Reviewer #2 (Public review):

Summary:

This manuscript presents a new approach for non-invasive, MRI-based measurements of cerebral blood volume (CBV). Here, the authors use ferumoxytol, a high-contrast agent, and apply specific sequences to infer CBV. The authors then move to statistically compare measured regional CBV with the known distribution of different types of neurons, markers of metabolic load, and others. While the presented methodology captures an estimated 30% of the vasculature, the authors corroborated previous findings regarding the lack of vascular compartmentalization around functional neuronal units in the primary visual cortex.

Strengths:

Non-invasive methodology geared to map vascular properties in vivo.

Implementation of a highly sensitive approach for measuring blood volume.

Ability to map vascular structural and functional vascular metrics to other types of published data.

Weaknesses:

The key issue here is the underlying assumption about the appropriate spatial sampling frequency needed to capture the architecture of the brain vasculature. Namely, ~7 penetrating vessels / mm2 as derived from Weber et al 2008 (Cer Cor). The cited work begins by characterizing the spacing of penetrating arteries and ascending veins using a vascular cast of 7 monkeys (Macaca mulatta, same as in the current paper). The ~7 penetrating vessels / mm2 are computed by dividing the total number of identified vessels by the area imaged. The problem here is that all measurements were made in a "non-volumetric" manner and only in V1. Extrapolating from here to the entire brain seems like an over-assumption, particularly given the region-dependent heterogeneity that the current paper reports.

Reviewer #2 (Public review):

Summary:

In this study by Sánchez-León and colleagues, the authors attempted to determine the influence of neuronal orientation on the efficacy of cerebellar tDCS in modulating neural activity. To do this, the authors made recordings from Purkinje cells, the primary output neurons of the cerebellar cortex, and determined the inter-dependency between the orientation of these cells and the changes in their firing rate during cerebellar tDCS application.

Strengths:

(1) A major strength is the in vivo nature of this study. Being able to simultaneously record neural activity and apply exogenous electrical current to the brain during both an anesthetized state and during wakefulness in these animals provides important insight into the physiological underpinnings of tDCS.

(2) The authors provide evidence that tDCS can modulate neural activity in multiple cell types. For example, there is a similar pattern of modulation in Purkinje cells and non-Purkinje cells (excitatory and inhibitory interneurons). Together, these data provide wholistic insight into how tDCS can affect activity across different populations of cells, which has important implications for basic neuroscience, but also clinical populations where there may be non-uniform or staged effects of neurological disease on these various cell types.

(3) There is a systematic investigation into the effects of tDCS on neural activity across multiple regions of the cerebellum. The authors demonstrate that the pattern of modulation is dependent on the target region. These findings have important implications for determining the expected neuromodulatory effects of tDCS when applying this technique over different target regions non-invasively in animals and humans.

Weaknesses:

(1) In the introduction, there is a lack of context regarding why neuronal orientation might be a critical factor influencing the responsiveness to tDCS. The authors allude to in vitro studies that have shown neuronal orientation to be relevant for the effects of tDCS on neural activity but do not expand on why this might be the case. These points could be better understood by informing the reader about the uniformity/non-uniformity of the induced electric field by tDCS. In addition, there is a lack of an a priori hypothesis. For example, would the authors have expected that neuronal orientation parallel or perpendicular to the electrical field to be related to the effects of tDCS on neural activity?

(2) It is unclear how specific stimulation parameters were determined. First, how were the tDCS intensities used in the present experiments determined/selected, and how does the relative strength of this induced electric field equate to the intensities used non-invasively during tDCS experiments in humans? Second, there is also a fundamental difference in the pattern of application used here (e.g., 15 s pulses separated by 10 s of no stimulation) compared to human studies (e.g., 10-20 min of constant stimulation).

(3) In their first experiment, the authors measure the electric field strength at increasing depths during increasing stimulation intensities. However, it appears that an alternating current rather than a direct current, which is usually employed in tDCS protocols, was used. There is a lack of rationale regarding why the alternating current was used for this component. Typically, this technique is more commonly used for entraining/boosting neural oscillations compared to studies using tDCS which aim to increase or decrease neural activity in general.

Reviewer #2 (Public review):

Summary:

The authors aimed to investigate how noradrenergic and glucocorticoid activity after retrieval influence subsequent memory recall with a 24-hour interval, by using a controlled three-day fMRI study involving pharmacological manipulation. They found that noradrenergic activity after retrieval selectively impairs subsequent memory recall, depending on hippocampal and cortical reactivation during retrieval.

Overall, there are several significant strengths of this well-written manuscript.

Strengths:

(1) The study is methodologically rigorous, employing a well-structured three-day experimental design that includes fMRI imaging, pharmacological interventions, and controlled memory tests.

(2) The use of pharmacological agents (i.e., hydrocortisone and yohimbine) to manipulate glucocorticoid and noradrenergic activity is a significant strength.

(3) The clear distinction between online and offline neural reactivation using MVPA and RSA approaches provides valuable insights into how memory dynamics are influenced by noradrenergic and glucocorticoid activity distinctly.

Weaknesses:

(1) One potential limitation is the reliance on distinct pharmacodynamics of hydrocortisone and yohimbine, which may complicate the interpretation of the results.

(2) Another point related above, individual differences in pharmacological responses, physiological and cortisol measures may contribute to memory recall on Day 3.

(3) Median-splitting approach for reaction times and hippocampal activity should better be justified.

Reviewer #2 (Public review):

Summary:

Feddersen & Bramkamp determined important characteristics of how MinD protein binds/dissociates to/from the membrane, and dimerizes in relation to its ATPase activity. The presented data clearly shows the differences in function of MinD homologs from B. subtilis and E. coli.

Strengths:

The work presents well-executed experiments that lead to interesting conclusions and a new model of how Min system works during B. subtilis mid-cell division. Importantly, this model is supported by in vitro characterization of well-chosen mutants in the functional domains of MinD. Outstandingly, most of the in vitro data are confirmed by single-molecule localization microscopy.

Weaknesses:

The authors immobilized liposomes, for which they used E. coli total lipids, to measure ATPase activity and liposome association and dissociation of B. subtilis MinD. For these experiments would be more suitable to use B. subtilis total lipids as more biologically relevant data could be gained.

Although the work is in detail and nicely compares the function of B. subtilis Min system with E. coli Min system, it lacks the comparison of the Min system function in other rod-shaped Gram-positive bacteria. I would suggest including in the Discussion the complexity of other Min systems. Especially, this complexity is seen in other rod-shaped and spore formers such as Clostridial species in which one of these Min systems or both are present, an oscillating E. coli Min system type and more static as in B. subtilis.

Reviewer #2 (Public Review):

Summary:

This paper evaluates the effect of COVID-19 booster vaccination on reinfection in Shanghai, China among individuals who received primary COVID-19 vaccination followed by initial infection, during an Omicron wave.

Strengths:

A large database is collated from electronic vaccination and infection records. Nearly 200,000 individuals are included in the analysis and 24% became reinfected.

Weaknesses:

The authors have revised the manuscript and have provided satisfactory responses to my prior comments.

Reviewer #2 (Public review):

Summary:

This manuscript reports analyses of fMRI data from infants and toddlers watching naturalistic movies. Visual areas in the infant brain show distinct functions, consistent with previous studies using resting state and awake task-based infant fMRI. The pattern of activity in visual regions contains some features predicted by the regions' retinotopic responses. The revised version of the manuscript provides additional validation of the methodology and clarifies the claims. As a result, the data provide clear support for the claims.

Strengths:

The authors have collected a unique dataset: the same individual infants both watched naturalistic animations and a specific retinotopy task. Using these data positions the authors show that activity evoked by movies, in infants' visual areas, is correlated with the regions' retinopic response. The revised manuscript validates this methodology, using adult data. The revised manuscript also shows that an infant's movie-watching data is not sufficient or optimal to predict their visual areas' retinotopic responses; anatomical alignment with a group of previous participants provides more accurate prediction of a new participant's retinotopic response.

Weaknesses:

A key step in the analysis of the movie-watching data is the selection of independent components of the movie evoked response, by a trained researcher, that resemble retinotopic spatial patterns. While the researcher is unlikely to be biased by this infant's own retinotopy , as the authors argue, the researcher is actively looking for ICs that resemble average patterns of retinotopic response. So, how likely is it that ICs that resemble retinotopic organization arise by chance (i.e. in noise) in infant fMRI data? I do not see an analysis that addresses this question. With apologies if I missed it.

Reviewer #2 (Public review):

Summary:

The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.

Strengths:

The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in vitro and in vivo (patient samples). Activation and inhibition of the TRPV4 channel can modulate the cell volume and cell mobility. These results unveil a novel mechanism of mechanical sensing in cancer cells, potentially offering new avenues for therapeutic intervention targeting cancer metastasis by modulating TRPV4 activity. This is a very comprehensive study, and the data presented in the paper are clear and convincing. The study represents a very important advance in our understanding of the mechanical biology of cancer.

Weaknesses:

However, I do think that there are several additional experiments that could strengthen the conclusions of this work. A critical limitation is the absence of genetic ablation of the TRPV4 gene to confirm its essential role in the response to cell crowding.

Reviewer #2 (Public review):

This manuscript reports a series of studies that sought to identify a biological basis for morphine-induced social deficits. This goal has important translational implications and is, at present, incompletely understood in the field. The extant literature points to changes in periventricular CRF and oxytocin neurons as critical substrates for morphine to alter social behavior. The experiments utilize mice, administered morphine prior to a sociability assay. Both male and female mice show reduced sociability in this procedure. Pretreatment with the CRF1 receptor antagonist, antalarmin, clearly abolished the morphine effect in males, and the data are compelling. Consistently, CRF1-/- male mice appeared to be spared of the effect of morphine (while wild-type and het mice had reduced sociability). The same experiment was reported as non-feasible in females due to the effect of dose on exploratory behavior per se. Seeking a neural correlate of the behavioral pharmacology, acute cell-attached recordings of PVN neurons were made in acute slices from mice pretreated with morphine or anatalarmin. Morphine increased firing frequencies, and both antalarmin and CRF1-/- mice were spared of this effect. Increasing confidence that this is a CRF1 mediated effect, there is a gene deletion dose effect where het's had an intermediate response to morphine. In general, these experiments are well-designed and sufficiently powered to support the authors' inferences. A final experiment repeated the cell-attached recordings with later immunohistochemical verification of the recorded cells as oxytocin or vasopressin positive. Here the data are more nuanced. The majority of sampled cells were positive for both oxytocin and vasopressin, in cells obtained from males, morphine pretreatment increased firing in this population and was CRF1 dependent, however in females the effect of morphine was more modest without sensitivity to CRF1. Given that only ~8 cells were only immunoreactive for oxytocin, it may be premature to attribute the changes in behavior and physiology strictly to oxytocinergic neurons. In sum, the data provide convincing behavioral pharmacological evidence and a regional (and possibly cellular) correlation of these effects suggesting that morphine leads to sociality deficits via CRF interacting with oxytocin in the hypothalamus. While this hypothesis remains plausible, the current data do not go so far as directly testing this mechanism in a site or cell-specific way. With regard to the presentation of these data and their interpretation, the manuscript does not sufficiently draw a clear link between mu-opioid receptors, their action on CRF neurons of the PVN, and the synaptic connectivity to oxytocin neurons. Importantly, sex, cell, and site-specific variations in the CRF are well established (see Valentino & Bangasser) yet these are not reviewed nor are hypotheses regarding sex differences articulated at the outset. The manuscript would have more impact on the field if the implications of the sex-specific effects evident here were incorporated into a larger literature.

With regards to the model proposed in the discussion, it seems that there is an assumption that ip morphine or antalarmin have specific effects on the PVN and that these mediate behavior - but this is impossible to assume and there are many meaningful alternatives (for example, both MOR and CRF modulation of the raphe or accumbens are worth exploration). While it is up to the authors to conduct additional studies, a demonstration that the physiology findings are in fact specific to the PVN would greatly increase confidence that the pharmacology is localized here. Similarly, direct infusion of antalarmin to the PVN, or cell-specific manipulation of OT neurons (OT-cre mice with inhibitory dreadds) combined with morphine pre-exposure would really tie the correlative data together for a strong mechanistic interpretation.

Because the work is framed as informing a clinical problem, the discussion might have increased impact if the authors describe how the acute effects of CRF1 antagonists and morphine might change as a result of repeated use or withdrawal.

Reviewer #2 (Public Review):

Summary:

This paper identifies a novel SARS-CoV-2 epitope that measures host-virus interactions that have clinical correlations and can act as a signature of infection. In doing so, the authors present a novel structure-driven epitope profiling pipeline that allows them to rapidly iterate through multiple possible peptide epitope candidates for directly measuring host-virus binding. With this approach, the authors identify an IgM antibody response driven by the N-terminus of the Membrane protein of SARS-CoV-2, and demonstrate that epitope is directly correlative with cell-level measurements of infection, and can even act as a clinical signature of infection. The findings are significant to those interested in epitope identification and present a unique step forward for incorporating structural data in an iterative screening approach. The study itself presents some unique connections between the models presented, the IgM being generated, and clinical outcomes, but the claim that these IgM levels are indicative of anything more than past infection will require further detailed analysis.

Strengths:

(1) The methodological approach presented in this study is incredibly powerful and shows major promise to identify other peptide epitopes of proteins for antibody profiling. The simplicity of the methodological approach to string together established protocols and measurements offers a unique elegant promise that this is a generalizable method to many other systems and disease contexts.

(2) The clever use of a SASA metric to study and identify each of the major components demonstrates how structural information is a powerful way to approach identifying and nominating candidate peptides.

(3) This paper spans an exciting range of structural data to clinical-derived measurements, demonstrating the powerful possibilities that can arise from connecting structural biophysical data to clinical measurements to build generalized pipelines or models

Weaknesses:

(1) While the authors use SASA as a great way to screen peptides based on the presumption that SASA can act as a measure of the stability of protein folding, there are many caveats that may come with this measurement that can reduce generalizability. Assessing SASA per residue is a high variance metric that requires many additional layers of further analysis to make inferences about peptide stability. Further, since proteins are inherently dynamic, alternative configurations may yield fluctuating SASA values that inherently bias and introduce noise into the results. It would be useful to compare these SASA metrics for peptides to other structural measures often associated with protein stability used in the literature, such as Radius of Gyration, Hydrodynamic Radius, Secondary Structure degree, etc.

(2) In Figure 3G, the author put forth that IgM ELISA results and whole spike IgG correlate with one another. While it is clear that IgM for M1 and IgM for spike S1' subunit both correlate similarly to whole spike IgG levels, the correlation in both cases is incredibly weak, with whole spike IgG fluctuating widely across a narrow range of IgM for M1 values. This interpretation is also contradicted by 3G's best-fit lines that would have a large residual value to the data. Lastly, the Pearson correlation values for both correlations are misleading here as Pearson correlation indicates the strength and direction of two linear variables. This means that any dataset will inherently have a Pearson r value of ~0.40 but one may not be predictive of the other. It would be better for the authors to instead use measures such as Spearman R or additional statistical analysis like histogramming to demonstrate this coupling.

(3) It is not clear from the text if the authors are the first to use LASSO models to correlate IgM levels with infection scores in patients. LASSO-based logistic regressions are powerful tools used widely in statistical approaches to measure the association between two variables. However, there is a lack of citations indicating that the authors' approach is based on previous efforts and matches the best practice in generating these models on clinical data. It would be useful to add citations to indicate that this approach is following established statistical best practices in line with the field. If the use of the LASSO approach is novel, it would be key to mention this and highlight why the authors feel a LASSO model is the appropriate approach here.

(4) The authors demonstrate in Figure 5 that their IgM levels are very clearly correlative with a history of SARS-CoV-2 infection, and provides another avenue for the detection of prior infections. However, these claims are extended to compare to direct symptoms such as fatigue, depression, and quality of life. Specifically, the authors claim that IgM persistence is correlated with lower quality of life and stress-indicative symptoms. However, Figure 5D contradicts this, highlighting that both persistent and non-persistent IgM groups have similar trends and patterns in fatigue, depression, and quality of life. The authors should reexamine this interpretation of their data, and revisit if there are alternative analyses that may indicate where persistent and non-persistent IgM groups separate.

(5) One under-discussed component of this paper is the potential for sequence variation impacting IgM generation and detection. With resistance being a consistent issue amongst infectious diseases and immune evasion, it may be useful to discuss the possible sequence variance seen in the M protein sequence of M1, as well as to see if the IgM levels induced upon M1 presentation can be separated out from their existing analyses (it may not be!). Regardless, it would be useful for the authors to consider the potential for sequence variation in the M1 peptide and its downstream effects.

Reviewer #3 (Public review):

Since its first experimental report in 2017 (Patel et al. Science 2017), there have been several studies on the phenomenon in which ATP functions as a biological hydrotrope of protein aggregates. In this manuscript, by conducting molecular dynamics simulations of three different proteins, Trp-cage, Abeta40 monomer, and Abeta40 dimer at concentrations of ATP (0.1, 0.5 M), which are higher than those at cellular condition (a few mM), Sarkar et al. find that the amphiphilic nature of ATP, arising from its molecular structure consisting of phosphate group (PG), sugar ring, and aromatic base, enables it to interact with proteins in a protein-specific manner and prevents their aggregation and solubilize if they aggregate. The authors also point out that in comparison with NaXS, which is the traditional chemical hydrotrope, ATP is more efficient in solubilizing protein aggregates because of its amphiphilic nature.

Trp-cage, featured with hydrophobic core in its native state, is denatured at high ATP concentration. The authors show that the aromatic base group (purine group) of ATP is responsible for inducing the denaturation of helical motif in the native state.

For Abeta40, which can be classified as an IDP with charged residues, it is shown that ATP disrupts the salt bridge (D23-K28) required for the stability of beta-turn formation.

By showing that ATP can disassemble preformed protein oligomers (Abeta40 dimer), the authors suggest that ATP is "potent enough to disassemble existing protein droplets, maintaining proper cellular homeostasis," and enhancing solubility.

Overall, the message of the paper is clear and straightforward to follow. In addition to the previous studies in the literature on this subject. (J. Am. Chem. Soc. 2021, 143, 31, 11982-11993; J. Phys. Chem. B 2022, 126, 42, 8486-8494; J. Phys. Chem. B 2021, 125, 28, 7717-7731; J. Phys. Chem. B 2020, 124, 1, 210-223), the study, which tested using MD simulations whether ATP is a solubilizer of protein aggregates, deserves some attention from the community and is worth publishing.

Weakness

My only major concern is that the simulations were performed at unusually high ATP concentrations (100 and 500 mM of ATP), whereas the real cellular concentration of ATP is 1-5 mM.

I was wondering if there is any report on a titration curve of protein aggregates against ATP, and what is the transition mid-point of ATP-induced solubility of protein aggregates. For instance, urea or GdmCl have long been known as the non-specific denaturants of proteins, and it has been well experimented that their transition mid-points of protein unfolding are in the range of ~(1 - 6) M depending on the proteins.

The authors responded to my comment on ATP concentration that because of the computational issue in all-atom simulations, they had no option but to employ mM-protein concentrations instead of micromolar concentrations, thus requiring 1000-folds higher ATP concentration, which is at least in accordance with the protein/ATP stoichiometry. However, I believe this is an issue common to all the researchers conducting MD simulations. Even if the system is in the same stoichiometric ratio, it is never clear to me (is it still dilute enough?) whether the mechanism of solubilization of aggregate at 1000 fold higher concentration of ATP remains identical to the actual process.

Reviewer #2 (Public review):

Summary:

The authors generated analogs consisting of modified neurotensin (NT) peptides capable of binding to low density lipoprotein (LDL) and NT receptors. Their lead analog was further evaluated for additional validation as a novel therapeutic. The putative mechanism of action for NT in its antiseizure activity is hypothermia, and as therapeutic hypothermia has been demonstrated in epilepsy, NT analogs may confer antiseizure activity and avoid the negative effects of induced hypothermia.

Strengths:

The authors demonstrate an innovative approach, i.e. using LDLR as a means of transport into the brain, that may extend to other compounds. They systematically validate their approach and its potential through binding, brain penetration, in vivo antiseizure efficacy, and neuroprotection studies.

Weaknesses:

Tolerability studies are warranted, given the mechanism of action and the potential narrow therapeutic index. In vivo studies were used to assess efficacy of the peptide conjugate analogs in the mouse KA model. However, it would be beneficial to have shown tolerability in naïve animals to better understand the therapeutic potential of this approach.

Mice may be particularly sensitive to hypothermia. It would be beneficial to show similar effects in a rat model.

Reviewer #2 (Public review):

Based on reviewer feedback, Das and Menon have made several modifications to their manuscript, including a revised Introduction with a reframed motivation (now more oriented around the role of tripartite network in memory operations), new control analyses (as requested by Reviewers, including an updated and more appropriate baseline period and a control region, the IFG), an assessment of narrowband phase synchronization (as requested), as well as updates for clarity throughout the Methods section.

While I believe the authors have been responsive to reviewer feedback, and these modifications do enhance the manuscript, I have a few suggestions for how these new analyses could be made more statistically robust and better contextualized against the main findings of the manuscript. I continue to have some reservations about a tendency for their data to be overinterpreted, and for conclusions to be drawn more strongly than the data actually warrant.

(1) Clarifying the new control analyses. The authors have been responsive to our feedback and implemented several new analyses. The use of a pre-task baseline period and a control brain region (IFG) definitively help to contextualize their results, and the findings shown in the revision do suggest that (1) relative to a pre-task baseline, directed interactions from the AI are stronger and (2) relative to a nearby region, the IFG, the AI exhibits greater outward-directed influence.

However, it is difficult to draw strong quantitative conclusions from the analyses as presented, because they do not directly statistically contrast the effect in question (directed interactions with the FPN and DMN) between two conditions (e.g. during baseline vs. during memory encoding/retrieval). As I understand it, in their main figures the authors ask, "Is there statistically greater influence from the AI to the DMN/FPN in one direction versus another?" And in the AI they show greater "outward" PTE than "inward" PTE from other networks during encoding/retrieval. The balance of directed information favors an outward influence from the AI to DMN/FPN.

But in their new analyses, they simply show that the degree of "outward" PTE is greater during task relative to baseline in (almost) all tasks. I believe a more appropriately matched analysis would be to quantify the inward/outward balance during task states, quantify the inward/outward balance during rest states, and then directly statistically compare the two. It could be that the relative balance of directed information flow is non-significantly changed between task and rest states, which would be important to know.

Likewise, a similar principle applies to their IFG analysis. They show that the IFG tends to have an "inward" balance of influence from the DMN/FPN (the opposite of the AIs effect), but this does not directly answer whether the AI occupies a statistically unique position in terms of the magnitude of its influence on other regions. More appropriate, as I suggest above, would be to quantify the relative balance inward/outward influence, both for the IFG and the AI, and then directly compare those two quantities. (Given the inversion of the direction of effect, this is likely to be a significant result, but I think it deserves a careful approach regardless.)

(2) Consider additional control regions. The authors justify their choice of IFG as a control region very well. In my original comments, I perhaps should have been more clear that the most compelling control analyses here would be to subject every region of the brain outside these networks (with good coverage) to the same analysis, quantify the degree of inward/outward balance, and then see how the magnitude of the AI effect stacks up against all possible other options. If the assertion is that the AI plays a uniquely important role in these memory processes, showing how its influence stacks up against all possible "competitors" would be a very compelling demonstration of their argument.

(3) Reporting of successful vs. unsuccessful memory results. I apologize if I was not clear in my original comment (2.7, pg. 13 of the response document) regarding successful vs. unsuccessful memory. The fact that no significant difference was found in PTE between successful/unsuccessful memory is a very important finding that adds valuable context to the rest of the manuscript. I believe it deserves a figure, at least in the Supplement, so that readers can visualize the extent of the effect in successful/unsuccessful trials. This is especially important now that the manuscript has been reframed to focus more directly on claims regarding episodic memory processing; if that is indeed the focus, and their central analysis does not show a significant effect conditionalized on the success of memory encoding/retrieval, it is important that readers can see these data directly.

(4) Claims regarding causal relationships in the brain. I understand that the authors have defined "causal" in a specific way in the context of their manuscript; I do believe that as a matter of clear and transparent scientific communication, the authors nonetheless bear a responsibility to appreciate how this word may be erroneously interpreted/overinterpreted and I would urge further review of the manuscript to tone down claims of causality. Reflective of this, I was very surprised that even as both reviewers remarked on the need to use the word "causal" with extreme caution, the authors added it to the title in their revised manuscript.

Reviewer #2 (Public Review):

Summary:

This paper from the Barford lab describes medium/high-resolution cryo-EM structures of three versions of the S. cerevisiae anaphase-promoting complex/cyclosome (APC/C):

(1) the recombinant apo complex purified from insect cells,

(2) the apo complex phosphorylated in vitro by cyclin-dependent kinase, and

(3) an active APC/C-Cdh1-substrate ternary complex.

