Review):

Summary:

This paper makes important contributions to the structural analysis of the DNA replication-linked nucleosome assembly machine termed Chromatin Assembly Factor-1 (CAF-1). The authors focus on the interplay of domains that bind DNA, histones and replication clamp protein PCNA.

Strengths:<br /> The authors analyze soluble complexes containing full-length versions of all three fission yeast CAF-1 subunits, an important accomplishment given that many previous structural and biophysical studies have focused on truncated complexes. New data here supports previous experiments indicating that the KER domain is a long alpha helix that binds DNA. Via NMR, the authors discover structural changes at the histone binding site, defined here with high resolution. Most strikingly, the experiments here show that for the S. pombe CAF-1 complex, that the WHD domain at the C-terminus of the large subunit lacks DNA binding activity observed in the human and budding yeast homologs, indicating a surprising divergence in the evolution of this complex. Together, these are important contributions to the understanding of how the CAF-1 complex works.

Weaknesses:<br /> 1. Given the strong structural predication about the roles of residues L359 and F380 (Fig. 2f), mutation of these residues would be the definitive test of their contribution to histone binding.

2. Could it be that the apparent lack of histone deposition by the delta-WHD mutant complex occurs because this mutant complex is unstable when added to the Xenopus extract?

Reviewer #1 (Public Review):

The study investigates the role of PARP-1 in transcriptional regulation. Biochemical and ChIP-seq analyses demonstrate specific binding of PARP-1 to active histone marks, particularly H4K20me, in polytene chromosomes of Drosophila third instar larvae. Under heat stress conditions, PARP-1's dynamic repositioning from the Hsp70 promoter to its gene body is observed, facilitating gene activation. PARP-1, in conjunction with PR-Set7, plays a crucial role in the activation of Hsp70 and a subset of heat shock genes, coinciding with an increase in H4K20me1 levels at these gene loci. This study proposes that H4K20me1 is a key facilitator of PARP-1 binding and gene regulation. However, there are several critical concerns that are yet to be addressed. The experimental validation and demonstration of results in the main manuscript are scant. Recent developments in the area are omitted, as an important publication hasn't been discussed anywhere in the work (PMID: 36434141). The proposed mechanism operates quite selectively, and any extrapolations require intensive scientific evidence.

Major Comments:

1. PARP1 hypomorphic mutant validation data must be provided at RNA levels as the authors have mentioned about its global reduction in RNA levels.

2. The authors should provide immunoblot data for global Poly (ADP) ribosylation levels in PARP1 hypomorphic mutant condition as compared to the control. They must also provide the complete details of the mouse anti-pADPr antibody used in their immunoblot in Figure 5B.

3. PR-Set7 mutant validation results should be provided in the main manuscript, as done by the authors using qRT-PCR. Also, immunoblot data for the PR-set7 null condition should be supplemented in the main manuscript as the authors have already mentioned their anti-PR-Set7 (Rabbit, 1:1000, Novus Biologicals, 44710002) antibody in the materials and methods section.

4. The authors have probably missed out on a very important recent report (PMID: 36434141), suggesting the antagonistic nature of the PARP1 and PR-SET7 association. In light of these important observations, the authors must check for the levels of PR-Set7 in PARP1 hypomorphic conditions.

5. Also, the results of the aforementioned study should be adequately discussed in the present study along with its implications in the same.

6. Gene transcriptional activation requires open chromatin and RNA polymerase II binding to the promoter. Since, differentially expressed genes in both PR-Set7 null and PARP1 hypomorph mutants, co-enriched with PARP-1 and H4K20me1 were mainly upregulated, the authors should provide RNA polymerase II occupancy data of these genes via RNA-Pol II ChIP-seq to further attest their claims.

7. As discussed in Figure 4, the authors found transcriptional activation of group B genes even after a significant reduction of H3K20me1 in their gene body after heat shock. Given the dynamic equilibrium shift in epigenetic marks that regulate gene expression and their locus-specific transcriptional regulation, the authors should further look for the enrichment of other epigenetic marks and even H4K20me1 specific demethylases such as PHF8 (PMID: 20622854), and their cross-talk with PARP1 to further bridge the missing links of this tale. This will add more depth to this work.

Reviewer #1 (Public Review):

Summary:<br /> Ngoune et al. present compelling evidence that Slender cells are challenged to infect tsetse flies. They explore the experimental context of a recent important paper in the field, Schuster et al., that presents evidence suggesting the proliferative Slender bloodstream T.brucei can infect juvenile tsetse flies. Schuster et al. was disruptive to the widely accepted paradigm that the Stumpy bloodstream form is solely responsible for tsetse infection and T.brucei transmission potential.

Evidence presented here shows that in all cases, Stumpy form parasites are exponentially more capable of infecting tsetse flies. They further show that Slender cells do not infect mature flies.

However, they raise questions of immature tsetse immunological potential and field transmission potential that their experiments do not address. Specifically, they do not show that teneral tsetse flies are immunocompromised, that tsetse flies must be immunocompromised for Slender infection nor that younger teneral tsetse infection is not pertinent to field transmission.

Strengths:<br /> Experimental Design is precise and elegant, outcomes are convincing. Discussion is compelling and important to the field. This is a timely piece that adds important data to a critical discussion of host:parasite interactions, of relevance to all parasite transmission.

Weaknesses:<br /> As above, the authors dispute the biological relevance of teneral tsetse infection in the wild, without offering evidence to the contrary. Statements need to be softened for claims regarding immunological competence or relevance to field transmission.

Joint Public Review:

The authors develop reporter constructs in E. coli that are repressed by the mammalian Musashi-1 (MSI-1) RNA-binding protein. Using a set of rigorously controlled experiments, the authors convincingly show that MSI-1 can be directed to control translation, and that translational control by MSI-1 can be modulated allosterically by oleic acid. This is a potentially useful tool for synthetic biologists, with the advantage over transcriptional regulation that one gene in an operon could be targeted. The authors' MSI-1-regulated reporter constructs could also be useful for mechanistic studies of MSI-1.

