Reviewer #2 (Public Review):

Summary:

This manuscript mainly studied the biological effect of tenascin XB (TNXB) on hemophilic arthropathy (HA) progression. Using bioinformatic and histopathological approaches, the authors identified the novel candidate gene TNXB for HA. Next, authors showed that TNXB knockdown lead to chondrocyte apoptosis, matrix degeneration and subchondral bone loss in vivo/vitro. Furthermore, AKT agonist promoted extracellular matrix synthesis and prevented apoptosis in TNXB knockdown chondrocytes.

Strengths:

In general, this study significantly advances our understanding of HA pathogenesis. The authors utilize comprehensive experimental strategies to demonstrate the role of TNXB in cartilage degeneration associated with HA. The results are clearly presented, and the conclusions appear appropriate.

Weaknesses:

Additional clarification is required regarding the gender of the F8-/- mouse in the study. Is the mouse male or female?

Reviewer #2 (Public Review):

Summary:

This study takes a new approach to studying the role of corticofugal projections from auditory cortex to inferior colliculus. The authors performed two-photon imaging of cortico-recipient IC neurons during a click detection task in mice with and without lesions of auditory cortex. In both groups of animals, they observed similar task performance and relatively small differences in the encoding of task-response variables in the IC population. They conclude that non-cortical inputs to the IC provide can substantial task-related modulation, at least when AC is absent.

Strengths:

This study provides valuable new insight into big and challenging questions around top-down modulation of activity in the IC. The approach here is novel and appears to have been executed thoughtfully. Thus, it should be of interest to the community.

Weaknesses:

There are however, substantial concerns about the interpretation of the findings and limitations to the current analysis. In particular, Analysis of single unit activity is absent, making interpretation of population clusters and decoding less interpretable. These concerns should be addressed to make sure that the results can be interpreted clearly in an active field that already contains a number of confusing and possibly contradictory findings.

Reviewer #2 (Public Review):

Summary:

The authors' report describes a novel vaccine platform derived from a newly discovered organelle called a migrasome. First, the authors address a technical hurdle in using migrasomes as a vaccine platform. Natural migrasome formation occurs at low levels and is labor intensive, however, by understanding the molecular underpinning of migrasome formation, the authors have designed a method to make engineered migrasomes from cultured, cells at higher yields utilizing a robust process. These engineered migrasomes behave like natural migrasomes. Next, the authors immunized mice with migrasomes that either expressed a model peptide or the SARS-CoV-2 spike protein. Antibodies against the spike protein were raised that could be boosted by a 2nd vaccination and these antibodies were functional as assessed by an in vitro pseudoviral assay. This new vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA that require very stringent storage conditions.

Strengths:

The authors present very robust studies detailing the biology behind migrasome formation and this fundamental understanding was used to form engineered migrasomes, which makes it possible to utilize migrasomes as a vaccine platform. The characterization of engineered migrasomes is thorough and establishes comparability with naturally occurring migrasomes. The biophysical characterization of the migrasomes is well done including thermal stability and characterization of the particle size (important characterizations for a good vaccine).

Weaknesses:

With a new vaccine platform technology, it would be nice to compare them head-to-head against a proven technology. The authors would improve the manuscript if they made some comparisons to other vaccine platforms such as a SARS-CoV-2 mRNA vaccine or even an adjuvanted recombinant spike protein. This would demonstrate a migrasome-based vaccine could elicit responses comparable to a proven vaccine technology. Additionally, understanding the integrity of the antigens expressed in their migrasomes could be useful. This could be done by looking at functional monoclonal antibodies binding to their migrasomes in a confocal microscopy experiment.

Reviewer #2 (Public Review):

Summary:

The aim of the study was to understand how cells of the skin communicate across dermal layers. The research group has previously demonstrated that cellular connections called airinemes contribute to this communication. The current work builds upon this knowledge by showing that differentiated keratinocytes also use cytonemes, specialized signaling filopodia, to communicate with undifferentiated keratinocytes. They show that cytonemes are the more abundant type of cellular extension used for communication between the differentiated keratinocyte layer and the undifferentiated keratinocytes. Disruption of cytoneme formation led to the expansion of the undifferentiated keratinocytes into the periderm, mimicking skin diseases like psoriasis. The authors go on to show that disruption of cytonemes results in perturbations in Notch signaling between the differentiated keratinocytes of the periderm and the underlying proliferating undifferentiated keratinocytes. Further, the authors show that Interleukin-17, also known to drive psoriasis, can restrict the formation of periderm cytonemes, possibly through the inhibition of Cdc42 expression. This work suggests that cytoneme-mediated Notch signaling plays a central role in normal epidermal regulation. The authors propose that disruption of cytoneme function may be an underlying cause of various human skin diseases.

Strengths:

The authors provide strong evidence that periderm keratinocytes cytonemes contain the notch ligand DeltaC to promote Notch activation in the underlying intermediate layer to regulate accurate epidermal maintenance.

Weaknesses:

The impact of the study would be increased if the mechanism by which Interlukin-17 and Cdc42 collaborate to regulate cytonemes was defined. Experiments measuring Cdc42 activity, rather than just measuring expression, would strengthen the conclusions.

Reviewer #2 (Public Review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects.

While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identification and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives.

In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

DOI: 10.1038/s41467-023-43550-2

Resource: BDSC_16671

Curator: @maulamb

SciCrunch record: RRID:BDSC_16671

What is this?

DOI: 10.1038/s41467-023-43550-2

Resource: (BDSC Cat# 9575,RRID:BDSC_9575)

Curator: @maulamb

SciCrunch record: RRID:BDSC_9575

What is this?

DOI: 10.1038/s41467-023-43550-2

Resource: (BDSC Cat# 1309,RRID:BDSC_1309)

Curator: @maulamb

SciCrunch record: RRID:BDSC_1309

What is this?

DOI: 10.1038/s41467-023-43550-2

Resource: (BDSC Cat# 27656,RRID:BDSC_27656)

Curator: @maulamb

SciCrunch record: RRID:BDSC_27656

What is this?

Reviewer #2 (Public Review):

Summary:

The authors carry out a careful and rigorous quantitative analysis of RecB transcript and protein levels at baseline and in response to DNA damage. Using single-molecule FISH and Halo-tagging in order to achieve sensitive measurements, they provide evidence that enhanced RecB protein levels in response to DNA damage are achieved through a post-transcriptional mechanism mediated by the Sm-like RNA binding protein, Hfq. In terms of biological relevance, the authors suggest that this mechanism provides a way to control the optimum level of RecB expression as both deletion and over-expression are deleterious. In addition, the proposed mechanism provides a new framework for understanding how transcriptional noise can be suppressed at the protein level.

Strengths:

Strengths of the manuscript include the rigorous approaches and orthogonal evidence to support the core conclusions, for example, the evidence that altering either Hhq or its recognition sequence on the RNA similarly enhance the protein to RNA ratio of RecB. The writing is clear and the experiments are well-controlled. The modeling approaches provide essential context to interpret the data, particularly given the small numbers of molecules per cell. The interpretations are careful and well supported.

Weaknesses:

The authors make a compelling case for the biological need to exquisitely control RecB levels, which they suggest is achieved by the pathway they have uncovered and described in this work. However, this conclusion is largely inferred as the authors only investigate the effect on cell survival in response to (high levels of) DNA damage and in response to two perturbations - genetic knock-out or over-expression, both of which are likely more dramatic than the range of expression levels observed in unstimulated and DNA damage conditions.

Reviewer #2 (Public Review):

The authors have done well to address the points raised in my previous review.

The updated version of this manuscript retains the technical competence of the first, but with important changes that make the analysis more legible and results better contextualized. Specifically, the discussion is richer, the interpretation of the results is more nuanced, the terminology is more precise, and issues of clarity related to the methodology and results have been resolved.

Broad caveats remain about the nature of authorship, and who we should expect to be quoted in science journalism. Namely, who is the lead author? Ideally, the corresponding author would be included as well, or else some bibliometric definition of the most senior author on the byline. However, the authors' approach here is certainly adequate, and they did well to incorporate discussion of authorship and the scholarly division of labour in their discussion.

In sum, I find the article greatly improved and a competent analysis into the unequal use of quotations in scientific journalism.

Investigativer Hintergrundbericht zum Aufbau von LNG-Kapazitäten durch Russland in der Arktis. Das Projekt Arctic LNG 2 ist nicht nur klimaschädlich, sondern auch katastrophal für die lokalen Ökosysteme. Europa bezieht mehr LNG aus Russland als vor der Invasion der kompletten Ukraine. Russland nutzte auch 2023 westliches Knowhow und trotz der Sanktionen gelieferte Ausrüstung vor allem der deutschen Firma Linde. https://www.derstandard.at/story/3000000191618/wie-westliche-konzerne-ein-russisches-prestigeprojekt-auch-nach-kriegsbeginn-ermoeglichten

Ausführlicher Bericht über die Konflikte um das neue LNG-Terminal CP2 an der Küste von Louisiana. https://www.nytimes.com/2023/12/26/climate/cp2-natural-gas-export-louisiana.html

Reviewer #3 (Public Review):

Liang and colleagues set out to test whether the human brain uses distance and grid-like codes in social knowledge using a design where participants had to navigate in a two-dimensional social space based on competence and warmth during an fMRI scan. They showed that participants were able to navigate the social space and found distance-based codes as well as grid-like codes in various brain regions, and the grid-like code correlated with behavior (reaction times).

On the whole, the experiment is designed appropriately for testing for distant-based and grid-like codes, and is relatively well powered for this type of study, with a large amount of behavioral training per participant. They revealed that a number of brain regions correlated positively or negatively with distance in the social space, and found grid-like codes in the frontal polar cortex and posterior medial entorhinal cortex, the latter in line with prior findings on grid-like activity in entorhinal cortex. The current paper seems quite similar conceptually and in design to previous work, most notably Park et al., 2021, Nature Neuroscience.

(1) The authors claim that this study provides evidence that humans use a spatial / grid code for abstract knowledge like social knowledge.

This data does specifically not add anything new to this argument. As with almost all studies that test for a grid code in a similar "conceptual" space (not only the current study), the problem is that, when the space is not a uniform, square/circular space, and 2-dimensional then there is no reason the code will be perfectly grid like, i.e., show six-fold symmetry. In real world scenarios of social space (as well as navigation, semantic concepts), it must be higher dimensional - or at least more than two dimensional. It is unclear if this generalizes to larger spaces where not all part of the space is relevant. Modelling work from Tim Behrens' lab (e.g., Whittington et al., 2020) and Bradley Love's lab (e.g., Mok & Love, 2019) have shown/argued this to be the case. In experimental work, like in mazes from the Mosers' labs (e.g., Derdikman et al., 2009), or trapezoid environments from the O'Keefe lab (Krupic et al., 2015), there are distortions in mEC cells, and would not pass as grid cells in terms of the six-fold symmetry criterion.

After revision, the authors now discuss some of this and the limitations and notes that future work is required to address the problem.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

(2) lines145-149: "The results showed that WT Huh7 cell cultured in high glucose medium exhibited a much higher infective rate than those in low glucose medium. However, CTSL KO Huh7 cells maintained a low infective rate of SARS-CoV-2 regardless of glucose or insulin levels (Fig. 3f-h). Therefore, hyperglycemia enhanced SARS-CoV-2 infection dependent on CTSL." However, this evidence may be insufficient to support the claim that hyperglycemia enhances SARS-CoV-2 infection dependent on CTSL. The human hepatoma cell line Huh7 might not be an ideal model to validate the authors' hypothesis regarding high blood glucose promoting SARS-CoV-2 infection through CTSL.

(3) The Abstract and Introduction sections lack effective organization.

In this revised version of the study, the authors have addressed my concerns by providing additional experiments, references and discussing further the points of controversy. I think that the authors have made improvements to the manuscript.

Reviewer #2 (Public Review):

Summary:

Liu and colleagues describe the transcriptional changes observed during chloramphenicol-induced surface mobility of Bacillus subtilis. Practically, they describe that numerous transcriptional regulatory pathways are influenced by the subinhibitory concentration of a translational inhibitor and some of these regulatory changes might contribute to the induction of sliding. Nevertheless, how such translational stress is translated to induction of sliding remains undetermined. The authors clearly describe their aim (line 457): "Our goal for this study was to gain insight into how B. subtilis mobilizes a colony in response to subinhibitory exposure to translation inhibitors.", this is unfortunately not solved here, only the authors characterize the transcriptional landscape differences.

Strengths:

The very thorough analysis of transcriptional changes in the wild type and codY mutant strains is appreciated, and there are definitely a plethora of changes observed related to several global transcriptional regulators in B. subtilis. I compliment the authors for this very detailed and thorough description of transcriptional changes.

Weaknesses:

While the transcriptional changes are well and carefully described, the discussion practically interprets the correlations as causations. I am not disputing that the authors are not on the correct path with their assumptions, but their conclusions are not supported by direct experimental data, especially on (1) translational stress directly inducing mobility and (2) division of labor.

Major 1:

The authors conclude that their results point towards a putative mechanism, e.g. line 460 "which suggests translation stress is a trigger for colony mobilization"; however, no experiment demonstrates this aspect. The authors do not test ppGpp-related stress (mutants in ppGpp-related genes, or mutating the functional domain of CodY), nor do they directly connect ppGpp levels dynamics with induction of subsequent pathways. Again, I understand that the authors are on the right path to connect these pathways and identify what is causing mobility induction, but no direct data is represented, solely the transcriptional changes, therefore remains slightly descriptive.

The statement in the chapter title (line 474) is not demonstrated directly and should be revised. Similarly, in line 476, the authors claim that their "data supports a model", but "support" would require direct experimental data on this aspect.

The authors even clearly indicate in lines 504-506 that they do not reveal the direct mechanism, but the rest of the discussion delivers statements that do not consider the lack of direct data.

Major 2:

Line 427: "The results are consistent with a division of metabolic labor among cells in the expanding population" - the data shows heterogeneity, but the direct division of labor is not demonstrated.

Line 442: So in this case, the proposed division of labor is disrupted in the codY mutant (no inner localisation), and hence expansion appears, suggesting a lack of a putative division of labor is not necessary for induced mobility. On the contrary, there could be heterogeneous gene expression, division of labor requires demonstration of fitness benefit from such interaction.

Division of labor assumes that a mixture of mutants would complement full sliding dynamics, and this could be easily demonstrated by fluorescent labeled cells that should be organized in a similar fashion to those observed with luciferase reporters (pucA mutant on the outer ring, while pdhA mutant interior colony part). Without such experimental demonstration, the authors can only conclude spatially heterogeneous gene expresstion without clear functional contribution to subinhibitory chrolamphenociol-induced surface mobility.

Again, the authors' statement in line 472 "reveal a regulated, spatiotemporal division of metabolism" is not demonstrated by experiment, but spatial heterogeneity is revealed here.<br /> The statement in the Discussion chapter (line 499) is also not demonstrated by experimental data: "Metabolic coordination enables surface expansion of mobilitzed B. subtilis"

Line 550: while I agree with the authors' statement that these functions work cooperatively as demonstrated by van Gestel and colleagues (2015 PloS Biol), the exploitation of these shared goods is not quantitatively equivalent, see Jautzus et al 2022 ISME J (DOI: 10.1038/s41396-022-01279-8).

In summary: the two major conclusions of the manuscript are unfortunately not demonstrated, the presented transcriptional data delivers suggestions, supported with specific mutants displaying certain phenotypes (lack of mobility induction or constitutive mobility without inducer), but it remains unclear how translational stress induces mobility and whether the transcriptional heterogeneity detected directly contributes to metabolic division of labor.

The authors should present direct evidence on the major concerns: how translational stress induces surface mobility (using ppGpp synthesis and turnover mutants and specific CdoY mutant lacking ppGpp sensing) and whether the metabolic division of labor contributes to induced surface mobility (mixing mutants and following their distribution).

Reviewer #3 (Public Review):

In this study, Ruan et al. investigate the role of the IQCH gene in spermatogenesis, focusing on its interaction with calmodulin and its regulation of RNA-binding proteins. The authors examined sperm from a male infertility patient with an inherited IQCH mutation as well as Iqch CRISPR knockout mice. The authors found that both human and mouse sperm exhibited structural and morphogenetic defects in multiple structures, leading to reduced fertility in Ichq-knockout male mice. Molecular analyses such as mass spectrometry and immunoprecipitation indicated that RNA-binding proteins are likely targets of IQCH, with the authors focusing on the RNA-binding protein HNRPAB as a critical regulator of testicular mRNAs. The authors used in vitro cell culture models to demonstrate an interaction between IQCH and calmodulin, in addition to showing that this interaction via the IQ motif of IQCH is required for IQCH's function in promoting HNRPAB expression. In sum, the authors concluded that IQCH promotes male fertility by binding to calmodulin and controlling HNRPAB expression to regulate the expression of essential mRNAs for spermatogenesis. These findings provide new insight into molecular mechanisms underlying spermatogenesis and how important factors for sperm morphogenesis and function are regulated.