The focus of the paper is on comparing similarities and differences between S. cerevisiae and human APC/C structures, mechanisms of activation by coactivator, and regulation by phosphorylation. The authors find that the overall structures of S. cerevisiae and human APC/C are remarkably similar, including the binding sites and orientation for the substrate-recruiting coactivator, Cdh1. In addition, the mechanism of Cdh1 inhibition by phosphorylation appears conserved across kingdoms. However, key differences were also observed that reveal divergence in APC/C mechanisms that are important for researchers in this field to know. Specifically, the mechanism of APC/C-Cdc20 activation by mitotic phosphorylation appears to be different, due to the absence of the key Apc1 autoinhibition loop in the S. cerevisiae complex. In addition, the conformational activation of human APC/C by coactivator binding was not observed in the S. cerevisiae complex, implying that stimulation of E2 binding must occur via a different mechanism in this species.

Strengths:

Consistent with the numerous prior cryo-EM structures of human APC/C from the Barford lab, the technical quality of the structure models is a major strength of this work. In addition, the detailed comparison of similarities and differences between the two species will be a very valuable resource for the scientific community. The manuscript is written very well and allows readers lacking expertise in cryo-EM to understand the important aspects of the conservation of APC/C structure and mechanism across kingdoms.

Weaknesses:

The lack of experimentation in this work to test some of the putative differences in APC/C mechanism (e.g. stimulation of E2 binding by coactivator and stimulation of activity by mitotic phosphorylation) could be considered a weakness. Nonetheless, the authors do a nice job explaining how the structure interpretations imply these differences likely exist, and this work sets the stage nicely for future studies to understand these differences at a mechanistic level. There is enough value in having the S. cerevisiae structure models and the comparison to the human structures, without any additional experimentation.

The validation of APC/C phosphorylation in the unphosphorylated and hyperphosphorylated states is not very robust. Given the lack of significant effects of phosphorylation on APC/C structure observed here (compared to the human complex), this becomes important. A list of phosphorylation sites identified by mass spec before and after in vitro phosphorylation is provided but lacks quantitative information. This list indicates that a significant number of phosphorylation sites are detected in the purified APC/C prior to reaction with purified kinases. Many more sites are detected after in vitro kinase reaction, but it isn't clear how extensively any of the sites are modified. There is reason for caution then, in accepting the conclusions that structures of unphosphorylated and hyperphosphorylated APC/C from S. cerevisiae are nearly identical.

Reviewer #2 (Public Review):

The authors fully addressed my concerns and made appropriate changes in the manuscript. The quality of the manuscript is now significantly improved.

Reviewer #2 (Public review):

Summary:

In their manuscript, Lin et al. present a comprehensive single-cell analysis of tea plant roots. They measured the transcriptomes of 10,435 cells from tea plant root tips, leading to the identification and annotation of 8 distinct cell clusters using marker genes. Through this dataset, they delved into the cell-type-specific expression profiles of genes crucial for the biosynthesis, transport, and storage of theanine, revealing potential multicellular compartmentalization in theanine biosynthesis pathways. Furthermore, their findings highlight CsLBD37 as a novel transcription factor with dual regulatory roles in both theanine biosynthesis and lateral root development.

Strengths:

This manuscript provides the first single-cell dataset analysis of roots of the tea plants. It also enables detailed analysis of the specific expression patterns of the gene involved in theanine biosynthesis. Some of these gene expression patterns in roots were further validated through in-situ RT-PCR. Additionally, a novel TF gene CsLBD37's role in regulating theanine biosynthesis was identified through their analysis.

Weaknesses:

The revised manuscript has addressed the concerns raised during the initial review.

Reviewer #2 (Public review):

Summary:

This study by Sun et al. presents a role for the S. pombe MAP kinase Pmk1 in the activation of the Spindle Assembly Checkpoint (SAC) via controlling the protein levels of APC/C activator Cdc20 (Slp1 in S. pombe). The data presented in the manuscript is thorough and convincing. The authors have shown that Pmk1 binds and phosphorylates Slp1, promoting its ubiquitination and subsequent degradation. Since Cdc20 is an activator of APC/C, which promotes anaphase entry, constitutive Pmk1 activation leads to an increased percentage of metaphase-arrested cells. The authors have used genetic and environmental stress conditions to modulate MAP kinase signalling and demonstrate their effect on APC/C activation. This work provides evidence for the role of MAP kinases in cell cycle regulation in S. pombe and opens avenues for exploration of similar regulation in other eukaryotes.

Strengths:

The authors have done a very comprehensive experimental analysis to support their hypothesis. The data is well represented, and including a model in every figure summarizes the data well.

Weaknesses:

As mentioned in the comments, the manuscript does not establish that MAP kinase activity leads to genome stability when cells are subjected to genotoxic stressors. That would establish the importance of this pathway for checkpoint activation.

Reviewer #2 (Public review):

Summary:

The authors show that A. japonicus calcitonins (AjCT1 and AjCT2) activate not only the calcitonin/calcitonin-like receptor but also activate the two PDF receptors, ex vivo. They also explore secondary messenger pathways that are recruited following receptor activation. They determine the source of CT1 and CT2 using qPCR and in situ hybridization and finally test the effects of these peptides on tissue contractions, feeding, and growth. This study provides solid evidence that CT1 and CT2 act as ligands for calcitonin receptors; however, evidence supporting cross-talk between CT peptides and PDF receptors is only based on ex vivo experiments.

Strengths:

This is the first study to report the pharmacological characterization of CT receptors in an echinoderm. Multiple lines of evidence in cell culture (receptor internalization and secondary messenger pathways) support this conclusion.

Weaknesses:

The authors claim that A. japonicus CTs activate "PDF" receptors and suggest that this cross-talk is evolutionarily ancient since a similar phenomenon also exists in the fly Drosophila melanogaster. These conclusions are not fully supported for several reasons. The authors perform phylogenetic analysis to show that the two "PDF" receptors form an independent clade. This clade is sister to the clade comprising CT receptors. This phylogenetic analysis suffers from several issues. Firstly, the phylogenies lack bootstrap support. Secondly, the resolution of the phylogeny is poor because representative members from diverse phyla have not been included. For instance, insect or other protostomian PDF receptors have not been included so how can the authors distinguish between "PDF" receptors or another group of CT receptors? Thirdly, no in vivo evidence has been presented to support that CT can activate "PDF" receptors in vivo.

The source of CT which mediates the effects on longitudinal muscles and intestine is unclear. Is it autocrine or paracrine signaling by CT from the same tissue or is it long-range hormonal signaling?

Pharmacology experiments showing the effects of CT1 and CT2 on ACh-induced contractions were performed. Sample traces have been provided but no traces with ACh alone have been included. How long do ACh-induced contractions persist? These controls are necessary to differentiate between the eventual decay of ACh effects and relaxation induced by CT1 and CT2. The traces also do not reflect the results portrayed in dose-response curves. For instance, in Figure 6B, maximum relaxation is reported for 10-6M. Yet, the trace hardly shows any difference before and after the addition of 10-6M peptide. The maximum effect in the trace appears to be after the addition of 10-8M peptide.

I am unsure how differences in wet mass indicate feeding and growth differences since no justification has been provided. Couldn't wet mass also be influenced by differences in osmotic balance, a key function of calcitonin-like peptides in protostomian invertebrates? The statistical comparisons have not been included in Figure 7B.

While the authors succeeded in knocking down CT, the physiological effects of reduced CT signaling were not examined.

Reviewer #2 (Public review):

Summary:

The study by VanBelzen et. al. compares chromatin immunoprecipitation (ChIP-seq) and chromatin endogenous cleavage sequencing (ChEC-seq2) to examine RNA polymerase II (RNAPII) binding patterns in yeast. While ChIP-seq shows RNAPII enrichment mainly over transcribed regions, ChEC-seq2 highlights RNAPII binding at promoters and upstream activating sequences (UASs), suggesting it captures distinct RNAPII populations that the authors speculate are linked more tightly to active transcription. The authors develop a stochastic model for RNAPII kinetics using ChEC-seq2 data, revealing insights into transcription regulation and the role of the nuclear pore complex in stabilizing promoter-associated RNAPII. The study suggests that ChEC-seq2 identifies regulatory events that ChIP-seq may overlook.

Strengths:

(1) This is a carefully crafted study that adds significantly to existing literature in this area. Transgenic MNase fusions with endogenous Rpb1 and Rpb3 subunits were carefully performed, and complemented by fusions with several additional proteins that help the authors to dissect the transcription cycle. Both the S. cerevisiae lines and the sequencing data are likely to be of significant use to the community.

(2) The validation of ChEC-seq2 and its comparison with ChIP-seq is highly valuable technical information for the community.

(3) The kinetic modeling appears to be thoughtfully done.

Weaknesses:

(1) The term "nascent transcription" is all too often used interchangeably for NET-seq, PRO-seq, 4sU-seq, and other assays that often provide different types of information. The authors should make it clear their use of the term refers to SLAM-seq data.

(2) The authors do not perform any comparison to run-on (PRO-seq) data. My impression is that the distribution of PRO-seq signal in S. cerevisiae agrees better with the distribution the authors observe by ChIP-seq. PRO-seq only captures RNAPII that is engaged and actively transcribing. If PRO-seq does indeed provide a similar profile as ChIP-seq, wouldn't this indicate that the high frequency of association between RNAPII and either the promoter or UAS reflects RNAPII that has not yet started transcription elongation? Perhaps this could help sort out what types of activities are occurring at the UAS (which does not appear to require a full PIC) or at the promoter (which does)?

Reviewer #2 (Public Review):

In the current manuscript Li et al., study the preservation of the regional identity during the process of astrocyte generation from pluripotent stem cells. More precisely, this work investigates if neural progenitor cells patterned for the ventral midbrain give rise to astrocytes with conserved regional specification, which could reflect the astrocytic heterogeneity in the brain. To this end, the authors utilized a previously generated reporter iPSC line in which the expression of introduced blue fluorescence protein (BFP) is subjacent to the activation of LMXA1, a ventral midbrain floor plate marker. The study reports that following a defined patterning protocol based on SHH and FGF8, over 90% of d19 cells, corresponding to a neural progenitor stage, acquired the midbrain floor plate identity. However, during the subsequent astrogenic induction and glial progenitor expansion, this identity is gradually lost, supposedly due to the growth advantage of cells deriving from the residual LMX1A- neural progenitors. Contrariwise, if the LMX1A+ progenitors were purified, regional identity would be maintained throughout the astrocytic generation and incur an early astrogenic switch and maturation of derived astrocytes. By using single-cell RNA sequencing, the authors further identified distinct transcriptomic signatures on the astrocytic progeny of LMX1A- and LMX1A- progenitors.

Strengths and weaknesses:

(1) The main model utilized was engineered from the KOLF2 human iPSC line into an elegant LMX1A-reporter line based on the expression of BFP. This results in an attractive model for studies tracing the fate of LMX1A cells. However, consideration should be given to the fact that the parental line, exhibits a splice disruption in the COL3A1 gene encoding type III collagen (Pantazis 2022, doi:10.1016/j.stem.2022.11.004 ), which has been identified as being enriched in certain ventral astrocytic populations (Bradley 2019, doi:10.1242/dev.170910).

(2) The authors argue that the depletion of BFP seen in the unsorted population immediately after the onset of astrogenic induction is due to the growth advantage of the derivatives of the residual LMX1A- population. However, no objective data supporting this idea is provided, and one could also hypothesize that the residual LMX1A- cells could affect the overall LMX1A expression in the culture through negative paracrine regulation. Therefore, cell cycle or proliferation studies of these cells are needed to prove the authors' assumption. Furthermore, on line 124 it is stated that: "Interestingly, the sorted BFP+ cells exhibited similar population growth rate to that of unsorted cultures...". In the face of the suggested growth disadvantage of those cells, this statement needs clarification.

(3) Regarding the fidelity of the model system, it is not clear to me how the TagBFP expression was detected in the BFP+ population supposedly in d87 and d136 pooled astrocytes (Fig S6C) while no LMX1A expression was observed in the same cells (Fig S6F).

(4) The generated single-cell RNASeq dataset is extremely valuable. However, given the number of conditions included in this study (i.e. early vs late astrocytes, BFP+ vs BFP-, sorted vs unsorted, plus non-patterned and neuronal samples) the resulting analysis lacks detail. For instance, from a developmental perspective and to better grasp the functional significance of astrocytic heterogeneity, it would be interesting to map the identified clusters to early vs late populations and to the BFP status. Moreover, although comprehensive, Figure S7 is complex to understand given that citations rather than the reference populations are depicted.

(5) Do the authors have any consideration regarding the morphology of the astrocytes obtained in this study? None of the late astrocyte images depict a prototypical stellate morphology, which is reported in many other studies involving the generation of iPSC-derived astrocytes and which is associated with the maturity status of the cell.

Reviewer #2 (Public review):

Summary:

The manuscript by García-Vázquez et al identifies the G2 and S phases expressed protein 1(GTSE1) as a substrate of the CycD-CDK4/6 complex. CycD-CDK4/6 is a key regulator of the G1/S cell cycle restriction point, which commits cells to enter a new cell cycle. This kinase is also an important therapeutic cancer target by approved drugs including Palbocyclib. Identification of substrates of CycD-CDK4/6 can therefore provide insights into cell cycle regulation and the mechanism of action of cancer therapeutics. A previous study identified GTSE1 as a target of CycB-Cdk1 but this appears to be the first study to address the phosphorylation of the protein by Cdk4/6.

The authors identified GTSE1 by mining an existing proteomic dataset that is elevated in AMBRA1 knockout cells. The AMBRA1 complex normally targets D cyclins for degradation. From this list, they then identified proteins that contain a CDK4/6 consensus phosphorylation site and were responsive to treatment with Palbocyclib.

The authors show CycD-CDK4/6 overexpression induces a shift in GTSE1 on phostag gels that can be reversed by Palbocyclib. In vitro kinase assays also showed phosphorylation by CDK4. The phosphorylation sites were then identified by mutagenizing the predicted sites and phostag got to see which eliminated the shift.

The authors go on to show that phosphorylation of GTSE1 affects the steady state level of the protein. Moreover, they show that expression and phosphorylation of GTSE1 confer a growth advantage on tumor cells and correlate with poor prognosis in patients.

Strengths:

The biochemical and mutagenesis evidence presented convincingly show that the GTSE1 protein is indeed a target of the CycD-CDK4 kinase. The follow-up experiments begin to show that the phosphorylation state of the protein affects function and has an impact on patient outcomes.

Weaknesses:

It is not clear at which stage in the cell cycle GTSE1 is being phosphorylated and how this is affecting the cell cycle. Considering that the protein is also phosphorylated during mitosis by CycB-Cdk1, it is unclear which phosphorylation events may be regulating the protein.

Reviewer #2 (Public review):

Summary:

This manuscript by Hosack and Arce-McShane examines the directional tuning of neurons in macaque primary motor (MIo) and somatosensory (SIo) cortex. The neural basis of tongue control is far less studied than, for example, forelimb movements, partly because the tongue's kinematics and kinetics are difficult to measure. A major technical advantage of this study is using biplanar video-radiography, processed with modern motion tracking analysis software, to track the movement of the tongue inside the oral cavity. Compared to prior work, the behaviors are more naturalistic behaviors (feeding and licking water from one of three spouts), although the animals were still head-fixed.

The study's main findings are that:

• A majority of neurons in MIo and a (somewhat smaller) percentage of SIo modulated their firing rates during tongue movements, with different modulations depending on the direction of movement (i.e., exhibited directional tuning). Examining the statistics of tuning across neurons, there was anisotropy (e.g., more neurons preferring anterior movement) and a lateral bias in which tongue direction neurons preferred that was consistent with the innervation patterns of tongue control muscles (although with some inconsistency between monkeys).

• Consistent with this encoding, tongue position could be decoded with moderate accuracy even from small ensembles of ~28 neurons.

• There were differences observed in the proportion and extent of directional tuning between the feeding and licking behaviors, with stronger tuning overall during licking. This potentially suggests behavioral context-dependent encoding.

• The authors then went one step further and used a bilateral nerve block to the sensory inputs (trigeminal nerve) from the tongue. This impaired the precision of tongue movements and resulted in an apparent reduction and change in neural tuning in Mio and SIo.

Strengths:

The data are difficult to obtain and appear to have been rigorously measured, and provide a valuable contribution to this under-explored subfield of sensorimotor neuroscience. The analyses adopt well-established methods, especially from the arm motor control literature, and represent a natural starting point for characterizing tongue 3D direction tuning.

Weaknesses:

There are alternative explanations for some of the interpretations, but those interpretations are described in a way that clearly distinguishes results from interpretations, and readers can make their own assessments. Some of these limitations are described in more detail below.

One weakness of the current study is that there is substantial variability in results between monkeys, and that only one session of data per monkey/condition is analyzed (8 sessions total). This raises the concern that the results could be idiosyncratic. The Methods mention that other datasets were collected, but not analyzed because the imaging pre-processing is very labor-intensive. While I recognize that time is precious, I do think in this case the manuscript would be substantially strengthened by showing that the results are similar on other sessions.

This study focuses on describing directional tuning using the preferred direction (PD) / cosine tuning model popularized by Georgopoulous and colleagues for understanding neural control of arm reaching in the 1980s. This is a reasonable starting point and a decent first-order description of neural tuning. However, the arm motor control field has moved far past that viewpoint, and in some ways, an over-fixation on static representational encoding models and PDs held that field back for many years. The manuscript benefits from drawing the readers' attention (perhaps in their Discussion) that PDs are a very simple starting point for characterizing how cortical activity relates to kinematics, but that there is likely much richer population-level dynamical structure and that a more mechanistic, control-focused analytical framework may be fruitful. A good review of this evolution in the arm field can be found in Vyas S, Golub MD, Sussillo D, Shenoy K. 2020. Computation Through Neural Population Dynamics. Annual Review of Neuroscience. 43(1):249-75

Can the authors explain (or at least speculate) why there was such a large difference in behavioral effect due to nerve block between the two monkeys (Figure 7)?

Do the analyses showing a decrease in tuning after nerve block take into account the changes (and sometimes reduction in variability) of the kinematics between these conditions? In other words, if you subsampled trials to have similar distributions of kinematics between Control and Block conditions, does the effect hold true? The extreme scenario to illustrate my concern is that if Block conditions resulted in all identical movements (which of course they don't), the tuning analysis would find no tuned neurons. The lack of change in decoding accuracy is another yellow flag that there may be a methodological explanation for the decreased tuning result.

The manuscript states that "Our results suggest that the somatosensory cortex may be less involved than the motor areas during feeding, possibly because it is a more ingrained and stereotyped behavior as opposed to tongue protrusion or drinking tasks". Could an alternative explanation be more statistical/technical in nature: that during feeding, there will be more variability in exactly what somatosensation afferent signals are being received from trial to trial (because slight differences in kinematics can have large differences in exactly where the tongue is and the where/when/how of what parts of it are touching other parts of the oral cavity)? This variability could "smear out" the apparent tuning using these types of trial-averaged analyses. Given how important proprioception and somatosensation are for not biting the tongue or choking, the speculation that somatosensory cortical activity is suppressed during feedback is very counter-intuitive to this reviewer.

for - inter-level awareness - Micheal Levin - metaphor - 2 neurons conversing with each other

Reviewer #2 (Public review):

In recent years, lots of researchers tried to explore the existence of new acetyltransferase and deacetylase by using specific antibody enrichment technologies and high resolution mass spectrometry. Here is an example for this effort. Yuqian Wang et al. studied a novel Zn2+- and NAD+-independent KDAC protein, AhCobQ, in Aeromonas hydrophila. They studied the biological function of AhCobQ by using biochemistry method and MS identification technology to confirm it. These results extended our understanding of the regulatory mechanism of bacterial lysine acetylation modifications. However, I find this conclusion is a little speculative, and unfortunately, it also doesn't totally support the conclusion as the authors provided.

Reviewer #2 (Public review):

Summary:

This revised article has characterized the mouse Schlemm's canal expression profile using a comprehensive approach based on sorted SEC, LEC, and BEC total RNA-Seq, scRNA-Seq, and snRNA-Seq to enrich the selection of SECs. The revised study has successfully profiled genome-wide gene expression using sorted SECs, demonstrating that SECs have a closer similarity to LECs than BECs. The combined scRNA- and snRNA-Seq data with deep coverage of gene expression led to the successful identification of many novel biomarkers for inner wall SECs, outer wall SECs, collector channel ECs, and pericytes. In addition, the study also identified two novel states of inner wall SECs separated by new markers. The study provides significant novel information about the biology and expression profile of SECs in the inner and outer walls. It is of great significance to have this novel, convincing, and comprehensive study led by leading researchers published in this journal. The revision has improved the clarity and significance of the study with more details.

Strengths:

This is a comprehensive study using various data to support the expression characterization of mouse SECs. First, the study profiled genome-wide expression using sorted SECs, LECs, and BECs from the same tissue/organ to identify the similarities and differences among the three types of cells. Second, snRNA-Seq was applied to enrich the number of SECs from mouse ocular tissues significantly. Increased sampling of SECs and other cells led to more comprehensive coverage and characterization of cells, including pericytes. Third, the combined scRNA- and snRNA-Seq data analyses increase the power to further characterize the subtle differences within SECs, leading to identifying the expression markers of Inner and Outer wall SECs, collector channel ECs, and distal region cells. Fourth, the identified unique markers were validated for RNA and protein expression in mouse ocular tissues. Fifth, the study explored how the IOP- and glaucoma-associated genes are expressed in the ScRNA- and snRNA-Seq data, providing potential connections of these GWAS genes with IOP and glaucoma. Sixth, the initial pathway and network analyses generated exciting hypotheses that could be tested in other independent studies.

Weaknesses:

The authors have addressed most of the previous comments by adding more details about the protocol and additional discussions. Several comments requiring additional experimental data have been addressed as future directions, such as protein validation, RNA expression validation in human samples, and GWAS-identified IOP genes.

Comments on the latest version:

The authors have addressed previous comments responsively. The authors have suggested several experiments to be completed in the future since these could be time-consuming with human samples. The revised article is with better clarity and clearer significance. No additional comments.

Reviewer #2 (Public review):

Summary:

kTMP is a novel method of stimulating the brain using electromagnetic fields. It has potential benefits over existing technology because it is a safe and easy technology. It explores a range of brain frequencies that has not been explored in depth before (2-5kHz) and thus offers new opportunities.

Strengths:

This work relied on standard methods and was carefully and conservatively performed.

Weaknesses:

There were few weaknesses. The sham condition was prepared as well as could be done, but sham is always challenging in a treatment with sound and sensation, and with knowledgeable operators. New technology, also, is very exciting to subjects and it is difficult to achieve a natural experiment. These difficulties are related to the technology, however, and not to the execution of these experiments..

Reviewer #2 (Public review):

The authors carried out the current studies with the justification that the biochemical mechanisms that lead to alcohol addiction are incompletely understood. The topic and question addressed here are impactful and indeed deserve further research. To this end, a metabolomics approach toward investigating the metabolic effects of alcohol use disorder and the effect of alcohol withdrawal in AUD subjects is valuable. However, this work is primarily descriptive in nature, and these data alone do not meet the stated goal of investigating biochemical mechanisms of alcohol addiction. The current work's most significant limitation is the cross-sectional study design, though inadequate description and citation of the underlying methodological approaches also hampers interest.

Most of the data are cross-sectional in study design, i.e., alcohol use disorder vs controls. However, it is well established that there is a high degree of interpersonal variation with metabolism, and further, there is somewhat high intra-personal variation in metabolism over time. This means that the relatively small cohort of subjects is unlikely to just reflect the broader condition of interest (AUD/withdrawal). The authors report a comparison of a later time-point after alcohol withdrawal (T2) vs the AUD condition. Nonetheless, without replicate time points from the control subjects it is difficult to assess how much of these changes are due to withdrawal vs the intra-personal variation described above. Overall, insufficient experimental context exists to interpret these findings into a biological understanding. For example, while several metabolites are linked with AUD and associated with microbiome or host metabolism based on existing literature, it is unclear from the current study what function these changes have concerning AUD, if any. The authors also argue that alcohol withdrawal shifts the AUD plasma metabolic fingerprint towards healthy controls (line 153). However, this is hard to assess based on the provided plots since the direction of change of the orange data subset considers AUD T2 vs. T1. In contrast, AUD T2 vs. Control would represent the claimed shift. To substantiate these claims, the authors would better support their argument by showing this comparison in all experimental groups (including control subjects) in their multi-dimensional model (e.g., PCA). The authors attempt to extend the significance of their findings by assessing post-mortem brain tissues from AUD subjects; however, the finding that many of the metabolites changed in T2/T1 are also found in AUD brain tissues is interesting but does not strongly support the authors' claims that these metabolites are markers of AUD (line 173). Concerning the plasma cohort itself, it is unclear how the authors assessed for compliance with alcohol withdrawal or whether the subjects' blood-alcohol levels were independently verified.