The authors initial construct design led to only weak regulation by MSI-1, presumably because the MSI-1 binding sites were not suitably positioned to repress translation initiation. A more rationally designed construct led to considerably greater repression. A minor weakness of the paper is that the authors used their initial, weakly regulated construct to assess the effect of MSI-1 binding site mutations and for their mathematical modeling; these experiments would be better suited to the more strongly regulated construct.

Reviewer #1 (Public Review):

Summary:<br /> The authors aim to address a critical challenge in the field of bioinformatics: the accurate and efficient identification of protein binding sites from sequences. Their work seeks to overcome the limitations of current methods, which largely depend on multiple sequence alignments or experimental protein structures, by introducing GPSite, a multi-task network designed to predict binding residues of various molecules on proteins using ESMFold.

Strengths:<br /> 1. Benchmarking. The authors provide a comprehensive benchmark against multiple methods, showcasing the performances of a large number of methods in various scenarios.

2. Accessibility and Ease of Use. GPSite is highlighted as a freely accessible tool with user-friendly features on its website, enhancing its potential for widespread adoption in the research community.

Weaknesses:<br /> 1. Lack of Novelty. The method primarily combines existing approaches and lacks significant technical innovation. This raises concerns about the original contribution of the work in terms of methodological development. Moreover, the paper reproduces results and analyses already presented in previous literature, without providing novel analysis or interpretation. This further diminishes the contribution of this paper to advancing knowledge in the field.

2. Benchmark Discrepancies. The variation in benchmark results, especially between initial comparisons and those with PeSTo. GPSite achieves a PR AUC of 0.484 on the global benchmark but a PR AUC of 0.61 on the benchmark against PeSTo. For consistency, PeSTo should be included in the benchmark against all other methods. It suggests potential issues with the benchmark set or the stability of the method. This inconsistency needs to be addressed to validate the reliability of the results.

3. Interface Definition Ambiguity. There is a lack of clarity in defining the interface for the binding site predictions. Different methods are trained using varying criteria (surfaces in MaSIF-site, distance thresholds in ScanNet). The authors do not adequately address how GPSite's definition aligns with or differs from these standards and how this issue was addressed. It could indicate that the comparison of those methods is unreliable and unfair.

While GPSite demonstrates the potential to surpass state-of-the-art methods in protein binding site prediction, the evidence supporting these claims seems incomplete. The lack of methodological novelty and the unresolved questions in benchmark consistency and interface definition somewhat undermine the confidence in the results. Therefore, it's not entirely clear if the authors have fully achieved their aims as outlined.

The work is useful for the field, especially in disease mechanism elucidation and novel drug design. The availability of genome-scale binding residue annotations GPSite offers is a significant advancement. However, the utility of this tool could be hampered by the aforementioned weaknesses unless they are adequately addressed.

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

Summary:<br /> In this study, the authors investigate a very interesting but often overlooked aspect of abstract vs. concrete processing in language. Specifically, they study if the differences in processing of abstract vs. concrete concepts in the brain are static or dependent on the (visual) context in which the words occur. This study takes a two-step approach to investigate how context might affect the perception of concepts. First, the authors analyze if concrete concepts, expectedly, activate more sensory systems while abstract concepts activate higher-order processing regions. Second, they measure the contextual situatedness vs. displacement of each word with respect to the visual scenes it is spoken in and then evaluate if this contextual measure correlates with more activation in the sensory vs. higher-order regions respectively.

Strengths:<br /> This study raises a pertinent and understudied question in language neuroscience. It also combines both computational and meta-analytic approaches.

Weaknesses:<br /> Overall, the study had many intermediary steps that required manual subsection / random sampling and variable choices (like the time lag of analysis) with almost no visualization and interpretation of how these choices affect the observed results. The approach was also roundabout.

Peaks and Valleys Analysis:<br /> 1. Doesn't this method assume that the features used to describe each word, like valence or arousal, will be linearly different for the peaks and valleys? What about non-linear interactions between the features and how they might modulate the response?<br /> 2. Doesn't it also assume that the response to a word is infinitesimal and not spread across time? How does the chosen time window of analysis interact with the HRF? From the main figures and Figures S2-S3 there seem to be differences based on the timelag.<br /> 3. Were the group-averaged responses used for this analysis?<br /> 4. Why don't the other terms identified in Figure 5 show any correspondence to the expected categories? What does this mean? Can the authors also situate their results with respect to prior findings as well as visualize how stable these results are at the individual voxel or participant level? It would also be useful to visualize example time courses that demonstrate the peaks and valleys.

Estimating contextual situatedness:<br /> 1. Doesn't this limit the analyses to "visual" contexts only? And more so, frequently recognized visual objects?<br /> 2. The measure of situatedness is the cosine similarity of GloVE vectors that depend on word co-occurrence while the vectors themselves represent objects isolated by the visual recognition models. Expectedly, "science" and the label "book" or "animal" and the label "dog" will be close. But can the authors provide examples of context displacement? I wonder if this just picks up on instances where the identified object in the scene is unrelated to the word. How do the authors ensure that it is a displacement of context as opposed to the two words just being unrelated? This also has a consequence on deciding the temporal cutoff for consideration (2 seconds).<br /> 3. While the introduction motivated the problem of context situatedness purely linguistically, the actual methods look at the relationship between recognized objects in the visual scene and the words. Can word surprisal or another language-based metric be used in place of the visual labeling? Also, it is not clear how the process identified in (2) above would come up with a high situatedness score for abstract concepts like "truth".<br /> 4. It is a bit hard to see the overlapping regions in Figures 6A-C. Would it be possible to show pairs instead of triples? Like "abstract across context" vs. "abstract displaced"? Without that, and given (2) above, the results are not yet clear. Moreover, what happens in the "overlapping" regions of Figure 3?