The strengths of the study include the use of mouse and human samples, which demonstrate a likely relevance of the mouse model to humans; the use of multiple biochemical techniques to address the molecular mechanisms involved; the development of a new CRISPR mouse model; ample controls; and clearly displayed results. Assays are done rigorously and in a quantitative manner. Overall, the claims made by the authors in this manuscript are well-supported by the data provided.

Reviewer #3 (Public Review):

The work proposes a model of neural information processing based on a 'synergistic global workspace,' which processes information in three principal steps: a gatekeeping step (information gathering), an information integration step, and finally, a broadcasting step. They provided an interpretation of the reduced human consciousness states in terms of the proposed model of brain information processing, which could be helpful to be implemented in other states of consciousness. The manuscript is well-organized, and the results are important and could be interesting for a broad range of literature, suggesting interesting new ideas for the field to explore.

Reviewer #2 (Public Review):

Summary:

The main conclusion of the manuscript is that the presence of linker Histone H1 protects Arabidopsis pericentromeric heterochromatic regions and longer transposable elements from encroachment by other repressive pathways. The manuscript focuses on the RNA-dependent DNA-methylation (RdDM) pathway but indirectly finds that other pathways must also be ectopically enriched.

Strengths:

The authors present diverse sets of genomic data comparing Arabidopsis wild-type and h1 mutant background allowing an analysis of differential recruitment of RdDM component NPRE1, which is related to changes in DNA methylation and H1 coverage. The manuscript also contains recruitment data for SUVH1 in wild-type and h1 mutant backgrounds.<br /> Furthermore, the authors make use of a line that recruits NRPE1 ectopically to show that H1 occupancy is not altered because of this recruitment. These data clearly show that there is a hierarchy in which DNA-methylation is impacted by presence of H1 while H1 distribution is independent of DNA-methylation.

Weaknesses:

The manuscript is driven by a strong and reasonable hypothesis that absence of H1 results increased access of chromatin binding factors and that this explains how the RdDM machinery is restricted from encroaching heterochromatic regions, which are particularly enriched in H1. Indeed, increased binding of NPRE1 at pericentromeric sites is observed; however, the major DNA-methylation changes at these sites are symmetric and not related to the RdDM pathway. Thus, the authors propose that many factors redistribute, which is again reasonable. The authors show redistribution of SUVH1 and relate their data to a previous report showing redistribution of the PcG machinery in H1 depletion mutants (Teano et al. in Cell reports (Volume 42, Issue 8, 29 August 2023), but the manuscript provides limited mechanistic insight as to why there is a strong increase in heterochromatin symmetric DNA-methylation.

Reviewer #2 (Public Review):

This paper uses a novel maze design to explore mouse navigation behaviour in an automated analogue of the Barnes maze. A major strength is the novel and clever experimental design which rotates the floor and intramaze cues before the start of each new trial, allowing the previous goal location to become the next starting position. The modelling sampling a Markov chain of navigation strategies is elegant, appropriate and solid, appearing to capture the behavioural data well. This work provides a valuable contribution and I'm excited to see further developments, such as neural correlates of the different strategies and switches between them.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Jiang et al., explore the role of neurexins at glycinergic MNTB-LSO synapses. The authors utilize elegant and compelling ex vivo slice electrophysiology to assess how the genetic conditional deletion of Nrxns1-3 impacts inhibitory glycinergic synaptic transmission and found that TKO of neurexins reduced electrically and optically evoked IPSC amplitudes, slowed optically evoked IPSC kinetics and reduced presynaptic release probability. The authors use classic approaches including reduced [Ca2+] in ACSF and EGTA chelation to propose that changes in these evoked properties are likely driven by the loss of calcium channel coupling. Intriguingly, while evoked transmission was impaired, the authors reported that spontaneous IPSC frequency was increased, due to an increase in the number of synapses in LSO. Overall, this manuscript provides important insight into the role of neurexins at the glycinergic MNTB-LSO synapse and further emphasizes the need for continued study of both the non-redundant and redundant roles of neurexins.

The authors have addressed all of my previous concerns.

Reviewer #2 (Public Review):

Weng et al. perform a comprehensive study of gene expression changes in young and old animals, in wild-type and daf-2 insulin receptor mutants, in the whole animal and specifically in the nervous system. Using this data, they identify gene families that are correlated with neuronal ageing, as well as a distinct set of genes that are upregulated in neurons of aged daf-2 mutants. This is particularly interesting as daf-2 mutants show both extended lifespan and healthier neurons in aged animals, reflected by better learning/memory in older animals compared with wild-type controls. Indeed, knockdown of several of these upregulated genes resulted in poorer learning and memory. In addition, the authors showed that several genes upregulated during ageing in wild-type neurons also contribute to learning and memory; specifically, knockdown of these genes in young animals resulted in improved memory. This indicates that (at least in this small number of cases), genes that show increased transcript levels with age in the nervous system somehow suppress memory, potentially by having damaging effects on neuronal health.

Finally, from a resource perspective, the neuronal transcriptome provided here will be very useful for C. elegans researchers as it adds to other existing datasets by providing the transcriptome of older animals (animals at day 8 of adulthood) and demonstrating the benefits of performing tissue-specific RNAseq instead of whole-animal sequencing.

The work presented here is of high quality and the authors present convincing evidence supporting their conclusions. I only have a few comments/suggestions:

(1) Do the genes identified to decrease learning/memory capacity in daf-2 animals (Figure 4d/e) also impact neuronal health? daf-2 mutant worms show delayed onset of age-related changes to neuron structure (Tank et al., 2011, J Neurosci). Does knockdown of the genes shown to affect learning also affect neuron structure during ageing, potentially one mechanism through which they modulate learning/memory?

(2) The learning and memory assay data presented in this study uses the butanone olfactory learning paradigm, which is well established by the same group. Have the authors tried other learning assays when testing for learning/memory changes after knockdown of candidate genes? Depending on the expression pattern of these genes, they may have more or less of an effect on olfactory learning versus for e.g. gustatory or mechanosensory-based learning.

(3) A comment on the 'compensatory vs dysregulatory' model as stated by the authors on page 7 - I understand that this model presents the two main options, but perhaps this is slightly too simplistic: gene expression that rises during ageing may be detrimental for memory (= dysregulatory), but at the same time may also be beneficial other physiological roles in other tissues (=compensatory).

Comments on revised version:

I am satisfied with how the authors have addressed all my comments/suggestions.

Reviewer #2 (Public Review):

Severe leptospirosis in humans and some mammals often meet death in the endpoint. In this article, authors explored the role of the gut microbiota in severe leptospirosis. They found that Leptospira infection promoted a dysbiotic gut microbiota with an expansion of Proteobacteria and LPS neutralization therapy synergized with antileptospiral therapy significantly improved the survival rates in severe leptospirosis. This study is well-organized and has potentially important clinical implications not only for severe leptospirosis but also for other gut-damaged infections.

Reviewer #3 (Public Review):

This study uses a range of methods to characterize heterogeneous neural populations within the nucleus incertus (NI). The authors focus on two major populations, expressing gsc2 and rln3a, and present convincing evidence that these cells have different patterns of connectivity, calcium activity and effects on behavior. Although the study does not go as far as clarifying the role of NI in any specific neural computation or aspect of behavioral control, the findings will be valuable in support of future endeavors to do so. In particular, the authors have made two beautiful knock-in lines that recapitulate endogenous expression pattern of gsc2 and rln3a which will be a powerful tool to study the roles of the relevant NI cells. Experiments are well done, data are high quality and most claims are well supported. In this revised version, the authors have added additional analysis that has clarified their results and strengthened some of the claims.

Two points of note:

• The data very clearly show different patterns of neurites for gsc2 and rln3a neurons in the IPN and the authors interpret these are being axonal arbors. However, they do not rule out the possibility that some of the processes might be dendritic in nature. Of relevance to this point, they cite a recent study (Petrucco et al. 2023) that confirmed that, as in other species, tegmental neurons in zebrafish extend spatially segregated dendritic as well as axonal arbors into IPN, and the authors speculate that these GABAergic tegmental cells might in fact be part of NI.

• Although the gsc2 and rln3a populations show differences in calcium activity, there is not as clear a dichotomy as stated in the abstract. For example, both populations clearly respond to electric shocks, albeit with different response time courses.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

(2) Visual homogeneity (at least given the current from) is an unnecessary term. It is similar to distractor heterogeneity/distractor variability/distractor statics in literature. However, the authors attempt to claim it as a novel concept. The title is "visual homogeneity computations in the brain enable solving generic visual tasks". The last sentence of the abstract is "a NOVEL IMAGE PROPERTY, visual homogeneity, is encoded in a localized brain region, to solve generic visual tasks". In the significance, it is mentioned that "we show that these tasks can be solved using a simple property WE DEFINE as visual homogeneity". If the authors agree that visual homogeneity is not new, I suggest a complete rewrite of the title, abstract, significance, and introduction.

(3) Also, "solving generic tasks" is another overstatement. The oddball search tasks, same-different tasks, and symmetric tasks are only a small subset of many visual tasks. Can this "quantitative model" solve motion direction judgment tasks, visual working memory tasks? Perhaps so, but at least this manuscript provides no such evidence. On line 291, it says "we have proposed that visual homogeneity can be used to solve any task that requires discriminating between homogeneous and heterogeneous displays". I think this is a good statement. A title that says "XXXX enable solving discrimination tasks with multi-component displays" is more acceptable. The phrase "generic tasks" is certainly an exaggeration.

(4) If I understand it correctly, one of the key findings of this paper is "the response times for target-present searches were positively correlated with visual homogeneity. By contrast, the response times for target-absent searches were negatively correlated with visual homogeneity" (lines 204-207). I think the authors have already acknowledged that the positive correlation is not surprising at all because it reflects the classic target-distractor similarity effect. But the authors claim that the negative correlations in target-absent searches is the true novel finding.

(5) I would like to make it clear that this negative correlation is not new either. The seminal paper by Duncan and Humphreys (1989) has clearly stated that "difficulty increases with increased similarity of targets to nontargets and decreased similarity between nontargets" (the sentence in their abstract). Here, "similarity between nontargets" is the same as the visual homogeneity defined here. Similar effects have been shown in Duncan (1989) and Nagy, Neriani, and Young (2005). See also the inconsistent results in Nagy& Thomas, 2003, Vicent, Baddeley, Troscianko&Gilchrist, 2009.<br /> More recently, Wei Ji Ma has systematically investigated the effects of heterogeneous distractors in visual search. I think the introduction part of Wei Ji Ma's paper (2020) provides a nice summary of this line of research.

I am surprised that these references are not mentioned at all in this manuscript (except Duncan and Humphreys, 1989).

(6) If the key contribution is the quantitative model, the study should be organized in a different way. Although the findings of positive and negative correlations are not novel, it is still good to propose new models to explain classic phenomena. I would like to mention the three studies by Wei Ji Ma (see below). In these studies, Bayesian observer models were established to account for trial-by-trial behavioral responses. These computational models can also account for the set-size effect, behavior in both localization and detection tasks. I see much more scientific rigor in their studies. Going back to the quantitative model in this paper, I am wondering whether the model can provide any qualitative prediction beyond the positive and negative correlations? Can the model make qualitative predictions that differ from those of Wei Ji's model? If not, can the authors show that the model can quantitatively better account for the data than existing Bayesian models? We should evaluate a model either qualitatively or quantitatively.

(7) In my opinion, one of the advantages of this study is the fMRI dataset, which is valuable because previous studies did not collect fMRI data. The key contribution may be the novel brain region associated with display heterogeneity. If this is the case, I would suggest using a more parametric way to measure this region. For example, one can use Gabor stimuli and systematically manipulate the variations of multiple Gabor stimuli, the same logic also applies to motion direction. If this study uses static Gabor, random dot motion, object images that span from low-level to high-level visual stimuli, and consistently shows that the stimulus heterogeneity is encoded in one brain region, I would say this finding is valuable. But this sounds like another experiment. In other words, it is insufficient to claim a new brain region given the current form of the manuscript.

REFERENCES<br /> - Duncan, J., & Humphreys, G. W. (1989). Visual search and stimulus similarity. Psychological Review, 96(3), 433-458. doi: 10.1037/0033-295x.96.3.433<br /> - Duncan, J. (1989). Boundary conditions on parallel processing in human vision. Perception, 18(4), 457-469. doi: 10.1068/p180457<br /> - Nagy, A. L., Neriani, K. E., & Young, T. L. (2005). Effects of target and distractor heterogeneity on search for a color target. Vision Research, 45(14), 1885-1899. doi: 10.1016/j.visres.2005.01.007<br /> - Nagy, A. L., & Thomas, G. (2003). Distractor heterogeneity, attention, and color in visual search. Vision Research, 43(14), 1541-1552. doi: 10.1016/s0042-6989(03)00234-7<br /> - Vincent, B., Baddeley, R., Troscianko, T., & Gilchrist, I. (2009). Optimal feature integration in visual search. Journal of Vision, 9(5), 15-15. doi: 10.1167/9.5.15<br /> - Singh, A., Mihali, A., Chou, W. C., & Ma, W. J. (2023). A Computational Approach to Search in Visual Working Memory.<br /> - Mihali, A., & Ma, W. J. (2020). The psychophysics of visual search with heterogeneous distractors. BioRxiv, 2020-08.<br /> - Calder-Travis, J., & Ma, W. J. (2020). Explaining the effects of distractor statistics in visual search. Journal of Vision, 20(13), 11-11.

Reviewer #2 (Public Review):

Summary:

Regalado et al. studied how an extended motivational state, necessary for maintaining behavioural drive despite unrewarding experiences, could be encoded in the ACC and its potential causal implications for learning discriminatory behaviour and avoiding unrewarding stimuli. They designed a self-initiated learning task and identified bulk neural responses tuned specifically to reward delivery as well as trial initiation. Interestingly, in both cases, neural activity precedes behavioural onset, indicating the encoding of a motivational signal. To investigate the neural encoding of motivational signals during unrewarded, distracting stimuli presentation, they created a discrimination task by introducing 'no reward' cues, during which animals need to learn not to reduce running speed and not engage in licking. Interestingly, with mice learning to increase running speed and reduce licking rates after 'no reward' cues, the preceding ACC activity also gradually increased. Importantly, only the increase in running speed after 'no reward' cues was impaired upon optogenetic inhibition of ACC activity during early training, linking the extended motivational signal in ACC and learning to maximise rewards by actively avoiding distracting and unrewarded stimuli. Such motivational signals could also be observed in OFC-ACC projecting neurons. Especially the continuous ramping of activity upon repeated 'non-reward' cues, which could be exclusively observed in the 'fast learner' subgroup, provides an interesting concept of how an extended motivational signal necessary for learning avoidance of unrewarded stimuli could be implemented in ACC. The shift in the temporal activity of initially reward-responsive neurons towards the preceding 'no reward' cue, provides a potential mechanism linking extended motivation to reward maximisation. This mechanism seems to be particularly important in periods of persistent 'non-reward' cues, as demonstrated in the impairment of running speed increase after two consecutive 'non-reward' cues.

Appraisal:

The authors provide convincing experimental evidence to support their claims of an extended motivational signal encoded in the ACC that is implemented by OFC-ACC signalling and critically involved in learning avoidance of unrewarded stimuli. The newly designed task seems appropriate to identify correlates of relevant cognitive and behavioural variables (e.g. sustained motivation). The combination of recording Ca2+ transients (bulk as well as longitudinal single neuron recordings) to identify potential neural responses and subsequent evaluation of their causal role in establishing and maintaining this persistent motivational state using opto- and pharmacogenetic manipulations is generally accepted.

Impact:

The findings will be valuable for further research on the impact of motivational states on behaviour and cognition. The authors provided a promising concept of how persistent motivational states could be maintained, as well as established a novel, reproducible task assay. While experimental methods used are currently state-of-the-art, theoretical analysis seems to be incomplete/not extensive.

Reviewer #2 (Public Review):

Summary:

The authors present a new model for animal pose estimation. The core feature they highlight is the model's stability compared to existing models in terms of keypoint drift. The authors test this model across a range of new and existing datasets. The authors also test the model with two mice in the same arena. For the single animal datasets the authors show a decrease in sudden jumps in keypoint detection and the number of undetected keypoints compared with DeepLabCut and SLEAP. Overall average accuracy, as measured by root mean squared error, generally shows similar but sometimes superior performance to DeepLabCut and better performance compared to SLEAP. The authors confusingly don't quantify the performance of pose estimation in the multi (two) animal case instead focusing on detecting individual identity. This multi-animal model is not compared with the model performance of the multi-animal mode of DeepLabCut or SLEAP.