The second area of concern is the lack of description of the analytical methodology, the lack of metabolite identification validation evidence, and related statistical questions. The authors cite reference #59 regarding the general methodology. However, this reference from their group is a tutorial/review/protocol focused resource paper and it needs to be clarified how specific critical steps were actually applied to the current plasma study samples, given the range of descriptions provided in the citations. The authors report a variety of interesting metabolites, including their primary fragment intensities, which is appreciated (Supp Table 3), but no MS2 matching scores are provided for level 2 or 3 hits. Further, level 1 hits under their definition are validated by an in-house standard, but not supporting data are provided other than this categorization. Finally, a common risk in such descriptive studies is finding spurious associations, especially considering the many factors as described in the current work. These include AUD, depression, anxiety, craving, withdrawal, etc. The authors describe the use of BH correction for multiple-hypothesis testing. Still, this approach only accounts for the many possible metabolite association tests within each comparison (such as metabolites vs. depression) and does not account for the multi-variate comparisons to the many behavior/clinical factors described above. The authors should employ one of several common strategies, such as linear mixed effects models for these types of multi-variate assessments.

Revised Review after Resubmission:

I thank the authors for their responses and revisions to the figures and data and their clarifications of their results and study goals. However, based on this updated information, it is now more apparent that the paper falls into the common trap of descriptive studies where insufficient experimental design was considered to test the association in question robustly. Further, follow-up initiatives are lacking to test the findings by other experimental means. Despite the authors' responses, the paper still fails to convert or interpret the metabolomics findings into any new biological understanding or meaningfully testable hypotheses, and the results remain descriptive in nature with significant caveats.

The authors clarify that their study's "goal was not to investigate the biochemical mechanisms of AUD but how metabolomics could contribute to the psychological alterations of AUD." However, the 2nd sentence of the abstract remains as follows: "The biochemical mechanisms that lead to this disorder are not yet fully understood, and in this respect, metabolomics represents a promising approach to decipher metabolic events related to AUD."This leads the reader to conclude that the purpose of the current study is to use metabolomics to address this gap, despite their later clarification. In the revised response, the authors walk back their claims of these goals, yet the manuscript text and data is largely unchanged in the revision. The serious caveats pointed out by several reviewers concerning the study as reported significantly reduces the utility of the described findings for the broader scientific community, and the authors largely downplay these limitations without addressing the underlying issues.

The authors also clarified in their response that the study's key purpose of the study is to assess "correlations between the blood metabolome and psychological symptoms developed in AUD patients." This goal is dubious as the vast majority of metabolites are not psychoactive, and it is implausible that the metabolome would affect mental state or vice versa. More biological frameworks and citations are needed for this paradigm. The soundness of the goal is further questioned by the study's simplistic design and the authors' admission that "In this discovery-based approach, the aim was to discover potential candidates linked with psychological symptoms for subsequent work to evaluate causality." Yet, the authors side-step the point about the risk of finding spurious associations and decline to control this risk using widely-accepted approaches such as multi-variate correction, instead continuing to use only BH correction for multiple hypothesis testing. The reviewers previously pointed out that BH correction only accounts for the many possible metabolite association tests within each comparison (such as metabolites vs depression). However, it does not account for the multi-variate comparisons to the many behavior/clinical factors. This issue is ignored in the response because the study's goal is hypothesis generating. Instead, the authors focused their responses on the issue of causality which was not the central point of the criticism.

Further, the authors employ mainly systemic plasma analyses unlikely to reflect brain biochemistry. The authors deny that the purpose of including the post-mortem brain tissue data was to demonstrate that "metabolites significantly correlated with the psychological symptoms - and present in the central nervous system (frontal cortex or CSF) - are "markers of AUD," yet if this is not the goal, the structure of the experiment, and the value of these data, is unclear. Another reviewer pointed out that it is difficult to control cross-sectional post-mortem tissue due to a lack of suitable controls, and the authors again side-step the question by citing the lack of suitable controls and the impossibility of "healthy controls" in post-mortem samples. This is true, but this lack of technical feasibility and the confounding factor of CVD/lipid metabolism does not justify the weak experimental design in this respect. Therefore, it remains unclear what can be understood from these data, given the limitations.

Finally, the authors acknowledge the limitation in their revision that they did not assess a second-time point in the control cohort of samples which could have been used to tease apart intra-personal variation from AUD-associated changes during alcohol-abstinence. Unfortunately, this is not a small caveat to simply acknowledge in the discussion section; it severely limits the interpretation and utility of the reported data more broadly, and the authors do not address this underlying problem.

Reviewer #2 (Public review):

Summary:

This research provides compelling and detailed evidence showing that aging influences intrinsic membrane properties of peripheral sympathetic motor neurons, which become hyperexcitable. The authors found that sympathetic motor neurons from old mice exhibit increased firing rates (spontaneous and evoked), more depolarized membrane resting potential, and increased rheobase. Furthermore, the study investigates cellular mechanisms underlying age-associated hyperexcitability and shows solid evidence supporting that a decreased activity of KCNQ2/3 channels during aging is a major contributor to the increased excitability of sympathetic old neurons. The conclusions of this paper are supported by the data.

Strengths:

Detailed and rigorous analysis of electrical responses of peripheral sympathetic motor neurons using electrophysiology (perforated patch and whole-cell recordings). The study identifies a decreased KCNQ2/3 current as a cellular mechanism behind age-induced hyperexcitability in sympathetic motor neurons.

Weaknesses:

The revised version of the manuscript has addressed all my concerns.

Reviewer #2 (Public review):

Summary:

The study explores a new strategy of lysin-derived antimicrobial peptide-primed screening to find peptidoglycan hydrolases from bacterial proteomes. Using this strategy authors identified five peptidoglycan hydrolases from A. baumannii. They further tested their antimicrobial activities on various Gram positive and Gram-negative pathogens.

Strengths:

Overall, the study is good and adds new members to the peptidoglycan hydrolases family. The authors also show that these lysins have bactericidal activities against both Gram-positive and Gram-negative bacteria. The crystal structure data is good, reveals different thermostablility to the peptidoglycan hydrolases. Structural data also reveals that PhAb10 and PHAb11 form thermostable dimer and data is corroborated by generating variant protein defective in supporting intermolecular bond pairs. The mice bacterial infection shows promise for the use of these hydrolases as antimicrobial agents.

Weaknesses:

While the authors have employed various mechanisms to justify their findings, some aspects are still unclear. Only CFU has been used to test bactericidal activity. This should also be corroborated by live/dead assay. Moreover, SEM or TEM analysis would reveal the effect of these peptidoglycan hydrolases on Gram-negative /Gram-positive cell envelopes. The authors claim that these hydrolases are similar to T4 lysozyme, but they have not correlated their findings with already published findings on T4 lysozyme. T4 lysozyme has C-terminal amphipathic helix with antimicrobial properties. Moreover, heat, denatured lysozyme also shows enhanced bactericidal activity due to the formation of hydrophobic dimeric forms, which are inserted in the membrane. Authors also observe that heat denatured PHAb10 and PHAb11 have bactericidal activity but no enzymatic activity. These findings should be corroborated by studying the effect of these holoenzymes/ truncated peptides on bacterial cell membranes. Also, a quantitative peptidoglycan cleavage assay should be performed in addition to halo assay. Including these details would make the work more comprehensive.

Revised version: The authors have addressed most of the questions in the revised version of the paper.

Reviewer #2 (Public review):

Summary:

In this study, the authors aimed to delineate the antimicrobial activity of linalool and tried to investigate the mode of action of linalool against S. parasitica infection. One of the main focuses of this work was to identify the in vitro and in vivo mechanisms associated with the protective role of linalool against S. parasitica infection.

Strengths:

(1) The authors have used a variety of techniques to prove their hypothesis.

(2) An adequate number of replicates were used in their studies.

(3) Their findings showed a protective role of linalool against oomycetes and makes it an attractive future antibiotic in the aquaculture industry.

Weaknesses:

There are several weaknesses in this manuscript.

(1) The authors have taken for granted that the readers already know the experiments/assays used in the manuscript. There was not enough explanation for the figures as well as figure legends.

(2) The authors missed adding the serial numbers to the references.

(3) The introduction section does not provide adequate rationale for their work, rather it is focused more on the assays done.

(4) Full forms are missing in many places (both in the text and figure legends), also the resolution of the figures is not good. In some figures, the font size is too small.

(5) There is much mislabeling of the figure panels in the main text. A detailed explanation of why and how they did the experiments and how the results were interpreted is missing.

(6) There is not enough experimental data to support their hypothesis on the mechanism of action of linalool. Most of the data comes from pathway analysis, and experimental validation is missing.

Overall, the conclusions drawn by the authors are partially justified by the data. Importantly, this paper has discovered the novelty of the compound linalool as a potent antimicrobial agent and might open up future possibilities to use this compound in the aquaculture industry.

Reviewer #2 (Public review):

The authors presented evidence from various in vivo and in vitro experiments demonstrating the mutual interaction between CCL5 and astrocytic miR-342-5p in the ipsilateral core of cerebral ischemia. However, miR-342-5p was downregulated only late after MCAO (D3-7). Additionally, this downregulation was observed not only in the ipsilateral core but also in the ipsilateral penumbra and contralateral sides. Therefore, it is not convincing that the upregulation of CCL5 in the ipsilateral core at later time points (D3 and D7) is attributable to the decreased expression of miR-342-5p. In particular, infarct injury was already evident within a short time period (say 24 h) following MCAO.

(1) The temporal and spatial expression patterns of miR-324-5p do not match those of CCL-5, especially at D1 and D3 (see Figure 1C, 1D). Despite the inverse relationship between miR-324-5p and CCL-5 expression at D7 after MCAO, what was the purpose of administering miR-324-5p agomir (or antagomir) at D1 post-MCAO? If the connection cannot be clearly established, the conclusion reached at the end will be difficult to accept.

(2) Would administering miR-342-5p or anti-CCL5 at later time points (e.g., after D3) reduce infarct size or improve functional recovery? If this is not the case, the effect of CCL5 on neuronal cell damage (infarct size formation) must occur within a very short time after MCAO. Additionally, if the increased CCL5 expression is due to the downregulation of miR-342-5p, its impact would likely be less significant.

(3) While the study offers valuable insights into the roles of CCL5 and its connection with the regulation of miR-342-5p (though this connection is somewhat weak), it is recommended that the authors explore potential translational applications of these findings.

Overall, given the experimental designs and results, it is difficult to support the conclusions drawn in the manuscript.

Reviewer #2 (Public Review):

Summary:

The paper aims to investigate the synergies between desiccation chaperones and small molecule cosolutes, and describe its mechanistic basis. The paper reports that IDP chaperones have stronger synergies with the cosolutes they coexist with, and in one case suggests that this is related to oligomerization propensity of the IDP.

Strengths:

The authors have done a good job improving the paper. The study uses a lot of orthogonal methods and the experiments are technically well done. They are addressing a new question that has not really been addressed previously.

Weaknesses:

The conclusions are still based on a few examples and only partial correlations. However, this is now acknowledged by the authors and the conclusions are presented with appropriate caveats.

Reviewer #2 (Public review):

Sztangierska et al. have investigated the impact of the nucleotide exchange (NEF) factor Hsp110 on the Hsp70-dependent dissolution of amorphous aggregates in the presence of representative members of two classes of J-domain protein.<br /> The authors find that the nucleotide exchange factor of the Hsp110 family, sse1, stimulates the disaggregation activity of yeast Hsp70, ssa1, in particular in the presence of the J-domain protein sis1. Linking chaperone-substrate interactions as determined by biolayer interferometry (BLI) to activity assays, they show that sse1 facilitates the loading of more ssa1 onto the aggregate substrate and propose that this is due to active remodelling of the protein aggregate which exposes more chaperone binding sites and thus facilitates reactivation. This study highlights two important facets of Hsp70 biology: different Hsp70 functions rely on the functional cooperation of specific co-chaperone combinations and the stoichiometry of the different players of the Hsp70 system is an important parameter in tuning Hsp70 chaperone activity.

Strengths:

The manuscript presents a systematic analysis of the functional cooperation of sse1 with a class B J-domain protein sis1 in the disaggregation of two different model aggregate substrates, allowing the authors to draw more general conclusions about Hsp70 disaggregation activity.

The authors can pinpoint the role of sse1 to the initial remodeling of aggregates, rather than the later stages of refolding, highlighting the functional specificity of Hsp70 co-chaperones.

They demonstrate the competitive nature of binding to ssa1 between sse1 and sis1 which can explain the poisoning of Hsp70 chaperone activities observed at high NEF concentrations.

Weaknesses:

While structural requirements have been identified that allow sse1, in cooperation with sis1, to facilitates the loading of Hsp70 on the amorphous aggregate substrate, how this is achieved on a mechanistic level remains an open question.

Reviewer #2 (Public review):

Summary

The authors present a method that allows for the identification and localization of molecular machinery at chemical synapses in unstained, unfixed native brain tissue slices. They believe that this approach will provide a 3D structural basis for understanding different mechanisms of synaptic transmission, plasticity, and development. To achieve this, the group used genetically engineered mouse lines and generated thin brain slices that underwent high-pressure freezing (HPF) and focused ion beam (FIB) milling. Utilizing cryo-electron tomography (cryo-ET) and integrating it with cryo-fluorescence microscopy, they achieved micrometer resolution in identifying the glutamatergic synapses along with nanometer resolution to locate AMPA receptors GluA2-subunits using Fab-AuNP conjugates. The findings are summarized with detailed examples of successfully prepared substrates for cryo-ET, specific morphological identification and localization, and the detailed structural organization of excitatory synapses, including synaptic vesicle clusters close to the postsynaptic density and in the cleft.

Strengths

The study advances previous work that used cultured neurons or synaptosomes. Combining cryo-electron tomography (cryo-ET) with fluorescence-guided targeting and labeling with Fab-AuNP conjugates enabled the study of synapses and molecular structures in their native environment without chemical fixation or staining. This preserves their near-native state, offering high specificity and resolution. The methods developed are mostly generalizable, allowing adaptation for identifying and localizing other key molecules at glutamatergic synapses and potentially useful for studying a variety of synapses and cellular structures beyond the scope of this research.

Weaknesses

The preparation and imaging techniques are complex and require highly specialized equipment and expertise, potentially limiting their accessibility and widespread adoption.

Additionally, the methods might need further modifications/tweaks to study other types of synapses or molecular structures effectively.

The reliance on genetically engineered mouse lines and monoclonal, high-affinity antibodies/Fab fragments to specifically label receptors/proteins would limit the wider employment of these methods.

Reviewer #2 (Public review):

Summary:

Yang and colleagues developed a new in vitro blood-brain barrier model that is relatively simple yet outperforms previous models. By incorporating a neuroblastoma cell line, they demonstrated increased electrical resistance and decreased permeability to small molecules

Strengths:

The authors initially elucidated the soluble mediator responsible for enhancing endothelial functionality, namely GDNF. Subsequently, they elucidated the mechanisms by which GDNF upregulates the expression of VE-cadherin and Claudin-5. They further validated these findings in vivo, and demonstrated predictive value for molecular permeability as well. The study is meticulously conducted and easily comprehensible. The conclusions are firmly supported by the data, and the objectives are successfully achieved. This research is poised to advance future investigations in BBB permeability, leakage, dysfunction, disease modeling, and drug delivery, particularly in high-throughput experiments. I anticipate an enthusiastic reception from the community interested in this area. While other studies have produced similar results with tri-cultures (PMID: 25630899), this study notably enhances electrical resistance compared to previous attempts.

Weaknesses:

The power of this system lies in its simplicity, which is enough to study BBB permeability. However, it also lacks some other important cell-cell interactions such as those involving pericytes. Nonetheless, this is still a valuable tool for high throughput screening.

As with many other similar systems, it has lower TEER values compared to the in vivo counterpart, this is an issue that researchers in the field will have to address in future studies

Reviewer #2 (Public review):

Summary:

The authors investigated if obesity is associated with elevated working memory deficits. Prior theorizing would suggest that individuals with a higher BMI would be worse at working memory updating, potentially due to impaired dopaminergic signaling in the striatum. However, the authors find that higher BMI was associated with worse working memory performance, irrespective of having to ignore or update new information. To further explore the putative dopaminergic mechanisms, participants are stratified according to genetic polymorphisms in COMT, Taq1A, DARPP and C957T and the ratio of the amino acids phenylalanine and tyrosine, all implicated in dopamine-signaling. They find that carrying specific alleles of Taq1A and DARPP, but not of COMT and C957T, mitigated the otherwise negative relationship between BMI and working memory for updating, but not for maintenance.

The authors put forward several possible mechanistic explanations of these observations, including imbalances in the striatal go/no-go dopamine pathways. However, only future, more direct measures of dopamine signaling can provide a confirmation of these hypotheses.

Strengths:

Differentiating between working memory maintenance (ignoring) and updating is a powerful way to get a deeper insight into specific working memory deficits in individuals with obesity. This way of assessing working memory could potentially be applied to various populations at risk for cognitive or working memory deficits.

By pooling data from three studies, the authors reached a relatively large sample of 320 participants, which enables the assessment of more subtle effects on working memory, including the differentiation between updating and ignoring.

Working memory gating has long implicated striatal dopamine signaling. This paper shows that a specific combination of a high BMI and specific dopamine-related genotypes can selectively moderate working memory updating. More insight into how these risk factors interact can ultimately lead to more tailor-made treatments.

Weaknesses:

The introduction mentions that specific alleles can alter dopamine signaling in various ways. However, the authors are less clear on how they expect these alterations to subsequently affect working memory updating and maintenance in the current study. While I understand that the complexity of these mechanisms might make it challenging to form specific predictions, it would be helpful if the authors acknowledged this uncertainty and clarified that their analyses are exploratory in nature, and they will therefore refrain from any directional hypotheses regarding the genotypes.

The majority of participants seems to fall within the normal BMI-range, whereas the interaction between BMI and genetic variations or amino acid ratio particularly surfaces at higher BMI. As genetic variations are usually associated with small effect sizes, the effective sample size, although large for a behavioral analysis only, might have been too small to detect meaningful effects of particular alleles of COMT and C957T.

The relationships between genetic variations, BMI and specific disturbances in dopamine signaling are complex, as compensating mechanisms might be at play to mitigate any detrimental effects. Future studies that apply more direct measures or manipulations of dopaminergic processes could therefore aid in mechanistically explaining the results.

Reviewer #2 (Public Review):

Summary:

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 that ameliorate disease-specific phenotypes in vitro.

Strengths:

This manuscript engaged in an important topic and yielded some interesting data.

Reviewer #3 (Public review):

Previous work (Chouhan et al., 2022) from the Sehgal group investigated the relationship between sleep and long-term memory formation by dissecting the role of mushroom body intrinsic neurons, extrinsic neurons, and output neurons during sleep-dependent and sleep-independent memory consolidation. In this manuscript, Li et al., profiled transcriptome in the anterior-posterior (ap) α'/β' neurons and identified genes that are differentially expressed after training in fed condition, which supports sleep-dependent memory formation. By knocking down candidate genes systematically, the authors identified Polr1F and Regnase-1 as two important hits that play potential roles in sleep and memory formation. What is the function of sleep and how to create a memory are two long-standing questions in science. The present study used a new approach to identify novel components that may link sleep and memory consolidation in a specific type of neuron. Importantly, these components implicated that RNA processing may play a role in these processes.

While I am enthusiastic about the innovative approach employed to identify RNA processing genes involved in sleep regulation and memory consolidation, I feel that the data presented in the manuscript is insufficient to support the claim that these two genes establish a definitive link between sleep and memory consolidation. First, the developmental role of Regnase-1 in reducing sleep remains unclear because knocking down Regnase-1 using the GeneSwitch system produced neither acute nor chronic sleep loss phenotype. In the revised manuscript, the author used the Gal80ts to restrict the knockdown of Regnase-1 in adult animals and concluded that Regnase-1 RNAi appears to affect sleep through development. Conducting overexpression experiments of Regnase-1 would lend some credibility to the phenotypes, however, this is not pursued in the revised manuscript. Second, while constitutive Regnase-1 knockdown produced robust phenotypes for both sleep-dependent and sleep-independent memory, it also led to a severe short-term memory phenotype. This raises the possibility that flies with constitutive Regnase-1 knockdown are poor learners, thereby having little memory to consolidate. The defect in learning could be simply caused by chronic sleep loss before training. Thus, this set of results does not substantiate a strong link between sleep and long-term memory consolidation. Lastly, the discussion on the sequential function of training, sleep, and RNA processing on memory consolidation appears speculative based on the present data.

Reviewer #2 (Public review):

Summary:

The authors used a combination of anchored hybrid enrichment and Sanger sequencing to construct a phylogenomic data set for the weevil family Belidae. Using evidence from fossils and previous studies they are able to estimate a phylogenetic tree with a range of dates for each node - a timetree. They use this to reconstruct the history of the belids' geographic distributions and associations with their hostplants. They infer that the belids' association with conifers pre-dates the rise of the angiosperms. They offer an interpretation of belid history in terms of the breakup of Gondwanaland, but acknowledge that they cannot rule out alternative interpretations that invoke dispersal.

Strengths:

The strength of any molecular-phylogenetic study hinges on four things: the extent of the sampling of taxa; the extent of the sampling of loci (DNA sequences) per genome; the quality of the analysis; and - most subjectively - the importance and interest of the evolutionary questions the study allows the authors to address. The first two of these, sampling of taxa and loci, impose a tradeoff: with finite resources, do you add more taxa or more loci? The authors follow a reasonable compromise here, obtaining a solid anchored-enrichment phylogenomic data set (423 genes, >97 kpb) for 33 taxa, but also doing additional analyses that included 13 additional taxa from which only Sanger sequencing data from 4 genes was available. The taxon sampling was pretty solid, including all 7 tribes and a majority of genera in the group. The analyses also seemed to be solid - exemplary, even, given the data available.

This leaves the subjective question of how interesting the results are. The very scale of the task that faces systematists in general, and beetle systematists in particular, presents a daunting challenge to the reader's attention: there are so many taxa, and even a sophisticated reader may never have heard of any of them. Thus it's often the case that such studies are ignored by virtually everyone outside a tiny cadre of fellow specialists. The authors of the present study make an unusually strong case for the broader interest and importance of their investigation and of its focal taxon, the belid weevils.

The belids are of special interest because - in a world churning with change and upheaval, geologically and evolutionarily - relatively little seems to have been going on with them, at least with some of them, for the last hundred million years or so. The authors make a good case that the Araucaria-feeding belid lineages found in present-day Australasia and South America have been feeding on Araucaria continuously since the days when it was a dominant tree taxon nearly worldwide, before it was largely replaced by angiosperms. Thus these lineages plausibly offer a modern glimpse of an ancient ecological community.

Comments on current version:

The MS was already in pretty good shape last time around, and the authors have made most of the minor revisions and copy-edits suggested by the reviewers. There may be a few remaining points of disagreement with the reviewers, but these seem to be minor matters of opinion and nothing that ought to delay publication.

Reviewer #2 (Public Review):

Summary:

The authors identify the root cap as an important key region for establishing microbial symbioses with roots. By highlighting for the first time the crucial importance of tight regulation of a specific form of programmed cell death of root cap cells and the clearance of their cell corpses, they start unraveling the molecular mechanisms and its regulation at the root cap (e.g. by identifying an important transcription factor) for the establishment of symbioses with fungi (and potentially also bacterial microbiomes).

Strengths:

It is often believed that the recruitment of plant microbiomes occurs from bulk soil to rhizosphere to endosphere. These authors demonstrate that we have to re-think microbiome assembly as a process starting and regulated at the root tip and proceeding along the root axis.

Comments on revised version:

The authors have addressed all critical points in their revision.

Reviewer #2 (Public review):

Summary:

This manuscript examines expression of orexin receptors in midbrain - with a focus on dopamine neurons - and uses several fairly sophisticated manipulation techniques to explore the role of this peptide neurotransmitter in reward-related behaviors. Specifically, in situ hybridization is used to show that dopamine neurons predominantly express orexin receptor 1 subtype and then go on to delete this receptor in dopamine transporter-expressing using a transgenic strategy. Ex vivo calcium imaging of midbrain neurons is used to show that, in the absence of this receptor, orexin is no longer able to excite dopamine neurons of the substantia nigra.

The authors proceed to use this same model to study the effect of orexin receptor 1 deletion on a series of behavioral tests, namely, novelty-induced locomotion and exploration, anxiety-related behavior, preference for sweet solutions, cocaine-induced conditioned place preference, and energy metabolism. Of these, the most consistent effects are seen in the tests of novelty-induced locomotion and exploration in which the mice with orexin 1 receptor deletion are observed to show greater levels of exploration, relative to wild-type, when placed in a novel environment, an effect that is augmented after icv administration of orexin.