Miscellaneous comments:<br /> 1. In Figure 3, it is surprising that the "concrete-only" regions dominate the angular gyrus and we see an overrepresentation of this category over "abstract-only". Can the authors place their findings in the context of other studies?<br /> 2. The following line (Pg 21) regarding the necessary differences in time for the two categories was not clear. How does this fall out from the analysis method?<br /> 3. Both categories overlap **(though necessarily at different time points)** in regions typically associated with word processing.

Reviewer #1 (Public Review):

The manuscript by Wagstyl et al. describes an extensive analysis of gene expression in the human cerebral cortex and the association with a large variety of maps capturing many of its microscopic and macroscopic properties. The core methodological contribution is the computation of continuous maps of gene expression for >20k genes, which are being shared with the community. The manuscript is a demonstration of several ways in which these maps can be used to relate gene expression with histological features of the human cortex, cytoarchitecture, folding, function, development and disease risk. The main scientific contribution is to provide data and tools to help substantiate the idea of the genetic regulation of multi-scale aspects of the organisation of the human brain. The manuscript is dense, but clearly written and beautifully illustrated.

Reviewer #1 (Public Review):

Despite numerous studies on quinidine therapies for epilepsies associated with GOF mutant variants of Slack, there is no consensus on its utility due to contradictory results. In this study Yuan et al. investigated the role of different sodium selective ion channels on the sensitization of Slack to quinidine block. The study employed electrophysiological approaches, FRET studies, genetically modified proteins and biochemistry to demonstrate that Nav1.6 N- and C-tail interacts with Slack's C-terminus and significantly increases Slack sensitivity to quinidine blockade in vitro and in vivo. This finding inspired the authors to investigate whether they could rescue Slack GOF mutant variants by simply disrupting the interaction between Slack and Nav1.6. They find that the isolated C-terminus of Slack can reduce the current amplitude of Slack GOF mutant variants co-expressed with Nav1.6 in HEK cells and prevent Slack induced seizures in mouse models of epilepsy. This study adds to the growing list of channels that are modulated by protein-protein interactions, and is of great value for future therapeutic strategies.

Reviewer #1 (Public Review):

In this preprint, Zhang et al. describe a new tool for mapping the connectivity of mouse neurons. Essentially, the tool leverages the known peculiar infection capabilities of Rabies virus: once injected into a specific site in the brain, this virus has the capability to "walk upstream" the neural circuits, both within cells and across cells: on one hand, the virus can enter from a nerve terminal and infect retrogradely the cell body of the same cell (retrograde transport). On the other hand, the virus can also sometimes spread to the presynaptic partners of the initial target cells, via retrograde viral transmission.

Similarly to previously published approaches with other viruses, the authors engineer a complex library of viral variants, each carrying a unique sequence ('barcode'), so they can uniquely label and distinguish independent infection events and their specific presynaptic connections, and show that it is possible to read these barcodes in-situ, producing spatial connectivity maps. They also show that it is possible to read these barcodes together with endogenous mRNAs, and that this allows spatial mapping of cell types together with anatomical connectivity.

The main novelty of this work lies in the combined use of rabies virus for retrograde labeling together with barcoding and in-situ readout. Previous studies had used rabies virus for retrograde labeling, albeit with low multiplexing capabilities, so only a handful of circuits could be traced at the same time. Other studies had instead used barcoded viral libraries for connectivity mapping, but mostly focused on the use of different viruses for labeling individual projections (anterograde tracing) and never used a retrograde-infective virus.

The authors creatively merge these two bits of technology into a powerful genetic tool, and extensively and convincingly validate its performance against known anatomical knowledge. The authors also do a very good job at highlighting and discussing potential points of failure in the methods.

Unresolved questions, which more broadly affect also other viral-labeling methods, are for example how to deal with uneven tropism (ie. if the virus is unable or inefficient in infecting some specific parts of the brain), or how to prevent the cytotoxicity induced by the high levels of viral replication and expression, which will tend to produce "no source networks", neural circuits whose initial cell can't be identified because it's dead. This last point is particularly relevant for in-situ based approaches: while high expression levels are desirable for the particular barcode detection chemistry the authors chose to use (gap-filling), they are also potentially detrimental for cell survival, and risk producing extensive cell death (which indeed the authors single out as a detectable pitfall in their analysis). This is likely to be one the major optimisation space for future implementations of this barcoding approach.

Overall the paper is well balanced, the data are well presented and the conclusions are strongly supported by the data. Impact-wise, the method is definitely going to be very useful for the neurobiology community.

Reviewer #2 (Public Review):

Summary: In the revised manuscript, the authors aim to investigate brain-wide activation patterns following administration of the anesthetics ketamine and isoflurane, and conduct comparative analysis of these patterns to understand shared and distinct mechanisms of these two anesthetics. To this end, they perform Fos immunohistochemistry in perfused brain sections to label active nuclei, use a custom pipeline to register images to the ABA framework and quantify Fos+ nuclei, and perform multiple complementary analyses to compare activation patterns across groups.

In the latest revision, the authors have made some changes in response to our previous comments on how to fix the analyses. However, the revised analyses were not changed correctly and remain flawed in several fundamental ways.

Critical problems:

(1) Before one can perform higher level analyses such as hiearchal cluster or network hub (or PC) analysis, it is fundamental to validate that you have significant differences of the raw Fos expression values in the first place. First of all, this means showing figures with the raw data (Fos expression levels) in some form in Figures 2 and 3 before showing the higher level analyses in Figures 4 and 5; this is currently switched around. Second and most importantly, when you have a large number of brain areas with large differences in mean values and variance, you need to account for this in a meaningful way. Changing to log values is a step in the right direction for mean values but does not account well for differences in variance. Indeed, considering the large variances in brain areas with high mean values and variance, it is a little difficult to believe that all brain regions, especially brain areas with low mean values, passed corrections for multiple comparisons test. We suggested Z-scores relative to control values for each brain region; this would have accounted for wide differences in mean values and variance, but this was not done. Overall, validation of anesthesia-induced differences in Fos expression levels is not yet shown.

