6,243 Matching Annotations
  1. Feb 2024
    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors address an important outstanding question: what forces are the primary drivers of evolutionary rate covariation? Exploration of this topic is important because it is currently difficult to interpret the functional/mechanistic implications of evolutionary covariation. These analyses also speak to the predictive power (and limits) of evolutionary rate covariation. This study reinforces the existing paradigm that covariation is driven by a varied/mixed set of interaction-types that all fall under the umbrella explanation of 'co-functional interactions'.

      Strengths:<br /> Very smart experimental design that leverages individual protein domains for increased resolution.

      Weaknesses:<br /> Nuanced and sometimes inconclusive results that are difficult to capture in a short title/abstract statement.

      EDIT: The authors have done a satisfactory job of honing their language to get the nuanced ideas across clearly. The added scholarship and theoretical discussion they added strengthen the impact of the manuscript. The revised edition addresses my concerns.

    1. Reviewer #3 (Public Review):

      The study focuses on in vivo and in vitro cellular responses intranasal instillation of glycoforms and mutants of SARS-CoV2 spike trimer or spike bearing VLP in mice. Collectively, the experiments suggest that SARS-CoV2 spike has pro-inflammatory roles through increase M1 macrophage associated cytokines and induction of neutrophil netosis/necrosis, a proinflammatory cell death pathway. These effects seem largely independent of hACE2 interaction and partly depend upon interactions with SIGLECs on macrophages and neutrophils. A strength of the study is that a number sophisticated methods are used, including intravital microscopy in the cramaster and liver as well as acute lung slice models, to look at uptake of the spike proteins and immune cell dynamics. The weakness is that some of the reagents maybe contaminated with uncharacterized glycoforms and some important controls, such as control spike protein and control VLP are unevenly applied or not included. The authors have revised the manuscript through some improvements in the writing, but the survey nature and suggestive level of evidence is still a weakness. The study calls attention to sources of proinflammatory activity in the SARS CoV2 spike that may involve some carbohydrate interactions.

    1. Reviewer #2 (Public Review):

      This manuscript reports the discovery and analysis of a large protein complex that controls mating type and sexual reproduction of the model ciliate Tetrahymena thermophila. In contrast to many organisms that have two mating types or two sexes, Tetrahymena is multi-sexual with 7 distinct mating types. Previous studies identified the mating type locus, which encodes two transmembrane proteins called MTA and MTB that determine the specificity of mating type interactions. In this study, mutants are generated in the MTA and MTB genes and mutant isolates are studied for mating properties. Cells missing either MTA or MTB failed to co-stimulate wild-type cells of different mating types. Moreover, a mixture of mutants lacking MTA or MTB also failed to stimulate. These observations support the conclusion that MTA and MTB may form a complex that directs mating-type identity. To address this, the proteins were epitope-tagged and subjected to IP-MS analysis. This revealed that MTA and MTB are in a physical complex, and also revealed a series of 6 other proteins (MRC1-6) that together with MTA/B form the mating type recognition complex (MTRC). All 8 proteins feature predicted transmembrane domains, three feature GFR domains, and two are predicted to function as calcium transporters. The authors went on to demonstrate that components of the MTRC are localized on the cell surface but not in the cilia. They also presented findings that support the conclusion that the mating type-specific region of the MTA and MTB genes can influence both self- and non-self-recognition in mating.

      Taken together, the findings presented are interesting and extend our understanding of how organisms with more than two mating types/sexes may be specified. The identification of the six-protein MRC complex is quite intriguing. It would seem important that the function of at least one of these subunits be analyzed by gene deletion and phenotyping, similar to the findings presented here for the MTA and MTB mutants. A straightforward prediction might be that a deletion of any subunit of the MRC complex would result in a sterile phenotype. The manuscript was very well written and a pleasure to read.

    1. Langes Interview mit Hans Joachim Schellnhuber im Standard, under anderem zu Kipppunkten und der Möglichkeit, dass wir uns schon auf dem Weg in ein „neues Klimaregime“ befinden. Schellnhuber geht davon aus, dass auch das 2°-Ziel überschritten werden wird. Der „Königsweg“, um der Atmosphäre danach wieder CO<sub>2</sub> zu entziehen, sei der weltweite Ersatz von Zement durch Holz beim Bauen, den er als Direktor des IIASA vor allem erforschen wolle. Die Wahrscheinlichkeit dafür, dass „noch alles gutgehen" werde, sei gering. https://www.derstandard.at/story/3000000204635/klimaforscher-schellnhuber-werden-auch-ueber-das-zwei-grad-ziel-hinausschiessen

    1. RRID:ZFIN_ZDB-ALT-130409-2

      DOI: 10.7554/eLife.42455

      Resource: (ZFIN Cat# ZDB-ALT-130409-2,RRID:ZFIN_ZDB-ALT-130409-2)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-130409-2


      What is this?

    2. RRID:ZFIN_ZDB-ALT-130624-2

      DOI: 10.7554/eLife.42455

      Resource: (ZFIN Cat# ZDB-ALT-130624-2,RRID:ZFIN_ZDB-ALT-130624-2)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-130624-2


      What is this?

    3. RRID:ZFIN_ZDB-ALT-110520-2

      DOI: 10.7554/eLife.42455

      Resource: (ZFIN Cat# ZDB-ALT-110520-2,RRID:ZFIN_ZDB-ALT-110520-2)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-110520-2


      What is this?

    1. Reviewer #2 (Public Review):

      Summary:<br /> This work describes a new pharmacological targeting approach to inhibit selective functions of the ubiquitously expressed chemokine receptor CXCR4, a potential target of immunomodulatory or anti-cancer treatments. Overall, the results build a strong case for the potential of this new compound to target specific functions of CXCR4, particularly linked to tumorigenesis. However, a more thorough evaluation of the function of the compound as well as future studies in mammalian model systems are needed to better assess the promise of the compound.

      Strengths:<br /> The work elegantly utilizes in silico drug modelling to propose new small molecule compounds with specific features. This way, the authors designed compound AGR1.137, which abolishes ligand-induced CXCR4 receptor nanoclustering and the subsequent directed cell migration without affecting ligand binding itself or some other ligand-induced signaling pathways. The authors have used a relatively broad set of experiments to validate and demonstrate the effects of the drug. Importantly, the authors also test AGR1.137 in vivo, using a zebrafish model of tumorigenesis and metastasis. A relatively strong inhibitory effect of the compound is reported.

      Weaknesses:<br /> The data would be significantly strengthened by adding kinetics and titration of concentrations. This is particularly important as it is the first description of these particular compounds and would help to evaluate the potency and possible side effects of the drug.

      The authors carry out single-molecule tracking experiments to analyze nanoclustering of CXCR4 upon ligand binding. This complex data is presented in a sub-optimal manner. Representative images of the data should be included together with more thorough analysis tools like autocorrelation function or mean square displacement to get a more conclusive view of receptor clustering and the effects of the compound.

      In the in vivo tumorigenesis experiments, again more kinetics and different concentrations of the drug would generate more convincing data. Also, the individual data points should be visualized to allow full evaluation of the data, throughout the experiments.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study aims to demonstrate that E. coli can acquire rapid antibiotic resistance mutations in the absence of a DNA damage response. To investigate this, the authors employed a sophisticated experimental framework based on a modified Adaptive Laboratory Evolution (ALE) workflow. This workflow involves numerous steps culminating in the measurement of antibiotic resistance. The study presents evidence that a recA strain develops ampicillin resistance mutations more quickly than the wild-type, as shown by measuring the Minimum Inhibitory Concentration (MIC) and mutation frequency. Whole-genome sequencing of 15 recA- colonies resistant to ampicillin revealed predominantly inactivation of genes involved in the multi-drug efflux pump system, whereas, in the wild-type, mutations appear to enhance the activity of the chromosomal ampC cryptic promoter. By analyzing mutants involved in the SOS response, including a lexA3 mutant incapable of inducing the SOS response, the authors conclude that the rapid evolution of antibiotic resistance occurs in an SOS-independent manner when recA is absent.

      Furthermore, RNA sequencing (RNA-seq) of the four experimental conditions suggests that genes related to antioxidative responses drive the swift evolution of antibiotic resistance in the recA- strain.

      Weaknesses:<br /> However, a potential limitation of this study is the experimental design used to determine the 'rapid' evolution of antibiotic resistance. It may introduce a significant bottleneck in selecting ampicillin-resistant mutants early on. A recA mutant could be more susceptible to ampicillin than the wild-type, and only resistant mutants might survive after 8 hours, potentially leading to their enrichment in subsequent steps. To address this concern, it would be critical to perform a survival analysis at various time points (0h, 2h, 4h, 6h, and 8h) during ampicillin treatment for both recA and wild-type strains, ensuring there is no difference in viability.

      The observation that promoter mutations are absent in recA strains could be explained by previous research indicating that amplification of the AmpC genes is a mechanism for E. coli resistance to ampicillin, which does not occur in a recA-deficient background (PMID# 19474201).

      The section describing Figure 3 is poorly articulated, and the conclusions drawn are apparent. The inability of a recA strain to induce the SOS response is well-documented (lines 210 and 278). The data suggest that merely blocking SOS induction is insufficient to cause 'rapid' evolution in their experimental conditions. To investigate whether SOS response can be induced independently of lexA cleavage by recA, alternative experiments, such as those using a sulA-GFP fusion, might be more informative.

      In Figure 4E, the lack of increased SulA gene expression in the wild-type strain treated with ampicillin is unexpected, given that SulA is an SOS-regulated gene. The fact that polA (Pol I) is going down should be taken into account in the interpretation of Figures 2D and 2E.

      The connection between compromised DNA repair, the accumulation of Reactive Oxygen Species (ROS) based on RNA-seq data, and accelerated evolution is merely speculative at this point and not experimentally established.

    1. Reviewer #3 (Public Review):

      Bae et al. described the key roles of pericytes in cavernous tissues in diabetic erectile dysfunction using both mouse and human single-cell transcriptomic analysis. Erectile dysfunction (ED) is caused by dysfunction of the cavernous tissue and affects a significant proportion of men aged 40-70. The most common treatment for ED is phosphodiesterase 5 inhibitors; however, these are less effective in patients with diabetic ED. Therefore, there is an unmet need for a better understanding of the cavernous microenvironment, cell-cell communications in patients with diabetic ED, and the development of new therapeutic treatments to improve the quality of life.

      Pericytes are mesenchymal-derived mural cells that directly interact with capillary endothelial cells (ECs). They play a vital role in the pathogenesis of erectile function as their interactions with ECs are essential for penile erection. Loss of pericytes has been associated with diabetic retinopathy, cancer, and Alzheimer's disease and has been investigated in relation to the permeability of cavernous blood vessels and neurovascular regeneration in the authors' previous studies. This manuscript explores the mechanisms underlying the effect of diabetes on pericyte dysfunction in ED. Additionally, the cellular landscape of cavernous tissues and cell type-specific transcriptional changes were carefully examined using both mouse and human single-cell RNA sequencing in diabetic ED. The novelty of this work lies in the identification of a newly identified pericyte (PC)-specific marker, LBH, in mouse and human cavernous tissues, which distinguishes pericytes from smooth muscle cells. LBH not only serves as a cavernous pericyte marker, but its expression level is also reduced in diabetic conditions. The LBH-interacting proteins (Cryab and Vim) were further identified in mouse cavernous pericytes, indicating that these signaling interactions are critical for maintaining normal pericyte function. Overall, this study demonstrates the novel marker of pericytes and highlights the critical role of pericytes in diabetic ED.

      Comments on revised version:

      Bae and colleagues substantially improved the data quality and revised their manuscript "Pericytes contribute to pulmonary vascular remodeling via HIF2a signaling". While these revisions clarify some of the concerns raised, others remain. In my view, the following question must be addressed.

      In my prior question on #3, I completely disagree with the statement that "identified cells with pericyte-like characteristics in the walls of large blood vessels". The staining that authors provided for LBH, was clearly stained for SMCs, not pericytes. Per Fig 2E, the authors are correct that LBH is colocalized with SMA+ cells( SMCs). However, the red signal from LBH clearly stains endothelial cells. In the rest of 2E and 2D, LBH is CD31- and their location suggests LBH stained for SMCs in the Aorta, Kidney vasculature, Dorsal vein, and Dorsal Artery.

    1. Reviewer #2 (Public Review):

      Summary: The authors seek to elucidate the early evolution of cnidarians through computer modeling of fluid flow in the oral region of very small, putative medusozoan polyps. They propose that the evolutionary advent of the free-swimming medusoid life stage was preceded by a sessile benthic life stage equipped with circular muscles that originally functioned to facilitate feeding and that later became co-opted for locomotion through jet propulsion.

      Strengths: Assumptions of the modeling exercise laid out clearly; interpretations of the results of the model runs in terms of functional morphology plausible. An intriguing investigation that should stimulate further discussion and research.

      Weaknesses: Speculation on the origin of the medusoid life stage in cnidarians heavily dependent on prior assumptions concerning the soft part anatomy and material properties of the skeleton of the modeled fossil organism that may be open to alternative interpretations. Logically, of course, the hypothesis that cnidarian medusae originated from benthic polyps must be evaluated along with the alternative hypotheses that the medusa came first and that the ancestral cnidarian exhibited both life stages.

    1. Reviewer #2 (Public Review):

      Summary and strengths:

      1) The work provides significant insights because usually non-significant studies can be considered replicated by their null replications as well. The work discuss and provide data demonstrating that when analyzing studies with p > 0.05 for the result to be replicated, equivalence tests and bayes factor approaches are more suitable, since studies can be underpowered even if replications use larger samples than their original studies in general. Non-significant p-values are highly expected even with 80% of power for a true effect.

      2) The evidence used features methods and analyses more rigorous than current state-of-the-art research on replicability.

      Weaknesses:<br /> I am satisfied with the revisions made by the authors in response to my initial suggestions, as well as their subsequent responses to my observations throughout the reviewing process.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this study, the authors present a robust pipeline that integrates high-content phenotypic imaging of a targeted pool of 366 CRISPRi-screened genes with in situ sequencing of single cells, achieving a resolution for 1.3 million cells. The application of this pipeline on the U2OS cell line effectively screens for nuclear and actin morphology changes. One study's strength lies in the utilization of a barcode system, enabling efficient imaging and genotype determination for 85% of cells. The authors employ two distinct approaches to delineate phenotypic changes. In the first approach, cells are characterized by approximately 1,000 morphological features, with dimensionality reduction via PCA using 25 principal components and a novel image sampling method called VIEWED (Visual Interpretation of Embedding by Constrained Walkthrough Sampling). The second approach employs a deep learning technique, specifically the Beta-variational encoder, to identify morphological differences, offering a generative AI approach for visualizing interpreted distinctions learned through the algorithm. While the Beta-variational encoder is deemed simpler to use and interpret, the classical PCA approach demonstrates superiority due to its heightened sensitivity in identifying more genes with phenotypic changes. Both methods, however, successfully identify shared phenotypic gene hits, showing consistent replication across multiple individual guides for each gene hit. Key phenotypic clusters are identified and replicated similarly by both the conventional PCA feature approach and the Beta-variational encoder approach.

      Strengths:<br /> - A novel barcode methodology for efficient genotyping via in situ sequencing, minimizing rounds of imaging and genotyping 85% of cells.<br /> - Use of a beta variational autoencoder, generative AI approach to facilitate detection of morphological change in cells, gene hits, and phenotypic gene clusters.

      Weaknesses:<br /> Although the outcome is reproduced with 3 gRNA/gene, no biological replicate is presented and is as such limiting on convincing on reproducibility of the phenotypic detection approach.

      The presented work is highly compelling as it employs an optical pooled CRISPRi screen, showcasing the capability to conduct pool screening beyond the typical frequency count of guides with the next-generation sequencing approach, effectively establishing a direct link between cell images and guide RNAs in the pool screen approach. This achievement, typically associated with arrayed screens, sets the study apart. Moreover, the study offers captivating images of individual cells that vividly portray convincing phenotypic changes. Additionally, the work effectively highlights the potency of generative AI in interpreting cell phenotypic changes detected by the algorithm. This aspect of the study is particularly relevant in the present time, as it introduces a potentially highly valuable methodology. Overall, the research provides a robust demonstration of innovative techniques and methodologies, contributing significantly to the field.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This interesting study challenges a dogma regarding the link between bacterial metabolism decrease and tolerance to aminoglycosides (AG). The authors demonstrate that mutants well-known for being tolerant to AG, such as those of complexes I and II, are not so due to a decrease in the proton motive force (PMF) and thus antibiotic uptake, as previously reported in the literature.

      Strengths:<br /> This is a complete study. These results are surprising and are based on various read-outs, such as ATP levels, pH measurement, membrane potential, and the uptake of fluorophore-labeled gentamicin. Utilizing a proteomic approach, the authors show instead that in tolerant mutants, there is a decrease in the levels of proteins associated with ribosomes (targets of AG), causing tolerance.

      Weaknesses:<br /> The use of a single high concentration of aminoglycoside: my main comment on this study concerns the use of an AG concentration well above the MIC (50 µg/ml or 25 µg/ml for uptake experiments), which is 10 times higher than previously used concentrations (Kohanski, Taber) in study showing a link with PMF. This significant difference may explain the discrepancies in results. Indeed, a high concentration of AG can mask the effects of a metabolic disruption and lead to less specific uptake. However, this concentration highlights a second molecular level of tolerance. Adding experiments using lower concentrations (we propose 5 µg/ml to compare with the literature) would provide a more comprehensive understanding of AG tolerance mechanisms during a decrease in metabolism.

      Another suggestion would be to test iron limitation (using an iron chelator as DIP), which has been shown to induce AG tolerance. Can the authors demonstrate if this iron limitation leads to a decrease in ribosomal proteins? This experiment would validate their hypothesis in the case of a positive result. Otherwise, it would help distinguish two types of molecular mechanisms for AG tolerance during a metabolic disruption: (i) PMF and uptake at low concentrations, (ii) ribosomal proteins at high concentrations.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors tried to understand the mechanism of how a drug candidate, VLZ, works on a receptor, 5-HTR1A, by activating the SRC/MAPK pathway to promote the formation of platelets.

      Strengths:<br /> The authors used both computational and experimental methods. This definitely saves time and funds to find a useful drug candidate and its therapeutic marker in the subfield of platelets reduction in cancer patients. The authors achieved the aim of explaining the mechanism of VLZ in improving thrombocytopenia by using two cell lines and two animal models.

      Weaknesses:<br /> Only two cell lines, HEL and Meg-01 cells, were evaluated in this study. However, using more cell lines is really depending on the workflow and the grant situations of the current research team.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The paper sets out to understand the mechanisms underlying the colonization and degradation of marine particles using a natural Vibrio isolate as a model. The data are measurements of motility and gene expression using microfluidic devices and RNA sequencing. The results reveal that degradation products of alginate do stimulate motility but not chemotaxis. The evidence for these claims is strong. The story of how particle degradation occurs through colonization and dispersal has modest support in the data. A quantitative description of these dynamics awaits future studies.

      Strengths:<br /> The microfluidic and transcriptional measurements are the central strengths of the paper as they allow the delineation of phenotypes at the cellular and molecular levels in the presence of polymer and byproducts of polymer degradation.

      Weaknesses:<br /> The explanation of the microfluidics measurements is somewhat confusing but I think this could be easily remedied. The quantitative interpretation of the dispersal data could also be improved and I'm not clear if the data support the claim made.

    1. we—are the beginningour work is today:A mugA floor brushBootsA catalogAnd when one person in his laboratory set upA squar

      I see this quote as a direct declaration of war against technology as a whole. Going back to analog roots of brushes, well-worn boots, and a magazine. All things needed in order to set up the grid behind art, a guideline of human creativity.

    2. We say that the world’s magnificence has been enriched by a new beauty:the beauty of speed.

      I feel this rule of the manifesto of futurism still holds up to this day. News, ads, and tabloids come at us faster than ever. There is always something to turn our eyes to and whenever the 15 minutes of fame and attention are up, we move on to the next thing. It is a constant, overstimulating cycle of content.

    3. We, however, are satisfied if in our bookthe lyric and epic evolution of our times is given shape.

      Each era has their own version of what the world should follow and believe. It is an constant evolving movement.

    4. We will glorify war—the world’s only hygiene—militarism, patriotism, thedestructive gesture of freedom-bringers, beautiful ideas worth dying for, andscorn for woman

      Pro-violence, very problematic

    5. previously—Engineers relaxed with artnow—Artists relax with technology1 For a detailed discussion ofRodchenko’s belief in theideal Soviet citizen, see VictorMargolin, The Struggle forUtopia: Rodchenko, Lissitzky,Moholy-Nagy, 1917–1946(Chicago: university of ChicagoPress, 1998).

      progressive way of thinking

    1. Reviewer #2 (Public Review):

      Summary

      The authors proposed a toolset Photo-SynthSeg to the software FreeSurfer which performs 3D reconstruction and high-resolution 3D segmentation on a stack of coronal dissection photographs of brain tissues. To prove the performance of the toolset, three experiments were conducted, including volumetric comparison of brain tissues on AD and HC groups from MADRC, quantitative evaluation of segmentation on UW-ADRC and quantitative evaluation of 3D reconstruction on HCP digitally sliced MRI data.

      Strengths

      To guarantee successful workflow of the toolset, the authors clearly mentioned the prerequisites of dissection photograph acquisition, such as fiducials or rulers in the photos and tissue placement of brain slices with more than one connected component. The quantitative evaluation of segmentation and reconstruction on synthetic and real data demonstrates the accuracy of the methodology. Also, the successful application of this toolset on two brain banks with different slice thicknesses, tissue processing and photograph settings demonstrates its robustness. By working with tools of the SynthSeg pipeline, Photo-SynthSeg could further support volumetric cortex parcellation. The toolset also benefits from its adaptability of different 3D references, such as surface scan, ex vivo MRI and even probabilistic atlas, suiting the needs for different brain banks.

      Weaknesses

      Certain weaknesses are already covered in the manuscript. Cortical tissue segmentation could be further improved. The quantitative evaluation of 3D reconstruction is quite optimistic due to random affine transformations. Manual edits of slice segmentation task are still required and take a couple of minutes per photograph. Finally, the current toolset only accepts coronal brain slices and should adapt to axial or sagittal slices in future work.

    1. Reviewer #2 (Public Review):

      Summary:

      This manuscript by Xu et al., is an interesting study aiming to identify novel features of macaque cortical development. This study serves as a valuable atlas of single cell data during macaque neurogenesis, which extends the developmental stages previously explored. Overall, the authors have achieved their aim of collecting a comprehensive dataset of macaque cortical neurogenesis and have identified a few unknown features of macaque development.

      Strengths:

      The authors have accumulated a robust dataset of developmental time points and have applied a variety of informatic approaches to interrogate this dataset. One interesting finding in this study is the expression of previously unknown receptors on macaque oRG cells. Another novel aspect of this paper is the temporal dissection of neocortical development across species. The identification that the regulome looks quite different, despite similar expression of transcription factors in discrete cell types, is intriguing.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This paper focuses on an interesting question that has puzzled psychologists for decades, that is, why do people demonstrate a mix of uncertainty approach and avoidance behavior, given the fact that reducing uncertainty could always gain information and seems beneficial? This paper designed a novel task to demonstrate behavioral signatures of uncertainty approaching and avoidance during the exploration phase within the same task at both a within-subject and between-subject level. On the algorithmic level, this paper compared four different implementations of uncertainty-guided exploration and found that the model sensitive to relative uncertainty provides the best fit for human behavior compared to its counterparts using expected information gain or past exposure. This paper then links people's uncertainty attitude with accuracy and finds that uncertainty avoidance during exploration does not impair task performance, implying that uncertainty avoidance may be the output of a resource-rational decision-making process. To examine this account, this paper uses reaction time as an independent proxy of costly deliberation and shows that people deliberate shorter when engaging in repetitive choice, which presumably saves cognitive resources. Finally, the paper shows that people's tendency to engage in repetitive choice correlates with their tendency to avoid uncertainty, which supports the argument that avoiding uncertainty could be a strategy developed under the constraint of limited cognitive resources.

      Strengths:<br /> One of the highlights of this paper, as mentioned in the previous paragraph, is that the authors can establish the existence of the uncertainty approach and avoidance behavior within the same task whereas previous work usually focuses on one of them. This dissociation allows the authors to examine what situational factor is related to the emergence of the act of avoiding uncertainty, and extract parameters describing participants' attitude towards uncertainty during baseline as well as during situations where uncertainty avoidance is more common. Besides documenting the existence of uncertainty avoidance behavior, this paper also tried to explain this behavior by proposing under the resource rational framework and has carefully quantified different aspects (e.g., accuracy; choice speed) of participants' behavior as well as examined their relationships. Though more experiments are needed to fully understand human uncertainty avoidance behavior, this paper has provided both empirical and theoretical contributions toward a mechanistic understanding of how people balance approaching and avoiding uncertainty.

      Weaknesses:<br /> I have a couple of concerns related to this paper. First, there seems to exist an anti-correlation between total uncertainty and absolute relative uncertainty (Figure 5 panel C, \delta uncertainty is restricted to a small range when total uncertainty is high). It seems to be a natural product of the exploration process since the high total uncertainty phase is usually the period where the participant knows little about either option, leading to a less distinguishable relative uncertainty. However, it remains unknown whether the documented uncertainty avoidance still applies when extrapolating to larger absolute relative uncertainty. It would be great if the experiment allows for a manipulation of uncertainty in the middle of the experiment (e.g., introducing a new deck/informing that one deck has been updated). Relatedly, the current 'threshold' of uncertainty avoidance behavior, if I understand correctly, is found by empirically fitting participants' data. This brings the question: can we predict when people will demonstrate uncertainty avoidance behavior before collecting any data? Or, is it possible that by measuring some metrics related to cognitive cost sensitivity, we could predict the proportion of choices that participants will show uncertainty-avoidant behavior? Finally, regarding the analysis of different behavior patterns in the game, it seems that the authors try to link repetitive behavior, uncertainty attitude, and accuracy together by testing the correlation between the two of them. I wonder whether other multivariate statistical methods e.g., mediation analysis, will be better suited for this purpose.

    1. Reviewer #2 (Public Review):

      The authors demonstrate convincingly the potential of single mesodermal cells, removed from zebrafish embryos, to show cell-autonomous oscillatory signaling dynamics and differentiation. Their main conclusion is that a cell-autonomous timer operates in these cells and that additional external signals are integrated to tune cellular dynamics. Combined, this is underlying the precision required for proper embryonic segmentation, in vivo. I think this work stands out for its very thorough, quantitative, single-cell real-time imaging approach, both in vitro and also in vivo. A very significant progress and investment in method development, at the level of the imaging setup and also image analysis, was required to achieve this highly demanding task. This work provides new insight into the biology underlying embryo axis segmentation.<br /> The work is very well presented and accessible. I think most of the conclusions are well supported. Here a my comments and suggestions:

      1) The authors state that "We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding remarkable agreement."

      I think this statement needs to be better placed in context. In absolute terms, the period of oscillations and the timing of differentiation are actually very different in vitro, compared to in vitro. While oscillations have a period of ~30 minutes in vivo, oscillations take twice as long in vitro. Likewise, while the last oscillation is seen after 143 minutes in vivo, the timing of differentiation is very significantly prolonged, i.e.more than doubled, to 373min in vitro (Supplementary Figure 1-9). I understand what the authors mean with 'remarkable agreement', but this statement is at the risk of being misleading. I think the in vitro to in vivo differences (in absolute time scales) needs to be stated more explicitly. In fact, the drastic change in absolute timescales, while preserving the relative ones,i.e. the number of oscillations a cell is showing before onset of differentiation remains relatively invariant, is a remarkable finding that I think merits more consideration (see below).

      2) One timer vs. many timers<br /> The authors show that the oscillation clock slowing down and the timing of differentiation, i.e. the time it takes to activate the gene mesp, are in principle dissociable processes. In physiological conditions, these are however linked. We are hence dealing with several processes, each controlled in time (and hereby space). Rather than suggesting the presence of 'a timer', I think the presence of multiple timing mechanisms would reflect the phenomenology better. I would hence suggest separating the questions more consistently, for instance into the following three:<br /> a. what underlies the slowing down of oscillations?<br /> b. what controls the timing of onset of differentiation?<br /> c. and finally, how are these processes linked?

      Currently, these are discussed somewhat interchangeably, for instance here: "Other models posit that the slowing of Her oscillations arise due to an increase of time-delays in the negative feedback loop of the core clock circuit (Yabe, Uriu, and Takada 2023; Ay et al. 2014), suggesting that factors influencing the duration of pre-mRNA splicing, translation, or nuclear transport may be relevant. Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock."(page 14). In the first part, the slowing down of oscillations is discussed and then the authors conclude on 'the timer', which however is the one timing differentiation, not the slowing down. I think this could be somewhat misleading.

      3) From this and previous studies, we learn/know that without clock oscillations, the onset of differentiation still occurs. For instance in clock mutant embryos (mouse, zebrafish), mesp onset is still occurring, albeit slightly delayed and not in a periodic but smooth progression. This timing of differentiation can occur without a clock and it is this timer the authors refer to "Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock." (page 14). This 'timer' is related to what has been previously termed 'the wavefront' in the classic Clock and Wavefront model from 1976, i.e. a "timing gradient' and smooth progression of cellular change. The experimental evidence showing it is cell-autonomous by the time it has been laid down,, using single cell measurements, is an important finding, and I would suggest to connect it more clearly to the concept of a wavefront, as per model from 1976.

      4) Regarding question a., clearly, the timer for the slowing down of oscillations is operating in single cells, an important finding of this study. It is remarkable to note in this context that while the overall, absolute timescale of slowing down is entirely changed by going from in vivo to in vitro, the relative slowing down of oscillations, per cycle, is very much comparable, both in vivo and in vivo. To me, while this study does not address the nature of this timer directly, the findings imply that the cell-autonomous timer that controls slowing down is, in fact, linked to the oscillations themselves. We have previously discussed such a timer, i.e. a 'self-referential oscillator' mechanism (in mouse embryos, see Lauschke et al., 2013) and it seems the new exciting findings shown here in zebrafish provide important additional evidence in this direction. I would suggest commenting on this potential conceptual link, especially for those readers interested to see general patterns.

      5) Regarding question c., i.e. how the two timing mechanisms are functionally linked, I think concluding that "Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock." (page 14), might be a bit of an oversimplification. It is correct that the timer of differentiation is operating without a clock, however, physiologically, the link to the clock (and hence the dependence of the timescale of clock slowing down), is also evident. As the author states, without clock input, the precision of when and where differentiation occurs is impacted. I would hence emphasize the need to answer question c., more clearly, not to give the impression that the timing of differentiation does not integrate the clock, which above statement could be interpreted to say.

      6) A very interesting finding presented here is that in some rare examples, the arrest of oscillations and onset of differentiation (i.e. mesp) can become dissociated. Again, this shows we deal here with interacting, but independent modules. Just as a comment, there is an interesting medaka mutant, called doppelkorn (Elmasri et al. 2004), which shows a reminiscent phenotype "the Medaka dpk mutant shows an expansion of the her7 expression domain, with apparently normal mesp expression levels in the anterior PSM.". The authors might want to refer to this potential in vivo analogue to their single cell phenotype.

      7) One strength of the presented in vitro system is that it enables precise control and experimental perturbations. A very informative set of experiments would be to test the dependence of the cell-autonomous timing mechanisms (plural) seen in isolated cells on ongoing signalling cues, for instance via Fgf and Wnt signaling. The inhibition of these pathways with well-characterised inhibitors, in single cells, would provide important additional insight into the nature of the timing mechanisms, their dependence on signaling and potentially even into how these timers are functionally interdependent.

    1. Reviewer #3 (Public Review):

      Summary:

      1. Grandits and colleagues were trying to develop a new tool to accelerate pharmacological studies by using neural networks to emulate the human ventricular cardiomyocyte action potential (AP). The AP is a complex electrical signal that governs the heartbeat, and it is important to accurately model the effects of drugs on the AP to assess their safety and efficacy. Traditional biophysical simulations of the AP are computationally expensive and time-consuming. The authors hypothesized that neural network emulators could be trained to predict the AP with high accuracy and that these emulators could also be used to quickly and accurately predict the effects of drugs on the AP.

      Strengths:

      2. One of the study's major strengths is that the authors use a large and high-quality dataset to train their neural network emulator. The dataset includes a wide range of APs, including normal and abnormal APs exhibiting EADs. This ensures that the emulator is robust and can be used to predict the AP for a variety of different conditions.

      Another major strength of the study is that the authors demonstrate that their neural network emulator can be used to accelerate pharmacological studies. For example, they use the emulator to predict the effects of a set of known arrhythmogenic drugs on the AP. The emulator is able to predict the effects of these drugs, even though it had not been trained on these drugs specifically.

      Weaknesses:

      One weakness of the study is that it is important to validate neural network emulators against experimental data to ensure that they are accurate and reliable. The authors do this to some extent, but further validation would be beneficial. In particular for the inverse problem, where the estimation of pharmacological parameters very challenging and led to particularly large inaccuracies.

      Additional context:

      4. The work by Grandits et al. has the potential to revolutionize the way that pharmacological studies are conducted. Neural network emulation has the promise to reduce the time and cost of drug development and to improve the safety and efficacy of new drugs. The methods and data presented in the paper are useful to the community because they provide a starting point for other researchers to develop and improve neural network emulators for the human ventricular cardiomyocyte AP. The authors have made their code and data publicly available, which will facilitate further research in this area.

      5. It is important to note that neural network emulation is still a relatively new approach, and there are some challenges that need to be addressed before it can be widely adopted in the pharmaceutical industry. For example, neural network emulators need to be trained on large and high-quality datasets. Additionally, it is important to validate neural network emulators against experimental data to ensure that they are accurate and reliable. Despite these challenges, the potential benefits of neural network emulation for pharmacological studies are significant. As neural network emulation technology continues to develop, it is likely to become a valuable tool for drug discovery and development.

    1. Reviewer #2 (Public Review):

      Summary:

      The malaria parasite Plasmodium develops into oocysts and sporozoites inside Anopheles mosquitoes, in a process called sporogony. Sporozoites invade the insect salivary glands in order to be transmitted during a blood meal. An important question regarding malaria transmission is whether all mosquitoes harboring Plasmodium parasites are equally infectious. In this paper, the authors investigated the progression of P. falciparum sporozoite development in Anopheles mosquitoes, using a sensitive qPCR method to quantify sporozoites and an artificial skin system to probe for parasite expelling. They assessed the association between oocyst burden, salivary gland infection intensity, and sporozoites expelled.

      The data show that higher sporozoite loads are associated with earlier colonization of salivary glands and a higher prevalence of sporozoite-positive salivary glands and that higher salivary gland sporozoite burdens are associated with higher numbers of expelled sporozoites. Intriguingly, there is no clear association between salivary gland burdens and the prevalence of expelling, suggesting that most infections reach a sufficient threshold to allow parasite expelling during a mosquito bite. This important observation suggests that low-density gametocyte carriers, although less likely to infect mosquitoes, could nevertheless contribute to malaria transmission.

      Strengths:

      The paper is well written and the work is well conducted. The authors used two experimental models, one using cultured P. falciparum gametocytes and An. stephensi mosquitoes, and the other one using natural gametocyte infections in a field setup with An. coluzzii mosquitoes. Both studies gave similar results, reinforcing the validity of the observations. Parasite quantification relies on a robust and sensitive qPCR method, and parasite expelling was assessed using an innovative experimental setup based on artificial skin.

      Weaknesses:

      There is no clear association between the prevalence of sporozoite expelling and the parasite burden. However, high total sporozoite burdens are associated with earlier and more efficient colonization of the salivary glands, and higher salivary gland burdens are associated with higher numbers of expelled sporozoites. While these observations suggest that highly infected mosquitoes could transmit/expel parasites earlier, this is not directly addressed in the study. In addition, whether all expelled sporozoites are equally infectious is unknown. The central question, i.e. whether all infected mosquitoes are equally infectious, therefore remains open.

    1. Reviewer #2 (Public Review):

      Dipasree Hajra et al demonstrated that Salmonella was able to modulate the expression of Sirtuins (Sirt1 and Sirt3) and regulate the metabolic switch in both host and Salmonella, promoting its pathogenesis. The authors found Salmonella infection induced high levels of Sirt1 and Sirt3 in macrophages, which were skewed toward the M2 phenotype allowing Salmonella to hyper-proliferate. Mechanistically, Sirt1 and Sirt3 regulated the acetylation of HIF-1alpha and PDHA1, therefore mediating Salmonella-induced host metabolic shift in the infected macrophages. Interestingly, Sirt1 and Sirt3-driven host metabolic switch also had an effect on the metabolic profile of Salmonella. Counterintuitively, inhibition of Sirt1/3 led to increased pathogen burdens in an in vivo mouse model. Overall, this is a well-designed study. There are a few comments below that would further strengthen the current study.

      Major comments:<br /> In the in vivo study (lines 436-446) - the authors noticed increased pathogen burden in the EX-527 or the 3TYP-treated mice cohorts but decreased pathogen burden within the F4/80+ macrophage population. What are the other cell types that have increased pathogen burden in splenocytes from EX-527 or the 3TYP treated? Can this be further explored and explained?

      While the authors indicated that IL-6 cytokine storm and elevated ROS production could result in bacterial dissemination in vivo, one could also argue that Sirt1/3 inhibitors might have an impact on gut function and/or gut microbiota (PMID: 22115311). Did Sirt1/3 inhibitors also lead to increased pathogen burdens in the gut? If so, the potential effect of these in vivo treatments on gut microbiota/colonization resistance should be discussed.

      Minor comment:<br /> Sirt1 has been shown to be degraded during Salmonella infection (PMID: 28192515), which is different from the current study. An explanation should be provided for this.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors present a report of a large Pseudomonas aeruginosa hospital outbreak affecting more than 80 patients with first sampling dates in 2011 that stretched over more than 10 years and was only identified through genomic surveillance in 2020. The outbreak strain was assigned to the sequence type 621, an ST that has been associated with carpabapenem resistance across the globe. Ongoing transmission coincided with both increasing resistance without acquisition of carbapenemase genes as well as the convergence of mutations towards a host-adapted lifestyle.

      Strengths:<br /> The convincing genomic analyses indicate spread throughout the hospital since the beginning of the century and provide important benchmark findings for future comparison.

      The sampling was based on all organisms sent to the Multidrug-resistant Organism Repository and Surveillance Network across the U.S. Military Health System.

      Using sequencing data from patient and environmental samples for phylogenetic and transmission analyses as well as determining recurring mutations in outbreak isolates allows for insights into the evolution of potentially harmful pathogens with the ultimate aim of reducing their spread in hospitals.

      Weaknesses:<br /> The epidemiological information was limited and the sampling methodology was inconsistent, thus complicating the inference of exact transmission routes. Epidemiological data relevant to this analysis include information on the reason for sampling, patient admission and discharge data, and underlying frequency of sampling and sampling results in relation to patient turnover.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this work, Hu and colleagues investigate telomerase-independent survival in Saccharomyces cerevisiae strains engineered to have different chromosome numbers. The authors describe the molecular patterns of survival that change with fewer chromosomes and that differ from the well-described canonical Type I and Type II, including chromosome circularization and other atypical outcomes. They then take advantage of the strain with 3 chromosomes to examine the effect of deleting all the subtelomeric elements, called X and Y'. For most of the tested phenotypes, they find no significant effect of the absence of X- and Y'-element, and show that they are not essential for survivor formation. They speculate that X- and Y'-elements are remnants of ancient telomere maintenance mechanisms.

      Strengths:<br /> This work advances our understanding of the telomerase-independent strategies available to the cell by altering the structure of the genome and of the subtelomeres, a feat that was enabled by the set of strains they engineered previously. By using strains with non-standard genome structures, several alternative survival mechanisms are uncovered, revealing the diversity and plasticity of telomere maintenance mechanisms. Overall, the conclusions are well supported by the data, with adequate sample sizes for investigating survivors. The assessment of the genetic requirements for survivors in strains with different chromosome numbers greatly improved the quality of this work. The molecular analyses based on Southern blots are also very well-conducted.

      Weaknesses:<br /> The authors discovered alternative telomerase-independent survival strategies beyond the well-described type I and II (including circularization, type X and atypical, as they called them) at play in the context of reduced number of chromosomes. Their work provides a molecular and a partial genetic characterization of these survival pathways. A more thorough analysis of the frequency of each type of survivors and their genetic requirements would have advanced our understanding or the diversity of survival strategies in the absence of telomerase. However, as noted by the authors, the quantification of the rate of emergence of survivors (and their subtypes) is very difficult to achieve. This comment is therefore not meant as a criticism but rather as a perspective on exciting future research avenues.

    1. Reviewer #3 (Public Review):

      The authors have done a fine job of updating the manuscript and it is substantially improved. In particular, the paragraphs towards the end of the Introduction and Discussion are vastly improved. The last paragraph of the Introduction now clearly explicates the hypotheses (save one minor point of confusion). The limitations section of the Discussion is also very helpful and fair. However, there are still areas where claims need to be tempered.

      Major criticisms<br /> • The results still do not lead to the conclusion that the angular gyrus is causally involved in insight-driven memory configuration. Although the authors do state that other regions such as the hippocampus may have contributed to the pattern of results, there is still no evidence of target engagement or a link between target engagement and the behavioral results. Thus, while the results support that cTBS to the angular gyrus affects insight-driven memory configuration, it is a strong overstep to say that the angular gyrus is causally involved in insight-driven memory reconfiguration. In particular, this applies to both the title and the last line of the Abstract. In relation to this, have the authors conducted any target engagement analyses? It seems like a good starting point would be to identify the censor closest to the stimulation site in each individual, Hjorth transforms the signal of that sensor by subtracting the average of the surrounding sensors to increase signal localization, and then measure the effects of stimulation on theta power. Presumably, we would expect that cTBS would decrease theta power relative to sham stimulation. Although this isn't the only type of analysis that could at least partially confirm target engagement, there needs to be some sort of formal analysis to maintain the claims of the title and last line of the Abstract.<br /> • The authors removed the mentions of "inhibitory stimulation" from the manuscript to their credit, but a rigorous and fair treatment of the effects of cTBS is still lacking, and it is still unclear why cTBS to the angular gyrus would cause an inhibitory effect in the first place. The authors state that

      "Previous evidence has demonstrated the inhibitory effect of cTBS on the targeted brain region under stimulation (Huang et al., 2005; Jannati et al., 2023). Nonetheless, the effects of cTBS appear to vary based on the targeted region, with cTBS to parietal regions demonstrating the capability to enhance hippocampal connectivity (Hermiller et al., 2019, 2020)."

      The inhibitory effects of motor cortex cTBS s on corticospinal excitability in nine subjects from the Huang paper and the Jannati review (not a primary source) do not constitute sufficient evidence to hypothesize an inhibitory effect on insight-driven memory reconfiguration. The second sentence provides much more sufficient evidence that parietal stimulation should have some sort of a facilitatory effect, but this is simply glossed over without an explanation of why cTBS to the parietal cortex should inhibit insight-driven memory reconfiguration. Pilot data showing such inhibitory effects or a body of evidence showing inhibitory effects of angular gyrus stimulation on closely-related areas of cognition would have given reason to believe this. However, without these, an a priori assumption that parietal cTBS would be inhibitory seems highly debatable and paints the results as provisional, rather than confirmatory"

    1. Reviewer #3 (Public Review):

      The findings of Bo Yu and colleagues titled "Identification of fallopian tube microbiota and its association with ovarian cancer: a prospective study of intraoperative swab collections from 187 patients" describes the identification of the fallopian tube microbiome and relationship with ovarian cancer. The studies are highly rigorous obtaining specimens from the fallopian tube, ovarian surfaces, paracolic gutter of patients of known or suspected ovarian cancer or benign tumor patients. The investigators took great care to insure there was no or limited contamination including test the surgical suite air, as the test locations are from low abundance microbiota. The findings provide evidence that the microbiota in the fallopian tube, especially in ovarian cancer has similarities to gut microbial communities. This is a potentially novel observation.

      The studies investigate the microbiome of >1000 swabs from 81 ovarian cancer and 106 non-cancer patients. The sites collected are low biomass microbiota making the study particularly challenging. The studies provide descriptive evidence that the ovarian cancer fallopian tube microbiota contain species that are similar to the gut microbiota. In contrast the fallopian tube microbiota of non-cancer patients that exhibit more similarity to the uterine/cervical microbiota. This may be a relevant observation but is highly descriptive with limited insights on the functional relevance.

      The data indicate the presence of low biomass FT microbiota. The findings support the existence of FT microbiota in ovarian cancer that appears to be related to gut microbial species. While interesting, there is no insights on how and why these microbial species are found in the FT. The studies only identify the species but there is no transcriptomic analysis to provide an indication on whether the bacteria are activating DNA damage pathways. This is an interesting observation that requires more insights to address how these bacteria reach the fallopian tube and a related question is whether these bacteria are found in the peritoneum.

      An additional concern is whether these data can be used to develop biomarkers of disease and early detection of disease.

    1. Reviewer #2 (Public Review):

      Summary:

      The study provides valuable and compelling evidence that while activation of the mTOR cascade confers some similarities in alterations in cell size, mTOR pathway activation, cortical lamination, baseline firing properties, and synaptic activity, there are distinctions that could account for clinical differences in seizure and epilepsy phenotypes in patients harboring these mutations. These findings could have important implications going forward as we design clinical therapeutic strategies to modulate mTOR activity in these individuals to treat seizures.

      This study presents a valuable finding on the role that distinct mTOR pathway genes play in altered cell shape, cortical laminar migration, and cellular excitability in the mouse medial prefrontal cortex (mPFC). The evidence supporting the claims of the authors is solid, although analysis of the role of the mTORC2 pathway and consideration of distinct metabolic states i.e., amino acid levels would have strengthened the study. The work will be of interest to neuroscientists working on human epilepsy. These genes have each been assayed in previous independent studies and thus the direct comparison is what provides the innovation and interest.