In the final part of the paper, the authors use PET imaging to compare brain-wide activity patterns in the mutant mice compared to wildtype. They find differences in several areas both under control conditions (i.e., after injection of saline) as well as after injection of orexin. They focus in on changes in dorsal bed nucleus of stria terminalis (dBNST) and the lateral paragigantocellular nucleus (LPGi) and perform analysis of the dopaminergic projections to these areas. They provide anatomical evidence that these regions are innervated by dopamine fibers from midbrain, are activated by orexin in control, but not mutant mice, and that dopamine receptors are present. Thus, they argue these anatomical data support the hypothesis that behavioral effects of orexin receptor 1 deletion in dopamine neurons are due to changes in dopamine signaling in these areas.

Strengths:

Understanding how orexin interacts with the dopamine system is an important question and this paper contains several novel findings along these lines. Specifically:

(1) Distribution of orexin receptor subtypes in VTA and SN is explored thoroughly.<br /> (2) Use of the genetic model that knocks out a specific orexin receptor subtype from dopamine-transporter-expressing neurons is a useful model and helps to narrow down the behavioral significance of this interaction.<br /> (3) PET studies showing how central administration of orexin evokes dopamine release across the brain is intriguing, especially that two key areas are pursued - BNST and LPGi - where the dopamine projection is not as well described/understood.

Weaknesses:

The role of the orexin-dopamine interaction is not explored in enough detail. The manuscript presents several related findings, but the combination of anatomy and manipulation studies do not quite tell a cogent story. Ideally, one would like to see the authors focus on a specific behavioral parameter and show that one of their final target areas (dBNST or LPGi) was responsible or at least correlated with this behavioral readout.

In many places in the Results, insufficient explanation and statistical reporting is provided. Throughout the Results - especially in the section on behavior although not restricted to this part - statements are made without statistical tests presented to back up the claims, e.g., "Compared to controls, Ox1RΔDAT 143 mice did not show significant changes in spontaneous locomotor activity in home cages" (L143) and "In a hole-board test, female Ox1RΔDAT mice showed increased nose pokes into the holes in early (1st and 2nd) sessions compared to control mice" (L151). In other places, ANOVAs are mentioned but full results including main effects and interactions are not described in detail, e.g., in F3-S3, only a single p-value is presented and it is difficult to know if this is the interaction term or a post hoc test (L205). These and all other statements need statistics included in the text as support. Addition of these statistical details was also requested by the editor.

In the presentation of reward processing this is particularly important as no statistical tests are shown to demonstrate that controls show a cocaine-induced preference or a sucrose preference. Here, one option would be to perform one-sample t-tests showing that the data were different to zero (no preference). As it is, the claim that "Both of the control and Ox1RΔDAT groups showed a preference for cocaine injection" is not yet statistically supported.

Reviewer #2 (Public review):

Summary:

The study of Rollenhagen et al. examines the ultrastructural features of Layer 1 of the human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as far as possible from the epilepsy focus, and as such considered to be non-epileptic. The analyses included 4 patients with different ages, sex, medication, and onset of epilepsy. The manuscript is a follow-on study with 3 previous publications from the same authors on different layers of the temporal cortex:

Layer 4 - Yakoubi et al 2019 eLife<br /> Layer 5 - Yakoubi et al 2019 Cerebral Cortex<br /> Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex.

They find, that the L1 synaptic boutons mainly have a single active zone, a very large pool of synaptic vesicles, and are mostly devoid of astrocytic coverage.

Strengths:

The manuscript is well-written and easy to read. The Results section gives a detailed set of figures showing many morphological parameters of synaptic boutons and glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in the human brain are still very limited, the current manuscript has substantial relevance.

The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analysis is clear and precise.

Weaknesses:

One of the main findings of this paper is that "low degree of astrocytic coverage of L1 SBs suggests that glutamate spillover and as a consequence synaptic cross-talk may occur at the majority of synaptic complexes in L1". However, the authors only quantified the volume ratio of astrocytes in all 6 layers, which is not necessarily the same as the glial coverage of synapses. In order to strengthen this statement, the authors could provide 3D data (that they have from the aligned serial sections) detailing the percentage of synapses that have glial processes in close proximity to the synaptic cleft, that would prevent spillover.

A specific statement is missing on whether only glutamatergic boutons were analysed in this MS, or GABAergic boutons were also included. There is a statement, that they can be distinguished from glutamatergic ones, but it would be useful to state it clearly in the Abstract, Results, and Methods section what sort of boutons were analysed. Also, what is the percentage of those boutons from the total bouton population in L1?

Synaptic vesicle diameter (that has been established to be ~40nm independent of species) can properly be measured with EM tomography only, as it provides the possibility to find the largest diameter of every given vesicle. Measuring it in 50 nm thick sections results in underestimation (just like here the values are ~25 nm) as the measured diameter will be smaller than the true diameter if the vesicle is not cut in the middle, (which is the least probable scenario). The authors have the EM tomography data set for measuring the vesicle diameter properly.

It is a bit misleading to call vesicle populations at certain arbitrary distances from the presynaptic active zone as readily releasable pool, recycling pool, and resting pool, as these are functional categories, and cannot directly be translated to vesicles at certain distances. Indeed, it is debated whether the morphologically docked vesicles are the ones, that are readily releasable, as further molecular steps, such as proper priming are also a prerequisite for release.

Tissue shrinkage due to aldehyde fixation is a well-documented phenomenon that needs compensation when dealing with density values. The authors cite Korogod et al 2015 - which actually draws attention to the problem comparing aldehyde fixed and non-fixed tissue, still the data is non-compensated in the manuscript. Since all the previous publications from this lab are based on aldehyde fixed non-compensated data, and for this sake, this dataset should be kept as it is for comparative purposes, it would be important to provide a scaling factor applicable to be able to compare these data to other publications.

Reviewer #3 (Public review):

Summary:

How is it that animals find learned food locations in their daily life? Do they use landmarks to home in on these learned locations or do they learn a path based on self-motion (turn left, take ten steps forward, turn right, etc.). This study carefully examines this question in a well designed behavioral apparatus. A key finding is that to support the observed behavior in the hidden food arena, mice appear to not use the distal cues that are present in the environment for performing this task. Removal of such cues did not change the learning rate, for example. In a clever analysis of whether the resulting cognitive map based on self-motion cues could allow a mouse to take a shortcut, it was found that indeed they are. The work nicely shows the evolution of the rodent's learning of the task, and the role of active sensing in the targeted reduction of uncertainty of food location proximal to its expected location.

Strengths:

A convincing demonstration that mice can synthesize a cognitive map for the finding of a static reward using body frame-based cues. Showing that uncertainty of final target location is resolved by an active sensing process of probing holes proximal to the expected location. Showing that changing the position of entry into the arena rotates the anticipated location of the reward in a manner consistent with failure to use distal cues.

Weaknesses:

The task is low stakes, and thus the failure to use distal cues at most costs the animal a delay in finding the food; this delay is likely unimportant to the animal, and the pre-training procedure is likely to make it clear to the animal's that distal cues are unreliable even if desirable to use. Thus, it is unclear whether this result would generalize to a situation where the animal may be under some time pressure, urgency due to food (or water) restriction, or due to predatory threat, or situations where distal cues are reliable. In such cases, the use of distal cues to make locating the reward robust to changing start locations may be more likely to be observed.

Reviewer #2 (Public review):

The article focuses on the study of Magnaporthe oryzae, the fungal pathogen responsible for rice blast disease, which poses a significant threat to global food security. The research delves into the infection mechanisms of the pathogen, particularly the role of penetration pegs and the formation of a penetration ring in the invasion process. The study highlights the persistent localization of Ppe1 and its homologs to the penetration ring, suggesting its function as a structural feature that facilitates the transition of penetration pegs into invasive hyphae. The article provides a thorough examination of the infection process of M. oryzae, from the attachment of conidia to the development of appressoria and the formation of invasive hyphae. The discovery of the penetration ring as a structural element that aids in the invasion process is a significant contribution to the understanding of plant-pathogen interactions. The experimental methods are well-documented, allowing for reproducibility and validation of the results.

Reviewer #2 (Public Review):

Summary:

The manuscript emphasizes a phylogenetic conservation of the hippocampal region and primary sensory cortical regions in mammalian species. The authors then propose that the evident species-specific differences in behavior and memory-related functions may be due to differences in type and amount of cortico-hippocampal connectivity.

Strengths:

The authors are well-established researchers with a long history of excellent results and publications. The question (co-influence of cortical and hippocampal connections) is potentially interesting.

Weaknesses:

The treatment is very broad and macro scale, ignoring the likelihood that hippocampal-cortical connectivity and behavioral outcomes result from multiple differences at a more micro-scale. The designated "mammalian" sample is also broad. Thus, it can appear incomplete as a sample, and incompletely discussed.

Reviewer #2 (Public review):

Summary:

This paper provides an ingenious experimental test of an efficient coding objective based on optimization as a task success. The key idea is that different tasks (estimation vs discrimination) will, under the proposed model, lead to a different scaling between the encoding precision and the width of the prior distribution. Empirical evidence in two tasks involving number perception supports this idea.

Strengths:

- The paper provides an elegant test of a prediction made by a certain class of efficient coding models previously investigated theoretically by the authors.

The results in experiments and modeling suggest that competing efficient coding models, optimizing mutual information alone, may be incomplete by missing the role of the task.

Weaknesses:

- The claims would be more strongly validated if data were present at more than two widths in the discrimination experiment.

- A very strong prediction of the model -- which determines encoding entirely from prior and task -- is that Fisher Information is uniform throughout the range, strongly at odds with the traditional assumption of imprecision increasing with the numerosity (Weber/Fechner law). This prediction should be checked against the data collected. It may not be trivial to determine this in the Estimation experiment, but should be feasible in the Discrimination experiment in the Wide condition: Is there really no difference in discriminability at numbers close to 10 vs numbers close to 90? Figure 2 collapses over those, so it's not evident whether such a difference holds or not. I'd have loved to look into this in reviewing, but the authors have not yet made their data publicly available - I strongly encourage them to do so.

Importantly, the inverse u-shaped pattern in Figure 1 is itself compatible with a Weber's-law-based encoding, as shown by simulation in Figure 5d in Hahn&Wei [1]. This suggests a potential competing variant account, in apparent qualitative agreement with the findings reported: the encoding is compatible with Fisher's law, and only a single scalar, the magnitude of sensory noise, is optimized for the task for the loss function (3). As this account would be substantially more in line with traditional accounts of numerosity perception - while still exhibiting task-dependence of encoding as proposed by the authors - it would be worth investigating if it can be ruled out based on the data gathered for this paper.

References:

[1] Hahn & Wei, A unifying theory explains seemingly contradictory biases in perceptual estimation, Nature Neuroscience 2024

Reviewer #2 (Public review):

Summary:

In their study, Cooper et al. investigated the spontaneous fluctuations in extracellular 5-HT release in the CA1 region of the hippocampus using GRAB5-HT3.0. Their findings revealed the presence of ultra-low frequency (less than 0.05 Hz) oscillations in 5-HT levels during both NREM sleep and wakefulness. The phase of these 5-HT oscillations was found to be related to the timing of hippocampal ripples, microarousals, electromyogram (EMG) activity, and hippocampal-cortical coherence. In particular, ripples were observed to occur with greater frequency during the descending phase of 5-HT oscillations, and stronger ripples were noted to occur in proximity to the 5-HT peak during NREM. Microarousal and EMG peaks occurred with greater frequency during the ascending phase of 5-HT oscillations. Additionally, the strongest coherence between the hippocampus and cortex was observed during the ascending phase of 5-HT oscillations. These patterns were observed in both NREM sleep and the awake state, with a greater prevalence in NREM. The authors posit that 5-HT oscillations may temporally segregate internal processing (e.g., memory consolidation) and responsiveness to external stimuli in the brain.

Strengths:

The findings of this research are novel and intriguing. Slow brain oscillations lasting tens of seconds have been suggested to exist, but to my knowledge they have never been analyzed in such a clear way. Furthermore, although it is likely that ultra-slow neuromodulator oscillations exist, this is the first report of such oscillations, and the greatest strength of this study is that it has clarified this phenomenon both statistically and phenomenologically.

Weaknesses:

As with any paper, this one has some limitations. While there is no particular need to pursue them, I will describe ten of them below, including future directions:

(1) Contralateral recordings: 5-HT levels and electrophysiological recordings were obtained from opposite hemispheres due to technical limitations. Ipsilateral simultaneous recordings may show more direct relationships.

(2) Sample size: The number of mice used in the experiments is relatively small (n=6). Validation with a larger sample size would be desirable.

(3) Lack of causality: The observed associations show correlations, not direct causal relationships, between 5-HT oscillations and neural activity patterns.

(4) Limited behavioral states: The study focuses primarily on sleep and quiet wakefulness. Investigation of 5-HT oscillations during a wider range of behavioral states (e.g., exploratory behavior, learning tasks) may provide a more complete understanding.

(5) Generalizability to other brain regions: The study focuses on the CA1 region of the hippocampus. It's unclear whether similar 5-HT oscillation patterns exist in other brain regions.

(6) Long-term effects not assessed: Long-term effects of ultra-low 5-HT oscillations (e.g., on memory consolidation or learning) were not assessed.

(7) Possible species differences: It's uncertain whether the findings in mice apply to other mammals, including humans.

(8) Technical limitations: The temporal resolution and sensitivity of the GRAB5-HT3.0 sensor may not capture faster 5-HT dynamics.

(9) Interactions with other neuromodulators: The study does not explore interactions with other neuromodulators (e.g., norepinephrine, acetylcholine) or their potential ultraslow oscillations.

(10) Limited exploration of functional significance: While the study suggests a potential role for 5-HT oscillations in memory consolidation and arousal, direct tests of these functional implications are not included.

Reviewer #2 (Public review):

Summary:

The authors provide valuable findings characterizing a Prosapip1 conditional knockout mouse and the effects of knockout on hippocampal excitatory transmission, NMDAR transmission, and several learning behaviors. Furthermore, the authors selectively and conditionally knockout Prosapip1 in the dorsal hippocampus and show that it is required for the same spatial learning and memory assessed in the conditional knockout mice. The study uncovers how Prosapip1 is involved PSD organization and is a functional and critical player in dorsal Hippocampal LTP via its interaction with GluN2B subunits.

Strengths:

The study is well-controlled and detailed, and the data in the paper match the conclusions.

Weaknesses:

Some statistical information is lacking.

Reviewer #2 (Public review):

Summary:

In this paper, the authors investigate the role of NMDA-receptors in recurrent processing. In doing so, the authors present data from two studies, where they attempt to decode different stimulus features, namely contrast, collinearity, and illusory contours. The latter of which the authors claim relies uniquely on recurrent processing. Therefore, to test whether NMDA receptors are particularly involved in recurrent processing they administer a NMDA-antagonist to see whether the decoding of illusory contours is specifically perturbed, and leaves the decoding of other features intact. They further aim to disentangle the role of NMDA-receptors by manipulating visibility and task relevance of the decoded features

In the first experiment, the authors decode two targets, the first was always presented clearly, the second's visibility was manipulated by presenting it after a short lag rather than a long lag (inducing attentional blink), as well as masking the target on half the trials. First, they find for target 1 clear evidence for the NMDA-receptor increasing (rather than decreasing) decoding performance of illusory contours. They move on to analyse target 2 to explore the manipulations of lag and masking. Here they find that masking reduced decoding of all three stimulus features, but only the lag reduced decoding of illusory contours. Importantly, the NMDA-antagonist improved decoding only in the unmasked, long lag condition, in the cluster analyses. However, the interaction with the lag condition was not significant, and the effect on decoding was primarily present in the later decoding time window, and not significant when exploring the peak of the decoding time window.

The second experiment was highly similar, but got rid of the lag manipulation, and replaced it with a manipulation of task relevance. Notably, masking did not abolish the decoding of illusory contours completely, in contrast to the first experiment. More importantly, they find that the NMDA-receptor now clearly increases decoding of illusory contours, particularly when the illusory contours are not masked. No effect of task relevance is found.

Taken together the authors state that evidence is found for NMDA-receptors role in recurrent processing.

Strengths:

This is an interesting study using state-of-the-art methods in combination with drug manipulation to study recurrent processing. Their analysis methods are state-of-the-art, and the question that they are trying to address is topical and interesting to a wide research audience, encompassing both researchers interested in visual perception and consciousness, as well as those interested in perturbed vision as found in psychiatric disorders.

Weaknesses:

The experimental design is somewhat complicated, which can make it difficult to match the authors' claims to the actual evidence that is provided. I have some reservations about the paper which are born out of a few issues.<br /> (1) The title, abstract, and introduction hide their counterintuitive finding of increased decoding, presumably as it was unexpected.<br /> (2) Their analysis choices are sometimes unclear, making it difficult to assess whether the analyses are sensible.<br /> (3) The appropriate tests for the interactions that the authors claim they found are often lacking.

To start off, I think the reader is being a bit tricked when reading the paper. Perhaps my priors are too strong, but I assumed, just like the authors, that NMDA-receptors would disrupt recurrent processing, in line with previous work. However, due to the continuous use of the ambiguous word 'affected' rather than the more clear increased or perturbed recurrent processing, the reader is left guessing what is actually found. That's until they read the results and discussion finding that decoding is actually improved. This seems like a really big deal, and I strongly urge the authors to reword their title, abstract, and introduction to make clear they hypothesized a disruption in decoding in the illusion condition, but found the opposite, namely an increase in decoding. I want to encourage the authors that this is still a fascinating finding.

Apologies if I have missed it, but it is not clear to me whether participants were given the drug or placebo during the localiser task. If they are given the drug this makes me question the logic of their analysis approach. How can one study the presence of a process, if their very means of detecting that process (the localiser) was disrupted in the first place? If participants were not given a drug during the localiser task, please make that clear. I'll proceed with the rest of my comments assuming the latter is the case. But if the former, please note that I am not sure how to interpret their findings in this paper.

The main purpose of the paper is to study recurrent processing. The extent to which this study achieves this aim is completely dependent to what extent we can interpret decoding of illusory contours as uniquely capturing recurrent processing. While I am sure illusory contours rely on recurrent processing, it does not follow that decoding of illusory contours capture recurrent processing alone. Indeed, if the drug selectively manipulates recurrent processing, it's not obvious to me why the authors find the interaction with masking in experiment 2. Recurrent processing seems to still be happening in the masked condition, but is not affected by the NMDA-receptor here, so where does that leave us in interpreting the role of NMDA-receptors in recurrent processing? If the authors can not strengthen the claim that the effects are completely driven by affecting recurrent processing, I suggest that the paper will shift its focus to making claims about the encoding of illusory contours, rather than making primary claims about recurrent processing.

An additional claim is being made with regards to the effects of the drug manipulation. The authors state that this effect is only present when the stimulus is 1) consciously accessed, and 2) attended. The evidence for claim 1 is not supported by experiment 1, as the masking manipulation did not interact in the cluster-analyses, and the analyses focussing on the peak of the timing window do not show a significant effect either. There is evidence for this claim coming from experiment 2 as masking interacts with the drug condition. Evidence for the second claim (about task relevance) is not presented, as there is no interaction with the task condition. A classical error seems to be made here, where interactions are not properly tested. Instead, the presence of a significant effect in one condition but not the other is taken as sufficient evidence for an interaction, which is not appropriate. I therefore urge the authors to dampen the claim about the importance of attending to the decoded features. Alternatively, I suggest the authors run their interactions of interest on the time-courses and conduct the appropriate cluster-based analyses.

How were the length of the peak-timing windows established in Figure 1E? My understanding is that this forms the training-time window for the further decoding analyses, so it is important to justify why they have different lengths, and how they are determined. The same goes for the peak AUC time windows for the interaction analyses. A number of claims in the paper rely on the interactions found in these post-hoc analyses, so the 223- to 323 time window needs justification.

Reviewer #2 (Public review):

Summary:

I reviewed the manuscript titled "Translational Control in the Spinal Cord Regulates Gene Expression and Pain Hypersensitivity in the Chronic Phase of Neuropathic Pain." This manuscript compares transcription and translation in the spinal cord during the acute and chronic phases of neuropathic pain induced by surgical nerve injury. The authors chose to focus their investigation on translation in the chronic phase due to its greater impact on gene expression in the spinal cord compared to transcription.

(1) The study is significant because the molecular mechanisms underlying chronic pain remain elusive. The role of translational regulation in the spinal cord has not been investigated in neuroplasticity and chronic pain mouse models. The manuscript is innovative and technically robust. The authors employed several cutting-edge techniques such as Rio-seq, TRAP-seq, slice electrophysiology, and viral approaches. Despite the technical complexity, the manuscript is well-written. The authors demonstrated that inhibition of eIF4E alleviates pain hypersensitivity, that de novo protein synthesis is more pronounced in inhibitory interneurons, and that manipulating mTOR-eIF4E pathways alters mechanical sensitivity and neuroplasticity.

(2) Strengths: innovation (conceptual and technical levels), data support the conclusions.

Weakness:

Confusion about the sex of the animals. It is unclear whether eIF4E ASO affects translation and which cells. It is not determined that modulating translation in PV+ neurons impacts neuropathic pain behaviors.

Reviewer #2 (Public Review):

The manuscript by Dearlove et al. entitled "DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids" reports a novel activity of a DELTEX E3 ligase family member, DTX3L, which can conjugate ubiquitin to the 3' hydroxyl of single-stranded oligonucleotides via an ester linkage. The findings that unmodified oligonucleotides can act as substrates for direct ubiquitylation and the identification of DTX3 as the enzyme capable of performing such oligonucleotide modification are novel, intriguing, and impactful because they represent a significant expansion of our view of the ubiquitin biology. The authors perform a detailed and diligent biochemical characterization of this novel activity, and key claims made in the article are well supported by experimental data. However, the studies leave room for some healthy skepticism about the physiological significance of the unique activity of DTX3 and DTX3L described by the authors because DTX3/DTX3L can also robustly attach ubiquitin to the ADP ribose moiety of NAD or ADP-ribosylated substrates. The study could be strengthened by a more direct and quantitative comparison between ubiquitylation of unmodified oligonucleotides by DTX3/DTX3L with the ubiquitylation of ADP-ribose, the activity that DTX3 and DTX3L share with the other members of the DELTEX family.

Comment on revised version:

In my opinion, reviewers' comments are constructively addressed by the authors in the revised manuscript, which further strengthens the revised submission and makes it an important contribution to the field. Specifically, the authors perform a direct quantitative comparison of two distinct ubiquitylation substrates, unmodified oligonucleotides and fluorescently labeled NADH and report that kcat/Km is 5-fold higher for unmodified oligos compared to NADH. This observation suggests that ubiquitylation of unmodified oligos is not a minor artifactual side reaction in vitro and that unmodified oligonucleotides may very well turn out to be the physiological substrates of the enzyme. However, the true identity of the physiological substrates and the functionally relevant modification site(s) remain to be established in further studies.

Reviewer #2 (Public review):

In this article, Tian et al present a convincing analysis of the molecular mechanisms underpinning TIPE-mediated regulation of glycolysis and tumor growth in melanoma. The authors begin by confirming TIPE expression in melanoma cell lines and identify "high" and "low" expressing models for functional analysis. They show that TIPE depletion slows tumour growth in vivo, and using both knockdown and over expression approaches, show that this is associated with changes in glycolysis in vitro. Compelling data using multiple independent approaches is presented to support an interaction between TIPE and the glycolysis regulator PKM2, and over-expression of TIPE promoted nuclear translocation of PKM2 dimers. Mechanistically, the authors also demonstrate that PKM2 is required for TIPE-mediated activation of HIF1a transcriptional activity, as assessed using an HRE-promoter reporter assay, and that TIPE-mediated PKM2 dimerization is p-ERK dependent. Finally, the dependence of TIPE activity on PKM2 dimerization was demonstrated on tumor growth in vivo and in regulation of glycolysis in vitro, and ectopic expression of HIF1a could rescue inhibition of PKM2 dimerization in TIPE overexpressing cells and reduced induction of general cancer stem cell markers, showing a clear role for HIF1a in this pathway.