(2) Let's assume for a moment that the raw Fos expression analyses indicate significant differences. They used hierarchal cluster analyses as a rationale for examining 53 brain areas in all subsequent analyses of Fos expression following isoflurane versus home cage or ketamine versus saline. Instead, the authors changed to 201 brain areas with no validated rationale other than effectively saying 'we wanted to look at more brain areas'. And then later, when they examined raw Fos expression values in Figures 4 and 5, they assess 43 brain areas for ketamine and 20 brain areas for isoflurane, without any rationale for why choosing these numbers of brain areas. This is a particularly big problem when they are trying to compare effects of isoflurane versus ketamine on Fos expression in these brain areas - they did not compare the same brain areas.

Less critical comments:

(3) The explanation of hierarchical level's in lines 90-95 did not make sense.

(4) I am still perplexed by why the authors consider the prelimbic and infralimbic cortex 'neuroendocrine' brain areas in the abstract. In contrast, the prelimbic and infralimbic were described better in the introduction as "associated information processing" areas.

5- It looks like overall Fos levels in the control group Home (ISO) are a magnitude (~10-fold) lower than those in the control group Saline (KET) across all regions shown. This large difference seems unlikely to be due to a biologically driven effect and seems more likely to be due to a technical issue, such as differences in staining or imaging between experiments. The authors discuss this issue but did not answer whether the Homecage-ISO experiment or at least the Fos labeling and imaging performed at the same time as for the Saline-Ketamine experiment?

Reviewer #1 (Public Review):

Summary:<br /> The authors ran a series of experiments with separate subject populations, different stimuli, and on two different MRI scanners (one 3T, one 7T) to establish a scenes-selective region on the intraparietal gyrus that they decided to name PIGS. I think that IPA (intraparietal place area) would also have been a good choice with an allusion to a beverage rather than a domestic animal. The authors show that PIGS can be detected robustly through a series of experiments. They anatomically and functionally separate PIGS from nearby V6, which encodes optic flow. The authors determined that PIGS encodes ego-motion.

Strengths:<br /> The robust detection of PIGS in several experiments with different sets of participants and on different scanners makes these results convincing. The functional differentiation is well executed.

Weaknesses:<br /> The distinction of PIGS from nearby OPA, which has also been implied in navigation and ego-motion, is not as clear as it could be.

Impact:<br /> Overall, this is a valuable contribution to the cognitive neuroscience of the visual system. It shows that there is still room for discovering details of visual processing, given recent advances in scanning technology, statistical methods, and larger sample sizes.

Reviewer #1 (Public Review):

Summary:<br /> The present study evaluates the role of visual experience in shaping functional correlations between extrastriate visual cortex and frontal regions. The authors used fMRI to assess "resting-state" temporal correlations in three groups: sighted adults, congenitally blind adults, and neonates. Previous research has already demonstrated differences in functional correlations between visual and frontal regions in sighted compared to early blind individuals. The novel contribution of the current study lies in the inclusion of an infant dataset, which allows for an assessment of the developmental origins of these differences.

The main results of the study reveal that correlations between prefrontal and visual regions are more prominent in the blind and infant groups, with the blind group exhibiting greater lateralization. Conversely, correlations between visual and somato-motor cortices are more prominent in sighted adults. Based on these data, the authors conclude that visual experience plays an instructive role in shaping these cortical networks. This study provides valuable insights into the impact of visual experience on the development of functional connectivity in the brain.

Strengths:<br /> The dissociations in functional correlations observed among the sighted adult, congenitally blind, and neonate groups provide strong support for the study's main conclusion regarding experience-driven changes in functional connectivity profiles between visual and frontal regions.

In general, the findings in sighted adult and congenitally blind groups replicate previous studies and enhance the confidence in the reliability and robustness of the current results.

Split-half analysis provides a good measure of robustness in the infant data.

Weaknesses:<br /> There is some ambiguity in determining which aspects of these networks are shaped by experience.

This uncertainty is compounded by notable differences in data acquisition and preprocessing methods, which could result in varying signal quality across groups. Variations in signal quality may, in turn, have an impact on the observed correlation patterns.

The study's findings could benefit from being situated within a broader debate surrounding the instructive versus permissive roles of experience in the development of visual circuits.

Reviewer #1 (Public Review):

Summary:

Songbirds provide a tractable system to examine neural mechanisms of sequence generation and variability. In past work, the projection from LMAN to RA (output of the anterior forebrain pathway) was shown to be critical for driving vocal variability during babbling, learning, and adulthood. LMAN is immediately adjacent to MMAN, which projects to HVC. MMAN is less well understood but, anatomically, appears to resemble LMAN in that it is the cortical output of a BG-thalamocortical loop. Because it projects to HVC, a major sequence generator for both syllable phonology and sequence, a strong prediction would be that MMAN drives sequence variability in the same way that LMAN drives phonological variability. This hypothesis predicts that MMAN lesions in a Bengalese finch would reduce sequence variability. Here, the authors test this hypothesis. They provide a surprising and important result that is well motivated and well analyzed: MMAN lesions increase sequence variability - this is exactly the opposite result from what would be predicted based on the functions of LMAN.

Strengths:

1. A very important and surprising result shows that lesions of a frontal projection from MMAN to HVC, a sequence generator for birdsong, increase syntactical variability.

2. The choice of Bengalese finches, which have complex transition structures, to examine the mechanisms of sequence generation, enabled this important discovery.

3. The idea that frontal outputs of BG-cortical loops can generate vocal variability comes from lesions/inactivations of a parallel pathway from LMAN to RA. The difference between MMAN and LMAN functions is striking and important.

Weaknesses:

1. If more attention was paid to how syllable phonology was (or was not) affected by MMAN lesions then the claims could be stronger around the specific effects on sequence.

Reviewer #1 (Public Review):

Summary:<br /> The authors investigated causal inference in the visual domain through a set of carefully designed experiments, and sound statistical analysis. They suggest the early visual system has a crucial contribution to computations supporting causal inference.

Strengths:<br /> I believe the authors target an important problem (causal inference) with carefully chosen tools and methods. Their analysis rightly implies the specialization of visual routines for causal inference and the crucial contribution of early visual systems to perform this computation. I believe this is a novel contribution and their data and analysis are in the right direction.