      The manuscript by Nguyen and colleagues attempts to define both the common and differential roles of mTOR pathway genes, both by gene knockout (KO) and activation, on cortical neuronal size, cortical lamination, and excitability. They focused on 5 genes that have been linked to human malformations of cortical development (MCD) and epilepsy: RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO. The RhebY35L, mTORS2215Y are known and pathogenic human gain-of-function variants. Each of these genes is known to modulate the activity of mTORC1 and either KO or activation will lead to abnormal and persistent hyperactivation of mTOR activity. Using in utero electroporation they transfected plasmids containing these gene constructs into fetal mouse brains at E15.5 and then assessed neuronal shape and size, laminar positioning, spontaneous activity, synaptic activity, and expression of a novel voltage-gated potassium channel (HCN4) at varying time postnatally e.g., P7-9 (neonates) and P28-43 (young adults).

      The study clearly achieves its stated aims i.e., that disruption of each of five distinct mTOR pathway genes, Rheb, mTOR, Depdc5, Pten, and Tsc1, individually impacts pyramidal neuron development and electrophysiological function in the mouse mPFC. The data from each of the 5 genes provides strong support to the notion that mTOR pathway gene mutations yield the unifying clinical parcellation of mTORopathies, likely as a consequence of mTOR pathway activation. The data also provide interesting evidence that subtle or even overt differences in clinical phenotypes between RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO in humans could be due to effects of these genes either on mTOR or on yet to be defined alternative pathways. Assuredly follow-up studies to examine how Rheb, mTOR, Dedpdc5, Pten, and Tsc1 engage with other protein binding partners or other pathways will be warranted in future studies.

      Strengths:

      The investigators demonstrate that gene KO or activation leads to common changes in cell size (enlargement) though with different effects across each gene subtype suggesting distinct genetic effects despite a common effect on mTOR signaling. The major effect was seen in forebrain neurons expressing mTORS2215Y. They also report gene-specific effects of each mTOR pathway gene on cortical lamination. For example, while RhebY35L, mTORS2215Y, Dedpdc5KO, and Tsc1KO significantly disrupted laminar positioning of neurons in layer 2/3, PtenKO had minimal effects on laminar positioning. This finding is intriguing since it means that simply activating mTOR during fetal brain development will not necessarily alter cortical lamination and that an increase in cell size by itself doesn't disrupt laminar fidelity. To verify that the expression of plasmids led to mTORC1 hyperactivation, phosphorylated levels of S6 (i.e., p-S6), a downstream substrate of mTORC1, were assayed by immunohistochemistry in P28-43 mice. Expression of the RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO plasmids all led to significantly increased p-S6 staining intensity, supporting that the expression of each of these plasmids leads to increased mTORC1 signaling.

      Whole-cell current- and voltage-clamp recordings were performed in P25-P51 mice in acute brain slice preparations. Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to decreased depolarization-induced excitability, but only RhebY35L, mTORS2215Y, and Tsc1KO expression led to depolarized resting membrane potentials. Interestingly, expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to the abnormal presence of HCN4 channels with variations in functional expression suggesting a common cellular mechanism that could confer excitability. Treatment with rapamycin, an mTOR inhibitor, reversed the expression changes in HCN4. Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to different impacts on sEPSC properties. Effects of treatment with the selective HCN channel blocker zatebradine on hyperpolarization-induced inward currents in mTORS2215Y neurons confirmed the identity of ΔI as Ih.

      Overall the data presented provides a convincing and compelling direct comparison of the roles that select mTOR pathway genes play on brain development and network excitability. It is critical to directly compare these gene effects in mouse models because although these genes are part of the mTOR pathway and clearly cause augmentation of mTOR activation, there are mechanistic differences in how these gees modify mTOR and how they interact with other proteins and phenotypic differences in humans harboring mutations in these same genes.

    1. Reviewer #2 (Public Review):

      Summary: The authors have previously demonstrated that the E3 ligase PDLIM2 inhibits NF-kB and STAT3 and is epigenetically repressed in human lung cancers (Sun et al. Nat. Comm. 2019 10: 5324); therefore, PDLIM2 is a tumor suppressor in lung cancer. In this manuscript, they follow up on their previous findings and show that expression of PDLIM2 is downregulated in human lung cancers by both genetic deletion and promoter methylation. They further describe a novel approach to restore the expression of PDLIM2 in mouse lung tumors by systemically administering PDLIM2 plasmids encapsulated in nanoparticles (termed "nanoPDLIM2"). The nanoPDLIM2 approach was shown to exhibit efficacy with low toxicity in a urethane-induced mouse lung cancer model. The authors further demonstrated synergy of nanoPDLIM2 with chemotherapy and PD-1 blockade immunotherapy. The combination therapy of nanoPDLIM2, chemotherapy and immunotherapy proved most effective with complete tumor remission in 60% of mice. Mechanistically, nanoPDLIM2 upregulated MHC-I expression, enhanced CD4/CD8 T cell activation and tumor infiltration, and suppressed MDR1 induction and nuclear expression of STAT3, RelA and prosurvival genes in tumors. Overall, this study is important because it reinforces the critical roles of PDLIM2 in suppressing lung cancer, and also identifies a potential approach to restoring PDLIM2 expression in lung tumors. The experiments were well executed; the data are convincing and support the conclusions made by the authors.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Liao and colleagues generated tagged SMAD1 and SMAD5 mouse models and identified genome occupancy of these two factors in the uterus of these mice using the CUT&RUN assay. The authors used integrative bioinformatic approaches to identify putative SMAD1/5 direct downstream target genes and to catalog the SMAD1/5 and PGR genome co-localization pattern. The role of SMAD1/5 on stromal decidualization was assayed in vitro on primary human endometrial stromal cells. The new mouse models offer opportunities to further dissect SMAD1 and SMAD5 functions without the limitation from SMAD antibodies, which is significant. The CUT&RUN data further support the usefulness of these mouse models for this purpose.

      Strengths:<br /> The strength of this study is the novelty of new mouse models and the valuable cistromic data derived from these mice. Overall the present manuscript is an excellent resource paper for the field of reproductive biology.

      Weaknesses:<br /> The weakness of the present version of the manuscript includes the self-limited data analysis approaches such as the proximal promoter based bioinformatic filter and an outdated method on inferring the cell type composition. Evidence was provided for potential associations between SMAD1/5 and other major transcription factors. However, causal effects of SMAD1/5 on the genome occupancy of other major uterine transcription factors were discussed but not experimentally examined in the present manuscript, which is understandable.

      For data in Figure 2B, the current manuscript fails to elaborate the common and distinct features between clusters 1 and 3 as well as the biological significance of having two separate clusters for SMAD1. In addition, Figure S1A shows overlapping genome occupancy between SMAD1 and SMAD5, which is not clearly demonstrated in Figure 2B.

      For data in Figure 5A, the result description does not provide adequate information to guide readers to full understanding of the data. The biological meaning behind the three PR clusters is not stated nor speculated. Moreover, Figure 5A and Figure S1B are inherently connected but fail to be adequately described in the main text.

    1. Reviewer #2 (Public Review):

      This is a genome-wide association study of COVID-19 in individuals of admixed American ancestry (AMR) recruited from Brazil, Colombia, Ecuador, Mexico, Paraguay, and Spain. After quality control and admixture analysis, a total of 3,512 individuals were interrogated for 10,671,028 genetic variants (genotyped + imputed). The genetic association results for these cohorts were meta-analyzed with the results from The Host Genetics Initiative (HGI), involving 3,077 cases and 66,686 controls. The authors found two novel genetic loci associated with COVID-19 at 2q24.2 (rs13003835) and 11q14.1 (rs77599934), and other two independent signals at 3p21.31 (rs35731912) and 6p21.1 (rs2477820) already reported as associated with COVID-19 in previous GWASs. Additional meta-analysis with other HGI studies also suggested risk variants near CREBBP, ZBTB7A, and CASC20 genes.

      Strengths:<br /> These findings rely on state-of-the-art methods in the field of Statistical Genomics and help to address the issue of a low number of GWASs in non-European populations, ultimately contributing to reducing health inequalities across the globe.

      Weaknesses:<br /> There is no replication cohort, as acknowledged by the authors (page 29, line 587), and no experimental validation to assess the biological effect of putative causal variants/genes. Thus, the study provides good evidence of association, rather than causation, between the genetic variants and COVID-19. Lastly, I consider it crucial to report the results for the SCOURGE Latin American GWAS, in addition to its meta-analysis with HGI results, since HGI data has a different phenotype scheme (Hospitalized COVID vs Population) compared to SCOURGE (Hospitalized COVID vs Non-hospitalized COVID).

    1. Reviewer #2 (Public Review):

      DNA gyrase is an essential enzyme in bacteria that regulates DNA topology and has the unique property to introduce negative supercoils into DNA. This enzyme contains 2 subunits GyrA and GyrB, which forms an A2B2 heterotetramer that associates with DNA and hydrolyzes ATP. The molecular structure of the A2B2 assembly is composed of 3 dimeric interfaces, called gates, which allow the cleavage and transport of DNA double stranded molecules through the gates, in order to perform DNA topology simplification.<br /> The article by Germe et al. questions the existence and possible mechanism for subunit exchange in the bacterial DNA gyrase complex.

      The complexes are purified as a dimer of GyrA and a fusion of GyrB and GyrA (GyrBA), encoded by different plasmids, to allow the introduction of targeted mutations on one side only of the complex. The conclusion drawn by the authors is that subunit exchange does happen in vitro, favored by DNA binding and wrapping. They propose that the accumulation of gyrase in higher-order oligomers can favor rapid subunit exchange between two active gyrase complexes brought into proximity. This study is nicely illustrated with diagrams that explain the possible mechanism.

      The authors are also debating the conclusions of a previous article by Gubaev, Weidlich et al 2016 (https://doi.org/10.1093/nar/gkw740). Gubaev et al. originally used this strategy of complex reconstitution to propose a nicking-closing mechanism for the introduction of negative supercoils by DNA gyrase, an alternative mechanism that precludes DNA strand passage, previously established in the field. Germe et al. propose that the detected negative supercoiling activity in this earlier study may be due to the subunit swapping of the recombinant protein with the endogenous enzyme.

      Strengths

      The mix of gyrase subunits is plausible, this mechanism has been suggested by Ideka et al, 2004 and also for the human Top2 isoforms with the formation of Top2a/Top2b hybrids being identified in HeLa cells (doi: 10.1073/pnas.93.16.8288).<br /> Germe et al have used extensive and solid biochemical experiments, together with thorough experimental controls, involving :<br /> - the purification of gyrase subunits including mutants with domain deletion, subunit fusion or point mutations.<br /> - DNA relaxation, cleavage and supercoiling assays<br /> - biophysical characterization in solution (size exclusion chromatography, mass photometry, mass spectrometry)

      Together the combination of experimental approaches provides convincing evidence for subunit swapping in gyrase in vitro, despite the technical limitations of standard biochemistry applied to such a complex macromolecule.

      Weaknesses

      The conclusions of this study could be strengthened by in vivo data to identify subunit swapping in the bacteria. Indeed, if shown in vivo, together with this biochemical evidence, this mechanism could have a substantial impact on our understanding of bacterial physiology and resistance to drugs. These in vivo perspectives are beyond the scope of the present in vitro investigation but are however explained by the authors.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study examined the possible affect of spike-wave discharges (SWDs) on the response to visual or somatosensory stimulation using fMRI and EEG. This is a significant topic because SWDs often are called seizures and because there is non-responsiveness at this time, it would be logical that responses to sensory stimulation are reduced. On the other hand, in rodents with SWDs, sensory stimulation (a noise, for example) often terminates the SWD/seizure.

      In humans, these periods of SWDs are due to thalamocortical oscillations. A certain percentage of the normal population can have SWDs in response to photic stimulation at specific frequencies. Other individuals develop SWDs without stimulation. They disrupt consciousness. Individuals have an absent look, or "absence", which is called absence epilepsy.

      The authors use a rat model to study the responses to stimulation of the visual or somatosensory systems during and in between SWDs. They report that the response to stimulation is reduced during the SWDs. While some data show this nicely, the authors also report on lines 396-8 "When comparing statistical responses between both states, significant changes (p<0.05, cluster-) were noticed in somatosensory auditory frontal..., with these regions being less activated in interictal state (see also Figure 4). That statement is at odds with their conclusion. I do not see that this issue was addressed.

      They also conclude that stimulation slows the pathways activated by the stimulus. I do not see any data proving this. It would require repeated assessments of the pathways in time. This issue was not addressed.

      The authors also study the hemodynamic response function (HRF) and it is not clear what conclusions can be made from the data. This is still an issue. No conclusions appear to be possible to make.

      Finally, the authors use a model to analyze the data. This model is novel and while that is a strength, its validation is unclear. The authors did not add any validation of their model.

      Strengths:<br /> Use of fMRI and EEG to study SWDs in rats.

      Weaknesses:<br /> Several aspects of the Methods and Results were improved but some are still are unclear.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study builds upon previous work that demonstrated that brain injury results in leakage of albumin across the blood brain barrier, resulting in activation of TGF-beta in astrocytes. Consequently, this leads to decreased glutamate uptake, reduced buffering of extracellular potassium and hyperexcitability. This study asks whether such a process can play a physiological role in cortical plasticity. They first show that stimulation of a forelimb for 30 minutes in a rat results in leakage of the blood brain barrier and extravasation of albumin on the contralateral but not ipsilateral cortex. The authors propose that the leakage is dependent upon neuronal excitability and is associated with an enhancement of excitatory transmission. Inhibiting the transport of albumin or the activation of TGF-beta prevents the enhancement of excitatory transmission. In addition, gene expression associated with TGF-beta activation, synaptic plasticity and extracellular matrix are enhanced on the "stimulated" hemisphere. That this may translate to humans is demonstrated by a break down in the blood brain barrier following activation of brain areas through a motor task.

      Strengths:<br /> This study is novel and the results are potentially important as they demonstrate an unexpected break down of the blood brain barrier with physiological activity and this may serve a physiological purpose, affecting synaptic plasticity.

      The strengths of the study are:<br /> 1) The use of an in vivo model with multiple methods to investigate the blood brain barrier response to a forelimb stimulation.<br /> 2) The determination of a potential functional role for the observed leakage of the blood brain barrier from both a genetic and electrophysiological view point<br /> 3) The demonstration that inhibiting different points in the putative pathway from activation of the cortex to transport of albumin and activation of the TGF-beta pathway, the effect on synaptic enhancement could be prevented.<br /> 4) Preliminary experiments demonstrating a similar observation of activity dependent break down of the blood brain barrier in humans.

      Weaknesses:<br /> The authors adequately addressed most of my points. A few remain:<br /> 1) Although the reviewers have addressed the possible effects of anaesthesia on neuro-vascular coupling. They have not mentioned or addressed the possible effects of ketamine (an NMDA receptor antagonist) on synaptic plasticity. Indeed, the low percentage of SEP increase following potentiation (10-20%) could perhaps be explained by partial block of NMDA receptors by ketamine.<br /> 2) The experimental paradigms remain unclear to me. Now, it appears that drugs are applied for 50 minutes and that the stimulation occurs during the "washout period". The more conventional approach would be to have the drug application during the stimulation period to determine if the drugs occlude or enhance the effects of stimulation and then washout the drugs. The problem is that drugs variably washout at different rates depending upon their lipid solubility.<br /> 3) It is still not clear to what extent the experimenters and those doing the analysis were blinded to group. If one or both were blind to group, then please put this in the methods.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors are studying the behavioral response to pathogen exposure. They and others have previously describe the role that the G-protein coupled receptors in the nervous system plays in detecting pathogens, and initiating behavioral patterns (e.g. avoidance/learned avoidance) that minimize contact. The authors study this problem in C. elegans, which is amenable to genetic and cellular manipulations and allow the authors to define cellular and signaling mechanisms. This paper extends the original idea to now implicate signaling and transcriptional pathways within a particular neuron (ASJ) and the gut in mediating avoidance behaviour.

      Strengths:<br /> The work is rigorous and elegant and the data are convincing. The authors make superb use of mutant strains in C. elegans, as well tissue specific gene inactivation and expression and genetic methods of cell ablation. to demonstrate how a gene, NPR15 controls behavioral changes in pathogen infection. The results suggest that ASJ neurons and the gut mediate such effects. I expect the paper will constitute an important contribution to our understanding of how the nervous system coordinates immune and behavioral responses to infection.

    1. Reviewer #2 (Public Review):

      This paper describes the results of a set of complementary and convergent experiments aimed at describing roles for the non-selective cation channels NALCN and TRPC6 in mediating subthreshold inward depolarizing currents and action potential generation in VTA DA neurons under normal physiological conditions. In general, the authors have responded satisfactorily to reviewer comments, and the revised manuscript is improved. The manuscript could still benefit from additional revision, including the following:

      1. From the previous review, we mentioned that " 'The HCN' as written in line 69 is a bit misleading, as HCN channels in the heart and brain are different members of a family of channels, although as written in the text, it seems that they are identical." This is still the case (now line 73).

      2. The authors state in line 112 that "most of the experiments were also repeated in female mice" - this is true in the case of most electrophysiological experiments, although not behavioral experiments. Authors should amend the statement in line 112 and clarify in the Discussion section which findings are generalizable between sexes; e.g.:<br /> a. Discussion of HCN contribution to VTA DA activity (beginning line 453) should clarify male mice.<br /> b. Similarly, any discussion of behavioral findings should clarify male mice.

      3. The authors' statement in lines 179-183 ("In contrast, fewer GABAergic neuronal markers (Glutamic acid decarboxylase, GAD1/2 and vesicular GABA transporter, VGAT) co-expressed with the DA neurons, which is consistent with previous studies that VTA DA neurons co-expressing GABAergic neuronal markers mainly project to the lateral habenula") is a little confusing - as stated, it seems that the authors are confirming DA/GABA coexpression in VTA-LHb neurons, which is not the case.

      4. Additional information could be included in the Methods section description of Western Blotting procedures - e.g., what thickness of tissue and what size gauge were used to dissect VTA for these experiments?

    1. Reviewer #2 (Public Review):

      The manuscript investigates the function of basal forebrain cholinergic axons in mouse primary visual cortex (V1) during locomotion using two-photon calcium imaging in head-fixed mice. Cholinergic modulation has previously been proposed to mediate the effects of locomotion on V1 responses. The manuscript concludes that the activity of basal forebrain cholinergic axons in visual cortex provides a signal which is more correlated with binary locomotion state than locomotion velocity of the animal. Cholinergic axons did not seem to respond to grating stimuli or visuomotor prediction error. Optogenetic stimulation of these axons increased the amplitude of responses to visual stimuli and decreased the response latency of layer 5 excitatory neurons, but not layer 2/3 neurons. Moreover, optogenetic or chemogenetic stimulation of cholinergic inputs reduced pairwise correlation of neuronal responses. These results provide insight into the role of cholinergic modulation to visual cortex and demonstrate that it affects different layers of visual cortex in a distinct manner. The experiments are well executed and the data appear to be of high quality. However, further analyses are required to fully support some of the study's conclusions.

      The manuscript concludes that cholinergic axons convey a binary locomotion signal and are not tuned to running speed. Getting head-fixed animals to run at the speeds typical of freely moving animals can require training, which was not undertaken in this study. Consequently, the typically low running velocity of mice is a potential limitation of this study.

      The analyses of the effects of locomotion and stimulation of cholinergic inputs present grand averages of responses across all neurons, and therefore may mask heterogeneity across layer 2/3 and layer 5 neurons.

    1. Reviewer #2 (Public Review):

      The authors phototag DA and GABA neurons in the VTA in mice performing a t-maze task, and report choice-specific responses in the delay period of a memory-guided task, more so that in a variant task w/o a memory component. Overall, I found the results convincing. While showing responses that are choice selective in DA neurons is not entirely novel (e.g. Morris et al NN 2006, Parker et al NN 2016), the fact that this feature is stronger when there is a memory requirement is an interesting and a novel observation.

    1. Reviewer #2 (Public Review):

      In this manuscript, Yao et al. present a series of experiments aiming at generating a cellular atlas of the human hippocampus across aging, and how it may be affected by injury, in particular, stroke. Although the aim of the study is interesting and relevant for a larger audience, due to the ongoing controversy around the existence of adult hippocampal neurogenesis in humans, a number or technical weaknesses result in a poor support for many of the conclusions made from the results of these experiments.<br /> In particular, a recent meta analysis of five previous studies applying similar techniques to human samples has identified different aspects of sample size as main determinants of the statistical power needed to make significant conclusions. Some of this aspects are the number of nuclei sequenced and subject stratification. These two aspects are of concern in Yao's study. First, the number of sequenced nuclei is lower than the calculated numbers of nuclei required for detecting rare cell types. However, Yao et al. report succeeding in detecting rare populations, including several types of neural stem cells in different proliferation states, which have been demonstrated to be extremely scarce by previous studies. It would be very interesting to read how the authors interpret these differences. Secondly, the number of donors included in some of the groups is extremely low (n=1) and the miscellaneous information provided about the donors is practically inexistent. As individual factors such as chronic conditions, medication, lifestyle parameters, etc... are considered determinant for the variability of adult hippocampal neurogenesis levels across individuals, this represents a series limitation of the current study. Overall, several technical weaknesses severely limit the relevance of this study and the ability of the authors to achieve their experimental aims.

      After a first review round, the manuscript is still lacking a clear discussion of its several technical limitations, which will help the audience to grasp the relevance of the findings. In particular, detailed information about individual patients health status and relevant lifestyle parameters that may have affected it is lacking. The authors make the point themselves that the discrepancies among studies might be caused by health state differences across hippocampi, which subsequently lead to different degrees of hippocampal neurogenesis.". So, even in the authors own interpretation this is a serious limitation to the manuscript, that however out of the authors control, impacts on the quality of their findings.

    1. Reviewer #2 (Public Review):

      Summary:

      This study aims to test auditory confounds during transcranial ultrasound stimulation (TUS) protocols that rely on audible frequencies. In several experiments, the authors show that a commonly observed suppression of motor-evoked potentials (MEP) during TUS can be explained by acoustic stimulation. For instance, not only target TUS, but also stimulation of a control site and acoustic stimulation led to suppressed MEP.

      The authors have convincingly addressed all of my comments and provided useful additional details. I believe that this is a strong study that will impact the field. Thanks also for making the sound stimuli open-source.

    1. Reviewer #2 (Public Review):

      The authors describe what they assert to be a very unusual trigeminal nuclear complex in the brainstem of elephants, and based on this, follow with many speculations about how the trigeminal nuclear complex, as identified by them, might be organized in terms of the sensory capacity of the elephant trunk.

      The identification of the trigeminal nuclear complex/inferior olivary nuclear complex in the elephant brainstem is the central pillar of this manuscript from which everything else follows, and if this is incorrect, then the entire manuscript fails, and all the associated speculations become completely unsupported.

      The authors note that what they identify as the trigeminal nuclear complex has been identified as the inferior olivary nuclear complex by other authors, citing Shoshani et al. (2006; 10.1016/j.brainresbull.2006.03.016) and Maseko et al (2013; 10.1159/000352004), but fail to cite either Verhaart and Kramer (1958; PMID 13841799) or Verhaart (1962; 10.1515/9783112519882-001). These four studies are in agreement, but the current study differs.

      Let's assume for the moment that the four previous studies are all incorrect and the current study is correct. This would mean that the entire architecture and organization of the elephant brainstem is significantly rearranged in comparison to ALL other mammals, including humans, previously studied (e.g. Kappers et al. 1965, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Volume 1 pp. 668-695) and the closely related manatee (10.1002/ar.20573). This rearrangement necessitates that the trigeminal nuclei would have had to "migrate" and shorten rostrocaudally, specifically and only, from the lateral aspect of the brainstem where these nuclei extend from the pons through to the cervical spinal cord (e.g. the Paxinos and Watson rat brain atlases), the to the spatially restricted ventromedial region of specifically and only the rostral medulla oblongata. According to the current paper, the inferior olivary complex of the elephant is very small and located lateral to their trigeminal nuclear complex, and the region from where the trigeminal nuclei are located by others appears to be just "lateral nuclei" with no suggestion of what might be there instead.

      Such an extraordinary rearrangement of brainstem nuclei would require a major transformation in the manner in which the mutations, patterning, and expression of genes and associated molecules during development occur. Such a major change is likely to lead to lethal phenotypes, making such a transformation extremely unlikely. Variations in mammalian brainstem anatomy are most commonly associated with quantitative changes rather than qualitative changes (10.1016/B978-0-12-804042-3.00045-2).

      The impetus for the identification of the unusual brainstem trigeminal nuclei in the current study rests upon a previous study from the same laboratory (10.1016/j.cub.2021.12.051) that estimated that the number of axons contained in the infraorbital branch of the trigeminal nerve that innervate the sensory surfaces of the trunk is approximately 400 000. Is this number unusual? In a much smaller mammal with a highly specialized trigeminal system, the platypus, the number of axons innervating the sensory surface of the platypus bill skin comes to 1 344 000 (10.1159/000113185). Yet, there is no complex rearrangement of the brainstem trigeminal nuclei in the brain of the developing or adult platypus (Ashwell, 2013, Neurobiology of Monotremes), despite the brainstem trigeminal nuclei being very large in the platypus (10.1159/000067195). Even in other large-brained mammals, such as large whales that do not have a trunk, the number of axons in the trigeminal nerve ranges between 400,000 and 500,000 (10.1007/978-3-319-47829-6_988-1). The lack of comparative support for the argument forwarded in the previous and current study from this laboratory, and that the comparative data indicates that the brainstem nuclei do not change in the manner suggested in the elephant, argues against the identification of the trigeminal nuclei as outlined in the current study. Moreover, the comparative studies undermine the prior claim of the authors, informing the current study, that "the elephant trigeminal ganglion ... point to a high degree of tactile specialization in elephants" (10.1016/j.cub.2021.12.051). While clearly, the elephant has tactile sensitivity in the trunk, it is questionable as to whether what has been observed in elephants is indeed "truly extraordinary".

      But let's look more specifically at the justification outlined in the current study to support their identification of the unusually located trigeminal sensory nuclei of the brainstem.

      (1) Intense cytochrome oxidase reactivity.<br /> (2) Large size of the putative trunk module.<br /> (3) Elongation of the putative trunk module.<br /> (4) The arrangement of these putative modules corresponds to elephant head anatomy.<br /> (5) Myelin stripes within the putative trunk module that apparently match trunk folds.<br /> (6) Location apparently matches other mammals.<br /> (7) Repetitive modular organization apparently similar to other mammals.<br /> (8) The inferior olive described by other authors lacks the lamellated appearance of this structure in other mammals.

      Let's examine these justifications more closely.

      (1) Cytochrome oxidase histochemistry is typically used as an indicative marker of neuronal energy metabolism. The authors indicate, based on the "truly extraordinary" somatosensory capacities of the elephant trunk, that any nuclei processing this tactile information should be highly metabolically active, and thus should react intensely when stained for cytochrome oxidase. We are told in the methods section that the protocols used are described by Purkart et al (2022) and Kaufmann et al (2022). In neither of these cited papers is there any description, nor mention, of the cytochrome oxidase histochemistry methodology, thus we have no idea of how this histochemical staining was done. To obtain the best results for cytochrome oxidase histochemistry, the tissue is either processed very rapidly after buffer perfusion to remove blood or in recently perfusion-fixed tissue (e.g., 10.1016/0165-0270(93)90122-8). Given: (1) the presumably long post-mortem interval between death and fixation - "it often takes days to dissect elephants"; (2) subsequent fixation of the brains in 4% paraformaldehyde for "several weeks"; (3) The intense cytochrome oxidase reactivity in the inferior olivary complex of the laboratory rat (Gonzalez-Lima, 1998, Cytochrome oxidase in neuronal metabolism and Alzheimer's diseases); and (4) The lack of any comparative images from other stained portions of the elephant brainstem; it is difficult to support the justification as forwarded by the authors. The histochemical staining observed is likely background reactivity from the use of diaminobenzidine in the staining protocol. Thus, this first justification is unsupported.

      Justifications (2), (3), and (4) are sequelae from justification (1). In this sense, they do not count as justifications, but rather unsupported extensions.

      (4) and (5) These are interesting justifications, as the paper has clear internal contradictions, and (5) is a sequelae of (4). The reader is led to the concept that the myelin tracts divide the nuclei into sub-modules that match the folding of the skin on the elephant trunk. One would then readily presume that these myelin tracts are in the incoming sensory axons from the trigeminal nerve. However, the authors note that this is not the case: "Our observations on trunk module myelin stripes are at odds with this view of myelin. Specifically, myelin stripes show no tapering (which we would expect if axons divert off into the tissue). More than that, there is no correlation between myelin stripe thickness (which presumably correlates with axon numbers) and trigeminal module neuron numbers. Thus, there are numerous myelinated axons, where we observe few or no trigeminal neurons. These observations are incompatible with the idea that myelin stripes form an axonal 'supply' system or that their prime function is to connect neurons. What do myelin stripe axons do, if they do not connect neurons? We suggest that myelin stripes serve to separate rather than connect neurons." So, we are left with the observation that the myelin stripes do not pass afferent trigeminal sensory information from the "truly extraordinary" trunk skin somatic sensory system, and rather function as units that separate neurons - but to what end? It appears that the myelin stripes are more likely to be efferent axonal bundles leaving the nuclei (to form the olivocerebellar tract). This justification is unsupported.

      (6) The authors indicate that the location of these nuclei matches that of the trigeminal nuclei in other mammals. This is not supported in any way. In ALL other mammals in which the trigeminal nuclei of the brainstem have been reported they are found in the lateral aspect of the brainstem, bordered laterally by the spinal trigeminal tract. This is most readily seen and accessible in the Paxinos and Watson rat brain atlases. The authors indicate that the trigeminal nuclei are medial to the facial nerve nucleus, but in every other species, the trigeminal sensory nuclei are found lateral to the facial nerve nucleus. This is most salient when examining a close relative, the manatee (10.1002/ar.20573), where the location of the inferior olive and the trigeminal nuclei matches that described by Maseko et al (2013) for the African elephant. This justification is not supported.

      (7) The dual to quadruple repetition of rostrocaudal modules within the putative trigeminal nucleus as identified by the authors relies on the fact that in the neurotypical mammal, there are several trigeminal sensory nuclei arranged in a column running from the pons to the cervical spinal cord, these include (nomenclature from Paxinos and Watson in roughly rostral to caudal order) the Pr5VL, Pr5DM, Sp5O, Sp5I, and Sp5C. However, these nuclei are all located far from the midline and lateral to the facial nerve nucleus, unlike what the authors describe in the elephants. These rostrocaudal modules are expanded upon in Figure 2, and it is apparent from what is shown that the authors are attributing other brainstem nuclei to the putative trigeminal nuclei to confirm their conclusion. For example, what they identify as the inferior olive in Figure 2D is likely the lateral reticular nucleus as identified by Maseko et al (2013). This justification is not supported.

      (8) In primates and related species, there is a distinct banded appearance of the inferior olive, but what has been termed the inferior olive in the elephant by other authors does not have this appearance, rather, and specifically, the largest nuclear mass in the region (termed the principal nucleus of the inferior olive by Maseko et al, 2013, but Pr5, the principal trigeminal nucleus in the current paper) overshadows the partial banded appearance of the remaining nuclei in the region (but also drawn by the authors of the current paper). Thus, what is at debate here is whether the principal nucleus of the inferior olive can take on a nuclear shape rather than evince a banded appearance. The authors of this paper use this variance as justification that this cluster of nuclei could not possibly be the inferior olive. Such a "semi-nuclear/banded" arrangement of the inferior olive is seen in, for example, giraffe (10.1016/j.jchemneu.2007.05.003), domestic dog, polar bear, and most specifically the manatee (a close relative of the elephant) (brainmuseum.org; 10.1002/ar.20573). This justification is not supported.

      Thus, all the justifications forwarded by the authors are unsupported. Based on methodological concerns, prior comparative mammalian neuroanatomy, and prior studies in the elephant and closely related species, the authors fail to support their notion that what was previously termed the inferior olive in the elephant is actually the trigeminal sensory nuclei. Given this failure, the justifications provided above that are sequelae also fail. In this sense, the entire manuscript and all the sequelae are not supported.

      What the authors have not done is to trace the pathway of the large trigeminal nerve in the elephant brainstem, as was done by Maseko et al (2013), which clearly shows the internal pathways of this nerve, from the branch that leads to the fifth mesencephalic nucleus adjacent to the periventricular grey matter, through to the spinal trigeminal tract that extends from the pons to the spinal cord in a manner very similar to all other mammals. Nor have they shown how the supposed trigeminal information reaches the putative trigeminal nuclei in the ventromedial rostral medulla oblongata. These are but two examples of many specific lines of evidence that would be required to support their conclusions. Clearly, tract tracing methods, such as cholera toxin tracing of peripheral nerves cannot be done in elephants, thus the neuroanatomy must be done properly and with attention to detail to support the major changes indicated by the authors.

      So what are these "bumps" in the elephant brainstem?

      Four previous authors indicate that these bumps are the inferior olivary nuclear complex. Can this be supported?

      The inferior olivary nuclear complex acts "as a relay station between the spinal cord (n.b. trigeminal input does reach the spinal cord via the spinal trigeminal tract) and the cerebellum, integrating motor and sensory information to provide feedback and training to cerebellar neurons" (https://www.ncbi.nlm.nih.gov/books/NBK542242/). The inferior olivary nuclear complex is located dorsal and medial to the pyramidal tracts (which were not labelled in the current study by the authors but are clearly present in Fig. 1C and 2A) in the ventromedial aspect of the rostral medulla oblongata. This is precisely where previous authors have identified the inferior olivary nuclear complex and what the current authors assign to their putative trigeminal nuclei. The neurons of the inferior olivary nuclei project, via the olivocerebellar tract to the cerebellum to terminate in the climbing fibres of the cerebellar cortex.

      Elephants have the largest (relative and absolute) cerebellum of all mammals (10.1002/ar.22425), this cerebellum contains 257 x109 neurons (10.3389/fnana.2014.00046; three times more than the entire human brain, 10.3389/neuro.09.031.2009). Each of these neurons appears to be more structurally complex than the homologous neurons in other mammals (10.1159/000345565; 10.1007/s00429-010-0288-3). In the African elephant, the neurons of the inferior olivary nuclear complex are described by Maseko et al (2013) as being both calbindin and calretinin immunoreactive. Climbing fibres in the cerebellar cortex of the African elephant are clearly calretinin immunopositive and also are likely to contain calbindin (10.1159/000345565). Given this, would it be surprising that the inferior olivary nuclear complex of the elephant is enlarged enough to create a very distinct bump in exactly the same place where these nuclei are identified in other mammals?

      What about the myelin stripes? These are most likely to be the origin of the olivocerebellar tract and probably only have a coincidental relationship with the trunk. Thus, given what we know, the inferior olivary nuclear complex as described in other studies, and the putative trigeminal nuclear complex as described in the current study, is the elephant inferior olivary nuclear complex. It is not what the authors believe it to be, and they do not provide any evidence that discounts the previous studies. The authors are quite simply put, wrong. All the speculations that flow from this major neuroanatomical error are therefore science fiction rather than useful additions to the scientific literature.

      What do the authors actually have?<br /> The authors have interesting data, based on their Golgi staining and analysis, of the inferior olivary nuclear complex in the elephant.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors used a short hairpin RNA technique strategy to elucidate the functional activity of neurons in the retrotrapezoid nucleus (RTN), a critical brainstem region for central chemoreception. Dysfunction in this area is associated with the neuropathology of congenital central hypoventilation syndrome (CCHS). The subsequent examination of these rats aimed to shed light on the intricate aspects of RTN and its implications for central chemoreception and disorders like CCHS in adults. They found that using the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of Phox2b expression was observed in Nmb neurons. In addition, Phox2b knockdown did not affect breathing in room air or under hypoxia, but the hypercapnia ventilatory response was significantly impaired. They concluded that Phox2b in the adult brain has an important role in CO2 chemoreception. They thought that their findings provided new evidence for mechanisms related to CCHS neuropathology. The conclusions of this paper are well supported by data, but careful discussion seems to be required for comparison with the results of various previous studies performed by different genetic strategies for the RTN neurons.

      Strengths:<br /> The most exciting aspect of this work is the modelling of the Phox2b knockdown in one element of the central neuronal circuit mediating respiratory reflexes, that is in the RTN. To date, mutations in the PHOX2B gene are commonly associated with most patients diagnosed with CCHS, a disease characterized by hypoventilation and absence of chemoreflexes, in the neonatal period, which in severe cases can lead to respiratory arrest during sleep. In the present study, the authors demonstrated that the role of Phox2b extends beyond the developmental period, and its reduction in CCHS may contribute to the respiratory impairment observed in this disorder.

      Weaknesses:<br /> Whereas the most exciting part of this work is the knockdown of the Phox2b in the RTN in adult rodents, the weakness of this study is the lack of a clear physiological, developmental, and anatomical distinction between this approach and similar studies already reported elsewhere (Ruffault et al., 2015, DOI: 10.7554/eLife.07051; Ramanantsoa et al., 2011, DOI: 10.1523/JNEUROSCI.1721-11.2011; Huang et al., 2017, DOI: 10.1016/j.neuron.2012.06.027; Hernandez-Miranda et al., 2018, DOI: 10.1073/pnas.1813520115; Ferreira et al., 2022 DOI: 10.7554/eLife.73130; Takakura et al., 2008 DOI: 10.1113/jphysiol.2008.153163; Basting et al., 2015 DOI: 10.1523/JNEUROSCI.2923-14.2015; Marina et al., 2010 DOI: 10.1523/JNEUROSCI.3141-10.2010). In addition, several conclusions presented in this work are not directly supported by the provided data.

    1. Reviewer #2 (Public Review):

      Summary: The study by Cullen et al presents intriguing data regarding the contribution of mTOR complex 1 (mTORC1) versus mTORC2 or both in Pten-null induced macrocephaly and epileptiform activity. The role of mTORC2 in mTORopathies, and in particular Pten loss-off-function (LOF)-induced pathology and seizures, is understudied and controversial. In addition, recent data provided evidence against the role of mTORC1 in PtenLOF-induced seizures. To address these controversies and the contribution off these mTOR complexes in PtenLOF-induced pathology and seizures, the authors injected a AAV9-Cre into the cortex of conditional single, double and triple transgenic mice at postnatal day 0 to remove Pten, Pten+Raptor or Rictor, and Pten+raptor+rictor. Raptor and Rictor are essentially binding partners of mTORC1 and mTORC2, respectively. One major finding is that despite preventing the mild macrocephaly and increased cell size, Raptor knockout (KO, decrease mTORC1 activity) did not prevent the occurrence of seizures and the rate of SWD event, and aggravated seizure duration. Similarly, Rictor KO (decreased mTORC2 activity) partially prevented the mild macrocephaly and increased cell size but did not prevent the occurrence of seizures and did not affect seizure duration. However, Rictor KO reduced the rate of SWD events. Finally, the pathology and seizure/SWD activity were fully prevented in the double KO. These data suggest the contribution of both increased mTORC1 and mTORC2 in the pathology and epileptic activity of Pten LOF mice, emphasizing the importance of blocking both complexes for seizure treatment. Whether these data apply to other mTORopathies due to Tsc1, Tsc2, mTOR, AKT or other gene variants remain to be examined.

      Strengths: The strengths are as follow: 1) they address an important and controversial question that has clinical application, 2) the study uses a reliable and relatively easy method to KO specific genes in cortical neurons, based on AAV9 injections in pups. 2) they perform careful video-EEG analyses correlated with some aspects of cellular pathology.

      Weaknesses: the study has nevertheless a few weaknesses: 1) the conclusions are perhaps a bit overstated. The data do not show that increased mTORC1 or mTORC2 are sufficient to cause epilepsy. But the data clearly show that both increased mTORC1 and mTORC2 activity contribute to the pathology and seizure activity and as such are necessary for seizures to occur. 2) the data related to the EEG would benefit from having more mice. Adding more mice would have help determine whether there is a decrease in seizure activity with the Rictor or Raptor KO. 3) it would have been interesting to examine the impact of mTORC2 and mTORC1 overexpression related to point #1 above.

      The authors properly addressed my comments. Number 3 above was only a suggestion that could be a follow-up in another study.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The idea that various clinical conditions may be associated, at least partially, with a disrupted corollary discharge mechanism has been present for a long time.

      In this paper, the authors draw a link between sensory overload, a characteristic of autism spectrum disorder, and a disturbance in the corollary discharge mechanism. The authors substantiate their hypothesis with strong evidence from both the motor and perceptual domains. As a result, they broaden the clinical relevance of the corollary discharge mechanism to encompass autism spectrum disorder.

      The authors write:<br /> "Imagine a scenario in which you're watching a video of a fast-moving car on a bumpy road. As the car hits a pothole, your eyes naturally make quick, involuntary saccades to keep the car in your visual field. Without a functional efference copy system, your brain would have difficulty accurately determining the current position of your eye in space, which in turn affects its ability to anticipate where the car should appear after each eye movement."

      I appreciate the use of examples to clarify the concept of efference copy. However, I believe this example is more related to a gain-field mechanism, informing the system about the position of the eye with respect to the head, rather than an example of efference copy per se.

      Without an efference copy mechanism, the brain would have trouble accurately determining where the eyes will be in space after an eye movement, and it will have trouble predicting the sensory consequences of the eye movement. However it can be argued that the gain-field mechanism would be sufficient to inform the brain about the current position of the eyes with respect to the head.

      The authors write:<br /> "In the double-step paradigm, two consecutive saccades are made to briefly displayed targets 21, 22. The first saccade occurs without visual references, relying on internal updating to determine the eye's position."

      Maybe I have missed something, but in the double-step paradigm the first saccade can occur without the help of visual references if no visual feedback is present, that is, when saccades are performed in total darkness. Was this the case for this experiment? I could not find details about room conditions in the methods. Please provide further details.

      In case saccades were not performed in total darkness, then the first saccade can be based on the remembered location of the first target presented, which can be derived from the retinotopic trace of the first stimuli, as well as the contribution from the surroundings, that is: the remembered relative location of the first target with respect to the screen border along the horizontal meridian (i.e. allocentric cues).

      A similar logic could be applied to the second saccade. If the second saccade were based only on the retinotopic trace, without updating, then it would go up and 45 deg to the right, based on the example shown in Figure 1. With appropriate updating, the second saccade would go straight up. However, if saccades were not performed in total darkness, then the location of the second target could also be derived from its relationship with the surroundings (for example, the remembered distance from screen borders, i.e. allocentric cues).

      If saccades were not performed in total darkness, the results shown in Figures 2 and 3 could then be related to i) differences in motor updating between AQ score groups; ii) differences in the use of allocentric cues between AQ score groups; iii) a combination of i) and ii). I believe this is a point worth mentioning in the discussion."

      The authors write:<br /> "According to theories of saccadic suppression, an efference copy is necessary to predict the occurrence of a saccade."

      I would also refer to alternative accounts, where saccadic suppression appears to arise as early as the retina, due to the interaction between the visual shift introduced by the eye movement, and the retinal signal associated with the probe used to measure saccadic suppression. This could potentially account for the scaling of saccadic suppression magnitude with saccade amplitude.

      Idrees, S., Baumann, M.P., Franke, F., Münch, T.A. and Hafed, Z.M., 2020. Perceptual saccadic suppression starts in the retina. Nature communications, 11(1), p.1977.

    1. Reviewer #2 (Public Review):

      The authors repeated a previous behavioural study on the effects of overnight fasting on avoidance and extinction learning in healthy female participants in the 3T MRI scanner. Previous behavioural findings were replicated only in part. Fasting related changes of fMRI signals were less than expected.

      This paper is not without interest. Anxiety disorders are very frequent, and there is still a need to better understand ways to improve extinction and reduced avoidance. The authors follow up on previous observations of their group using overnight fasting. The findings, however, were largely negative, and it is difficult to tell how robust the observed positive findings are. The paradigm did not work as well as expected in the MR scanner.

      Introduction/main hypothesis: The reviewer does not understand why a smaller reward prediction error should result in faster extinction learning? The opposite should be the case. Plus, how much of a reward prediction error is expected in the CS- condition in extinction training? Here the US omission is expected. The reviewer may miss a key concept of the study.

      Results: A major part of the behavioural data of a previous pure behavioural study was not reproduced (avoidance learning), plus many of the MRI findings did not show a difference between the fasting and re-feed groups. Given the large amount of comparisons it makes one wonder how robust the presented findings are. The advances to the field are therefore limited.

    1. Reviewer #2 (Public Review):

      This short manuscript by Zhu et al. describes an investigation into the role of gamma protocadherins in synaptic connectivity in the mouse cerebral cortex. First, the authors conduct a single-cell RNA-seq survey of postnatal day 11 mouse cortical neurons, using an adapted 10X Genomics method to capture the 5' sequences that are necessary to identify individual gamma protocadherin isoforms (all 22 transcripts share the same three 3' "constant" exons, so standard 3'-biased methods can't distinguish them). This method adaptation is an advance for examining individual clustered protocadherin transcripts, and it is helpful to publish the method in this manuscript. The results largely confirm what was known from other approaches, which is that a few of the 19 A and B subtype gamma protocadherins are expressed in an apparently stochastic and combinatorial fashion in each cortical neuron, while the 3 C subtype genes are expressed by most neurons. Second, using elegant paired electrophysiological recordings, the authors show that in gamma protocadherin knockout cortical slices, the likelihood of two neurons on layers 2/3 being synaptically connected is increased. That suggests that gamma protocadherins generally inhibit synaptic connectivity in the cortex; again, this has been reported previously using morphological assays, but it is helpful to see it confirmed here with physiology. Finally, the authors use an impressive sequential in utero electroporation method to provide evidence that the degree of isoform matching between two neurons negatively regulates their reciprocal synaptic connectivity. These are difficult experiments to do, and while some caveats remain (e.g., lack of demonstration of protein levels in electroporated neurons, lack of resolution of the differences between the present results and those of other papers, a focus on C4 rather than C3 or C5 when considering the highly expressed C-type isoforms), the main result is consistent. Strengths of this manuscript include the impressive methodology and improved demonstration of the previously-reported finding that gamma protocadherins work via homophilic matching to put a brake on synapse formation in the cortex. Because of the unique organization and expression pattern of the gamma protocadherins, it is not likely that these results will be broadly applicable to the general understanding of the role of cell adhesion molecules in synapse development. However, the methodology, which is now better described, should be applicable more broadly and the improved demonstration of the role of gamma protocadherin's negative role in cortical synaptogenesis is helpful in confirming earlier studies. There are several differences between the results here and those of other papers on the cortex, as well as those examining other neuronal populations such as spinal cord. The present findings do not resolve them, but adopting genetic approaches rather than overexpression in the future should help.