The detailed mechanistic analysis of TIPE mediated regulation of PKM2 to control aerobic glycolysis and tumor growth is a major strength of the study and provides new insights into the molecular mechanisms that underpin the Warburg effect in melanoma cells. The main conclusions of this paper are well supported by data, however further investigation of a potential oncogenic effect of TIPE in melanoma patients is warranted to support the tumor promoting role of TIPE identified in the experimental models. Analysis of patient samples showed a significant increase in TIPE protein levels in primary melanoma compared to benign skin tumours, and a further increase upon metastatic progression. Moreover, TIPE levels correlate with proliferation (Ki67) and hypoxia gene sets in the TCGA melanoma patient dataset. However, the authors note in the discussion that high TIPE expression associates with better survival outcomes in the TCGA melanoma patients and these data should be included in this paper. Further investigation of how TIPE-mediated regulation of glycolysis contributes to melanoma progression is warranted to confirm the authors claims of a potential oncogenic function. Regardless, the new insights into the molecular mechanisms underpinning TIPE-mediated aerobic glycolysis in melanoma are convincing and will likely generate interest in the cancer metabolism field.

Reviewer #2 (Public review):

Summary:

The manuscript by Ferling et al. describes how phagocytosis of IgG but not PS-opsonized targets induces the cells to round up and disassemble their podosomes. The mechanism downstream of the FcR is then dissected. The authors show that RhoA-mediated actin polymerization is involved, as well as actin nucleators of the Formin family, but not ROCK or Myosin II. ERM proteins and ROS production play a role in podosome loss and RhoA activation. Similar observations were made after cells were put in contact with Candida albicans or with soluble LPS.

Strengths:

The manuscript is of very good scientific standards, based on solid cell biology and biochemistry approaches, both in a murine macrophage cell line and in murine primary macrophages. It reaches the criteria for a significant advance in the field.

Reviewer #2 (Public review):

The paper provides a comprehensive analysis of the importance of livestock abortion surveillance in Tanzania. The authors aim to highlight the significance of this surveillance system in identifying disease priorities and guiding interventions to mitigate the impact of livestock abortions on both animal and human health.

Summary:

The paper begins by discussing the context of livestock farming in Tanzania and the significant economic and social impact of livestock abortions. The authors then present a detailed overview of the livestock abortion surveillance system in Tanzania, including its objectives, methods, and data collection process. They analyze the data collected from this surveillance system over a specific period to identify the major causes of livestock abortions and assess their public health implications.

Evaluation:

Overall, this paper provides valuable insights into the importance of livestock abortion surveillance as a tool for disease prioritization and intervention planning in Tanzania. The authors effectively demonstrate the utility of this surveillance system in identifying emerging diseases, monitoring disease trends, and informing evidence-based interventions to control and prevent livestock abortions.

Strengths:

(1) Clear Objective: The paper clearly articulates its objective of highlighting the value of livestock abortion surveillance in Tanzania.

(2) Comprehensive Analysis: The authors provide a thorough analysis of the surveillance system, including its methodology, data collection process, and findings as seen in the supplementary files.

(3) Practical Implications: The paper discusses the practical implications of the surveillance system for disease control and public health interventions in Tanzania.

(4) Well-Structured: The paper is well-organized, with clear sections and subheadings that facilitate understanding and navigation.

All suggestions made for improvement of the manuscript have been appropriately effected.

Final Recommendation:

Overall, this paper makes a significant contribution to the literature on livestock abortion surveillance and its implications for disease control in Tanzania.

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 occurs 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 impact the sensitivity od RDTs, and knowing these patterns and geographic distribution makes it possible to make better decisions for malaria control.

Comments on latest version:

The authors answered all my questions.

Reviewer #2 (Public review):

The authors now say the main take-home for their work is (1) they have established methods for linkage mapping with scRNA-seq and that these (2) "can help gain insights about the genotype-phenotype map at a broader scale." My opinion in this revision is much the same as it was in the first round: I agree that they have met the first goal, and the second theme has been so well explored by other literature that I'm not convinced the authors' results meet the bar for novelty and impact. To my mind, success for this manuscript would be to support the claim that the scRNA-seq approach helps "reveal hidden components of the yeast genotype-to-phenotype map." I'm not sure the authors have achieved this. I agree that the new Figure 3 is a nice addition-a result that apparently hasn't been reported elsewhere (30% of growth trait variation can't be explained by expression). The caveats are that this is a negative result that needs to be interpreted with caution; and that it would be useful for the authors to clarify whether the ability to do this calculation is a product of the scRNA-seq method per se or whether they could have used any bulk eQTL study for it. Beside this, I regret to say that I still find that the results in the revision recapitulate what the bulk eQTL literature has already found, especially for the authors' focal yeast cross: heritability, expression hotspots, the role of cis and trans-acting variation, etc.

Likewise, when in the first round of review I recommended that the authors repeat their analyses on previous bulk RNA-seq data from Albert et al., my point was to lead the authors to a means to provide rigorous, compelling justification for the scRNA-seq approach. The response to reviewers and the text (starting on line 413) says the comparison in its current form doesn't serve this purpose because Albert et al. studied fewer segregants. Wouldn't down-sampling the current data set allow a fair comparison? Again, to my mind what the current manuscript needs is concrete evidence that the scRNA-seq method per se affords truly better insights relative to what has come before.

I also recommend that the authors take care to improve the main text for readability and professionalism. It would benefit from further structural revision throughout (especially in the figure captions) to allow high-impact conclusions to be highlighted and low-impact material to be eliminated. Figure 4 and the results text sections from line 319 onward could be edited for concision or perhaps moved to supplementary if they obscure the authors' case for the scRNA-seq approach. The text could also benefit from copy editing (e.g. three clauses starting with "while" in the paragraph starting on line 456; "od ratio" on line 415). I appreciate the authors' work on the discussion, including posing big picture questions for the field (lines 426-429), but I don't see how they have anything to do with the current scRNA-seq method.

Reviewer #2 (Public review):

In this manuscript, Ridout et al. present an intriguing extension of beta cell mass-focused models for diabetes. Their model incorporates reversible glucose-dependent inactivation of beta cell mass, which can trigger sudden-onset hyperglycemia due to bistability in beta cell mass dynamics. Notably, this hyperglycemia can be reversed with insulin treatment. The model is simple, elegant, and thought-provoking.

Concerning the grounding in experimental phenomenology, it would be beneficial to identify specific experiments to strengthen the model. In particular, what evidence supports reversible beta cell inactivation? This could potentially be tested in mice, for instance, by using an inducible beta cell reporter, treating the animals with high glucose levels, and then measuring the phenotype of the marked cells. Such experiments, if they exist, would make the motivation for the model more compelling. For quantitative experiments, the authors should be more specific about the features of beta cell dysfunction in KPD. Does the dysfunction manifest in fasting glucose, glycemic responses, or both? Is there a "pre-KPD" condition? What is known about the disease's timescale?

The authors should also consider whether their model could apply to other conditions besides KPD. For example, the phenomenology seems similar to the "honeymoon" phase of T1D. Making a strong case for the model in this scenario would be fascinating.

Reviewer #2 (Public review):

Summary:

The authors aimed to characterize the cellular phenotype and spatial relationship of cell types infiltrating the islets of Langerhans in human T1D using CODEX, a multiplexed examination of cellular markers

Strengths:

Major strengths of this study are the use of pancreas tissue from well-characterized tissue donors, and the use of CODEX, a state-of-the-art detection technique of extensive characterization and spatial characterization of cell types and cellular interactions. The authors have achieved their aims with the identification of the heterogeneity of the CD8+ T cell populations in insulitis, the identification of a vasculature phenotype and other markers that may mark insulitis-prone islets, and the characterization of tertiary lymphoid structures in the acinar tissue of the pancreas. These findings are very likely to have a positive impact on our understanding (conceptual advance) of the cellular factors involved in T1D pathogenesis which the field requires to make progress in therapeutics.

Weaknesses:

A major limitation of the study is the cohort size, which the authors directly state. However, this study provides avenues of inquiry for researchers to gain further understanding of the pathological process in human T1D.

Reviewer #3 (Public review):

In this manuscript, Rossato and colleagues present a method for real-time decoding of EMG into putative single motor units. Their manuscript details a variety of decision points in their code and data collection pipeline that lead to a final result of recording on the order of ~10 putative motor units per muscle in human males. Overall the manuscript is highly restricted in its potential utility but may be of interest to aficionados. For those outside the field of human or nonhuman primate EMG, these methods will be of limited interest.

Comment on revised version

The revised manuscript has thoroughly and responsively addressed the concerns and suggestions raised in the first review. I think the method will be of use to the field and fits well within the purview of eLife's publications on methods development.

Reviewer #2 (Public review):

Summary:

In this study, Liu et al. explore the interplay between G-quadruplexes (G4s) and R-loops. The authors developed novel techniques, HepG4-seq and HBD-seq, to capture and map these nucleic acid structures genome-wide in human HEK293 cells and mouse embryonic stem cells (mESCs). They identified dynamic, cell-type-specific distributions of co-localized G4s and R-loops, which predominantly localize at active promoters and enhancers of transcriptionally active genes. Furthermore, they assessed the role of helicase Dhx9 in regulating these structures and their impact on gene expression and cellular functions.

The manuscript provides a detailed catalogue of the genome-wide distribution of G4s and R-loops. However, the conceptual advance and the physiological relevance of the findings are not obvious. Overall, the impact of the work on the field is limited to the utility of the presented methods and datasets.

Strengths:<br /> (1) The development and optimization of HepG4-seq and HBD-seq offer novel methods to map native G4s and R-loops.<br /> (2) The study provides extensive data on the distribution of G4s and R-loops, highlighting their co-localization in human and mouse cells.<br /> (3) The study consolidates the role of Dhx9 in modulating these structures and explores its impact on mESC self-renewal and differentiation.

Comments on revised version:

In this revised manuscript, Liu et al. address most of the previous concerns raised by this reviewer. Namely, the comparison between the novel methods and existing ones is an important addition.

Reviewer #3 (Public review):

The revised manuscript adds some new relevant analyses. It still, however, is unclear which timescales of motions the method refers to and there is confusion about whether the model can predict "slower motions". While the authors answer some of my points, others are left unanswered. That is of course the authors' prerogative, and readers will in any case be able to read the reviewer comments. I am not sure it is productive to add further comments at this point.

Below are my comments from the first round of review:

The manuscript by Qin and Zhou presents an approach to predict dynamical properties of an intrinsically disordered protein (IDP) from sequence alone. In particular, the authors train a simple (but useful) machine learning model to predict (rescaled) NMR R2 values from sequence. Although these R2 rates only probe some aspects of IDR dynamics and the method does not provide insight into the molecular aspects of processes that lead to perturbed dynamics, the method can be useful to guide experiments.

A strength of the work is that the authors train their model on an observable that directly relates to protein dynamics. They also analyse a relatively broad set of proteins which means that one can see actual variation in accuracy across the proteins.

A weakness of the work is that it is not always clear what the measured R2 rates mean. In some cases, these may include both fast and slow motions (intrinsic R2 rates and exchange contributions). This in turn means that it is actually not clear what the authors are predicting. The work would also be strengthened by making the code available (in addition to the webservice), and by making it easier to compare the accuracy on the training and testing data.

Reviewer #3 (Public review):

Summary:

In this ambitious paper, the authors develop an unparalleled community resource of insect genome regulatory annotations spanning five insect orders. They employ their previously-developed SCRMshaw method for computational cross-species enhancer prediction, drawing on available training datasets of validated enhancer sequence and expression from Drosophila melanogaster, which had been previously shown to perform well across select holometabolous insects (representing 160-345MY divergence). In this work they expand regulatory sequence annotation to 33 insect genomes spanning Holometabola and Hemiptera, which is even more distantly related to the fly model. They perform multiple downstream analyses of sets of predicted enhancers to assess the true-positive rate of predictions; the independent comparisons of real predictions with simulated predictions and with chromatin accessibility data, as well as the functional validation through reporter gene analysis strengthen their conclusions that their annotation pipeline achieves a high true-positive rate and can be used across long divergence times to computationally annotate regulatory genome regions, an ability that has been largely inaccessible for non-model insects and now is possible across the many newly-sequenced insect scaffold-level genomes.

Strengths:

This work fills a large gap in current methods and resources for predicting regulatory regions of the genome, a task that has long lagged behind that of coding region prediction and analysis.

Despite technical constraints in working outside of well-developed model insect systems, the authors creatively draw on existing resources to scaffold a pipeline and independently assess likelihood of prediction validity.

The established database will be a welcome community resource in its current state, and even more so as the authors continue to expand their annotations to more insect genomes as they indicate. Their available analysis pipeline itself will be useful to the community as well for research groups that may want to undertake their own regulatory genome annotation.

Weaknesses:

The work here is limited by the field-wide lack of an independently validated set of tissue specific enhancers that could be used to directly benchmark this pipeline. The prediction of true positive enhancer identification rates and in vivo reporter gene assays offer some insight into the rates of successful prediction, but the output of SCRMshaw regulatory annotation should be regarded as another prediction-generating tool.

Reviewer #2 (Public review):

Golluscio et al. address one of the mechanisms of IKs (KCNQ1/KCNE1) channel upregulation by polyunsaturated fatty acids (PUFAs). PUFAs are known to upregulate KCNQ1 and KCNQ1/KCNE1 channels through two mechanisms: one shifts the voltage dependence in a negative direction, and the other increases the maximum conductance (Gmax). While the first mechanism is known to affect the voltage sensor equilibrium through a charge effect, the second mechanism is less understood. Using single-channel recordings and mutagenesis at putative PUFA binding sites, they successfully demonstrate that the selectivity filter is stabilized in a conducting state by PUFA binding, and that this is the mechanism by which PUFAs increase Gmax. Their single-channel recordings are straightforward and clearly show that the selectivity filter tends to become conductive upon PUFA binding. Since PUFAs are potential therapeutic reagents for cardiac arrhythmias such as long QT syndrome, their findings are beneficial for future research and applications of these compounds.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors provided a proof of concept that they can identify and mutate a cholesterol-binding site of a high-interest class B receptor, the GLP-1R, and functionally characterize the impact of this mutation on receptor behavior in the membrane and downstream signaling with the intent that similar methods can be useful to optimize small molecules that as ligands or allosteric modulators of GLP-1R can improve the therapeutic tools targeting this signaling system.

Strengths:

The majority of results on receptor behavior are elucidated in INS-1 cells expressing the wt or mutant GLP-1R, with one experiment translating the findings to primary mouse beta-cells. I think this paper lays a very strong foundation to characterize this mutation and does a good job discussing how complex cholesterol-receptor interactions can be (ie lower cholesterol binding to V229A GLP-1R, yet increased segregation to lipid rafts). Table 1 and Figure 9 are very beneficial to summarize the findings. The lower interaction with cholesterol and lower membrane diffusion in V229A GLP-1R resembles the reduced diffusion of wt GLP-1R with simv-induced cholesterol reductions, although by presumably decreasing the cholesterol available to interact with wt GLP-1R. This could be interesting to see if lowering cholesterol alters other behaviors of wt GLP-1R that look similar to V229A GLP-1R. I further wonder if the authors expect that increased cholesterol content of islets (with loading of MβCD saturated with cholesterol or high-cholesterol diets) would elevate baseline GLP-1R membrane diffusion, and if a more broad relationship can be drawn between GLP-1R membrane movement and downstream signaling.

Weaknesses:

I think there are no obvious weaknesses in this manuscript and overall, I believe the authors achieved their aims and have demonstrated the importance of cholesterol interactions on GLP-1R functioning in beta-cells. I think this paper will be of interest to many physiologists who may not be familiar with many of the techniques used in this paper and the authors largely do a good job explaining the goals of using each method in the results section. The intent of some methods, for example the Laurdan probe studies, are better expanded in the discussion. I found it unclear what exactly was being measured to assess 'receptor activity' in Fig 7E and F.

Certainly many follow-up experiments are possible from these initial findings and of primary interest is how this mutation affects insulin homeostasis in vivo under different physiological conditions. One of the biggest pathologies in insulin homeostasis in obesity/t2d is an elevation of baseline insulin release (as modeled in Fig 1E) that renders the fold-change in glucose stimulated insulin levels lower and physiologically less effective. No difference in primary mouse islet baseline insulin secretion was seen here but I wonder if this mutation would ameliorate diet-induced baseline hyperinsulinemia.

I would have liked to see the actual islet cholesterol content after 5wks high-cholesterol diet measured to correlate increased cholesterol load with diminished glucose-stimulated inulin. While not necessary for this paper, a comparison of islet cholesterol content after this cholesterol diet vs the more typical 60% HFD used in obesity research would be beneficial for GLP-1 physiology research broadly to take these findings into consideration with model choice.

Another area to further investigate is does this mutation alter ex4 interaction/affinity/time of binding to GLP-1 or are all of the described findings due to changes in behavior and function of the receptor?

Lastly, I wonder if V229A would have the same impact in a different cell type, especially in neurons? How similar are the cholesterol profiles of beta-cells and neurons? How this mutation (and future developed small molecules) may affect satiation, gut motility, and especially nausea, are of high translational interest. The comparison is drawn in the discussion between this mutation and ex4-phe1 to have biased agonism towards Gs over beta-arrestin signaling. Ex4-phe1 lowered pica behavior (a proxy for nausea) in the authors previously co-authored paper on ex4-phe1 (PMID 29686402) and I think drawing a parallel for this mutation or modification of cholesterol binding to potentially mitigate nausea is worth highlighting.

Reviewer #2 (Public review):

Summary:

This study tests a plausible and intriguing hypothesis that one cause of the differences in the neural underpinnings of concrete and abstract words is differences in their grounding in the current sensory context. The authors reasoned that, in this case, an abstract word presented with a relevant visual scene would be processed in a more similar way to a concrete word. Typically, abstract and concrete words are tested in isolation. In contrast, this study takes advantage of naturalistic movie stimuli to assess the neural effects of concreteness in both abstract and concrete words (the speech within the film), when the visual context is more or less tied to the word meaning (measured as the similarity between the word co-occurrence-based vector for the spoken word and the average of this vector across all present objects). This novel approach allows a test of the dynamic nature of abstract and concrete word processing, and as such provides a useful perspective accounting for differences in processing these word types.

The measure of contextual situatedness (how related a spoken word is to the average of the visually presented objects in a scene) is an interesting approach allowing parametric variation within naturalistic stimuli, which is a potential strength of the study. Additionally, the authors use an interesting peak and valley method and provide a rationale for this approach. This provided additional temporal information on the processing of spoken concrete and abstract words.

The authors predicted that sensory areas would be more active for concrete words, affective areas for abstract and language areas would be involved in both. The use of reverse inference to interpret areas such as the inferior frontal gyrus post hoc, as sensory, affective or language related deserves some caution. It is also important to remember that the different areas identified for each comparison do not necessarily have the same roles. As the number of clusters may therefore be a misleading way to assess the relationship of these areas with the sensory terms, the relationship between each area and the different sensory terms is provided in the supplemental to allow more nuanced interpretation. The study could benefit from being better situated in the prior literature on context and concrete vs abstract regional differences. Overall, the authors successfully demonstrate the context-dependent nature of abstract and concrete word processing.

Reviewer #2 (Public review):

Summary:

Yang and colleagues used a Hidden Markov Model (HMM) on whole-night fMRI to isolate sleep and wake brain states in a data-driven fashion. They identify more brain states (21) than the five sleep/wake stages described in conventional PSG-based sleep staging, show that the identified brain states are stable across nights, and characterize the brain states in terms of which networks they primarily engage.

Strengths:

This work's primary strengths are its dataset of two nights of whole-night concurrent EEG-fMRI (including REM sleep), and its sound methodology.

Weaknesses:

Weaknesses are its small sample size, and limited attempts at relating the identified fMRI brain states back to EEG.

General appraisal:

The paper's conclusions are generally well-supported, but additional analyses could improve the work further.<br /> The authors' main focus lies in identifying fMRI-based brain states, and they succeed at demonstrating both the presence and robustness of these states in terms of cross-night stability. Additional characterization of brain states in terms of which networks these brain states primarily engage adds additional insights.

A missed opportunity remains the absence of more analyses relating the HMM states back to EEG. While the authors show how power in different EEG bands varies with HMM state (Supplementary Figures 10 and 11) it would be much more informative to show the complete EEG spectra for each of the 21 HMM states, organized by PSG-based sleep/wake state. This would enable answering how EEG spectra of, say, different N2-related HMM states compare. Similarly, it is presently unclear whether anything noticeable happens within the EEG timecourse at the moment of an HMM class switch (particularly when the PSG stage remains stable). Such analyses might have shown that fMRI-based brain states map onto familiar EEG substates, or reveal novel EEG changes that have so far gone unnoticed. Furthermore, if band-specific analyses are to be performed, care should be taken to specify bands in accordance with the dominant sleep EEG features (e.g., slow oscillation and sigma/spindle bands are currently missing).

Reviewer #2 (Public Review):

The authors aimed to develop and validate a machine-learning driven neural network capable of automatic scoring of the Rey-Osterrieth Complex Figure. They aimed to further assess the robustness of the model to various parameters such as tilt and perspective shift in real drawings. The authors leveraged the use of a huge sample of lay workers in scoring figures and also a large sample of trained clinicians to score a subsample of figures. Overall, the authors found their model to have exceptional accuracy and perform similarly to crowdsourced workers and clinicians with, in some cases, less degree of error/score dispersion than clinicians.

Reviewer #2 (Public review):

Summary:

The authors investigate changes in theta-gamma phase amplitude coupling, and action potential entrainment to theta following traumatic brain injury (TBI). Both phenomena are widely hypothesized to be important for cognition, and the authors report deficits in both after TBI. The manuscript is well-written, the figures are well-constructed, and the author's use of high-level analysis methods for TBI EEG data collected from awake, behaving animals is welcome.

Major Comments:

- The animal n's are small (4 sham and 5 injured). In Figure 3, for instance, one wonders if panels D and E might have shown significant differences if more animals had been recorded.

- The text focuses on deficits in the theta and gamma bands, but the reduction in power appears to be broadband (see Figure 1F, especially Pyramidal cell layer panel). Therefore, the overall decrease in broadband (in the injured population) must be normalized between sham and injured animals before a selective comparison between sham and injured animals can be conducted. That is the only way that selective narrow bands i.e., theta and low gamma can be compared between the two cohorts. A brief discussion of the significance of a broadband decrease would be appreciated.

Reviewer #2 (Public review):

Summary:

LRRK2 has previously been shown to affect cilia formation and stability both in vitro and in vivo, in striatal cholinergic interneurons, in both transgenic mice and in human post-mortem brain samples from subjects carrying one of the LRRK2 pathogenic mutations: G2019S. In the current study, Brahmia and colleagues have conducted a comprehensive assessment of G2019S knock-in mice to address some gaps in the field, specifically: extending analysis to additional cholinergic neurons across 3 time points and determining the functional consequences of the ciliation deficits. They find that primary cilia are lost in all cholinergic neurons, with basal forebrain cholinergic neurons displaying an early onset (in 4-5-month-old mice) compared with other regions. They also show early dystrophic changes in cholinergic axons derived from basal forebrain and brainstem cholinergic neurons and age-dependent cholinergic cell loss in select forebrain and brainstem nuclei.

Strengths:

This is a comprehensive and careful analysis of ciliary deficits and their downstream consequences, which we assume are deficits in innervation and cell loss.

Weaknesses:

This study is observational and does not address the underlying mechanisms. The data on pRab12, although downstream of LRRK2, does not clearly address this and, instead, raises more questions than answers: e.g., is there really differentiation from Rab10 and its phosphorylation or is it primarily due to the limitations of pRab10 antibodies with regards to the lack of suitability of this antibody in mouse brain sections (could immunoblots on brain punches have been performed to overcome this?). Are Rab10, Rab12, and LRRK2 expressed at different levels in the vulnerable cell types? Plenty of recent high-quality single-cell/single nuclear RNA-seq data could have been used to address such a fundamental question. LRRK2 small molecule inhibitors are available and progressing in the clinic. They could/should have been used to demonstrate the LRRK2 dependence, reversibility, and timing of therapeutic intervention. The authors suggest that the mouse data mirror (and potentially explain) the cholinergic loss in PD patient brains, but this is not measured in the current work (the authors do acknowledge this limitation and suggest that this is an important further study). There are some recent human data (Khan et al 2024 PMID: 38293195, BioRxiv, which the authors cite) showing loss of primary cilia and cholinergic neurons in sporadic PD (no evidence of aberrant LRRK2 activity) and, interestingly, this is not further exacerbated in G2019S carriers, which may suggest a more complex underlying mechanism.

Reviewer #2 (Public review):

Summary:

Griesius et al. investigate the dendritic integration properties of two types of inhibitory interneurons in the hippocampus: those that express NDNF+ and those that express somatostatin. They found that both neurons showed supralinear synaptic integration in the dendrites, blocked by NMDA receptor blockers but not by blockers of Na+ channels. These experiments are critically overdue and very important because knowing how inhibitory neurons are engaged by excitatory synaptic input has important implications for all theories involving these inhibitory neurons.

Strengths:

(1) Determined the dendritic integration properties of two fundamental types of inhibitory interneurons.

(2) Convincing demonstration that supra-threshold integration in both cell types depends on NMDA receptors but not on Na+ channels.