Strengths:

The major strength of the paper is successfully demonstrating a model that is less likely to have incorrect large keypoint jumps compared to existing methods. As noted in the paper, this should lead to easier-to-interpret descriptions of pose and behavior to use in the context of a range of biological experimental workflows.

Weaknesses:

There are two main types of weaknesses in this paper. The first is a tendency to make unsubstantiated claims that suggest either model performance that is untested or misrepresents the presented data, or suggest excessively large gaps in current SOTA capabilities. One obvious example is in the abstract when the authors state ADPT "significantly outperforms the existing deep-learning methods, such as DeepLabCut, SLEAP, and DeepPoseKit." All tests in the rest of the paper, however, only discuss performance with DeepLabCut and SLEAP, not DeepPoseKit. At this point, there are many animal pose estimation models so it's fine they didn't compare against DeepPoseKit, but they shouldn't act like they did. Similar odd presentation of results are statements like "Our method exhibited an impressive prediction speed of 90{plus minus}4 frames per second (fps), faster than DeepLabCut (44{plus minus}2 fps) and equivalent to SLEAP (106{plus minus}4 fps)." Why is 90{plus minus}4 fps considered "equivalent to SLEAP (106{plus minus}4 fps)" and not slower? I agree they are similar but they are not the same. The paper's point of view of what is "equivalent" changes when describing how "On the single-fly dataset, ADPT excelled with an average mAP of 92.83%, surpassing both DeepLabCut and SLEAP (Figure 5B)" When one looks at Figure 5B, however, ADPT and DeepLabCut look identical. Beyond this, oddly only ADPT has uncertainty bars (no mention of what uncertainty is being quantified) and in fact, the bars overlap with the values corresponding to SLEAP and DeepPoseKit. In terms of making claims that seem to stretch the gaps in the current state of the field, the paper makes some seemingly odd and uncited statements like "Concerns about the safety of deep learning have largely limited the application of deep learning-based tools in behavioral analysis and slowed down the development of ethology" and "So far, deep learning pose estimation has not achieved the reliability of classical kinematic gait analysis" without specifying which classical gait analysis is being referred to. Certainly, existing tools like DeepLabCut and SLEAP are already widely cited and used for research.

The other main weakness in the paper is the validation of the multi-animal pose estimation. The core point of the paper is pose estimation and anti-drift performance and yet there is no validation of either of these things relating to multi-animal video. All that is quantified is the ability to track individual identity with a relatively limited dataset of 10 mice IDs with only two in the same arena (and see note about train and validation splits below). While individual tracking is an important task, that literature is not engaged with (i.e. papers like Walter and Couzin, eLife, 2021: https://doi.org/10.7554/eLife.64000) and the results in this paper aren't novel compared to that field's state of the art. On the other hand, while multi-animal pose estimation is also an important problem the paper doesn't engage with those results either. The two methods already used for comparison in the paper, SLEAP and DeepPoseKit, already have multi-animal modes and multi-animal annotated datasets but none of that is tested or engaged with in the paper. The paper notes many existing approaches are two-step methods, but, for practitioners, the difference is not enough to warrant a lack of comparison. The authors state that "The evaluation of our social tracking capability was performed by visualizing the predicted video data (see supplement Videos 3 and 4)." While the authors report success maintaining mouse ID, when one actually watches the key points in the video of the two mice (only a single minute was used for validation) the pose estimation is relatively poor with tails rarely being detected and many pose issues when the mice get close to each other.

Finally, particularly in the methods section, there were a number of places where what was actually done wasn't clear. For example in describing the network architecture, the authors say "Subsequently, network separately process these features in three branches, compute features at scale of one-fourth, one-eight and one-sixteenth, and generate one-eight scale features using convolution layer or deconvolution layer." Does only the one-eight branch have deconvolution or do the other branches also? Similarly, for the speed test, the authors say "Here we evaluate the inference speed of ADPT. We compared it with DeepLabCut and SLEAP on mouse videos at 1288 x 964 resolution", but in the methods section they say "The image inputs of ADPT were resized to a size that can be trained on the computer. For mouse images, it was reduced to half of the original size." Were different image sizes used for training and validation? Or Did ADPT not use 1288 x 964 resolution images as input which would obviously have major implications for the speed comparison? Similarly, for the individual ID experiments, the authors say "In this experiment, we used videos featuring different identified mice, allocating 80% of the data for model training and the remaining 20% for accuracy validation." Were frames from each video randomly assigned to the training or validation sets? Frames from the same video are very correlated (two frames could be just 1/30th of a second different from each other), and so if training and validation frames are interspersed with each other validation performance doesn't indicate much about performance on more realistic use cases (i.e. using models trained during the first part of an experiment to maintain ids throughout the rest of it.)

Reviewer #2 (Public Review):

Summary:

The paper from Li et al shows a mechanism by which axons can change direction during development. They use the sLNv neurons as a model. They find that the appearance of a new group of neurons (DNs) during post-embryonic proliferation secretes netrins and repels horizontally towards the midline, the axonal tip of the LNvs.

Strengths:

The experiments are well done and the results are conclusive.

Weaknesses:

The novelty of the study is overstated, and the background is understated. Both things need to be revised.

Reviewer #2 (Public Review):

Summary:

Rashid and colleagues demonstrate a novel hippocampal lateral septal circuit that is important for social recognition and drives the exploration of novel conspecifics. Their study spans from neural tracing to close-loop optogenetic experiments with clever controls and conditions to provide compelling evidence for their conclusion. They demonstrate that downstream of the hippocampal septal circuit, septal projections to the ventral tegmental area are necessary for general novelty discrimination. The study opens an avenue to study these circuits further to uncover the plasticity and synaptic mechanisms regulating social novelty preference.

Strengths:

Chemogenetic and optogenetic experiments have excellent behavioral controls. The synaptic tracing provides important information that informs the narrative of experiments presented and invites future studies to investigate the effects of septal input on dopaminergic activity.

Weaknesses:

There are unclear methodological important details for circuit manipulation experiments and analyses where multiple measures are needed but missing. Based on the legends, the chemogenetic experiment is done in a within-animal design. That is the same mouse receives SAL and CNO. However, the data is not presented in a within-animal manner such that we can distinguish if the behavior of the same animal changes with drug treatment. Similarly, the methods specify that the optogenetic manipulations were done in three different conditions, but the analyses do not report within-animal changes across conditions nor account for multiple measures within subjects. Finally, it is unclear if the order of drug treatment and conditions were counterbalanced across subjects.

Reviewer #2 (Public Review):

Summary:

In this work, the authors show that GABAergic neurons play a role in sensing mitochondrial stress and regulating organismal aging. Thus, disrupting the mitochondrial mitochondria function in GABAergic neurons induces resistance to thermal and paraquat stresses, promotes longevity, and affects reproduction. This mechanism is regulated by the iron-sulfur subunit of complex III of the mitochondrial electron transport chain, ISP-1, and a mitochondrial quality control m-AAA protease, SPG-7, which in turn requires DAF-16/FoxO activity in GABAergic neurons.

Strengths:

A strength of this work is that the authors identify the specific site where mitochondrial stress promotes health and longevity, i.e., GABAergic neurons. In addition, the paper corroborates the findings with the appropriate experiments. How neuronal regulation of mitochondrial function impacts systemic health and aging is of interest to cell biology and neuroscience fields.

Weaknesses:

The entire paper is based on tissue-specific RNAi in GABAergic neurons, which was achieved using two different conditions of RNAi (although not for all experiments). However, multiple studies have shown deficiencies in the tissue-specific RNAi in C. elegans, especially for the rde-1(ne219) mutant used in this study. Therefore, it is necessary to repeat critical experiments by rescuing the isp-1 or spg-7 mutants in GABAergic neurons. Additionally, it is clear in the paper that perturbing mitochondrial function requires DAF-16/FoxO activity in GABAergic neurons to promote longevity, yet the downstream cellular pathways are not described.

Reviewer #2 (Public Review):

Summary:

In the manuscript, Xiang Mou and Daoyun JI investigate how ACC neurons activated by observational learning communicate with the hippocampus. They assess this line of communication through a complex behavioral technique, in vivo electrophysiology, pharmacological approaches, and data analytical techniques. Firstly, the authors find that observational performance is dependent on the ACC, and that the ACC possesses neurons that show side selectivity (trajectory-related) in both the observation box when shuttling to reward, and during subsequent maze running, shuttling to the corresponding same side for reward. The side-selective activation appears stronger for correct trials compared to error trials specifically during observation of Demo rats. They compare how the CA1 of the hippocampus encodes these two environments and find that ACC side-selective neurons show a correlation with side-selective CA1 ensembles during maze behavior, water consumption, and sharp-wave ripples.

Strengths:

Overall, the paper provides strong evidence that ACC neurons are activated by observational learning and that this activation seems to be correlated with CA1 activity.

Weaknesses:

Concerns, however, surround the strength of evidence that links ACC and CA1 activity during observational learning. Only weak correlations between the two regions are shown, and it is unclear if the ACC may lead to CA1 activity or vice versa. It is possible that these processes reflect two parallel pathways. Without manipulation of ACC, it is difficult to assess whether ACC activity influences hippocampal replay.

Reviewer #2 (Public Review):

Summary:

In this study, the authors aim to identify the nuclear genome-encoded transcription factors that regulate mtDNA maintenance and mitochondrial biogenesis. They started with an RNAi screening in developing Drosophila eyes with reduced mtDNA content and identified a number of putative candidate genes. Subsequently, using ChIP-seq data, they built a potential regulatory network that could govern mitochondrial biogenesis. Next, they focused on a candidate gene, CG1603, for further characterization. Based on the expression of different markers, such as TFAM and SDHA, in the RNAi and OE clones in the midgut cells, they argue that CG1603 promotes mitochondrial biogenesis and the expression of ETC complex genes. Then, they used a mutant of CG1603 and showed that both mtDNA levels and mitochondrial protein levels were reduced. Using clonal analyses, they further show a reduction in mitochondrial biogenesis and membrane potential upon loss of CG1603. They made a reporter line of CG1603, showed that the protein is localized to the mitochondria, and binds to polytene chromosomes in the salivary gland. Based on the RNA-seq results from the mutants and the ChIP data, the authors argue that the nucleus-encoded mitochondrial genes that are downregulated >2 folds in the CG1603 mutants and that are bound by CG1603 are related to ETC biogenesis. Finally, they show that YL-1, another candidate in the network, is an upstream regulator of CG1603.

Strengths:

This is a valuable study, which identifies a potential regulator and a network of nucleus-encoded transcription factors that regulate mitochondrial biogenesis. Through in-vivo and in-vitro experimental evidence, the authors identify the role of CG1603 in this process. The screening strategy was smart, and the follow-up experiments were nicely executed.

Weaknesses:

Some additional experiments showing the effects of CG1603 loss on ETC integrity and functionality would strengthen the work.

Reviewer #2 (Public Review):

The study by Deganutti and co-workers is a methodological report on an adaptive sampling approach, multiple walker supervised molecular dynamics (mwSuMD), which represents an improved version of the previous SuMD.

Case-studies concern complex conformational transitions in a number of G protein Coupled Receptors (GPCRs) involving long time-scale motions such as binding-unbinding and collective motions of domains or portions. GPCRs are specialized GEFs (guanine nucleotide exchange factors) of heterotrimeric Gα proteins of the Ras GTPase superfamily. They constitute the largest superfamily of membrane proteins and are of central biomedical relevance as privileged targets of currently marketed drugs.

MwSuMD was exploited to address:<br /> (1) Binding and unbinding of the arginine-vasopressin (AVP) cyclic peptide agonist to the V2 vasopressin receptor (V2R);<br /> (2) Molecular recognition of the β2-adrenergic receptor (β2-AR) and heterotrimeric GDP-bound Gs protein;<br /> (3) Molecular recognition of the A1-adenosine receptor (A1R) and palmitoylated and geranylgeranylated membrane-anchored heterotrimeric GDP-bound Gi protein;<br /> (4) The whole process of GDP release from membrane-anchored heterotrimeric Gs following interaction with the glucagon-like peptide 1 receptor (GLP1R), converted to the active state following interaction with the orthosteric non-peptide agonist danuglipron;<br /> (5) The heterodimerization of D2 dopamine and A2A adenosine receptors (D2R and A2AR, respectively) and binding to a bi-valent ligand.

The mwSuMD method is solid and valuable, has wide applicability, and is compatible with the most world-widely used MD engines. It may be of interest to the computational structural biology community.

The huge amount of high-resolution data on GPCRs makes those systems suitable, although challenging, for method validation and development.

While the approach is less energy-biased than other enhanced sampling methods, knowledge, at the atomic detail, of binding sites/interfaces and conformational states is needed to define the supervised metrics, the higher the resolution of such metrics is the more accurate the outcome is expected to be. The definition of the metrics is a user- and system-dependent process.

The too many and ambitious case-studies undermine the accuracy of the output and reduce the important details needed for a methodological report. In some cases, the available CryoEM structures could have been exploited better.

The most consistent example concerns AVP binding/unbinding to V2R. The consistency with CryoEM data decreases with an increase in the complexity of the simulated process and involved molecular systems (e.g. receptor recognition by membrane-anchored G protein and the process of nucleotide exchange starting from agonist recognition by an inactive-state receptor). The last example, GPCR hetero-dimerization, and binding to a bi-valent ligand, is the most speculative one as it does not rely on high-resolution structural data for metrics supervision.

Reviewer #2 (Public Review):

Summary:

The current article presents a new type of analytical approach to the sequential organisation of whale coda units.

Strengths:

The detailed description of the internal temporal structure of whale codas is something that has been thus far lacking.

Weaknesses:

It is unclear how the insight gained from these analyses differs or adds to the voluminous available literature on how codas varies between whale groups and populations. It provides new details, but what new aspects have been learned, or what features of variation seem to be only revealed by this new approach?<br /> The theoretical basis and concepts of the paper are problematical and indeed, hamper potentially the insights into whale communication that the methods could offer. Some aspects of the results are also overstated.

Reviewer #2 (Public Review):

Summary:

The manuscript by Djebar et al investigated the role and the underlying mechanism of the ciliary transition zone protein Rpgrip1l in zebrafish spinal alignment. They showed that rpgrip1l mutant zebrafish develop a nearly full penetrance of body curvature at juvenile stages. The mutant fish have cilia defects associated with ventricular dilations and loss of the Reissner fibers. Scoliosis onset and progression are also strongly associated with astrogliosis and neuroinflammation, and anti-inflammatory drug treatment prevents scoliosis in mutant zebrafish, suggesting a novel pathogenic mechanism for human idiopathic scoliosis. This study is quite comprehensive with high-quality data, and the manuscript is well written, providing important information on how the ciliary transition zone protein functions in maintaining the zebrafish body axis straightness.

Strengths:

Very clear and comprehensive analysis of the mutant zebrafish.

Weaknesses:

(1) In Figures 1D-G, magnified high-resolution pictures are required to show there are indeed no vertebral malformations.

(2) Are the transcriptome data and proteomic data consistent? Consistent targets in both analyses should be highlighted.

(3) What is the role of Anxa2 in neuroinflammation? Is increased Anxa2 expression in rpgrip1l mutant zebrafish reduced after anti-inflammatory drug treatment? What is the expression level of anxa2 in cep290 mutant zebrafish?

(4) More background about Rpgrip1l should be provided in the introduction, particularly the past studies of the mammalian homolog of Rpgrip11, if there are any.

(5) Is there any human disease associated with Rpgrip1l? Do these patients have scoliosis phenotype?

(6) A summary diagram at the end would be helpful for understanding the main findings.

Reviewer #2 (Public Review):

Summary:

The authors provide compelling evidence that stimulation of epidermal cells in Drosophila larvae results in the stimulation of sensory neurons that evoke a variety of behavioral responses. Further, the authors demonstrate that epidermal cells are inherently mechanoresponsive and implicate a role for store-operated calcium entry (mediated by Stim and Orai) in the communication to sensory neurons.

Strengths:

The study represents a significant advance in our understanding of mechanosensation. Multiple strengths are noted. First, the genetic analyses presented in the paper are thorough with appropriate consideration to potential confounds. Second, behavioral studies are complemented by sophisticated optogenetics and imaging studies. Third, identification of roles for store-operated calcium entry is intriguing. Lastly, conservation of these pathways in vertebrates raise the possibility that the described axis is also functional in vertebrates.