    1. Reviewer #2 (Public Review):

      Summary:

      We often have prior expectations about how the sensory world will change, but it remains an open question as to how these expectations are integrated into perceptual decisions. In particular, scientists have debated whether prior knowledge principally changes the decisions we make about the perceptual world, or directly alters our perceptual encoding of incoming sensory evidence.

      The authors aimed to shed light on this conundrum by using a novel psychophysical task while measuring EEG signals that have previously been linked to either the sensory encoding or response selection phase of perceptual choice. The results convincingly demonstrate that both features of perceptual decision making are modulated by prior expectations - but that these biases in neural process emerge over different time courses (i.e., decisional signals are shaped early in learning, but biases in sensory processing are slower to emerge).

      Another interesting observation unearthed in the study - though not strictly linked to this perceptual/decisional puzzle - is that neural signatures of focused attention are exaggerated on trials where participants are given neutral (i.e. uninformative) cues. This is consistent with the idea that observers are more attentive to incoming sensory evidence when they cannot rely on their expectations.

      In general, I think the study makes a strong contribution to the literature, and does an excellent job of separating 'perceiving' from 'responding'. More perhaps could have been done though to separate 'perceiving' and 'responding' from 'deciding' (see below).

      Strengths:

      The work is executed expertly and focuses cleverly on two features of the EEG signals that can be closely connected to specific loci of the perceptual decision making process - the SSVEP which connects closely to sensory (visual) encoding, and Mu-Beta lateralisation which connects closely to movement preparation. This is a very appropriate design choice given the authors' research question.

      Another advantage of the design is the use of an unusually long training regime (i.e., for humans) - which makes it possible to probe the emergence of different expectation biases in the brain over different timecourses, and in a way that may be more comparable to work with nonhuman animals (who are routinely trained for much longer than humans).

      Weaknesses:

      In my view, the principal shortcoming of this study is that the experimental task confounds expectations about stimulus identity with expectations about to-be-performed responses. That is, cues in the task don't just tell participants what they will (probably) see, but what they (probably) should do.

      In many respects, this feature of the paradigm might seem inevitable, as if specific stimuli are not connected to specific responses, it is not possible to observe motor preparation of this kind (e.g., de Lange, Rahnev, Donner & Lau, 2013 - JoN).

      However, the theoretical models that the authors focus on (e.g., drift diffusion models) are models of decision (i.e., commitment to a proposition about the world) as much as they are models of choice (i.e., commitment to action). Expectation researchers interested in these models are often interested in asking whether predictions influence perceptual processing, perceptual decision and/or response selection stages (e.g., Feuerriegel, Blom & Hoogendorn, 2021 - Cortex), and other researchers have shown that parameters like drift bias and start point bias can be shifted in paradigms where observers cannot possibly prepare a response (e.g., Thomas, Yon, de Lange & Press, 2020 - Psych Sci).

      The present paradigm used by Walsh et al makes it possible to disentangle sensory processing from later decisional processes, but it blurs together the processes of deciding about the stimulus and choosing/initiating the response. This ultimately limits the insights we can draw from this study - as it remains unclear whether rapid changes in motor preparation we see reflect rapid acquisition of new decision criterion or simple cue-action learning. I think this would be important for comprehensively testing the models the authors target - and a good avenue for future work.

      In revising the manuscript after an initial round of revisions, the authors have done a good job of acknowledging these complexities - and I don't think that any of these outstanding scientific puzzles detract from the value of the paper as a whole.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study examined the longitudinal brain-behaviour link between attentional neural filtering and listening behaviour among a sample of aging individuals. The results based on the latent change score modeling showed that neither attentional neural filtering at T1 nor its T1-T2 change predicted individual two-year listening performance change. The findings suggest that neural filtering and listening behaviour may follow independent developmental trajectories. This study focuses on an interesting topic and has the potential to contribute a better understanding of the neurobiological mechanisms of successful communication across the lifespan.

      Strengths:<br /> Although research suggests that speech comprehension is neurally supported by an attention-guided filter mechanism, the evidence on their causal association is limited. This study addresses this gap by testing longitudinal stability of neural filtering as a neural mechanism upholding listening performance, potentially shedding lights on translational efforts aiming at the preservation of speech comprehension abilities among aging individuals.

      The latent change score modeling approach is appropriately used as a tool to examine key developmental questions and distinguish the complex processes underlying lifespan development in brain and behaviour with longitudinal data.

      Weaknesses:<br /> Although the paper does have strengths in principle, the weaknesses of the paper are that the findings are merely based on a single listening task. Since both neural and behavioral indicators are derived from the same task, the results may be applicable only to this specific task, and it is difficult to extrapolate them to cognitive and listening abilities measured by the other tasks. Therefore, more listening tasks are required to comprehensively measure speech comprehension and neural markers.

      The age span of the sample is relatively large. Although no longitudinal change from T1 to T2 was found at the group-level, from the cross-sectional and longitudinal change results (see Figure 3), individuals of different age groups showed different development pattern. Particularly, individuals over the age of 70 show a clear downward trend in both neural filtering index and accuracy. Therefore, different results may be found based on different age groups, especially older groups. However, due to sample limitations, this study was unable to examine whether age has a moderating effect on this brain-behaviour link.

      In the Dichotic listening task, valid and invalid cues were manipulated. According to the task description, the former could invoke selective attention, whereas the latter could invoke divided attention. It is possible that under the two conditions, the neural filtering index may reflect different underlying cognitive processes, and thus may differ in its predictive effect on behavioral performance. The author could perform a more in-depth data analysis on indicators under different conditions.

    1. Reviewer #2 (Public Review):

      Summary: This paper aims to achieve a better understanding of how the antigenic or genetic compositions of the dominant influenza A viruses in circulation at a given time are related to key features of seasonal influenza epidemics in the US. To this end, the authors analyse an extensive dataset with a range of statistical, data science and machine learning methods. They find that the key drivers of influenza A epidemiological dynamics are interference between influenza A subtypes and genetic divergence, relative to the previous one or two seasons, in a broader range of antigenically related sites than previously thought.

      Strengths: A thorough investigation of a large and complex dataset.

      Weaknesses: The dataset covers a 21 year period which is substantial by epidemiological standards, but quite small from a statistical or machine learning perspective. In particular, it was not possible to follow the usual process and test predictive performance of the random forest model with an independent dataset.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, the authors introduced ADSE, a SELEX-based protocol to explore the mechanism of emergency of species. They used DNA hybridization (to the bait pool, "resources") as the driving force for selection and quantitatively investigated the factors that may contribute to the survival during generation evolve (progress of SELEX cycle), revealing that besides individual-resource binding, the inter- and intra-individual interactions were also important features along with mutualism and parasitism.

      Strengths:<br /> The design of using pure biochemical affinity assay to study eco-evolution is interesting, providing an important viewpoint to partly explain the molecular mechanism of evolution.

    1. Reviewer #3 (Public Review):

      The main findings of this study are as follows: (1) The authors defined "metabolism-type" and "kinase-type" in unclassified sporadic PCC patients through the single-cell transcriptomics-based differentially expressed genes and functional enrichment analyses. (2) They identified the limitation of Pheochromocytoma of the Adrenal gland Scaled Score (PASS) system and suggested the combination of molecular diagnostic methods like scRNA-seq with pathological tools like PASS in aiding the clinical evaluation of PCCs. (3) Analysis of the PCC microenvironment revealed a lack of immune cell infiltration in both metabolism-type and kinase-type PCCs, while only the kinase-type PCC patient exhibited the low expression of HLA-Ⅰ that potentially regulated by RET, providing clues for the combined therapy with kinase inhibitors and immunotherapy in kinase-type PCC patients.

      The main strength of this manuscript is that it involves scRNA-seq analysis of an extremely rare tumor type-PCCs, which presents a single-cell transcriptomics-based molecular classification and microenvironment characterization of PCCs and further provides clues for potential therapeutic strategies to treat PCCs. The authors also validated the scRNA-seq analysis results (such as the expression levels of marker genes and the distribution of immune cells in the PCC microenvironment) with immunocytochemistry and multispectral immunofluorescent staining. In summary, the findings in this manuscript are quite interesting and significant, which will potentially be valuable for the molecular classification of PCCs.

    1. Reviewer #2 (Public Review):

      The manuscript of "IQCH regulates spermatogenesis by interacting with CaM to promote RNA-binding proteins' expression" from Ruan et al. identified a homozygous variant affect the splicing of IQCH in two infertile men from a Chinese family. The authors also generated a Iqch knockout mouse model to confirm the abnormal sperm phenotypes associated with IQCH deficiency. Further molecular biological assays supported the important role and mechanism of IQCH in spermatogenesis. This manuscript is informative for the clinical and basic research of male infertility.

    1. Reviewer #2 (Public Review):

      The authors provide a compelling data to demonstrate that the Notch-related transcription factor RBP-J can influence the number of circulating and recruited monocytes. The authors first delete the Rbpj gene in the myeloid lineage (Lyz2) and show that, as a proportion, only Ly6Clo monocytes are increased in the blood. The authors then attempted to identify why these cells were increased in proportion but ruled out proliferation or reduced apoptosis. Next, they investigated the gene signature of Rbpj null monocytes using RNA-sequencing and identified elevated Ccr2 as a defining feature. Crossing the Rbpj null mice to Ccr2 null mice showed reduced numbers of Ly6Clo monocytes compared with Rbpj null alone. Finally, the authors identify that an increased burden of blood Ly6Clo monocytes is correlated with increased lung recruitment and expansion of lung interstitial macrophages.

      The main conclusion of the authors, that there is a 'cell intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes' is strongly supported by the data. However, other claims and aspects of the study require clarification and further analysis of the data generated.

      Strengths<br /> The paper is well written and structured logically. The major strength of this study is the multiple technically challenging methods used to reinforce the main finding (e.g. parabiosis, adoptive transfer). The finding reinforces the fact that we still know little about how immune cell subsets are maintained in situ, and this study opens the way for novel future work. Importantly, the authors have generated an RNA-sequencing dataset that will prove invaluable for identifying the mechanism - they have promised public access to this data via GEO - it is expected this will be made accessible upon publication.

      Weaknesses - The main weakness of the study, is that although the main result is solidly supported, as written it is mostly descriptive in nature. For instance, there is no given mechanism by which RBP-J increases Ly6Clo monocytes. The authors conclude this is dependent on CCR2, however CCR2 deletion has a global effect on monocyte numbers and importantly in this study, it does not remove the Ly6Clo bias of cell proportions, if anything it seems to enhance the difference between the ly6C low and high populations in Rbpj null mice (figure 5C). This oversight in data interpretation likely occurred because: i) this experiment is missing a potentially important control (Lyz2cre/cre Ccr2RFP/RFP or RBP-J variations), and ii) lack of statistical comparisons between Ly6Clow and high subsets (e.g. two-way ANOVA design). In general, there seemed to be a focus on the Ly6C low cells, where the mechanism may be more identifiable in their precursors - likely the Ly6C high monocytes. Furthermore, the lack of this mechanism and data comparison may also be important, because it is possible that RBP-J signalling merely maintains the expression of Ly6C, rather than controls non-classical monocyte differentiation. In this case the comparison made for the sequencing data would be between Ly6C low non classical monocytes and 'artificially' Ly6C low classical monocytes. The basis of a population based on one marker is currently a widespread flaw in the field.

      Other specific weaknesses were identified (note these are in addition to the more important comments above):<br /> 1) The confirmation of knockout in supplemental figure 1A shows only a two third knockdown when this should be almost totally gone. The authors have confirmed this is perhaps poor primer design and cite a study which shows almost complete reduction in protein levels (though this could be made more clear).<br /> 2) Many figures (e.g. 1A) only show proportional data (%) when the addition of cell numbers would also be informative - for example, what if Ly6Chigh cells were decreasing, thus artificially increasing the proportion of Ly6Clo cells? Looking at figure 7B - where cell numbers are shown, it is clear that cell proportion differences often do not match number data - here RBP-J knockout also increases Ly6C high cells in number (but not %).<br /> 3) It was noted previously that many figures only have an n of 1 or 2 (e.g. 2B, 2C), the authors clarified that some of these displayed one dot to represent an experiment of multiple n.<br /> 4) There is incomplete analysis (e.g. Network analysis, comparison to subset-restricted gene expression) and interpretation of RNA-sequencing results (figure 4), additionally the difference between the genotypes in both monocyte subsets would provide a more complete picture and potentially reveal mechanisms<br /> 6) The experiments in figure 5 are missing a control (Lyz2cre/cre Ccr2RFP/RFP or the Rbpj+/+ versions) and may have been misinterpreted. For example if the control (RBP-J WT, CCR2 KO) was used then it would almost certainly show falling Ly6C low numbers compared to RBP-J WT CCR2 WT, but RBP-J KO CCR2 KO would still have more Ly6c low monocytes than RBP-J WT, CCR2 KO - meaning that the RBP-J function is independent of CCR2. I.e. Ly6c low numbers are mostly dependent on CCR2 but this is irrespective of RBP-J. Explained in another way, the normal ratio of Ly6C high to low is around 1.5 Ly6Chigh cells for every one Ly6Clow cell, this is flipped in the RBP-J knockout to 1 high to 1.25 low (the main finding of the paper), but when CCR2 is removed it actually becomes 1 high to 5 low (actual numbers 0.2% vs around 1%) - in which case all CCR2 removal is doing is lowering the number of monocytes and RBP-J's mechanism is independent of CCR2.<br /> 7) Figure 6 was difficult to interpret because of the lack of shown gating strategy. The authors state they copied the strategy from Schyns et al. however in order to review this correctly the authors should show a supplemental figure of their own gating.<br /> 8) Figure 7 has the same problem as figure 5, but this time has the correct control. CCR2 ablation has a global suppression of monocyte numbers however the increased ly6c low monocyte ratio is most likely still present in the DKO mice - the lower numbers reduce the clarity of the data. Additionally in Lung IM macrophages depletion of CCR2 in the DKO only had a partial effect in some cell types - so CCR2 is playing a role, but it is not fully dependent. A good comparison would be if they blocked PU.1 expression - the effect of RBP-J would also be muted but it doesn't mean anything in terms of mechanism. Statements about the origin of the cells may need to be removed due to lack of compelling evidence.<br /> 9) Even after being notified and acknowledging the study, the authors still have not referred to or cited a similar 2020 study in their manuscript. This study also investigated myeloid deletion of Rbpj (Zhang et al. 2020 - https://doi.org/10.1096/fj.201903086RR). Zhang et al identified that Ly6Clo alveolar macrophages were decreased in this model - it is intriguing to synthesise these two studies and hypothesise that the ly6c low monocytes steal the lung niche, but this was not discussed. The authors also indicated they looked at AM but saw no difference - perhaps they should look specifically at Ly6Clow AMs in their data to compare with this study?

    1. Reviewer #2 (Public Review):

      The study employs quantitative metabolomic and lipidomic analyses to scrutinize tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples from renal cell carcinoma (RCC) patients. The authors delve into the intricate world of renal cell carcinoma and its tumor microenvironment, shedding light on the factors that shape nutrient availability in both cancerous and adjacent normal tissues. The authors prove that non-cancer-driven tissue factors play a dominant role in shaping nutrient availability in RCC. This finding opens up new avenues for research, suggesting that the tumor microenvironment is profoundly influenced by factors beyond the presence of cancer cells. This study not only contributes valuable insights into RCC metabolism but also prompts a reevaluation of the factors governing nutrient availability in tumor microenvironments more broadly. Overall, it represents a significant step forward in our understanding of the intricate interplay between cancer and its surrounding milieu.

      The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. Since the authors exclusively employed samples from RCC patients and did not include kidney interstitial fluid and plasma samples from healthy individuals, we cannot accurately assess the true significance and applicability of the results until the role of cancer cells in reshaping KIF is understood. In essence, some metabolite levels in the tumor interstitial fluid did not show an increase or decrease compared to the adjacent normal kidney interstitial fluid. However, the levels of these metabolites in both TIF and KIF might be higher or lower than those in kidney interstitial fluid from healthy individuals, and the roles of these metabolites should not be overlooked. Similar concerns extend to plasma levels, emphasizing the importance of metabolites that synchronously change in RCC TIF, KIF, and plasma-whether elevated or reduced.

    1. Reviewer #2 (Public Review):

      In this manuscript, the authors uncover a variety of macromolecular Drosha complexes in NSCs and propose that they might exert specific functions in adult neurogenesis. This is an interesting and important area of research, the proteomics data are very useful, and the manuscript is well written and easy to understand. Overall, this manuscript has many strengths. The authors identify 165 proteins, several of them enriched in NSCs, and potentially specific for miRNA dependent or independent Drosha macromolecular complexes. Moreover, the authors convincingly show that Safb1 binds and post-transcriptionally destabilizes NFIB transcript in complex with Drosha, in vitro. With that said, most of the functional evidence are based on Safb1 overexpression in vitro, and in some cases with immortalized cell lines. This is a major limitation of the study. Further experiments should be done to convincingly demonstrate that Safb1 regulates cell fate determination in adult neural stem cells by enhancing Drosha cleavage of NFIB mRNA.

    1. Reviewer #2 (Public Review):

      The authors introduce MetaPathPredict, a method that infers the presence of functional units of gene sets, such as a set of genes coding enzymes for a common metabolic pathway, from a pool of genes or genetic sequences. MetaPathPredict employs a stacked ensemble of neural networks, each trained for a specific pathway, to consider mutual information between pathways.

      In predicting the presence of metabolic pathways in incomplete genomes, MetaPathPredict outperforms alternative naive classifiers and single neural network methods. These results demonstrate the effectiveness of a stacked ensemble of neural networks in exploiting mutual information between metabolic pathways.

    1. Reviewer #3 (Public Review):

      Studying the late development of neural circuits in relation to developmental changes in behaviour is clearly of great interest, particularly during the period of adolescence when a number of developmental abnormalities can be revealed. This is however not an easy task, since there are many concurrent changes that occur simultaneously during this developmental making it difficult to establish causality rather than correlation.

      The study focuses on behavioural and circuit changes that occur between juvenile and adulthood focusing in the prefrontal cortex and on its descending projections to the brainstem raphe nuclei. Because the pathway from the frontal cortex to serotonin raphe neurons has been involved in behavioural and stress control, exerting a top-down control on impulsive behavior, there is a good justification to focus on the development of this pathway during a period that is thought to correspond to adolescence.

      The authors identified a behavioral change in foraging strategy, which they term persistence. They find that adults tend to be more persistent than juveniles in an exploration for reward. To analyse the maturation of the prefrontal to raphe circuit they use a genetic approach (the Rbp4 promoter which drives expression in layer 5 cortical neurons) recording the synaptic drive elicited by stimulation of the axons arriving into the raphe area. They find that this maturation starts very late in the late adolescent period. They then study the effects of ablation of the layer 5 Rbp4 neurons in adults and find that adult animals have a behavior that is more similar to that of the juveniles. They then conclude that cortical prefrontal projections to the raphe are involved in the control of this behavior.

      The study is interesting in showing this new behavioural test quantifying developmental changes in exploratory behavior and indicating that some pathways derived mainly from the frontal cortex continue to mature late. However, there are a number of issues regarding the specificity of the genetic approach used. This makes it difficult to be convinced that the behaviour is related to changes in the cortico-raphe circuit.

    1. Reviewer #2 (Public Review):

      Using a single-cell omics approach combined with spatial proteomics and genetic fate mapping, Kayvanjoo et al found that fetal liver (FL) macrophages cluster into distinct yolk sac-derived subpopulations and that some of the HSCs in FL preferentially associate with one of the identified macrophage subpopulations. FLs lacking macrophages show a delay in erythropoiesis. The authors also try to identify a role of macrophages for HSCs function in FL, and claim that macrophages affect myeloid differentiation of HSCs. Experimental support for the function of macrophages on HSCs remains weak. Taken together, their data provide a precise map of FL macrophage subpopulations, which is novel and will serve the field well.

    1. Reviewer #2 (Public Review):

      This manuscript reports the results of an ancillary study of a prospective trial assessing the effects of androgen deprivation therapy (ADT) with Dagarelix (a GnRH antagonist) on body composition in patients with prostate cancer. An interesting relationship between FSH levels, that were suppressed by Dagarelix treatment, and body composition parameters (particularly fat body mass) was described after 12 months of therapy. Therefore, the authors conclude that FSH could be a promising marker to monitor the risk of sarcopenic obesity and cardiovascular complications in prostate cancer patients undergoing ADT. As acknowledged by the Authors the main limitation of the study is the limited sample of patients. However, since testosterone levels were not assessed it is not possible to firmly establish whether the changes in fat mass observed with treatment are directly or indirectly associated with a reduction in FSH (and therefore in the latter case mediated by testosterone). Moreover, it is not clear whether the effect of the change in FSH levels during the study and the body composition parameters achieved at 12 months was evaluated (instead of assessing the relationship between FSH changes and changes in body composition parameters). Finally, tests on bone muscle mass and strength were not performed, so the hypothesis that variation of FSH levels in prostate cancer patients in ADT may affect sarcopenia remains speculative.

    1. Reviewer #2 (Public Review):

      The Authors demonstrate compelling genetic evidence that the region that harbors rs6740960 plays a role in both normal craniofacial development risk for craniofacial disease. They show strong evidence that the conserved element harboring this variant is tested for LacZ reporter activity in the developing mouse that is has activity in relevant tissues. They perform several assays to demonstrate a physical link between this enhancer region to a specific target gene, PKDCC, in both cranial neural crest cells and differentiated chondrocytes. Removal of a single copy of the enhancer has little effect on PKDCC expression in CNCCs but strong impacts in chondryocytes. H1 derived cells that are heterozygous at the variant above show strong bias in H3K27ac signals in chondrocytes. The researchers then go on to recharacterize a PKDCC knockout mouse to show that it has craniofacial defects. They use modern micro-CT and analysis techniques to demonstrate subtle changes in jaw and skull structure in PKDCC heterozygous mice and confirm many of the phenotypes that were described by Kinoshita et al 2009. Overall these results point to dosage of PKDCC in craniofacial development with changes in skull shape and susceptibility to orofacial clefting. However the epigenomic differences presented in Figure 2B that serve as the foundation for the rest of the work do not agree with previously published work by this group (Prescot et al 2015). The researchers claim "enrichment of the coactivator p300 and of the active chromatin mark H3K27ac at this region is higher in the chimpanzee CNCCs as compared to human, suggesting that this non-coding element may have higher regulatory activity in the chimp. However this region was not identified in the top 1000 biased enhancer regions provided in the supplement of the Prescott et al 2015 paper. The authors do not indicate any statistical significance and largely rely on signal tracks that have not been corrected for input controls to make this conclusion. The in vivo assay for enhancer activity while excellent at demonstrating where an enhancer can be active is not well suited to quantitative comparisons. Furthermore the researchers claim that the mouse orthologous sequence is not active in the assay despite strong H3K27ac and other enhancer related signals in developing mouse craniofacial tissues as available from the Mouse Encode Project. This calls into questions whether this assay is informative at all if the native sequence which shows functionally conserved activity is not active in the mouse embryo. Lastly the authors only consider this region as a potential enhancer and not any other type of regulatory sequence. GENCODE gene annotations demonstrate a potential lncRNA (LINC02898 /ENST00000378711.2) that is directly adjacent to the region marked by this variant. This could be a promoter for an RNA that regulates PKDCC in cis. Inspection of gene expression data from a recent preprint Yankee et al 2022 as well as Prescot et al data available from the recount3 database indeed indicate RNA signal from both CNCCs and primary human tissue consistent with this annotation. The Mundlos lab has demonstrated similar regulatory mechanisms through lncRNA Maenli at the En1 locus that result in limb abnormalities.

    1. Reviewer #2 (Public Review):

      This study follows up on previous work from this group, and others, relating paternal diet to changes in sperm epigenetics, and offspring phenotypes. The authors focus on paternal diet (high-fat diet versus a control chow), sperm chromatin, and molecular changes in the placenta associated with offspring development.

      The text is well written and the figures are generally well presented and clear. The sperm epigenetic analyses and analysis of the placenta epigenetics and gene expression are generally well performed. The study provides new insight into how paternally mediated intergenerational epigenetic inheritance could involve placenta-embryo signaling.

      A major weakness is that the high-fat diet used was from a different manufacturer than the control (lower fat) diet. Therefore, it is difficult to judge whether the effects are due to a change in fat levels, or the many other molecules that are likely to differ in chow between different manufacturers. Other weaknesses include lack of methodological detail in parts, low n values for some experiments, and the need for more mechanistic data.

      Whilst the authors may have achieved their aims, more data is needed to inform a potential mechanism.

      This study adds to our understanding of how changes in paternal diet may alter sperm epigenetics and offspring development. The novelty is in the link to gene expression in the placenta associated with offspring development in utero.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Verma et al. provide a short technical report showing that endogenously tagged dynein and dynactin molecules localize to growing microtubule plus-ends and also move processively along microtubules in cells. The data are convincing, and the imaging and movies very nicely demonstrate their claims. I don't have any large technical concerns about the work. It is perhaps not surprising that dynein-dynactin complexes behave this way in cells due to other reports on the topic, but the current data are among some of the nicest direct demonstrations of this phenomenon. It may be somewhat controversial since a separate group has reported that dynein does not move processively in mammalian cells (https://www.biorxiv.org/content/10.1101/2021.04.05.438428v3). Because of this, it might be nice for the authors to comment on this discrepancy in the field, although the aforementioned work is still in pre-print form.

      Strengths:<br /> Using state-of-the-art methods to endogenously tag dynein/dynactin subunits and performing live-cell imaging is convincing and useful for the field.

      Weaknesses:<br /> The claims are perhaps not surprising or novel given the extensive data already published in the field. However, there aren't many similar studies using endogenously tagged subunits to date.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper that uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

      Strengths:<br /> One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full-length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

      Weaknesses:<br /> While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figures 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

    1. Reviewer #2 (Public Review):

      Summary:

      In this manuscript, Benner et al. interrogate the transcriptional regulator OVO to identify its targets in the Drosophila germline. The authors perform ChIP-seq in the adult ovary and identify established as well as novel OVO binding motifs in potential transcriptional targets of OVO. Through additional bioinformatic analysis of existing ATAC-seq, CAGE-seq, and histone methylation data, the authors confirm previous reports that OVO is enriched at transcription start sites and suggest that OVO does not act as part of the core RNA polymerase complex. Benner et al. then perform bulk RNA-seq in OVO mutant and "wildtype" (GAL4 mediated expression of OVO under the control of the ovo promoter in OVO mutants) ovaries to identify genes that are differentially expressed in the presence of OVO. This analysis supports previous reports that OVO likely acts at transcription start sites as a transcriptional activator. While the authors propose that OVO activates the expression of genes that are important for egg integrity, maturation, and for embryonic development (nanos, gcl, pgc, bicoid), this hypothesis is based on correlation and is not supported by in vivo analysis of the respective OVO binding sites in some of the key genes. A temporal resolution for OVO's role during germline development and egg chamber maturation in the ovary is also missing. Together, this manuscript contains relevant ChIP-seq and RNA-seq datasets of OVO targets in the Drosophila ovary alongside thorough bioinformatic analysis but lacks important in vivo experimental evidence that would validate the high-quality datasets.

      Strengths:

      The manuscript contains relevant ChIP-seq and RNA-seq datasets of OVO targets in the Drosophila ovary alongside thorough bioinformatic analysis

      Weaknesses:

      1. The authors propose that OVO acts as a positive regulator of essential germline genes, such as those necessary for egg integrity/maturation and embryonic/germline development. Much of this hypothesis is based on GO term analysis (and supported by the authors' ChIP-seq data). However accurate interpretation of GO term enrichment is highly dependent on using the correct background gene set. What control gene set did the authors use to perform GO term analysis (the information was not in the materials and methods)? If a background gene set was not previously specified, it is essential to perform the analysis with the appropriate background gene set. For this analysis, the total set of genes that were identified in the authors' RNA-seq of OVO-positive ovaries would be an ideal control gene set for which to perform GO term analysis. Alternatively, the total set of genes identified in previous scRNA-seq analysis of ovaries (see Rust et al., 2020, Slaidina et al., 2021 among others) would also be an appropriate control gene set for which to perform GO term analysis. If indeed GO term analysis of the genes bound by OVO compared to all genes expressed in the ovary still produces an enrichment of genes essential for embryonic development and egg integrity, then this hypothesis can be considered.

      2. The authors provide important bioinformatic analysis of new and existing datasets that suggest OVO binds to specific motifs in the promoter regions of certain germline genes. While the bioinformatic analysis of these data is thorough and appropriate, the authors do not perform any in vivo validation of these datasets to support their hypotheses. The authors should choose a few important potential OVO targets based on their analysis, such as gcl, nanos, or bicoid (as these genes have well-studied phenotypes in embryogenesis), and perform functional analysis of the OVO binding site in their promoter regions. This may include creating CRISPR lines that do not contain the OVO binding site in the target gene promoter, or reporter lines with and without the OVO binding site, to test if OVO binding is essential for the transcription/function of the candidate genes.

      3. The authors perform de novo motif analysis to identify novel OVO binding motifs in their ChIP-seq dataset. Motif analysis can be significantly strengthened by comparing DNA sequences within peaks, to sequences that are just outside of peak regions, thereby generating motifs that are specific to peak regions compared to other regions of the promoter/genome. For example, taking the 200 nt sequence on either side of an OVO peak could be used as a negative control sequence set. What control sequence set did the authors use as for their de novo motif analysis? More detail on this is necessary in the materials and methods section. Re-analysis with an appropriate negative control sequence set is suggested if not previously performed.

      4. The authors mention that OVO binding (based on their ChIP-seq data) is highly associated with increased gene expression (lines 433-434). How many of the 3,094 peaks (conservative OVO binding sites), and what percentage of those peaks, are associated with a significant increase in gene expression from the RNA-seq data? How many are associated with a decrease in gene expression? This information should be added to the results section.

      5. The authors mention that a change in endogenous OVO expression cannot be determined from the RNA-seq data due to the expression of the OVO-B cDNA rescue construct. Can the authors see a change in endogenous OVO expression based on the presence/absence of OVO introns in their RNA-seq dataset? While intronic sequences are relatively rare in RNA-seq, even a 0.1% capture rate of intronic sequence is likely to be enough to determine the change in endogenous OVO expression in the rescue construct compared to the OVO null.

      6. The authors conclude with a model of how OVO may participate in the activation of transcription in embryonic pole cells. However, the authors did not carry out any experiments with pole cells that would support/test such a model. It may be more useful to end with a model that describes OVO's role in oogenesis, which is the experimental focus of themanuscript.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors of this manuscript are interested in discovering and functionally characterizing genes that might cause obesity. To find such genes, they conducted a forward genetic screen in mice, selecting strains which displayed increased body weight and adiposity. They found a strain, with germ-line deficiency in the gene Spag7, which displayed significantly increased body weight, fat mass, and adipose depot sizes manifesting after the onset of adulthood (20 weeks). The mice also display decreased organ sizes, leading to decreased lean body mass. The increased adiposity was traced to decreased energy expenditure at both room temperature and thermoneutrality, correlating with decreased locomotor activity and muscle atrophy. Major metabolic abnormalities such as impaired glucose tolerance and insulin sensitivity also accompanied the phenotype. Unexpectedly, when the authors generated an inducible, whole body knockout mouse using a globally expressed Cre-ERT2 along with a globally floxed Spag7, and induced Spag7 knockout before the onset of obesity, none of the phenotypes seen in the original strain were recapitulated. The authors trace this discrepancy to the major effect of Spag7 being on placental development.

      Strengths:

      Strengths of the manuscript are its inherently unbiased approach, using a forward genetic screen to discover previously unknown genes linked to obesity phenotypes. Another strong aspect of the work was the generation of an independent, complementary, strain consisting of an inducible knockout model, in which the deficiency of the gene could be assessed in a more granular form. This approach enabled the discovery of Spag7 as a gene involved in the establishment of the mature placenta, which determines the metabolic fate of the offspring. Additional strengths include the extensive array of physiological parameters measured, which provided a deep understanding of the whole-body metabolic phenotype and pinpointed its likely origin to muscle energetic dysfunction.

      Weaknesses:

      Weaknesses that can be raised are the lack of molecular mechanistic understanding of the numerous phenotypic observations. For example, the specific role of Spag7 to promote placental development remains unclear. Also, the reason why placental developmental abnormalities lead to muscle dysfunction, and whether indeed the entire metabolic phenotype of the offspring can be attributed solely to decreased muscle energetics is not fully explored.

      Overall, the authors achieved a remarkable success in identifying genes associated with development of obesity and metabolic disease, discovering the role of Spag7 in placental development, and highlighting the fundamental role of in-utero development in setting future metabolic state of the offspring.

      Comments on revised version:

      I have no further comments on my assessment of this interesting paper.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this work, the authors sought to 1) establish a method for measuring muscle fiber subcellular structure (myofibrils) using common, non-specialized laboratory techniques and equipment, and 2) use this method to provide evidence on whether loading-induced muscle fiber growth was the result of myofibril growth (of existing myofibrils) or myofbrillogenesis (creation of new myofibrils) in mice and humans. The latter is a fundamental question in the muscle field. The authors succeeded in their aims and provided useful methods for the muscle field and detailed insight into muscle fiber hypertrophy; specifically, that loading-induced muscle fiber hypertrophy may be driven mostly by myofibrillogenesis.

      Strengths:<br /> 1) The usage of murine and human samples to provide evidence on myofibril hypertrophy vs myofibrillogenesis.<br /> 2) A nice historical perspective on myofibrillogenesis in skeletal muscle.<br /> 3) The description of a useful and tractable IHC imaging method for the muscle biology field supported by extensive validation against electron microscopy.<br /> 4) Fundamental information on how myofiber hypertrophy ensues.

      Weaknesses:

      - The usage of young growing mice (8-10 weeks) versus adult mice (>4 months) in the murine mechanical overload experiments, as well as no consideration for biological sex. The former point is partly curtailed by the adult human data that is provided (male only). Still, the usage of adult mice would be preferable for these experiments given that maturational growth may somehow affect the outcomes. For the latter point, it is not clear whether male or female mice were used.

    1. Reviewer #2 (Public Review):

      Making state-of-the-art (super-resolution) microscopy widely available has been the subject of many publications in recent years as correctly referenced in the manuscript. By advocating the ideas of open-microscopy and trying to replace expensive, scientific-grade components such as lasers, cameras, objectives, and stages with cost-effective alternatives, interested researchers nowadays have a number of different frameworks to choose from. In the iteration of the theme presented here, the authors used the existing modular UC2 framework, which consists of 3D printable building blocks, and combined a cheapish laser, detector and x,y,(z) stage with expensive filters/dichroics and an expensive high-end objective (>15k Euros).

      The choice of using the UC2 framework has the advantage, that the individual building blocks can be 3D printed, although it should be mentioned that the authors used injection-moulded blocks that will have a limited availability if not offered commercially by a third party. The strength of the manuscript is the tight integration of the hardware and the software (namely the implementations of imSwitch as a GUI to control data acquisition, OS SMLM algorithms for fast sub-pixel localisation and access to Napari).

      The presented experimental data is convincing, demonstrating (1) extended live cell imaging both using bright-field and fluorescence in the incubator, (2) single-particle tracking of quantum dots, and (3) and STORM measurements in cells stained against tubulin.

      For the revised (current) version of the manuscript, the authors further polished the manuscript and, more importantly, added plenty of information on the GitHub page that should make it significantly easier for interested researchers to replicate the instrument.

      Overall, this is compelling work that is helping to make super-resolved microscopy more accessible.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The article by Shuai et al. describes a comprehensive collection of over 800 split-GAL4 and split-LexA drivers, covering approximately 300 cell types in Drosophila, aimed at advancing the understanding of associative learning. The mushroom body (MB) in the insect brain is central to associative learning, with Kenyon cells (KCs) as primary intrinsic neurons and dopaminergic neurons (DANs) and MB output neurons (MBONs) forming compartmental zones for memory storage and behavior modulation. This study focuses on characterizing sensory input as well as direct upstream connections to the MB both anatomically and, to some extent, behaviorally. Genetic access to specific, sparsely expressed cell types is crucial for investigating the impact of single cells on computational and functional aspects within the circuitry. As such, this new and extensive collection significantly extends the range of targeted cell types related to the MB and will be an outstanding resource to elucidate MB-related processes in the future.

      Strengths:<br /> The work by Shuai et al. provides novel and essential resources to study MB-related processes and beyond. The resulting tools are publicly available and, together with the linked information, will be foundational for many future studies. The importance and impact of this tool development approach, along with previous ones, for the field cannot be overstated. One of many interesting aspects arises from the anatomical analysis of cell types that are less stereotypical across flies. These discoveries might open new avenues for future investigations into how such asymmetry and individuality arise from development and other factors, and how it impacts the computations performed by the circuitry that contains these elements.

      Weaknesses:<br /> Providing such an array of tools leaves little to complain about. However, despite the comprehensive genetic access to diverse sensory pathways and MB-connected cell types, the manuscript could be improved by discussing its limitations. For example, the projection neurons from the visual system seem to be underrepresented in the tools produced (or almost absent). A discussion of these omissions could help prevent misunderstandings. Additionally, more details on the screening process, particularly the selection of candidate split halves and stable split-GAL4 lines, would provide valuable insights into the methodology and the collection's completeness.

    1. Reviewer #2 (Public Review):

      Summary:

      The study investigates whether speech and music processing involve specific or shared brain networks. Using intracranial EEG recordings from 18 epilepsy patients, it examines neural responses to speech and music. The authors found that most neural activity is shared between speech and music processing, without specific regional brain selectivity. Furthermore, domain-selective responses to speech or music are limited to frequency-specific coherent oscillations. The findings challenge the notion of anatomically distinct regions for different cognitive functions in the auditory process.

      Strengths:

      1. This study uses a relatively large corpus of intracranial EEG data, which provides high spatiotemporal resolution neural recordings, allowing for more precise and dynamic analysis of brain responses. The use of continuous speech and music enhances ecological validity compared to artificial or segmented stimuli.

      2. This study uses multiple frequency bands in addition to just high-frequency activity (HFA), which has been the focus of many existing studies in the literature. This allows for a more comprehensive analysis of neural processing across the entire spectrum. The heterogeneity across different frequency bands also indicates that different frequency components of the neural activity may reflect different underlying neural computations.

      3. This study also adds empirical evidence towards distributed representation versus domain-specificity. It challenges the traditional view of highly specialized, anatomically distinct regions for different cognitive functions. Instead, the study suggests a more integrated and overlapping neural network for processing complex stimuli like speech and music.

      Weaknesses:

      While this study is overall convincing, there are still some weaknesses in the methods and analyses that limit the implication of the work.

      The study's main approach, focusing primarily on the grand comparison of response amplitudes between speech and music, may overlook intricate details in neural coding. Speech and music are not entirely orthogonal with each other at different levels of analysis: at the high-level abstraction, these are two different categories of cognitive processes; at the low-level acoustics, they overlap a lot; at intermediate levels, they may also share similar features. The selected musical stimuli, incorporating both vocals and multiple instrumental sounds, raise questions about the specificity of neural activation. For instance, it's unclear if the vocal elements in music and speech engage identical neural circuits. Additionally, the study doesn't adequately address whether purely melodic elements in music correlate with intonations in speech at a neural level. A more granular analysis, dissecting stimuli into distinct features like pitch, phonetics, timbre, and linguistic elements, could unveil more nuanced shared, and unique neural processes between speech and music. Prior research indicates potential overlap in neural coding for certain intermediate features in speech and music (Sankaran et al. 2023), suggesting that a simple averaged response comparison might not fully capture the complexity of neural encoding. Further delineation of phonetic, melodic, linguistic, and other coding, along with an analysis of how different informational aspects (phonetic, linguistic, melodic, etc) are represented in shared neural activities, could enhance our understanding of these processes and strengthen the study's conclusions.

      The paper's emphasis on shared and overlapping neural activity, as observed through sEEG electrodes, provides valuable insights. It is probably true that domain-specificity for speech and music does not exist at such a macro scale. However, it's important to consider that each electrode records from a large neuronal population, encompassing thousands of neurons. This broad recording scope might mask more granular, non-overlapping feature representations at the single neuron level. Thus, while the study suggests shared neural underpinnings for speech and music perception at a macroscopic level, it cannot definitively rule out the possibility of distinct, non-overlapping neural representations at the microscale of local neuronal circuits for features that are distinctly associated with speech and music. This distinction is crucial for fully understanding the neural mechanisms underlying speech and music perception that merit future endeavors with more advanced large-scale neuronal recordings.

      While classifying electrodes into 3 categories provides valuable insights, it may not fully capture the complexity of the neural response distribution to speech and music. A more nuanced and continuous approach could reveal subtler gradations in neural response, rather than imposing categorical boundaries. This could be done by computing continuous metrics, like unique variances explained by each category, or ratio-based statistics, etc. Incorporating such a continuum could enhance our understanding of the neural representation of speech and music, providing a more detailed and comprehensive picture of cortical processing.

    1. Reviewer #2 (Public Review):

      In this paper, Phan et al. investigate the properties of human HP1 paralogs, their interactions and abilities to undergo liquid-liquid phase separation. For this, they use a coarse-grained computational approach (validated with additional all-atom simulations) which allows to explore complex mixtures. Matching (wet-lab) experimental results, HP1 beta (HP1b) exhibits different properties from HP1 alpha and gamma (HP1a,g), in that it does not phase separate. Using domain switch experiments, the authors determine that the more negatively charged hinge in HP1b, compared to HP1a and HP1g, is mainly responsible for this effect. Exploring heterotypic complexes, mixtures between HP1 subtypes and DNA, the authors further show that HP1a can serve as a scaffold for HP1b to enter into condensed phases and that DNA can further stabilize phase separated compartments. Most interestingly, they show that a multicomponent mixture containing DNA, and HP1a and HP1b generates spatial separation between the HP1 paralogs: due to increased negative charge of DNA within the condensates, HP1b is pushed out and accumulates at the phase boundary. This represents an example how complex assemblies could form in the cell.

      Overall, this is purely computational work, which however builds on extensive experimental results (including from the authors). The methods showcase how coarse-grained models can be employed to generate and test hypotheses how proteins can condense. Applied to HP1 proteins, the results from this tour-de-force study are consistent and convincing, within the experimental constraints. Moreover, the authors generate further models to test experimentally, in particular in light of multicomponent mixtures. Finally, the authors clearly discuss the computational methods, assumptions and limits of the methodology, which makes this a strong contribution to our understanding of biophysical basis of condensate formation in gene regulation.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors were trying to identify and characterize the intrinsic factors that control the process of cell aging of bone marrow mesenchymal stromal cells (BMSCs), which is believed to be related to osteoporosis.

      Strengths:<br /> The method is reasonable. The concept and methods used in this work can be easily extended to other systems and cells to study their aging process. It is also interesting to further examine if PCBP2 functions as an intrinsic aging factor in these other cell types.

      The results are solid, supporting the claims and conclusions. The authors successfully identified and characterized PCBP2 as one of the intrinsic aging factors for BMSC cells.

      Weaknesses:<br /> It is unclear if PCBP2 can also function as an intrinsic factor for BMSC cells in female individuals. More work may be needed to further dissect the mechanism of how PCBP2 impacts FGF2 expression. Could PCBP2 impact the FGF2 expression independent of ROS?

      Additional context that would help readers interpret or understand the significance of the work:<br /> In the current work, the authors studied the aging process of BMSC cells, which are related to osteoporosis. Aging processes also impact many other cell types and their function, such as in muscle, skin, and the brain.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The story of the co-evolution of TTX-bearing newts and their independently evolved TTX-resistant garter snake predators is a classic in evolutionary ecology/physiology. Over the years specific amino acid substitutions in the muscle-expressing (and other) sodium channels have been identified and the behavioral assays of snake crawling performance have indicated that the attainment of TTX-resistance comes at a cost in mobility. One previous study also examined how the amino acid mutations affected the biophysics of Nav channel properties. The present paper starts with this foundation and builds by adding in details and making causal connections across multiple snake populations with different degrees of resistance. The addition of muscle physiology bridges the gap between organismal performance and sodium channel biophysics. Moving in the other direction, examining molecular models of Nav channel structure and energetics allows a deep understanding of how amino acid substitutions affect channel properties. In the end, a clear picture is painted from molecular to organismal levels in two different parallel evolutions of TTX resistance.

      Strengths:<br /> This study is a tour de force. It is clearly written, and nicely illustrated, and the methods and procedures are meticulously documented.

      Weaknesses:<br /> One caveat is that the Nav channels used to test mutations in expression systems are rat channels engineered with TTX-resisting substitutions observed in snake populations. The ideal experiment would have been to use the snake channels. While the rat channels appear to be a good substitute for the snake channels and the authors have taken pains to show that the important amino acids are conserved between garter snakes and rats, the authors might explain why they did not use snake channels.