Weaknesses:

It is unknown whether highly clustered synaptic input, as used in this study (and several previous studies), occurs physiologically.

Reviewer #2 (Public review):

Summary:

The authors investigated DG neuronal activity at the population and single-cell level across sleep/wake periods. They found an infraslow oscillation (0.01-0.03 Hz) in both granule cells (GC) and mossy cells (MC) during NREM sleep.

The important findings are:

(1) The antiparallel temporal dynamics of DG neuron activities and serotonin neuron activities/extracellular serotonin levels during NREM sleep, and

(2) The GC Htr1a-mediated GC infraslow oscillation.

Strengths:

(1) The combination of polysomnography, Ca-fiber photometry, two-photon microscopy, and gene depletion is technically sound. The coincidence of microarousals and dips in DG population activity is convincing. The dip in activity in upregulated cells is responsible for the dip at the population level.

(2) DG GCs express excitatory Htr4 and Htr7 in addition to inhibitory Htr1a, but deletion of Htr1a is sufficient to disrupt DG GC infraslow oscillation, supporting the importance of Htr1a in DG activity during NREM sleep.

Weaknesses:

(1) The current data set and analysis are insufficient to interpret the observation correctly.

a. In Figure 1A, during NREM, the peaks and troughs of GC population activities seem to gradually decrease over time. Please address this point.

b. In Figure 1F, about 30% of Ca dips coincided with MA (EMG increase) and 60% of Ca dips did not coincide with EMG increase. If this is true, the readers can find 8 Ca dips which are not associated with MAs from Figure 1E. If MAs were clustered, please describe this properly.

c. In Figure 1F, the legend stated the percentage during NREM. If the authors want to include the percentage of wake and REM, please show the traces with Ca dips during wake and REM. This concern applies to all pie charts provided by the authors.

d. In Figure 1C, please provide line plots connecting the same session. This request applies to all related figures.

e. In Figure 2C, the significant increase during REM and the same level during NREM are not convincing. In Figure 2A, the several EMG increasing bouts do not appear to be MA, but rather wakefulness, because the duration of the EMG increase is greater than 15 seconds. Therefore, it is possible that the wake bouts were mixed with NREM bouts, leading to the decrease of Ca activity during NREM. In fact, In Figure 2E, the 4th MA bout seems to be the wake bout because the EMG increase lasts more than 15 seconds.

f. Figure 5D REM data are interesting because the DRN activity is stably silenced during REM. The varied correlation means the varied DG activity during REM. The authors need to address it.

g. In Figure 6, the authors should show the impact of DG Htr1a knockdown on sleep/wake structure including the frequency of MAs. I agree with the impact of Htr1a on DG ISO, but possible changes in sleep bout may induce the DG ISO disturbance.

(2) It is acceptable that DG Htr1a KO induces the reduced freezing in the CFC test (Figure 6E, F), but it is too much of a stretch that the disruption of DG ISO causes impaired fear memory. There should be a correlation.

(3) It is necessary to describe the extent of AAV-Cre infection. The authors injected AAV into the dorsal DG (AP -1.9 mm), but the histology shows the ventral DG (Supplementary Figure 4), which reduces the reliability of this study.

Reviewer #2 (Public review):

This manuscript builds on the authors' earlier work, most recently Wong et al. 2019, in which they showed the importance of the perirhinal cortex (PRh) during the first-order conditioning stage of sensory preconditioning. Sensory preconditioning requires learning between two neutral stimuli (S2-S1) and subsequent development of a conditioned response to one of the neutral stimuli after pairing of the other stimulus with a motivationally relevant unconditioned stimulus (S1-US). One highly debated question regarding the mechanisms of learning of sensory preconditioning has been whether conditioned responses evoked by the indirectly trained stimulus (S2) occur through a mediated representation at the time of the first-order US training, or whether the conditioned responses develop through a chained evoked representation (S2--> S1 --> US) at the time of test. The authors' prior findings provided strong evidence for PRh being involved in mediated learning during the first-order training. They showed that protein synthesis was required during the first-order S1-US learning to support the conditioned response to the indirectly trained stimulus (S2) at the test.

One question remaining following the previous paper was whether certain conditions may promote a chaining mechanism over mediated learning, as there is some evidence for chained representations at the time of the test. In this paper, the authors directly address this important question and find unambiguous results that the extent of training during the preconditioning stage impacts the involvement of PRh during the first-order conditioning or stage 2. They show that putative blockade of synaptic changes in PRh, using an NMDA antagonist, disrupts responding to the preconditioned cue at test during shorter duration preconditioning training (8 trials), but not during extended training (32 trials). They also show that this is the case for communication between the PRh and BLA during the same stage of training using a contralateral inactivation approach. This confirms their previous findings in 2019 of connectivity between these regions for the short-duration training, while they observe here for the first time that this is not the case for extended training. Finally, they show that with extended training, communication between BLA and the PRh is required at the final test of the preconditioned stimulus, but not for the short duration training.

The results are clear and extremely consistent across experiments within this paper as well as with earlier work. The experiments here are thorough, and well-conceived, and address an important and highly debated question in the field regarding the neural and psychological mechanisms underlying sensory preconditioning. This work is highly impactful for the field as the debate over mediated versus chaining mechanisms has been an important topic for more than 70 years.

Reviewer #2 (Public review):

Summary:

This study by Tardiff, Kang & Gold seeks to: i) develop a normative account of how observers should adapt their decision-making across environments with different levels of correlation between successive pairs of observations, and ii) assess whether human decisions in such environments are consistent with this normative model.

The authors first demonstrate that, in the range of environments under consideration here, an observer with full knowledge of the generative statistics should take both the magnitude and sign of the underlying correlation into account when assigning weight in their decisions to new observations: stronger negative correlations should translate into stronger weighting (due to the greater information furnished by an anticorrelated generative source), while stronger positive correlations should translate into weaker weighting (due to the greater redundancy of information provided by a positively correlated generative source). The authors then report an empirical study in which human participants performed a perceptual decision-making task requiring accumulation of information provided by pairs of perceptual samples, under different levels of pairwise correlation. They describe a nuanced pattern of results with effects of correlation being largely restricted to response times and not choice accuracy, which could partly be captured through fits of their normative model (in this implementation, an extension of the well-known drift-diffusion model) to the participants' behaviour while allowing for mis-estimation of the underlying correlations.

Strengths:

As the authors point out in their very well-written paper, appropriate weighting of information gathered in correlated environments has important consequences for real-world decision-making. Yet, while this function has been well studied for 'high-level' (e.g. economic) decisions, how we account for correlations when making simple perceptual decisions on well-controlled behavioural tasks has not been investigated. As such, this study addresses an important and timely question that will be of broad interest to psychologists and neuroscientists. The computational approach to arrive at normative principles for evidence weighting across environments with different levels of correlation is very elegant, makes strong connections with prior work in different decision-making contexts, and should serve as a valuable reference point for future studies in this domain. The empirical study is well designed and executed, and the modelling approach applied to these data showcases a deep understanding of relationships between different parameters of the drift-diffusion model and its application to this setting. Another strength of the study is that it is preregistered.

Weaknesses:

In my view, the major weaknesses of the study center on the narrow focus and subsequent interpretation of the modelling applied to the empirical data. I elaborate on each below:

Modelling interpretation: the authors' preference for fitting and interpreting the observed behavioural effects primarily in terms of raising or lowering the decision bound is not well motivated and will potentially be confusing for readers, for several reasons. First, the entire study is conceived, in the Introduction and first part of the Results at least, as an investigation of appropriate adjustments of evidence weighting in the face of varying correlations. The authors do describe how changes in the scaling of the evidence in the drift-diffusion model are mathematically equivalent to changes in the decision bound - but this comes amidst a lengthy treatment of the interaction between different parameters of the model and aspects of the current task which I must admit to finding challenging to follow, and the motivation behind shifting the focus to bound adjustments remained quite opaque. Second, and more seriously, bound adjustments of the form modelled here do not seem to be a viable candidate for producing behavioural effects of varying correlations on this task. As the authors state toward the end of the Introduction, the decision bound is typically conceived of as being "predefined" - that is, set before a trial begins, at a level that should strike an appropriate balance between producing fast and accurate decisions. There is an abundance of evidence now that bounds can change over the course of a trial - but typically these changes are considered to be consistently applied in response to learned, predictable constraints imposed by a particular task (e.g. response deadlines, varying evidence strengths). In the present case, however, the critical consideration is that the correlation conditions were randomly interleaved across trials and were not signaled to participants in advance of each trial - and as such, what correlation the participant would encounter on an upcoming trial could not be predicted. It is unclear, then, how participants are meant to have implemented the bound adjustments prescribed by the model fits. At best, participants needed to form estimates of the correlation strength/direction (only possible by observing several pairs of samples in sequence) as each trial unfolded, and they might have dynamically adjusted their bounds (e.g. collapsing at a different rate across correlation conditions) in the process. But this is very different from the modelling approach that was taken. In general, then, I view the emphasis on bound adjustment as the candidate mechanism for producing the observed behavioural effects to be unjustified (see also next point).

Modelling focus: Related to the previous point, it is stated that participants' choice and RT patterns across correlation conditions were qualitatively consistent with bound adjustments (p.20), but evidence for this claim is limited. Bound adjustments imply effects on both accuracy and RTs, but the data here show either only effects on RTs, or RT effects mixed with accuracy trends that are in the opposite direction to what would be expected from bound adjustment (i.e. slower RT with a trend toward diminished accuracy in the strong negative correlation condition; Figure 3b). Allowing both drift rate and bound to vary with correlation conditions allowed the model to provide a better account of the data in the strong correlation conditions - but from what I can tell this is not consistent with the authors' preregistered hypotheses, and they rely on a posthoc explanation that is necessarily speculative and cannot presently be tested (that the diminished drift rates for higher negative correlations are due to imperfect mapping between subjective evidence strength and the experimenter-controlled adjustment to objective evidence strengths to account for effects of correlations). In my opinion, there are other candidate explanations for the observed effects that could be tested but lie outside of the relatively narrow focus of the current modelling efforts. Both explanations arise from aspects of the task, which are not mutually exclusive. The first is that an interesting aspect of this task, which contrasts with most common 'univariate' perceptual decision-making tasks, is that participants need to integrate two pieces of information at a time, which may or may not require an additional computational step (e.g. averaging of two spatial locations before adding a single quantum of evidence to the building decision variable). There is abundant evidence that such intermediate computations on the evidence can give rise to certain forms of bias in the way that evidence is accumulated (e.g. 'selective integration' as outlined in Usher et al., 2019, Current Directions in Psychological Science; Luyckx et al., 2020, Cerebral Cortex) which may affect RTs and/or accuracy on the current task. The second candidate explanation is that participants in the current study were only given 200 ms to process and accumulate each pair of evidence samples, which may create a processing bottleneck causing certain pairs or individual samples to be missed (and which, assuming fixed decision bounds, would presumably selectively affect RT and not accuracy). If I were to speculate, I would say that both factors could be exacerbated in the negative correlation conditions, where pairs of samples will on average be more 'conflicting' (i.e. further apart) and, speculatively, more challenging to process in the limited time available here to participants. Such possibilities could be tested through, for example, an interrogation paradigm version of the current task which would allow the impact of individual pairs of evidence samples to be more straightforwardly assessed; and by assessing the impact of varying inter-sample intervals on the behavioural effects reported presently.

მაშინ,როდესაც საქმე გვაქვს შრომის გენდერულ დაყოფასთან ანუ პოლიტიკაში ჩართულ ქალთ მიმართ ძალადობასთან,გამოკითხვების ეს პროცენტული მაჩვენებელი არ გამორიცხავს იმ ფაქტს,რომ ის ქალები,რომლებიც უარყოფენ ძალადობის შემთხვევებს არ არიან ფსიქოლოგიური თუ მორალური ბულინგის მსხვერპლნი,რადგან ამის აღიარება მათ საფრთხისა და შიშის გრძნობას აღურავს

Breaking down former President Donald Trump’s rambling linguistic style by [[Steve Inskeep]]

for - biodiversity - safe earth system boundaries - 2 measures - intact natural ecosystems - ecosystems modified by human pressures - question - quantification of biodiversity tipping points at various scales

question - quantification of biodiversity tipping points at various scales - As ecologist David Suzuki often says, economy depends on ecology, not the other way around - Is there quantification at different potential tipping points for extinction for biodiversity at different scales and localities?

Reviewer #2 (Public review):

Chen et al. investigated the regulatory mechanism of bacterial colonization in the intestinal mucus layer in mice and its implications to 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 were appropriately designed and executed with precision. The revised manuscript represents a significant improvement over the initial version. The inclusion of new, higher-resolution images provides stronger support for the conclusions drawn. Additionally, statistical analyses of the imaging data, as recommended, have been integrated. The authors have effectively addressed the majority of the reviewers' suggestions and criticisms, making this version well-suited for publication.

Reviewer #2 (Public Review):

Through RNA analysis, Xie et al found LncRNA Snhg3 was one of the most down-regulated Snhgs by high fat diet (HFD) in mouse liver. Consequently, the authors sought to examine the mechanism through which Snhg3 is involved in the progression of metabolic dysfunction-associated fatty liver diseases (MASLD) in HFD-induced obese (DIO) mice. Interestingly, liver-specific Sngh3 knockout reduced, while Sngh3 over-expression potentiated fatty liver in mice on a HFD. Using the RNA pull-down approach, the authors identified SND1 as a potential Sngh3 interacting protein. SND1 is a component of the RNA-induced silencing complex (RISC). The authors found that Sngh3 increased SND1 ubiquitination to enhance SND1 protein stability, which then reduced the level of repressive chromatin H3K27me3 on PPARg promoter. The upregulation of PPARg, a lipogenic transcription factor, thus contributed to hepatic fat accumulation.

The authors propose a signaling cascade that explains how LncRNA sngh3 may promote hepatic steatosis. Multiple molecular approaches have been employed to identify molecular targets of the proposed mechanism, which is a strength of the study.

Reviewer #2 (Public review):

The manuscript by Carbo et al. reports a novel role for the MltG homolog AgmT in gliding motility in M. xanthus. The authors conclusively show that AgmT is a cell wall lytic enzyme (likely a lytic transglycosylase), its lytic activity is required for gliding motility, and that its activity is required for proper binding of a component of the motility apparatus to the cell wall. The data are generally well-controlled. The marked strength of the manuscript includes the detailed characterization of AgmT as a cell wall lytic enzyme, and the careful dissection of its role in motility. Using multiple lines of evidence, the authors conclusively show that AgmT does not directly associate with the motility complexes, but that instead its absence (or the overexpression of its active site mutant) results in failure of focal adhesion complexes to properly interact with the cell wall.

Reviewer #2 (Public review):

Summary:

The authors show that a spiking network model with clustered connectivity produces intrinsic spike sequences when driven with an ramping input, which are recapitulated in the absence of input. This behavior is only seen for some network parameters (neuron cluster participation and number of clusters in the network), which correspond to those that produce a small world network. By changing the strength of ramping input to each network cluster, the network can show different sequences.

Strengths:

A strength of the paper is the direct comparison between the properties of the model and neural data.

Weaknesses:

My main critique of the paper relates to the form of the input to the network. Specifically, it's unclear how much the results depend on the choice of a one-dimensional environment with ramping input. While this is an elegant idealization that allows the authors to explore the representation and replay properties of their model, it is a strong and highly non-physiological constraint. In order to address this concern, the authors would need to test the spatial tuning of their network in 2-dimensional environments, and with different kinds of input from a population of neurons that have a range of degree of spatial tuning and physiological plausibility. A method for systematically producing input with varying degrees of spatial tuning in both 1D and 2D environments has been previously used in (Fang et al 2023, eLife, see Figures 4 and 5), which could be readily adapted for the current study; and behaviorally plausible trajectories in 2D can be produced using the RatInABox package (George et al 2022, bioRxiv), which can also generate e.g. grid cell-like activity that could be used as physiologically plausible input to the network.

Reviewer #2 (Public review):

Dierks et al. investigate the impact of m6A RNA modifications on the mRNA life cycle, exploring the links between transcription, cytoplasmic RNA degradation, and subcellular RNA localization. Using transcriptome-wide data and mechanistic modelling of RNA metabolism, the authors demonstrate that a simplified model of m6A primarily affecting cytoplasmic RNA stability is sufficient to explain the nuclear-cytoplasmic distribution of methylated RNAs and the dynamic changes in m6A levels upon perturbation. Based on multiple lines of evidence, they propose that passive mechanisms based on the restricted decay of methylated transcripts in the cytoplasm play a primary role in shaping condition-specific m6A patterns and m6A dynamics. The authors support their hypothesis with multiple large-scale datasets and targeted perturbation experiments. Overall, the authors present compelling evidence for their model which has the potential to explain and consolidate previous observations on different m6A functions, including m6A-mediated RNA export.

Reviewer #2 (Public Review):

Summary:

Yang et al. present an article investigating the spatiotemporal atlas of NFATc1+ and PDGFR-α+ cells within the dental and periodontal mesenchyme. The study explores their capacity for progeny cell generation and their relationships - both inclusive and hierarchical - under homeostatic conditions. Utilizing the Cre/loxP-Dre/Rox system to construct tool mice, combined with tissue transparency and continuous tissue slicing for 3D reconstruction, the researchers effectively mapped the distribution of NFATc1+ and PDGFR-α+ cells. Additionally, in conjunction with DTA mice, the study provides preliminary validation of the impact of PDGFR-α+ cells on dental pulp and periodontal tissues. Primarily, this study offers an in-situ distribution atlas for NFATc1+ and PDGFR-α+ cells but provides limited information regarding their origin, fate differentiation, and functionality.

Strengths:

(1) Tissue transparency techniques and continuous tissue slicing for 3D reconstruction, combined with transgenic mice, provide high-quality images and rich, reliable data.<br /> (2) The Cre/loxP and Dre/Rox systems used by the researchers are powerful and innovative.<br /> (3) The IR1 lineage tracing model is significantly important for investigating cellular differentiation pathways.<br /> (4) This study provides effective spatial distribution information of NFATc1+/PDGFR-α+ cell populations in the dental and periodontal tissues of adult mice.

Weaknesses:

(1) In the functional experiment section, the investigation into the role of NFATc1+/PDGFR-α+ cell populations is somewhat lacking.

(2) The author mentions that 3D reconstruction of consecutive tissue slices can provide more detailed information on cell distribution, so what is the significance of using tissue-clearing techniques in this article?

(3) After reading the entire article, it is confusing whether the purpose of the article is to explore the distribution and function of NFATc1+/PDGFR-α+ cells in teeth and periodontal tissues, or to compare the differences between tissue clearing techniques and 3D reconstruction of continuous histological slices using NFATc1+/PDGFR-α+ cells?

(4) The researchers did not provide a clear definition of the cell types of NFATc1+/PDGFR-α+ cells in teeth and periodontal tissues.

(5) In studies related to long bones, the author defines the NFATc1+/PDGFR-α+ cell population as SSCs, which as a stem cell group should play an important role in tooth development or injury repair. However, the distribution patterns and functions of the NFATc1+/PDGFR-α+ cell population in these two conditions have not been discussed in this study.

Reviewer #2 (Public review):

Summary:

The authors propose a methodology to perform causal (temporal) discovery. The approach appears to be robust and is tested in the different scenarios: one related with live-cell imaging data, and another one using synthetic (mathematically defined) time series data. They compare the performance of their findings against another well-know method by using metrics like F-score, precision and recall,

Strengths:

Performance, robustness, the text is clear and concise, The authors provide the code to review.

Weaknesses:

One concern could be the applicability of the method in other areas like climate, economy. For those areas, public data are available and might be interesting to test how the method performs with this kind of data.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors provide evidence that posttranslational modification of synuclein by N-acetylation increases clustering of synaptic vesicles in vitro. When using liposomes the authors found that while clustering is enhanced by the presence of either lysophosphatidylcholine (LPC) or phosphatidylcholine in the membrane, N-acetylation enhanced clustering only in the presence of LPC. Enhancement of binding was also observed when LPC micelles were used, which was corroborated by increased intra/intermolecular cross-linking of N-acetylated synuclein in the presence of LPC.

Strengths:

It is known for many years that synuclein binds to synaptic vesicles but the physiological role of this interaction is still debated. The strength of this manuscript is clearly in the structural characterization of the interaction of synuclein and lipids (involving NMR-spectroscopy) showing that the N-terminal 100 residues of synuclein are involved in LPC-interaction, and the demonstration that N-acetylation enhances the interaction between synuclein and LPC.

Weaknesses:

Lysophosphatides form detergent-like micelles that destabilize membranes, with their steady-state concentrations in native membranes generally being a lot lower than in the experiments reported here. Since no difference in binding between the N-acetylated and unmodified form was observed when the acidic phospholipid phosphatidylserine was included. It remains unclear to which extent binding to LPC is physiologically relevant, particularly in the light of recent reports from other laboratories showing that synuclein may interact with liquid-liquid phases of synapsin I, or associate with the unfolded regions of VAMP that both were reported to cause vesicle clustering.

Reviewer #2 (Public review):

Summary:

The manuscript by David et al. describes a novel image segmentation method, implementing Local Moran's method, which determines whether the value of a datapoint or a pixel is randomly distributed among all values, in differentiating pixel clusters from the background noise. The study includes several proof-of-concept analyses to validate the power of the new approach, revealing that implementation of Local Moran's method in image segmentation is superior to threshold-based segmentation methods commonly used in analyzing confocal images in neuroanatomical studies.

Strengths:

Several proof-of-concept experiments are performed to confirm the sensitivity and validity of the proposed method. Using composed images with varying levels of background noise and analyzing them in parallel with the Local Moran's or a Threshold-Based Method (TBM), the study is able to compare these approaches directly and reveal their relative power in isolating clustered pixels.

Similarly, dual immuno-electron microscopy was used to test the biological relevance of a colocalization that was revealed by Local Moran's segmentation approach on dual-fluorescent labeled tissue using immuno-markers of the axon terminal and a membrane-protein (Figure 5). The EM revealed that the two markers were present in terminals and their post-synaptic partners, respectively. This is a strong approach to verify the validity of the new approach for determining object-based colocalization in fluorescent microscopy.

The methods section is clear in explaining the rationale and the steps of the new method (however, see the weaknesses section). Figures are appropriate and effective in illustrating the methods and the results of the study. The writing is clear; the references are appropriate and useful.

Weaknesses:

While the steps of the mathematical calculations to implement Local Moran's principles for analyzing high-resolution images are clearly written, the manuscript currently does not provide a computation tool that could facilitate easy implementation of the method by other researchers. Without a user-friendly tool, such as an ImageJ plugin or a code, the use of the method developed by David et al by other investigators may remain limited.

This weakness is eliminated in the revision, which now provides the approach as a Matlab tool.

Reviewer #2 (Public Review):

Summary:

Alison G. Barber et al. reports the function of Msi2 in mouse models of non-small cell lung cancer. The expression of Msi2 in normal lung was evaluated using a knockin reporter allele. Msi2 expressing cells were found to be around 30-40% in normal lung epithelium without a strong bias in subsets of lung cells. Knocking out Msi2 in a KrasG12D and P53 KO model reduced lung cancer initiation. Knocking down Msi2 in established lung cancer cells reduced in vitro sphere formation and in vivo xenograft. Finally, the authors identified several genes whose expression was downregulated by Msi2 knockdown. Knocking down four of these genes, including Ptgds, Arl2bp, hRnf157, and Syt11, each with a single shRNA, reduced lung sphere formation in vitro, suggesting their involvement in lung cancer.

Strengths:

This manuscript represents an interesting advance on the role of Msi2 in lung cancer. While some of the data (for example the knockdown effect of Msi2 in established lung cancer cells) corroborated previous findings, the study of Msi2 expression in normal lung and the characterization of the KO phenotype in lung cancer initiation are new and interesting.

Weaknesses:

Two areas can be further strengthened. Several conclusions are not fully supported by the existing data. The stable/dynamic nature of Msi2 expressing cells in lung would benefit from more detailed investigations for proper data interpretation.

(1) It will be interesting to determine whether Msi2+ cells are a relatively stable subset or rather the Msi2+ cells in lung is a dynamic concept that is transient or interconvertible. This is relevant to the interpretation of what Msi2 positivity really means.

(2) Does Kras mutation and/or p53 loss upregulate Msi2? This point and the point above are related to whether Msi2+ cells are truly more susceptible to tumorigenesis, as the authors suggested.

(3) The KO of Msi2 reducing tumor number and burden in the lung cancer initiation model is interesting. However, there are two alternative interpretations. First, it is possible that the Msi2 KO mice (without Kras activation and p53 loss) has reduced total lung cell numbers or altered percentage of stem cells. There is currently only one sentence citing data not shown on line 125, commenting that there is no difference in BASC and AT2 cell populations. It will be helpful that such data are shown and the effect of KO on overall lung mass or cellularity is clarified. Second, the phenotype may also be due to a difference in the efficiencies of cre on Kras and p53 in the Msi2 WT and KO mice.