Weaknesses:

The study has a few conceptual weaknesses that are arguably minor. The involvement of store-operated calcium entry implicates ER calcium store release. Whether mechanical stimulation evokes ER calcium release in epidermal cells and how this might come about (e.g., which ER calcium channels, roles for calcium-induced calcium release etc.) remains unaddressed. On a related note, the kinetics of store-operated calcium entry is very distinct from that required for SV release. The link between SOC and epidermal cells-neuron transmission is not reconciled. Finally, it is not clear how optogenetic stimulation of epidermal cells results in the activation of SOC.

Reviewer #2 (Public Review):

The manuscript titled, "Identification of a Musashi2 translocation as a novel oncogene in myeloid leukemia" by Spinler et al. studies the functional role of the translocation t(7;17)(p15;q23), resulting in MSI2/HOXA9 fusion gene, as a secondary driver in bcCML. MSI2-HOXA9 forced expression along with BCR-ABL enhances colony formation and leads to a more aggressive disease in vivo. Depletion of the RNA binding domain RRM1 or RRM2 of MSI2 led to a significant reduction in colony formation, with RRM1 depletion specifically impacting differentiation and blast cell counts. Mechanistically, the authors find that MSI2-HOXA9 aberrantly localizes to the nucleus, elevating the expression of mitochondrial polymerase Polrmt, thereby leading to upregulation of mitochondrial components and enhancing mitochondrial function and basal respiration. Overall, this study examines how the rare MSI2-HOXA9 fusion gene can act as a novel cooperating oncogene and could serve as a secondary hit in the progression of CML to blast crisis.

Strengths:

(1) Demonstration that MSI2-HOXA9 contributes to oncogenesis in the BCR-ABL context.

(2) Development of a novel cooperativity model for BCR-ABL and provides additional supporting data for the role of MSI2 in leukemogenesis.

(3) Evidence that MSI2-HOXA9 acts uniquely compared to MSI2 alone through nuclear vs. cytoplasmic localization and activation of mitochondrial polymerase Polrmt.

Weaknesses:

(1) MSI2-HOXA9 fusion is extremely rare as it has been only found in a handful of patients and it is not clear whether other MSI2 fusions function in a similar manner.

(2) The mechanism needs to be strengthened since MSI2 alone or the HOXA9 mutant may not be linked to the mitochondrial mechanism.

(3) It is not clear that the mitochondrial pathway is sufficient for the MSI2-HOXA9 oncogenic mechanism.

Reviewer #2 (Public Review):

Through RNA analysis, Xie et al found LncRNA Snhg3 was one of the most down-regulated Snhgs by a 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 was reduced, while Sngh3 over-expression potentiated fatty liver in mice on an 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. There are, however, several potential issues to consider before jumping to a conclusion.

(1) First of all, it's important to ensure the robustness and rigor of each study. The manuscript was not carefully put together. The image qualities for several figures were poor, making it difficult for the readers to evaluate the results with confidence. The biological replicates and numbers of experimental repeats for cell-based assays were not described. When possible, the entire immunoblot imaging used for quantification should be presented (rather than showing n=1 representative). There were multiple mislabels in figure panels or figure legends (e.g., Figure 2I, Figure 2K, and Figure 3K). The b-actin immunoblot image was reused in Figure 4J, Figure 5G, and Figure 7B with different exposure times. These might be from the same cohort of mice. If the immunoblots were run at different times, the loading control should be included on the same blot as well.

(2) The authors can do a better job in explaining the logic for how they came up with the potential function of each component of the signaling cascade. Sngh3 is down-regulated by HFD. However, the evidence presented indicates its involvement in promoting steatosis. In Figure 1C, one would expect PPARg expression to be up-regulated (when Sngh3 was down-regulated). If so, the physiological observation conflicts with the proposed mechanism. In addition, SND1 is known to regulate RNA/miRNA processing. How do the authors rule out this potential mechanism? How about the hosting snoRNA, Snord17? Does it involve the progression of NASLD?

(3) The role of PPARg in fatty liver diseases might be a rodent-specific phenomenon. PPARg agonist treatment in humans may actually reduce ectopic fat deposition by increasing fat storage in adipose tissues. The relevance of the findings to human diseases should be discussed.

Reviewer #2 (Public Review):

Summary:

The authors provide an interesting observation that ER-targeted excess misfolded proteins localize to the nucleus within membrane-entrapped vesicles for further quality control during cell division. This is useful information indicating transient nuclear compartmentalization as a quality control strategy for misfolded ER proteins in mitotic cells, although endogenous substrates of this pathway are yet to be identified.

Strengths:

This microscopy-based study reports unique membrane-based compartments of ER-targeted misfolded proteins within the nucleus. Quarantining aggregating proteins in membrane-less compartments is a widely accepted protein quality control mechanism. This work highlights the importance of membrane-bound quarantining strategies for aggregating proteins. These observations open up multiple questions on proteostasis biology. How do these membrane-bound bodies enter the nucleus? How are the single-layer membranes formed? How exactly are these membrane-bound aggregates degraded? Are similar membrane-bound nuclear deposits present in post-mitotic cells that are relevant in age-related proteostasis diseases? Etc. Thus, the observations reported here are potentially interesting.

Weaknesses:

This study, like many other studies, used a set of model misfolding-prone proteins to uncover the interesting nuclear-compartment-based quality control of ER proteins. The endogenous ER-proteins that reach a similar stage of overdose of misfolding during ER stress remain unknown.

The mechanism of disaggregation of membrane-trapped misfolded proteins is unclear. Do these come out of the membrane traps? The authors report a few vesicles in living cells. This may suggest that membrane-untrapped proteins are disaggregated while trapped proteins remain aggregates within membranes.

The authors figure out the involvement of proteasome and Hsp70 during the disaggregation process. However, the detailed mechanisms including the ubiquitin ligases are not identified. Also, is the protein ubiquitinated at this stage?

This paper suffers from a lack of cellular biochemistry. Western blots confirming the solubility and insolubility of the misfolded proteins are required. This will also help to calculate the specific activity of luciferase more accurately than estimating the fluorescence intensities of soluble and aggregated/compartmentalized proteins. Microscopy suggested the dissolution of the membrane-based compartments and probably disaggregation of the protein. This data should be substantiated using Western blots. Degradation can only be confirmed by Western blots. The authors should try time course experiments to correlate with microscopy data. Cycloheximide chase experiments will be useful.

The cell models express the ER-targeted misfolded proteins constitutively that may already reprogram the proteostasis. The authors may try one experiment with inducible overexpression.

It is clear that a saturating dose of ER-targeted misfolded proteins activates the pathway. The authors performed a few RT-PCR experiments to indicate the proteostasis-sensitivity. Proteome-based experiments will be better to substantiate proteostasis saturation.

The authors should immunostain the nuclear compartments for other ER-membrane resident proteins that span either the bilayer or a single layer. The data may be discussed.

All microscopy figures should include control cells with similarly aggregating proteins or without aggregates as appropriate. For example, is the nuclear-targeted FlucDM-EGFP similarly entrapped? A control experiment will be interesting. Expression of control proteins should be estimated by western blots.

There are few more points that may be out of the scope of the manuscript. For example, how do these compartments enter the nucleus? Whether similar entry mechanisms/events are ever reported? What do the authors speculate? Also, the bilayer membrane becomes a single layer. This is potentially interesting and should be discussed with probable mechanisms. Also, do these nuclear compartments interfere with transcription and thereby deregulate cell division? What about post-mitotic cells? Similar deposits may be potentially toxic in the absence of cell division. All these may be discussed.

Reviewer #2 (Public Review):

This important work will be of interest to centriole and cilia cell biologists. It describes in detail how microtubules control multiple aspects of centriole amplification in brain multiciliated cells. This study provides a greater time-resolved and molecular proteomic mapping of the different steps involved, with or without microtubule disruption. Boudjema et al. show that microtubules are important throughout the centriole amplification process, from the early stages, where the procentrioles emerge from a pericentriolar "nest", through the growth stage where microtubules maintain the perinuclear localisation, to the detachment stage, where microtubules assist in perinuclear disengagement and apical migration. The results are generally well supported by the evidence, but the manuscript would benefit significantly from some heavy editing to introduce more niche terms, standardize abbreviations in text, and labels on figures to help bring the readers, especially non-specialists, along with them - increasing the accessibility of their work.

Reviewer #2 (Public Review):

Summary:

In this work, the authors investigate the role of the Superoxide disumutase 1 (Sod1) enzyme, which acts to reduce the reactive oxygen species load, in the Drosophila testis. They show that the knockdown of Sod1 in somatic cells impacts stem cell numbers both autonomously in the soma and non-autonomously in the germline. Somatic stem cell numbers are increased, while germline stem cells are decreased and differentiate prematurely. The authors then show that in somatic Sod1 knockdowns, several signalling pathways are disrupted and that these may be responsible at least in part for the phenotypes observed. Finally, over-expression of Sod1 in the soma results in opposite phenotypes, suggesting that ROS levels do play a role in maintaining the balance between both stem cell populations in the testis.

Strengths:

The main strength of this work is to show a previously unappreciated role for Sod1 (and presumably by extension of ROS) in the Drosophila testis and in the regulation of stem cell self-renewal and differentiation. The authors use multiple readouts to show that the knockdown of Sod1 in the soma increases the number of somatic cells and also drives a non-autonomous, premature differentiation of germ cells. They also quantify the early differentiation of the germline using two different methods. Importantly, overexpression of Sod1 produces opposite phenotypes to knockdown, strengthening the conclusions.

Weaknesses:

Although the data presented are interesting, an important weakness of the manuscript as it currently stands is that many statements are not fully supported by the data. In particular, the authors do not provide any evidence of "cell redox-pairs" as indicated in the manuscript title, nor of intercellular redox gradients, as stated in several places throughout. While the data are consistent with non-autonomous regulation of ROS levels, this would not constitute a gradient. However, and crucially, there is no evidence provided to show that Sod1 manipulation in the soma is affecting ROS levels in the germline and that any of the phenotypes observed are a consequence of elevated ROS in the germline, rather than indirect effects caused by dysregulation of somatic self-renewal and differentiation, which is known to impact the germline. Indeed, there are many published reports of autonomous manipulations in the soma that influence either germline stem cell number (eg PMID: 19797664 among others) or differentiation (eg PMID: 17629483). The latter example is particularly relevant as the authors show altered somatic ERK levels, and the role of somatic ERK in promoting germ cell development is well established (PMID: 11048722, 11048723,...). Thus, whether Sod1 plays any non-autonomous role in controlling germ cell fate through ROS in the germline directly, or whether the phenotypes observed can all be explained by autonomous effects on somatic cell behaviour is debatable, but the experiments presented here do not distinguish between these two hypotheses. The only evidence presented by the authors for a non-autonomous role of Sod1 is the expression of a GFP reporter for gstD1. The quantifications and images are not clear and do not show unambiguously that this reporter is expressed in germ cells. Indeed, the quantifications show overlap between somatic and germline markers, suggesting that either the images themselves or the way they are quantified does not allow the authors to distinguish between the two cell types. Similarly, the claim that somatic mitochondria are enriched at the CySC-GSC interface and that this distribution maintains the redox balance in the niche is not supported by any experimental data. CySCs are extremely thin cells and much of the space is occupied by the nucleus (PMID: 114676), therefore it is likely that mitochondria would be enriched at the periphery. A careful analysis would be necessary to show that this enrichment is specific to the interface with GSCs. Moreover, no experiments are conducted to test whether mitochondrial distribution in CySCs has any impact on GSCs. Finally, no experiments are conducted to show definitively that the phenotypes observed upon Sod1 knockdown are indeed due to increased ROS, while this claim is made several times in the text. At present, the data presented here can support a role for Sod1 in somatic CySCs, but much more caution is required in attributing this to either ROS or intercellular ROS signaling. Therefore, several claims made in the title and throughout the text are not supported by evidence.

Besides this central point, there are other areas that should be improved. In particular, the data using the Fucci reporter to show accelerated proliferation do not appear convincing. It would seem that the proportions of cells in each phase are roughly similar, just that there are more cycling cells. A careful analysis of these results would distinguish between these two and determine whether Sod1 knockdown simply impairs differentiation (and therefore results in more somatic cells proliferating) or whether it speeds up the cell cycle (resulting in an increased mitotic index as suggested, but this requires a ratio to be shown). Similarly, several quantifications are not clearly explained, making it hard to understand what is being measured. As an example, while the decrease in pERK in CySCs is clear from the image and matched in the quantification, the increase in cyst cells is not apparent from the fire LUT used. The change in fluorescence intensity therefore may be that more cells have active ERK, rather than an increase per cell (similar arguments apply to the quantifications for p4E-BP or Ptc). Therefore, it is hard to know whether Sod1 knockdown results in increased or decreased signaling in individual cells.

Impact of study:

Demonstrating intercellular communication through ROS and its importance in maintaining the balance between two stem cell populations would be a finding of interest to a broad field. However, it remains to be demonstrated that this is the case, and given this, this study will have a limited impact.

Reviewer #2 (Public Review):

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

The results show that HMMR is distributed as puncta on neurons using standard immunofluorescence and PLA. Depletion of HMMR reduced neurite length and extent of branching; reduced post-translational acetylation of neurite microtubules. Conversely, overexpression of HMMR increased resistance to nocodazole. The parameters of microtubule dynamics were also impacted by reduction or overexpression of HMMR. The authors discuss the possibility HMMR regulates neurite morphological changes via regulation of microtubule nucleation and dynamics.

Reviewer #3 (Public Review):

Summary:

The authors aim to demonstrate the effectiveness of their developed methodology, which utilizes super-resolution microscopy and single-molecule tracking in live cells on a high-throughput scale. Their study focuses on measuring the diffusion state of a molecule target, the estrogen receptor, in both ligand-bound and unbound forms in live cells. By showcasing the ability to screen 5067 compounds and measure the diffusive state of the estrogen receptor for each compound in live cells, they illustrate the capability and power of their methodology.

Readers are well introduced to the principles in the initial stages of the manuscript with highly convincing video examples. The methods and metrics used (fbound) are robust. The authors demonstrate high reproducibility of their screening method (R2=0.92). They also showcase the great sensitivity of their method in predicting the proliferation/viability state of cells (R2=0.84). The outcome of the screen is sound, with multiple compounds clustering identified in line with known estrogen receptor biology.

Reviewer #2 (Public Review):

The manuscript submitted by Sujeethkumar et al. describes an alternative approach to skeletal tissue repair using extracellular matrix (ECM) deposited by genetically modified mesenchymal stromal/stem cells. Here, they generate a loss of function mutations in VEGF or RUNX2 in a BMP2-overexpressing MSC line and define the differences in the resulting tissue-engineered constructs following seeding onto a type I collagen matrix in vitro, and following lyophilization and subcutaneous and orthotopic implantation into mice and rats. Some strengths of this manuscript are the establishment of a platform by which modifications in cell-derived ECM can be evaluated both in vitro and in vivo, the demonstration that genetic modification of cells results in complexity of in vitro cell-derived ECM that elicits quantifiable results, and the admirable goal to improve endogenous cartilage repair. However, I recommend the authors clarify their conclusions and add more information regarding reproducibility, which was one limitation of primary-cell-derived ECMs.

Overcoming the limitations of native/autologous/allogeneic ECMs such as complete decellularization and reduction of batch-to-batch variability was not specifically addressed in the data provided herein. For the maintenance of ECM organization and complexity following lyophilization, evidence of complete decellularization was not addressed, but could be easily evaluated using polarized light microscopy and quantification of human DNA for example in constructs pre and post-lyophilization. It would be ideal to see minimization of batch-to-batch variability using this approach, as mitigation of using a sole cell line is likely not sufficient (considering that the sole cell line-derived Matrigel does exhibit batch-to-batch and manufacturer-to-manufacturer variability).

I recommend adding details regarding experimental design and outcomes not initially considered. Inter- and intra-experimental reproducibility was not adequately addressed. The size of in vitro-derived cartilage pellets was not quantified, and it is not clear that more than one independent 'differentiation' was performed from each gene-edited MSC line to generate in vitro replicates and constructs that were implanted in vivo.

The use of descriptive language in describing conclusions may mislead the reader and should be modified accordingly throughout the manuscript. For example, although this reviewer agrees with the comparative statements made by the authors regarding parental and gene-edited MSC lines, non-quantifiable terms such as 'frank' 'superior' (example, line 242) are inappropriate and should rather be discussed in terms of significance. Another example is 'rich-collagenous matrix,' which was not substantiated by uniform immunostaining for type II collagen (line 189).