      The noise analysis seems like a reasonable way to get at the question of single-channel conductance. But why did the authors not just measure single channel conductance in patches as opposed to this much more complex and roundabout method? It is recommended that the authors discuss how noise analysis deals with the problem of having the number of open channels changing rapidly during activation and fast inactivation. Is this a potential problem for deriving the total number of channels?

    1. Reviewer #2 (Public Review):

      Summary:<br /> In short, this paper uses a previously published method, ReplicaDock, to improve predictions from AlphaFold-multimer. The method generated about 25% more acceptable predictions than AFm, but more important is improving an Antibody-antigen set, where more than 50% of the models become improved.

      When looking at the results in more detail, it is clear that for the models where the AFm models are good, the improvement is modest (or not at all). See, for instance, the blue dots in Figure 6. However, in the cases where AFm fails, the improvement is substantial (red dots in Figure 6), but no models reach a very high accuracy (Fnat ~0.5 compared to 0.8 for the good AFm models). So the paper could be summarized by claiming, "We apply ReplicaDock when AFm fails", instead of trying to sell the paper as an utterly novel pipeline. I must also say that I am surprised by the excellent performance of ReplicaDock - it seems to be a significant step ahead of other (not AlphaFold) docking methods, and from reading the original paper, that was unclear. Having a better benchmark of it alone (without AFm) would be very interesting.

      These results also highlight several questions I try to describe in the weakness section below. In short, they boil down to the fact that the authors must show how good/bad ReplicaDock is at all targets (not only the ones where AFm fails. In addition, I have several more technical comments.

      Strengths:<br /> Impressive increase in performance on AB-AG set (although a small set and no proteins).

      Weaknesses:<br /> The presentation is a bit hard to follow. The authors mix several measures (Fnat, iRMS, RMSDbound, etc). In addition, it is not always clear what is shown. For instance, in Figure 1, is the RMSD calculated for a single chain or the entire protein? I would suggest that the author replace all these measures with two: TM-score when evaluating the quality of a single chain and DockQ when evaluating the results for docking. This would provide a clearer picture of the performance. This applies to most figures and tables. For instance, Figure 9 could be shown as a distribution of DockQ scores.

      The improvements on the models where AFm is good are minimal (if at all), and it is unclear how global docking would perform on these targets, nor exactly why the plDDT<0.85 cutoff was chosen. To better understand the performance of ReplicaDock, the authors should therefore (i) run global and local docking on all targets and report the results, (ii) report the results if AlphaFold (not multimer) models of the chains were used as input to ReplicaDock (I would assume it is similar). These models can be downloaded from AlphaFoldDB.

      Further, it would be interesting to see if ReplicaDock could be combined with AFsample (or any other model to generate structural diversity) to improve performance further.

      The estimates of computing costs for the AFsample are incorrect (check what is presented in their paper). What are the computational costs for RepliaDock global docking?

      It is unclear strictly what sequences were used as input to the modelling. The authors should use full-length UniProt sequences if they were not done.

      The antibody-antigen dataset is small. It could easily be expanded to thousands of proteins. It would be interesting to know the performance of ReplicaDock on a more extensive set of Antibodies and nanobodies.

      Using pLDDT on the interface region to identify good/bas models is likely suboptimal. It was acceptable (as a part of the score) for AlphaFold-2.0 (monomer), but AFm behaves differently. Here, AFm provides a direct score to evaluate the quality of the interaction (ipTM or Ranking Confidence). The authors should use these to separate good/bad models (for global/local docking), or at least show that these scores are less good than the one they used.

    1. Reviewer #2 (Public Review):

      This work probes the control of the hox operon in the cyanobacterium Synechocystis, where this operon directs the synthesis of a bidirectional hydrogenase that functions to produce hydrogen. In assessing the control of the hox system, the authors focused on the relative contributions of cyAbrB2, alongside SigE (and to a lesser extent, SigA and cyAbrB1) under both aerobic and microoxic conditions. In mapping the binding sites of these different proteins, they discovered that cyAbrB2 bound many sites throughout the chromosome repressed many of its target genes, and preferentially bound regions that were (relatively) rich in AT-residues. These characteristics led the authors to consider that cyAbrB2 may function as a nucleoid-associated protein (NAP) in Synechocystis, given its functional similarities with other NAPs like H-NS. They assessed the local chromosome conformation in both wild-type and cyabrB2 mutant strains at multiple sites within a 40 kb window on either side of the hox locus, using a region within the hox operon as bait. They concluded that cyAbrB2 functions as a nucleoid-associated protein that influences the activity of SigE through its modulation of chromosome architecture.

      The authors approached their experiments carefully, and the data were generally very clearly presented and described.

      Based on the data presented, the authors make a strong case for cyAbrB2 as a nucleoid-associated protein, given the multiple ways in which it seems to function similarly to the well-studied Escherichia coli H-NS protein. It would be helpful to provide some additional commentary within the discussion around the similarities and differences of cyAbrB2 to other nucleoid-associated proteins, and possible mechanisms of cyAbrB2 control (post-translational modification; protein-protein interactions; etc.). The manuscript would also be strengthened with the inclusion of biochemical experiments probing the binding of cyAbrB2, particularly focussing on its oligomerization and DNA polymerization/bridging potential.

      Previous work had revealed a role for SigE in the control of hox cluster expression, which nicely justified its inclusion (and focus) in this study. However, the results of the SigA studies here suggested that SigA both strongly associated with the hox promoter, and its binding sites were shared more frequently than SigE with cyAbrB2. The focus on cyAbrB2 is also well-justified, given previous reports of its control of hox expression; however, it shares binding sites with an essential homologue cyAbrB1. Interestingly, while the B1 protein appears to bind similar sites, instead of repressing hox expression, it is known as an activator of this operon. It seems important to consider how cyAbrB1 activity might influence the results described here.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors have previously engineered an antibody fusion protein targeting ZNRF3/RNF43 ubiquitin ligases, which enhances Wnt signaling specifically in hepatocytes. This is achieved using RSPO2RA (ZNRF3/RNF43 ligand with F105R/F109A mutations which abolish its binding to LGRs) and ASGR1 (hepatocyte-specific cell surface molecule). In the current study, they have identified two new ASGR1 and ASGR1/2 antibodies (8M24 and 8G8), which also enhance Wnt signaling when fused to RSPO2RA antibody. These also lead to the degradation of ASGR1, demonstrating that protein degradation and signaling enhancement can be dually targeted with a single molecule.

      Strengths:<br /> The authors show crystal structures for binding of these antibodies to ASGR1/2, and hypothesize about why specificity is mediated through specific residues. They do not test these hypotheses.

      The authors demonstrate a sub-picomolar affinity of these antibodies for ASGR1/2, which should be powerful clinically.

      The authors demonstrate in hepatocyte cell lines that these function as mimetics, and that they do not function in HEK cells, which do not express ASGR1. They do not perform an exhaustive screen of all non-hepatocyte cells, nor do they test these molecules in vivo.

      Surprisingly, these molecules also induced loss of ASGR1, which the authors hypothesize is due to ubiquitination and degradation, initiated by the E3 ligases recruited to ASGR1. They demonstrate that inhibition of either the proteasome or lysosome abrogates this effect and that it is dependent on E1 ubiquitin ligases. They do not demonstrate direct ubiquitination of ASGR1 by ZNRF3/RNF43.

      Weaknesses:<br /> As co-listed with strengths above, the analysis is not always exhaustive but shows good preliminary findings for the field.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this work, Lao et al. develop an open-source software (OpenNucleome) for GPU-accelerated molecular dynamics simulation of the human nucleus accounting for chromatin, nucleoli, nuclear speckles, etc. Using this, the authors investigate the steady-state organization and dynamics of many of the nuclear components.

      Strengths:<br /> This is a comprehensive open-source tool to study several aspects of the nucleus, including chromatin organization, interactions with lamins and organization, and interactions with nuclear speckles and nucleoli. The model is built carefully, accounting for several important factors and optimizing the parameters iteratively to achieve experimentally known results. The authors have simulated the entire genome at 100kb resolution (which is a very good resolution to simulate and study the entire diploid genome) and predict several static quantities such as the radius of gyration and radial positions of all chromosomes, and time-dependent quantities like the mean-square displacement of important genomic regions.

      Weaknesses:<br /> One weakness of the model is that it has several parameters. Some of them are constrained by the experiments. However, the role of every parameter is not clear in the manuscript.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Pulfer A. et al. developed a deep learning-based apoptosis detection system named ADeS, which outperforms the currently available computational tools for in vitro automatic detection. Furthermore, ADeS can automatically identify apoptotic cells in vivo in intravital microscopy time-lapses, preventing manual labeling with potential biases. The authors trained and successfully evaluated ADeS in packed epithelial monolayers and T cells distributed in 3D collagen hydrogels. Moreover, in vivo, training and evaluation were performed on polymorphonucleated leukocytes in lymph nodes and spleen.

      Strengths:<br /> Pulfer A. et colleagues convincingly presented their results, thoroughly evaluated ADeS for potential toxicity assay, and compared its performance with available state-of-the-art tools.

      Weaknesses:<br /> The use of ADeS is still restricted to samples where cells are fluorescently labeled either in the cytoplasm or in the nucleus, which limits its use for in vitro toxicity assays that are performed on primary cells or organoids (e.g., iPSCs-derived systems) that are normally harder to transfect.

      In conclusion, ADeS will be a useful tool to improve output quality and accelerate the evaluation of assays in several research areas with basic and applied aims.

    1. Reviewer #2 (Public Review):

      Summary<br /> This paper has three parts. The first part applied a coarse-grained model with proteome partition to calculate cell growth under respiration and fermentation modes. The second part considered single-cell variability and performed population average to acquire an ensemble metabolic profile for acetate fermentation. The third part used model and simulation to compare experimental data in literature and obtained substantial consistency.

      Strengths and major contributions<br /> (i) The coarse-grained model considered specific metabolite groups and their inter-relations and acquired an analytical solution for this scenario. The "resolution" of this model is in between the Flux Balanced Analysis/whole-cell simulation and proteome partition analysis.

      (ii) The author considered single-cell level metabolic heterogeneity and calculated the ensemble average with explicit calculation. The results are consistent with known fermentation and growth phenomena qualitatively and can be quantitatively compared to experimental results.

      Weaknesses<br /> (i) If I am reading this paper correctly, the author's model predicts binary (or "digital") outcomes of single-cell metabolism, that is, after growth rate optimization, each cell will adopt either "respiration mode" or "fermentation mode" (as illustrated in Figure Appendix - Figure 1 C, D). Due to variability enzyme activity k_i^{cat} and critical growth rate λ_C, each cell under the same nutrient condition could have either respiration or fermentation, but the choice is binary.

      The binary choice at the single-cell level is inconsistent with our current understanding of metabolism. If a cell only uses fermentation mode (as shown in Appendix - Figure 1C), it could generate enough energy but not be able to have enough metabolic fluxes to feed into the TCA cycle. That is, under pure fermentation mode, the cell cannot expand the pool of TCA cycle metabolites and hence cannot grow.

      This caveat also appears in the model in Appendix (S25) that assumes J_E = r_E*J_{BM} where r_E is a constant. From my understanding, r_E can be different between respiration and fermentation modes (at least for real cells) and hence it is inappropriate to conclude that cells using fermentation, which generates enough energy, can also generate a balanced biomass.

      (ii) The minor weakness of this model is that it assumes a priori that each cell chooses its metabolic strategy based on energy efficiency. This is an interesting assumption but there is no known biochemical pathway that directly executes this mechanism. In evolution, growth rate is more frequently considered for metabolic optimization. In Flux Balanced Analysis, one could have multiple objective functions including biomass synthesis, energy generation, entropy production, etc. Therefore, the author would need to justify this assumption and propose a reasonable biochemical mechanism for cells to sense and regulate their energy efficiency.

      My feeling is that the mathematical structure of this model could be correct, but the single-cell interpretation for the ensemble averaging has issues. Each cell could potentially adopt partial respiration and partial fermentation at the same time and have temporal variability in its metabolic mode as well. With the modification of the optimization scheme, the author could have a revised model that avoids the caveat mentioned above.

      Discussion and impact for the field<br /> Proteome partition models and Flux Balanced Analysis are both commonly used mathematical models that emphasize different parts of cellular physiology. This paper has ingredients for both, and I expect after revision it will bridge our understanding of the whole cell.

    1. Reviewer #2 (Public Review):

      In the current study, Latchoumane and collaborators focus on the Cav3.1 calcium channels in the mediodorsal thalamic nucleus as critical players in the regulation of brain-states and ethanol resistance in mice. By combining behavioural, electrophysiological, and genetic techniques, they report three main findings. First, KO Cav3.1 mice exhibit resistance to ethanol-induced sedation and sustained tonic firing in thalamocortical units. Second, knocked-down Cav3.1 mice reproduce the same behaviour when the mediodorsal, but not the ventrobasal, thalamic nucleus is targeted. Third, either optogenetic or electric stimulation of the mediodorsal thalamus reduces ethanol-induced sedation in control animals.

      Overall, the study is well designed and performed, correctly controlled for confounds, and properly analysed. Nonetheless, it is important to address some aspects of the report. The results support the conclusions of the study. These results are likely to be relevant in the field of systems neuroscience, as they increase the molecular evidence showing how the thalamus regulates brain states.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Animals exhibit different speeds of locomotion. In vertebrates, this is thought to be implemented by different groups of spinal interneurons and motor neurons. A fundamental assumption in the field has been that neural mechanisms that generate and sustain the rhythm at different locomotor speeds are the same. In this study, the authors challenge this view. Using rigorous in vivo electrophysiology during fictive locomotion combined with genetics, the authors provide a detailed analysis of cellular and synaptic properties of different subtypes of spinal V2a neurons that play a crucial role in rhythm generation. Importantly, they are able to show that speed-related subsets of V2a neurons have distinct cellular and synaptic properties and may utilize different mechanisms to implement different locomotor speeds.

      Strengths:<br /> The authors fully utilize the zebrafish model system and solid electrophysiological analyses to study the active and passive properties of speed-related V2a subsets. Identification of the V2a subtype is based directly on their recruitment at different locomotor speeds and not on indirect markers like soma size, D-V position etc. Throughout the article, the authors have cleverly used standard electrophysiological tests and analysis to tease out different neuronal properties and link it to natural activity. For example, in Figures 2 and 4, the authors make comparisons of V2a spiking with current steps and during fictive swims showing spike rates measured with current steps are physiologically relevant and observed during natural recruitment. The experiments done are rigorous and well-controlled.

      Weaknesses:<br /> The authors claim that a primary result of their study is that reciprocal inhibition is important for rhythmogenesis at fast speeds while recurrent inhibition is key at slow speeds. This is shown in Figure 6, however, the authors do not show any statistical tests for this claim. The authors also do not show any conclusive evidence that reciprocal inhibition is required for rhythmogenesis at fast speeds and vice versa for slow speeds. Additional experiments or modeling studies that conclusively show the necessity of these different inhibitory sources to the generation of different rhythms would be needed to strengthen this claim.

      The authors do a great job of teasing out cellular and synaptic properties in the different V2a subsets, however, it is not clear if or how these match the final output. For example, V2aD neurons are tonic or bursting for fast and slow speeds respectively but it is not intuitive how these cellular properties would influence phasic excitation and inhibition these neurons receive.

      It is not clear from the discussion why having different mechanisms of rhythm generation at different speeds could be an important circuit design. The authors use anguilliform and carangiform modes of swimming to denote fast and slow speeds but there are differences in these movements other than speed, like rostrocaudal coordination. The frequency and pattern of these movements are linked and warrant more discussion.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this work, Kashefi et al. investigate the planning of sequential reaching movements and how the additional information about future reaches affects planning and execution. This study, carried out with human subjects, extends a body of research in sequential movements to ask important questions: How many future reaches can you plan in advance? And how do those future plans interact with each other?

      The authors designed several experiments to address these questions, finding that information about future targets makes reaches more efficient in both timing and path curvature. Further, with some clever target jump manipulations, the authors show that plans for a distant future reach can influence plans for a near future reach, suggesting that the planning for multiple future reaches is not independent. Lastly, the authors show that information about future targets is acquired parafoveally--that is, subjects tend to fixate mainly on the target they are about to reach to, acquiring future target information by paying attention to targets outside the fixation point.

      The study opens up exciting questions about how this kind of multi-target planning is implemented in the brain. As the authors note in the manuscript, previous work in monkeys showed that preparatory neural activity for a future reaching movement can occur simultaneously with a current reaching movement, but that study was limited to the monkey only knowing about two future targets. It would be quite interesting to see how neural activity partitions preparatory activity for a third future target, given that this study shows that the third target's planning may interact with the second target's planning.

      Strengths:<br /> A major strength of this study is that the experiments and analyses are designed to answer complementary questions, which together form a relatively complete picture of how subjects act on future target information. This complete description of a complex behavior will be a boon to future work in understanding the neural control of sequential, compound movements.

      Weaknesses:<br /> I found no real glaring weaknesses with the paper, though I do wish that there had been some more discussion of what happens to planning with longer dwell times in target. In the later parts of the manuscript, the authors mention that the co-articulation result (where reaches are curved to make future target acquisition more efficient) was less evident for longer dwell times, likely because for longer dwell times, the subject needs to fully stop in target before moving to the next one. This result made me wonder if the future plan interaction effect (tested with the target jumps) would have been affected by dwell time. As far as I can tell, the target jump portion only dealt with the shorter dwell times, but if the authors had longer dwell time data for these experiments, I would appreciate seeing the results and interpretations.

      Beyond this, the authors also mentioned in the results and discussion the idea of "neural resources" being assigned to replan movements, but it's not clear to me what this might actually mean concretely. I wonder if the authors have a toy model in mind for what this kind of resource reassignment could mean. I realize it would likely be quite speculative, but I would greatly appreciate a description or some sort of intuition if possible.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Here Jeong et al., use a combination of theoretical and experimental approaches to define molecular contexts that support specific chromatin conformations. They seek to define features that are associated with TADs that are retained after cohesin depletion (the authors refer to these TADs as P-TADs). They were motivated by differences between single cell data, which suggest that some TADs can be maintained in the absence of cohesin, whereas ensemble HiC data suggest complete loss of TADs. By reananalyzing a number of HiC datasets from different cell types, the authors observe that in ensemble methods, a significant subset of TADs are retained. They observe that P-TADs are associated with mismatches in epigenetic state across TAD boundaries. They further observe that "physical boundaries" are associated with P-TAD maintenance. Their structure/simulation based approach appears to be a powerful means to generate 3D structures from ensemble HiC data, and provide chromosome conformations that mimic the data from single-cell based experiments. Their results also challenge current dogma in the field about epigenetic state being more related to compartment formation rather than TAD boundaries. Their analysis is particularly important because limited amounts of imaging data are presently available for defining chromosome structure at the single-molecule level, however, vast amounts of HiC and ChIP-seq data are available. By using HiC data to generate high quality simulated structural data, they overcome this limitation. Overall, this manuscript is important for understanding chromosome organization, particularly for contacts that do not require cohesin for their maintenance, and for understanding how different levels of chromosome organization may be interconnected. I cannot comment on the validity of the provided simulation methods and hope that another reviewer is qualified to do this.

      Specific comments<br /> -It is unclear what defines a physical barrier. From reading the text and the methods, it is not entirely clear to me how the authors have designated sites of physical barriers. It may help to define this on pg 7, second to last paragraph, when the authors first describe instances of P-TAD maintenance in the absence of epigenetic mismatch.

      -Figure 7 adds an interesting take to their approach. Here the authors use microC data to analyze promoter-enhancer/promoter-promoter contacts. These data are included as part of the discussion. I think this data could be incorporated into the main text, particularly because it provides a biological context where P-TADs would have a rather critical role.

      -Figure 3a- the numbers here do not match the text (page 6, second to last paragraph). The numbers have been flipped for either chromosome 10 or chromosome 13 in the text or the figures.

      In the revision, the authors have sufficiently addressed my specific concerns from above.

    1. Reviewer #2 (Public Review):

      The responses to the comments and changes in the manuscript are convincing, especially the secretion patterns of high and low secreting cells are interesting and reassuring. The only criticism I still have is that most observations are already published in the previous paper by the same authors.

      Summary:<br /> In their manuscript titled "Stimulation-induced cytokine polyfunctionality as a dynamic concept," the authors investigate the dynamic nature of polyfunctional cytokine responses to established stimulants. The authors use their previously published single-cell encapsulation droplet-microfluidic platform to analyse the response of peripheral blood mononuclear cells (PBMCs) to different stimulants and measure the secretion dynamics of individual cytokines. This assay shows that polyfunctionality in cytokine responses is a complex but short-lived phenomenon that decreases with prolonged stimulation times. The study finds that polyfunctional cells predominantly display elevated cytokine concentrations with similar secretion patterns but higher secretion levels compared to their monocytokine-secreting counterparts. The method is promising to analyse the correlation between the secretion dynamics of different cytokines in primary samples and heterogeneous cell populations.

      Strengths:<br /> This method provides single-cell-resolved and dynamic cytokine concentration information, which might be used to identify "fingerprints" of secretion patterns for selected cytokines. When extending the available data to more than one donor, this might be the basis for a diagnostic tool. The combination of established droplet microfluidics with an epi-fluorescence microscope-based readout makes it convincing that the method is transferable to other labs. Specifically, the dynamic analysis of cytokine concentrations is interesting, and the differences or similarities in secretion timepoints might be missed with end-point methods. The authors convincingly show that they detect up to three different cytokines in single cells.

      Weaknesses:<br /> The conclusions of the study are based on samples from a single donor, which makes the conclusions on secretion patterns difficult to interpret. The choice of cytokines is explained, but the justification of the groupings of the antibodies into the two panels is missing. It would further be helpful to discuss how the single cell incubation might affect the secretion dynamics vs. the influence of co-culture of all cell types during the 24 h activation. The authors compare average secretion rates and levels. However, the right panel in Fig. 6 looks like there might be two different populations of mono- or polyfuntional cells that have two secretion rates. As the authors have single-cell data, I would find the separation into these populations more meaningful than comparing the mean values. In line with this comment, comparing the mean values for these cytokines instead of the mean of the populations with distinct secretion properties might actually show stronger differences than the authors report here.Is the plateau of the cytokine concentration caused by the fluorescence signal saturating the camera, saturation of the magnetic beads, exhaustion of the fluorescent antibodies, or constant cytokine concentrations? The high number of non-CSCs and the limited number of droplets decrease the statistical power of the method. The authors discuss their choice to use PBMCs and not solely T cells, but this aspect is missing in the discussion.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Let-7 family miRNAs are largely redundant in function, and originate from multiple genomic loci ("clusters"). Erice et al demonstrate that two individual clusters (let7afd and let7bc2) in mice regulate the generation of IL-17 producing CD8 T cells in vitro and in vivo in a model of emphysema. These cells also express higher levels of the IL-17-inducing transcription factor RORgt, encoded by Rorc, which the authors demonstrate to be a direct target of let-7. Since multiple let-7 family miRNAs are downregulated in T cells and lung tissue in emphysema, these data support a model in which reduced let-7 allows increased IL-17 production by T cells, contributing to disease pathogenesis.

      Strengths:<br /> The inclusion of miRNA and pri-miRNA expression data from sorted human lung T cells as well as mouse T cells from an emphysema model is a strength.

      The study includes complementary loss of function and gain of function experimental systems to test the effect of altered let-7 function, though it should be noted that these involved different let-7 family members and did not yield simple, complementary results for all experimental outcomes.

      The most important finding is that deletion of just one let-7 cluster ("Let7bc2") is sufficient to exacerbate emphysema in the nCB and CS models.

      Weaknesses:<br /> The functional analyses are unusually focused on IL-17 producing CD8 T cells, but it is not made clear whether these cells are an important player in emphysema pathogenesis in the nCB and CS models. The data shown reveal that they are far less numerous than IL-17-producing CD4 T cells. It is also notable that the Figure 1 expression data from human subjects used sorted CD4+ T cells. And as the author mentioned, prior work on let-7 showed that it regulated Th17 (CD4) responses.

      Compared with Let7bc2 deletion, Let7afd deletion had a much larger effect on IL17 production by CD8 T cells in vitro, and it also had a larger effect on RORgt expression in untreated mice in vivo, especially in the lung. It would be valuable to more thoroughly characterize the let7afd mice. RORgt expression should be shown in the in vitro assays. In the results, the authors state that let7afdLOF mice "did not exhibit lung histopathology nor inflammatory changes" up to 6 months of age. Similarly, it is stated in the conclusion that "the let-7afdLOF mice ... did not exhibit changes in Tc17/Th17 subpopulations" in vivo. All these data should be shown, and if no baseline changes are apparent, then I also recommend challenging these mice with nCB and/or cigarette smoke.

      This brings up the larger issue of redundancy among the let-7 family members and genomic clusters. This should be discussed, including some explanation of the relative expression of each mature family member in T cells, and how that maps to the clusters studied here (and those that were not investigated). It would also be helpful to explain the relationship between mouse Let7bc2 and human Let7a3b, since Let7bc2 is the primary focus of emphysema experiments in this manuscript.

      This is especially important because the study of individual let-7 clusters is the core novelty of this body of work, as described in the first paragraph of the discussion. The regulation of let-7 expression has been reported before and its functional role has been investigated with a variety of tools.

      Let7g overexpression caused a marked reduction in Rorgt expression in T cells at baseline and in the setting of nCB challenge, and it reduced the frequency of IL17+ producing CD8 T cells in the lung to baseline levels. Yet there was no change in the MLI measurement of histopathology. Is this a robust result? The responses in the experiment shown in Fig. 6C-D are quite muted compared to those shown in Figure 2. The latter also shows a larger number of replicates, and it is unclear whether the data in 6D include measurement from all of the mice tested (e.g. pooled from 2 small experiments) or only mice from one experiment.

      Although RORgt is a great candidate to have direct effects on IL-17 expression, the mechanistic understanding of let-7 action on T cell differentiation and cytokine production is limited to this single target. As noted in the discussion, others have identified cytokine receptor targets that may play a role, but it is also likely others among the many targets of let-7 also contribute.

    1. Reviewer #2 (Public Review):

      This is a potentially interesting study addressing a possible scale-invariant log-normal characteristic of droplet size distribution in the phase separation behavior of biomolecular condensates. Some of the data presented are valuable and intriguing. However, as it stands, the validity and utility of this study are uncertain because there are serious deficiencies in the execution and presentation of the authors' results. Many of these shortcomings are fundamental, including a lack of clarity in the basic conceptual framework of the study, insufficient justification of the experimental setup, less-than-conclusive experimental evidence, and inadequate discussion of implications of the authors' findings to future experimental and theoretical studies of biomolecular condensates. Accordingly, this reviewer considers that the manuscript should undergo a major revision to address the following. In particular, the discussion should be significantly expanded by including references mentioned below as well as other references pertinent to the issues raised.

      1. The theoretical analysis in this study is based on experimental data on condensed droplet size distributions for FUS and α-synuclein. The size data for FUS droplet is indirect as it relies on the assumption that FUS droplet diameter is proportional to fluorescence intensity of labeled FUS (page 10 of manuscript), with fluorescence data adopted from a previously published work by another group (Kar et al. & Pappu, ref.27). Because fluorescence of a droplet is expected to be dependent upon the condensed-phase concentration of FUS, this proportional relationship, even if it holds, must also be modulated by FUS concentration in the droplet. Moreover, why should fluorescence be proportional to diameter but not the cross-sectional area or volume of the FUS droplet, which would be more intuitive? These issues should be clarified. A new measure by microscopy is used to determine the size distribution of condensed α-synuclein; but no microscopy image is shown. It is of critical importance that such raw data (for example microscopy images) be presented for the completeness and reproducibility of the experiment because the entire study relies on the soundness of these experimental measurements.

      2. Despite the authors' claim of a universal scaling relationship, the log-log scatter plots in Figure 1 (page 15 of the manuscript) exhibit significant deviations from linearity at low protein concentrations (ρ→0). Given this fact, is universal scaling really valid? Discussion of this behavior is conspicuously absent (except the statement that these data points are excluded in the fit). In any case, the possible origins of these deviations should be thoroughly discussed so that the regime of universal scaling can be properly delineated.

      3. Droplet size distribution most likely depends on the time duration after the preparation of the sample. For α-synuclein, "liquid droplet size characterisation images were captured 10 minutes post-liquid droplet formation" (page 9 of the manuscript). Why 10 minutes? Have the authors tried imaging at different time points and, if so, do the distributions at different time points remain essentially the same? If they are different, what is the criterion for focusing only on a particular time point? Information related to these questions should be provided.

      4. At least two well-known mechanisms can lead to the time-dependent distribution of liquid droplet sizes: (i) coalescence of droplets in spatial proximity to form a larger droplet, and (ii) Ostwald ripening, i.e., formation of larger droplets concomitant with the dissolution of smaller droplets without fusion of droplets. The implications of these mechanisms on the authors' droplet size distributions should be addressed. Indeed, maintaining a size distribution against these mechanisms in vivo often requires active suppression [Bressloff, Phys Rev E 101, 042804 (2020)] with possible involvement of chemical reactions [Kirschbaum & Zwicker, J R Soc Interface 18, 20210255 (2021)]. These considerations are central to the basic rationale of this study and therefore should be carefully tackled.

      5. If coalescence and/or Ostwald ripening do occur, given sufficient time after sample preparation, the condensed phase may become a single large "droplet" or a single liquid layer. Does this occur in the authors' experiments?

      6. It is unclear whether the authors aim to address the kinetic phenomenon of liquid droplet formation and evolution or equilibrium properties. The two types of phenomena appear to be conflated in the authors' narrative. Clarification is needed. If this work aims to address time-independent (or infinite-time) equilibrium properties, how are they expected to be related to droplet size distribution, which most likely is time-dependent?

      7. The relationship between the potentially time-dependent droplet size distribution and equilibrium properties of ρt and ρc (transition and critical concentrations, respectively) should be better spelled out. An added illustrative figure will be helpful.

      8. The authors comment that their findings appear to be inconsistent with Flory-Huggins theory because Flory-Huggins "characterizes droplet formation as a consequence of nucleation ..." (page 8 of the manuscript). Here, three issues need detailed clarification: (i) In what way does Flory-Huggins mandate nucleation? (ii) Why are the findings of apparent scale invariance inconsistent with nucleation? (iii) If liquid droplet formations do not arise from nucleation, what physical mechanism(s) is (are) envisioned by the authors to be underpinning the formation of condensed liquid droplets in protein phase separation?

      9. Are any of the authors' findings related to finite-system effects of phase separation [see, e.g., Nilsson & Irbäck, Phys Rev E 101, 022413 (2020)]?

      10. Since the authors are using their observation of an apparent scale-invariant droplet size distribution to evaluate phase separation theory, it is important to clarify whether their findings provide any constraint on the shape of coexistence curves (phase diagrams).

      11. More specifically, do the authors' findings suggest that the phase diagrams predicted by Flory-Huggins are invalid? Or, are they suggesting that even if the phase diagrams predicted by Flory-Huggins are empirically correct (if verified by experimental testing), they are underpinned by a free energy function different from that of Flory-Huggins? It is important to answer this question to clarify the implications of the authors' findings on equilibrium phase behaviors and the falsifiability of the implications.

      12. How about the implications of the authors' findings on other theories of protein phase separation that are based on interactions that are different from the short spatial range interactions treated by Flory-Huggins? For instance, it has been observed that whereas the Flory-Huggins-predicted phase diagrams always convex upward, phase diagrams for charged intrinsically disordered proteins with long spatial range Coulomb interactions exhibit a region that concave upward [Das et al., Phys Chem Chem Phys 20, 28558-28574 (2018)]. Can information be provided by the authors' findings regarding apparent scale-invariant droplet size distribution on the underlying interaction driving the protein molecules toward phase separation?

      13. Table S1 (page 4) and Table S2 (page 7) are mentioned in the text but these tables are not in the submitted files.

      14. The two systems studied (FUS and α-synuclein) have a single intrinsically disordered protein (IDP) component. It is not clear if the authors expect their claimed scaling relation to be applicable to systems with multiple IDP components and if so, why.

    1. Reviewer #2 (Public Review):

      Summary: In the mauscript entitled "The Intricate Relationship of G-Quadruplexes and Pathogenicity Islands: A Window into Bacterial Pathogenicity" Bo Lyu explored the interactions between guanine-quadruplex (G4) structures and pathogenicity islands (PAIs) in 89 bacterial genomes through rigorous computational approach. This paper handles an intriguing and complex topic in the field pathogenomics, it has the potential to contribute significantly to the understanding of G4-PAI interactions and bacterial pathogenicity.

      Strengths: Chosen research area and summarizing the results through neat illustrations

      Weaknesses: I did not find any significant ones.

    1. Reviewer #3 (Public Review):

      Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. Two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes; this manuscript confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors have conducted an exceptionally informative series of studies investigating the neural basis of interoception in transdiagnostic psychiatric symptoms. By comparing differential and overlapping neural activation during 'top-down' and 'bottom-up' interoceptive tasks, they reveal convergent activation largely localised to the ventral dysgranular subregion ('mid-insula'), which differs in extent between patients and controls, replicating and extending previous suggestions of this region as a central locus of disruption in psychiatric disorders. Their work also reveals different extents of divergent activation in the anterior insula during anticipation of interoceptive disruption. This substantially advances our previous knowledge of the anatomy of interoception and confirms theoretical predictions of the roles of different cytoarchitectural subregions of the insula in interoceptive dysfunction in mental health conditions.

      Strengths:<br /> The work is exceptional in terms of breadth and depth, making use of multiple imaging and analysis techniques which are non-standard and go well beyond what is known today. The study is statistically well-powered and the tasks are well-validated in the literature. To my knowledge, these functions of the insula in interoception and mental health have never been compared directly before, so the results are novel and informative for both basic science and psychiatry. The work is strongly theory-driven, building on and directly testing results from influential theories and previous studies. It is likely that the results will strengthen our theoretical models of interoception and advance psychiatric studies of the insula.

      Weaknesses:<br /> The study has three current limitations. (1) The interpretation of the resting-state data is not quite as clear-cut as the task-based data - as presented currently, changes could potentially represent fluctuations over time rather than following interoception specifically. In contrast, much stronger conclusions can be drawn from the authors' task-based data. (2) The transdiagnostic sample could be better characterised in terms of diagnostic information, and was almost entirely female; it is also unclear what the effect of psychotropic medications may have been on the results given the effects of (e.g.) serotonergic medication on the BOLD signal. (3) As the authors point out, there may have been task-specific preprocessing/analysis differences that influenced results, for example, due to physiological correction in one but not both tasks.

    1. Reviewer #2 (Public Review):

      Sasaki et al. investigated methods to entrain vasomotion in awake wild-type mice across multiple regions of the brain using a horizontally oscillating visual pattern which induces an optokinetic response (HOKR) eye movement. They found that spontaneous vasomotion could be detected in individual vessels of their wild-type mice through either a thinned cranial window or intact skull preparation using a widefield macro-zoom microscope. They showed that low-resolution autofluorescence signals coming from the brain parenchyma could be used to capture vasomotion activity using a macro-zoom microscope or optical fibre, as this signal correlates well with the intensity profile of fluorescently-labelled single vessels. They show that vasomotion can also be entrained across the cortical surface using an oscillating visual stimulus with a range of parameters (with varying temporal frequencies, amplitudes, or spatial cycles), and that the amplitude spectrum of the detected vasomotion frequency increases with repeated training sessions. The authors include some control experiments to rule out fluorescence fluctuations being due to artifacts of eye movement or screen luminance and attempt to demonstrate some functional benefit of vasomotion entraining as HOKR performance improves after repeat training. These data add in an interesting way to the current knowledge base on vasomotion, as the authors demonstrate the ability to entrain vasomotion across multiple brain areas and show some functional significance to vasomotion with regards to information processing as HOKR task performance correlates well with vascular oscillation amplitudes.

      The aims of the paper are mostly well supported by the data, but some streamlining of the data presentation would improve overall clarity. The third aim to establish the functional significance of vasomotion in relation to plasticity in information processing could be better supported by the inclusion of some additional control experiments. Specifically:

      1) The clarity and comprehensibility of the paper could be significantly enhanced by incorporating additional details in both the introduction and discussion sections. In the introduction, a succinct definition of the frequency range of vasomotion should be provided, as well as a better description of the horizontal optokinetic response (i.e. as they have in the results section in the first paragraph below the 'Entrainment of vasomotion with visual stimuli presentation' sub-heading). The discussion would benefit from the inclusion of a clear summary of the results presented at the start, and the inclusion of stronger justification (i.e. more citations) with regards to the speculation about vasomotion and neuronal plasticity (e.g. paragraph 5 includes no citations).

      2) The novel methods for detecting vasomotion using low-resolution imaging techniques are discussed across the first four figures, but this gets a little bit confusing to follow as the authors jump back and forth between the different imaging and analysis techniques they have employed to capture vasomotion. The data presentation could be better streamlined - for instance by presenting only the methods most relevant for the functional dataset (in Figures 5-7), with the additional information regarding the various controls to establish the use of autofluorescence intensity imaging as a valid method for capturing vasomotion reduced to fewer figure panels, or moved to supplementary figures so as to not detract from the main novel findings contributed in this study.

      3) The authors heavily rely on representative traces from individual vessels to illustrate their findings, particularly evident in Figures 1-4. While these traces offer a valuable visualization, augmenting their approach by presenting individual data points across the entire dataset, encompassing all animals and vessels, would significantly enhance the robustness of their claims. For instance, in Figures 1 and 2, where average basal and dilated traces are depicted for a representative vessel, supplementing these with graphs showcasing peak values across all measured vessels would enable the authors to convey a more holistic representation of their data. Or in Figure 3, where the amplitude spectrum is presented for individual Texas red fluorescence intensity changes in V1 across novice, trained, and expert mice, incorporating a summary graph featuring the amplitude spectrum value at 0.25Hz for each individual trace (across animals/imaging sessions), followed by statistical analysis, would fortify the strength of their assertions. Moreover, providing explicit details on sample sizes for each individual figure panel (where not a representative trace), including the number of animals or vessels/imaging sessions, would contribute to transparency and aid readers in assessing the generalisability of the findings.

      4) In the experiments where mice are classed as "novice", "trained" or "expert", the inclusion of the specific range of the number of training sessions for each category would improve replicability.

      5) The authors don't state whether mice were habituated to the imaging set-up prior to the first data collection, as head-fixation and restraint can be stress-inducing for animals, especially upon first exposure, which could impact their neurovascular coupling responses differentially in "novice" versus "trained" imaging sessions (e.g. see Han et al., 2020, DOI: https://doi.org/10.1523/JNEUROSCI.1553-20.2020). The stress associated with a tail vein injection prior to imaging could also partially explain why mice didn't learn very well if Texas Red was injected before the training session. If no habituation was conducted in these experiments, the study would benefit from the inclusion of some control experiments where "novice" responses were compared between habituated and non-habituated animals.

      6) The experiments regarding the brain-wide vasomotion entrainment across the cortical surface would benefit from some additional information about how brain regions were identified (e.g. particularly how V1 and V2 were distinguished given how close together they are).

      7) Whilst the authors show that HOKR task performance and vasomotion amplitude are increased with repeated training to provide some support to their aim of investigating the functional significance of vasomotion with regards to information processing plasticity, the inclusion of some additional control experiments would provide stronger evidence to address this aim. For instance, if vasomotion signalling is blocked or reduced (e.g. using optogenetics or in an AD mouse model where arteriole amyloid load restricts vasomotion capacity), does flocculus-dependent task performance (e.g. HOKR eye movements) still improve with repeated exposure to the external stimulus.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, the authors attempted to study mechanisms of transcription inhibition in cells treated with IR. They observed that, unlike histone chaperone HIRA-dependent transcription inhibition during UV-induced damage, IR-induced transcription inhibition does not depend on HIRA. Through the CRISPR/Cas9 screen, they identified protein neddylation is important for transcription inhibition. By sequencing nascent RNA, they observed that down-regulated transcripts upon IR treatment are largely highly transcribed genes including histone genes and rDNA.

      Strengths:<br /> The authors utilized comprehensive approaches to fill in the knowledge gap of IR-induced transcription inhibition.

      Weaknesses:<br /> it is not clear that inhibition of histone genes by IR is due to a reduction of S phase progression.

    1. Reviewer #2 (Public Review):

      Summary:

      There are two potential contributions made by this study, both of which are not fully supported by the data presented. First, that Twist-positive hepatocytes in the midlobular zone are derived from Twist2-expressing cells in embryonic livers via intermediate EpCAM-expressing cells. Second, that there is a population of hepatocytes with mesenchymal features that drive regeneration after various injuries. The concept that mid-lobular hepatocytes are more regenerative in adult injury settings has already been established and this paper further supports that body of knowledge.

      Strengths:

      There are copious scRNA-seq data that are supportive of the claims, but these analyses were not definitive.

      Weaknesses:

      1. There is not sufficient evidence to support the following assertion: "markers identified a mesenchymal-hepatocyte hybrid population (13.7% of total hepatocytes) that express signature genes of both lineages." Twist-Cre reporter mice mark hepatocytes and mesenchymal populations, but it is not clear whether or not this means that the hepatocyte population labeled by Twist is mesenchymal. It is very possible for hepatocytes to express mesenchymal genes without being a true hybrid population. There is not much evidence that zone 2 cells are a mix of hepatocyte and mesenchymal. The idea of a hybrid population needs to be defined. The definition probably needs to involve the concept that hybrid cells must have morphologic or functional features of mesenchymal cells, rather than just expressing some genes from each cell type.

      Related to this, the authors claim that co-expression of Twist and EpCAM in E10.5 liver cells might support the existence of a hepatomesenchymal cell type. This is possible, but one should note that adult hepatocytes can express EpCAM, especially during ductular reactions, so it is not necessarily a mesenchymal marker per se.

      2. The authors assert several times that Twist-Cre mice appear to have no effect on overall liver regeneration phenotypes. They use this to suggest a lack of an effect for heterozygous deletion of Twist by the Cre allele. It is still possible for these mice to have altered lineage tracing results. It is very difficult to rule this out. For example, Axin2-CreER mice did not have any overt liver function or regeneration phenotypes, but the lineage tracing results from these mice differed from other CreER mice.

      3. The central problem with this study is that the authors use a Cre strain and not a CreER strain. With a Cre strain, there could be new labeling of Twist-positive cells at multiple later time points. Thus, it is very difficult to assert that the Tomato-positive population at later time points are really descendants of the originally labeled population. It is very difficult to interpret the results of Cre-based lineage tracing experiments.

      With this technical limitation in mind, I do not think that there is enough evidence to support the assertion made on page 6: "These findings suggest that EpCAMlow progenitor cells give rise to hepatocytes and MCs." The authors use scRNA-seq trajectory analysis to come to the conclusion that mesenchymal cells give rise to hepatocytes between p1 and p14. Much more evidence is needed before the authors can arrive at this conclusion. It is much more likely that midlobular hepatocytes arise from other hepatocytes. To support their arguments, the authors would have to use a CreER line that exclusively labels mesenchymal cells in the liver, then lineage traces them until p14 to determine if they become hepatocytes. Without such an experiment, I do not think the current experiments are interpretable.

      4. The injury experiments are again limited in their interpretability because they do not use CreER. It is very possible that Twist is turned on after CCl4 or surgical injury, and thus new hepatocytes might activate Tomato. It is unclear if previously Tomato-positive midzone hepatocytes were proliferating to increase the Tomato positive population. The authors use expression-based studies to argue against ectopic activation of Twist, but it is very difficult to exclude Cre activation using these types of studies.

    1. Reviewer #2 (Public Review):

      Summary:

      Here, the authors show that neutral lipids play a role in spermatogenesis. Neutral lipids are components of lipid droplets, which are known to maintain lipid homeostasis, and to be involved in non-gonadal differentiation, survival, and energy. Lipid droplets are present in the testis in mice and Drosophila, but not much is known about the role of lipid droplets during spermatogenesis. The authors show that lipid droplets are present in early differentiating germ cells, and absent in spermatocytes. They further show a cell autonomous role for the lipase brummer in regulating lipid droplets and, in turn, spermatogenesis in the Drosophila testis. The data presented show that a relationship between lipid metabolism and spermatogenesis is congruous in mammals and flies, supporting Drosophila spermatogenesis as an effective model to uncover the role lipid droplets play in the testis.

      Strengths and weaknesses:

      The authors do a commendably thorough characterization of where lipid droplets are detected in normal testes: located in young somatic cells, and early differentiating germ cells. They use multiple control backgrounds in their analysis, including w[1118], Canton S, and Oregon R, which adds rigor to their interpretations. The authors employ markers that identify which lipid droplets are in somatic cells, and which are in germ cells. The authors use these markers to present measured distances of somatic and germ cell-derived lipid droplets from the hub. Because they can also measure the distance of somatic and germ cells with age-specific markers from the hub, these results allow the authors to correlate position of lipid droplets with the age of cells in which they are present. This analysis is clearly shown and well quantified.

      The quantification of lipid droplet distance from the hub is applied well in comparing brummer mutant testes to wild type controls. The authors measure the number of lipid droplets of specific diameters, and the spatial distribution of lipid droplets as a function of distance from the hub. These measurements quantitatively support their findings that lipid droplets are present in an expanded population of cells further from the hub in brummer mutants. The authors further quantify lipid droplets in germline clones of specified ages; the quantitative analysis here is displayed clearly and supports a cell autonomous role for brummer in regulating lipid droplets in spermatocytes.

      Data examining testis size and number of spermatids in brummer mutants clearly indicates the importance of regulating lipid droplets to spermatogenesis. The authors show beautiful images supported by rigorous quantification supporting their findings that brummer mutants have both smaller testes with fewer spermatids at both 29 and 25C. There is also significant data supporting defects in testis size, but not spermatid number, in 14-day-old brummer mutant animals compared to controls. Their analysis clearly shows an expanded region beyond the testis apex that includes younger germ cells, supporting a role for lipid droplets influencing germ cell differentiation during spermatogenesis.