(4) All shRNA experiments (for both Msi2 KD and the KD of candidate genes) utilized a single shRNA. This approach cannot exclude off-target effects of the shRNA.

(5) The technical details of the PDX experiment (Figure 4F) are not fully explained.

Reviewer #3 (Public Review):

Summary:<br /> Adamic and colleagues present fMRI data from ADE patients and a healthy control group acquired during two interoceptive tasks (attention and perturbation) from the same session. They report convergent activity within the granular and dysgranular insular cortex during both tasks, with a patient group-specific lateralisation effect. Furthermore, insular functional connectivity was found to be linked to disease severity.

Strengths:<br /> The study is well-designed and - despite some limitations noted by the authors - provides much-needed insight into the functional pathways of interoceptive processing in health and disease. The manuscript is clear, concise, and well-written.

Weaknesses:<br /> None remain after the authors' revision.

Reviewer #2 (Public review):

Summary:

This paper presents an interesting and useful analysis of grid cell heterogeneity, showing that the experimentally observed heterogeneity of spacing and orientation within a grid cell module can allow more accurate decoding of location from a single module.

Strengths:

I found the statistical analysis of the grid cell variability to be very systematic and convincing. I also found the evidence for enhanced decoding of location based on between-cell variability within a module to be convincing and important, supporting their conclusions.

Weaknesses:

(1) Even though theoreticians might have gotten the mistaken impression that grid cells are highly regular, this might be due to an overemphasis on regularity in a subset of papers. Most experimentalists working with grid cells know that many if not most grid cells show high variability of firing fields within a single neuron, though this analysis focuses on between neurons. In response to this comment, the reviewers should tone down and modify their statements about what are the current assumptions of the field (and if possible provide a short supplemental section with direct quotes from various papers that have made these assumptions).

(2) The authors state that "no characterization of the degree and robustness of variability in grid properties within individual modules has been performed." It is always dangerous to speak in absolute terms about what has been done in scientific studies. It is true that few studies have had the number of grid cells necessary to make comparisons within and between modules, but many studies have clearly shown the distribution of spacing in neuronal data (e.g. Hafting et al., 2005; Barry et al., 2007; Stensola et al., 2012; Hardcastle et al., 2015) so the variability has been visible in the data presentations. Also, most researchers in the field are well aware that highly consistent grid cells are much rarer than messy grid cells that have unevenly spaced firing fields. This doesn't hurt the importance of the paper, but they need to tone down their statements about the lack of previous awareness of variability (specific locations are noted in the specific comments).

(3) The methods section needs to have a separate subheading entitled: How grid cells were assigned to modules" that clearly describes how the grid cells were assigned to a module (i.e. was this done by Gardner et al., or done as part of this paper's post-processing?

Reviewer #2 (Public review):

Summary:

While bacteria have the ability to induce genes in response to specific stresses, they also use the General Stress Response (GSR) to deal with growth conditions that presumably include a larger range of stresses (for instance, stationary phase growth). The activation of GSR-specific sigma factors is frequently at the heart of the induction of a GSR. Given the range of stresses that can lead to GSR induction, the regulatory inputs are frequently complex. In B. subtilis, the stressosome, a multi-protein complex, contains a set of proteins that, upon appropriate stresses, initiate partner switching cascades that free the sigma B sigma factor from an anti-sigma. The focus here is on the mode of activation of RsbU, a serine/threonine phosphatase of the PPM family, leading to sigB activation. RbsT, a component of the degradosome interacts with RsbU upon stress, activating the phosphatase activity. Once active, RsbU dephosphorylates its target (RsbV, an anti-antisigma), which in turn binds the anti-sigma. The conclusion is that flexible linker domains upstream of the phosphatase domain are the target for activation, via binding of proteins to the N-terminal domain, resulting in a crossed-linker dimeric structure. The authors then use the information on RsbU to suggest that parallel approaches are used to activate PPM phosphatases for the GSR response in other bacteria. (Biology vs. Mechanism, evolution?)

Strengths and Weaknesses:

Many of these have to do with clarifying what was done and why. This includes the presentation and content of the figures.

One issue relates to the background and context. A bit more information on the stresses that release RsbT would be useful here. The authors might also consider a figure showing the major conclusions and parallels for SpoIIE activation and possibly other partner switches that are discussed, introducing the switch change more clearly to set the stage for the work here (and the generalization). There are a lot of players to keep track of.

Reviewer #2 (Public Review):

Summary:

Mutations in SUFU are implicated in SHH medulloblastoma (MB). SUFU modulates Shh signaling in a context-dependent manner, making its role in MB pathology complex and not fully understood. This study reports that elevated FGF5 levels are associated with a specific subtype of SHH MB, particularly in pediatric cases. The authors demonstrate that Sufu deletion in a mouse model leads to abnormal proliferation of granule cell precursors (GCPs) at the secondary fissure (region B), correlating with increased Fgf5 expression. Notably, pharmacological inhibition of FGFR restores normal cerebellar development in Sufu mutant mice.

Strengths:

The identification of increased FGF5 in subsets of MB is novel and a key strength of the paper.

Weaknesses:

The study appears incomplete despite the potential significance of these findings. The current paper does not fully establish the causal relationship between Fgf5 and abnormal cerebellar development, nor does it clarify its connection to SUFU-related MB. Some conclusions seem overstated, and the central question of whether FGFR inhibition can prevent tumor formation remains untested.

Reviewer #2 (Public review):

The regulation of protein function heavily relies on the dynamic changes in the shape and structure of proteins and their complexes. These changes are widespread and crucial. However, examining such alterations presents significant challenges, particularly when dealing with large protein complexes in conditions that mimic the natural cellular environment. Therefore, much emphasis has been put on developing novel methods to study protein structure, interactions, and dynamics. Crosslinking mass spectrometry (CSMS) has established itself as such a prominent tool in recent years. However, doing this in a quantitative manner to compare structural changes between conditions has proven to be challenging due to several technical difficulties during sample preparation. Luo and Ranish introduce a novel set of isobaric labeling reagents, called Qlinkers, to allow for a more straightforward and reliable way to detect structural changes between conditions by quantitative CSMS (qCSMS).

The authors do an excellent job describing the design choices of the isobaric crosslinkers and how they have been optimized to allow for efficient intra- and inter-protein crosslinking to provide relevant structural information. Next, they do a series of experiments to provide compelling evidence that the Qlinker strategy is well suited to detect structural changes between conditions by qCSMS. First, they confirm the quantitative power of the novel-developed isobaric crosslinkers by a controlled mixing experiment. Then they show that they can indeed recover known structural changes in a set of purified proteins (complexes) - starting with single subunit proteins up to a very large 0.5 MDa multi-subunit protein complex - the polII complex.

The authors give a very measured and fair assessment of this novel isobaric crosslinker and its potential power to contribute to the study of protein structure changes. They show that indeed their novel strategy picks up expected structural changes, changes in surface exposure of certain protein domains, changes within a single protein subunit but also changes in protein-protein interactions. However, they also point out that not all expected dynamic changes are captured and that there is still considerable room for improvement (many not limited to this crosslinker specifically but many crosslinkers used for CSMS).

Taken together the study presents a novel set of isobaric crosslinkers that indeed open up the opportunity to provide better qCSMS data, which will enable researchers to study dynamic changes in the shape and structure of proteins and their complexes. However, in its current form, the study some aspects of the study should be expanded upon in order for the research community to assess the true power of these isobaric crosslinkers. Specifically:

Although the authors do mention some of the current weaknesses of their isobaric crosslinkers and qCSMS in general, more detail would be extremely helpful. Throughout the article a few key numbers (or even discussions) that would allow one to better evaluate the sensitivity (and the applicability) of the method are missing. This includes:

(1) Throughout all the performed experiments it would be helpful to provide information on how many peptides are identified per experiment and how many have actually a crosslinker attached to it.

(2) Of all the potential lysines that can be modified - how many are actually modified? Do the authors have an estimate for that? It would be interesting to evaluate in a denatured sample the modification efficiency of the isobaric crosslinker (as an upper limit as here all lysines should be accessible) and then also in a native sample. For example, in the MBP experiment, the authors report the change of one mono-linked peptide in samples containing maltose relative to the one not containing maltose. The authors then give a great description of why this fits to known structural changes. What is missing here is a bit of what changes were expected overall and which ones the authors would have expected to pick up with their method and why have they not been picked up. For example, were they picked up as modified by the crosslinker but not differential? I think this is important to discuss appropriately throughout the manuscript to help the reader evaluate/estimate the potential sensitivity of the method. There are passages where the authors do an excellent job doing that - for example when they mention the missed site that they expected to see in the initial the polII experiments (lines 191 to 207). This kind of "power analysis" should be heavily discussed throughout the manuscript so that the reader is better informed of what sensitivity can be expected from applying this method.

(3) It would be very helpful to provide information on how much better (or not) the Qlinker approach works relative to label-free qCLMS. One is missing the reference to a potential qCLMS gold standard (data set) or if such a dataset is not readily available, maybe one of the experiments could be performed by label-free qCLMS. For example, one of the differential biosensor experiments would have been well suited.

Reviewer #2 (Public review):

Summary:

Excessive sucrose is a possible initial factor for the development of metabolic dysfunction-associated fatty liver disease (MAFLD). To investigate the possibility that intervention with JNK inhibitor could lead to the treatment of metabolic dysfunction caused by excessive sucrose intake, the authors performed multi-organ transcriptomics analysis (liver, visceral fat (vWAT), skeletal muscle, and brain) in a rat model of MAFLD induced by sucrose overtake (+ a selective JNK2 and JNK3 inhibitor (JNK-IN-5A) treatment). Their data suggested that changes in gene expression in the vWAT as well as in the liver contribute to the pathogenesis of their MAFLD model and revealed that the JNK inhibitor has a cross-organ therapeutic effect on it.

Strengths:

(1) It has been previously reported that inhibition of JNK signalling can contribute to the prevention of hepatic steatosis (HS) and related metabolic syndrome in other models, but the role of JNK signalling in the metabolic disruption caused by excessive intake of sucrose, a possible initial factor for the development of MAFLD, has not been well understood, and the authors have addressed this point.

(2) This study is also important because pharmacological therapy for MAFLD has not yet been established.

(3) By obtaining transcriptomic data in multiple organs and comprehensively analyzing the data using gene co-expression network (GCN) analysis and genome-scale metabolic models (GEM), the authors showed the multi-organ interaction in not only in the pathology of MAFLD caused by excessive sucrose intake but also in the treatment effects by JNK-IN-5A.

(4) Since JNK signalling has diverse physiological functions in many organs, the authors effectively assessed possible side effects with a view to the clinical application of JNK-IN-5A.

Weaknesses:

(1) The metabolic process activities were evaluated using RNA-seq results in Figure 7, but direct data such as metabolite measurements are lacking.

(2) There is a lack of consistency in the data between JNK-IN-5A_D1 and _D2, and there is no sufficient data-based explanation for why the effects observed in D1 were inconsistent in the D2 samples.

(3) Although it is valuable that the authors were able to suggest the possibility of JNK inhibitor as a therapeutic strategy for MAFLD, the evaluation of the therapeutic effect was limited to the evaluation of plasma TG, LDH, and gene expression changes. As there was no evaluation of liver tissue images, it is unclear what changes were brought about in the liver by the excessive sucrose intake and the treatment with JNK-IN-5A.

Reviewer #2 (Public review):

Summary:<br /> The authors goal is to develop a more accurate system that reports TDP-43 activity as a splicing regulator. Prior to this, most methods employed western blotting or QPCR-based assays to determine whether targets of TDP-43 were up or down-regulated. The problem with that is the sensitivity. This approach uses an ectopic delivered construct containing splicing elements from CFTR and UNC13A (two known splicing targets) fused to a GFP reporter. Not only does it report TDP-43 function well, but it operates at extremely sensitive TDP-43 levels, requiring only picomolar TDP-43 knockdown for detection. This reporter should supersede the use of current TDP-43 activity assays, it's cost-effective, rapid and reliable.

Strengths:<br /> In general, the experiments are convincing and well designed. The rigor, number of samples and statistics, and gradient of TDP-43 knockdown were all viewed as strengths. In addition, the use of multiple assays to confirm the splicing changes were viewed as complimentary (ie PCR and GFP-fluorescence) adding additional rigor. The final major strength I'll add is the very clever approach to tether TDP-43 to the loss of function cassette such that when TDP-43 is inactive it would autoregulate and induce wild-type TDP-43. This has many implications for the use of other genes, not just TDP-43, but also other protective factors that may need to be re-established upon TDP-43 loss of function.

Weaknesses:<br /> Admittedly, one needs to initially characterize the sensor and the use of cell lines is an obvious advantage, but it begs the question of whether this will work in neurons. Additional future experiments in primary neurons will be needed. The bulk analysis of GFP-positive cells is a bit crude. As mentioned in the manuscript, flow sorting would be an easy and obvious approach to get more accurate homogenous data. This is especially relevant since the GFP signal is quite heterogeneous in the image panels, for example, Figure 1C, meaning the siRNA is not fully penetrant. Therefore, stating that 1% TDP-43 knockdown achieves the desired sensor regulation might be misleading. Flow sorting would provide a much more accurate quantification of how subtle changes in TDP-43 protein levels track with GFP fluorescence.

Some panels in the manuscript would benefit from additional clarity to make the data easier to visualize. For example, Figure 2D and 2G could be presented in a more clear manner, possibly split into additional graphs since there are too many outputs. Sup Figure 2A image panels would benefit from being labeled, its difficult to tell what antibodies or fluorophores were used. Same with Figure 4B.

Figure 3 is an important addition to this manuscript and in general is convincing showing that TDP-43 loss of function mutants can alter the sensor. However, there is still wild-type endogenous TDP-43 in these cells, and it's unclear whether the 5FL mutant is acting as a dominant negative to deplete the total TDP-43 pool, which is what the data would suggest. This could have been clarified. Additional treatment with stressors that inactivate TDP-43 could be tested in future studies.

Overall, the authors definitely achieved their goals by developing a very sensitive readout for TDP-43 function. The results are convincing, rigorous, and support their main conclusions. There are some minor weaknesses listed above, chief of which is the use of flow sorting to improve the data analysis. But regardless, this study will have an immediate impact for those who need a rapid, reliable, and sensitive assessment of TDP-43 activity, and it will be particularly impactful once this reporter can be used in isolated primary cells (ie neurons) and in vivo in animal models. Since TDP-43 loss of function is thought to be a dominant pathological mechanism in ALS/FTD and likely many other disorders, having these types of sensors is a major boost to the field and will change our ability to see sub-threshold changes in TDP-43 function that might otherwise not be possible with current approaches.

Reviewer #2 (Public review):

Summary

This work investigates how multiple DNA elements combine to regulate gene expression. The authors use an episomal reporter assay which measures the transcriptional output of the reporter under the regulation of an enhancer-enhancer-promoter triple. The authors test all combinations of 8 promoters and 59 enhancers in this assay. There are two main findings: (1) enhancer pairs generally combine additively on reporter output (2) the extent to which enhancers increase reporter output over the promoter (individually and as enhancer-enhancer pairs) is inversely related to the intrinsic strength of the promoter. Both of these findings are interesting and are well supported by the data.

This study extends previous results on enhancer-promoter combinations to enhancer-enhancer-promoter triples. For example the near equivalence of Fig. 5b and Fig. S7b is intriguing. This experimental design also provides the ability to investigate the notion of selectivity (also commonly referred to as compatibility) between enhancer-enhancer pairs and promoters.

The authors note many limitations, including the selection of the elements and the size and spacing of the tested elements. Some of the enhancer-enhancer-promoter triples they test were also investigated by a different experimental design in Brosh et al 2023. Brosh et al observed non-additivity between these elements while this study did not. Ultimately we do not know which mechanisms produce the non-additivity that has been observed in native loci and which experimental designs would preserve such mechanisms.

Overall this is a nice experimental design and a great dataset for probing how enhancers and promoters combine to regulate gene expression. I have no major concerns, but I will try to clarify some methodological points I found confusing.

Methodology<br /> The following two comments are meant to help the reader understand the methodology/terminology used in this paper and how it relates to other similar studies.

The interpretation that "promoters scale enhancer signals in a non-linear manner" is potentially confusing. I believe that the authors use "non-linear" to refer to the slopes (represented by the letter 'b' in Fig. 5b) being not equal to 1. Given how the boost index is defined, this implies the relationship

Activity of EEP = (Activity of CCP) * (Average Linear Boost)^b

One potential source of confusion is that the Average Linear Boost term itself depends on the set of promoters that are assayed. Averaging across (many) promoters may alleviate this concern, in which case Average Linear Boost may be considered some form of intrinsic enhancer strength. If so, there is a correspondence between this terminology and the terminology presented in Bergman et al 2022. If b not equal to 1 refers to a non-linear scaling, then the reader may think that b=1 refers to a linear scaling. But if b=1, and the Average Linear Boost term is interpreted as intrinsic enhancer strength, then the equation above implies that the activity of EEP is equal to an intrinsic promoter strength times an intrinsic enhancer strength. This is essentially the relationship that is considered in Bergman et al 2022 and which is referred to in that paper as 'multiplicative'. The purpose of this comment is not to argue for what is the relationship that best explains the data, it is just to clarify the terminology.

Enhancer-promoter selectivity: As a follow-up to a previous study (Martinez-Ara et al, Molecular Cell 2022) the authors mention that the data in this study also shows that enhancers show selectivity for certain promoters. I found the methodology hard to follow, so this section of the review is meant to guide the reader in understanding how the authors define 'selectivity'. The authors consider an enhancer to be not selective if its 'boost index' is the same across a set of promoters. 'Boost index' is defined to be the ratio of the reporter output with the enhancer and promoter divided by the reporter output with just the promoter. Conceptually, I think that considering the boost index is a reasonable way to quantify selectivity. The authors use a frequentist approach to classify each enhancer as selective or not selective. The null hypothesis is that the boost index of the enhancer is equal across a set of promoters. This can be visualized in Fig. 2C where the null hypothesis is that the mean of each vertical distribution is equal. Note that in Figure S4b of this paper (and in Figure 4B of their 2022 paper) the within-group variance is not plotted. Statistical significance is assessed using a Welch F-test.

Reviewer #3 (Public Review):

This is an interesting manuscript that builds off of this group's previous work focused on the interface between Hsf1, heat shock protein (HSP) mRNA production, and 3D genome topology. Here the group subjects the yeast Saccharomyces cerevisiae to either heat stress (HS) or ethanol stress (ES) and examines Hsf1 and Pol II chromatin binding, Histone occupancy, Hsf1 condensates, HSP gene coalescence (by 3C and live cell imaging), and HSP mRNA expression (by RT-qPCR and live cell imaging). The manuscript is well written, and the experiments seem well done, and generally rigorous, with orthogonal approaches performed to support conclusions. The main findings are that both HS and ES result in Hsf1/Pol II-dependent intergenic interactions, along with formation of Hsf1 condensates. Yet, while HS results in rapid and strong induction of HSP gene expression and Hsf1 condensate resolution, ES result in slow and weak induction of HSP gene expression without Hsf1 condensate resolution. Thus, the conclusion is somewhat phenomenological - that the same transcription factor can drive distinct transcription, topologic, and phase-separation behavior in response to different types of stress.

Reviewer #2 (Public review):

This manuscript proposed a new link between the formation of chloroplast budding vesicles (Rubisco-containing bodies [RCBs]) and the development of chloroplast-associated autophagosomes. The authors' previous work demonstrated two types of autophagy pathways involved in chloroplast degradation, including piecemeal degradation of partial chloroplast and whole chloroplast degradation. However, the mechanisms underlying piecemeal degradation are largely unknown, particularly regarding the initiation and release of the budding structures. Here, the authors investigated the progression of piecemeal-type chloroplast trafficking by visualizing it with a high-resolution time-lapse microscope. They provide evidence that autophagosome formation is required for the initiation of chloroplast budding, and that stromule formation is not correlated with this process. In addition, the authors also demonstrated that the release of chloroplast-associated autophagosome is independent of a chloroplast division factor, DRP5b.

Overall, the findings are interesting, and in general, the experiments are very well executed.

Comments on revised version:

The authors have generally addressed all of my concerns (and the other reviewer's) and adapted the manuscript where necessary. The revised version has significantly improved the manuscript. From my perspective there are no further concerns.

Reviewer #3 (Public review):

Summary:

The authors found two endosomal fusion modes by live cell imaging of endosomes in yolk sac lateral endoderm cells of 8.5-day-old embryonic mice and described the fusion modes by mathematical models and simulations. They also showed that actin polymerization is involved in the regulation of one of the fusion modes.

Strengths:

The strength of this study is that the authors' claims are well supported by beautiful live cell images and theoretical models. By using specialized cells, yolk sac visceral endoderm cells, the live images of endosomal fusion, localization of actin-related molecules, and validation data from multiple inhibitor experiments are clear.

Weaknesses:

Although it would be out of scope of this study, there is no experimental verification of whether the mechanism of endosome fusion claimed by the authors occurs in general cells, so the article is limited to showing a phenomenon specific to yolk sac lateral endoderm cells. The methods used were very basic and solid. Most of the image analysis was performed manually, but the results were statistically tested.

Summary:

Seiichi Koike et al. studied two fusion models, explosive fusion, and bridge fusion, utilizing yolk sac visceral endoderm cells. They elucidated these two fusion models in vivo by employing mathematical modeling and incorporating fluctuations derived from actin dynamics as a key regulator for rapid homotypic fusion between late endosomes.

Strengths:

This study uncovered the role of actin dynamics in regulating the transition of fusion models in homotypic fusion between late endosomes and introduced a method for observing the fusion of single vesicles with two different targets.

Weaknesses:

The physiological significance of different fusion models is lacking.

Reviewer #2 (Public review):

Summary:

In this study, Geurts et al. investigated the effects of the catecholamine reuptake inhibitor methylphenidate (MPH) on value-based decision-making using a combination of aversive and appetitive Pavlovian to Instrumental Transfer (PIT) in a human cohort. Using an elegant behavioural design they showed a valence- and action-specific effects of Pavlovian cues on instrumental responses. Initial analyses show no effect of MPH on these processes. However the authors performed a more in-depth analysis and demonstrated that MPH actually modulates PIT in action-specific manner depending of individual working memory capacities. The authors interpret that as an effect on cognitive control of Pavlovian biasing of actions and decision-making more than an invigoration of motivational biases.

Strengths:

A major strength of this study is its experimental design. The elegant combination of appetitive and aversive Pavlovian learning with approach/avoidance instrumental actions allows to precisely investigate the different modulation of value-based decision making depending on the context and environmental stimuli. Important MPH is only administered after Pavlovian and instrumental learning, restricting the effect on PIT performance only. Finally, the use of a placebo-controlled crossover design allows within-comparisons between PIT effect under placebo and MPH and the investigation of the relationships between working memory abilities, PIT and MPH effects.

Weaknesses:

As authors stated in their discussion, this study is purely correlational and their conclusions could be strengthened by the addition of interesting (but time- and resource-consuming) neuroimaging work.<br /> The originality of this work compared to their previous published work using the same cohort could also be clarified at different stages of the article, as I initially wondered what was really novel. This point is much clearer in the discussion section.<br /> A point which, in my opinion, really requires clarification is when the working memory performance presented in Figure 2B has been determined. Was it under placebo (as I would guess) or under MPH? If it is the former, it would be also interesting to look at how MPH modulates working memory based on initial abilities.<br /> A final point is that it could be interesting to also discuss these results, not only regarding dopamine signalling, but also including potential effect of MPH on noradrenaline in frontal regions, considering the known role of this system in modulating behavioural flexibility.

Reviewer #2 (Public Review):

Summary:

The Mutational Hazard Hypothesis (MHH) is a very influential hypothesis in explaining the origins of genomic and other complexity that seem to entail the fixation of costly elements. Despite its influence, very few tests of the hypothesis have been offered, and most of these come with important caveats. This lack of empirical tests largely reflects the challenges of estimating crucial parameters.

The authors test the central contention of the MHH, namely that genome size follows effective population size (Ne). They martial a lot of genomic and comparative data, test the viability of their surrogates for Ne and genome size, and use correct methods (phylogenetically corrected correlation) to test the hypothesis. Strikingly, they not only find that Ne is not THE major determinant of genome size, as is argued by MHH, but that there is not even a marginally significant effect. This is remarkable, making this an important paper.

Strengths:

The hypothesis tested is of great importance.

The negative finding is of great importance for reevaluating the predictive power of the tested hypothesis.

The test is straightforward and clear.

The analysis is a technical tour-de-force, convincingly circumventing a number of challenges of mounting a true test of the hypothesis.