Weaknesses:<br /> In my humble opinion, a few aspects deserve more attention:

1. Causal inference (or causal detection) in the brain should be quite fundamental and quite important for human cognition/perception. Thus, the underlying computation and neural substrate might not be limited to the visual system (I don't mean the authors did claim that). In fact, to the best of my knowledge, multisensory integration is one of the best-studied perceptual phenomena that has been conceptualized as a causal inference problem. Assuming the causal inference in those studies (Shams 2012; Shams and Beierholm 2022; Kording et al. 2007; Aller and Noppeney 2018; Cao et al. 2019) (and many more e.g., by Shams and colleagues), and the current study might share some attributes, one expects some findings in those domains are transferable (at least to some degree) here as well. Most importantly, underlying neural correlates that have been suggested based on animal studies and invasive recording that has been already studied, might be relevant here as well. Perhaps the most relevant one is the recent work from the Harris group on mice (Coen et al. 2021). I should emphasize, that I don't claim they are necessarily relevant, but they can be relevant given their common roots in the problem of causal inference in the brain. This is a critical topic that the authors may want to discuss in their manuscript.

2. If I understood correctly, the authors are arguing pro a mere bottom-up contribution of early sensory areas for causal inference (for instance, when they wrote "the specialization of visual routines<br /> for the perception of causality at the level of individual motion directions raises the possibility that this function is located surprisingly early in the visual system *as opposed to a higher-level visual computation*."). Certainly, as the authors suggested, early sensory areas have a crucial contribution, however, it may not be limited to that. Recent studies progressively suggest perception as an active process that also weighs in strongly, the top-down cognitive contributions. For instance, the most simple cases of perception have been conceptualized along this line (Martin, Solms, and Sterzer 2021)<br /> and even some visual illusion (Safavi and Dayan 2022), and other extensions (Kay et al. 2023). Thus, I believe it would be helpful to extend the discussion on the top-down and cognitive contributions of causal inference (of course that can also be hinted at, based on recent developments). Even adaptation, which is central in this study can be influenced by top-down factors (Keller et al. 2017). I believe, based on other work of Rolfs and colleagues, this is also aligned with their overall perspective on vision.

3. The authors rightly implicate the neural substrate of causal inference in the early sensory system. Given their study is pure psychophysics, a more elaborate discussion based on other studies that used brain measurements is needed (in my opinion) to put into perspective this conclusion. In particular, as I mentioned in the first point, the authors mainly discuss the potential neural substrate of early vision, however much has been done about the role of higher-tier cortical areas in causal inference e.g., see (Cao et al. 2019; Coen et al. 2021).

There were many areas in this manuscript that I liked: clever questions, experimental design, and statistical analysis.

Bibliography<br /> \============

Aller, Mate, and Uta Noppeney. 2018. "To Integrate or Not to Integrate: Temporal Dynamics of Bayesian Causal Inference." Biorxiv, December, 504118. .

Cao, Yinan, Christopher Summerfield, Hame Park, Bruno Lucio Giordano, and Christoph Kayser. 2019. "Causal Inference in the Multisensory Brain." Neuron 102 (5): 1076-87.e8. .

Coen, Philip, Timothy P. H. Sit, Miles J. Wells, Matteo Carandini, and Kenneth D. Harris. 2021. "The Role of Frontal Cortex in Multisensory Decisions." Biorxiv, April. Cold Spring Harbor Laboratory, 2021.04.26.441250. .

Kay, Kendrick, Kathryn Bonnen, Rachel N. Denison, Mike J. Arcaro, and David L. Barack. 2023. "Tasks and Their Role in Visual Neuroscience." Neuron 111 (11). Elsevier: 1697-1713. .

Keller, Andreas J, Rachael Houlton, Björn M Kampa, Nicholas A Lesica, Thomas D Mrsic-Flogel, Georg B Keller, and Fritjof Helmchen. 2017. "Stimulus Relevance Modulates Contrast Adaptation in Visual Cortex." Elife 6. eLife Sciences Publications, Ltd: e21589.

Kording, K. P., U. Beierholm, W. J. Ma, S. Quartz, J. B. Tenenbaum, and L. Shams. 2007. "Causal Inference in Multisensory Perception." PloS One 2: e943. .

Martin, Joshua M., Mark Solms, and Philipp Sterzer. 2021. "Useful Misrepresentation: Perception as Embodied Proactive Inference." Trends Neurosci. 44 (8): 619-28. .

Safavi, Shervin, and Peter Dayan. 2022. "Multistability, Perceptual Value, and Internal Foraging." Neuron, August. .

Shams, L. 2012. "Early Integration and Bayesian Causal Inference in Multisensory Perception." In The Neural Bases of Multisensory Processes, edited by M. M. Murray and M. T. Wallace. Frontiers in<br /> Neuroscience. Boca Raton (FL).

Shams, Ladan, and Ulrik Beierholm. 2022. "Bayesian Causal Inference: A Unifying Neuroscience Theory." Neuroscience & Biobehavioral Reviews 137 (June): 104619. .

Reviewer #1 (Public Review):

Summary and strengths. This paper starts with an exceptionally fair and balanced introduction to a topic, the mirror neuron literature, which is often debated and prone to controversies even in the choice of the terminology. In my opinion, the authors made an excellent job in this regard, and I really appreciated it. Then, they propose a novel method to look at population dynamics to compare neural selectivity and alignment between execution and observation of actions performed with different types of grip.