      The authors present a series of data exploring a cell autonomous role for brummer in the germline, including clonal analysis and tissue specific manipulations. The clonal data indicating increased lipid droplets in spermatocyte clones, and a higher proportion of brummer mutant GSCs at the hub are convincing and supported by quantitation. The authors also show a tissue specific rescue of the brummer testis size phenotype by knocking down mdy specifically in germ cells, which is also supported by statistically significant quantitation. The authors present data examining the number of spermatocyte and post-meiotic clones 14 days after clonal induction. Their finding is significant with a p-value of 0.0496, which they acknowledge is less robust than their other data reported in this study, and could be a result of a low sample size. They indicate that future studies might validate these results with additional samples.

      The authors do a beautiful job of validating where they detect brummer-GFP by presenting their own pseudotime analysis of publicly available single cell RNA sequencing data. Their data is presented very clearly, and supports expression of brummer in older somatic and germline cells of the age when lipid droplets are normally not detected. The authors also present a thorough lipidomic analysis of animals lacking brummer to identify triglycerides as an important lipid droplet component regulating spermatogenesis.

      Impact:

      The authors present data supporting the broad significance of their findings across phyla. This data represents a key strength of this manuscript. The authors show that loss of a conserved triglyceride lipase impacts testis development and spermatogenesis, and that these impacts can be rescued by supplementing diet with medium-chain triglycerides. The authors point out that these findings represent a biological similarity between Drosophila and mice, supporting the relevance of the Drosophila testis as a model for understanding the role of lipid droplets in spermatogenesis. The connection buttresses the relevance of these findings and this model to a broad scientific community.

    1. Reviewer #2 (Public Review):

      This work investigates the mechanisms, patterns, and geographical distribution of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum. Rapid diagnostic tests (RDTs) detect P. falciparum histidine-rich protein 2 (PfHRP2) and its paralog PfHRP3 located in subtelomeric regions. However, laboratory and field isolates with deletions of pfhrp2 and pfhrp3 that can escape diagnosis by RDTs are spreading in some regions of Africa. They find that pfhrp2 deletions are less common and likely occur through chromosomal breakage with subsequent telomeric healing. Pfhrp3 deletions are more common and show three distinct patterns: loss of chromosome 13 from pfhrp3 to the telomere with evidence of telomere healing at breakpoint (Asia; Pattern 13-); duplication of a chromosome 5 segment containing pfhrp1 on chromosome 13 through non-allelic homologous recombination (NAHR) (Asia; Pattern 13-5++); and the most common pattern, duplication of a chromosome 11 segment on chromosome 13 through NAHR (Americas/Africa; Pattern 13-11++). The loss of these genes impacts the sensitivity of RDTs, and knowing these patterns and geographic distribution makes it possible to make better decisions for malaria control.

    1. Reviewer #2 (Public Review):

      The authors present an image-analysis pipeline for mother-machine data, i.e., for time-lapses of single bacterial cells growing for many generations in one-dimensional microfluidic channels. The pipeline is available as a plugin of the python-based image-analysis platform Napari. The tool comes with two different previously published methods to segment cells (classical image transformation and thresholding as well as UNet-based analysis), which compare qualitatively and quantitatively well with the results of widely accessible tools developed by others (BACNET, DelTA, Omnipose). The tool comes with a graphical user interface and example scripts, which should make it valuable for other mother-machine users, even if this has not been demonstrated yet.

      The authors also add a practical overview of how to prepare and conduct mother-machine experiments, citing their previous work, referring to detailed instructions on their github page, and giving more advice on how to load cells using centrifugation.

      Finally, the authors emphasize that machine-learning methods for image segmentation reproduce average quantities of training datasets, such as the length at birth or division. Therefore, differences in training can propagate to differences in measured average quantities. This result is not surprising but good to remember before interpreting absolute measurements of cell shape.

    1. Reviewer #2 (Public Review):

      This work by Knights et al., makes use of the Cam-CAN dataset to investigate functional compensation during a fluid processing task in older adults, in a fairly large sample of approximately 200 healthy adults ranging from 19 to 87. Using univariate methods, the authors identify two brain regions in which activity increases as a function of both age and performance and conduct further investigations to assess whether the activity of these regions provides information regarding task difficulty. The authors conclude that the cuneal cortex - a region of the brain previously implicated in visual attention - shows evidence of compensation in older adults.

      The conclusions of the paper are well supported by the data, and the authors use appropriate statistical analyses. The use of multivariate methods over the last 20 years has demonstrated many effects that would have been missed using more traditional univariate analysis techniques. The data set is also of an appropriate size, and as the authors note, fluid processing is an extremely important domain in the field of cognition in aging, due to its steep decline over aging. However, it might have been nice to see an analysis of a more crystallised intelligence task included too, as a contrast since this is an area that does not demonstrate such a decline (and perhaps continues to improve over aging).

    1. Reviewer #2 (Public Review):

      Summary:

      Bezares Calderon et al demonstrate that the planktonic larva of marine annelid Platynereis dumerii responds to increased pressure in the water column by swimming upward. The authors show the larvae do so via their ciliated photoreceptors that recruit serotoninergic motor neurons to elicit swimming via an increased ciliary beat frequency of the multiciliary band of their head.

      Strengths:

      The authors built original setups to increase water pressure and monitor behavior or calcium activity in the cells. Using their original setups, they combined behavioral and imaging experiments on wild type and mutant larvae for an opsin to show how photoreceptors encode the response to pressure and recruit in response serotoninergic motor neurons that increase the ciliary beating frequency of the multiciliary band in the head.

      Weaknesses:

      Technical note:<br /> The authors should use DF/F to quantify over time the calcium response in photoreceptors. Furthermore, they should show that there is no concern of motion artifact when the pressure changes - as it could be a concern.

      The authors have not shown<br /> 1- how the off response to decrease of pressure is mediated<br /> 2- which receptor/channel mediates in photoreceptors the response to increased pressure,<br /> 3- nor how the integration of light and pressure information is integrated by photoreceptors in order to guide the behavior of the larvae.

      These points are beyond the scope of the study. However, if possible within a short time frame, it would be really interesting to find out whether conflicting stimuli or converging stimuli (light & pressure) can cancel each other out or synergize. In particular since the authors cite unpublished results in the discussion: "Our unpublished results indeed suggest that green light determines the direction of swimming and can override upward swimming induced by pressure, which only influences the speed of swimming (LABC and GJ, unpublished)." Showing in one panel this very cool phenomenon would be exciting & open tons of questions for the field.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript titled "Identification of pharmacological inducers of a reversible hypometabolic state for whole organ preservation" reports the effects of delta opioid receptor activator SNC80 and its modified analog WB3 with ~1,000 times less delta opioid receptor binding activity on metabolic state.

      Strengths:<br /> This is an interesting study with potentially broad implications for organ preservation.

      Weaknesses:<br /> There are several limitations that raise concerns.

      1. The authors developed an analog of a known delta opioid receptor activator SNC80 with three orders of magnitude lesser binding with the delta opioid receptor WB3. This will likely reduce the undesirable effects of SNC80 while preserving the metabolic slowing needed for organ preservation. Yet, most experiments were done with SNC80, not the superior modification, WB3, shown in only a limited set of experiments, Figure 3.

      2. The heart is one of the most challenging organs to preserve, and some experiments are done to establish the metabolic effects of SNC80. However, the biodistribution study, shown in Figure 2, conspicuously omitted the heart.

      3. I do not understand the design of the electrophysiology and contractility experiments with the porcine hearts. How did you defibrillate the hearts after removal and establishing perfusion? Lines 173-175 on Page 7 state: "After defibrillation with epinephrine, the P and QRS waveforms were visible in ECGs from 3 of 4 SNC80-treated hearts (Table S1), suggesting that those hearts regain atrial and ventricular polarization." Please clarify. Defibrillation is done with an electric shock. Also, please show the ECG recordings to support your conclusions about "polarization." What did you mean by "polarization"? Depolarization? Repolarization? Or resting potential. To establish a normal physiological state, please show ECG waveforms and present data on basic ECG characteristics: heart rate, PQ and QT intervals, and P and QRS durations. I recommend perfusion of the porcine heart with WB3, not only SNC80.

      4. Pathology data also raises concerns. The histology images shown in Figure 4f are not quantified, and they show apparently higher levels of tissue disruption in SNC80-treated tissue vs vehicle-treated. The test (lines 169-171) confirms this concern: "In some hearts treated with SNC80, greater waviness of muscle fibers was observed, possibly indicating a state of muscle contraction." It will be helpful to measure markers of apoptosis and necrosis and to apply TTC viability staining.

      5. The apparent state of contracture suggests a higher degree of myocardial damage and a high intracellular calcium level in SNC80-treated hearts. The authors suggested that the sodium-calcium exchanger NCX is a possible target of SNC80 and could be responsible for the "hypometabolic state." However, NCX1 is critically important in the extrusion of cytosolic Ca2+ during the diastolic phase. Failure to remove excessive calcium and restore ionic homeostasis would lead to calcium overload and heart failure.

      6. I am surprised the authors did not consider using the gold standard assay for measuring mitochondrial function in cells by the Seahorse Cell Mito Stress Test.

    1. Reviewer #2 (Public Review):

      Summary:

      Invasive fungal infections are very difficult to treat with limited drug options. With the increasing concern of drug resistance, developing an antifungal vaccine is a high priority. In this study, the authors studied the metal metabolism in Candida albicans by testing some chelators, including EDTA, to block the metal acquisition and metabolism by the fungus. Interestingly, they found EDTA-treated yeast cells grew poorly in vitro and non-pathogenic in vivo in a murine model. Mice immunized by EDTA-treated Candida (CAET) were protected against challenge with wild-type Candida cells. RNA-Seq analysis to survey the gene expression profile in response to EDTA treatment in vitro revealed upregulation of genes in metal homeostasis and downregulation of ribosome biogenesis. They also revealed an induction of both pro- and anti-inflammatory cytokines involved in Th1, Th2 and Th17 host immune response in response to CAET immunization. Overall, this is an interesting study with translational potential.

      Strengths:

      The main strength of the report is that the authors identified a potential whole-cell live vaccine strain that can provide full protection against candidiasis. Abundant data both on in vitro phenotype, gene expression profile, and host immune response have been presented.

      Weaknesses:

      A weakness is that the immune mechanism of CAET-mediated host protection remains unclear. The immune data is somewhat confusing. The authors only checked cytokines and chemokines in blood. The immune response in infected tissues and antibody response may be investigated.

    1. Reviewer #2 (Public Review):

      Pial collateral vessels are anastomotic connections that cross-connect distal arterioles of the middle, anterior, and posterior cerebral arteries. With respect to ischemic stroke, good pial collateral flow positively correlates with decreased infarct volume and improved recovery; accordingly, optimizing collateral flow represents an important intervention for limiting stroke damage. The goal of this study was to determine the endothelial cell (EC) subtype(s) that contribute to the embryonic and neonatal development of pial collaterals and their expansion in response to stroke. To this end, the authors used lineage tracing methods in the mouse, labeling arterial endothelial cells (using Bmx-CreERT on switch line, R26mTmG) or venous and microvascular endothelial cells (using Vegfr3-CreERT on R26mTmG) and assessing pial collaterals via confocal microscopy. The authors convincingly demonstrate that arterial-lineage ECs comprise the majority of pial collateral ECs during development and in adulthood, with a minor contribution from pial plexus-derived microvascular ECs that decline over time. They also convincingly demonstrate that pial collateral outward remodeling after experimentally-induced stroke (distal middle cerebral artery occlusion, or dMCAO) involves, at least in part, local proliferation of arterial-lineage ECs. The latter is intriguing given that arterial ECs generally leave the cell cycle. While these conclusions are quite solid, some key details are missing that could improve analysis, and some important caveats are not addressed. Moreover, less convincing are mechanistic claims that pial collaterals form via a migratory process of "mosaic colonization" of a preexisting vessel.

      1. It is difficult to understand whether individual collaterals are truly mosaic vessels, or whether arterial or venous/microvascular lineage ECs predominate in any particular region of the pial collateral vasculature. This is due to a number of methodological reasons: arterial and venous/microvascular contributions to pial collaterals were assessed independently, only a few (and in some cases, just one) collaterals were analyzed in each mouse, and regionality/location of collaterals was not addressed. Additionally, the inefficiency and variability of EC labeling, especially with the Vegfr3-CreERT line (Fig. S1, ~6-30%), compounds this problem.

      2. The identification of "pre-collateral" vessels requires further support. The authors define these vessels by their connection to the feeding artery, their (often) larger diameter, and their more pronounced ICAM2 expression. While most of these criteria are demonstrated in Figure S3, it is not apparent how these vessels were defined in Figure 4, which lacks specific annotation of each of these identifying criteria. As the identification of these novel vessels is one of the key findings of this paper, a more robust method of unambiguously defining them is warranted.

      3. The conclusion that collateral-forming ECs migrate in the direction of flow into preexisting vessels is not well supported. The authors state that the presence of filopodial projections (Figure 4) supports this conclusion. However, filopodia number and directional polarization/orientation were not quantified, and "intercalation movements"/migration, per se, cannot be inferred from these static images.

      4. In Figure 5, the simplest explanation for relative Cx40 expression in different vessels is the absence (low expression) or presence (high expression) of flow. This figure provides little mechanistic insight beyond this already-known relationship, and it is unclear how many times this experiment was performed (there is no N, no quantification or correlation).

      5. There is no statistical analysis in this work. This is justified by the authors by their admission that the study is of a "descriptive nature and...exploratory design."

    1. Reviewer #2 (Public Review):

      The authors present here a mathematical and computational study of the topological/graph theory requirements to obtain sustained oscillations in neural network models. A first approach mathematically demonstrates that, for a given network of interconnected neural populations (understood in the sense of dynamical systems) requires an odd number of inhibitory populations to sustain oscillations. The authors extend this result via numerical simulations of (i) a simplified set of Wilson-Cowan networks, (ii) a simplified circuit of the cortico-basal ganglia network, and (iii) a more complex, spike-based neural network of basal ganglia network, which provides insight on experimental findings regarding abnormal synchrony levels in Parkinson's Disease (PD).

      The work elegantly and effectively combines a solid mathematical proof with careful numerical simulations at different levels of description, which is uncommon and provides additional layers of confidence to the study. Furthermore, the authors included detailed sections to provide intuition about the mathematical proof, which will be helpful for readers less inclined to the perusal of mathematical derivations. Its insightful and well-informed connection with a practical neuroscience problem, the presence of strong beta rhythms in PD, elevates the potential influence of the study and provides testable predictions.

      In its updated form, the authors have solved the most pressing issues of the study, by acknowledging the limitations of their work regarding the effects of delays in oscillations, and addressing some of these effects in new simulations. Although some interesting simulations are still not presented in the revised version, they could constitute the focus of future work to complement the conclusions presented here. The absence of explanations for some of the figures and panels has been corrected, and the issues with grammar and lack of clarity have been improved. This important work is therefore now improved.

    1. Reviewer #2 (Public Review):

      Summary:

      This study proposes visual homogeneity as a novel visual property that enables observers perform to several seemingly disparate visual tasks, such as finding an odd item, deciding if two items are the same, or judging if an object is symmetric. In Experiment 1, the reaction times on several objects were measured in human subjects. In Experiment 2, the visual homogeneity of each object was calculated based on the reaction time data. The visual homogeneity scores predicted reaction times. This value was also correlated with the BOLD signals in a specific region anterior to LO. Similar methods were used to analyze reaction time and fMRI data in a symmetry detection task. It is concluded that visual homogeneity is an important feature that enables observers to solve these two tasks.

      Strengths:

      1) The writing is very clear. The presentation of the study is informative.<br /> 2) This study includes several behavioral and fMRI experiments. I appreciate the scientific rigor of the authors.

      Weaknesses:

      1) My main concern with this paper is the way visual homogeneity is computed. On page 10, lines 188-192, it says: "we then asked if there is any point in this multidimensional representation such that distances from this point to the target-present and target-absent response vectors can accurately predict the target-present and target-absent response times with a positive and negative correlation respectively (see Methods)". This is also true for the symmetry detection task. If I understand correctly, the reference point in this perceptual space was found by deliberating satisfying the negative and positive correlations in response times. And then on page 10, lines 200-205, it shows that the positive and negative correlations actually exist. This logic is confusing. The positive and negative correlations emerge only because this method is optimized to do so. It seems more reasonable to identify the reference point of this perceptual space independently, without using the reaction time data. Otherwise, the inference process sounds circular. A simple way is to just use the mean point of all objects in Exp 1, without any optimization towards reaction time data.

      2) On page 11, lines 214-221. It says: "these findings are non-trivial for several reasons". However, the first reason is confusing. It is unclear to me why "it suggests that there are highly specific computations that can be performed on perceptual space to solve oddball tasks". In fact, these two sentences provide no specific explanation for the results.

      3) The second reason is interesting. Reaction times in target-present trials can be easily explained by target-distractor similarity. But why does reaction time vary substantially across target-absent stimuli? One possible explanation is that the objects that are distant from the feature distribution elicit shorter reaction times. Here, all objects constitute a statistical distribution in the feature (perceptual) space. There is certainly a mean of this distribution. Some objects look like outliers and these outliers elicit shorter reaction times in the target-absent trials because outlier detection is very salient.

      One might argue that the above account is merely a rephrasing of the idea of visual homogeneity proposed in this study. If so, feature saliency is not a new account. In other words, the idea of visual homogeneity is another way of reiterating the old feature saliency theory.

      4) One way to reject the feature saliency theory is to compare the reaction times of the objects that are very different from other objects (i.e., no surrounding objects in the perceptual space, e.g., the wheel in the lower right corner of Fig. 2B) with the objects that are surrounded by several similar objects (e.g., the horse in the upper part of Fig. 2B). Also, please choose the two objects with similar distance from the reference point. I predict that the latter will elicit longer reaction times because they can be easily confounded by surrounding similar objects (i.e., four-legged horses can be easily confounded by four-legged dogs). If the density of object distribution per se influences the visual homogeneity score, I would say that the "visual homogeneity" is essentially another way of describing the distributional density of the perceptual space.

      5) The searchlight analysis looks strange to me. One can easily perform a parametric modulation by setting visual homogeneity as the trial-by-trial parametric modulator and reaction times as a covariate. This parametric modulation produces a brain map with the correlation of every voxel in the brain. On page 17 lines 340-343, it is unclear to me what the "mean activation" is.

      Minor points:

      1) In the intro, it says: "using simple neural rules..." actually it is very confusing what "neural rules" are here. Better to change it to "computational principles" or "neural network models"??

      2) In the intro, it says: "while machine vision algorithms are extremely successful in solving feature-based tasks like object categorization (Serre, 2019), they struggle to solve these generic tasks (Kim et al., 2018; Ricci et al. 2021). These are not generic tasks. They are just a specific type of visual task-judging relationship between multiple objects. Moreover, a large number of studies in machine vision have shown that DNNs are capable of solving these tasks and even more difficult tasks. Two survey papers are listed here.

      Wu, Q., Teney, D., Wang, P., Shen, C., Dick, A., & Van Den Hengel, A. (2017). Visual question answering: A survey of methods and datasets. Computer Vision and Image Understanding, 163, 21-40.

      Małkiński, M., & Mańdziuk, J. (2022). Deep Learning Methods for Abstract Visual Reasoning: A Survey on Raven's Progressive Matrices. arXiv preprint arXiv:2201.12382.

    1. Reviewer #2 (Public Review):

      Summary:

      Chen et al., investigate the role of DCP1 paralogs in regulating RNA decay in human tissue culture cells. They assess the impact of the absence of DCP1a and/or DCP1b on the interaction of DCP2 with mRNA and other members of the decapping complex. In vitro RNA decay assays were performed to demonstrate that DCP1a/b plays a minor role in DCP2-mediated decapping and decay. The impacts of DCP1a and/or DCP1b knockout on the transcriptome and metabolome were determined.

      Strengths:

      Analysis of RNA abundance and metabolite differences in human tissue culture cells lacking DCP1a and/or DCP1b was performed.

      The protein-protein interactions between DCP2 and other members of the decapping machinery mediated by DCP1a and/or DCP1b were assessed.

      The functional role of DCP1a and/or DCP1b in mediating mRNA decapping/decay in human tissue culture cell extracts was determined.

      Human tissue culture cells lacking DCP1a and/or DCP1b appear to have altered metabolomes, however, the significance and meaning of these differences are not clear.

      Weaknesses:

      The direct targets of DCP1a and/or DCP1b were not determined as the analysis was restricted to RNA-seq to assess RNA abundance, which can be a result of direct or indirect regulation by DCP1a/b.

      P-bodies appear to be larger in human cells lacking DCP1a and DCP1b but a lack of image quantification prevents this conclusion from being drawn.

      The lack of details in the methodology and figure legends limit reader understanding.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors measure axon outgrowth rate, laminin adhesion strength, and actin rearward flow rate. They find that the axon outgrowth rate has a biphasic dependence on adhesion strength. In interpreting the results, they suggest that the results "imply that adhesion modulation is key to the regulation of axon guidance"; however, they measure elongation rate, not guidance.

      Strengths:

      The measurements of adhesion strength by laser-induced shock waves are reasonable as is the measurement of actin flow rates by speckle microscopy.

      Weaknesses:

      They only measure the length of the axons after 3 days and have no measurements of the actual rate of growth cone movements when they are moving. They do not measure the rate of actin growth at the leading edge to know its contribution to the extension rate. This is inadequate.

      These studies are unlikely to have an impact on the field because the measurement of axon growth rate at short times is missing.

    1. Reviewer #2 (Public Review):

      This work examines the roles of Gtr1/Gtr2 and Pib2 in activation of TORC1 in S cerevisiae and proposes they are non-redundant in activating TORC1. Previous work from many groups has suggested that the Gtr complex and Pib2 activate TORC1 in a parallel manner. One contribution of this study is the suggestion that using the standard readout(s) of TORC1 activation are not sufficient to assess the separate roles of these two components in the complex network of amino acid and starvation response signaling. The overall conclusion of the work, based on phosphoproteome analyses of deletion strains and comparison to rapamycin treatment, with some supporting experimentation, is that Pib2 signaling sustains the starvation response in poor amino acid/nitrogen sources, whereas the additional activation of the Gtr complex is required for the full spectrum of TORC1 effects on growth.

      At first, the authors recapitulate and extend studies on TORC1 inactivation using the Rps6 reporter. Here, Pib2 could inactivate TORC1 on glutamine starvation only if the Gtr complex is partially compromised. The authors speculated that Gtr and Pib2 do lead to different responses, but these cannot be detected by monitoring the phospho state of Rps6.

      The authors determined the phosphoproteome in wild type cells and a variety of knockout strains, in rich media and in the presence of rapamycin. The authors identified 175 phosphosites that are downregulated on rapamycin treatment, at least under these conditions. Many were dependent on both Pib2 and the Gtr complex but, of particular interest for this work , were the phosphosites on Ser33, that were dependent on the presence of Pib2 but not the Gtr complex. The authors noted that phosphosites not dependent on Pib2 or Gtr1/2 included Sch9 and other common readouts of TORC1 activation.

      Focusing on Ser33, the authors next show that rapamycin, amino acid and nitrogen starvation result in loss of Ser33 phosphorylation. Further analysis showed that the Ser33 phosphorylation status depends on the quality of the amino acid and nitrogen source.

      Then the authors use this to develop a model where TORC1 has three states depending on whether either Gtr1/2, or Pib2, or both are active in signaling to TORC1, depending on the nutrient state and quality of amino acids/nitrogen available. The new state is state III, where TORC1 is active to promote growth and the starvation response remains active, via the Npr1/Par32 branch. The remainder of the work involves developing tools to assess the growth (Sch9) and starvation (Par32) branches under various amino acid/nutrient states. While moving from media with an excess of all amino acids to glutamine or leucine led to only transient occupation of state III, the new state was already occupied when the cells were in a poor amino acid/nitrogen source and moved to a better one. In other words, the Pib2 signalling permitted aspects of a starvation response to be maintained in the background of a Sch9 growth signal.

      Finally, the authors address a puzzle: Sch9 phosphorylation does not have the dynamic range to account for the difference in growth rates of yeast cells in SC or proline medium. Tod6 was dephosphorylated in the absence of Gtr1/Gtr2 or Pib2 in the phosphoproteomics and is the likely connection, as it moves to the nucleus on growth on proline media (or on rapamycin), where it may control the chromatin accessibility of ribosome growth and biogenesis genes.

      Overall, the core of this work, the phosphoproteome analyses, convincingly demonstrates that activation of TORC1 relies on a nuanced interplay of signaling pathways and that to fully appreciate and dissect the consequences of the Gtr- and Pib2-responsive signaling pathways a more comprehensive range of readouts is required. The work elegantly shows a scenario where Pib2-based signaling is active, required to sustain some growth even when the amino acid/nitrogen mix is poor.

      There are some areas, however, where the work could be strengthened. The model proposed in this work is based on nuanced signaling responses to various states of nitrogen/amino acid starvation. However, the phosphoproteome was determined in a synthetic rich background, supplemented with rapamycin where relevant, and comparing the phosphoproteome of pib2 del and gtr1 del/gtr2 del to this. The phosphoproteome is by far the strongest data in this work suggesting multi-level regulation so an appropriately matched phosphoproteome condition screen would likely significantly substantiate the model: the conditions used might miss all the nuanced signaling responses the authors develop throughout the paper. Not unrelated, the authors show that Pib2 can transmit glutamine starvation signals to TORC1 in the presence of a partial Gtr1/2 complex (gtr1 del or gtr2 del) but not a complete deletion of the complex (Fig. 2). Similar to the above comment, the phosphoproteome was determined only with full loss of the gtr complex, and then only in a rich background, which may miss this entire branch of Pib2 signaling. Perhaps in support of this, Pib2Ser113 phosphorylation apparently decreased significantly on rapamycin treatment but not on loss of the Gtr complex (TableS1), whereas other Pib2 phospho sites were not similarly affected by rapamycin treatment. Adding to the notion of complexity, the other sites may themselves be subject to other signaling pathways that could regulate Pib2 - and these may change on nutrient starvation.

      The data showing the enrichment of Pib2 with Ser33 is weak (Fig. 5G, mostly because of the significant precipitation of Ser33 in the absence of Pib2), particularly without the contribution of the immunopurifications of Fig5S1. Assessing the binding of Ser3 may be a better candidate?

    1. Reviewer #2 (Public Review):

      "Chromatin Structure II: Stem-loops and circle-loops" by Ke*, Fujioka*, Schedl, and Jaynes reports a set of experiments and subsequent analyses focusing on the role of Drosophila boundary elements in shaping 3D genome structure and regulating gene expression. The authors primarily focus on the region of the fly genome containing the even skipped (eve) gene; eve is expressed in a canonical spatial pattern in fly embryos and its locus is flanked by the well-characterized neighbor of homie (nhomie) and homie boundary elements. The main focus of investigation is the orientation dependence of these boundary elements, which had been observed previously using reporter assays. In this study, the authors use Crispr/Cas9 editing followed by recombination-mediated cassette exchange to create a series of recombinant fly lines in which the nhomie boundary element is either replaced with exongenous sequence from phage 𝝀, an inversion of nhomie, or a copy of homie that has the same orientation as the endogenous homie sequence. The nhomie sequence is also regenerated in its native orientation to control for effects introduced by the transgenesis process.

      The authors then perform high-resolution Micro-C to analyze 3D structure and couple this with fluorescent and colorimetric RNA in situ hybridization experiments to measure the expression of eve and nearby genes during different stages of fly development. The major findings of these experiments are that total loss of boundary sequence (replacement with 𝝀 DNA) results in major 3D structure changes and the most prominent observed gene changes, while inversion of the nhomie boundary or replacement with homie resulted in more modest effects in terms of 3D structure and gene expression changes and a distinct pattern of gene expression change from the 𝝀 DNA replacement. As the samples in which the nhomie boundary is inverted or replaced with homie have similar Micro-C profiles at the eve locus and show similar patterns of a spurious gene activation relative to the control, the observed effects appear to be driven by the relative orientation of the nhomie and homie boundary elements to one another.

      Collectively, the findings reported in the manuscript are of broad interest to the 3D genome field. Although extensive work has gone into characterizing the patterns of 3D genome organization in a whole host of species, the underlying mechanisms that structure genomes and their functional consequences are still poorly understood. The perhaps best understood system, mechanistically, is the coordinated action of CTCF with the cohesin complex, which in vertebrates appears to shape 3D contact maps through a loop extrusion-pausing mechanism that relies on orientation-dependent sequence elements found at the boundaries of interacting chromatin loops. Despite having a CTCF paralog and cohesin, the Drosophila genome does not appear to be structure by loop extrusion-pausing. The identification of orientation-dependent elements with pronounced structural effects on genome folding thus may shed light on alternative mechanisms used to regulated genome structure, which in turn may yield insights into the significance of particular folding patterns.

      On the whole, this study is comprehensive and represents a useful contribution to the 3D genome field. The transgenic lines and Micro-C datasets generated in the course of the work will be valuable resources for the research community. Moreover, the manuscript, while dense in places, is generally clearly written and comprehensive in its description of the work. However, I have a number of comments and critiques of the manuscript, mainly centering on the framing of the experiments and presentation of the Micro-C results and on manner in which the data are analyzed and reported. They are as follows:

      Major Points:

      1. The authors motivate much of the introduction and results with hypothetical "stem loop" and "circle loop" models of chromosome confirmation, which they argue are reflected in the Micro-C data and help to explain the observed ISH patterns. While such structures may possibly form, the support for these specific models vs. the many alternatives is not in any way justified. For instance, no consideration is given to important biophysical properties such as persistence length, packing/scaling, and conformational entropy. As the biophysical properties of chromatin are a very trafficked topic both in terms of experimentation and computational modeling and generally considered in the analysis of chromosome conformation data, the study would be strengthened by acknowledgement of this body of work and more direct integration of its findings.

      2. Similar to Point 1, while there is a fair amount of discussion of how the observed results are or are not consistent with loop extrusion, there is no discussion of the biophysical forces that are thought to underly compartmentalization such as block-polymer co-segregation and their potential influence. I found this absence surprising, as it is generally accepted that A/B compartmentalization essentially can explain the contact maps observed in Drosophila and other non-vertebrate eukaryotes (Rowley, ..., Corces 2017; PMID 28826674). The manuscript would be strengthened by consideration of this phenomenon.

      3. The contact maps presented in the study represent many cells and distinct cell types. It is clear from single-cell Hi-C and multiplexed FISH experiments that chromosome conformation is highly variable even within populations of the same cell, let alone between cell types, with structures such as TADs being entirely absent at the single cell level and only appearing upon pseudobulking. It is difficult to square these observations with the models of relatively static structures depicted here. The authors should provide commentary on this point.

      4. The analysis of the Micro-C data appears to be largely qualitative. Key information about the number of reads sequenced, reaps mapped, and data quality are not presented. No quantitative framework for identifying features such as the "plumes" is described. The study and its findings would be strengthened by a more rigorous analysis of these rich datasets, including the use of systematic thresholds for calling patterns of organization in the data.

      5. Related to Point 4, the lack of quantitative details about the Micro-C data make it difficult to evaluate if the changes observed are due to biological or technical factors. It is essential that the authors provide quantitative means of controlling for factors like sampling depth, normalization, and data quality between the samples.

      6. The ISH effects reported are modest, especially in the case of the HCR. The details provided for how the imaging data were acquired and analyzed are minimal, which makes evaluating them challenging. It would strengthen the study to provide much more detail about the acquisition and analysis and to include depiction of intermediates in the analysis process, e.g. the showing segmentation of stripes.

    1. Reviewer #2 (Public Review):

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

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

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

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

      How does intrachromosomal homolog pairing impact the models proposed in this manuscript (Abed et al. 2019; Erceg et al., 2019). Several papers recently have shown that somatic homolog pairing is not uniform and shows significant variation across the genome with evidence for both tight pairing regions and loose pairing regions. Might loose pairing interactions have the capacity to alter the cis configuration of the eve locus?<br /> In summary, the transgenic experiments are extensive and elegant and fully support the authors' models. However, in my opinion, they do not completely rule out additional models at play, including extrusion-based mechanisms. Indeed, my major issue is the limited conceptual advance in this manuscript. The authors essentially repeat many of their previous work and analyses. The authors make no attempt to dissect the mechanism of this process by modifying extrusion components directly. Some discussion of Rollins et al., 1999 on the discovery of Nipped-B and its role in enhancer-promoter communication should also be made to reconcile their conclusions in the proposed absence of extrusion events.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The manuscript by Wang and colleagues provided mechanistic insights into SCARF1 and its interactions with the lipoprotein ligands. The authors reported two crystal structures of the N-terminal fragments of SCARF1 ectodomain (ECD). On the basis of the structural analysis, the authors further investigated the interactions between SCARF1 and modified LDLs using cell-based assays and biochemical experiments. Together with the two structures and supporting data, this work provided new insights into the diverse mechanisms of scavenger receptors and especially the crucial role of SCARF1 in lipid metabolism.

      Strengths:<br /> The authors started by determining the crystal structures of two fragments of SCARF1 ECD. The superposition of the two high-resolution structures, together with the predicted model by AlphaFold, revealed that the ECD of SCARF1 adopts a long-curved conformation with multiple EGF-like domains arranged in tandem. Non-crystallographic and crystallographic two-fold symmetries were observed in crystals of f1 and f2 respectively, indicating the formation of SCARF1 homodimers. Structural analysis identified critical residues involved in dimerization, which were validated through mutational experiments. In addition, the authors conducted flow cytometry and confocal experiments to characterize cellular interactions of SCARF1 with lipoproteins. The results revealed the vital role of the 133-221aa region in the binding between SCARF1 and modified LDLs. Moreover, four arginine residues were identified as crucial for modified LDL recognition, highlighting the contribution of charge interactions in SCARF1-lipoprotein binding. The lipoprotein binding region is further validated by designing SCARF1/SCARF2 chimeric molecules. Interestingly, the interaction between SCARF1 and modified LDLs could be inhibited by teichoic acid, indicating potential overlap in or sharing of binding sites on SCARF1 ECD.

      The author employed a nice collection of techniques, namely crystallographic, SEC, DLS, flow cytometry, ELISA, and confocal imaging. The experiments are technically sound and the results are clearly written, with a few concerns as outlined below. Overall, this research represents an advancement in the mechanistic investigation of SCARF1 and its interaction with ligands. The role of scavenger receptors is critical in lipid homeostasis, making this work of interest.

    1. Reviewer #2 (Public Review):

      In bacteria and mammals, metabolically generated aldehydes become toxic at high concentrations because they irreversibly modify the free amino group of various essential biological macromolecules. However, these aldehydes can be present in extremely high amounts in archaea and plants without causing major toxic side effects. This fact suggests that archaea and plants have evolved specialized mechanisms to prevent the harmful effects of aldehyde accumulation.

      In this manuscript, the authors show that the plant enzyme DTD2, originating from archaea, functions as a D-aminoacyl-tRNA deacylase. This enzyme effectively removes stable D-aminoacyl adducts from tRNAs, enabling these molecules to be recycled for translation. Furthermore, they demonstrate that DTD2 serves as a broad detoxifier for various aldehydes in vivo, extending its function beyond acetaldehyde, as previously believed. Finally, the authors suggest a potential application of their findings by showing that the absence of DTD2 renders plants more susceptible to reactive aldehydes, while its overexpression provides protection against them.

      Overall, this study provides a molecular explanation for the remarkable efficiency of plants in handling reactive aldehydes. However, direct evidence that translation is impaired in plants lacking DTD2 experience is currently lacking. Furthermore, because root morphology of DTD2-overexpressing plants appears to differ from that of WT, a thorough phenotypic analysis of DTD2-overexpressing plants will be essential to accurately assess the potential translational application of this enzyme for engineering stress-tolerant plants.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors aimed to uncover what role, if any, the UFD1/NPL4 complex might play in the innate immune responses of the nematode C. elegans. The authors find that loss of the complex renders animals more sensitive to both pathogenic and non-pathogenic bacteria. However, there appears to be a complex interplay with known innate immune pathways since the loss of UFD1/NPL4 actually results in increased survival of animals lacking the canonical innate immune pathways.

      Strengths:<br /> The authors perform robust genetic analysis to exclude and include possible mechanisms by which the UFD1/NPL4 pathway acts in the innate immune response.

      Weaknesses:<br /> The argument that the loss of the UFD1/NPL4 complex triggers a response that mimics that of an intracellular pathogen has not been thoroughly investigated. Additionally, the finding of a role of the GATA transcription factor, ELT-2, in this response is suggestive, but experiments showing sufficiency in the context of loss of the UFD1/NPL4 complex need to be explored.

    1. Reviewer #2 (Public Review):

      In this paper, Paladini and colleagues investigate the concerted motions within the Abl kinase that control its conformational transition between the active (disassembled) and inactive (assembled state). This work follows their previously published findings that binding of the type II inhibitor, imatinib to the active site of Abl, leads to kinase core disassembly via the force imposed by the P-loop and other regions of the N-lobe on the SH3 domain. Interestingly, imatinib-induced disassembly is prevented when an allosteric inhibitor, asciminib, binds to the myristate-binding pocket. Key to asciminib and myristate binding are motions of helix I, located in the C-lobe, and thus, helix I is hypothesized to be the sensor of the imatinib-induced changes. Specifically, bending of helix I upon engagement of myristate or asciminib was postulated to be important for re-assembly of the autoinhibited Abl core, and thus, reducing the "force" with which kinase N-lobe pushes against the SH2 domain upon binding imatinib.

      The authors use NMR to measure conformational transitions in the several 15N-labeled Abl kinase constructs that display different degrees of helix I truncations. This analysis is slightly limited by the instability of the constructs that carry truncations beyond the helix I "bend". Nevertheless, it is sufficient to establish that truncation of helix I that removes its fragment, which is in contact with myristate or asciminib ligands, results in loss of the ability of helix I to impose "force" on the SH2 domain that results in kinase core disassembly, even in the presence of imatinib binding. In the absence of this force, the allosteric coupling between the helix I/SH2 and KD/SH3 interfaces is compromised. Principle component analysis is used to analyze the NMR data, and it is very clear and convincing.

      A compelling evidence in support of the proposed allosteric mechanism comes from the analysis of the E528K disease mutation, identified in the Abl1 malformation syndrome. The authors show that this mutant, poised to break a salt bridge formed between E528 in the C-terminal portion of helix I and R479 on the kinase domain, increases helix I outward motions resulting in core disassembly and higher Abl kinase activity. Together, these results reinforce that helix I motions are central to the mechanism of kinase activation via core disassembly.

    1. ZFIN: ZDB-ALT-110912-2

      DOI: 10.1016/j.neuron.2024.01.001

      Resource: ZFIN_ZDB-ALT-110912-2

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-110912-2


      What is this?

    2. ZFIN: ZDB-ALT-090116-2

      DOI: 10.1016/j.neuron.2024.01.001

      Resource: (ZFIN Cat# ZDB-ALT-090116-2,RRID:ZFIN_ZDB-ALT-090116-2)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-090116-2


      What is this?

    3. ZFIN: ZDB-ALT-141023-2

      DOI: 10.1016/j.neuron.2024.01.001

      Resource: (ZFIN Cat# ZDB-ALT-141023-2,RRID:ZFIN_ZDB-ALT-141023-2)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-141023-2


      What is this?

    1. Reviewer #2 (Public Review):

      This is an interesting followup study that uses long read sequencing to examine previously constructed mutation accumulation lines between wild populations of S. cerevisiae and S. paradoxus. They also complement this work with reporter assays in hybrid backgrounds. The authors are attempting to test the hypothesis that hybridization leads to genome shock and unrestrained transposition. The paper largely confirms previous results (suggesting hybridization does not increase transposition) that are well cited and discussed in the paper, both from this group and from the Smukowski Heil/Dunham group but extends them to a new set of species/hybrids and with some additional resolution via the long read sequencing. The paper is well written and clear and I have no serious complaints.

      In the abstract, the authors make three primary claims:

      Structural variation plays a strong role in TE load.<br /> Transposition plays only a minor role in shaping the TE landscape in MA lines.<br /> Transposition rates are not increased by hybridization but are affected by genotype specific factors.

      Comments on revised submission:

      I found all three claims supported, albeit with some minor questions. Those questions were answered by the authors in revision. I appreciate the authors revisions and feel the paper is now in better shape than upon the original submission.

    1. Reviewer #3 (Public Review):

      Summary:<br /> In this study, the researchers used ancient environmental DNA (aeDNA) retrieved from sediment cores, from two lakes in the Arctic, on the Yamal peninsula, in Siberia. The dating of one of the cores, showed that the sediment layers were very recent (ranging between the years 2019 - 1895). From this core they sequenced 23 libraries which were enriched for mammal mitochondrial genomes. They found a high proportion of two species that have been extinct for thousands of years, the mammoth and the woolly rhinoceros. The highest proportion of mammoth reads were found in very young layer (~81 years old) and as this initial finding does not match the temporal occurrence of the species, they confirmed the identification with several other methods. Additionally, they applied a different dating method on some samples and found that the aging of the samples was not completely congruent. The authors suggest the that the presence of these two Pleistocene megafauna in such recent sediment layers is a consequence of physical processes, specific to the study site, and that the high quality of the aeDNA recovered is a result of permafrost preservation.

      The strengths of the study are in the rigorous confirmation of the identification of the taxa with four different PCR and sequencing techniques being used, the initial enrichment panel, and then subsequent metabarcoding PCRs, and taxa specific PCR for COI and cytB. Along with the ancient DNA protocol applied, this is therefore very convincing that the DNA detected in the samples is indeed from the Pleistocene mammals. Additionally, two methods were used to age the sediment cores, and although the depth of the samples tested do not overlap, they give reasonable ages (apart from the anomalous sample) and all together these are robust results.

      There is now an analysis supporting the idea that there are multiple individual mammoths in the sample as well as a figure to display the locations of the haplotypes. The authors also confirm that the woolly rhinocerous did not recover enough sequences for analysis. The aims have been clarified and no longer states that they are looking at mammal biodiversity through time, so the papers focus is now more specifically on just the mammoth. But a supplementary table of the reads from common mammals has been added.

      Overall the results support that there has been some movement of DNA throughout the sediment core which may impact the dating of the last occurrence of particular extinct taxa. As highlighted, though the geological processes by which this may have arisen are specific to this particular lake and may not be broadly relevant, therefore highlighting that knowledge of each system is important to understanding DNA distribution.

    1. Reviewer #3 (Public Review):

      In this paper by Keramidioti et al, the authors have characterized a polyclonal antibody from rabbit, which was raised against a peptide of the intracellular domain of the Hydra Cadherin. This antibody unexpectedly recognizes presumably all neurons in the Hydra polyp but the specificity of the antibody was not investigated. Regardless, the antibody can be used to visualize and study the nerve net under a variety of conditions. The authors find that the endodermal and ectodermal nerve net do not make any contacts through the mesoglea, in contrast to earlier assumptions and data. They show that ectodermal neurons make close contacts to the myoepithelial muscles, in contrast to the endodermal muscles. Furthermore, they show that tentacle endoderm surprisingly does not have any neurons. Finally, a very nice tool to visualize the connections between the neurons is the staining of mosaic nGreen transgenic lines. This showed that the neurites align in parallel forming bundles of neurites over longer stretches, in particular in the ectoderm, which offers a mechanism how new neurons are added laterally to the existing nerve net. This has important implications about the way the neurons might communicate with each other.

      Taken together, this paper adds to our knowledge of the Hydra nerve net and provides a new experimental tool. Although most of the study is rather descriptive the pictures are of spectacular quality, providing fascinating new insights into the arrangement and topology of the nerve net.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Dubicka et al. in their paper entitled " Biocalcification in porcelaneous foraminifera" suggest that in contrast to the traditionally claimed two different modes of test calcification by rotallid and porcelaneous miliolid formaminifera, both groups produce calcareous tests via the intravesicular mineral precursors (Mg-rich amorphous calcium carbonate). These precursors are proposed to be supplied by endocytosed seawater and deposited in situ as mesocrystals formed at the site of new wall formation within the organic matrix. The authors did not observe the calcification of the needles within the transported vesicles, which challenges the previous model of miliolid mineralization. Although the authors argue that these two groups of foraminifera utilize the same calcification mechanism, they also suggest that these calcification pathways evolved independently in the Paleozoic.

      Strengths:<br /> The authors document various unknown aspects of calcification of Pseudolachlanella eburnea and elucidate some poorly explained phenomena (e.g., translucent properties of the freshly formed test) however there are several problematic observations/interpretations which in my opinion should be carefully addressed.

      Weaknesses:<br /> 1. The authors (line 122) suggest that "characteristic autofluorescence indicates the carbonate content of the vesicles (Fig. S2), which are considered to be Mg-ACCs (amorphous MgCaCO3) (Fig. 2, Movies S4 and S5)". Figure S2 which the authors refer to shows only broken sections of organic sheath at different stages of mineralization. Movie S4 shows that only in a few regions some vesicles exhibit red autofluorescence interpreted as Mg-ACC (S5 is missing but probably the authors were referring to S3). In their previous paper (Dubicka et al 2023: Heliyon), the authors used exactly the same methodology to suggest that these are intracellularly formed Mg-rich amorphous calcium carbonate particles that transform into a stable mineral phase in rotaliid Aphistegina lessonii. However, in Figure 1D (Dubicka et al 2023) the apparently carbonate-loaded vesicles show the same red autofluorescence as the test, whereas in their current paper, no evidence of autofluorescence of Mg-ACC grains accumulated within the "gel-like" organic matrix is given. The S3 and S4 movies show circulation of various fluorescing components, but no initial phase of test formation is observable (numerous mineral grains embedded within the organic matrix - Figures 3A and B - should be clearly observed also as autofluorescence of the whole layer). Thus the crucial argument supporting the calcification model (Figure 5) is missing. There is no support for the following interpretation (lines 199-203) "The existence of intracellular, vesicular intermediate amorphous phase (Mg-ACC pools), which supply successive doses of carbonate material to shell production, was supported by autofluorescence (excitation at 405 nm; Fig. 2; Movies S3 and S4; see Dubicka et al., 2023) and a high content of Ca and Mg quantified from the area of cytoplasm by SEM-EDS analysis (Fig. S6)."