Weaknesses:

I note no particular strengths, but I believe the paper could be further strengthened in three major ways.

(1) The authors should note that the hypothesis that they are testing is larger than the MHH. The MHH hypothesis says that<br /> (i) low-Ne species have more junk in their genomes and<br /> (ii) this is because junk tends to be costly because of increased mutation rate to nulls, relative to competing non/less-junky alleles.

The current results reject not just the compound (i+ii) MHH hypothesis, but in fact any hypothesis that relies on i. This is notably a (much) more important rejection. Indeed, whereas MHH relies on particular constructions of increased mutation rates of varying plausibility, the more general hypothesis i includes any imaginable or proposed cost to the extra sequence (replication costs, background transcription, costs of transposition, ectopic expression of neighboring genes, recombination between homologous elements, misaligning during meiosis, reduced organismal function from nuclear expansion, the list goes on and on). For those who find the MHH dubious on its merits, focusing this paper on the MHH reduces its impact - the larger hypothesis that the small costs of extra sequence dictate the fates of different organisms' genomes is, in my opinion, a much more important and plausible hypothesis, and thus the current rejection is more important than the authors let on.

(2) In addition to the authors' careful logical and mathematical description of their work, they should take more time to show the intuition that arises from their data. In particular, just by looking at Figure 1b one can see what is wrong with the non-phylogenetically-corrected correlations that MHH's supporters use. That figure shows that mammals, many of which have small Ne, have large genomes regardless of their Ne, which suggests that the coincidence of large genomes and frequently small Ne in this lineage is just that, a coincidence, not a causal relationship. Similarly, insects by and large have large Ne, regardless of their genome size. Insects, many of which have large genomes, have large Ne regardless of their genome size, again suggesting that the coincidence of this lineage of generally large Ne and smaller genomes is not causal. Given that these two lineages are abundant on earth in addition to being overrepresented among available genomes (and were even more overrepresented when the foundational MHH papers collected available genomes), it begins to emerge how one can easily end up with a spurious non-phylogenetically corrected correlation: grab a few insects, grab a few mammals, and you get a correlation. Notably, the same holds for lineages not included here but that are highly represented in our databases (and all the more so 20 years ago): yeasts related to S. cerevisiae (generally small genomes and large median Ne despite variation) and angiosperms (generally large genomes (compared to most eukaryotes) and small median Ne despite variation). Pointing these clear points out will help non-specialists to understand why the current analysis is not merely a they-said-them-said case, but offers an explanation for why the current authors' conclusions differ from the MHH's supporters and moreover explain what is wrong with the MHH's supporters' arguments.

(3) A third way in which the paper is more important than the authors let on is in the striking degree of the failure of MHH here. MHH does not merely claim that Ne is one contributor to genome size among many; it claims that Ne is THE major contributor, which is a much, much stronger claim. That no evidence exists in the current data for even the small claim is a remarkable failure of the actual MHH hypothesis: the possibility is quite remote that Ne is THE major contributor but that one cannot even find a marginally significant correlation in a huge correlation analysis deriving from a lot of challenging bioinformatic work. Thus this is an extremely strong rejection of the MHH. The MHH is extremely influential and yet very challenging to test clearly. Frankly, the authors would be doing the field a disservice if they did not more strongly state the degree of importance of this finding.

Reviewer #2 (Public review):

Summary:

Fei, Lu, Shi, et al. present a thorough evaluation of the immune cell landscape in pre-eclamptic human placentas by single-cell multi-omics methodologies compared to normal control placentas. Based on their findings of elevated frequencies of inflammatory macrophages and memory-like Th17 cells, they employ adoptive cell transfer mouse models to interrogate the coordination and function of these cell types in pre-eclampsia immunopathology. They demonstrate the putative role of the IGF1-IGF1R axis as the key pathway by which inflammatory macrophages in the placenta skew CD4+ T cells towards an inflammatory IL-17A-secreting phenotype that may drive tissue damage, vascular dysfunction, and elevated blood pressure in pre-eclampsia, leaving researchers with potential translational opportunities to pursue this pathway in this indication.

They present a major advance to the field in their profiling of human placental immune cells from pre-eclampsia patients where most extant single-cell atlases focus on term versus preterm placenta, or largely examine trophoblast biology with a much rarer subset of immune cells. While the authors present vast amounts of data at both the protein and RNA transcript level, we, the reviewers, feel this manuscript is still in need of much more clarity in its main messaging, and more discretion in including only key data that supports this main message most effectively.

Strengths:

(1) This study combines human and mouse analyses and allows for some amount of mechanistic insight into the role of pro-inflammatory and anti-inflammatory macrophages in the pathogenesis of pre-eclampsia (PE), and their interaction with Th17 cells.

(2) Importantly, they do this using matched cohorts across normal pregnancy and common PE comorbidities like gestation diabetes (GDM).

(3) The authors have developed clear translational opportunities from these "big data" studies by moving to pursue potential IGF1-based interventions.

Weaknesses:

(1) Clearly the authors generated vast amounts of multi-omic data using CyTOF and single-cell RNA-seq (scRNA-seq), but their central message becomes muddled very quickly. The reader has to do a lot of work to follow the authors' multiple lines of inquiry rather than smoothly following along with their unified rationale. The title description tells fairly little about the substance of the study. The manuscript is very challenging to follow. The paper would benefit from substantial reorganizations and editing for grammatical and spelling errors. For example, RUPP is introduced in Figure 4 but in the text not defined or even talked about what it is until Figure 6. (The figure comparing pro- and anti-inflammatory macrophages does not add much to the manuscript as this is an expected finding).

(2) The methods lack critical detail about how human placenta samples were processed. The maternal-fetal interface is a highly heterogeneous tissue environment and care must be taken to ensure proper focus on maternal or fetal cells of origin. Lacking this detail in the present manuscript, there are many unanswered questions about the nature of the immune cells analyzed. It is impossible to figure out which part of the placental unit is analyzed for the human or mouse data. Is this the decidua, the placental villi, or the fetal membranes? This is of key importance to the central findings of the manuscript as the immune makeup of these compartments is very different. Or is this analyzed as the entirety of the placenta, which would be a mix of these compartments and significantly less exciting?

(3) Similarly, methods lack any detail about the analysis of the CyTOF and scRNAseq data, much more detail needs to be added here. How were these clustered, what was the QC for scRNAseq data, etc? The two small paragraphs lack any detail.

(4) There is also insufficient detail presented about the quantities or proportions of various cell populations. For example, gdT cells represent very small proportions of the CyTOF plots shown in Figures 1B, 1C, & 1E, yet in Figures 2I, 2K, & 2K there are many gdT cells shown in subcluster analysis without a description of how many cells are actually represented, and where they came from. How were biological replicates normalized for fair statistical comparison between groups?

(5) The figures themselves are very tricky to follow. The clusters are numbered rather than identified by what the authors think they are, the numbers are so small, that they are challenging to read. The paper would be significantly improved if the clusters were clearly labeled and identified. All the heatmaps and the abundance of clusters should be in separate supplementary figures.

(6) The authors should take additional care when constructing figures that their biological replicates (and all replicates) are accurately represented. Figure 2H-2K shows N=10 data points for the normal pregnant (NP) samples when clearly their Table 1 and test denote they only studied N=9 normal subjects.

(7) There is little to no evaluation of regulatory T cells (Tregs) which are well known to undergird maternal tolerance of the fetus, and which are well known to have overlapping developmental trajectory with RORgt+ Th17 cells. We recommend the authors evaluate whether the loss of Treg function, quantity, or quality leaves CD4+ effector T cells more unrestrained in their effect on PE phenotypes. References should include, accordingly: PMCID: PMC6448013 / DOI: 10.3389/fimmu.2019.00478; PMC4700932 / DOI: 10.1126/science.aaa9420.

(8) In discussing gMDSCs in Figure 3, the authors have missed key opportunities to evaluate bona fide Neutrophils. We recommend they conduct FACS or CyTOF staining including CD66b if they have additional tissues or cells available. Please refer to this helpful review article that highlights key points of distinguishing human MDSC from neutrophils: https://doi.org/10.1038/s41577-024-01062-0. This will both help the evaluation of potentially regulatory myeloid cells that may suppress effector T cells as well as aid in understanding at the end of the study if IL-17 produced by CD4+ Th17 cells might recruit neutrophils to the placenta and cause ROS immunopathology and fetal resorption.

(9) Depletion of macrophages using several different methodologies (PLX3397, or clodronate liposomes) should be accompanied by supplementary data showing the efficiency of depletion, especially within tissue compartments of interest (uterine horns, placenta). The clodronate piece is not at all discussed in the main text. Both should be addressed in much more detail.

(10) There are many heatmaps and tSNE / UMAP plots with unhelpful labels and no statistical tests applied. Many of these plots (e.g. Figure 7) could be moved to supplemental figures or pared down and combined with existing main figures to help the authors streamline and unify their message.

(11) There are claims that this study fills a gap that "only one report has provided an overall analysis of immune cells in the human placental villi in the presence and absence of spontaneous labor at term by scRNA-seq (Miller 2022)" (lines 362-364), yet this study itself does not exhaustively study all immune cell subsets...that's a monumental task, even with the two multi-omic methods used in this paper. There are several other datasets that have performed similar analyses and should be referenced.

(12) Inappropriate statistical tests are used in many of the analyses. Figures 1-2 use the Shapiro-Wilk test, which is a test of "goodness of fit", to compare unpaired groups. A Kruskal-Wallis or other nonparametric t-test is much more appropriate. In other instances, there is no mention of statistical tests (Figures 6-7) at all. Appropriate tests should be added throughout.

Reviewer #2 (Public review):

Summary:<br /> Biomechanical forces, such as blood flow, are crucial for organ formation, including heart development. This study by Shuo Chen et al. aims to understand how cardiac cells respond to these forces. They used zebrafish as a model organism due to its unique strengths, such as the ability to survive without heartbeats, and conducted transcriptomic analysis on hearts with impaired contractility. They thereby identified id2b as a gene regulated by blood flow and is crucial for proper heart development, in particular, for the regulation of myocardial contractility and valve formation. Using both in situ hybridization and transgenic fish they showed that id2b is specifically expressed in the endocardium, and its expression is affected by both pharmacological and genetic perturbations of contraction. They further generated a null mutant of id2b to show that loss of id2b results in heart malformation and early lethality in zebrafish. Atrioventricular (AV) and excitation-contraction coupling were also impaired in id2b mutants. Mechanistically, they demonstrate that Id2b interacts with the transcription factor Tcf3b to restrict its activity. When id2b is deleted, the repressor activity of Tcf3b is enhanced, leading to suppression of the expression of nrg1 (neuregulin 1), a key factor for heart development. Importantly, injecting tcf3b morpholino into id2b-/- embryos partially restores the reduced heart rate. Moreover, treatment of zebrafish embryos with the Erbb2 inhibitor AG1478 results in decreased heart rate, in line with a model in which Id2b modulates heart development via the Nrg1/Erbb2 axis. The research identifies id2b as a biomechanical signaling-sensitive gene in endocardial cells that mediates communication between the endocardium and myocardium, which is essential for heart morphogenesis and function.

Strengths:<br /> The study provides novel insights into the molecular mechanisms by which biomechanical forces influence heart development and highlights the importance of id2b in this process.

Weaknesses:<br /> The claims are in general well supported by experimental evidence, but the following aspects may benefit from further investigation:

(1) In Figure 1C, the heatmap demonstrates the up-regulated and down-regulated genes upon tricane-induced cardiac arrest. Aside from the down-regulation of id2b expression, it was also evident that id2a expression was up-regulated. As a predicted paralog of id2b, it would be interesting to see whether the up-regulation of id2a in response to tricane treatment was a compensatory response to the down-regulation of id2b expression.

(2) The study mentioned that id2b is tightly regulated by the flow-sensitive primary cilia-klf2 signaling axis; however aside from showing the reduced expression of id2b in klf2a and klf2b mutants, there was no further evidence to solidify the functional link between id2b and klf2. It would therefore be ideal, in the present study, to demonstrate how Klf2, which is a transcriptional regulator, transduces biomechanical stimuli to Id2b.

(3) The authors showed the physical interaction between ectopically expressed FLAG-Id2b and HA-Tcf3b in HEK293T cells. Although the constructs being expressed are of zebrafish origin, it would be nice to show in vivo that the two proteins interact.

Reviewer #2 (Public Review):

Summary:<br /> The study wanted to functionally identify individual DANs that mediate larval olfactory<br /> learning. Then search for DAN-specific driver strains that mark single dopaminergic neurons, which subsequently can be used to target genetic manipulations of those neurons. 56 GAL4 drivers identifying dopaminergic neurons were found (Table 1) and three of them drive the expression of GFP to a single dopaminergic neuron in the third-instar larval brain hemisphere. The DAN driver R76F02-AD;R55C10-DBD appears to drive the expression to a dopaminergic neuron innervating the lower peduncle (LP), which would be DAN-c1.<br /> Split-GFP reconstitution across synaptic partners (GRASP) technique was used to investigate the "direct" synaptic connections from DANs to the mushroom body. Potential synaptic contact between DAN-c1 and MB neurons (at the lower peduncle) were detected.<br /> Then single odor associative learning was performed and thermogenetic tools were used (Shi-ts1 and TrpA1). When trained at 34{degree sign}C, the complete inactivation of dopamine release from DAN-c1 with Shibirets1 impaired aversive learning (Figure 2h), while Shibirets1 did not affect learning when trained at room temperature (22{degree sign}C). When paired with a gustatory stimulus (QUI or SUC), activation of DAN-c1 during training impairs both aversive and appetitive learning (Figure 2k).<br /> They examined the expression pattern of D2R in fly brains and were found in dopaminergic neurons and the mushroom body (Figure 3). To inspect whether the pattern of GFP signals indeed reflected the expression of D2R, three D2R enhancer driver strains (R72C04, R72C08, and R72D03-GAL4) were crossed with the GFP-tagged D2R strain.<br /> D2R knockdown (UAS-RNAi) in dopaminergic neurons driven by TH-GAL4 impaired larval aversive learning. Using a microRNA strain (UAS-D2R-miR), a similar deficit was observed. Crossing the GFP-tagged D2R strain with a DAN-c1-mCherry strain demonstrated the expression of D2R in DAN-c1 (Figure 4a). Knockdown of D2R in DAN-c1 impaired aversive learning with the odorant pentyl acetate, while appetitive learning was unaffected (Figure 4e). Sensory and motor functions appear not affected by D2R suppression.<br /> To exclude possible chronic effects of D2R knockdown during development, optogenetics was applied at distinct stages of the learning protocol. ChR2 was expressed in DAN-c1, and blue light was applied at distinct stages of the learning protocol. Optogenetic activation of DAN-c1 during training impaired aversive learning, not appetitive learning (Figure 5b-d).<br /> Knockdown of D2Rs in MB neurons by D2R-miR impaired both appetitive and aversive learning (Figure 6a). Activation of MBNs during training impairs both larval aversive and appetitive learning.<br /> Finally, based on the data the authors propose a model where the effective learning requires a balanced level of activity between D1R and D2R (Figure 7).

Strengths:<br /> The work is well written, clear, and concise. They use well documented strategies to examine GAL4 drivers with expression in a single DAN, behavioral performance in larvae with distinct genetic tools including those to do thermo and optogenetics in behaving flies. Altogether, the study was able to expand our understanding of the role of D2R in DAN-c1 and MB neurons in the larva brain.

Weaknesses:<br /> Is not completely clear how the system DAN-c1, MB neurons and Behavioral performance work. We can be quite sure that DAN-c1;Shits1 were reducing dopamine release and impairing aversive memory (Figure 2h). Similarly, DAN-c1;ChR2 were increasing dopamine release and also impaired aversive memory (Figure 5b). However, is not clear what is happening with DAN-c1;TrpA1 (Figure 2K). In this case the thermos-induction appears to impair the behavioral performance of all three conditions (QUI, DW and SUC) and the behavior is quite distinct from the increase and decrease of dopamine tone (Figure 2h and 5b).

The study successfully examined the role of D2R in DAN-c1 and MB neurons in olfactory conditioning. The conclusions are well supported by the data, with the exception of the claim that dopamine release from DAN-c1 is sufficient for aversive learning in the absence of unconditional stimulus (Figure 2K). Alternatively, the authors need to provide a better explanation of this point.<br /> The study provides insight into the role of D2R in associative learning expanding our understanding and might be a reference similar to previous key findings (Qi and Lee, 2014, https://doi.org/10.3390/biology3040831).

Reviewer #2 (Public Review):

Summary:

Ning and colleagues present studies supporting a role for breast carcinoma amplified sequence 2 (Bcas2) in positively regulating primitive wave hematopoiesis through amplification of beta-catenin-dependent (canonical) Wnt signaling. The authors present compelling evidence that zebrafish bcas2 is expressed at the right time and place to be involved in primitive hematopoiesis, that there are primitive hematopoietic defects in hetero- and homozygous mutant and knockdown embryos, that Bcas2 mechanistically positively regulates canonical Wnt signaling, and that Bcas2 is required for nuclear retention of B-cat through physical interaction involving armadillo repeats 9-12 of B-cat and the coiled-coil domains of Bcas2. Overall, the data and writing are clean, clear, and compelling. This study is a first-rate analysis of a strong phenotype with highly supportive mechanistic data. The findings shed light on the controversial question of whether, when, and how canonical Wnt signaling may be involved in hematopoietic development. We detail some minor concerns and questions below, which if answered, we believe would strengthen the overall story and resolve some puzzling features of the phenotype. Notwithstanding these minor concerns, we believe this is an exceptionally well-executed and interesting manuscript.

Strengths:

(1) The study features clear and compelling phenotypes and results.

(2) The manuscript narrative exposition and writing are clear and compelling.

(3) The authors have attended to important technical nuances sometimes overlooked, for example, focusing on different pools of cytosolic or nuclear b-catenin.

(4) The study sheds light on a controversial subject: regulation of hematopoietic development by canonical Wnt signaling and presents clear evidence of a role.

(5) The authors present evidence of phylogenetic conservation of the pathway.

Weaknesses:

(1) The authors present compelling data that Bcas2 regulates nuclear retention of B-cat through physical association involving binding between the Bcas2 CC domains and B-cat arm repeats 9-12. Transcriptional activation of Wnt target genes by B-cat requires physical association between B-cat and Tcf/Lef family DNA binding factors involving key interactions in Arm repeats 2-9 (Graham et al., Cell 2000). Mutually exclusive binding by B-cat regulatory factors, such as ICAT that prevent Tcf-binding is a documented mechanism (e.g. Graham et al., Mol Cell 2002). It would appear - based on the arm repeat usage by Bcas2 (repeats 9-12)-that Bcas2 and Tcf binding might not be mutually exclusive, which would support their model that Bcas2 physical association with B-cat to retain it in the nucleus would be compatible with co-activation of genes by allowing association with Tcf. It might be nice to attempt a three-way co-IP of these factors showing that B-cat can still bind Tcf in the presence of Bcas2, or at least speculate on the plausibility of the three-way interaction.

(2) A major way that canonical Wnt signaling regulates hematopoietic development is through regulation of the LPM hematopoietic competence territories by activating expression of cdx1a, cdx4, and their downstream targets hoxb5a and hoxa9a (Davidson et al., Nature 2003; Davidson et al., Dev Biol 2006; Pilon et al., Dev Biol 2006; Wang et al., PNAS 2008). Could the authors assess (in situ) the expression of cdx1a, cdx4, hoxb5a, and hoxa9a in the bcas2 mutants?

(3) The authors show compellingly that even heterozygous loss of bcas2 has strong Wnt-inhibitory effects. If Bcas2 is required for canonical Wnt signaling and bcas2 is expressed ubiquitously from the 1-cell stage through at least the beginning of gastrulation, why do bcas2 KO embryos not have morphological axis specification defects consistent with loss of early Wnt signaling, like loss of head (early), or brain anteriorization (later)? Could the authors provide some comments on this puzzle? Or if they do see any canonical Wnt signaling patterning defects in het- or homozygous embryos, could they describe and/or present them?

Reviewer #3 (Public Review):

Summary:

Well-illustrated new material is documented for Acanthomeridion, a formerly incompletely known Cambrian arthropod. The formerly known facial sutures are proposed be associated with ventral plates that the authors homologise with the free cheeks of trilobites (although also testing alternative homologies). An update of a published phylogenetic dataset permits reconsideration of whether dorsal ecdysial sutures have a single or multiple origins in trilobites and their relatives.

Strengths:

Documentation of an ontogenetic series makes a sound case that the proposed diagnostic characters of a second species of Acanthomeridion are variation within a single species. New microtomographic data shed light on appendage morphology that was not formerly known. The new data on ventral plates and their association with the ecdysial sutures are valuable in underpinning homologies with trilobites.

Reviewer #2 (Public Review):

In this manuscript, Yang et al. present a modeling framework to understand the pattern of response biases and variance observed in delayed-response orientation estimation tasks. They combine a series of modeling approaches to show that coupled sensory-memory networks are in a better position than single-area models to support experimentally observed delay-dependent response bias and variance in cardinal compared to oblique orientations. These errors can emerge from a population-code approach that implements efficient coding and Bayesian inference principles and is coupled to a memory module that introduces random maintenance errors. A biological implementation of such operation is found when coupling two neural network modules, a sensory module with connectivity inhomogeneities that reflect environment priors, and a memory module with strong homogeneous connectivity that sustains continuous ring attractor function. Comparison with single-network solutions that combine both connectivity inhomogeneities and memory attractors shows that two-area models can more easily reproduce the patterns of errors observed experimentally.

Strengths:

The model provides an integration of two modeling approaches to the computational bases of behavioral biases: one based on Bayesian and efficient coding principles, and one based on attractor dynamics. These two perspectives are not usually integrated consistently in existing studies, which this manuscript beautifully achieves. This is a conceptual advancement, especially because it brings together the perceptual and memory components of common laboratory tasks.

The proposed two-area model provides a biologically plausible implementation of efficient coding and Bayesian inference principles, which interact seamlessly with a memory buffer to produce a complex pattern of delay-dependent response errors. No previous model had achieved this.

Weaknesses:

The correspondence between the various computational models is not clearly shown. It is not easy to see clearly this correspondence because network function is illustrated with different representations for different models. In particular, the Bayesian model of Figure 2 is illustrated with population responses for different stimuli and delays, while the attractor models of Figure 3 and 4 are illustrated with neuronal tuning curves but not population activity.

The proposed model has stronger feedback than feedforward connections between the sensory and memory modules (J_f = 0.1 and J_b = 0.25). This is not the common assumption when thinking about hierarchical processing in the brain. The manuscript argues that error patterns remain similar as long as the product of J_f and J_b is constant, so it is unclear why the authors preferred this network example as opposed to one with J_b = 0.1 and J_f = 0.25.

Reviewer #2 (Public review):

In this study, Ninagawa et al., sheds light on UGGT's role in ER quality control of glycoproteins. By utilizing UGGT1/UGGT2 DKO , they demonstrate that several model misfolded glycoproteins undergo early degradation. One such substrate is ATF6alpha where its premature degradation hampers the cell's ability to mount an ER stress response.

This study convincingly demonstrates that many unstable misfolded glycoproteins undergo accelerated degradation without UGGTs. Also, this study provides evidence of a "tug of war" model involving UGGTs (pulling glycoproteins to being refolded) and EDEMs (pulling glycoproteins to ERAD).

The study explores the physiological role of UGGT, particularly examining the impact of ATF6α in UGGT knockout cells' stress response. The authors further investigate the physiological consequences of accelerated ATF6α degradation, convincingly demonstrating that cells are sensitive to ER stress in the absence of UGGTs and unable to mount an adequate ER stress response.

These findings offer significant new insights into the ERAD field, highlighting UGGT1 as a crucial component in maintaining ER protein homeostasis. This represents a major advancement in our understanding of the field.

Reviewer #2 (Public review):

Summary:

Together with the known anatomical connectivity, molecular atlasses paves the way toward functional maps of the nervous system of C. elegans. Along with the analysis of previous scRNA sequencing and reporter strains, new expression patterns are generated for hermaphrodite and males based on CRISPR-knocked-in GFP reporter strains and the use of the color-coded Neuropal strain to accurately identify neurons. Beyond a map of the known neurotransmitters (GABA, Acetylcholine, Glutamate, dopamine, serotonin, tyramine, octopamine), the atlas also identifies neurons likely using betaine and suggests sets of neurons employing new unknown monoaminergic transmission, or using exclusively peptidergic neurotransmission.

Strengths:

The use of CRISPR reporter alleles and of the Neuropal strain to assign neurotransmitter usage to each neuron is much more rigourous than previous analysis and reveal intriguing differences between scRNA seq, fosmid reporter and CRISPR knock-in approaches. The differences between approaches are discussed.

Weaknesses:

All have been addressed.

Reviewer #2 (Public Review):