I have similar recommendations regarding conclusive statements from the rat implantation model, which was appropriately used for the purpose of evaluating the response of native skeletal cells to the different cell-derived ECMs. Interpretations of these results should be described with more accuracy. For example, increased TRAP staining does not indicate reduced active bone formation (line 237). Many would not conclude that GAGs were retained in the RUNX2-KO line graft subchondral region based on the histology. Quantification of % chondral regeneration using histology is not accurate as it is greatly influenced by the location in the defect from which the section was taken. Chondral regeneration is usually semi-quantified from gross observations of the cartilage surface immediately following excision. The statements regarding integration (example line 290) are not founded by histological evidence, which should show high magnification of the periphery of the graft adjacent to the native tissue.

Reviewer #2 (Public Review):

Summary:

The authors investigated the effects of the timing of dietary occasions on weight loss and well-being with the aim of explaining if a consistent, timely alignment of dietary occasions throughout the days of the week could improve weight management and overall well-being. The authors attributed these outcomes to a timely alignment of dietary occasions with the body's own circadian rhythms. However, the only evidence the authors provided for this hypothesis is the assumption that the individual timing of dietary occasions of the study participants identified before the intervention reflects the body's own circadian rhythms. This concept is rooted in understanding of dietary cues as a zeitgeber for the circadian system, potentially leading to more efficient energy use and weight management. Furthermore, the primary outcome, body weight loss, was self-reported by the study participants.

Strengths:

The innovative focus of the study on the timing of dietary occasions rather than daily energy intake or diet composition presents a fresh perspective in dietary intervention research. The feasibility of the diet plan, developed based on individual profiles of the timing of dietary occasions identified before the intervention, marks a significant step towards personalised nutrition.

Weaknesses:

Several methodological issues detract from the study's credibility, including unclear definitions not widely recognized in nutrition or dietetics (e.g., "caloric event"), lack of comprehensive data on body composition, and potential confounders not accounted for (e.g., age range, menstrual cycle, shift work, unmatched cohorts, inclusion of individuals with normal weight, overweight, and obesity). The primary outcome's reliance on self-reported body weight and subsequent measurement biases further undermines the reliability of the findings. Additionally, the absence of registration in clinical trial registries, such as the EU Clinical Trials Register or clinicaltrials.gov, and the multiple testing of hypotheses which were not listed a priori in the research protocol published on the German Register of Clinical Trials impede the study's transparency and reproducibility.

Achievement of Objectives and Support for Conclusions:

The study's objectives were partially met; however, the interpretation of the effects of meal timing on weight loss is compromised by the weaknesses mentioned above. The evidence only partially supports some of the claims due to methodological flaws and unstructured data analysis.

Impact and Utility:

Despite its innovative approach, significant methodological and analytical shortcomings limit the study's utility. If these issues were addressed, the research could have meaningful implications for dietary interventions and metabolic research. The concept of timing of dietary occasions in sync with circadian rhythms holds promise but requires further rigorous investigation.

Reviewer #2 (Public Review):

Summary:

In this manuscript the authors have generated a single-cell atlas of the miracidium, the first free-living stage of an important human parasite, Schistosoma mansoni. Miracidia develop from eggs produced in the mammalian (human) host and are released into freshwater, where they can infect the parasite's intermediate snail host to continue the life cycle. This study adds to the growing single-cell resources that have already been generated for other life-cycle stages and, thus, provides a useful resource for the field.

Strengths:

Beyond generating lists of genes that are differentially expressed in different cell types, the authors validated many of the cluster-defining genes using in situ hybridization chain reaction. In addition to providing the field with markers for many of the cell types in the parasite at this stage, the authors use these markers to count the total number of various cell types in the organism. Because the authors realized that their cell isolation protocols were biasing the cell types they were sequencing, they applied a second method to help them recover additional cell types.

Schistosomes have ZW sex chromosomes and the authors make the interesting observation that the stem cells at this stage are already expressing sex (i.e. W)-specific genes.

Weaknesses:

The sample sizes upon which the in situ hybridization results and cell counts are based are either not stated (in most cases) or are very small (n=3). This lack of clarity about biological replicates and sample sizes makes it difficult for the reader to assess the robustness of the results and the extremely small sample sizes (when provided) are a missed opportunity to explore the variability of the system, or lack thereof.

Although assigning transcripts to a given cell type is usually straightforward via in situ experiments, the authors fail to consider the potential difficulty of assigning the appropriate nuclei to cells with long cytoplasmic extensions, like neurons. In the absence of multiple markers and a better understanding of the nervous system, it seems likely that the authors have overestimated the number of neurons and misassigned other cell types based on their proximity to neural projections.

The conclusion that germline genes are expressed in the miracidia stem cells seems greatly overstated in the absence of any follow-up validation. The expression scales for genes like eled and boule are more than 3 orders of magnitude smaller than those used for any of the robustly expressed genes presented throughout the paper. These scales are undefined, so it isn't entirely clear what they represent, but neither of these genes is detected at levels remotely high (or statistically significant) enough to survive filters for cluster-defining genes. Given that germ cells often develop early in embryogenesis and arrest the cell cycle until later in development, and that these transcripts reveal no unspliced forms, it seems plausible that the authors are detecting some maternally supplied transcripts that have yet to be completely degraded.

Reviewer #2 (Public Review):

Immunogenic cell death (ICD) can lead to the release of factors such as DAMPs which promote an adaptive immune response. In the context of cancer, there is clear evidence of anti-tumour benefits as a result of ICD, perhaps induced by chemotherapy.

Lilong et al used TCGA data to explore whether a previously published 34 gene 'ICD-related' signature could stratify bladder cancer patients by prognosis and ultimately predict patient survival. The gene signature contains many genes involved in inflammation and immunity (IFNg, IL6, TNF, IL17A, TLR4, CD8B, etc) and those related to ICD (such as CALR, HMGB1, HSP, NLRP3, etc). The authors divide patients into 'ICD-high' and '-low' based on the expression of this gene set and find that 'ICD-high' is associated with longer survival in TCGA bladder cancer data. The authors further argue that ICD-high group responds better to PD1 therapies. From this 34-gene signature, it appears that LASSO regularisation and Cox analysis identifies a four-gene 'risk' signature (CALR, IL1R1, IFNB1, IFNG) which is associated with shorter patient survival and lower immunotherapy response rates. This is the primary finding. Their methodology is very similar to a publication in 2021 in Frontiers in Immunology instead in the context of head and neck squamous cell carcinoma. This paper is not referenced.

In terms of the strengths of the work, it is certainly plausible that the author's four gene signature has an association with survival in bladder cancer, at least based on the two datasets studied. However, the relatedness of their findings to ICD is unconvincing, and glaring omissions from the manuscript in terms of methods limit confidence in the work. The authors show a potential association with bladder cancer patient survival and their four gene signatures, but substantial revisions are required for this to be appropriately evidenced.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Ghazi et reported that inhibition of KRASG12C signaling increases autophagy in KRASG12C-expressing lung cancer cells. Moreover, the combination of DCC 3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C-driven lung cancer cell proliferation in vitro and tumor growth in vivo. Additionally, in genetically engineered mouse models of KRASG12C-driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Additionally, this study found that LKB1 deficiency diminishes the sensitivity of KRASG12C/LKB1Null-driven lung cancer to the combination treatment, perhaps through the emergence of mixed adeno/squamous cell carcinomas and mucinous adenocarcinomas.

Strengths:

Both human cancer cells and mouse models were employed in this study to illustrate that inhibiting ULK1/2 could enhance the responsiveness of KRASG12C lung cancer to sotorasib. This research holds translational importance.

Weaknesses:

Additional validation of certain data is necessary.

(1) mCherry-EGFP-LC3 reporter was used to assess autophagy flux in Figure 1A. Please explain how autophagy status (high, medium, and low) was defined. It's also suggested to show WB of LC3 processing in different treatments as in Figure 1A at 48 hours.

(2) For Figures 1J, K, and L, please provide immunohistochemistry (IHC) images demonstrating RAS downstream signaling blockade by sotorasib and autophagy blockade by DCC 3116 in tumors.

(3) Given that both DCC 3116 and ULK1K46N exhibit the ability to inhibit autophagy and synergize with sotorasib in inhibiting cell proliferation, in addition to demonstrating decreased levels of pATG13 via ELISA assay, please include Western blot analyses of LC3 or p62 to confirm the blockade of autophagy by DCC 3116 and ULK1K46N in Figure 1 & Figure 2.

(4) Since adenocarcinomas, adenosquamous carcinomas (ASC), and mucinous adenocarcinomas were detected in KL lung tumors, please conduct immunohistochemistry (IHC) to detect these tumors, including markers such as p63, SOX2, Katrine 5.

(5) Please provide the sample size (n) for each treatment group in the survival study (Figure 4E). It appears that all mice were sacrificed for tumor burden analysis in Figure 4F. However, there doesn't seem to be a significant difference among the treatment groups in Figure 4F, which contrasts with the survival analysis in Figure 4E. It is suggested to increase the sample size in each treatment group to reduce variation.

(6) In KP mice (Figure 5), it seems that a single treatment alone is sufficient to inhibit established KP lung tumor growth. Combination treatment does not further enhance anti-tumor efficacy. Therefore, this result doesn't support the conclusion generated from human cancer cell lines. Please discuss.

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Reviewer #2 (Public Review):

Summary:

Bates TA. et al. studied the biochemical characteristics of ESAT-6, a major virulence factor of Mycobacterium tuberculosis (Mtb), as part of the heterodimer with CFP10, a molecular chaperon of ESAT-6, as in homodimer and in homotetramer using recombinant ESAT-6 and CFP10 expressed in E. coli by applying several biochemical assays including Biolayer Interferometry (BLI) assay. The main findings show that ESAT-6 forms a tight interaction with CFP10 as a heterodimer at neutral pH, and ESAT-6 forms homodimer and even tetramer based larger molecular aggregates at acidic pH. Although the discussion of the potential problems associated with the contamination of ESAT-6 preparations with ASB-14 during the LPS removal step is interesting, but this research does not test the potential impact of residual ASB-14 contaminant on the biochemical behavior ESAT-6-CFP10 heterodimer and ESAT-6 homodimer or tetramer and their hemolytic activity in comparison with the ones without ASB-14. The main strength of this study is the generation of ESAT-6 specific nanobodies and demonstration of its anti-tuberculosis efficiency in THP-1 cell line infected with Mtb strains with reporter genes.

Strengths:

Generation and demonstration of the anti-ESAT-6 nanobodies against tuberculosis infection in cell line based Mtb infection model. Probably identifying potential anti-ESAT-6 nanobody interacting amino acid residues of ESAT-6 is critical in understanding their effects on ESAT-6 mediated membrane lytic activity.

Weaknesses:

Although the biochemistry studies provide quantitative data about the interactions of ESAT-6 with its molecular chaperon CFP10 and the interaction of ESAT-6 homodimer and tetramers, the novel information from these studies are minimal.

Reviewer #2 (Public Review):

In this work, the authors uncovered the effects of DNA dilution on E. coli, including a decrease in growth rate and a significant change in proteome composition. The authors demonstrated that the decline in growth rate is due to the reduction of active ribosomes and active RNA polymerases because of the limited DNA copy numbers. They further showed that the change in the DNA-to-volume ratio leads to concentration changes in almost 60% of proteins, and these changes mainly stem from the change in the mRNA levels.

Reviewer #2 (Public Review):

Summary:

The authors try to achieve a method of protection against pathogenic strains using saprophytic species. It is undeniable that the saprophytic species, despite not causing the disease, activates an immune response. However, based on these results, using the saprophytic species does not significantly impact the animal's infection by a virulent species.

Strengths:

Exposure to the saprophytic strain before the virulent strain reduces animal weight loss, reduces tissue kidney damage, and increases cellular response in mice.

Weaknesses:

Even after the challenge with the saprophyte strain, kidney colonization and the release of bacteria through urine continue. Moreover, the authors need to determine the impact on survival if the experiment ends on the 15th.

Reviewer #2 (Public Review):

Summary:

This manuscript by Alexander et al describes a careful and rigorous application of multiomics to mouse primordial germ cells (PGCs) and their surrounding gonadal cells during the period of sex differentiation.

Strengths:

In thoughtfully designed figures, the authors identify both known and new candidate gene regulatory networks in differentiating XX and XY PGCs and sex-specific interactions of PGCs with supporting cells. In XY germ cells, novel findings include the predicted set of TFs regulating Bnc2, which is known to promote mitotic arrest, as well as the TFs POU6F1/2 and FOXK2 and their predicted targets that function in mitosis and signal transduction. In XX germ cells, the authors deconstruct the regulation of the premeiotic replication regulator Stra8, which reveals TFs involved in meiosis, retinoic acid signaling, pluripotency, and epigenetics among predictions; this finding, along with evidence supporting the regulatory potential of retinoic acid receptors in meiotic gene expression is an important addition to the debate over the necessity of retinoic acid in XX meiotic initiation. In addition, a self-regulatory network of other TFs is hypothesized in XX differentiating PGCs, including TFAP2c, TCF5, ZFX, MGA, and NR6A1, which is predicted to turn on meiotic and Wnt signaling targets. Finally, analysis of PGC-support cell interactions during sex differentiation reveals more interactions in XX, via WNTs and BMPs, as well as some new signaling pathways that predominate in XY PGCs including ephrins, CADM1, Desert Hedgehog, and matrix metalloproteases. This dataset will be an excellent resource for the community, motivating functional studies and serving as a discovery platform.

Weaknesses:

My one major concern is that the conclusion that PGC sex differentiation (as read out by transcription) involves chromatin priming is overstated. The evidence presented in the figures includes a select handful of genes including Porcn, Rimbp1, Stra8, and Bnc2 for which chromatin accessibility precedes expression. Given that the authors performed all of their comparisons between XX versus XY datasets at each timepoint, have they missed an important comparison that would be a more direct test of chromatin priming: between timepoints for each sex? Furthermore, it remains possible that common mechanisms of differentiation to XX and XY could be missing from this analysis that focused on sex-specific differences.

Reviewer #2 (Public Review):

Summary:

Mistri et al explore the role of SLAM-SAP signaling in the developmental programming of innate-like gd T cell subsets. Using proteo-genomics, they determined that abrogation of SLAM-SAP signaling altered that programming, reducing some IL-17-producing subsets, including a novel Vγ4 γδT1 subset, and diverting gdTCR-expressing precursors to the ab fate. Altogether, this is a very thorough, thoughtfully interpreted study that adds significantly to our understanding of the contribution of the SLAM-SAP pathway to lineage specification. A particularly interesting element is the role of SLAM-SAP in preventing gd17 progenitors from switching fates and adopting the ab fate.

One thing to keep in mind in assessing the ultimate fate of the "ab wannabe cells" is that mechanisms exist to silence the gd TCR as cells differentiate to the DP stage and so their presence as diverted DP cells may not be evident by staining for gdTCR expression - and will only be evident transcriptomically.

Strengths:

This is an exceedingly well-designed and thorough study that significantly enriches our understanding of gd T cell development.

Weaknesses:

There are no major weaknesses identified by this reviewer.

Reviewer #3 (Public Review):

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

The paper is well written, easy to follow and the conclusions drawn are supported by the evidence presented. The analyses and conclusions are interesting and will be of value to virologists, cell biologists, and biophysicists studying SARS-CoV-2 function and assembly.

Reviewer #2 (Public Review):

Summary:

The origin of plasmatoid dendritic cells and their subclasses continues to be a debated field, akin to any immune cell field that is determined through the expression of surface markers (relative to clear subclass separation based on functional biology and experimentation). In this context, in this manuscript by Araujo et al, the authors attempt to demonstrate that a subtype of pDCs comes from lymphoid origin due to the presence of some B cell gene expression markers. They nomenclature these cells as B-pDCs. Strikingly, pDCs function via expression of IFNa where as B-pDCs do not express IFNa - thereby raising the question of what are their physiological or pathophysiological properties. B-pDCs also express AXL, a marker not seen in mouse pDCs but observed in human pDCs. Overall, using a combination of gene expression profiling of immune cells isolated from mice via RNA-seq and single-cell profiling the authors propose that B-pDCs are a novel subtype of pDCs in mice that were not previously identified and characterized.

Weaknesses:

My two points of discussion about this manuscript are as follows.

(1) How new are these observations that pDCs could also originate from common lymphoid progenitors. This fact has been previously outlined by many laboratories including Shigematsu et al, Immunity 2004. These studies in the manuscript can be considered new based on the single-cell profiling presented, only if the further characterization of the isolated B-pDCs is performed at the functional biology level. Overlapping gene expression profiles are often seen in developing immune cell types- especially when only evaluated at the RNA expression level- and can lead to cell type complexity (and identification of new cell types) that are not biologically and functionally relevant.

(2) The authors hardly perform any experiments to interrogate the function of these B-pDCs. The discussion on this topic can be enhanced. Ideally, some biological experiments would confirm that B-pDCs are important.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors provide structural analysis of the NHL domain for C. elegans NHL-2 and provide functional analysis of the NHL RNA binding domain. Their data support a model in which NHL-2 binding to mRNA targets through U rich motifs to promote miRISC regulation of translation and mRNA stability.