      2. The authors suggest that "no organic matter was detected between the needles of the porcelain structures (Figures 3E; 3E; S4C, and S5A)". Such a suggestion, which is highly unusual considering that biogenic minerals almost by definition contain various organic components, was made based only on FE-SEM observation. The authors should either provide clearcut evidence of the lack of organic matter (unlikely) or may suggest that intense calcium carbonate precipitation within organic matrix gel ultimately results in a decrease of the amount of the organic phase (but not its complete elimination), alike the pure calcium carbonate crystals are separated from the remaining liquid with impurities ("mother liquor"). On the other hand, if (249-250) "organic matrix involved in the biomineralization of foraminiferal shells may contain collagen-like networks", such "laminar" organization of the organic matrix may partly explain the arrangement of carbonate fibers parallel to the surface as observed in Fig. 3E1.

      3. The author's observations indeed do not show the formation of individual skeletal crystallites within intracellular vesicles, however, do not explain either what is the structure of individual skeletal crystallites and how they are formed. Especially, what are the structures observed in polarized light (and interpreted as calcite crystallites) by De Nooijer et al. 2009? The author's explanation of the process (lines 213-216) is not particularly convincing "we suspect that the OM was removed from the test wall and recycled by the cell itself".

      4. The following passage (lines 296-304) which deals with the concept of mesocrystals is not supported by the authors' methodology or observations. The authors state that miliolid needles "assembled with calcite nanoparticles, are unique examples of biogenic mesocrystals (see Cölfen and Antonietti, 2005), forming distinct geometric shapes limited by planar crystalline faces" (later in the same passage the authors say that "mesocrystals are common biogenic components in the skeletons of marine organisms" (are they thus unique or are they common)? It is my suggestion to completely eliminate this concept here until various crystallographic details of the miliolid test formation are well documented.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Cheng et al. explore the development of the arteries that form the Circle of Willis and investigate how blood flow pulsatility influences vascular smooth muscle cell (VSMC) differentiation. Using live confocal imaging of the developing zebrafish, the authors show that endothelial cells in the Circle of Willis arteries transition from venous to arterial identity between 54 hours post-fertilization (hpf) and 3 days post-fertilization (dpf), and that this coincides with pdgfrb+ mural cell progenitor differentiation into acta2+ arterial VSMCs. They find that the anterior portions of the Circle of Willis, including the internal carotid arteries (CaDI), establish acta2 expression earlier than posterior aspects, likely due to faster flow rate and increased pulsatility through the CaDI. Then, using computational fluid dynamics, an in vitro co-culture assay, and genetic and drug manipulations of blood flow, the authors provide evidence that pdgfrb+ differentiation is dependent upon pulsatile blood flow and klf2a activation. The results add to our understanding of vascular development and suggest that deficits in pulsatile flow could be potential drivers of arteriopathies.

      Strengths:<br /> 1) Longitudinal confocal imaging of live developing zebrafish makes the timeline of arterial development in the Circle of Willis easy to understand. This is a strong approach to studying how vascular networks are altered with genetic and pharmacological manipulations.

      2) Rigorous use of multiple techniques to test the hypothesis that pulsatile blood flow is required for smooth muscle cell differentiation. The microangiography experiment, in vitro co-culture assay, and genetic and drug manipulations of heart rate at various developmental time points yield outcomes that are consistent with the hypothesis.

    1. Reviewer #2 (Public Review):

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

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

      Weaknesses:<br /> The multi-component systems described here function in the context of large oligomeric complexes. Some of the single chain AF2 predictions shown in this work are not compatible, for example, with homohexameric complex formation due to incompatible orientation of domains. The recent advances in protein structure prediction, in particular AlphaFold2 (AF2) multimer, now allow us to confidently probe potential protein-protein interactions and protein complex formation. This predictive power could be exploited here to produce a better glimpse of these multimeric protein systems. It can also provide a more sound explanation for some of the observed differences amongst different McrBC types.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors describe a novel ML approach to predict binding between MHC-bound peptides and T-Cell receptors. Such approaches are particularly useful for predicting the binding of peptide sequences with low similarity when compared to existing data sets. The authors focus on improving dataset quality and optimizing model architecture to achieve a pan-specific predictive model in hopes of achieving a high precision model for novel peptide sequences.

      Strengths:<br /> Since assuring the quality of training datasets is the first major step in any ML training project, the extensive human curation and computational analysis and enhancements made in this manuscript represent a major contribution to the field. Moreover, the systematic approach to testing redundancy reduction and data augmentation is exemplary, and will significantly help future research in the field.

      The authors also highlight how their model can identify outliers and how that can be used to improve the model around known sequences, which can help the creation and optimization of future datasets for peptide binding.

      The new models presented here are novel and built using paired α/β TCR sequence data to predict peptide-specific TCR binding, and have been extensively and rigorously tested.

      Weaknesses:<br /> Achieving an accurate pan-specific model is an ambitious goal, and the authors have significant difficulties when trying to achieve non-random performance for prediction of TCR binding to novel peptides. This is the most challenging task for this kind of model, but also the most desirable when applying such models to biotechnological and bioengineering projects.

      The manuscript is a highly technical and extremely detailed computational work, which can make the achievements and impact of the work hard to parse for application-oriented researchers, and still hard to translate to real-world use-cases for TCR specificity predictions.

    1. Reviewer #2 (Public Review):

      Summary:

      Langenbacher at el. examine the requirement of Rtf1, a component of the PAF1C, which regulates transcriptional pausing in cardiac development. The authors first confirm their previous morphant study with newly generated rtf1 mutant alleles, which recapitulate the defects in cardiac progenitor and differentiation gene expression observed previously in morphants. They then examine the conservation of Rtf1 in mouse embryos and embryonic stem cell-derived cardiomyocytes. Conditional loss of Rtf1 in mesodermal lineages and depletion in murine ESCs demonstrates a failure to turn on cardiac progenitor and differentiation marker genes, supporting conservation of Rtf1 in promoting cardiac development. The authors subsequently employ bulk RNA-seq on flow-sorted hand2:GFP+ cells and multiomic single-cell RNA-seq on whole Rtf1-depleted embryos at the 10-12 stage. These experiments corroborate that genes associated with cardiac and muscle development are lost. Furthermore, the differentiation trajectories suggest that the expression of genes associated with cardiac maturation is not initiated. Structure-function analysis supports that the Plus3 domain is necessary for its function in promoting cardiac progenitor formation. ChIP-seq for RNA Pol II on 10-12 somite stage embryos suggests that Rtf1 is required for proper promoter pausing. This defect can partially be rescued through use of a pharmacological inhibitor for Cdk9, which inhibits elongation, can partially restore elongation in rtf1 mutants.

      Strengths:

      Many aspects of the data are strong, which support the basic conclusions of the authors that Rtf1 is required for transcriptional pausing and has a conserved requirement in vertebrate cardiac development. Areas of strength include the genetic data supporting the conserved requirement for Rtf1 in promoting cardiac development, the complementary bulk and single-cell RNA-sequencing approaches providing some insight into the gene expression changes of the cardiac progenitors, the structure-function analysis supporting the requirement of the Plus3 domain, and the pharmacological epistasis combined with the RNA Pol II ChIP-seq, supporting the mechanism implicating Cdk9 in the Rtf1 dependent mechanism of RNA Pol II pausing.

      Weaknesses:

      While most of the basic conclusions are supported by the data, there are a number of analyses that are confusing as to why they chose to perform the experiments the way they did and some places where the interpretations presently do not support the interpretations. One of the conclusions is that the phenotype affects the maturation of the cardiomyocytes and they are arresting in an immature state. However, this seems to be mostly derived from picking a few candidates from the single cell data in Fig. 6. If that were the case, wouldn't the expectation be to observe relatively normal expression of earlier marker genes required for specification, such as Nkx2.5 and Gata5/6? The in situ expression analysis from fish and mice (Fig. 2 and Fig. 3) and bulk RNA-seq (Fig. 5) seems to suggest that there are pretty early specification and differentiation defects. While some genes associated with cardiac development are not changed, many of these are not specific to cardiomyocyte progenitors and expressed broadly throughout the ALPM. Similarly, it is not clear why a consistent set of cardiac progenitor genes (for instance mef2ca, nkx2.5, and tbx20) was analyzed for all the experiments, in particular with the single cell analysis.

      The point of the multiomic analysis is confusing. RNA- and ATAC-seq were apparently done at the same time. Yet, the focus of the analysis that is presented is on a small part of the RNA-seq data. This data set could have been more thoroughly analyzed, particularly in light of how chromatin changes may be associated with the transcriptional pausing. This seems to be a lost opportunity. Additionally, how the single cell data is covered in Supplemental Fig. 2 and 3 is confusing. There is no indication of what the different clusters are in the Figure or the legend.

      While the effect of Rtf1 loss on cardiomyocyte markers is certainly dramatic, it is not clear how well the mutant fish have been analyzed and how specific the effect is to this population. It is interpreted that the effects on cardiomyocytes are not due to "transfating" of other cell fates, yet supplemental Fig. 4 shows numerous effects on potentially adjacent cell populations. Minimally, additional data needs to be provided showing the live fish at these stages and marker analysis to support these statements. In some images, it is not clear the embryos are the same stage (one can see pigmentation in the eyes of controls that is not in the mutants/morphants), causing some concern about developmental delay in the mutants.

      With respect to the transcriptional pausing defects in the Rtf1 deficient embryos, it is not clear from the data how this effect relates to the expression of the cardiac markers. This could have been directly analyzed with some additional sequencing, such as PRO-seq, which would provide a direct analysis of transcriptional elongation.

      Some additional minor issues include the rationale that sequence conservation suggests an important requirement of a gene (line 137), which there are many examples this isn't the case, referencing figures panels out of order in Figs. 4, 7, and 8) as described in the text, and using the morphants for some experiments, such as the rescue, that could have been done in a blinded manner with the mutants.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The widely distributed pannexin 1 (PANX1) is an ATP-permeable channel that plays an important role in intercellular communication and has been implicated in various pathophysiological processes and diseases. Previous studies have demonstrated that PANX1 can be phosphorylated at two molecular sites via the non-receptor kinase Src, thereby leading to channel opening and ATP release. In this paper, the authors used a variety of methods to detect tyrosine phosphorylation modification of PANX1 channel protein, however, their results showed that commercially available antibodies against the two phosphorylation sites used in previous studies did not work well, in other words, phosphorylation changes in PANX1 could not be detected by those antibodies. Therefore, the authors call for the re-examination and evaluation of previous research results.

      Strengths:<br /> In general, this is a meticulous study, using different detection methods and different expression systems.

    1. Reviewer #2 (Public Review):

      The mechanism of microtubule formation, stabilization, and organization in neurites is important for neuronal function. In this manuscript, the authors examine the phenotype of neurons following alteration in the level of the protein HMMR, a microtubule-associated protein with established roles in mitosis. Neurite morphology is measured as well as microtubule stability and dynamic parameters using standard assays. A binding partner of HMMR, TPX2, is localized. The results support a role for HMMR in neurons.

      The work presented in this manuscript seeks to determine if a MAP called HMMR contributes to microtubule dynamics in neurons. Several steps, including validation of the RNAi, additional statistical analysis, use of cells at the same age in culture, and better documentation in figures, would increase the impact of the work.

      In many places, the data can be improved which might make the story more convincing. As presented, the results show that HMMR is distributed as puncta on neurons with data coming from a single HMMR antibody, and some background staining that was not discussed. In the discussion the authors state that HMMR impacts microtubule stability, which was evaluated by the presence of post-translational modification and resistance to nocodazole; the data are suggestive but not entirely convincing. The discussion also states that HMMR increases the "amount" of growing microtubules which was measured as the frequency of comet appearance. The authors did not comment on how the number of growing microtubules results in the observed morphological changes.

    1. Reviewer #2 (Public Review):

      The authors provide evidence for the early events of the lipopeptide daptomycin inserting into bacterial membranes. The authors utilize several biochemical and biophysical methods to characterize the nature of daptomycin interactions with a diverse set of phospholipids. The authors found that daptomycin, when complexed with calcium ions, can transiently interact with the headgroups of anionic phospholipids. In particular, the authors found that daptomycin rapidly interacts with the headgroup of cardiolipin and that this interaction is reversible and dependent on calcium. The authors provide evidence supporting previously published data that daptomycin interacts with phosphatidylglycerol (PG) with high affinity in a 1:2 ratio. The authors showed that this interaction includes both a calcium-dependent headgroup interaction (denoted the pre-insertion complex) and a distinct, irreversible interaction that is likely occurring between the hydrophobic tail of daptomycin with the tails of the PG molecules (denoted the quaternary complex of daptomycin, calcium, and 2 PG). The authors also isolated a daptomycin-containing complex from Bacillus subtilis cells following exposure to daptomycin and calcium. PG was identified from the isolated complex, albeit with a different acyl chain length from that used in vitro. Taken together, these data deepen our understanding of the stages of daptomycin interaction and intercalation in a membrane and can contribute to translational research on the development of structural analogs that could augment the efficacy of daptomycin treatment.

      The authors have provided sufficient evidence to support a very specific interaction between daptomycin and PG, but their conclusions drawn from the data are exceedingly broad. In particular, the role of lipid II and lipid II precursors in the insertion and flipping events of daptomycin in the membrane are only briefly addressed despite the recently described pivotal role assigned to lipid II in the formation of a membrane-active daptomycin complex (Grein et al. Nature Communications 2020). While the authors put forth an intriguing and probable hypothesis that there are potentially multiple complexes and conformations of daptomycin as it incorporates within the membrane, the strength of the study's results and conclusions lies in its examination of the early headgroup interactions and distinctive PG interaction rather than the later events of daptomycin insertion in the membrane. The in vivo data presented supports the authors' model, but the conclusions do not address critical differences between the two very different systems i.e., in the behavior of micelles versus cell bilayer membranes.

    1. Reviewer #2 (Public Review):

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

    1. Reviewer 2 (Public Review):

      With the data presented in this manuscript, the authors help complete the set of high resolution HER2- associated complex heterodimer structures as well as HER4 homodimer structures in the presence of NRG1b and BTC. Purification of HER2-HER4 heterodimers appears to be inherently challenging due to the propensity of HER4 to form homodimers. The authors have used an effective scheme to isolate these HER2-HER4 heterodimers and have employed graphene-oxide grid chemistry to presumably overcome the issues of low sample yield for solving cryo-EM structures of these complexes. The authors conclude HER2-HER4 heterodimers with either ligand is conformationally homogeneous relative to the HER4 homodimers. The HER2-HER4 heterodimers also appear to be better stabilized compared to other published HER2 heterodimers. The ability to model glycans in the context of HER4 homodimers is exciting to see and provides a strong rationale for the stability of these structures. Overall, the work is of great interest and the methods described in this work would benefit a wide variety of structural biology projects.

    1. Reviewer #2 (Public Review):

      Li et al present a method to extract "behaviorally relevant" signals from neural activity. The method is meant to solve a problem which likely has high utility for neuroscience researchers. There are numerous existing methods to achieve this goal some of which the authors compare their method to-thankfully, the revised version includes one of the major previous omissions (TNDM). However, I still believe that d-VAE is a promising approach that has its own advantages. Still, I have issues with the paper as-is. The authors have made relatively few modifications to the text based on my previous comments, and the responses have largely just dismissed my feedback and restated claims from the paper. Nearly all of my previous comments remain relevant for this revised manuscript. As such, they have done little to assuage my concerns, the most important of which I will restate here using the labels/notation (Q1, Q2, etc) from the reviewer response.

      Q1) I still remain unconvinced that the core findings of the paper are "unexpected". In the response to my previous Specific Comment #1, they say "We use the term 'unexpected' due to the disparity between our findings and the prior understanding concerning neural encoding and decoding." However, they provide no citations or grounding for why they make those claims. What prior understanding makes it unexpected that encoding is more complex than decoding given the entropy, sparseness, and high dimensionality of neural signals (the "encoding") compared to the smoothness and low dimensionality of typical behavioural signals (the "decoding")?

      Q2) I still take issue with the premise that signals in the brain are "irrelevant" simply because they do not correlate with a fixed temporal lag with a particular behavioural feature hand-chosen by the experimenter. In the response to my previous review, the authors say "we employ terms like 'behaviorally-relevant' and 'behaviorally-irrelevant' only regarding behavioral variables of interest measured within a given task, such as arm kinematics during a motor control task.". This is just a restatement of their definition, not a response to my concern, and does not address my concern that the method requires a fixed temporal lag and continual decoding/encoding. My example of reward signals remains. There is a huge body of literature dating back to the 70s on the linear relationships between neural and activity and arm kinematics; in a sense, the authors have chosen the "variable of interest" that proves their point. This all ties back to the previous comment: this is mostly expected, not unexpected, when relating apparently-stochastic, discrete action potential events to smoothly varying limb kinematics.

      Q5) The authors seem to have missed the spirit of my critique: to say "linear readout is performed in motor cortex" is an over-interpretation of what their model can show.

      Q7) Agreeing with my critique is not sufficient; please provide the data or simulations that provides the context for the reference in the fano factor. I believe my critique is still valid.

      Q8) Thank you for comparing to TNDM, it's a useful benchmark.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The dominant paradigm in the past decade for modeling the ventral visual stream's response to images has been to train deep neural networks on object classification tasks and regress neural responses from units of these networks. While object classification performance is correlated to the variance explained in the neural data, this approach has recently hit a plateau of variance explained, beyond which increases in classification performance do not yield improvements in neural predictivity. This suggests that classification performance may not be a sufficient objective for building better models of the ventral stream. Lindsey & Issa study the role of factorization in predicting neural responses to images, where factorization is the degree to which variables such as object pose and lighting are represented independently in orthogonal subspaces. They propose factorization as a candidate objective for breaking through the plateau suffered by models trained only on object classification. They claim that (i) maintaining these non-class variables in a factorized manner yields better neural predictivity than ignoring non-class information entirely, and (ii) factorization may be a representational strategy used by the brain.

      The first of these claims is supported by their data. The second claim does not seem well-supported, and the usefulness of their observations is not entirely clear.

      Strengths:<br /> This paper challenges the dominant approach to modeling neural responses in the ventral stream, which itself is valuable for diversifying the space of ideas.

      This paper uses a wide variety of datasets, spanning multiple brain areas and species. The results are consistent across the datasets, which is a great sign of robustness.

      The paper uses a large set of models from many prior works. This is impressively thorough and rigorous.

      The authors are very transparent, particularly in the supplementary material, showing results on all datasets. This is excellent practice.

      Weaknesses:<br /> 1. The primary weakness of this paper is a lack of clarity about what exactly is the contribution. I see two main interpretations: (1-A) As introducing a heuristic for predicting neural responses that improve over-classification accuracy, and (1-B) as a model of the brain's representational strategy. These two interpretations are distinct goals, each of which is valuable. However, I don't think the paper in its current form supports either of them very well:

      (1-A) Heuristic for neural predictivity. The claim here is that by optimizing for factorization, we could improve models' neural predictivity to break through the current predictivity plateau. To frame the paper in this way, the key contribution should be a new heuristic that correlates with neural predictivity better than classification accuracy. The paper currently does not do this. The main piece of evidence that factorization may yield a more useful heuristic than classification accuracy alone comes from Figure 5. However, in Figure 5 it seems that factorization along some factors is more useful than others, and different linear combinations of factorization and classification may be best for different data. There is no single heuristic presented and defended. If the authors want to frame this paper as a new heuristic for neural predictivity, I recommend the authors present and defend a specific heuristic that others can use, e.g. [K * factorization_of_pose + classification] for some constant K, and show that (i) this correlates with neural predictivity better than classification alone, and (ii) this can be used to build models with higher neural predictivity. For (ii), they could fine-tune a state-of-the-art model to improve this heuristic and show that doing so achieves a new state-of-the-art neural predictivity. That would be convincing evidence that their contribution is useful.

      (1-B) Model of representation in the brain. The claim here is that factorization is a general principle of representation in the brain. However, neural predictivity is not a suitable metric for this, because (i) neural predictivity allows arbitrary linear decoders, hence is invariant to the orthogonality requirement of factorization, and (ii) neural predictivity does not match the network representation to the brain representation. A better metric is representational dissimilarity matrices. However, the RDM results in Figure S4 actually seem to show that factorization does not do a very good job of predicting neural similarity (though the comparison to classification accuracy is not shown), which suggests that factorization may not be a general principle of the brain. If the authors want to frame the paper in terms of discovering a general principle of the brain, I suggest they use a metric (or suite of metrics) of brain similarity that is sensitive to the desiderata of factorization, e.g. doesn't apply arbitrary linear transformations, and compare to classification accuracy in addition to invariance.

      Overall, I suggest the authors clarify exactly what their claim is, then focus on that claim and present results to justify it. If neither of the claims above can be supported by evidence, then this paper still has value as an idea that they spent effort trying to test, but they should not suggest these claims in the paper. In that case, it may also be possible to increase the value of the contribution by characterizing how the structure of class-free variable representations impacts correlation with neural fit, instead of just comparing existence vs absence (invariance) of this information. For example, evaluate the degree to which local or global orthogonality matters, or the degree to which curvature of the embedding matters.

      2. I think the comparison to invariance, which is pervasive throughout the paper, is not very informative. First, it is not surprising that invariance is more weakly correlated with neural predictivity than factorization, because invariant representations lose information compared to factorized representations. Second, there has long been extensive evidence that responses throughout the ventral stream are not invariant to the factors the authors consider, so we already knew that invariance is not a good characterization of ventral stream data.

      3. The formalization of the factorization metric is not particularly elegant, because it relies on computing top K principal components for the other-parameter space, where K is arbitrarily chosen as 10. While the authors do show that in their datasets the results are not very sensitive to K (Figure S5), that is not guaranteed to be the case in general. I suggest the authors try to come up with a formalization that doesn't have arbitrary constants. For example, one possibility that comes to mind is E[delta_a x delta_b], where 'x' is the normalized cross product, delta_a, and delta_b are deltas in representation space induced by perturbations of factors a and b, and the expectation is taken over all base points and deltas. This is just the first thing that comes to mind, and I'm sure the authors can come up with something better. The literature on disentangling metrics in machine learning may be useful for ideas on measuring factorization.

      4. The authors defined the term "factorization" according to their metric. I think introducing this new term is not necessary and can be confusing because the term "factorization" is vague and used by different researchers in different ways. Perhaps a better term is "orthogonality", because that is clear and seems to be what the authors' metric is measuring.

      5. One general weakness of the factorization paradigm is the reliance on a choice of factors. This is a subjective choice and becomes an issue as you scale to more complex images where the choice of factors is not obvious. While this choice of factors cannot be avoided, I suggest the authors add two things: First, an analysis of how sensitive the results are to the choice of factors (e.g. transform the basis set of factors and re-run the metric); second, include some discussion about how factors may be chosen in general (e.g. based on temporal statistics of the world, independent components analysis, or something else).

    1. Reviewer #2 (Public Review):

      High-resolution functional magnetic resonance imaging (fMRI) at ultra-high magnetic field strengths (7 T and above) can potentially study cortical functioning at the mesoscopic scale, i.e., at the spatial scale of cortical columns and layers. The authors of the study entitled "Mesoscale functional organization and connectivity of color, disparity, and naturalistic texture in human second visual area" remarkably show the current possibilities of high-resolution fMRI methods by studying the columnar and laminar organization for the processing of color, binocular disparity, and naturalistic texture in human secondary visual cortex (V2).

      The study could robustly show color-selective and disparity-selective stripes in human V2. While this was already demonstrated in several in vivo studies using fMRI (Nasr et al., 2016, J Neurosci, 36, 1841-1857; Dumoulin et al., 2017, Sci Rep, 7, 733; Tootell et al., 2021, Cereb Cortex, 31, 1163-1181; Navarro et al., 2021, NeuroImage, 225, 117520; Kennedy et al., 2023, Prog Neurobiol, 220, 102374; Haenelt et al., 2023, eLife, 12, e78756), the strength, in my opinion, of the current study is three-fold:

      1. Previous studies mainly focused on the columnar architecture of the stripe architecture in V2, neglecting any information across cortical depth. This study included a laminar analysis, which showcases the current possibilities of high-resolution fMRI methods that target the cortical local circuitry at the mesoscopic level.

      2. The successful mapping of color-selective and disparity-selective stripes in V2 was corroborated by an innovative connectivity analysis, which shows the expected higher connectivity of color-selective clusters in V2 with area V4 and binocular disparity with area V3ab.

      3. Furthermore, in addition to color-selective and disparity-selective stripes in V2 that were already shown in several studies at the columnar level (but without a laminar analysis), this study included naturalistic textures and analyzed the mesoscopic processing in V2. As expected, they showed greater sensitivity for texture selectivity in higher-order areas such as V4 and V3ab. In addition, due to the laminar analysis, feedforward and feedback connectivity were shown to be differentiable, demonstrating that feedback processes from higher-order areas rather drive texture processing in V2.

      Overall, the study shows interesting results that are valuable for the general neuroscientific community. In addition, the manuscript is understandable and clearly written.

      However, a few points might be worth discussing:

      1. In lines 162-163, it is stated that no clear columnar organization exists for naturalistic texture processing in V2. In my opinion, this should be rephrased. As far as I understand, Figure 2B refers to the analysis used to support the conclusion. The left and middle bar plots only show a circular analysis since ROIs were based on the color and disparity contrast used to define thin and thick stripes. The interesting graph is the right plot, which shows no statistically significant overlap of texture processing with thin, thick, and pale stripe ROIs. It should be pointed out that this analysis does not dismiss a columnar organization per se but instead only supports the conclusion of no coincidence with the CO-stripe architecture.

      2. In Figure 3, cortical depth-dependent analyses are presented for color, disparity, and texture processing. I acknowledge that the authors took care of venous effects by excluding outlier voxels. However, the GE-BOLD signal at high magnetic fields is still biased to extravascular contributions from around larger veins. Therefore, the highest color selectivity in superficial layers might also result from the bias to draining veins and might not be of neuronal origin. Furthermore, it is interesting that cortical profiles with the highest selectivity in superficial layers show overall higher selectivity across cortical depth. Could the missing increase toward the pial surface in other profiles result from the ROI definition or overall smaller signal changes (effect size) of selected voxels? At least, a more careful interpretation and discussion would be helpful for the reader.

      3. I was slightly surprised that no retinotopy data was acquired. The ROI definition in the manuscript was based on a retinotopy atlas plus manual stripe segmentation of single columns. Both steps have disadvantages because they neglect individual differences and are based on subjective assessment. A few points might be worth discussing: (1) In lines 467-468, the authors state that V2 was defined based on the extent of stripes. This classical definition of area V2 was questioned by a recent publication (Nasr et al., 2016, J Neurosci, 36, 1841-1857), which showed that stripes might extend into V3. Could this have been a problem in the present analysis, e.g., in the connectivity analysis? (2) The manual segmentation depends on the chosen threshold value, which is inevitably arbitrary. Which value was used?

      4. The use of 1-mm isotropic voxels is relatively coarse for cortical depth-dependent analyses, especially in the early visual cortex, which is highly convoluted and has a small cortical thickness. For example, most layer-fMRI studies use a voxel size of around isotropic 0.8 mm, which has half the voxel volume of 1 mm isotropic voxels. With increasing voxel volume, partial volume effects become more pronounced. For example, partial volume with CSF might confound the analysis by introducing pulsatility effects.

      5. The SVM analysis included a feature selection step stated in lines 531-533. Although this step is reasonable for the training of a machine learning classifier, it would be interesting to know if the authors think this step could have reintroduced some bias to remaining draining vein contributions.

    1. Reviewer #2 (Public Review):

      Summary:<br /> According to the sensory recruitment model, the contents of working memory (WM) are maintained by activity in the same sensory cortical regions responsible for processing perceptual inputs. A strong version of the sensory recruitment model predicts that stimulus-specific activity patterns measured in sensory brain areas during WM storage should be identical to those measured during perceptual processing. Previous research casts doubt on this hypothesis, but little is known about how stimulus-specific activity patterns during perception and memory differ. Through clever experimental design and rigorous analyses, Duan & Curtis convincingly demonstrate that stimulus-specific representations of remembered items are highly abstracted versions of representations measured during perceptual processing and that these abstracted representations are immune to aperture biases that contribute to fMRI feature decoding. The paper provides converging evidence that neural states responsible for representing information during perception and WM are fundamentally different, and provides a potential explanation for this difference.

      Strengths:<br /> 1. The generation of stimuli with matching vs. orthogonal orientations and aperture biases is clever and sets up a straightforward test regarding whether and how aperture biases contribute to orientation decoding during perception and WM. The demonstration that orientation decoding during perception is driven primarily by aperture bias while during WM it is driven primarily by orientation is compelling.

      2. The paper suggests a reason why orientation decoding during WM might be immune to aperture biases: by weighting multivoxel patterns measured during WM storage by spatial population receptive field estimates from a different task the authors show that remembered - but not actively viewed - orientations form "line-like" patterns in retinotopic cortical space.

      Weaknesses:<br /> 1. The paper tests a strong version of the sensory recruitment model, where neural states representing information during WM are presumed to be identical to neural states representing the same information during perceptual processing. As the paper acknowledges, there is already ample reason to doubt this prediction (see, e.g., earlier work by Kok & de Lange, Curr Biol 2014; Bloem et al., Psych Sci, 2018; Rademaker et al., Nat Neurosci, 2019; among others). Still, the demonstration that orientation decoding during WM is immune to aperture biases known to drive orientation decoding during perception makes for a compelling demonstration.

      2. Earlier work by the same group has reported line-like representations of orientations during memory storage but not during perception (e.g., Kwak & Curtis, Neuron, 2022). It's nice to see that result replicated during explicit perceptual and WM tasks in the current study, but I question whether the findings provide fundamental new insights into the neural bases of WM. That would require a model or explanation describing how stimulus-specific activation patterns measured during perception are transformed into the "line-like" patterns seen during WM, which the authors acknowledge is an important goal for future research.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Pisanski and colleagues map regions of the brainstem that produce the rhythm for active expiratory breathing movements and influence their motor patterns. While the neural origins of inspiration are very well understood, the neural bases for expiration lag considerably. The problem is important and new knowledge pertaining to the neural origins of expiration is welcome.

      The authors perturb the parafacial lateral (pFL) respiratory group of the brainstem with microinjection of bicuculline, to elucidate how disinhibition in specific locations of the pFL influences active expiration (and breathing in general) in anesthetized rats. They provide valuable, if not definitive, evidence that the borders of the pFL appear to extend more rostrally than previously appreciated. Prior research suggests that the expiratory pFL exists at the caudal pole of the facial cranial nucleus (VIIc). Here, the authors show that its borders probably extend as much as 1 mm rostral to VIIc. The evidence is convincing albeit with caveats.

      Strengths:<br /> The authors achieve their aim in terms of showing that the borders of the expiratory pFL are not well understood at present and that it (the pFL) extends more rostrally. The results support that point. The data are strong enough to cause many respiratory neurobiologists to look at the sites rostral to the VIIc for expiratory rhythmogenic neurons and characterize their properties and mechanisms. At present my view is that most respiratory neurobiologists overlook the regions rostral to VIIc in their studies of expiratory rhythm and pattern.

      Weaknesses:<br /> The injection of bicuculline has indiscriminate effects on excitatory and inhibitory neurons, and the parafacial region is populated by excitatory neurons that are expiratory rhythmogenic and GABA and glycinergic neurons whose roles in producing active expiration are contradictory (Flor et al. J Physiol, 2020, DOI: 10.1113/JP280243). It remains unclear how the microinjections of bicuculline differentially affect all three populations. A more selective approach would be able to disinhibit the populations separately. Nevertheless, for the main point at hand, the data do suggest that we should reconsider the borders of the expiratory pFL nucleus and begin to examine its physiology up to 1 mm rostral to VIIc.

      The control experiment showed that bicuculline microinjections induced cFos expression in the pFL, which is good, but again we don't know which neurons were disinhibited: glutamatergic, GABAergic, or glycinergic.

      The manuscript characterizes how bicuculline microinjections affect breathing parameters such as tidal volume, frequency, ventilation, inspiratory and expiratory time, as well as oxygen consumption. Those aspects of the manuscript are a bit tedious and sometimes overanalyzed. Plus, there was no predictive framework established at the outset for how one should expect disinhibition to affect breathing parameters. In other words, if the authors are seeking to map the pFL borders, then why analyze the breathing patterns so much? Does doing so provide more insight into the borders of pFL? I did not think it was compellingly argued.

      Further, lines 382-386 make a point about decreasing inspiratory time even though the data do not meet the statistical threshold.

      In lines 386-395, the reporting appears to reach significance (line 388) but not reach significance (line 389). I had trouble making sense of that disparity.

      The other statistical hiccups include "tended towards significance" (line 454), "were found to only reach significance for a short portion of the response" (line 486-7), "did not reach the level of significance" (line 506), which gives one the sense of cherry picking or over-analysis. Frankly, this reviewer finds the paper much more compelling when just asking whether the microinjections evoke active expiration. If yes, then the site is probably part of the pFL.

      I encourage the authors to consider the fickleness of p-values in general and urge them to consider not just p but also effect size.

    1. Reviewer #2 (Public Review)

      Summary:<br /> This paper by Maddox et al. presents the results of a study of Ca channel function in mouse cone photoreceptor synaptic terminals. It builds on earlier work by the same authors (Maddox et al. 2020 in eLife) which demonstrated that a non-conducting but voltage-sensing variant of Cav1.4 (G369i knock-in, or KI) could substitute for WT Cav1.4 to promote relatively normal rod synapse development despite an inability to support Ca2+-dependent glutamatergic transmission to postsynaptic bipolar cells. Cav1.4 knock-out (KO) rod synapses, however, were completely disorganized, indicating that the presence of Cav1.4 protein is critical for synaptic organization. Here, the authors extend their study of the G369i-KI retina to demonstrate that G369i-KI cones develop working (though disrupted and sometimes aberrant) synapses that support some visual function owing to compensatory expression of Cav3-containing Ca channels that can mediate some Ca2+-dependent transmission from cones to postsynaptic cells. This compensatory expression of a low voltage-activated Ca conductance was not noted previously (Maddox et al. 2020) in G369i-KI rods.

      Strengths:<br /> In all, this is a scientifically sound study that shows obvious differences between synaptic terminal morphology and organization, macroscopic Ca currents, transmission to postsynaptic horizontal and bipolar cells (with whole-cell recording and ERG, respectively), and visually-guided behavior in experimental groups.

      Weaknesses:<br /> The major criticism that I have of the study is that it infers Ca channel molecular composition based solely on pharmacological analysis, which, as the authors note, is confounded by the cross-reactivity of many of the "specific" channel-type antagonists. The authors note that Cav3 mRNAs have been found in cones, but here, they do not perform any analysis to examine Cav3 transcript expression after G369i-KI nor do they examine Ca channel transcript expression in monkey or squirrel cones, which serve as controls of sorts for the G369i-KI (i.e. like WT mouse cones, cones of these other species do not seem to exhibit LVA Ca currents).

      Secondarily, in Maddox et al. 2020, the authors raise the possibility that G369i-KI, by virtue of having a functional voltage-sensing domain-might couple to intracellular Ca2+ stores, and it seems appropriate that this possibility be considered experimentally here.

      As a minor point: the authors might wish to note - in comparison to another retinal ribbon synapse-that Zhang et al. 2022 (in J. Neuroscience) performed a study of mouse rod bipolar cells found a number of LVA and HVA Ca conductances in addition to the typical L-type conductance mediated by Cav1-containing channels.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript proposes a modeling approach to capture nonlinear processes of photocurrents in mammalian (mouse, primate) rod and cone photoreceptors. The ultimate goal is to separate these nonlinearities at the level of photocurrent from subsequent nonlinear processing that occurs in retinal circuitry. The authors devised a strategy to generate stimuli that cancel the major nonlinearities in photocurrents. For example, modified stimuli would generate genuine sinusoidal modulation of the photocurrent, whereas a sinusoidal stimulus would not (i.e., because of asymmetries in the photocurrent to light vs. dark changes); and modified stimuli that could cancel the effects of light adaptation at the photocurrent level. Using these modified stimuli, one could record downstream neurons, knowing that any nonlinearities that emerge must happen post-photocurrent. This could be a useful method for separating nonlinear mechanisms across different stages of retinal processing, although there are some apparent limitations to the overall strategy.

      Strengths:<br /> 1. This is a very quantitative and thoughtful approach and addresses a long-standing problem in the field: determining the location of nonlinearities within a complex circuit, including asymmetric responses to different polarities of contrast, adaptation, etc.<br /> 2. The study presents data for two primary models of mammalian retina, mouse, and primate, and shows that the basic strategy works in each case.<br /> 3. Ideally, the present results would generalize to the work in other labs and possibly other sensory systems. How easy would this be? Would one lab have to be able to record both receptor and post-receptor neurons? Would in vitro recordings be useful for interpreting in vivo studies? It would be useful to comment on how well the current strategy could be generalized.

      Weaknesses:<br /> 1. The model is limited to describing photoreceptor responses at the level of photocurrents, as opposed to the output of the cell, which takes into account voltage-dependent mechanisms, horizontal cell feedback, etc., as the authors acknowledge. How would one distinguish nonlinearities that emerge at the level of post-photocurrent processing within the photoreceptor as opposed to downstream mechanisms? It would seem as if one is back to the earlier approach, recording at multiple levels of the circuit (e.g., Dunn et al., 2006, 2007).<br /> 2. It would have been nice to see additional confirmations of the approach beyond what is presented in Figure 9. This is limited by the sample (n = 1 horizontal cell) and the number of conditions (1). It would have been interesting to at least see the same test at a dimmer light level, where the major adaptation mechanisms are supposed to occur beyond the photoreceptors (Dunn et al., 2007).

  2. Jan 2024
    1. Reviewer #2 (Public Review):

      Summary:<br /> The present study addresses the role of enkephalins, which are specifically expressed by regulatory T cells (Treg), in sensory perception in mice. The authors used a combination of transcriptomic databases available online to characterize the molecular signature of Treg. The proenkephalin gene Penk is among the most enriched transcripts, suggesting that Treg plays an analgesic role through the release of endogenous opioids. In addition, in silico analysis suggests that Penk is regulated by the TNFR superfamily; this being experimentally confirmed. Using flow cytometry analysis, the authors then show that Penk is mostly expressed in Treg of the skin and colon, compared to other immune cells. Finally, genetic conditional excision of Penk, selectively in Treg, results in heat hypersensitivity, as assessed by behavior analysis.

      Strengths:<br /> The manuscript is clear and reveals a previously unappreciated role of enkephalins, as released by immune cells, in sensory perception. The rationale in this manuscript is easy to follow, and conclusions are well supported by data.

      Weaknesses:<br /> The sensory deficit of Penk cKO appears to be quite limited compared to control littermates.

    1. Reviewer #2 (Public Review):

      Understanding how the LC/noradrenaline system controls basic cognitive processes is important and timely. This study aims to understand the role Locus Coeurelus /noradrenaline system in extinction of conditioned responding. The authors used a discriminative appetitive procedure to show that photoexcitation of noradrenergic neurons of the Locus Coeruleus has no effect on the performance during extinction but impacts expression of extinguished responding through a decreased spontaneous recovery. This study is appropriately designed and the results are well analysed. Therefore, it provides an important and timely addition to the field

    1. Reviewer #2 (Public Review):

      Summary<br /> The manuscript by Galicia et al describes the structure of the bacterial GTPyS-bound CtRoco protein in the presence of nanobodies. The major relevance of this study is in the fact that the CtRoco protein is a homolog of the human LRRK2 protein with mutations that are associated with Parkinson's disease. The structure and activation mechanisms of these proteins are very complex and not well understood. Especially lacking is a structure of the protein in the GTP-bound state. Previously the authors have shown that two conformational nanobodies can be used to bring/stabilize the protein in a monomer-GTPyS-bound state. In this manuscript, the authors use these nanobodies to obtain the GTPyS-bound structure and importantly discuss their results in the context of the mammalian LRRK2 activation mechanism and mutations leading to Parkinson's disease. The work is well performed and clearly described. In general, the conclusions on the structure are reasonable and well-discussed in the context of the LRRK2 activation mechanism.

      Strengths:<br /> The strong points are the innovative use of nanobodies to stabilize the otherwise flexible protein and the new GTPyS-bound structure that helps enormously in understanding the activation cycle of these proteins.

      Weakness:<br /> The strong point of the use of nanobodies is also a potential weak point; these nanobodies may have induced some conformational changes in a part of the protein that will not be present in a GTPyS-bound protein in the absence of nanobodies.

      Two major points need further attention.

      1. Several parts of the protein are very flexible during the monomer-dimer activity cycle. This flexibility is crucial for protein function, but obviously hampers structure resolution. Forced experiments to reduce flexibility may allow better structure resolution, but at the same time may impede the activation cycle. Therefore, careful experiments and interpretation are very critical for this type of work. This especially relates to the influence of the nanobodies on the structure that may not occur during the "normal" monomer-dimer activation cycle in the absence of the nanobodies (see also point 2). So what is the evidence that the nanobody-bound GTPyS-bound state is biochemically a reliable representative of the "normal" GTP-bound state in the absence of nanobodies, and therefore the obtained structure can be confidentially used to interpret the activation mechanism as done in the manuscript.

      2. The obtained structure with two nanobodies reveals that the nanobodies NbRoco1 and NbRoco2 bind to parts of the protein by which a dimer is impossible, respectively to a0-helix of the linker between Roc-COR and LRR, and to the cavity of the LRR that in the dimer binds to the dimerizing domain CORB. It is likely the open monomer GTP-bound structure is recognized by the nanobodies in the camelid, suggesting that overall the open monomer structure is a true GTP-bound state. However, it is also likely that the binding energy of the nanobody is used to stabilize the monomer structure. It is not automatically obvious that in the details the obtained nonobody-Roco-GTPyS structure will be identical to the "normal" Roco-GTPyS structure. What is the influence of nanobody-binding on the conformation of the domains where they bind; the binding energy may be used to stabilize a conformation that is not present in the absence of the nanobody. For instance, NbRoco1 binds to the a0 helix of the linker; what is here the "normal" active state of the Roco protein, and is e.g. the angle between RocCOR and LRR also rotated by 135 degrees? Furthermore, nanobody NbRoco2 in the LRR domain is expected to stabilize the LRR domain; it may allow a position of the LRR domain relative to the rest of the protein that is not present without nanobody in the LRR domain. I am convinced that the observed open structure is a correct representation of the active state, but many important details have to be supported by e,g, their CX-MS experiments, and in the end probably need confirmation by more structures of other active Roco proteins or confirmation by a more dynamic sampling of the active states by e.g. molecular dynamics or NMR.

    1. Reviewer #2 (Public Review):

      In their manuscript, Medina and colleagues investigate transcriptional differences between mild and severe SARS-CoV-2 infections. Their analyses are very comprehensive incorporating a multitude of bioinformatics tools ranging from PCA plots, GSEA and DEG analysis, protein-protein interaction network, and weighted correlation network analyses. They conclude that in mild COVID-19 infection NK cell functionality is compromised and this is connected to cytokine interactions and Th1/Th2 cell differentiation pathways cross-talk, bridging the innate and the adaptive arms of the immune system.

      The authors successfully recruited participants with both mild and severe COVID-19 between November 2020 to May 2021. The analyzed cohort is gender and acceptably age-matched and the results reported are promising. Signatures associated with NK cell cytotoxicity in mild and neutrophil functions in the severe group during acute infection are the chief findings reported in this manuscript.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study by Vijay and colleagues addresses a clinically important, and often overlooked aspect of Tb treatment. Detecting for variations in the level of antibiotic tolerance amongst otherwise antibiotic-susceptible isolates is difficult to routinely screen for, and consequently not performed. The authors, present a convincing argument that indeed, there is significant variation in the susceptibility of isoniazid-resistant strains to killing by rifampicin, in some cases at the same tolerance levels as bona fide resistant strains. On the whole, the study is easy to follow and the results are justified. This work should be of interest to the wider TB community at both a clinical and basic level.

      Weaknesses:<br /> The manuscript is long, repetitive in places, and the figures could use some amending to improve clarity (this could be a me-specific issue as they look ok on my screen, yet the colour is poor when printed).

      It would have been great to have seen some correlation between increased rifampicin tolerance and treatment outcome, although I'm not sure if this data is available to the researchers. I agree with the researchers the use of a single media condition is a limitation. However, this is true of a lot of studies.

    1. Reviewer #2 (Public Review):

      Chen et al. investigated the regulatory mechanism of bacterial colonization in the intestinal mucus layer in mice and its implications for intestinal diseases. They demonstrated that Chi3l1 is a protein produced and secreted by intestinal epithelial cells into the mucus layer upon response to the gut microbiota, which has a turnover effect on facilitating the colonization of gram-positive bacteria in the mucosa. The data also indicate that Chi3l1 interacts with the peptidoglycan of the bacteria cell wall, supporting the colonization of beneficial bacteria strains such as Lactobacillus, and that deficiency in Chi3l1 predisposes mice to colitis. The inclusion of a small but pertinent piece of human data added to solidify their findings in mice.

      Overall, the experiments performed were appropriate and well executed, but the data analysis is incomplete and needs to be extended. Also, additional experiments are necessary for clarification and stronger support for their conclusions.