Weakness. Unfortunately, the goal and findings within this well-described framework are less clear to me. The authors aimed to investigate, using a novel analytic approach, whether and to what extent a match exists between population codes and neural dynamics when a monkey performs an action or observes it performed by an experimenter. This motivation stems from the fact that the general evidence in the literature is that the match between visual and motor selectivity of mirror neuron responses is essentially at a chance level. While the approach devised by the author is generally well-described and understandable, the main result obtained confirms this general finding of a lack of matching between the two contexts in 2 out of the three monkeys. Nevertheless, the authors claim that the patterns associated with execution and observation can be re-aligned with canonical correlation, indicating that these distinct neural representations show dynamical similarity that may enable the nervous system to recognize particular actions. This final conclusion is hardly acceptable to me, and constitutes my major concern, at least without a more explicit explanation: how do we know that this additional operation can be performed by the brain? Is this a computational trick to artificially align something that is naturally non-aligned, or can it capture something real and useful?<br /> Based on the accumulated evidence on space-constrained coding of others' actions by mirror neurons (e.g., Caggiano et al. 2009; Maranesi et al. 2017), recent evidence also cited by the authors (Pomper et al. 2023), and the most recent views supported even by the first author of the original discovery (i.e., Vittorio Gallese, see Bonini et al. 2022 on TICS), it seems that one of the main functions of these cells, especially in monkeys, might be to prepare actions and motor responses during social interaction rather than recognizing the actions of others - something that visual brain areas could easily do better than motor ones in most situations. In this perspective, and given the absence of causal evidence so far, the lack of visuo-motor congruence is a potentially relevant feature of the mechanism rather than something to be computationally cracked at all costs.

Specific comments on Results/Methods:<br /> I can understand, based on the authors' hypothesis, that they employed an ANOVA to preliminarily test whether and which of the recorded neurons fit their definition of "mirror neurons". However, given the emphasis on the population level, and the consolidated finding of highly different execution and observation responses, I think it could be interesting to apply the same analysis on (at least also) the whole recorded neuronal population, without any preselection-based on a single neuron statistic. Such preselection of mirror neurons could influence the results of EXE-OBS comparisons since all the neurons activated only during EXE or OBS are excluded. Related to this point, the authors could report the total number of recorded neurons per monkey/session, so that also the fraction of neurons fitting their definition of mirror neuron is explicit.<br /> Furthermore, the comparison of the dynamics of the classification accuracy in figures 4 and 5, and therefore the underlying assumption of subspaces shift in execution and observation, respectively, reveal substantial similarities between monkeys despite the different contexts, which are clearly greater than the similarities among neural subspaces shifts across task epochs: to me, this suggests that the main result is driven by the selected neural populations in different monkeys/implants rather than by an essential property of the neuronal dynamics valid across animals. Could the author comment on this issue? This could easily explain the "strange" result reported in figure 6 for monkey T.

Reviewer #1 (Public Review):

The manuscript by Hariani et al. presents experiments designed to improve our understanding of the connectivity and computational role of Unipolar Brush Cells (UBCs) within the cerebellar cortex, primarily lobes IX and X. The authors develop and cross several genetic lines of mice that express distinct fluorophores in subsets of UBCs, combined with immunocytochemistry that also distinguishes subtypes of UBCs, and they use confocal microscopy and electrophysiology to characterize the electrical and synaptic properties of subsets of so-labelled cells, and their synaptic connectivity within the cerebellar cortex. The authors then generate a computer model to test possible computational functions of such interconnected UBCs.

Using these approaches, the authors report that:<br /> 1) GRP-driven TDtomato is expressed exclusively in a subset (20%) of ON-UBCs, defined electrophysiologically (excited by mossy fiber afferent stimulation via activation of UBC AMPA and mGluR1 receptors) and immunocytochemically by their expression of mGluR1.

2) UBCs ID'd/tagged by mCitrine expression in Brainbow mouse line P079 is expressed in a similar minority subset of OFF-UBCs defined electrophysiologically (inhibited by mossy fiber afferent stimulation via activation of UBC mGluR2 receptors) and immunocytochemically by their expression of Calretinin. However, such mCitrine expression was also detected in some mGluR1 positive UBCs, which may not have shown up electrophysiologically because of the weaker fluorophore expression without antibody amplification.

3) Confocal analysis of crossed lines of mice (GRP X P079) stained with antibodies to mGluR1 and calretinin documented the existence of all possible permutations of interconnectivity between cells (ON-ON, ON-OFF, OFF-OFF, OFF-ON), but their overall abundance was low, and neither their absolute or relative abundance was quantified.

4) A computational model (NEURON ) indicated that the presence of an intermediary UBC (in a polysynaptic circuit from MF to UBC to UBC) could prolong bursts (MF-ON-ON), prolong pauses (MF-ON-OFF), cause a delayed burst (MF-OFF-OFF), cause a delayed pause (MF-OFF-ON) relative to solely MF to UBC synapses which would simply exhibit long bursts (MF-ON) or long pauses (MF-OFF).

The authors thus conclude that the pattern of interconnected UBCs provides an extended and more nuanced pattern of firing within the cerebellar cortex that could mediate longer lasting sensorimotor responses.

The cerebellum's long known role in motor skills and reflexes, and associated disorders, combined with our nascent understanding of its role in cognitive, emotional, and appetitive processing, makes understanding its circuitry and processing functions of broad interest to the neuroscience and biomedical community. The focus on UBCs, which are largely restricted to vestibular lobes of the cerebellum reduces the breadth of likely interest somewhat. The overall design of specific experiments is rigorous and the use of fluorophore expressing mouse lines is creative. The data that is presented and the writing are clear.

Reviewer #1 (Public Review):

Summary:<br /> In this paper, the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage-activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:<br /> The experiments are comprehensive and clearly described, both in the text and in the figures. Statistical analyses are provided throughout.

Weaknesses:<br /> None.

Joint Public Review:

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

Reviewer #1 (Public Review):

Summary:<br /> The authors originally investigated the function of p53 isoforms with an alternative C-terminus encoded by the Alternatively Spliced (AS) exon in place of exon 11 encoding the canonical "α" C-terminal domain. For this purpose, the authors create a mouse model with a specific deletion of the AS exon.