Strengths:

The authors present convincing data to describe the structure of the NHL-2 NHL domain along with functional analysis that supports an important role for two amino acids that are required for RNA binding activity. The function of these two amino acids were further studied through phenotypic assays to analyze their contribution to miRNA mediated regulation through the let-7 pathway. These data support an important role for RNA binding activity of NHL-2 in the regulation of miRNA dependent pathways. Genetic interactions support a role for the eIF4E binding protein IFET-1 in the miRISC activity.

Weaknesses:

The use of phenotypic assays to monitor let-7 pathway activity could be better explained so that the reader can more easily follow the significance of changes in alae formation or col-19::gfp expression.

The challenges of comparing expression levels using extrachromosomal arrays should be acknowledged.

The figure legends need to be revised to more clearly and accurately explain what is shown in the figures.

Reviewer #2 (Public Review):

Summary:

In this manuscript by Cannarsa et. al., the authors describe the engineering of a light-entrainable synthetic biological oscillator in bacteria. It is based on an upgraded version of one of the first synthetic circuits to be constructed, the repressilator. The authors sought to make this oscillator entrainable by an external forcing signal, analogous to the way natural biological oscillators (like the circadian clock) are synchronized. They reasoned that an optogenetic system would provide a convenient and flexible means of manipulation. To this end, the authors exploited the CcaS-CcaA light-switchable system, which allows activation and deactivation of transcription by green and red light, respectively. They used this system to make the expression of one of the repressilator's transcription factors (lacI) light-controlled, from a construct separated from the main repressilator plasmid. This way, under red light the oscillator runs freely, but exposure to green light causes overexpression of the lacI, pushing the system into a specific state. Consequently, returning to red light will restore the oscillations from the same phase in all cells, effectively synchronizing the cell population.

After demonstrating the functionality of the basic concept, the authors combined modeling and experiments to show how periodic exposure to green light enables efficient entrainment, and how the frequency of the forcing signal affects the oscillatory behavior (detuning).

This work provides an important demonstration of engineering tunability into a foundational genetic circuit, expands the synthetic biology toolbox, and provides a platform to address critical questions about synchronization in biological oscillators. Due to the flexibility of the experimental system, it is also expected to provide a fertile ground for future testing of theoretical predictions regarding non-linear oscillators.

Strengths:

* The study provides a simple and elegant mechanism for the entertainment of a synthetic oscillator. The design relies on optogenetic proteins, which enable efficient experimentation compared to alternative approaches (like using chemical inducers). This way, a static culture (without microfluidics or change of growth media) can be easily exposed to flexible temporal sequences of the zeitgeber, and continuously measured through time.

* The study makes use of both plate-reader-based population-level readout and mother-machine single-cell measurements. Synchronization through entrainment is a single cell level phenomenon, but with a clear population-level manifestation. Thus, this experimental approach combination provides a strong validation to their system. At the same time, differences between the readout from the two systems have emerged, and provided a further opportunity for model refinement and testing.

* The authors correctly identified the main optimization goal, namely the effective leakiness of their construct even under red light. Then, they successfully overcame this issue using synthetic biology approaches.

* The work is supported by a simplified model of the repressilator, which provides a convenient analytical and numerical means to draw testable predictions. The model predictions are well aligned with the experimental evidence.

Weaknesses:

* Even after optimizing the expression level of the light-sensitive gene, the system is very sensitive, i.e., a very short exposure is sufficient to elicit the strongest entertainment. This limited dynamic range might hamper some model testing and future usage.

* As a result of the previous point, the system is entrained by transiently "breaking" the oscillator: each pulse of green light represents a Hopf bifurcation into a single attractor. it means that the system cannot oscillate in constant green light. In comparison, this is generally not the case for natural zeitgebers like light and temperature for the circadian rhythms. Extreme values might prevent oscillations (not necessarily due to breaking the core oscillator), but usually, free running is possible in a wide range of constant conditions. In some cases, the free-running period length will vary as a function of the constant value.

While the approach presented in this manuscript is valid, a comprehensive analysis of more subtle modes of repressilator entrainment could also be of value.

* The entire work makes use of a single intensity and single duration of the green pulse to force entrainment. While the model has clear predictions for how those modalities should affect entrainment, none of the experiments attempted to validate those predictions.

Reviewer #2 (Public Review):

Studying Apteronotus leptorhynchus (the weakly electric brown ghost knifefish), the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. Chirping is a behavior that has been well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that could have a great impact on the field. The authors do provide convincing evidence that chirps may function in homeoactive sensing. However, their evidence arguing against a role for chirps in communication is not as strong, and fails to sufficiently consider the evidence from a large body of existing research. Ultimately, the manuscript presents very interesting data that is sure to stimulate discussion and follow-up studies, but it suffers from dismissing evidence in support of, or consistent with, a communicative function for chirps. The authors do acknowledge that chirps could function as both a communication and homeactive sensing signal, but it seems clear they wish to argue against the former and for the latter, and the evidence is not yet there to support this.

In the introduction, the authors state, "Since both chirps and positional parameters (such as size, orientation or motion) can only be detected as perturbations of the beat, and via the same electroreceptors, the inputs relaying both types of information are inevitably interfering." I disagree with this statement, which seems to be a key assumption. Both of these features certainly modulate the activity of electroreceptors, but that does not mean those modulations are ambiguous as to their source. You do not know whether the two types of modulations can be unambiguously decoded from electroreceptor afferent population activity.

My biggest issue with this manuscript is that it is much too strong in dismissing evidence that chirping correlates with context. In your behavioral observations, you found sex differences in chirping as well as differences between freely interacting and physically separated fish. Chirps tended to occur in close proximity to another fish. Your model of chirp variability found that environmental experience, social experience, and beat frequency (DF) are the most important factors explaining chirp variability. Are these not all considered behavioral or social context? Beat frequency (DF) in particular is heavily downplayed as being a part of "context" but it is a crucial part of the context, as it provides information about the identity of the fish you're interacting with. The authors show quite convincingly that the types of chirps produced do not vary with these contexts, but chirp rates do.

Further, in your playback experiments, fish responded differently to small vs. large DFs, males chirped more than females, type 2 chirps became more frequent throughout a playback, and rises tended to occur at the end of a playback. These are all examples of context-dependent behavior.

In the results, the authors state, "Overall, the majority of chirps were produced by male subjects, in comparable amounts regardless of environmental experience (resident, intruder or equal; Figure S1A,C), social status (dominant or subordinate; Figure S1B) or social experience (novel or experienced; Figure S1D)." This is not what is shown in Figure S1. S1A shows clear differences between resident vs. intruder males, S1B shows clear differences between dominant vs. subordinate males, and S1D shows clear differences between naïve and experienced males. The analysis shown in Figure 2 would seem to support this. Indeed, the authors state, "Overall, this analysis indicated that environmental and social experience, together with beat frequency (DF) are the most important factors explaining chirp variability."

The choice of chirp type varied widely between individuals but was relatively consistent within individuals across trials of the same experiment. The authors interpret this to mean that chirping does not vary with internal state, but is it not likely that the internal states of individuals are stable under stable conditions, and that individuals may differ in these internal states across the same conditions? Stable differences in communication signals between individuals are frequently interpreted as reflecting differences between those individuals in certain characteristics, which are being communicated by these signals.

I am not convinced of the conclusion drawn by the analysis of chirp transitions. The transition matrices show plenty of 1-2 and 2-1 transitions occurring. Further, the cross-correlation analysis only shows that chirp timing between individuals is not phase-locked at these small timescales. It is entirely possible that chirp rates are correlated between interacting individuals, even if their precise timing is not. Further, it is not clear to me how "transitions" were defined. The methods do not make this clear, and it is not clear to me how you can have zero chirp transitions between two individuals when those two individuals are both generating chirps throughout an interaction.

In the results, "Although all chirp types were used during aggressive interactions, these seemed to be rather less frequent in the immediate surround of the chirps (Figure 6A)." A lack of precise temporal correlation on short timescales does not mean there is no association between the two behaviors. An increased rate of chirping during aggression is still a correlation between the two behaviors, even if chirps and specific aggressive behaviors are not tightly time-locked.

In summary, it is simply too strong to say that chirping does not correlate with context, or to claim that there is convincing evidence arguing against a communication function of chirps. Importantly, however, this does not detract from your exciting and well-supported hypothesis that chirping functions in homeoactive sensing. A given EOD behavior could serve both communication and homeoactive sensing. I actually suspect this is quite common in electric fish (both gymnotiforms and mormyrids), and perhaps in other actively sensing species such as echolocating animals. The two are not mutually exclusive.

Reviewer #2 (Public Review):

Summary: In Deconstructing Complexity: A Computational Topology Approach to Trajectory Inference in the Human Thymus with tviblindi, Stuchly et al. propose a new trajectory inference algorithm called tviblindi and a visualization algorithm called vaevictis for single-cell data. The paper utilizes novel and exciting ideas from computational topology coupled with random walk simulations to align single cells onto a continuum. The authors validate the utility of their approach largely using simulated data and establish known protein expression dynamics along CD4/CD8 T cell development in thymus using mass cytometry data. The authors also apply their method to track Treg development in single-cell RNA-sequencing data of human thymus.

The technical crux of the method is as follows: The authors provide an interactive tool to align single cells along a continuum axis. The method uses expected hitting time (given a user input start cell) to obtain a pseudotime alignment of cells. The pseudotime gives an orientation/direction for each cell, which is then used to simulate random walks. The random walks are then arranged/clustered based on the sparse region in the data they navigate using persistent homology.

Strengths:<br /> The notion of using persistent homology to group random walks to identify trajectories in the data is novel.<br /> The strength of the method lies in the implementation details that make computationally demanding ideas such as persistent homology more tractable for large scale single-cell data. This enables the authors to make the method more user friendly and interactive allowing real-time user query with the data.

Weaknesses:<br /> The interactive nature of the tool is also a weakness, by allowing for user bias leading to possible overfitting for a specific data.

The main weakness of the method is lack of benchmarking the method on real data and comparison to other methods. Trajectory inference is a very crowded field with many highly successful and widely used algorithms, the two most relevant ones (closest to this manuscript) are not only not benchmarked against, but also not sited. Including those that specifically use persistent homology to discover trajectories (Rizvi et.al. published Nat Biotech 2017). Including those that specifically implement the idea of simulating random walks to identify stable states in single-cell data (e.g. CellRank published in Lange et.al Nat Meth 2022), as well as many trajectory algorithms that take alternative approaches. The paper has much less benchmarking, demonstration on real data and comparison to the very many other previous trajectory algorithms published before it. Generally speaking, in a crowded field of previously published trajectory methods, I do not think this one approach will compete well against prior work (especially due to its inability to handle the noise typical in real world data (as was even demonstrated in the little bit of application to real world data provided).

Beyond general lack of benchmarking there are two issues that give me particular concern. As previously mentioned, the algorithm is highly susceptible to user bias and overfitting. The paper gives the example (Figure 4) of a trajectory which mistakenly shows that cells may pass from an apoptotic phase to a different developmental stage. To circumvent this mistake, the authors propose the interactive version of tviblindi that allows users to zoom in (increase resolution) and identify that there are in fact two trajectories in one. In this case, the authors show how the author can fix a mistake when the answer is known. However, the point of trajectory inference is to discover the unknown. With so much interactive options for the user to guide the result, the method is more user/bias driven than data-driven. So a rigorous and quantitative discussion of robustness of the method, as well as how to ensure data-driven inference and avoid over-fitting would be useful.

Second, the paper discusses the benefit of tviblindi operating in the original high dimensions of the data. This is perhaps adequate for mass cytometry data where there is less of an issue of dropouts and the proteins may be chosen to be large independent. But in the context of single-cell RNA-sequencing data, the massive undersampling of mRNA, as well as high degree of noise (e.g. ambient RNA), introduces very large degree of noise so that modeling data in the original high dimensions leads to methods being fit to the noise. Therefore ALL other methods for trajectory inference work in a lower dimension, for very good reason, otherwise one is learning noise rather than signal. It would be great to have a discussion on the feasibility of the method as is for such noisy data and provide users with guidance. We note that the example scRNA-seq data included in the paper is denoised using imputation, which will likely result in the trajectory inference being oversmoothed as well.

Reviewer #2 (Public Review):

Summary:

The authors conducted a comparative analysis of four networks, varying in the presence of excitatory assemblies and the architecture of inhibitory cell assembly connectivity. They found that co-tuned E-I assemblies provide network stability and a continuous representation of input patterns (on locally constrained manifolds), contrasting with networks with global inhibition that result in attractor networks.

Strengths:

The findings presented in this paper are very interesting and cutting-edge. The manuscript effectively conveys the message and presents a creative way to represent high-dimensional inputs and network responses. Particularly, the result regarding the projection of input patterns onto local manifolds and continuous representation of input/memory is very Intriguing and novel. Both computational and experimental neuroscientists would find value in reading the paper.

Weaknesses:

Intuitively, classification (decodability) in discrete attractor networks is much better than in networks that have continuous representations. This could also be shown in Figure 5B, along with the performance of the random and tuned E-I networks. The latter networks have the advantage of providing network stability compared to the Scaled I network, but at the cost of reduced network salience and, therefore, reduced input decodability. The authors may consider designing a decoder to quantify and compare the classification performance of all four networks.

Networks featuring E/I assemblies could potentially represent multistable attractors by exploring the parameter space for their reciprocal connectivity and connectivity with the rest of the network. However, for co-tuned E-I networks, the scope for achieving multistability is relatively constrained compared to networks employing global or lateral inhibition between assemblies. It would be good if the authors mentioned this in the discussion. Also, the fact that reciprocal inhibition increases network stability has been shown before and should be cited in the statements addressing network stability (e.g., some of the citations in the manuscript, including Rost et al. 2018, Lagzi & Fairhall 2022, and Vogels et al. 2011 have shown this).

Providing raster plots of the pDp network for familiar and novel inputs would help with understanding the claims regarding continuous versus discrete representation of inputs, allowing readers to visualize the activity patterns of the four different networks. (similar to Figure 1B).

Reviewer #2 (Public Review):

Summary:

This manuscript reports interesting findings about the navigational behavior of mice. The authors have dissected this behavior in various components using a sophisticated behavioral maze and statistical analysis of the data. ​

Strengths:

The results are solid and they support the main conclusions, which will be of considerable value to many scientists.

Weaknesses:

Figure 1: In some trials the mice seem to be doing thigmotaxis, walking along the perimeter of the maze. This is perhaps due to the fear of the open arena. But, these paths along the perimeter would significantly influence all metrics of navigation, e.g. the distance or time to reward. Perhaps analysis can be done that treats such behavior separately and the factors it out from the paths that are away from the perimeter. 

Figure 1c: the color axis seems unusual. Red colors indicate less frequently visited regions (less than 25%) and white corresponds to more frequently visited places (>25%)? Why use such a binary measure instead of a graded map as commonly done?

Some figures use linear scale and others use logarithmic scale. Is there a scientific justification? For example, average latency is on a log scale and average speed is on a linear scale, but both quantify the same behavior. The y-axis in panel 1-I is much wider than the data. Is there a reason for this? Or can the authors zoom into the y-axis so that the reader can discern any pattern?<br /> <br /> 1F shows no significant reduction in distance to reward. Does that mean there is no improvement with experience and all the improvement in the latency is due to increasing running speed with experience?

Figure 3: The distance traveled was reduced by nearly 10-fold and speed increased by by about 3fold. So, the time to reach the reward should decrease by only 3 fold (t=d/v) but that too reduced by 10fold. How does one reconcile the 3fold difference between the expected and observed values? 

Figure 4: The reader is confused about the use of a binary color scheme here for the checking behavior: gray for a large amount of checking, and pink for small. But, there is a large ellipse that is gray and there are smaller circles that are also gray, but these two gray areas mean very different things as far as the reader can tell. Is that so? Why not show the entire graded colormap of checking probability instead of such a seemingly arbitrary binary depiction? 

Figure 4C: What would explain the large amount of checking behavior at the perimeter? Does that occur predominantly during thigmotaxis? 

Was there a correlation between the amount of time spent by the animals in a part of the maze and the amount of reward checking? Previous studies have shown that the two behaviors are often positively correlated, e.g. reference 20 in the manuscript.  How does this fit with the path integration hypothesis? 

"Scratches and odor trails were eliminated by washing and rotating the maze floor between trials." Can one eliminate scratches by just washing the maze floor? Rotation of the maze floor between trials can make these cues unreliable or variable but will not eliminate them. Ditto for odor cues.