      1) Images are of great quality but lack proper quantification and statistical analysis. Statements such as "substantial increase of Chi3l1 expression in SPF mice" (Fig.1A), "reduced levels of Firmicutes in the colon lumen of IECChil1" (Fig.3F), "Chil1-/- had much lower colonization of E.faecalis" (Fig.4G), or "deletion of Chi3l1 significantly reduced mucus layer thickness" (Supplemental Figure 3A-B) are subjective. Since many conclusions were based on imaging data, the authors must provide reliable measures for comparison between conditions, as long as possible, such as fluorescence intensity, area, density, etc, as well as plots and statistical analysis.

      2) In the fecal/Lactobacillus transplantation experiments, oral gavage of Lactobacillus to IECChil1 mice ameliorated the colitis phenotype, by preventing colon length reduction, weight loss, and colon inflammation. These findings seem to go against the notion that Chi3l1 is necessary for the colonization of Lactobacillus in the intestinal mucosa. The authors could speculate on how Lactobacillus administration is still beneficial in the absence of Chi3l1. Perhaps, additional data showing the localization of the orally administered bacteria in the gut of Chi3l1 deficient mice would clarify whether Lactobacillus are more successfully colonizing other regions of the gut, but not the mucus layer. Alternatively, later time points of 2% DSS challenge, after Lactobacillus transplantation, would suggest whether the gut colonization by Lactobacillus and therefore the milder colitis phenotype, is sustained for longer periods in the absence of Chi3l1.

    1. Reviewer #2 (Public Review):

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

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

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

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

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

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

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

    1. Reviewer #2 (Public Review):

      In the manuscript entitled "Linking the evolution of two prefrontal brain regions to social and foraging challenges in primates" the authors measure the volume of the frontal pole (FP, related to metacognition) and the dorsolateral prefrontal cortex (DLPFC, related to working memory) in 16 primate species to evaluate the influence of socio-ecological factors on the size of these cortical regions. The authors select 11 socio-ecological variables and use a phylogenetic generalized least squares (PGLS) approach to evaluate the joint influence of these socio-ecological variables on the neuro-anatomical variability of FP and DLPFC across the 16 selected primate species; in this way, the authors take into account the phylogenetic relations across primate species in their attempt to discover the the influence of socio-ecological variables on FP and DLPF evolution.

      The authors run their studies on brains collected from 1920 to 1970 and preserved in formalin solution. Also, they obtained data from the Mussée National d´Histoire Naturelle in Paris and from the Allen Brain Institute in California. The main findings consist in showing that the volume of the FP, the DLPFC, and the Rest of the Brain (ROB) across the 16 selected primate species is related to three socio-ecological variables: body mass, daily traveled distance, and population density. The authors conclude that metacognition and working memory are critical for foraging in primates and that FP volume is more sensitive to social constraints than DLPFC volume.

      The topic addressed in the present manuscript is relevant for understanding human brain evolution from the point of view of primate research, which, unfortunately, is a shrinking field in neuroscience. But the experimental design has two major weak points: the absence of lissencephalic primates among the selected species and the delimitation of FP and DLPFC. Also, a general theoretical and experimental frame linking evolution (phylogeny) and development (ontogeny) is lacking.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, the authors aim to demonstrate that cardiac glycosides restore autophagy flux in an iPSC-derived mDA neuronal model of WDR45 deficiency. They established a patient-derived induced pluripotent stem cell (iPSC)-based midbrain dopaminergic (mDA) neuronal model and performed a medium-throughput drug screen using high-content imaging-based IF analysis. Several compounds were identified to ameliorate disease-specific phenotypes in vitro.

      Strengths:<br /> This manuscript engaged in an important topic and yielded some interesting data.

      Weaknesses:<br /> This manuscript failed to provide solid evidence to support the conclusion.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study examines the pattern of responses produced by the combination of left-eye and right-eye signals in V1. For this, they used calcium imaging of neurons in V1 of awake, fixating monkeys. They take advantage of calcium imaging, which yields large populations of neurons in each field of view. With their data set, they observe how response magnitude relates to ocular dominance across the entire population. They analyze carefully how the relationship changed as the visual stimulus switched from contra-eye only, ipsi-eye only, and binocular. As expected, the contra-eye-dominated neurons responded strongly with a contra-eye-only stimulus. The ipsi-eye-dominated neurons responded strongly with an ipsi-eye-only stimulus. The surprise was responses to a binocular stimulus. The responses were similarly weak across the entire population, regardless of each neuron's ocular dominance. They conclude that this pattern of responses could be explained by interocular divisive normalization, followed by binocular summation.

      Strengths:<br /> A major strength of this work is that the model-fitting was done on a large population of simultaneously recorded neurons. This approach is an advancement over previous work, which did model-fitting on individual neurons. The fitted model in the manuscript represents the pattern observed across the large population in V1, and washes out any particular property of individual neurons. Given the large neuronal population from which the conclusion was drawn, the authors provide solid evidence supporting their conclusion. They also observed consistency across 5 fields of view.

      The experiments were designed and executed appropriately to test their hypothesis. Their data support their conclusion.

      Weaknesses:<br /> One weakness of their study is that calcium signals can exaggerate the nonlinear properties of neurons. Calcium imaging renders poor responses poorer and strong responses stronger, compared to single-unit recording. In particular, the dramatic change in the population response between monocular stimulation and binocular stimulation could actually be less pronounced when measured with single-unit recording methods. This means their choice of recording method could have accidentally exaggerated the evidence of their finding.

      The implication of their finding is that strong ocular dominance is the result of release from interocular suppression by a monocular stimulus, rather than the lack of binocular combination as many traditional studies have assumed. This could significantly advance our understanding of the binocular combination circuitry of V1. The entire population of neurons could be part of a binocular combination circuitry present in V1.

    1. Reviewer #2 (Public Review):

      The authors set out to draw further links between neural patterns observed at "rest" during fMRI, with their related thought content and personality traits. More specifically, they approached this with a "tri-partite network" view in mind, whereby the ventral attention network (VAN), the dorsal attention network (DAN), and the default mode network (DMN) are proposed to play a special role in ongoing conscious thought. They used a gradients approach to determine the low dimensional organisation of these networks. In concert, using PCA they reduced thought patterns captured at four time points during the scan, as well as traits captured from a large battery of questionnaires.

      The main findings were that specific thought and trait components were related to variations in the organisation of the tri-partite networks, with respect to cortical gradients.

      Strengths of the methods/results: Having a long (1 hr) resting state MRI session, which could be broken down into four separate scanning/sampling components is a strength. Importantly, the authors could show (via intra-class correlation coefficients) the similarity of thoughts and connectivity gradients across the entire session. Not only did this approach increase the richness of the data available to them, it speaks in an interesting way to the stability of these measures. The inclusion of both thought patterns during scanning along with trait-level dispositional factors is most certainly a strength, as many studies will often include either/or of these, rather than trying to reconcile across. Of the two main findings, the finding that detailed self-generated thought was associated with a decoupling of regions of DAN from regions in DMN was particularly compelling, in light of mounting literature from several fields that support this.

      Weaknesses of the methods/results: Considering the richness of the thought and personality data, I was a little surprised that only two main findings emerged (i.e., a relationship with trait introversion, and a relationship with the "specific internal" thought pattern). I wondered whether, at least in part and in relation to traits, this might stem from the large and varied set of questionnaires used to discern the traits. These questionnaires mostly comprised personality/mood, but some sampled things that do not fall into that category (e.g., musicality, internet addition, sleep), and some related directly to spontaneous thought properties (e.g., mind wandering, musical imagery). It would be interesting to see what relationships would emerge by being more selective in the traits measured, and in the tools to measure them.

      Taken together, the main findings are interesting enough. However, the real significance of this work, and its impact, lie in the richness of the approach: combing across fMRI, spontaneous thought, and trait-level factors. Triangulating these data has important potential for furthering our understanding of brain-behaviour relationship across different levels of organisation.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Previous research shows that humans tend to adjust learning in environments where stimulus-outcome contingencies become more volatile. This learning rate adaptation is impaired in some psychiatric disorders, such as depression and anxiety. In this study, the authors reanalyze previously published data on a reversal-learning task with two volatility levels. Through a new model, they provide some evidence for an alternative explanation whereby the learning rate adaptation is driven by different decision-making strategies and not learning deficits. In particular, they propose that adjusting learning can be explained by deviations from the optimal decision-making strategy (based on maximizing expected utility) due to response stickiness or focus on reward magnitude. Furthermore, a factor related to the general psychopathology of individuals with anxiety and depression negatively correlated with the weight on the optimal strategy and response stickiness, while it correlated positively with the magnitude strategy (a strategy that ignores the probability of outcome).

      Strengths:<br /> The main strength of the study is a novel and interesting explanation of an otherwise well-established finding in human reinforcement learning. This proposal is supported by rigorously conducted parameter retrieval and the comparison of the novel model to a wide range of previously published models.

      Weaknesses:<br /> My main concern is that the winning model (MOS6) does not have an error term (inverse temperature parameter beta is fixed to 8.804).

      1) It is not clear why the beta is not estimated and how were the values presented here chosen. It is reported as being an average value but it is not clear from which parameter estimation. Furthermore, with an average value for participants that would have lower values of inverse temperature (more stochastic behaviour) the model is likely overfitting.

      2) In the absence of a noise parameter, the model will have to classify behaviour that is not explained by the optimal strategy (where participants simply did not pay attention or were not motivated) as being due to one of the other two strategies.

      3) A model comparison among models with inverse temperature and variable subsets of the three strategies (EU + MO, EU + HA) would be interesting to see. Similarly, comparison of the MOS6 model to other models where the inverse temperature parameter is fixed to 8.804).

      This is an important limitation because the same simulation as with the MOS model in Figure 3b can be achieved by a more parsimonious (but less interesting) manipulation of the inverse temperature parameter.

      Furthermore, the claim that the EU represents an optimal strategy is a bit overstated. The EU strategy is the only one of the three that assumes participants learn about the stimulus-outcomes contingencies. Higher EU strategy utilisation will include participants that are more optimal (in maximum utility maximisation terms), but also those that just learned better and completely ignored the reward magnitude.

      Other minor issues that I have are the following:<br /> The mixture strategies model is an interesting proposal, but seems to be a very convoluted way to ask: to what degree are decisions of subjects affected by reward, what they've learned, and response stickiness? It seems to me that the same set of questions could be addressed with a simpler model that would define choice decisions through a softmax with a linear combination of the difference in rewards, the difference in probabilities, and a stickiness parameter.

      Learning rate adaptation was also shown with tasks where decision-making strategies play a less important role, such as the Predictive Inference task (see for instance Nassar et al, 2010). When discussing the merit of the findings of this study on learning rate adaptation across volatility blocks, this work would be essential to mention.

    1. Reviewer #2 (Public Review):

      In this study, Dietmar Funck and colleagues have made a significant breakthrough by identifying three isoforms of plant 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) as homo/arginine-6-hydroxylases, catalyzing the degradation of 6-hydroxyhomoarginine into 2-aminoadipate-6-semialdehyde (AASA) and guanidine. This discovery marks the very first confirmation of plant or eukaryotic enzymes capable of guanidine production.

      The authors selected three plant 2-ODD-C23 enzymes with the highest sequence similarity to bacterial guanidine-producing (EFE) enzymes. They proceeded to clone and express the recombinant enzymes in E coli, demonstrating capacity of all three Arabidopsis isoforms to produce guanidine. Additionally, by precise biochemical experiments, the authors established these three 2-ODD-C23 enzymes as homoarginine-6-hydroxylases (and arginine-hydroxylase for one of them). Furthermore, the authors utilized transgenic plants expressing GFP fusion proteins to show the cytoplasmic localization of all three 2-ODD-C23 enzymes. Most notably, using T-DNA mutant lines and CRISPR/Cas9-generated lines, along with combinations of them, they demonstrate the guanidine-producing capacity of each enzyme isoform in planta. These results provide robust evidence that these three 2-ODD-C23 Arabidopsis isoforms are indeed homoarginine-6-hydroxylases responsible for guanidine generation.<br /> The findings presented in this manuscript are a significant contribution for our understanding of plant biology, particularly given that this work is the first demonstration of enzymatic guanidine production in eukaryotic cells. However, there are a couple of concerns and potential ways for further investigation that the authors should (consider) incorporate.

      Firstly, the observation of cytoplasmic and nuclear GFP signals in the transgenic plants may also indicate cleaved GFP from the fusion proteins. Thus, the authors should perform a Western blot analysis to confirm the correct size of the 2-ODD-C23 fusion proteins in the transgenic protoplasts.

      Secondly, it may be worth measuring pipecolate (and proline?) levels under biotic stress conditions (particularly those that induce transcript changes of these enzymes, Fig S8). Given the results suggesting a potential regulation of the pathway by biotic stress conditions (eg. meJA), these experiments could provide valuable insights into the physiological role of guanidine-producing enzymes in plants. This additional analysis may give a significance of these enzymes in plant defense mechanisms.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study presents data from a broad range of methods (biochemical, EPR, SAXS, microscopy, etc.) on the large disordered protein FRQ relevant to circadian clocks and its interaction partners FRH and CK1, providing novel and fundamental insight into oligomerization state, local dynamics, and overall structure as a function of phosphorylation and association. Liquid-liquid phase separation is observed. These findings have bearings on the mechanistic understanding of circadian clocks, and on functional aspects of disordered proteins in general.

      Strengths:<br /> This is a thorough work that is well presented. The data are of overall high quality given the difficulty of working with an intrinsically disordered protein, and the conclusions are sufficiently circumspect and qualitative to not overinterpret the mostly low-resolution data.

      Weaknesses:<br /> None

    1. Reviewer #2 (Public Review):

      The paper is made of two parts. One deals with RNF146, the other with the development of compounds that may cause TEAD degradation. The two parts are rather unrelated to each other.

      The main limit of this work is the lack of evidence that TEAD factors are in fact regulated by the proteasome and ubiquitylation under endogenous conditions. Also lacking is the demonstration that TEADs are labile proteins to the extent that such quantitative regulation at the level of stability can impact on YAP-TAZ biology. Without these two elements, the relevance and physiological significance of all these data is lacking.

      As for the development of new inhibitors of TEAD, this is potentially very interesting but underdeveloped in this manuscript. Irrespectively, if TEAD is stable, these molecules are likely lead compounds of interest. If TEAD is unstable, as entertained in the first part of the paper, then these molecules are likely marginal.

      Here are a few other specific observations:

      1 The effect of MG is shown in a convoluted way, by MS. What about endogenous TEAD protein stability?

      2 The relevance of siRNF on YAP target genes of Fig.2D is not statistically significant.

      3 All assays are with protein overexpression and Ub-laddering

      4 An inconsistency exists on the only biological validation (only by overexpression) on the fly eye size. RNF gain in Fig4C is doing the opposite of what is expected from what is portrayed here as a YAP/TEAD inhibitor: RNF gain is shown to INCREASE eye size, phenocopying a Hippo loss of function phenotype. According to the model proposed, RNF addition should reduce eye size. The authors stated that " This is in contrast to the anti-growth effect of RNF-146 in the Hpo loss-of-function background and indicates RNF146 may regulate other genes/pathways controlling eye sizes besides its role as a negative regulator of Sd/yki activity". This raises questions on what the authors are really studying: why, according to the authors, these caveats should occur on the controls, and not when they study Hpo mutants?

      5 The role of TEAD inactivation on YAP function is already well known. Disappointingly no prior literature is cited. In any case, this is a mere control.

      6 The second part of the paper on the Development and Screening of pan-TEAD lipid pocket degraders is interesting but unconnected to the above. The degradation pathway it involves has nothing to do with the enzyme described in the first figures.

      7 The role of CIDE on YAP accessibility to Chromatin is superficially executed. Key controls are missing along with the connection with mechanisms and prior knowledge, of TEAD, YAP, chromatin, and other TEAD inhibitors, just to mention a few.

      8 The physiological relevance and the mechanistic interpretation of what should be in the ATAC seq in ovcar cells is missing.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, the authors conduct a detailed analysis of the molecular cues that control the guidance of bifurcated dorsal root ganglion axons in a key region of the spinal cord called the dorsal funiculus. This is a specific case of axon guidance that occurs in a precise way. The authors knew that Slit was important but many axons still target correctly in Slit knockouts, suggesting a role for other guidance factors. Netrin1 is also expressed in this region, so they looked at netrin mutants. The authors found axons outside the DREZ in the Ntn1 mutants, and they show by single-neuron genetic labeling that many of these come from DRG neurons. Quantified axonal tracing studies in Slit1/2, Ntn1, or triple mutant embryos support the idea that Slit and Ntr1 have distinct functions in guidance and that the effect of their loss is additive. Interestingly none of these knockouts affect bifurcation itself but rather the guidance of one or both of the bifurcated axon terminals. Knockout of the Slit receptors (Robo1/2) or the Netrin 1 receptor (DCC) in embryos causes similar guidance defects to loss of the ligands, providing additional confirmation of the requirement for both guidance pathways.

      Strengths:<br /> This study expands understanding of the role of the axon guidance factors Ntr1/DCC and Slit/Robo in a specific axon guidance decision. The strength of the study is the careful axonal labeling and quantification, which allows the authors to establish precise consequences of the loss of each guidance factor or receptor.

      Weaknesses:<br /> There are some places in the text where the discussion of these data is compared with other studies and models, but additional details would help clarify the arguments.

    1. Reviewer #2 (Public Review):

      Yu et al. investigated the structural landscape of 'secreted in xylem' (SIX) effector (virulence and avirulence) proteins from the plant-pathogenic fungus, Fusarium oxysporum f. sp. lycopersici (Fol), with the goal of better understanding effector function and recognition by host (tomato) immune receptors. In recent years, several experimental and computational studies have shown that many effector proteins of plant-associated fungi can be assigned to one of a few major structural families. In the study by Yu et al., X-ray crystallography was used to show that two avirulence effectors of Fol, Avr1 (SIX4) and Avr3 (SIX1), which are recognized by the tomato immune receptors I and I-3, respectively, form part of a new structural family, the Fol dual-domain (FOLD) family, found across three fungal divisions. Using AlphaFold2, an ab initio structural prediction tool, the authors then predicted the structures of all proteins within the Fol SIX effector repertoire (and other effector candidates) and provided evidence that two other effectors, SIX6 and SIX13, also belong to this family.

      In addition to identifying members of the FOLD family, structural prediction revealed that proteins of the Fol effector repertoire can largely be classified into a reduced set of structural families. Examples included four members of the ToxA-like family (including Avr2 (SIX3) and SIX8), as well as four members of a new family, Family 4 (including SIX5 and PSL1). Given previous studies had demonstrated that Avr2 (ToxA-like) and SIX5 (Family 4) interact and function together, and that the genes encoding these proteins are divergently transcribed, and because homologues of SIX8 (ToxA-like) and PSL1 (Family 4) from another Fusarium pathogen are functionally dependent on each other and, in the case of Fol, are encoded by genes that are next to each other in the genome, the authors hypothesized that SIX8 and PSL1 may also physically interact. In line with this, co-incubation of the SIX8 and PSL1 proteins, followed by size exclusion chromatography (SEC), gave elution and gel migration profiles consistent with interaction in the form of a heterodimer. AlphaFold2-Multimer modelling then suggested that this interaction was mediated through an intermolecular disulfide bond. Such a prediction was subsequently confirmed through mutational analysis of the relevant cysteine residue in each protein in conjunction with SEC.

      Finally, using a variant (homologue) of Avr1 from another Fusarium pathogen, as well as chimeric forms of this protein that integrated regions of Avr1 from Fol, Yu et al. determined through co-expression assays in Nicotiana benthamiana with the I immune receptor, as well as subsequent ion leakage assays, that the C-domain of Avr1 is recognized by the I immune receptor. Furthermore, through these assays, the authors were also able to show that surface-exposed residues in the C-domain enable Avr1 to evade recognition by a variant of the I receptor in Moneymaker tomato that does not provide resistance to Fol.

      Overall, the manuscript presents a large body of work that is well supported by the data. A key strength of the manuscript is the validation (benchmarking) of protein structures predicted using AlphaFold2, which is a first for large-scale effector structure prediction papers published to date. Another key strength is the use of large-scale effector structure predictions to make hypotheses about functional relationships or interactions that are then tested (i.e. the SIX8-PSL1 protein interaction and recognition of Avr1 by the I immune receptor). This testing again goes above and beyond the large-scale effector structure prediction papers published to date. Taken together, this showcases how experimental and computational experiments can be effectively combined to provide biologically relevant data for the plant protection and molecular plant-microbe interactions fields.

      In terms of weaknesses, the manuscript could have validated the SIX8-PSL1 protein interaction with in planta experiments, such as co-immunoprecipitation assays or co-localization experiments in conjunction with confocal microscopy, to provide support for the interaction in a plant setting. However, given what is already known about the Avr2-SIX5 interaction, these additional experiments are not crucial and could instead form part of a follow-up study.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Zhao et al. aimed to explore an important question-how to overcome resistance of hepatocellular carcinoma cells to radiotherapy. Given that immune-suppressive microenvironment is a major mechanism underlying resistance to radiotherapy, they reasoned that a drug that blocks PD-1/PD-L1 pathway could improve efficacy of radiation therapy and chose to investigate the effect of Nifuroxazide, an inhibitor of stat3 activation, on radiotherapy efficacy in treating hepatocellular carcinoma cells. From in vitro experiments, they find combination treatment (Nifuroxazide+ radiotherapy) increases apoptosis and reduces proliferation and migration, in comparison to radiotherapy alone. From in vivo experiments, they demonstrate that combined treatment reduces size and weight of tumors in vivo and enhances mice survival. These data indicate a better efficacy of combination therapy compared to radiotherapy alone. Moreover, they also determined the effect of combination therapy on tumor microenvironment as well as peripheral immune response. Specifically, they find that combination therapy increases infiltration of CD4+, CD8+ t cells and NK cells, activates CD8+ t cells, enhances polarization of M1 macrophages and decreases Treg cells in the tumor microenvironment. These changes in tumor microenvironment is consistent with reduced tumor growth by combination therapy. The most intriguing part of the study is the determination of effect of Nifuroxazide on PD-L1 expression in the context of radiotherapy. Considering Nifuroxazide is a stat3 activation inhibitor and stat3 inhibition leads to reduced expression of PD-L1, one would expect Nifuroxazide decreases PD-L1 expression through stat3. However, they find the effect of Nifuroxazide on PD-L1 is dependent on GSK3 mediated Proteasome pathways and independent of stat3, in the given experimental context. To determine the relevance to human hepatocellular carcinoma, they also measured the PD-L1 expression in human tumor tissues of HCC patients pre- and post-radiotherapy. The increased PD-L1 expression level in HCC after radiotherapy is impressive.<br /> Overall, the data are convincing and supportive to the conclusions.

      Strengths:<br /> 1) Novel finding: Identified novel mechanism underlying effect of Nifuroxazide on PD-L1 expression in hepatocellular carcinoma cells in the context of radiotherapy.<br /> 2) Comprehensive experimental approaches: using different approaches to prove same finding. For example, Fig4, both IHC and WB were used. Fig5. Both IF and WB were used.<br /> 3) Human disease relevance: Compared observations in mice with human tumor samples.

    1. Reviewer #2 (Public Review):

      This work provides a new tool (H3-Opt) for the prediction of antibody and nanobody structures, based on the combination of AlphaFold2 and a pre-trained protein language model, with a focus on predicting the challenging CDR-H3 loops with enhanced accuracy than previously developed approaches. This task is of high value for the development of new therapeutic antibodies. The paper provides an external validation consisting of 131 sequences, with further analysis of the results by segregating the test sets in three subsets of varying difficulty and comparison with other available methods. Furthermore, the approach was validated by comparing three experimentally solved 3D structures of anti-VEGF nanobodies with the H3-Opt predictions

      Strengths:

      The experimental design to train and validate the new approach has been clearly described, including the dataset compilation and its representative sampling into training, validation and test sets, and structure preparation. The results of the in silico validation are quite convincing and support the authors' conclusions.

      The datasets used to train and validate the tool and the code are made available by the authors, which ensures transparency and reproducibiity, and allows future benchmarking exercises with incoming new tools.

      Compared to AlphaFold2, the authors' optimization seems to produce better results for the most challenging subsets of the test set.

      Weaknesses:

      The comparison of affinity predictions derived from AlphaFold2 and H3-opt models, based on molecular dynamics simulations, should have been discussed in depth. In some cases, there are huge differences between the estimations from H3-opt models and those from experimental structures. It seems that the authors obtained average differences of the real delta, instead of average differences of the absolute value of the delta. This can be misleading, because high negative differences might be compensated by high positive differences when computing the mean value. Moreover, it would have been good for the authors to disclose the trajectories from the MD simulations.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript explores mechanisms by which STAT3 may regulate KRAS mutant cancers.

      In the first set of experiments, STAT3 GOF mutants diminished the transformation of p53-null mouse embryonic fibroblasts expressing endogenous mutant KRAS(G12D) (KP MEFs) and this was dependent on direct transcriptional activation induced by phosphorylated STAT3. It appears that this is mediated via a reduction in TGFb signaling such that knockout of either TGFBR2 or SMAD4 can phenocopy the effects of STAT3 GOF mutants in KP MEFs.

      In the next part of the paper, the authors used murine pancreatic ductal adenocarcinoma (PDAC)-derived cell lines bearing endogenous KRAS(G12D) and TP53(R172H) mutations (KPC) to determine the extent to which STAT3 may regulate KRAS dependency. They determined that KRAS and STAT3 KO both induced mesenchymal-like phenotypes and that TGFBR2 and SMAD4 KO induced epithelial phenotypes. The loss of STAT3 appeared to correlate with a KRAS-independent signature, and SMAD4/TGFBR2 KO could not induce epithelial phenotypes when STAT 3 was also knocked out.

      Strengths:<br /> Overall, this is an interesting paper that highlights the complicated interactions between KRAS, STAT3, and TGF beta signaling. The authors use multiple models and attempt to link data to patient cohorts.

      Weaknesses:<br /> While correlations are strong, the study would benefit from additional cause-and-effect type experiments. It would also be beneficial to better tie together the first and second parts of the paper.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In the current study, the authors investigated the role of loss of CTRP10 results in female obesity with preserved metabolic health. The overall conclusion is supported by the experimental data that CTRP10 negatively regulates body weight in females and that loss of CTRP10 results in benign obesity with largely preserved insulin sensitivity and metabolic health. The authors have shown the role of sex differences in the metabolically healthy obese (MHO) phenotype, which may increase the scope for research in this area.

      Strengths:<br /> The study provides a detailed idea of how genes are regulated in a sex-dependent manner.

      Weaknesses:<br /> Mechanistic details are missing.

    1. Reviewer #2 (Public Review):

      Summary

      In this work, Bartolome and colleagues develop a new approach to identify proteasome interacting proteins and substrates. The approach is based on fusing proteasome subunits with a biotin ligase that will label proteins that come in close physical distance of the ligase. These biotin-labeled proteins (or their resulting tryptic peptides) can be affinity purified using streptavidin and identified by mass spectrometry.

      This elegant solution was able to identify a large proportion of known proteasome interactors, as well as multiple potential new interactors. Combining this approach with a proteasome inhibitor allowed also for the enrichment of substrates, due to increased contact time between substrates and the proteasome. Again, the authors were able to identify novel substrates. Finally, the authors implemented this strategy in vivo, providing the hints for potential tissue-specific proteasome interactors.

      This novel strategy provides an additional approach to identify new proteasome substrates, which can be particularly powerful for low abundant proteins, e.g., transcription factors. The possibility to implement it in vivo in specific cell types opens the possibility for identifying proteasome interactors in small cell subpopulations or in subpopulations involved in disease.

      Strengths:

      The authors carefully characterized their genetically engineered proteasome-biotin ligase fusions to ensure that proteasome structure and activity was not altered. This is key to ensure that the proteins identified to interact with the proteasome reflect interactions that occur under physiological conditions.

      The authors implemented an algorithm that controls the false positive rate of the identified interactors of the proteasome. This is an important aspect to avoid spending time on the characterization of potential interactors that are just an artifact of the experimental setup.

      The addition of a proteasome inhibitor allowed the authors to identify substrates of the proteasome. Although there are other strategies to do this (e.g., affinity purification of Gly-Gly modified peptides, which is a marker for ubiquitination), this additional approach can highlight currently unknown substrates. One example are low abundance proteins, such as transcription factors.

      The overall strategy developed by the authors can be implemented in vivo, which opens for the possibility of determining cell type-specific proteasome interactors (and perhaps substrates).

      Weaknesses:

      There is a small proportion of the PSMA4-biotin ligase fusion that remains unassembled (i.e., not part of the functional proteasome) and that can contribute to a small proportion of false positive interactions.

    1. Reviewer #2 (Public Review):

      Zhang and Wei, et al. investigated the role of a centrosomal protein, CEP44, in regulating centrosomes and spindle integrity, with a focus on processes that may be dysregulated in breast cancer. The authors found that a breast cancer cell line, MDA-MB-436, lacks CEP44 protein and has amplified centrioles. CEP44 expression is reduced in samples from breast cancer patients. By super-resolution microscopy, the authors localize CEP44 to the proximal inner lumen of centrioles, as has also been previously shown by another group (Atorino et al 2020). Next, the authors investigate the role of CEP44 in centrosome regulation. They found that loss of CEP44 in HeLa cells results in extra puncta of CEP97 or Centrin-3, while ectopic overexpression of CEP44 in MDA-MB-436 cells reduces the number of CEP97 foci. Only one of the excess puncta in a CEP44-depleted HeLa cell recruits CEP164 or ODF2, indicating that extra foci were not the result of cytokinesis failure. In G1, most (~80%) of CEP44-depleted cells have 2 centrin foci, while in G2, a small population (~20%) have more than 4 centrin foci, and gamma-tubulin is recruited in foci in G2. The authors were able to observe centriole disengagement and amplification using live cell imaging. The authors propose that CEP44 acts in regulating centriole engagement by recruiting CEP57 and CEP57L1 to centrioles. The authors made CEP44 knockout cell lines using CRISPR and found that loss of CEP44 results in multipolar spindles, correlated with an increase in centriole amplification. Finally, the authors investigate the role of CEP44 at the mitotic spindle. The authors find that CEP44 localizes to spindles and is phosphorylated by Aurora A at G2/M on Ser324. Phosphorylation of CEP44 is required for its proper distribution between centrosomes and the spindle and microtubule stability within both spindles and interphase microtubules. Together, these studies shed light on the roles of CEP44 within centrosomes and spindles and will be of interest to cell biologists and cancer biologists studying cell division and centrosomes.

      The conclusions of this paper are only partially supported. The analyses could be improved to address the concerns about the major conclusions.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The manuscript analyzes the genetic requirement for DNA damage-induced cell cycle checkpoint induction and maintenance in budding yeast bearing one or two unrepairable DNA double-strand breaks using auxin-induced degradation (AID) of key DNA damage response (DDR) factors. The study paid particular attention to solving a puzzle regarding how yeast bearing two unrepaired DNA breaks fail to engage in "adaptation" whereas those with a single unrepairable break eventually resume cell cycling after a prolonged (up to 12 h) G2 arrest.

      The most novel findings are: 1. The genetic requirement for the entry to DDC and the maintenance are separable. For instance, Dun1 is partially required for the entry but not DDC maintenance whereas Chk1 is only required for maintenance. 2. Cells with two irreparable breaks respond to DDR only up to a certain time (~12 h post damage) and beyond this point, depend on spindle assembly checkpoint (SAC) and mitotic exit network (MEN) to halt cell cycling. 3. The authors also propose an interesting model that the location of DNA breaks and their distance to centromeres can lead to the triggering of SAC/MEN and dictate the duration of cell cycle arrest and their adaptability following DNA damage. The results thus provide the most compelling evidence on the role of SAC/MEN in DNA damage response and cell cycle arrest albeit its impact might be limited to the current experimental set-up or under conditions when DNA repair is severely deficient.

      Overall, the conclusion of the study is well supported by the elegant set of genetic experimental data and employed multiple readouts on DDC factor depletion on checkpoint integrity and cell cycle status. However, the study still relies heavily on Rad53 phosphorylation as the primary metric to assess checkpoint status. Since evidence exists the residual DDC still operates even when Rad53 phosphorylation is undetectable, additional readouts for DDC functions might be necessary to strengthen the study's conclusions. These and other concerns that need clarifications or further experimental validations are discussed below.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript shows detailed evidence of the role of cohesin regulators in rice meiosis and mitosis.

      Strengths:<br /> There is a very clear mechanism for its role during replication. The strength of the evidence and its novelty is very high. This paper makes a significant contribution to the body of knowledge on meiotic cohesion in a valuable plant model.

      Weaknesses:<br /> The authors did not consider creating heterozygous mutants for the replication fork.<br /> Moderate English language editing may be required.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors try to establish that there is an Abeta-dependent loss of nuclear pores early in Alzheimer's disease. To do so the authors compared different NUP proteins and assessed their function by analyzing nuclear leakage and resistance to induction of nuclear damage and the associated necroptosis. The authors use a mouse knockin for hAPP with familial Alzheimer's mutations to model amyloidosis related to Alzheimer's disease. Treatment with an inhibitor of beta-amyloid production partially rescued the loss of nuclear pore proteins in young KI neurons, implicating beta-amyloid in Nuclear Pore dysfunction, a mechanism already described in other neurodegenerative diseases but not in Alzheimer's disease.

      The conclusions of this paper related to familial AD are well supported by data but are not related to an aging decline in NUP function, where it is required to extend data analysis and one additional experiment.

      1. Adding statistics and comparisons between wild-type changes at different times/ages to determine if the nuclear pore changes with time in wild-type neurons. The images show differences in the Nuclear pore in neurons from the wild-type mice, with time in culture and age. However, a rigorous statistical analysis is lacking to address the impact of age/development on NUP function. Although the authors state that nuclear pore transport is reported to be altered in normal brain aging, the authors either did not design their experiments to account for the normal aging mechanisms or overlooked the analysis of their data in this light.

      2. Add experiments to assess the contribution of wild-type beta-amyloid accumulation with aging. It was described in 2012 (Guix FX, Wahle T, Vennekens K, Snellinx A, Chávez-Gutiérrez L, Ill-Raga G, Ramos-Fernandez E, Guardia-Laguarta C, Lleó A, Arimon M, Berezovska O, Muñoz FJ, Dotti CG, De Strooper B. 2012. Modification of γ-secretase by nitrosative stress links neuronal ageing to sporadic Alzheimer's disease. EMBO Mol Med 4:660-673, doi:10.1002/emmm.201200243) and 2021 (Burrinha T, Martinsson I, Gomes R, Terrasso AP, Gouras GK, Almeida CG. 2021. Upregulation of APP endocytosis by neuronal aging drives amyloid-dependent synapse loss. J Cell Sci 134. doi:10.1242/jcs.255752), 28 DIV neurons are senescent and accumulate beta-amyloid42. In addition, beta-amyloid 42 accumulates normally in the human brain (Baker-Nigh A, Vahedi S, Davis EG, Weintraub S, Bigio EH, Klein WL, Geula C. 2015. Neuronal amyloid-β accumulation within cholinergic basal forebrain in ageing and Alzheimer's disease. Brain 138:1722-1737. doi:10.1093/brain/awv024), thus, it would be important to determine if it contributes to NUP dysfunction. Unfortunately, the authors tested the Abeta contribution at div14 when wild-type Abeta accumulation was undetected. It would enrich the paper and allow the authors to conclude about normal aging if additional experiments were performed, namely, treating 28Div neurons with DAPT and assessing if NUP is restored.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This work investigates the enzymatic properties of lysostaphin (LSS) and LytM, two enzymes produced by Staphylococcus aureus and previously described as glycyl-glycyl endopeptidases. The authors use synthetic peptide substrates mimicking peptidoglycan fragments to determine the substrate specificity of both enzymes and identify the bonds they cleave.

      Strengths:<br /> - This work is addressing a real gap in our knowledge since very little information is available about the substrate specificity of peptidoglycan hydrolases.<br /> - The experimental strategy and its implementation are robust and provide a thorough analysis of LSS and LytM enzymatic activities. The results are very convincing and demonstrate that the enzymatic properties of the model enzymes studied need to be revisited.

      Weaknesses:<br /> - The manuscript is difficult to read in places and some figures are not always presented in a way that is easy to follow. This being said, the authors have made a good effort to present their experiments in an engaging manner. Some recommendations have been made to improve the current manuscript but these remain minor issues.

    1. Reviewer #2 (Public Review):

      The manuscript "Autoacetylation-mediated phase separation of TIP60 is critical for its functions" by Dubey S. et al reported that the acetyltransferase TIP60 undergoes phase separation in vitro and cell nuclei. The intrinsically disordered region (IDR) of TIP60, particularly K187 within the IDR, is critical for phase separation and nuclear import. The authors showed that K187 is autoacetylated, which is important for TIP60 nuclear localization and activity on histone H4. The authors did several experiments to examine the function of K187R mutants including chromatin binding, oligomerization, phase separation, and nuclear foci formation. However, the physiological relevance of these experiments is not clear since TIP60 K187R mutants do not get into nuclei. The authors also functionally tested the cancer-derived R188P mutant, which mimics K187R in nuclear localization, disruption of wound healing, and DNA damage repair. However, similar to K187R, the R188P mutant is also deficient in nuclear import, and therefore, its defects cannot be directly attributed to the disruption of the phase separation property of TIP60. The main deficiency of the manuscript is the lack of support for the conclusion that "autoacetylation-mediated phase separation of TIP60 is critical for its functions".

      This study offers some intriguing observations. However, the evidence supporting the primary conclusion, specifically regarding the necessity of the intrinsically disordered region (IDR) and K187ac of TIP60 for its phase separation and function in cells, lacks sufficient support and warrants more scrutiny. Additionally, certain aspects of the experimental design are perplexing and lack controls to exclude alternative interpretations. The manuscript can benefit from additional editing and proofreading to improve clarity.

    1. Reviewer #2 (Public Review):

      EZH2 is upregulated in most advanced cancers and has been investigated as a therapeutic target for many years. However, how EZH2 activity is regulated remains to be fully elucidated. In this study, Guo et al. provided a new mechanism for the regulation of EZH2. The authors demonstrated that the protein stability of EZH2 is dynamically regulated by lysine methylation-dependent proteolysis. Specifically, K20 of EZH2 is monomethylated by SET7 methyltransferase and demethylated by LSD1 demethylase. The methylated K20 is recognized by specific methyl-lysine reader L3MBTL3 to promote EZH2 for ubiquitin-dependent proteolysis by the CRL4DCAF5 ubiquitin E3 ligase complex, resulting in the dysregulation of EZH2/PRC2 activity and reduction of H3K27me3. The authors further found a methylation-phosphorylation switch existed in some cancer cells and this switch controls EZH2 stability and hematopoiesis.

      Overall, most conclusions of this paper are well-supported by the results presented, only some aspects of Figure 6 need to be extended. This work is of interest to biomedical researchers in the field of cancer epigenetics after minor revision.

    1. Reviewer #2 (Public Review):

      In a beautiful line of work, the authors have proposed the intriguing idea that activity patterns of neurons can fluctuate between representing one of multiple stimuli in its receptive field. This allows for time-multiplexing of information by neural populations. The idea was initially proposed by Caruso et al (2018) and tested for both auditory and visual stimuli and later extended in Jun et al (2022). The current study analyzes additional datasets to further extend the conclusions across multiple areas and different stimulus sets.

      Together with the earlier work, the current study provides solid evidence for the hypothesis that fluctuating activity patterns in neurons representing multiple stimuli may be a general phenomenon. This exciting possibility may have implications for the studies of perception, attention, decision-making, and other cognitive functions.

      In the current study, the claim that the fluctuating activity patterns may be a general phenomenon is supported by multiple data sets from area MT and face patches MF and AL in IT cortex, using multiple stimulus sets (moving dots and gratings for MT, and face-face and face-object pairs for IT cortex). The major strength of this study is the consistency of the results across these areas and stimulus sets.

      The description of the results would benefit from a better explanation of how low spike counts may influence the outcome of the analysis. Due to a smoothing procedure used for visualization, the spike counts for the paired stimuli (AB, black lines) shown in Figure 3a-b and Figure 4a-d go below 0. However, the actual spike count on a trial can not go below 0. The symmetric smoothing procedure may hide an underlying skewed distribution of spike counts that can only be positive. The statistical analysis is not performed on the smoothed distribution but on the actual spike counts, and the validity of the result is therefore not in question. However, the paper would benefit from 1) visualization of the unsmoothed trial counts, and 2) an explanation of how assumptions of symmetric/skewed distributions may affect the outcome.

      Overall, the authors have presented an interesting hypothesis that is supported by rigorous analysis, they clearly described the results, and they have given a fair discussion of what we can and cannot conclude from this dataset. This line of work deserves the attention of a broad audience within the field of neuroscience.

    1. Reviewer #2 (Public Review):

      The authors provide an analysis showing that the allele ages of putatively advantageous alleles tend to be older than those of neutral alleles. To do this, the authors first classify mutations as either neutral, advantageous or deleterious based on a metric called the 'evolutionary probability' which is correlated to the impact of selection acting on a mutation. Then, the authors quantify the age of the mutations using the GEVA method and they also quantify tc (the time of the ancestral node of the edge carrying the mutation). Interestingly, the authors find that advantageous mutations tend to have an older allele age and an older value of tc compared to neutral mutations. The authors posit some explanations for this result invoking the action of balancing selection.

      This is an interesting paper and its results could merit an important change in our conception of how we believe that natural selection is acting on the human genome. I have concerns about some of the analysis presented on this paper that have to do with two main factors: 1) Showing that the estimates of allele ages and tc are robust on the dataset presented (more on this topic here below). 2) Presenting more simulations or analytical theory where the authors can show that the models presented by the authors to explain the results indeed fit the data well. As an example, the authors could perform some simulations (likely using SLiM) under the balancing selection models posited by the authors and then show that they can produce data where the allele ages for deleterious, neutral and advantageous alleles have similar patterns to what is observed on the genomic dataset analyzed.

      Major concerns

      - What is the impact of multiple mutations on the same site on the estimates of allele ages with GEVA?

      - GEVA, which is one of the methods used by the authors, 'overestimates "intermediate" times and underestimates older times' according to Ragsdale and Thornton (2023) MBE. What is the impact of this effect for the analysis performed by the authors? Do RUNTC has any known biases on their estimate of tc?

      - Additionally what is the impact of phasing errors on the estimates of allele age presented by the authors?

    1. Reviewer #2 (Public Review):

      This is an interesting study examining the question of whether CSD sensitizes meningeal afferent sensory neurons leading to spontaneous activity or whether CSD sensitizes these neurons to mechanical stimulation related to locomotion. Using two-photon in vivo calcium imaging based on viral expression of GCaMP6 in the TG, awake mice on a running wheel were imaged following CSD induction by cortical pinprick. The CSD wave evoked a rise in intracellular calcium in many sensory neurons during the propagation of the wave but several patterns of afferent activity developed after the CSD. The minority of recorded neurons (10%) showed spontaneous activity while slightly larger numbers (20%) showed depression of activity, the latter pattern developed earlier than the former. The vast majority of neurons (70%) were unaffected by the CSD. CSD decreased the time spent running and the numbers of bouts per minute but each bout was unaffected by CSD. There also was no influence of CSD on the parameters referred to as meningeal deformation including scale, shear, and Z-shift. Using GLM, the authors then determine that there there is an increase in locomotion/deformation-related afferent activity in 51% of neurons, a decrease in 12% of neurons, and no change in 37%. GLM coefficients were increased for deformation related activity but not locomotion related activity after CSD. There also were an increase in afferents responsive to locomotion/deformation following CSD that were previously silent. This study shows that unlike prior reports, CSD does not lead to spontaneous activity in the majority of sensory neurons but that it increases sensitivity to mechanical deformation of the meninges. This has important implications for headache disorders like migraine where CSD is thought to contribute to the pathology in unclear ways with this new study suggesting that it may lead to increased mechanical sensitivity characteristic of migraine attacks.

    1. Reviewer #2 (Public Review):

      Summary:

      This study uses transcriptome sequence from a dioecious plant to compare evolutionary rates between genes with male- and female-biased expression and distinguish between relaxed selection and positive selection as causes for more rapid evolution. These questions have been explored in animals and algae, but few studies have investigated this in dioecious angiosperms, and none have so far identified faster rates of evolution in male-biased genes (though see Hough et al. 2014 https://doi.org/10.1073/pnas.1319227111).

      Strengths:

      The methods are appropriate to the questions asked. Both the sample size and the depth of sequencing are sufficient, and the methods used to estimate evolutionary rates and the strength of selection are appropriate. The data presented are consistent with faster evolution of genes with male-biased expression, due to both positive and relaxed selection.

      This is a useful contribution to understanding the effect of sex-biased expression in genetic evolution in plants. It demonstrates the range of variation in evolutionary rates and selective mechanisms, and provides further context to connect these patterns to potential explanatory factors in plant diversity such as the age of sex chromosomes and the developmental trajectories of male and female flowers.

      Weaknesses:

      The presence of sex chromosomes is a potential confounding factor, since there are different evolutionary expectations for X-linked, Y-linked, and autosomal genes. Attempting to distinguish transcripts on the sex chromosomes from autosomal transcripts could provide additional insight into the relative contributions of positive and relaxed selection.

    2. Reviewer #2 (Public Review):

      Summary:

      This study uses transcriptome sequence from a dioecious plant to compare evolutionary rates between genes with male- and female-biased expression and distinguish between relaxed selection and positive selection as causes for more rapid evolution. These questions have been explored in animals and algae, but few studies have investigated this in dioecious angiosperms, and none have so far identified faster rates of evolution in male-biased genes (though see Hough et al. 2014 https://doi.org/10.1073/pnas.1319227111).

      Strengths:

      The methods are appropriate to the questions asked. Both the sample size and the depth of sequencing are sufficient, and the methods used to estimate evolutionary rates and the strength of selection are appropriate. The data presented are consistent with faster evolution of genes with male-biased expression, due to both positive and relaxed selection.