Strengths:<br /> Interestingly, wt or p53ΔAS/ΔAS mouse embryonic fibroblasts did not differ in cell cycle control, expression of well-known p53 target genes, proliferation under hyperoxic conditions, or the growth of tumor xenografts. However, p53-AS isoforms were shown to confer male-specific protection against lymphomagenesis in Eμ-Myc transgenic mice, prone to highly penetrant B-cell lymphomas. In fact, p53ΔAS/ΔAS Eμ-Myc mice were less protected from developing B-cell lymphomas compared to WT counterparts. The important difference that the authors find between WT and p53ΔAS/ΔAS Eμ-Myc males is a higher number of immature B cells in p53ΔAS/ΔAS vs WT mice. Higher expression of Ackr4 and lower expression of Mt2 was found in p53+/+ Eμ-Myc males compared to p53ΔAS/ΔAS counterparts, suggesting that these two transcripts are in part regulators of B-cell lymphomagenesis and enrichment for immature B cells.

Weaknesses:<br /> The manuscript is interesting but the data are not so striking and are very correlative. The authors should add functional experiments to reinforce their hypotheses and to provide, beyond potential prognostic factors, any potential mechanism at the basis of the different rates of B-cell lymphomagenesis in males vs females individuals and in WT vs p53ΔAS/ΔAS Eμ-Myc males.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript "comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation" aims to provide insights into the mediators and mechanisms underlying tardigrade radiation tolerance. The authors start by assessing the effect of ionizing radiation (IR) on the tardigrade lab species, H. exemplaris, as well as the ability of this organism to recover from this stress - specifically, they look at DNA double and single-strand breaks. They go on to characterize the response of H. exemplaris and two other tardigrade species to IR at the transcriptomic level. Excitingly, the authors identify a novel gene/protein called TDR1 (tardigrade DNA damage response protein 1). They carefully assess the induction of expression/enrichment of this gene/protein using a combination of transcriptomics and biochemistry - even going so far as to use a translational inhibitor to confirm the de novo production of this protein. TDR1 binds DNA in vitro and co-localizes with DNA in tardigrades.

Reverse genetics in tardigrades is difficult, thus the authors use a heterologous system (human cells) to express TDR1 in. They find that when transiently expressed TDR1 helps improve human cell resistance to IR.

This work is a masterclass in integrative biology incorporating a holistic set of approaches spanning next-gen sequencing, organismal biology, biochemistry, and cell biology. I find very little to critique in their experimental approaches.

Strengths:<br /> 1. Use of trans/interdisciplinary approaches ('omics, molecular biology, biochemistry, organismal biology)<br /> 2. Careful probing of TDR1 expression/enrichment<br /> 3. Identification of a completely novel protein seemingly involved in tardigrade radio-tolerance.<br /> 4. Use of multiple, diverse, tardigrade species of 'omics comparison.

Weaknesses:<br /> 1. No reverse genetics in tardigrades - all insights into TDR1 function from heterologous cell culture system.<br /> 2. Weak discussion of Dsup's role in preventing DNA damage in light of DNA damage levels measured in this manuscript.<br /> 3. Missing sequence data which is essential for making a complete review of the work.

Overall, I find this to be one of the more compelling papers on tardigrade stress-tolerance I have read. I believe there are points still that the authors should address, but I think the editor would do well to give the authors a chance to address these points as I find this manuscript highly insightful and novel.

Reviewer #1 (Public Review):

Summary: Szathmary and colleagues explore the parabolic growth regime of replicator evolution. Parabolic growth occurs when nucleic acid strain separation is the rate-limiting step of the replication process which would have been the case for non-enzymatic replication of short oligonucleotide that could precede the emergence of ribozyme polymerases and helicases. The key result is that parabolic replication is conducive to the maintenance of genetic diversity, that is, the coexistence of numerous master sequences (the Gause principle does not apply). Another important finding is that there is no error threshold for parabolic replication except for the extreme case of zero fidelity.

Strengths:<br /> I find both the analytic and the numerical results to be quite convincing and well-described. The results of this work are potentially important because they reveal aspects of a realistic evolutionary scenario for the origin of replicators.

Weaknesses:<br /> There are no obvious technical weaknesses. It can be argued that the results represent an incremental advance because many aspects of parabolic replication have been explored previously (the relevant publications are properly cited). Obviously, the work is purely theoretical, experimental study of parabolic replication is due. In the opinion of this reviewer, though, these are understandable limitations that do not actually detract from the value of this work.

Reviewer #1 (Public Review):

In their manuscript, Arjun et al. investigate the role of the histone acetyltransferase Gcn5 in the control of drosophila blood cell homeostasis in the larval lymph gland. They use gcn5 zygotic mutants as well as targeted knock-down and over-expression of Gcn5 in various lymph gland populations to show that these modulations impact (in a rather haphazard manner) niche cell number, blood cell progenitor maintenance, plasmatocyte differentiation, crystal cell differentiation or DNA damage accumulation. Their results suggest that Gcn5 controls autophagy and they show that decreasing the expression of the autophagy machinery increases blood cell differentiation. Using drugs to modulate the mTOR pathway, they conclude that Gcn5 levels are regulated by mTOR but that the impact of this pathway on blood cell homeostasis can override Gcn5 function.

While the authors did a lot of experiments and good quantifications of the blood cell phenotypes, many results do not make much sense or do not bring valuable information about Gcn5 mode of action. Several conclusions of the manuscripts are not backed by solid data (e.g. that Gcn5 action is mediated by TFEB and the autophagy machinery) and different aspects of the literature are not well taken into consideration. Some results (such as the validation of the knockdown and overexpression of Gcn5) seem flawed. There are some concerns about the results obtained with gcn5 zygotic mutants and an interpretation of the phenotypes observed upon manipulation of Gcn5 expression in different cell types is missing.

Important revisions are needed to improve the quality of the manuscript and confirm the authors' findings.

Reviewer #1 (Public Review):

Summary:

This study describes all tangential neurons of the lobula plate (LOPs) of the fruit fly Drosophila melanogaster. Importantly, this is done in a complete manner, for the first time in any species. This means that for the first time, all neurons involved in transmitting wide-field optic flow information to the central brain are known. Exploiting known structure-function relations in these neurons (which are based on solid physiological data in different species of flies), the authors provide estimates of the physiological properties of all described neurons. Combined with transmitter predictions of these cells, this yields a full account of what information about wide-field motion is available to the central fly brain in order to derive behavioral commands from. The study goes one step further and includes anatomical descriptions and physiological property predictions for all major downstream target cells of LOPs.