"Possible odor gradient cues were eliminated by experiments where such gradients were prevented with vacuum fans (Fig. S6E)" What tests were done to ensure that these were *eliminated* versus just diminished? 

"Probe trials of fully trained mice resulted in trajectories and initial hole checking identical to that of regular trials thereby demonstrating that local odor cues are not essential for spatial learning." As far as the reader can tell, probe trials only eliminated the food odor cues but did not eliminate all other odors. If so, this conclusion can be modified accordingly. <br /> The interpretation of direction selectivity is a bit tricky. At different places in this manuscript, this is interpreted as a path integration signal that encodes goal location, including the Consync cells. However, studies show that (e.g. Acharya et al. 2016) direction selectivity in virtual reality is comparable to that during natural mazes, despite large differences in vestibular cues and spatial selectivity. How would one reconcile these observations with path integration interpretation? 

The manuscript would be improved if the speculations about place cells, grid cells, BTSP, etc. were pared down. I could easily imagine the outcome of these speculations to go the other way and some claims are not supported by data. "We note that the cited experiments were done with virtual movement constrained to 1D and in the presence of landmarks. It remains to be shown whether similar results are obtained in our unconstrained 2D maze and with only self-motion cues available." There are many studies that have measured the evolution of place cells in non-virtual mazes, look up papers from the 1990s. Reference 43 reports such results in a 2D virtual maze.

Reviewer #2 (Public Review):

The manuscript investigates the function of basal forebrain cholinergic axons in mouse primary visual cortex (V1) during locomotion using two-photon calcium imaging in head-fixed mice. Cholinergic modulation has previously been proposed to mediate the effects of locomotion on V1 responses. The manuscript concludes that the activity of basal forebrain cholinergic axons in visual cortex provides a signal which is more correlated with binary locomotion state than locomotion velocity of the animal and finds no evidence for modulation of cholinergic axons by locomotion velocity. Cholinergic axons did not seem to respond to grating stimuli or visuomotor prediction error. Optogenetic stimulation of these axons increased the amplitude of responses to visual stimuli and decreased the response latency of layer 5 excitatory neurons, but not layer 2/3 neurons. Moreover, optogenetic or chemogenetic stimulation of cholinergic inputs reduced pairwise correlation of neuronal responses. These results provide insight into the role of cholinergic modulation to visual cortex and demonstrate that it affects different layers of visual cortex in a distinct manner. The experiments are well executed and the data appear to be of high quality. However, further analyses may be required to fully support some of the study's conclusions. Specifically, the analyses of the effects of locomotion and stimulation of cholinergic inputs present grand averages of responses across all neurons, and therefore may mask heterogeneity across layer 2/3 and layer 5 neurons.

Reviewer #2 (Public Review):

Summary:

The current work describes a set of behavioral tasks to explore individual differences in the preferred perceptual and motor rhythms. Results show a consistent individual preference for a given perceptual and motor frequency across tasks and, while these were correlated, the latter is slower than the former one. Additionally, the adaptation accuracy to rate changes is proportional to the amount of rate variation and, crucially, the amount of adaptation decreases with age.

Strengths:

Experiments are carefully designed to measure individual preferred motor and perceptual tempo. Furthermore, the experimental design is validated by testing the consistency across tasks and test-retest, what makes the introduced paradigm a useful tool for future research.<br /> The obtained data is rigorously analyzed using a diverse set of tools, each adapted to the specificities across the different research questions and tasks.<br /> This study identifies several relevant behavioral features: (i) each individual shows a preferred and reliable motor and perceptual tempo and, while both are related, the motor is consistently slower than the pure perceptual one; (ii) the presence of hysteresis in the adaptation to rate variations; and (iii) the decrement of this adaptation with age. All these observations are valuable for the auditory-motor integration field of research, and they could potentially inform existing biophysical models to increase their descriptive power.

Weaknesses:

To get a better understanding of the mechanisms underlying the behavioral observations, it would have been useful to compare the observed pattern of results with simulations done with existing biophysical models. However, this point is addressed if the current study is read along with this other publication of the same research group: Kaya, E., & Henry, M. J. (2024, February 5). Modeling rhythm perception and temporal adaptation: top-down influences on a gradually decaying oscillator. https://doi.org/10.31234/osf.io/q9uvr

Reviewer #2 (Public Review):

This manuscript asks how different forms of selection affect the patterns of genetic diversity in microbial populations. One popular metric used to infer signatures of selection is dN/dS, the ratio of nonsynonymous to synonymous distances between two genomes. Previous observations across many bacterial species have found dN/dS decreases with dS, which is a proxy for the divergence time. The most common interpretation of this pattern was proposed by Rocha et al. (2006), who suggested the excess in nonsynonymous mutations on short divergence times represent transient deleterious mutations that have not yet been purged by selection.

In this study, the authors propose an alternative model based on the population structure of human gut bacteria, in which dN is dominated by selective sweeps of SNPs that revert previous mutations within local populations. The authors argue that contrary to standard population genetics models, which are based on the population dynamics of large eukaryotes, the large populations in the human gut mean that reversions may be quite common and may have a large impact on evolutionary dynamics. They show that such a model can fit the decrease of dN/dS in time at least as well as the purifying selection model.

Strengths

The main strength of the manuscript is to show that adaptive sweeps in gut microbial populations can lead to small dN/dS. While previous work has shown that using dN/dS to infer the strength of selection within a population is problematic (see Kryazhimskiy and Plotkin, 2008, cited in the paper) the particular mechanism proposed by the authors is new to my knowledge. In addition, despite the known caveats, dN/dS values are still routinely reported in studies of microbial evolution, and so their interpretation should be of considerable interest to the community.

The authors provide compelling justification for the importance of adaptive reversions and make a good case that these need to be carefully considered by future studies of microbial evolution. The authors show that their model can fit the data as well as the standard model based on purifying selection and the parameters they infer appear to be plausible given known data. More generally, I found the discussion on the implications of traditional population genetics models in the context of human gut bacteria to be a valuable contribution of the paper.

Weaknesses

The authors argue that the purifying selection model would predict a gradual loss in fitness via Muller's ratchet. This is true if recombination is ignored, but this assumption is inconsistent with the data from Garud, et al. (2019) cited in the manuscript, who showed a significant linkage decrease in the bacteria also used in this study.

I also found that the data analysis part of the paper added little new to what was previously known. Most of the data comes directly from the Garud et al. study and the analysis is very similar as well. Even if other appropriate data may not currently be available, I feel that more could be done to test specific predictions of the model with more careful analysis.

Finally, I found the description of the underlying assumptions of the model and the theoretical results difficult to understand. I could not, for example, relate the fitting parameters nloci and Tadapt to the simulations after reading the main text and the supplement. In addition, it was not clear to me if simulations involved actual hosts or how the changes in selection coefficients for different sites was implemented. Note that these are not simply issues of exposition since the specific implementation of the model could conceivably lead to different results. For example, if the environmental change is due to the colonization of a different host, it would presumably affect the selection coefficients at many sites at once and lead to clonal interference. Related to this point, it was also not clear that the weak mutation strong selection assumption is consistent with the microscopic parameters of the model. The authors also mention that "superspreading" may somehow make a difference to the probability of maintaining the least loaded class in the purifying selection model, but what they mean by this was not adequately explained.

Reviewer #2 (Public Review):

Summary:

The authors investigate replay (defined as sequential reactivation) and clustered reactivation during retrieval of an abstract cognitive map. Replay and clustered reactivation were analysed based on MEG recordings combined with a decoding approach. While the authors state to find evidence for both, replay and clustered reactivation during retrieval, replay was exclusively present in low performers. Further, the authors show that reactivation strength declined with an increasing graph distance.

Strengths:

The paper raises interesting research questions, i.e., replay vs. clustered reactivation and how that supports retrieval of cognitive maps. The paper is well written, well structured and easy to follow. The methodological approach is convincing and definitely suited to address the proposed research questions.

The paper is a great combination between replicating previous findings (Wimmer et al. 2020) with a new experimental approach but at the same time presenting novel evidence (reactivation strength declines as a function of graph distance).

What I also want to positively highlight is their general transparency. For example, they pre-registered this study but with a focus on a different part of the data and outlined this explicitly in the paper.

The paper has very interesting findings. However, there are some shortcomings, especially in the experimental design. These are shortly outlined below but are also openly and in detail discussed by the authors.

Weaknesses:

The individual findings are interesting. However, due to some shortcomings in the experimental design they cannot be profoundly related to each other. For example, the authors show that replay is present in low but not in high performers with the assumption that high performers tend to simultaneously reactivate items. But then, the authors do not investigate clustered reactivation (= simultaneous reactivation) as a function of performance due to a low number of retrieval trials and ceiling performance in most participants.<br /> As a consequence of the experimental design, some analyses are underpowered (very low number of trials, n = ~10, and for some analyses, very low number of participants, n = 14).

Reviewer #2 (Public Review):

The authors characterized the recombinase-based cumulative fate maps for vesicular glutamate transporters (Vglut1, Vglut2 and Vglut3) expression and compared those maps to their real-time expression profiles in central NA neurons by RNA in situ hybridization in adult mice. Authors have revealed a new and intriguing expression pattern for Vglut2, along with an entirely uncharted co-expression domain for Vglut3 within central noradrenergic neurons. Interestingly, and in contrast to previous studies, the authors demonstrated that glutamatergic signaling in central noradrenergic neurons does not exert any influence on breathing and metabolic control either under normoxic/normocapnic conditions or after chemoreflex stimulation. Also, they showed for the first-time the Vglut3-expressing NA population in C2/A2 nuclei. In addition, they were also able to demonstrate Vglut2 expression in anterior NA populations, such as LC neurons, by using more refined techniques, unlike previous studies.

A major strength of the study is the use of a set of techniques to investigate the participation of NA-based glutamatergic signaling in breathing and metabolic control. The authors provided a full characterization of the recombinase-based cumulative fate maps for Vglut transporters. They performed real-time mRNA expression of Vglut transporters in central NA neurons of adult mice. Further, they evaluated the effect of knocking down Vglut2 expression in NA neurons using a DBH-Cre; Vglut2cKO mice on breathing and control in unanesthetized mice. Finally, they injected the AAV virus containing Cre-dependent Td tomato into LC of v-Glut2 Cre mice to verify the VGlut2 expression in LC-NA neurons. A very positive aspect of the article is that the authors combined ventilation with metabolic measurements. This integration holds particular significance, especially when delving into the exploration of respiratory chemosensitivity. Furthermore, the sample size of the experiments is excellent.<br /> Despite the clear strengths of the paper, some weaknesses exist. It is not clear in the manuscript if the experiments were performed in males and females and if the data were combined. I believe that the study would have benefited from a more comprehensive analysis exploring the sex specific differences. The reason I think this is particularly relevant is the developmental disorders mentioned by the authors, such as SIDS and Rett syndrome, which could potentially arise from disruptions in central noradrenergic (NA) function, exhibit varying degrees of sex predominance. Moreover, some of the noradrenergic cell groups are sexually dimorphic. For instance, female Wistar rats exhibit a larger LC size and more LC-NA neurons than male subjects (Pinos et al., 2001; Garcia-Falgueras et al., 2005). More recently, a detailed transcriptional profiling investigation has unveiled the identities of over 3,000 genes in the LC. This revelation has highlighted significant sexual dimorphisms, with more than 100 genes exhibiting differential expression within LC-NA neurons at the transcript level. Furthermore, this investigation has convincingly showcased that these distinct gene expression patterns have the capacity to elicit disparate behavioral responses between sexes (Mulvey et al., 2018). Therefore, the authors should compare the fate maps, Vglut transporters in males and females, at least considering LC-NA neurons. Even in the absence of identified sex differences, this information retains significant importance.<br /> An important point well raised by the authors is that although suggestive, these experiments do not definitively rule out that NA-Vglut2 based glutamatergic signaling has a role in breathing control. Subsequent experiments will be necessary to validate this hypothesis.

An improvement could be made in terms of measuring body temperature. Opting for implanted sensors over rectal probes would circumvent the need to open the chamber, thereby preventing alterations in gas composition during respiratory measurements. Further, what happens to body temperature phenotype in these animals under different gas exposures? These data should be included in the Tables.

Is it plausible that another neurotransmitter within NA neurons might be released in higher amounts in DBH-Cre; Vglut2 cKO mice to compensate for the deficiency in glutamate and prevent changes in ventilation?

Continuing along the same line of inquiry is there a possibility that Vglut2 cKO from NA neurons not only eliminates glutamate release but also reduces NA release? A similar mechanism was previously found in VGLUT2 cKO from DA neurons in previous studies (Alsio et al., 2011; Fortin et al., 2012; Hnasko et al., 2010). Additionally, does glutamate play a role in the vesicular loading of NA? Therefore, could the lack of effect on breathing be explained by the lack of noradrenaline and not glutamate?

Reviewer #2 (Public Review):

Summary:

This is an interesting and well-performed study that develops a new modeling approach (MoA-HMM) to simultaneously characterize reinforcement learning parameters of different RL agents, as well as latent behavioral states that differ in the relative contributions of those agents to the animal's choices. They performed this study in rats trained to perform the two-step task. While the major advance of the paper is developing and rigorously validating this novel technical approach, there are also a number of interesting conceptual advances. For instance, humans performing the two-step task are thought to exhibit a trade-off between model-free and model-based strategies. However, the MoA-HMM did not reveal such a trade-off in the rats, but instead suggested a trade-off between model-based exploratory vs. exploitative strategies. Additionally, the firing rates of neurons in the orbitofrontal cortex (OFC) reflected latent behavioral states estimated from the HMM, suggesting that (1) characterizing dynamic behavioral strategies might help elucidate neural dynamics supporting behavior, and (2) OFC might reflect the contributions of one or a subset of RL agents that are preferentially active or engaged in particular states identified by the HMM.

Strengths:

The claims were generally well-supported by the data. The model was validated rigorously and was used to generate and test novel predictions about behavior and neural activity in OFC. The approach is likely to be generally useful for characterizing dynamic behavioral strategies.

Weaknesses:

There were a lot of typos and some figures were mis-referenced in the text and figure legends.

Reviewer #2 (Public Review):

Summary:

This research shows 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 KCNQ channels during aging is a major contributor to the increased excitability of sympathetic old neurons. All conclusions of this paper are well 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 decrease in KCNQ current as a cellular mechanism behind age-induced hyperexcitability in sympathetic motor neurons.

Weaknesses:

None, the revised version of the manuscript has addressed all my concerns.

Reviewer #2 (Public Review):

Summary:

The paper presents PPI-hotspot a method to predict PPI-hotspots. Overall, it could be useful but serious concerns about the validation and benchmarking of the methodology make it difficult to predict its reliability.

Strengths:

Develops an extended benchmark of hot-spots.

Weaknesses:

(1) Novelty seems to be just in the extended training set. Features and approaches have been used before.

(2) As far as I can tell the training and testing sets are the same. If I am correct, it is a fatal flaw.

(3) Comparisons should state that: SPOTONE is a sequence (only) based ML method that uses similar features but is trained on a smaller dataset. FTmap I think predicts binding sites, I don't understand how it can be compared with hot spots. Suggesting superiority by comparing with these methods is an overreach.

(4) Training in the same dataset as SPOTONE, and then comparing results in targets without structure could be valuable.

(5) The paper presents as validation of the prediction and experimental validation of hotspots in human eEF2. Several predictions were made but only one was confirmed, what was the overall success rate of this exercise?

Reviewer #2 (Public Review):

Summary:

This 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 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.

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:

A few minor weaknesses have been noted. First, since snRNA-Seq and scRNA-Seq generated different coverage of expressed genes in the cells, how did the combined analyses balance the un-equal sequencing coverage and missing data points in the snRNA-Seq data? Second, the RNA/protein validation of selected SEC molecular markers was done using mouse anterior segment tissues. It would be more helpful to examine whether these molecular markers for SECs could work well in human SECs. Third, the effort to characterize the GWAS-identified IOP- and glaucoma-associated genes is exciting but with limited new information. Additional work could be performed to prioritize these genes.

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, it 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 the 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 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. However, without replicative 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, there is not enough experimental context 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's 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 plots provided since the change in the direction of the orange data subset is considers AUD T2 vs T1. In contrast, AUD T2 vs Control would represent the claimed shift. To support these claims, the authors would better support their argument by showing this comparison as well as showing 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 present in AUD brain tissues is interesting; however, not strongly supporting of 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 need for more 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 is 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 are appreciated (Supplementary 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 no supporting data are provided besides this categorization. Finally, a common risk in such descriptive studies is finding spurious associations, especially considering many factors 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. However, this approach only accounts for the many possible metabolite association tests within each comparison (such as metabolites vs depression). It 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.