      This is a useful contribution to understanding the effect of sex-biased expression in genetic evolution in plants. It demonstrates the range of variation in evolutionary rates and selective mechanisms, and provides further context to connect these patterns to potential explanatory factors in plant diversity such as the age of sex chromosomes and the developmental trajectories of male and female flowers.

      Weaknesses:

      The presence of sex chromosomes is a potential confounding factor, since there are different evolutionary expectations for X-linked, Y-linked, and autosomal genes. Attempting to distinguish transcripts on the sex chromosomes from autosomal transcripts could provide additional insight into the relative contributions of positive and relaxed selection.

    1. Reviewer #2 (Public Review):

      In this study Hui Dong et al. identified and characterized two transporters of the monocarboxylate family, which they called Apcimplexan monocarboxylate 1 and 2 (AMC1/2) that the authors suggest are involved in the trafficking of metabolites in the non-photosynthetic plastid (apicoplast) of Toxoplasma gondii (the parasitic agent of human toxoplasmosis) to maintain parasite survival. To do so they first identified novel apicoplast transporters by conducting proximity-dependent protein labeling (TurboID), using the sole known apicoplast transporter (TgAPT) as a bait. They chose two out of the three MFS transporters identified by their screen based and protein sequence similarity and confirmed apicoplast localisation. They generated inducible knock down parasite strains for both AMC1 and AMC2, and confirmed that both transporters are essential for parasite intracellular survival, replication, and for the proper activity of key apicoplast pathways requiring pyruvate as carbon sources (FASII and MEP/DOXP). Then they show that deletion of each protein induces a loss of the apicoplast, more marked for AMC2 and affects its morphology both at its four surrounding membranes level and accumulation of material in the apicoplast stroma. The authors attempted to decipher the function of the transporters on metabolic functions of the apicoplast: (a) notably for IPP synthesis through the assessment of vesicle import allowed by IPP-based anchors, which was found to be affected in the mutants, as well as (b) apicoplast fatty acid synthesis by indirect assessment of vesicle import. However, none of them directly concluded on the actual function of the transporters. Furthermore heterologous complementation in bacterial system also failed to demonstrate the transporters' function.

      However, this study is very timely, as the apicoplast holds several important metabolic functions (FASII, IPP, LPA, Heme, Fe-S clusters...), which have been revealed and studied in depth but no further respective transporter have been identified thus far. hence, new studies that could reveal how the apicoplast can acquire and deliver all the key metabolites it deals with, will have strong impact for the parasitology community as well as for the plastid evolution communities. The current study is well initiated with appropriate approaches to identify two new putatively important apicoplast transporters, and showing how essential those are for parasite intracellular development and survival. However, in its current state, this is all the study provides at this point (i.e. essential apicoplast transporters disrupting apicoplast integrity, and indirectly its major functions, FASII and IPP, as any essential apicoplast protein disruption does). The study fails to deliver further message or function regarding AMC1 and 2, and thus validate their study. Currently the manuscript just describes how AMC1/2 deletion impacts parasite survival without answering the key question about them: what do they transport. The authors yet have to perform key experiments that would reveal their metabolic function. Ideally the authors would work further and determine the function of AMC1 and 2.

    1. one year ago finally when the project was set up in order to try to avoid this uh this crazy dynamic the name of the project is cicloter

      for - Cycle Terre - https://www.cycle-terre.eu/en/ - https://circulareconomy.europa.eu/platform/en/good-practices/cycle-terre-excavated-soil-urban-areas-becomes-construction-raw-material - https://www.sme-enterprize.com/sustainability-stories/environment/cycle-terre/

      description - The Cycle Terre project shifts perspectives - excavation material is no longer treated as waste to be disposed of, - but as a new raw building material for - compressed earth bricks - earth wall panels - earth coatings - https://www.cycle-terre.eu/mise-en-oeuvre/les-materiaux/ - The excavation of kilometers of tunnels to extend the Paris mass transit system will produce enormous amounts of raw feedstock for the Cycle Terre manufacturing plant.- 400 million tons!

    2. in general countries tend to excavate enormous volumes of earth and this earth is incredibly considered as a waste material

      for - circular economy - building - excavation waste - circular economy - construction - excavation waste - key insight - repurpose excavation waste as building material

      key insight - She makes an pretty important observation about the inefficiency of current linear construction process - The excavation part requires enormous amounts of energy, and the earth that is excavated is treated as waste that must be disposed of AT A COST! - Instead, with a paradigm shift of earth as a valuable building resource, the excavation PRODUCES the building materials! - This is precisely what BC Material's circular economy business model is and it makes total sense!<br /> - With a simple paradigm and perspective shift, waste is suddenly transformed into a resource! - waste2resource - waste-to-resource

      new meme - Waste-2-Resource

    1. Reviewer #3 (Public Review):

      The work by Ghasemahmad et al. has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the basolateral amygdala (BLA) while an animal listens to social vocalizations.

      Ghasemahmad et al. made changes to the manuscript that have significantly improved the work. In particular, the transparency in showing the underlying levels of Ach, DA, and 5HIAA is excellent. My previous concerns have been adequately addressed.

    1. Reviewer #2 (Public Review):

      This paper considers methods for statistical analysis of autocorrelated neural recording time series: an important question for neuroscience, that is underappreciated in the community. The paper makes a valuable contribution to this topic by comparing methods based on cross-validation and cyclic shift on simulated grid-cell data. My main suggestions regard clarity, which would greatly benefit from a more didactic approach: explaining the methods compared to the main text and providing more explanatory figures. But there are also some additional analyses that would strengthen the paper.

      There are two ways to build support for the validity of a statistical method: by mathematically proving that it is valid, or by empirically verifying it with simulated data where the correct answer is known. A mathematical proof removes all doubt to validity but empirical validation can still be useful even without proof, as it demonstrates that the method works in at least some circumstances. For empirical validation to be most convincing, it helps to also show some situations where the method doesn't work, ideally by varying a continuous parameter that reliably moves the simulation from a situation where it works to one where it doesn't. If the method works in all but extremely unrealistic cases, this builds confidence that it will work on real data.

      The main conclusion of this paper's simulations is that the cyclic shift method most often detects valid correlations, while still not exceeding the false positive rate expected for a valid test. Readers may take this paper as indicating that the circular shift method is safe in all circumstances, but this is not correct. The authors acknowledge that circular shift can sometimes be invalid, and have made modifications to mitigate the problem. But there is neither a mathematical proof that these mitigations work, nor an analysis of the circumstances under which they succeed and fail. I doubt a formal proof is possible since there are likely situations in which even the new methods give false positive results. So the authors should include an empirical test of their modified circular shift method as compared to plain circular shift in various simulations. To gain confidence in the new method it is important to characterize the situations where both methods succeed; where the new method succeeds but traditional cyclic shift gives false positive errors; and situations in which both fail. If situations where the new method fails are so unrealistic that they would never occur in real data, we can have better confidence in the method.

      The main contributions of the paper are the modifications to circular shifting and cross-validation that avoid problems of temporal contiguity, but these are only described in the Methods section. But this is a methods paper, so the description of the new methods should be in the main text, including explanatory figures currently in the Methods.

      The introduction presents two problems that can occur in neural data: autocorrelation, and omitted variables. However, it is not clear that the current methods help with the problem of omitted variables. In fact, I don't see how any analysis method could solve the problem of omitted variables. If an experimenter observes a correlation between X and Y, there is no way to know this isn't because a third variable Z correlates with X and influences Y, without any effect of X on Y. It is generally impossible to prove causation without making randomized manipulations of one variable; although some methods claim to infer causality by observing all variables that could possibly have a causal effect, this is unlikely to occur in neuroscience. In any case, the problem of omitted variables seems irrelevant to the current study and could be removed.

      The list of analysis methods mentioned in the first paragraph of the introduction (eg TDA, LVM) seems irrelevant: it is not clear how the methods evaluated here would be used to assess the significance of those methods. Better to stick to a description of how correlations are difficult to detect in autocorrelated signals, which is what the current methods address.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study discovered a neural mechanism that serves as a switch from rolling to fast crawling behaviors in Drosophila larvae. It addressed important open questions of how neural circuits determine the sequence of locomotor behaviors and how animals switch from one behavior to another. Overall, its results support the conclusions. The experimental approaches should be described more clearly.

      The escape behavior of Drosophila larvae includes rolling followed by fast crawling, where the neural mechanism of this sequence is unclear. The authors identified SeIN128, a group of descending neurons that facilitates rolling termination and shortens crawling latency. By investigating the EM connectome of larval CNS, they found that SeIN128 receives inputs from Basin-2 and A00c neurons, which are reported to facilitate rolling. SeIN128 makes reciprocal inhibitory synapses onto Basin-2 and A00c. Gad staining indicates that SeIN128 neurons are GABAergic, and inhibition of SeIN128 caused increased rolling probability and prolonged rolling. RNAi knockdown of GABA receptors in Basins further validated that SeIN128 inhibits Basins via GABAergic inputs. Lastly, the authors found that SeIN128 inhibits rolling induced by two types of Basin neurons, Basin-2 and Basin-4. Overall, SeIN128 forms a feedback inhibition ensemble that terminates rolling and shifts the animal to crawling.

      Strengths:<br /> - The question (i.e., the neural circuitry of action selection) addressed by this study is important.<br /> - Larval and adult Drosophila is a powerful model system in neuroscience study, with rich genetic tools, diverse behaviors, and well-studied nervous systems. This study makes good use of them.<br /> - The experiments, analyses, and results are mostly rigorous and support the major claims. This study combined multiple innovative approaches, such as automated, machine-learning-based behavioral assays, EM reconstruction of larval CNS neurons, and genetic manipulation of specific neurons.

      Weaknesses:<br /> - The description of methods and quantification for certain analyses are not clear or detailed enough for a comprehensive judgment of rigorousness, or for other scientists to repeat the experiments. This especially applies to the algorithm.<br /> - "Corkscrew-like rolling" is not an accurate term for larval rolling. The neuromuscular basis of rolling was recently studied by Cooney et. al., showing that rolling is the circumferential propagation of muscle activity where all segments contract similarly and synchronously.<br /> - The readability of the manuscript (text and figures) needs improvement, especially in making it understandable for a general audience. The addition of visual representations, simplifying the complex names of neurons, avoiding overall long sentences, and providing sufficient background introduction may help.

    1. Reviewer #2 (Public Review):

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

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

      Weaknesses:<br /> The paper provides a comprehensive account of visuomotor rotation paradigms, addressing incongruent behavioral results with a solid Bayesian integration model. However, its focus is narrowly confined to visuomotor rotation, leaving its applicability to broader motor learning paradigms, such as force field adaptation, saccadic adaptation, and de novo learning paradigms, uncertain. The paper's impact on the broader fields of neuroscience and cognitive science may be limited due to this specificity. While the paper excellently demonstrates that specific behavioral results in visuomotor rotation can be explained by Bayesian integration, a general computational principle, its contributions to other motor learning paradigms remain to be explored. The paper would benefit from a discussion on the model's generality and its limitations, particularly in relation to the undercompensating effects in other motor learning paradigms.

    1. Reviewer #3 (Public Review):

      Summary:

      The authors aim to provide a multidisciplinary resource on the structural and physiological organization of the hippocampal system and make the available experimental data available for further theoretical work, providing tools to do so in a very flexible and user-friendly way. Since this is a new version of an already existing data-resource, the authors certainly reach their aim and fulfil expectations that the reader might have. The content of the database is as good as the original data, collected from the published knowledge-database, sometimes with help of the original authors, and the overall quality depends further on how the data are curated by the team of authors and many others who helped them. That process is briefly described and more details are available in descriptions of previous versions and on the website. The data extraction, examples of how data can be used and the part on attempts to model the hippocampus are exiting and open doors to new and exciting research opportunities.

      Strengths:

      Excellent description with many outlined opportunities. Nicely illustrated and inviting to explore the online database. The database itself is easy to navigate and to access relevant information, allowing to do further research on the available data.

      Weaknesses:

      The figures are complex, containing a heavy information load. One needs some general knowlegde of the system in order to grasp the enormous potential of what is provided.

    1. Reviewer #2 (Public Review):

      Summary:

      The main purpose of this investigation was to 1) compare the effects of a single knockout (sKO) of Numb or a double knockout (dKO) of Numb and NumbL on ex-vivo physiological properties of the extensor digitorium longus (EDL) muscle in C57BL/6NCrl mice; and 2) analyze protein complexes isolated from C2C12 myotubes via immunoprecipitation and LC/MS/MS for potential Numb binding partners. The main findings are 1) the muscles from sKO and dKO were significantly weaker with little difference between the sKO and dKO lines, indicating the reduced force is mainly due to the inactivation of the Numb gene; and 2) there were 11 potential Numb binding proteins that were identified and cytoskeletal specific proteins including Septin 7.

      Strengths:

      Straight-forward yet elegant design to help determine the important role the Numb has in skeletal muscle.

      Weaknesses:

      There were a limited number of samples (3-6) that were used for the physiological experiments; however, there was a very large effect size in terms of differences in muscle tension development between the induced KO models and the controls.

    1. Reviewer #2 (Public Review):

      Summary: To create a robust and specific fluorescent sensor for aspartate.

      Strengths: Good quality characterisation in a range of environments and experimental conditions.

      Weaknesses: Sensor basically identical to iGluSnFR3, but nevertheless useful and specific. The results support the conclusions, and the paper is very straightforward. I think the work will be useful to people working on the effects of free aspartate in biology and given it is basically iGluSnFR3, which is widely used, should be very reproducible and reliable.

      Other context - it is a good quality study, although seems to be somewhat incremental.

    1. Reviewer #2 (Public Review):

      Summary:

      Deng et al. investigate, for the first time to my knowledge, the role that hippocampal dentate gyrus mossy cells play in Fragile X Syndrome. They provide compelling evidence that, in slice preparations from Fmr1 knockout mice, mossy cells are hypoactive due to increased Kv7 function whereas granule cells are hyperactive compared to slices from wild-type mice. They provide strong evidence that weakness of mossy cell-interneuron connections contribute to granule cell hyperexcitability, despite converse adaptations to mossy cell inputs. The authors show that application of the Kv7 inhibitor XE991 is able to rescue granule cell hyperexcitability back to wild-type baseline, supporting the overall conclusion that inhibition of Kv7 in the dentate may be a potential therapeutic approach for Fragile X Syndrome.

      Strengths:

      Thorough electrophysiological characterization of mossy cells in Fmr1 knockout mice, a novel finding.

      Their electrophysiological approach is quite rigorous: patched different neuron types (GC, MC, INs) one at a time within the dentate gyrus in FMR1 KO and WT, with and without 'circuit blockade' by pharmacologically inhibiting neurotransmission. This allows the most detailed characterization possible of passive membrane/intrinsic cell differences in dentate gyrus of Fmr1 knockout mice.

      Provide several examples showing the use of Kv7 inhibitor XE991 is able to rescue excitability of granule cell circuit in Fmr1 knockout mice (AP firing in intact circuit, postsynaptic current recordings, theta-gamma coupling stimulation)

      Weaknesses:

      Previously identified weaknesses have been addressed.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript provides microprobe serial oxygen isotope data from thin-sectioned modern and fossil orangutan teeth in an effort to reconstruct seasonality of rainfall in Borneo and Sumatra. The authors also explore the hypothesis that nursing could affect early tooth (first molar) isotope values. They find that all molars yield similar oxygen isotope values and therefore conclude that future research need not exclude use of first molars. With regard to seasonality, the modern orangutans yield similar results from both islands. The authors suggest differences between modern and fossil orangutan teeth.

      Strengths:<br /> The study employs a sampling method that captures serial isotope values within thin sections of teeth using a microprobe that provides much higher resolution than traditional hand-held drilling.

      Weaknesses:<br /> The study only examines six modern and six fossil orangutan individuals. Of those, only four modern individuals were samples across multiple molars.

    1. Reviewer #2 (Public Review):

      Summary:

      The manuscript by Rydhmer et al. proposes a new technology to survey insects. They deployed optical sensors in agricultural landscapes and contrast their results to those in classical malaise and sweep nets survey methodologies. They found the results of optical sensors to be comparable with classical survey methodologies. The authors discuss the pros and cons of their near-infrared sensor.

      Strengths:<br /> Contrasting the results of optical sensors with those obtained with classical malaise and sweep nets was a clever idea.

      Weaknesses:<br /> Maybe the first most important shortcoming is the lack of a larger question the new technology can help to answer. If the authors could frame their aims not only as a new tool to sample insects but maybe along the lines of a hypothesis to test in their (agricultural) field of research, this could be a more meaningful article.

      The second more important shortcoming is the lack of more complex analyses. The authors seem to be so fixed on counts of abundance and species that they miss the opportunity to look for more complex patterns in their data. The addition of a simple analysis like an NMDS (to test composition changes) could improve the manuscript significantly.

      The ecosystem process (granivory) assay is currently poorly contextualized and explained across the text; I was surprised to find this part in M&M without previous warning. It seems to me that adding this part could be a nice addition to the manuscript (see my comment above). But this needs to be explained better in all sections of the manuscript.

      As I think that addressing my previous points will reshape the manuscript in important ways, I refrain from giving more specific details at this point. But there are some! Maybe only to mention that Figures 4 and 6 would benefit from individual regressions by crop and Figure 5 from adding results from optical sensors.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This paper describes evolution experiments performed on yeast amino acid transporters aiming at the enlargement of the substrate range of these proteins. Yeast cells lacking 10 endogenous amino acid transporters and thus being strongly impaired to feed on amino acids were again complemented with amino acid transporters from yeast and grown on media with amino acids as the sole nitrogen source.

      In the first set of experiments, complementation was done with seven different yeast amino acid transporters, followed by measuring growth rates. Despite most of them have been described before in other experimental contexts, the authors could show that many of them have a broader substrate range than initially thought.

      Moving to the evolution experiments, the authors used the OrthoRep system to perform random mutagenesis of the transporter gene while it is actively expressed in yeast. The evolution experiments were conducted such that the medium would allow for poor/slow growth of cells expressing the wt transporters, but much better/faster growth if the amino acid transporter would mutate to efficiently take up a poorly transported (as in the case of citrulline and AGP1) or non-transported (as in case of Asp/Glu and PUT4) amino acid.

      This way and using Sanger sequencing of plasmids isolated from faster-growing clones, the authors identified a number of mutations that were repeatedly present in biological replicates. When these mutations were re-introduced into the transporter using site-directed mutagenesis, faster growth on the said amino acids was confirmed. Growth phenotype data were attempted to be confirmed by uptake experiments using radioactive amino acids; however, the radioactive uptake data and growth-dependent analyses do not fully match, hinting at the existence of further parameters than only amino acid uptake alone to impact the growth rates.

      When mapped to Alphafold prediction models on the transporters, the mutations mapped to the substrate permeation site, which suggests that the changes allow for more favourable molecular interactions with the newly transported amino acids.

      Finally, the authors compared the growth rates of the evolved transporter variants with those of the wt transporter and found that some variants exhibit a somewhat diminished capacity to transport its original range of amino acids, while other variants were as fit as the wt transporter in terms of uptake of its original range of amino acids.

      Based on these findings, the authors conclude that transporters can evolve novel substrates through generalist intermediates, either by increasing a weak activity or by establishing a new one.

      Strengths:<br /> The study provides evidence in favour of an evolutionary model, wherein a transporter can "learn" to translocate novel substrates without "forgetting" what it used to transport before. This evolutionary concept has been proposed for enzymes before, and this study shows that it also can be applied to transporters. The concept behind the study is easy to understand, i.e. improving growth by uptake of more amino acids as nitrogen source. In addition, the study contains a large and extensive characterization of the transporter variants, including growth assays and radioactive uptake measurements.

      Weaknesses:<br /> The authors took a genetic gain-of-function approach based on random mutagenesis of the transporter. While this has worked out for two transporters/substrate combinations, I wonder how comprehensive and general the insights are. In such approaches, it is difficult to know which mutation space is finally covered/tested. And information that can be gained from loss-of-function analyses is missed. The entire conclusions are grounded on a handful of variants analyzed. Accordingly, the outcome is somewhat anecdotal; in some cases, the fitness of the variants was changed and in others not. Highlighting the amino acid changes in the context of the structural models is interesting, but does not fully explain why the variants exhibit changed substrate ranges. Two important technical elements have not been studied in detail by the authors, but may well play a certain role in the interpretation of the results. Firstly, the authors did not quantify the amount of transporter being present on the cell surface; altered surface expression can impact uptake rates and thus growth rates. Secondly, the authors have not assessed whether overexpressing wt versus variant transporters has an impact on the growth rate per se. Overexpressing transporters from plasmids is quite a burden for the cells and often impacts growth rates. Variants may be more or less of a burden, an effect that may (or may also not) go hand in hand with increased/decreased surface production levels.

      And finally, I was somewhat missing an evolutionary analysis of these transporters to gain insights into whether the identified substitutions also occurred during natural evolution under real-life conditions.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This paper describes a new approach to detecting directed causal interactions between two genes without directly perturbing either gene. To check whether gene X influences gene Z, a reporter gene (Y) is engineered into the cell in such a way that (1) Y is under the same transcriptional control as X, and (2) Y does not influence Z. Then, under the null hypothesis that X does not affect Z, the authors derive an equation that describes the relationship between the covariance of X and Z and the covariance of Y and Z. Violation of this relationship can then be used to detect causality.

      The authors benchmark their approach experimentally in several synthetic circuits. In four positive control circuits, X is a TetR-YFP fusion protein that represses Z, which is an RFP reporter. The proposed approach detected the repression interaction in two or three of the positive control circuits. The authors constructed sixteen negative control circuit designs in which X was again TetR-YFP, but where Z was either a constitutively expressed reporter or simply the cellular growth rate. The proposed method detected a causal effect in two of the sixteen negative controls, which the authors argue is not a false positive, but due to an unexpected causal effect. Overall, these pilot studies, albeit in simplified scenarios, provide encouraging results.

      Strengths:<br /> The idea of a "no-causality control" in the context of detected directed gene interactions is a valuable conceptual advance that could potentially see play in a variety of settings where perturbation-based causality detection experiments are made difficult by practical considerations.

      By proving their mathematical result in the context of a continuous-time Markov chain, the authors use a more realistic model of the cell than, for instance, a set of deterministic ordinary differential equations.

      Caveats:<br /> The term "causally" is used in the main-text statement of the central theorem (Eq 2) without a definition of this term. This makes it difficult to fully understand the statement of the paper's central theorem without diving into the supplement.

      The basic argument of theorem 1 appears to rely on establishing that x(t) and y(t) are independent of their initial conditions. Yet, there appear to be some scenarios where this property breaks down:

      (1) Theorem 1 does not seem to hold in the edge case where R=beta=W=0, meaning that the components of interest do not vary with time, or perhaps vary in time only due to measurement noise. In this case x(t), y(t), and z(t) depend on x(0), y(0), and z(0). Since the distributions of x(0), y(0), and z(0) are unspecified, a counterexample to the theorem may be readily constructed by manipulating the covariance matrix of x(0), y(0), and z(0).

      (2) A similar problem may occur when transition probabilities decay with time. For example, suppose that again R=0 and X are degraded by a protease (B), but this protease is subject to its own first-order degradation. The deterministic version of this situation can be written, for example, dx/dt=-bx and db/dt=-b. In this system, x(t) approaches x(0)exp(-b(0)) for large t. Thus, as above, x(t) depends on x(0). If similar dynamics apply to the Y and Z genes, we can make all genes depend on their initial conditions, thus producing a pathology analogous to the above example.

      The reviewer does not know when such examples may occur in (bio)physical systems. Nevertheless, since one of the advantages of mathematics is the ability to correctly identify the domain of validity for a claim, the present work would be strengthened by "building a fence" around these edge cases, either by identifying the comprehensive set of such edge cases and explicitly prohibiting them in a stated assumption set, or by pointing out how the existing assumptions already exclude them.

    1. Reviewer #2 (Public Review):

      Summary:

      The manuscript by Bomba-Warczak et al. applied multi-isotope imaging mass spectrometry (MIMS) analysis to identify the long-lived proteins in mouse ovaries during reproductive aging, and found some proteins related to cytoskeletal and mitochondrial dynamics persisting for 10 months.

      Strengths:

      The manuscript provides a useful dataset about protein turnover during ovarian aging in mice.

      Weaknesses:

      The study is pretty descriptive and short of further new findings based on the dataset. In addition, some results such as the numbers of follicles and ovulated oocytes in aged mice are not consistent with the published literature, and the method for follicle counting is not accurate. The conclusions are not fully supported by the presented evidence.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Temporal binding, generally considered a timing illusion, results from actions triggering outcomes after a brief delay, distorting perceived timing. The present study investigates the relationship between attention and the perception of timing by employing a series of tasks involving auditory and visual stimuli. The results highlight the role of attention in event timing and the functional relevance of attention in outcome binding.

      Strengths:<br /> - Experimental Design: The manuscript details a well-structured sequence of experiments investigating the attention effect in outcome binding. Thoughtful variations in manipulation conditions and stimuli contribute to a thorough and meaningful investigation of the phenomenon.<br /> - Statistical Analysis: The manuscript employs a diverse set of statistical tests, demonstrating careful selection and execution. This statistical approach enhances the reliability of the reported findings.<br /> - Narrative Clarity: Both in-text descriptions and figures provide clear insights into the experiments and their results, facilitating readers in following the logic of the study.

      Weaknesses:<br /> - Conceptual Clarity: The manuscript aims to integrate key concepts in human cognitive functions, including attention, timing perception, and sensorimotor processes. However, before introducing experiments, there's a need for clearer definitions and explanations of these concepts and their known and unknown interrelationships. Given the complexity of attention, a more detailed discussion, including specific types and properties, would enhance reader comprehension.<br /> - Computational Modeling: The manuscript lacks clarity in explaining the model architecture and setup, and it's unclear if control comparisons were conducted. These details are critical for readers to properly interpret attention-related findings in the modeling section. Providing a clearer overview of these aspects will improve the overall understanding of the computational models used.

    1. Reviewer #3 (Public Review):

      Summary:

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

      Strengths:

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

    2. Reviewer #2 (Public Review):

      Summary:

      In this manuscript, Hariharan and colleagues present an elegant study regarding the mechanistic basis of sugar transport by the prototypical Na+-coupled transporter MelB. The authors identified a nanobody (Nb 725) that reduces melibiose binding but not Na+ binding. In vitro (ITC) experiments suggest that the conformation targeted by this nanobody is different from the published outward-open structures. They go on to solve the structure of this other conformational by cryo-EM using the Nanobody grafted with a fiducial marker and enhancer and, as predicted, capture a new conformation of MelB, namely the inward-open conformation. Through MD simulations and ITC measurements, they demonstrate that such state has a reduced affinity for sugar but that Na+ binding is mostly unaffected. A detailed observation and comparison between previously published structures in the outward-open conformation and this new conformational intermediate allows to strengthen and develop the mobile barrier hypothesis underpinning sugar transport. The conformational transition to the inward-facing state leads to the formation of a barrier on the extracellular side that directly affects the amino acid arrangement of the sugar binding site, leading to a decreased affinity that drives the direction of transport. In contrast, the Na+ binding remains the same. This structural data is complemented with dynamic insights from HDX-MS experiments conducted in the presence and absence of the Nb. These measurements highlight the overall protective effect of nanobody binding, consistent with the stabilization of one conformational intermediate.

      Strengths:

      The experimental strategy to isolate this elusive conformational intermediate is smart and well-executed. The biochemical and biophysical data were obtained in a lipid system (nanodiscs), which allows dismissing questions about detergent induced artefacts. The new conformation observed is of great interest and allows to have a better mechanistic understanding of ion-coupled sugar transport. The comparison between the two structures and the mobile barrier mechanism hypothesis is convincingly depicted and tested.

      Weaknesses:

      This is excellent experimental work. My recommendations stem mostly from concerns regarding the interpretation of the observed results. In particular, I am somewhat puzzled by the important role the authors give to the regulatory protein EIIa with little structural or biophysical data to back up their claims. The hypothesis that the conformation captured by the Nb is physiologically and functionally equivalent to that caused by EIIa binding is definitely a worthy hypothesis, but it is not an experimental result.

      Evidence in support could include a structure with EIIa bound. Since it does not bind at the same location as the Nb, it seems feasible. Or, the authors could have performed HDX-MS in the presence of EIIa to determine if the effect is similar to that of Nb_725 binding. In the absence of these experiments, discussion about EIIa should be limited. Along the same lines, I find it misleading to put in the abstract a sentence such as "It is the first structure of a major facilitator superfamily (MFS) transporter with experimentally determined cation binding, and also a structure mimicking the physiological regulatory state of MelB under the global regulator EIIAGlc of the glucose-specific phosphoenolpyruvate:phosphotransferase system." None of this is supported by the experimental work presented in this article: the Na+ is modelled (with great confidence, but still) and whether this structure mimics the physiological state of MelB bound to EIIa is not known. The results of the paper are strong and interesting enough per se, and there is no need to inflate them with hypothesis that belongs to the discussion section.

      I also note that the HDX-MS experiments do not distinguish between two conformational states, but rather an ensemble of states vs one state.

    1. Reviewer #3 (Public Review):

      The authors collected BALF samples from lung cancer patients newly diagnosed with PCP, DI-ILD or ICI-ILD. CyTOF was performed on these samples, using two different panels (T-cell and B-cell/myeloid cell panels). Results were collected, cleaned-up, manually gated and pre-processed prior to visualisation with manifold learning approaches t-SNE (in the form of viSNE) or UMAP, and analysed by CITRUS (hierarchical clustering followed by feature selection and regression) for population identification - all using Cytobank implementation - in an attempt to identify possible biomarkers for these disease states. By comparing cell abundances from CITRUS results and qualitative inspection of a small number of marker expressions, the authors claimed to have identified an expansion of CD16+ T-cell population in PCP cases and an increase in CD57+ CD8+ T-cells, FCRL5+ B-cells and CCR2+ CCR5+ CD14+ monocytes in ICI-ILD cases.

      By the authors' own admission, there is an absence of healthy donor samples and, perhaps as a result of retrospective experimental design and practical clinical reasons, also an absence of pre-treatment samples. The entire analysis effectively compares three yet-established disease states with no common baseline - what really constitutes a "biomarker" in such cases? These are very limited comparisons among three, and only these three, states.

      By including a new scRNA-Seq analysis using publicly available dataset, the authors addressed this fundamental problem. Though more thorough and numerical analysis would be appreciated for a deeper and more impactful analysis, this is adequate for the intended objectives of the study.

    1. Reviewer #2 (Public Review):

      The manuscript by Wang et al., follows up on the group's previous publication on KLF1 (EKLF) K47R mice and reduced susceptibility to tumorigenesis and increased life span (Shyu et al., Adv Sci (Weinh). Sep 2022;9(25):e2201409. doi:10.1002/advs.202201409). In the current manuscript, the authors have described these phenotypes in the context of age, gender, genetic background, and hematopoietic transplantation of bone marrow mononuclear cells. Despite the revisions, significant conceptual concerns still remain in the study that make the inferences in the manuscript less convincing. Major concerns are listed below.

      Major concerns:

      1. The authors mention more than once in the manuscript that KLF1 is expressed in range of blood cells including hematopoietic stem cells, megakaryocytes, T cells and NK cells. In the case of megakaryocytes, studies from multiple labs have shown that while EKLF is expressed megakaryocyte-erythroid progenitors, EKLF is important for the bipotential lineage decision of these progenitors, and its high expression promotes erythropoiesis, while its expression is antagonized during megakaryopoiesis. In the case of HSCs, the authors reference to their previous publication for KLF1's expression in these cells- however, in this study nor in the current study, there is no western blot documented to convincingly show that KLF1 protein is expressed at detectable levels in these cells. For T cells, the authors have referenced a study which is based on ectopic expression of KLF1. For NK cells, the authors reference bioGPS: however, upon inspection, this is also questionable. As part of the revision, the authors have provided western blots in supplemental figure S4. However, these blots are difficult to interpret, since the EKLF bands for NK cells, and T cells are very faint and since the positive control EKLF band from MEL erythroid cell lysates is oversaturated, to interpret the data clearly. Therefore, although a quantification is shown, the representative blot included for EKLF protein levels is not convincing.

      2. The current study rests on the premise that KLF1 is expressed in HSCs, NK cells and leukocytes, and the references cited are not sufficient to make this assumption, for the reasons mentioned in the first point. Therefore, the authors were asked to show both KLF1 mRNA and protein levels in these cells, and also compare them to the expression levels seen in KLF1 wild type erythroid cells along with knockout erythroid cells as controls, for context and specificity. The authors have now included western blots and mRNA levels and have compared it to MEL erythroid cells. This data raises additional questions. Overall, the mRNA levels in CD3+ T cells and B220+ B cells are approximately 3000 fold lower than MEL erythroid cells. Based on the information provided, although unclear, the assumption is that the MEL cell extracts are from undifferentiated cells. Therefore, this raises questions on the inference that the healthy aging phenotype is a result of cell intrinsic effects, since EKLF expression in these cells of interest is extremely low. This also allows for the consideration for potential systemic/indirect effects.

      3. In the discussion, the authors make broad inferences that go beyond the data shown in the manuscript. For example, they mention that the tumorigenesis resistance and long lifespan is most likely due to changes in transcription regulatory properties and changes in global gene expression profile of the mutant protein relative to WT leukocytes. And based on reduced mRNA levels of Pd-1 Pd-l1 genes in the CD3+ T cells and B220+ B cells from mutant mice, they "assert" that EKLF is an upstream regulator of these genes and regulates the transcriptomes of a diverse range of hematopoietic cells. The authors were asked to perform a ChIP assay to show whether WT EKLF binds on these genes in these cells, and whether this binding is reduced or abolished in the mutant cells, to substantiate the above statements. The authors have now included a ChIP assay in Figure S5. The data on WT EKLF and K74R EKLF on Pd-1 promoter shows that both forms of EKLF bind at similar levels. Therefore, the mechanism remains unclear, and there is insufficient discussion on how their data support cell intrinsic differences in transcriptional regulation between WT and mutant EKLF.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors sampled the B cell receptor repertoires of Cancers, their draining lymph nodes, and blood. They characterized the clonal makeup of all B cells sampled and then analyzed these clones to identify clonal overlap between tissues and clonal activation as expressed by their mutation level and CDR3 amino acid characteristics and length. They conclude that B cell clones from the Tumor interact more with their draining lymph node than with the blood and that there is less mutation/expansion/activation of B cell clones in Tumors. These conclusions are interesting but hard to verify due to the under-sampling and short sequencing reads as well as confusion as to when analysis is across all individuals or of select individuals.

      Strengths:

      The main strength of their analysis is that they take into account multiple characteristics of clonal expansion and activation and their different modes of visualization, especially of clonal expansion and overlap. The triangle plots once one gets used to them are very nice.

      Weaknesses:

      The data used appears inadequate for the conclusions reached. The authors' sample size of B cells is small and they do not address how it could be sufficient. at such low sampling rates, compounded by the palsmablast bias they mention, it is unclear if the overlap trends they observe show real trends. Analyzing only top clones by size does not solve this issue. As it could be that the top 100 clones of one tissue are much bigger than those of another and that all overlap trends are simply because the clones are bigger in one tissue or the other. i.e there is equal overlap of clones with blood but blood is not sufficiently sampled given its greater diversity and smaller clones. Similarly, the read length (150bp X2) is too short missing FWR1 and CDR1 and often parts of FWR2 if CDR3 is long. As the authors themselves note (and as was shown in (Zhang 2015 - PMC4811607) this makes mutation analysis difficult. It also makes the identification of V genes and thus clonal identification ambiguous. This issue becomes especially egregious when clones are mutated. Finally, it is not completely clear when the analysis is of single individuals or across all individuals. If it is the former the authors did not explain how they chose the individuals analyzed and if the latter then it is not clear from the figures which measurements belong to which individual (i.e they are mixing measurements from different people). For all these reasons while the authors make many interesting suggestions about the potential relationships of B cell repertoires in cancer tissues and their draining lymph nodes and how to characterize and visualize them, it is hard to assess any of their conclusions and specific results.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The study entitled "Different coexistence patterns between apex carnivores and mesocarnivores based on temporal, spatial, and dietary niche partitioning analysis in Qilian Mountain National Park, China" by Cong et al. addresses the compelling topic of carnivores' coexistence in a biodiversity hotspot in China. The study is interesting given it considers all three components affecting sympatric carnivores' distribution and co-occurrence, namely the temporal, the spatial, and the dietary partition within the carnivore guild. The authors have found that spatial co-occurrence is generally low, which represents the major strategy for coexistence, while there is temporal and dietary overlap. I also appreciated the huge sampling effort carried out for this study by the authors: they were able to deploy 280 camera trapping sites (which became 322 in the result section?) and collect a total of 480 scat samples. However, I have some concerns about the study on the non-consideration of the human dimension and potential anthropogenic disturbance that could affect the spatial and temporal distribution of carnivores, the choice of the statistical model to test co-occurrence, and the lack of clearly stated ecological hypotheses.

      Strengths:<br /> The strengths of the study are the investigation of all three major strategies that can mitigate carnivores' coexistence, therefore, the use of multiple monitoring techniques (both camera trapping and DNA metabarcoding) and the big dataset produced that consists of a very large sampled area with a noteworthy number of camera tap stations and many scat samples for each species.

      Weaknesses:<br /> I think that some parts of the manuscript should be written better and more clearly. A clear statement of the ecological hypotheses that could affect the partitioning among the carnivore guild is lacking. I think that the human component (thus anthropogenic disturbance) should have been considered more in the spatial analyses given it can influence the use of the environment by some carnivores. Additionally, a multi-species co-occurrence model would have been a more robust approach to test for spatial co-occurrence given it also considers imperfect detection.

      Temporal and dietary results are solid and this latter in particular highlights a big predation pressure on some prey species such as the pika. This implies important conservation and management implications for this species, and therefore for the trophic chain, given that i) the pika population should be conserved and ii) a potential poisoning campaign against small mammals could be incredibly dangerous also for mesocarnivores feeding on them due to secondary poisoning.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this study, Zhang and colleagues characterise the behaviour of mouse hematopoietic stem cells when cultured in PVA conditions, a recently published method for HSC expansion (Wilkinson et al., Nature, 2019), using multiome analysis (scRNA-seq and scATACseq in the same single cell) and extensive transplantation experiments. The latter are performed in several settings including barcoding and avoiding recipient conditioning. Collectively the authors identify several interesting properties of these cultures namely: 1) only very few cells within these cultures have long-term repopulation capacity, many others however have progenitor properties which can rescue mice from lethal myeloablation; 2) single cell characterisation by combined scRNAseq and scATACseq is not sufficient to identify cells with repopulation capacity; 3) expanded HSCs can be engrafted in unconditioned host and return to quiescence.

      The authors also confirm previous studies that EPCRhigh HSCs have better reconstitution capability than EPCRlow HSCs when transplanted.

      Strengths:<br /> The major strength of this manuscript is that it describes how functional HSCs are expanded in PVA cultures to a deeper extent that what has been done in the original publication. The authors are also mindful of considering the complexities of interpreting transplantation data. As these PVA cultures become more widely used by the HSC community, this manuscript is valuable as it provides a better understanding of the model and its limitations.

      Novelty aspects include:<br /> • The authors determined that small numbers of expanded HSCs enable transplantation into non-conditioned syngeneic recipients.<br /> • This is to my knowledge the first report characterising output of PVA cultures by multiome. This could be a very useful resource to the field.<br /> • They are also the first to my knowledge to use barcoding to quantify HSC repopulation capacity at the clonal level after PVA culture.<br /> • It is also useful to report that HSCs isolated from fetal livers do expand less than their adult counterparts in these PVA cultures.

      Weaknesses:<br /> • The analysis of the multiome experiment is limited. The authors do not discuss what cell types, other than functional or phenotypic HSCs are present in these cultures (are they mostly progenitors or bona fide mature cells?) and no quantifications are provided. It seems nonetheless that most cells in these cultures do not acquire differentiation markers. In addition, the functional experiments demosntrate very few retain transplantation capacity. Future works will have to investigate the nature of the bulk of the other cells in these cultures.<br /> • Barcoding experiments are technically elegant but do not bring particularly novel insights.<br /> • Number of mice analysed in certain experiments is fairly low (Figure 1 and 5).<br /> • The manuscript remains largely descriptive. While the data can be used to make useful recommendations to future users working with PVA cultures and in general with HSCs, those recommendations could be more clearly spelled out in the discussion.<br /> • The authors could have provided discussion of the other publications/preprints which have used these methods to date. This would have been useful for researchers who have not used this technique.

      Overall, the authors succeeded in providing a useful set of experiments to better interpret what type of HSCs are expanded in PVA cultures. More in depth mining of their bioinformatic data (by the authors or other groups) is likely to highlight other interesting/relevant aspects of HSC biology in relation to this expansion methodology.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, the membrane component of the sialic acid-specific TRAP transporter, SiaQM (HiSiaQM), from H. influenzae, is structurally characterized. TRAP transporters are substrate binding protein (SBP)-dependent secondary-active transporters, and HiSiaQM is the most comprehensively studied member of this family. While all previous work on fused TRAP transporter membrane proteins suggests that they are monomeric (including the previous structural characterization of HiSiaQM by a different group), a surprising finding from this work is the observation that HiSiaQM can form higher oligomers, consistent with it being a dimer. These higher oligomeric states were initially observed after extraction of the protein with LMNG detergent, but were also observed in DDM detergent, amphipol and nanodiscs using analytical ultracentrifugation (AUC). Structural characterization of dimeric HiSiaQM revealed 2 arrangements, a parallel and antiparallel arrangements, the latter of which is unlikely to be physiologically relevant.

      The higher resolution of this new structure of HiSiaQM (2.2-2.7 Å compared to 4.7 Å for the previous structure) facilitated the assignment of bound lipids at the dimer interface and a lipid molecule embedded in each of the protomers; allowed for a clearer refinement of the Na+ and putative substrate binding sites, which differ slightly from the previous structure; and produced better modelled side chains for the residues involved in the SBP:HiSiaQM interaction. The authors developed a useful AUC-based assay to determine the affinity for this interaction revealing an affinity of 65 µM. Finally, the authors make the very interesting observation that a sialic acid specific SBP from a different TRAP transporter can utilize HiSiaQM for transport, contrary to previous observations, revealing for the first time that TRAP membrane components can recognize multiple SBPs.

      Overall, this is a well written and presented manuscript detailing some interesting new observations about this interesting protein family. One of the main findings, that the protein can form a dimer, is supported by data, but the physiological relevance of this is questionable, and the possibility that this is artefactual has not been ruled out. Conclusions regarding the mechanistic importance of the lipid bindings sites is not currently supported by the data.

      Strengths:<br /> The main strength of this work is the increased resolution of HiSiaQM, which allows for much more precise assignment of side chains and their orientation. This will be of importance for subsequent mechanistic studies on the contributions of these residues to Na+ and sialic acid binding and conformational changes.<br /> The observation of the lipids, especially the lipid embedded near the fusion helix, is an intriguing observation, which lays the groundwork for future work to understand the lipid-dependence of these transporters.<br /> The development of the AUC-based approach to measure SBP affinity for the membrane component will likely prove be useful to future studies.

      Weaknesses:<br /> One of the main results from this work is the observation that HiSiaQM can form a dimer. Two arrangements were observed, parallel and antiparallel, the latter of which is almost certainly physiologically irrelevant as it would preclude essential interactions with the extracytoplasmic substrate binding protein. As acknowledged by the author, this non-physiological arrangement is likely a consequence of protein preparation (overexpression, extraction, purification, etc.). However, if one dismisses the antiparallel arrangement as non-relevant and an artefact of protein preparation, it is difficult for the parallel arrangement to maintain its credibility, as it was also processed in the same way. This is especially true when one considers that there is only 100 Å2 buried surface area in the parallel arrangement that does not involve any sidechains; it is difficult to envisage this as a specific interaction, e.g. compared to related proteins that have ~2000 Å2 buried surface area. Unless this dimerization is observed in a bacterial membrane at physiological protein concentrations, it is difficult to rule out the possibility that the observed dimerization is merely an artefact caused by the expression, purification and concentration of the protein.

      The manuscript contains some excellent structural analysis of this protein, whose higher resolution reveals some new and interesting insight. However, a weakness of the current work is a lack of validation of these observations using other approaches. For example, lipid interactions are observed in the structure that the authors claim is mechanistically important. However, without disrupting these interactions to look at the effect on transport, this conclusion is not supported. Similarly, the authors use their structure to predict residues that are important for the SBP:membrane protein interaction, and they develop an AUC-based binding assay to study this interaction, but they do not test their predictions using this approach.

    1. Reviewer #2 (Public Review):

      Summary:

      In this manuscript, the authors reported a study to uncover that β-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of Glucocorticoid-induced osteonecrosis of the femoral head (GONFH). In this study, they first observed abnormal osteogenesis and adipogenesis associated with decreased β-catenin in the necrotic femoral head of GONFH patients, but the exact pathological mechanisms of GONFH remain unknown. They then performed in vivo and in vitro studies to further revealed that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation bone marrow stromal cells (BMSCs) by inhibiting β-catenin signaling in glucocorticoid-induced GONFH rats, and specific deletion of β-catenin in Col2+ cells shifted BMSCs commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice.

      Strengths:

      This innovative study provides strong evidence supporting that β-catenin inhibition disrupts the homeostasis of osteogenic/adipogenic differentiation that contributes to the development of GONFH. This study also identifies an ideal genetic modified mouse model of GONFH. Overall, the experiment is logically designed, the figures are clear, and the data generated from humans and animals is abundant supporting their conclusions.

      Weaknesses:

      Lack of the discussion to explain how the Wnt agonist 1 works. There are several types of Wnt ligands. It is not clear if this agonist only targets Wnt1 or other Wnts as well? Also, why Wnt agonist 1 couldn't rescue the GONFH-like phenotype in β-cateninCol2ER mice needs to be discussed.