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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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."

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.

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.

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.

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.

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?

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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).

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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?

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Reviewer #2 (Public Review):

Strengths<br /> - the study combines different methods (pupillometry, RNNs, fMRI).<br /> - the study combines different viewpoints and fields of the scientific literature, including neuroscience, psychology, physics, dynamical systems.<br /> - This combination of methods and viewpoints is rarely done, it is thus very useful.<br /> - Overall well-written.

Weaknesses<br /> - The study relies on a report paradigm: participants report when they identify a switch in the item category. The sequence corresponds to the drawing of an object being gradually morphed into another object. Perceptual switches are therefore behaviorally relevant, and it is not clear whether the effect reported correspond to the perceptual switch per se, or the detection of an event that should change behavior (participant press a button indicating the perceived category, and thus switch buttons when they identify a perceptual change). The text mentions that motor actions are controlled for, but this fact only indicates that a motor action is performed on each trial (not only on the switch trial); there is still a motor change confounded with the switch. As a result, it is not clear whether the effect reported in pupil size, brain dynamics, and brain states is related to a perceptual change, or a decision process (to report this change).

- The study presents events that co-occur (perceptual switch, change in pupil size, energy landscape of brain dynamics) but we cannot identify the causes and consequences. Yet, the paper makes several claims about causality (e.g. in the abstract "neuromodulatory tone ... causally mediates perceptual switches", in the results "the system flattening the energy landscape ... facilitated an updating of the content of perception").

- Some effects may reflect the expectation of a perceptual switch, rather than the perceptual switch per se. Given the structure of the task, participants know that there will be a perceptual switch occurring once during a sequence of morphed drawings. This change is expected to occur roughly in the middle of the sequence, making early switches more surprising, and later switches less surprising. Differences in pupil response to early, medium, and late switches could reflect this expectation. The authors interpret this effect very differently ("the speed of a perceptual switch should be dependent on LC activity").

- The RNN is far more complex than needed for the task. It has two input units that indicate the level of evidence for the two categories being morphed, and it is trained to output the dominant category. A (non-recurrent) network with only these two units and an output unit whose activity is a sigmoid transform of the difference in the inputs can solve the task perfectly. The RNN activity is almost 1-dimensional probably for this reason. In addition, the difficult part of the computation done by the human brain in this task is already solved in the input that is provided to the network (the brain is not provided with the evidence level for each category, and in fact, it does not know in advance what the second category will be).

- Basic fMRI results are missing and would be useful, before using elaborate analyses. For instance, what are the regions that are more active when a switch is detected?

- The use of methods from physics may obscure some simple facts and simpler explanations. For instance, does the flatter energy landscape in the higher gain condition reflect a smaller number of states visited in the state space of the RNN because the activity of each unit gets in the saturation range? If correct, then it may be a more straightforward way of explaining the results.

- Some results are not as expected as the authors claim, at least in the current form of the paper. For instance, they show that, when trained to identify which of two inputs u1 and u2 is the largest (with u2=1-u1, starting with u1=1 and gradually decreasing u1), a higher gain results in the RNN reporting a switch in dominance before the true switch (e.g. when u1=0.6 and u2=0.4), and vice et versa with a lower gain. In other words, it seems to correspond to a change in criterion or bias in the RNN's decision. The authors should discuss more specifically how this result is related to previous studies and models on gain modulation. An alternative finding could have been that the network output is a more (or less) deterministic function of its inputs, but this aspect is not reported.

Reviewer #2 (Public Review):

This work presents a remarkably extensive set of experiments, assaying the interaction between methylation and expression across most CpG positions in the genome in two cell types. To this end, the authors use mSTARR-seq, a high-throughput method, which they have previously developed, where sequences are tested for their regulatory activity in two conditions (methylated and unmethylated) using a reporter gene. The authors use these data to study two aspects of DNA methylation: 1. Its effect on expression, and 2. Its interaction with the environment. Overall, they identify a small number of 600 bp windows that show regulatory potential, and a relatively large fraction of these show an effect of methylation on expression. In addition, the authors find regions exhibiting methylation-dependent response to two environmental stimuli (interferon alpha and glucocorticoid dexamethasone).

The questions the authors address represent some of the most central in functional genomics, and the method utilized is currently the best method to do so. The scope of this study is very impressive and I am certain that these data will become an important resource for the community. The authors are also able to report several important findings, including that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures.

Reviewer #2 (Public Review):

Summary: In this manuscript, the authors examined the role of the bile acid receptor TGR5 in the bone marrow under steady-state and stress hematopoiesis. They initially showed the expression of TGR5 in hematopoietic compartments and that loss of TGR5 doesn't impair steady-state hematopoiesis. They further demonstrated that TGR5 knockout significantly decreases BMAT, increases the APC population, and accelerates the recovery upon bone marrow transplantation.