Main strengths:

The paper is exceptional in three ways. First, it is the first comprehensive account of all tangential neurons of the lobula plate of an insect. This now provides the ground truth for similar studies in other insects. In particular, these results will allow neurons emerging in other species to be confidently described as novel/different from Drosophila, if they were not found in the current study. This is a major change from previously, when confidence in the non-existence of neuronal cell types in this system was impossible, as that system was not fully described.

Second, the rigorous prediction of physiological characteristics (flow-field encoding) in all anatomically described neurons provides a solid basis for system-wide modeling of optic flow encoding in Drosophila. Importantly, the presented physiological predictions include the downstream partner cells of the LOPs in the central brain, neurons for which only very few physiological descriptions exist, but which are essential for transforming optic flow input into behavioral outputs. This paper therefore opens a path towards closing the gap between sensory processing and behavior not only for a few identified and well-studied pathways, but for all wide-field motion processing that exists in a species.

Third, the connectomics work is not only based on one individual sample, but incorporates two EM volumes, analyzed with two different methods (manual tracing and auto segmentation/proofreading), using interhemispheric correspondence and inter-individual correspondence to validate the obtained neuron catalogue. Additionally, light microscopical data was used to validate the EM data. All of this provides exceptional levels of confidence in the presented results.

Main weaknesses:

While the authors compare their results with data from both larger flies and other work in Drosophila, a recent paper (Henning et al 2022) that presented novel data on the distribution of preferred motion directions in the fly lobula plate is not mentioned. This is unfortunate, as the claim of that paper is that the lobula plate contains six instead of four main tuning directions, both at the level of LOPs and T4/T5 input cells - a claim that could likely be directly confirmed or dismissed, or at least incorporated in the data presented in the current study. How would the flow-field predictions change if the data from Henning et al on T4 neurons was used as an input for the modeling rather than the classic four tuning directions?

While the authors nicely perform comparison to other fly species, a more general discussion of how the found cells relate to other insects, e.g. cells known from bees (e.g. Honkanen et al., 2023) or older work from locusts, could give the data more general relevance. While the comparison can likely not be done on a cell type level, given that the structure of the lobula complex is very different between those insects, the types of projections found and their physiologies, i.e. the overall patterns of how wide field motion is sent to the central brain, might be comparable and informative for highlighting general principles of motion processing.

Reviewer #1 (Public Review):

This study examined whether mitochondrial acyl-CoA thioesterase-2 (ACOT2) regulates mitochondrial matrix acyl-CoA levels. Acot2 deletion in murine skeletal muscle (SM) resulted in acyl-CoA build-up. When energy demand and pyruvate availability were elevated, a lack of ACOT2 activity promoted glucose oxidation. This preference for glucose over fatty acid oxidation was recapitulated in C2C12 myotubes with acute depletion of Acot2. In mice fed a high-fat diet, ACOT2 enabled the accretion of acyl-CoAs and ceramide derivatives in glycolytic SM, and this was associated with worse glucose homeostasis compared to when ACOT2 was absent. The authors suggest that ACOT2 supports CoASH availability to facilitate β-oxidation in glycolytic SM when lipid supply is modest. However, when lipid supply is high, ACOT2 enables acyl-CoA and lipid accumulation, CoASH sequestration, and poor glucose homeostasis. Thus, ACOT2 regulates matrix acyl-CoA concentration in glycolytic muscle, and its impact depends on lipid supply.

Based on the data provided in this study, the authors propose that ACOT2 regulates mitochondrial matrix acyl-CoA levels in white skeletal muscle to facilitate fatty acid oxidation β-oxidation. However, I do not believe the data supports this concept, since ACOT2 deletion actually increased fatty acid oxidation in the mitochondrial JO2 studies. In addition, there are some problems with the experimental data that the authors need to address. This includes the experimental conditions used to assess JO2 in the mitochondria, and not using Cre control mice.

Reviewer #1 (Public Review):

The authors focused on genetic variability in relation to insulin resistance. The used genetically different lines of mice and exposed them to the same diet. They found that genetic predisposition impacts the overall outcome of metabolic disturbances.

Reviewer #2 (Public Review):

The authors use a high-throughput sequencing-based enrichment assay to measure how individual amino acids substitutions in the Rep proteins of AAV change the production of AAV. The key experiment involved the creation of all possible single codon mutations of the AAV2 rep gene in a barcoded format, transfection of the library into HEK293T cells for production of AAV, and sequencing to see which rep variants were enriched in the viral particles produced from the library. As the library rep variants were flanked by inverted terminal repeats for packaging into viral particles, the authors could use high-throughput sequencing of the barcodes to determine how much each rep variant supported the production of AAV. The rep gene libraries were cleverly made through a cloning process that ensured each mutant was attached to an exactly known 20nt barcode included in each mutagenic oligo (and subsequently moved to the end of the library genes by another cloning step). This allowed the authors to confidently observe nearly all rep variants in their experiments, resulting in a comprehensive map between Rep protein variants and AAV production. The overall map should act as a useful guide for AAV engineering. Not only did certain variants improve AAV production by ~2-fold and show generality across AAV capsid serotypes, the map might be used to predict greater effects through combinations of mutations, especially if augmented by natural evolutionary datasets and statistical learning.

In interpreting the results of this study, the reader should bear in mind that what has been measured and validated in high throughput is the production of intact genome-containing AAVs. The authors also successfully show transduction for selected high production variants. This is important as the efficiency by which an AAV preparations transduce cells is most relevant property for gene therapy.

Overall, this is a well-executed and well-analyzed study. The results support the conclusions and claims of the work. I see this work as a useful resource for engineering recombinant AAVs to increase their production, which should have broad impact as the use of AAVs in gene therapy grows.