Strengths: The manuscript is well-structured and well-written.

Weaknesses: The mechanism is not clear, and additional studies need to be performed to support the authors' conclusion.

Reviewer #3 (Public Review):

Male infertility is an important health problem. Among pathologies with multiple morphological abnormalities of the flagellum (MMAF), only 50% of the patients have no identified genetic causes. It is thus primordial to find novel genes that cause the MMAF syndrome. In the current work, the authors follow up the identification of two patients with MMAF carrying a mutation in the CCDC146 gene. To understand how mutations in CCDC146 lead to male infertility, the authors generated two mouse models: a CCDC146-knockout mouse, and a knockin mouse in which the CCDC146 locus is tagged with an HA tag. Male CCDC146-knockout mice are infertile, which proves the causative role of this gene in the observed MMAF cases. Strikingly, animals develop no other obvious pathologies, thus underpinning the specific role of CCDC146 in male fertility. The authors have carefully characterised the subcellular roles of CCDC146 by using a combination of expansion and electron microscopy. They demonstrate that all microtubule-based organelles, such as the sperm manchette, the centrioles, as well as the sperm axonemes are defective when CCDC146 is absent. Their data show that CCDC146 is a microtubule-associated protein, and indicate, but do not prove beyond any doubt, that it could be a microtubule-inner protein (MIP).

This is a solid work that defines CCDC146 as a novel cause of male infertility. The authors have performed comprehensive phenotypic analysis to define the defects in CCDC146 knockout mice. The manuscript text is well written and easy to follow also for non-specialists. The introduction and discussion chapters contain important background information that allow to put the current work into the greater context of fertility research. Overall, this manuscript provides convincing evidence for CCDC146 being essential for male fertility and illustrates this with a large panel of phenotypic observations. Together, the work provides important first insights into the role of a so-far unexplored proteins, CCDC146, in spermatogenesis, thereby broadening the spectrum of genes involved in male infertility.

Reviewer #2 (Public Review):

Summary:<br /> In this study, the authors hypothesized that individuals with diabetes have elevated blood CTSL levels, which facilitates SARS-CoV-2 infection. The authors conducted in vitro experiments, revealing that elevated glucose levels promote SARS-CoV-2 infection in wild-type cells. In contrast, CTSL knockout cells show reduced susceptibility to high glucose-promoted effects. Additionally, the authors utilized lung tissue samples obtained from both diabetic and non-diabetic patients, along with db/db diabetic and control mice. Their findings indicate that diabetic conditions lead to an elevation in CTSL activity in both humans and mice.

Strengths:<br /> The authors have effectively met their research objectives, and their conclusions are supported by the data presented. Their findings suggest that high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum to the lysosome, potentially contributing to diabetic comorbidities and complications.

Weaknesses:<br /> 1. In Figure 1e, the authors measured plasma levels of COVID-19 related proteins, including ACE2, CTSL, and CTSB, in both diabetic and non-diabetic COVID-19 patients. Notably, only CTSL levels exhibited a significant increase in diabetic patients compared to non-diabetic patients, and these levels varied throughout the course of COVID-19. Given that the diabetes groups encompass both male and female patients, it is essential to ascertain whether the authors considered the potential impact of gender on CTSL levels. The diabetes groups comprised a higher percentage of male patients (61.3%) compared to the non-diabetes group, where males constituted only 38.7%.

2. Lines 145-149: "The results showed that WT Huh7 cell cultured in high glucose medium exhibited a much higher infective rate than those in low glucose medium. However, CTSL KO Huh7 cells maintained a low infective rate of SARS-CoV-2 regardless of glucose or insulin levels (Fig. 3f-h). Therefore, hyperglycemia enhanced SARS-CoV-2 infection dependent on CTSL." However, this evidence may be insufficient to support the claim that hyperglycemia enhances SARS-CoV-2 infection dependent on CTSL. The human hepatoma cell line Huh7 might not be an ideal model to validate the authors' hypothesis regarding high blood glucose promoting SARS-CoV-2 infection through CTSL.

3. The Abstract and Introduction sections lack effective organization.

Reviewer #2 (Public Review):

The present work analyzed the mitochondrial function and bioenergetics in the context of cancer cachexia induced by pancreatic cancer (PDAC). The authors used the KIC transgenic mice that spontaneously develop PDAC within 9-11 weeks of age. They deeply characterize bioenergetics in living mice by magnetic resonance (MR) and mitochondrial function/morphology mainly by oxygraphy and imaging on ex vivo muscles. By MR they found that phosphocreatine resynthesis and maximal oxidative capacity were reduced in the gastrocnemius muscle of tumor-bearing mice during the recovery phase after 6 minutes of 1 Hz electrical stimulation while pH was reduced in muscle during the stimulation time. By oxygraphy, the authors showed a decrease in basal respiration, proton leak, and maximal respiration in tumor-bearing mice that was associated with the decrease of complex I, II, and IV activity, a reduction of OXPHOS proteins, mitochondrial mass, mtDNA, and to several morphological alterations of mitochondrial shape. The authors performed transcriptomic and proteomic analyses to get insights into mitochondrial defects in the muscles of PDAC mice. By IPA analyses on transcriptomics, they found an increase in the signature of protein degradation, atrophy, and glycolysis and a downregulation of muscle function. Focusing on mitochondria they showed a downregulation mainly in OXPHOS, TCA cycle, and mitochondrial dynamics genes and upregulation of glycolysis, ROS defense, mitophagy, and amino acid metabolism. IPA analysis on proteomics revealed major changes in muscle contraction and metabolic pathways related to lipids, protein, nucleotide, and DNA metabolism. Focusing on mitochondria, the protein changes mainly were related to OXPHOS, TCA cycle, translation, and amino acid metabolism.

The major strength of the paper is the bioenergetics and mitochondrial characterization associated with the transcriptomic and proteomic analyses in PDAC mice that confirmed some published data of mitochondrial dysfunction but underlined some novel metabolic insights such as nucleotide metabolism.

There are minor weaknesses related to some analyses on mitochondrial proteins and to the fact that proteomic and transcriptomic comparison may be problematic in catabolic conditions because some gene expression is required to maintain or re-establish enzymes/proteins that are destroyed by the proteolytic systems (including the autophagy proteins and ubiquitin ligases). The authors should consider the following points.

Point1. The authors used the name sarcopenia as synonymous with muscle atrophy. However, sarcopenia clearly defines the disease state (disease code: ICD-10-CM (M62.84)) of excessive muscle loss and force drop during ageing (Ref: Anker SD et al. J Cachexia Sarcopenia Muscle 2016 Dec;7(5):512-514.). Therefore, the word sarcopenia must be used only when pathological age-related muscle loss is the subject of study. Sarcopenia can be present in cancer patients who also experience cachexia, however since the age of tumor-bearing mice in this study is 7-9 weeks old, the authors should refrain from using sarcopenia and instead replace it with the words muscle atrophy/ muscle wasting/muscle loss.

Point2. Most of the analyses of mitochondrial function are appropriate. However, the methodological approach to determining mitochondrial fusion and fission machinery shown in Fig. 5F is wrong. The correct way is to normalize the OPA1, MFn1/2 on mitochondrial proteins such as VDAC/porin. In fact, by loading the same amount of total protein (see actin in panel 5F) the difference between a normal and a muscle with enhanced protein breakdown is lost. In fact, we should expect a decrease in actin level in tumor-bearing mice with muscle atrophy while the blots clearly show the same level due to the normalization of protein content. Moreover, by loading the same amount of proteins in the gel, the atrophying muscle lysates become enriched in the proteins/organelles that are less affected by the proteolysis resulting in an artefactual increase. The correct way should be to lyse the whole muscle of control and tumor-bearing mice in an identical volume and to load in western blot the same volume between control cachectic muscles. Alternatively, the relative abundance of mitochondrial shaping proteins related to mitochondrial transmembrane or matrix proteins (mito mass) should compensate for the loading normalization. Because the authors showed elongated mitochondria despite mitophagy genes being up, fragmentation may be altered. Moreover, DNM1l gene is suppressed and therefore DRP1 protein must be analyzed. Finally, OPA 1 protein has different isoforms due to the action of proteases like OMA1, and YME1L that elicit different functions being the long one pro-fusion while the short ones do not. The authors must quantify the long and short isoforms of OPA1.

Point3. The comparison of proteomic and transcriptomic profiles to identify concordance or not is problematic when atrophy programs are induced. In fact, most of the transcriptional-dependent upregulation is to preserve/maintain/reestablish enzymes that are consumed during enhanced protein breakdown. For instance, the ubiquitin ligases when activated undergo autoubiquitination and proteasome degradation. The same happens for several autophagy-related genes belonging to the conjugation system (LC3, Gabarap), the cargo recognition pathways (e.g. Ubiquitin, p62/SQSTM1) and the selective autophagy system (e.g. BNIP3, PINK/PARKIN) and metabolic enzymes (e.g. GAPDH, lipin). Finally, in case identical amounts of proteins have been loaded in mass spec the issues rise in point 2 of selective enrichment should be considered. Therefore, when comparing proteomic and transcriptomic these issues should be considered in discussion.