Reviewer #2 (Public Review):

Summary:

The authors Fu et al., developed polymer models that combine loop extrusion with attractive interactions to best describe Hi-C population average data. They analyzed Hi-C data of the MYC locus as an example and developed an optimization strategy to extract the parameters that best fit this average Hi-C data.

Strengths:

The model has an intuitive nature and the authors masterfully fitted the model to predict relevant biology/Hi-C methodology parameters. This includes loop extrusion parameters, the need for self-interaction with specific energies, and the time and distance parameters expected for Hi-C capture.

Weaknesses:

(1) We are no longer in the age in which the community only has access to population average Hi-C. Why was only the population average Hi-C used in this study?

Can single-cell data: i.e. single-cell Hi-C/Dip-C data or chromatin tracing data (i.e. see Tan et al Science 2018 - for Dip-C, Bintu et al Science 2018, Su et al Cell 2020 for chromatin tracing, etc.) or even 2 color DNA FISH data (used here only as validation) better constrain these models? At the very least the simulations themselves could be used to answer this essential question.

I am expecting that the single-cell variance and overall distributions of distances between loci might better constrain the models, and the authors should at least comment on it.

(2) The authors claimed "Our parameter optimization can be adapted to build biophysical models of any locus of interest. Despite the model's simplicity, the best-fit simulations are sufficient to predict the contribution of loop extrusion and domain interactions, as well as single-cell variability from Hi-C data. Modeling dynamics enables testing mechanistic relationships between chromatin dynamics and transcription regulation. As more experimental results emerge to define simulation parameters, updates to the model should further increase its power." The focus on the Myc locus in this study is too narrow for this claim. I am expecting at least one more locus for testing the generality of this model.

Reviewer #2 (Public Review):

Summary:

The motivation for this study is to provide a comprehensive assessment of motor unit firing rate responses of entire pools during isometric contractions. The authors have used new quantitative methods to extract more unique motor units across contractions than prior studies. This was achieved by recording muscle fibre action potentials from four high-density surface electromyogram (HDsEMG) arrays (Caillet et al., 2023), quantifying residual EMG comparing the recorded and data-based simulation (Figure 1A-B), and developing a metric to compare the spatial identification for each motor unit (Figure 1D-E). From identified motor units, the authors have provided a detailed characterization of recruitment and firing rate responses during slow voluntary isometric contractions in the vastus lateralis and tibialis anterior muscles up to 80% of maximum intensity. In the lower limb, it is interesting how lower threshold motor units have firing rate responses that saturate, whereas higher threshold units that presumably produce higher muscle contractile forces continue to increase their firing rate. In many ways, these results agree with the rate coding of motor units in the extensor digitorum communis muscle (Monster and Chan, 1977). The paper is detailed, and the analyses are well explained. However, there are several points that I think should be addressed to strengthen the paper.

General comments:

(1) The authors claim they have measured the complete rate coding profiles of motor units in the vastus lateralis and tibialis anterior muscles. However, this study quantified rate coding during slow and prolonged voluntary isometric contractions whereas the function of rate coding during movements (Grimby and Hannerz, 1977) or more complex isometric contractions (Cutsem and Duchateau, 2005; Marshall et al., 2022) remains unexplored. For example, supraspinal inputs may not scale the same way across low and higher threshold motor units, or between muscles (Devanne et al., 1997), making the response of firing rates to increasing isometric contraction force less clear. Conceptually, the authors focus on the literature on intrinsic motoneurone properties, but in vivo, other possibilities are that descending supraspinal drive, spinal network dynamics, and afferent inputs have different effects across motor unit sizes, muscles, and types of contractions. Also, the influence from local muscles that act as synergists (e.g., vastii muscles for the vastus lateralis, and peroneal muscles that evert the foot for the tibialis anterior) or antagonists (coactivation during higher contraction intensities would stiffen the joint) may provide differential forms of proprioceptive feedback across motor pools.

(2) The evidence that the entire motor unit pool was recorded per muscle is not clear. There appears to be substantial residual EMG (Figure 1B), signal cancellation of smaller motor units (lines 172-176), some participants had fewer than 20 identified motor units, and contractions never went above 80% of MVC. Also, to my understanding, there remains no gold-standard in awake humans to estimate the total motor unit number in order to determine if the entire pool was decomposed. Furthermore, using four HDsEMG arrays also raises questions about how some channels were placed over non-target muscles, and if motor units were decomposed from surrounding synergists.

(3) The authors claim (Abstract L51; Discussion L376) that a commonly held view in the field is that rate coding is similar across motor units from the same pool. Perhaps this is in reference to some studies that have carefully assessed lower threshold motor units during lower force ramp contractions (e.g., Fuglevand et al., 2015; Revill and Fuglevand, 2017). However, a more complete integration of the literature exploring motor unit firing rate responses during rapid isometric contractions, comparing different muscles and contraction intensities would be helpful. From Figure 3, the range of rate coding in the tibialis anterior (~7-40 Hz) is greater than the vastus lateralis (~5-22 Hz) muscle across contraction levels. In agreement with other studies, the range of rate coding within some muscles is different than others (Kirk et al., 2021) and during maximal intensity (Bellemare et al., 1983) or rapid contractions (Desmedt and Godaux, 1978). Likewise, within a motor pool, there is a diversity of firing rate responses across motor units of different sizes as a function of isometric force (Monster and Chan, 1977; Desmedt and Godaux, 1977; Kukula and Clamann, 1981; Del Vecchio et al., 2019; Marshall et al., 2022). A strength of this paper is how firing rate responses are quantified across a wide range of motor unit recruitment thresholds and between two muscles. I suggest improving clarity for the general reader, especially in the motivation for testing two lower limb muscles, and elaborating on some of the functional implications.

Reviewer #2 (Public Review):

Summary:

Tóth and Bazeli et al. find rapamycin activates heterologously-expressed TRPM8 and dissociated sensory neurons in a TRPM8-dependent way with Ca2+-imaging. With electrophysiology and STTD-NMR, they confirmed the activation is through direct interaction with TRPM8. Using mutants and computational modeling, the authored localized the binding site to the groove between S4 and S5, different than the binding pocket of cooling agents such as menthol. The hydroxyl group on carbon 40 within the cyclohexane ring in rapamycin is indispensable for activation, while other rapalogs with its replacement, such as everolimus, still bind but cannot activate TRPM8. Overall, the findings provide new insights into TRPM8 functions and may indicate previously unknown physiological effects or therapeutic mechanisms of rapamycin.

Strengths:

The authors spent extensive effort on demonstrating that the interaction between TRPM8 and rapamycin is direct. The evidence is solid. In probing the binding site and the structural-function relationship, the authors combined computational simulation and functional experiments. It is very impressive to see that "within" a rapamycin molecule, the portion shared with everolimus is for "binding", while the hydroxyl group in the cyclohexane ring is for activation. Such detailed dissection represents a successful trial in the computational biology-facilitated, functional experiment-validated study of TRP channel structural-activity relationship. The research draws the attention of scientists, including those outside the TRP channel field, to previously neglected effects of rapamycin, and therefore the manuscript deserves broad readership.

Weaknesses:

The significance of the research could be improved by showing or discussing whether a similar binding pocket is present in other TRP channels, and hence rapalogs might bind to or activate these TRP channels. Additionally, while the finding on TRPM8 is novel, it is worthwhile to perform more comprehensive pharmacological characterization, including single-channel recording and a few more mutant studies to offer further insight into the mechanism of rapamycin binding to S4~S5 pocket driving channel opening. It is also necessary to know if rapalogs have independent or synergistic effects on top of other activators, including cooling agents and lower temperature, and their dependence on regulators such as PIP2.

Additional discussion that might be helpful:

The authors did confirm that rapamycin does not activate TRPV1, TRPA1 and TRPM3. But other TRP channels, particularly other structurally similar TRPM channels, should be discussed or tested. Alignment of the amino acid sequences or structures at the predicted binding pocket might predict some possible outcomes. In particular, rapamycin is known to activate TRPML1 in a PI(3,5)P2-dependent manner, which should be highlighted in comparison among TRP channels (PMID: 35131932, 31112550).

Reviewer #2 (Public Review):

Summary:

In this article, Wen et. al. describe the development of a 'proof-of-concept' bi-functional vector based on HSV-deltaICP-34.5's ability to purge latent HIV-1 and SIV genomes from cells. They show that co-infection of latent J-lat T-cell lines with an HSV-deltaICP-34.5 vector can reactivate HIV-1 from a latent state. Over- or stable expression of ICP 34.5 ORF in these cells can arrest latent HIV-1 genomes from transcription, even in the presence of latency reversal agents. ICP34.5 can co-IP with- and de-phosphorylate IKKa/b to block its interaction with NF-k/B transcription factor. Additionally, ICP34.5 can interact with HSF1 which was identified by mass-spec. Thus, the authors propose that the latency reversal effect of HSV-deltaICP-34.5 in co-infected JLat cells is due to modulatory effects on the IKKa/b-NF-kB and PP1-HSF-1 pathway.

Next, the authors cleverly construct a bifunctional HSV-based vector with deleted ICP34.5 and 47 ORFs to purge latency and avoid immunological refluxes, and additionally, expand the application of this construct as a vaccine by introducing SIV genes. They use this 'vaccine' in mouse models and show the expected SIV-immune responses. Experiments in rhesus macaques (RM), further elicit the potential for their approach to reactivate SIV genomes and at the same time block their replication by antibodies. What was interesting in the SIV experiments is that the dual-functional vector vaccine containing sPD1- and SIV Gag/Env ORFs effectively delayed SIV rebound in RMs and in some cases almost neutralized viral DNA copy detection in serum. Very promising indeed, however, there are some questions I wish the authors had explored to get answers to, detailed below.

Overall, this is an elegant and timely work demonstrating the feasibility of reducing virus rebound in animals, with the potential to expand to clinical studies. The work was well-written, and sections were clearly discussed.

Strengths:

The work is well designed, rationale explained, and written very clearly for lay readers.

Claims are adequately supported by evidence and well-designed experiments including controls.

Weaknesses:

(1) While the mechanism of ICP34.5 interaction and modulation of the NF-kB and HSF1 pathways are shown, this only proves ICP34.5 interactions but does not give away the mechanism of how the HSV-deltaICP-34.5 vector purges HIV-1 latency. What other components of the vector are required for latency reversal? Perhaps serial deletion experiments of the other ORFs in the HSV-deltaICP-34.5 vector might be revealing.

(2) The efficacy of the HSV vaccine vectors was evaluated in Rhesus Macaque model animals. Animals were chronically infected with SIV (a parent of HIV), treated with ART, challenged with bi-functional HSV vaccine or controls, and discontinued treatment, and the resulting virus burden and immune responses were monitored. The animals showed SIV Gag and Env-specific immune responses, and delayed virus rebound (however rebound is still there), and below-detection viral DNA copies. What would make a more convincing argument to this reviewer will be data to demonstrate that after the bi-functional vaccine, the animals show overall reduction in the number of circulating latent cells. The feasibility of obtaining such a result is not clearly demonstrated.

(3) The authors state that the reduced virus rebound detected following bi-functional vaccine delivery is due to latent genomes becoming activated and steady-state neutralization of these viruses by antibody response. This needs to be demonstrated. Perhaps cell-culture experiments from specimens taken from animals might help address this issue. In lab cultures one could create environments without antibody responses, under these conditions one would expect a higher level of viral loads to be released in response to the vaccine in question.

(4) How do the authors imagine neutralizing HIV-1 envelope epitopes by a similar strategy? A discussion of this point may also help.

(5) I thought the empty HSV-vector control also elicited somewhat delayed kinetics in virus rebound and neutralization, can the authors comment on why this is the case?

Reviewer #2 (Public Review):

Summary:

This paper derives the first three functional gradients in the left and right hippocampus across two datasets. These gradient maps are then compared to dopamine receptor maps obtained with PET, associated with age, and linked to memory. Results reveal links between dopamine maps and gradient 2, age with gradients 1 and 2, and memory performance.

Strengths:

This paper investigates how hippocampal gradients relate to aging, memory, and dopamine receptors, which are interesting and important questions. A strength of the paper is that some of the findings were replicated in a separate sample.

Weaknesses

The paper would benefit from added clarification on the number of models/comparisons for each test. Furthermore, it would be helpful to clarify whether or not multiple comparison correction was performed and - if so - what type or - if not - to provide a justification. The manuscript would furthermore benefit from code sharing and clarifying which results did/did not replicate.

Reviewer #3 (Public Review):

Summary:

This paper describes transcriptomes from three tardigrade species with or without treatment with ionizing radiation (IR). The authors show that IR produces numerous single strand and double strand breaks as expected and that these are substantially repaired within 4-8 hours. Treatment with IR induces strong upregulation of transcripts from numerous DNA repair proteins, and from the newly described protein TDR1 with homologs in both Hypsibioidea and Macrobiotoidea supefamilies. The authors show that TDR1 transcription produces newly translated TDR1 protein, which can bind DNA and co-localizes with DNA in the nucleus. At higher concentrations TDR appears to form aggregates with DNA, which might be relevant to a possible function in DNA damage repair. When introduced into human U2OS cells treated with the radiomimetic drug bleomycin, TDR1 reduces the number of double-strand breaks as detected by gamma H2AX spots. This paper will be of interest to the DNA repair field and to radiobiologists.

Strengths:

The paper is well-written and provides solid evidence of the upregulation of DNA repair enzymes after irradiation of tardigrades, as well as upregulation of the TRD1 protein. The reduction of gamma-H2A.X spots in U2OS cells after expression of TRD1 supports a role in a DNA damage.

Weaknesses:<br /> Genetic tools are still being developed in tardigrades, so there is no mutant phenotype to support a DNA repair function for TRD1, but this may be available soon.

Reviewer #2 (Public Review):

In this study, Huang et al. performed a scRNA-seq analysis of lung adenocarcinoma (LUAD) specimens from 9 human patients, including 5 who received neoadjuvant chemotherapy (NCT), and 4 without treatment (control). The new data was produced using 10 × Genomics technology and comprises 83622 cells, of which 50055 and 33567 cells were derived from the NCT and control groups, respectively. Data was processed via R Seurat package, and various downstream analyses were conducted, including CNV, GSVA, functional enrichment, cell-cell interaction, and pseudotime trajectory analyses. Additionally, the authors performed several experiments for in vitro and in vivo validation of their findings, such as immunohistochemistry, immunofluorescence, flow cytometry, and animal experiments.

The study extensively discusses the heterogeneity of cell populations in LUAD, comparing the samples with and without chemotherapy. However, there are several shortcomings that diminish the quality of this paper:

• The number of cells included in the dataset is limited, and the number of patients from different groups is low, which may reduce the attractiveness of the dataset for other researchers to reuse. Additionally, there is no metadata on patients' clinical characteristics, such as age, sex, history of smoking, etc., which would be valuable for future studies.<br /> • Several crucial details about the data analysis are missing: How many PCs were used for reduction? Which versions of Seurat/inferCNV/other packages were used? Why monocle2 was used and not monocle3 or other packages? Also, the authors use R version 3.6.1, and the current version is 4.3.2.<br /> • It seems that the authors may lack a fundamental understanding of scRNA-seq data processing and the functions of Seurat. For instance, they state, 'Next, we classified cell types through dimensional reduction and unsupervised clustering via the Seurat package.' However, dimensional reduction and unsupervised clustering are not methods for cell classification. Typically, cell types are classified using marker genes or other established methods.<br /> "Therefore, to identify subclusters within each of these nine major cell types, we performed principal component analysis" (Line 127). Principal component analysis is a method for dimensionality reduction, not cell clustering.<br /> The authors did not mention the normalization or scaling of the data, which are crucial steps in scRNA-seq data preprocessing.<br /> • Numerous style and grammar mistakes are present in the main text. For instance, certain sections of the methods are written in the present tense, suggesting that parts of a protocol were copied without text editing. Furthermore, some sections of the introduction are written in the past tense when the present tense would be more suitable. Clusters are inconsistently referred to by numbers or cell types, leading to confusion. Additionally, the authors frequently use the term "evolution" when describing trajectory analysis, which may not be appropriate. Overall, significant revisions to the main text are required.<br /> • Some figures are not mentioned in order or are not referenced in the text at all, such as Figure 5l (where it is also unclear how the authors selected the root cells). Additionally, many figures have text that is too small to be read without zooming in. Overall, the quality of the figures is inconsistent and sometimes very poor.<br /> • At times, the authors' statements are incomplete (ex. Lines 67-69, Line 177, Line 629, Lines 646-648 and 678).

The results section lacks clarity on several points:<br /> • The authors state that "myofibroblasts exclusively originated from the control group". However, pathways up-regulated in myofibroblasts (such as glycolysis) were enhanced after chemotherapy, as indicated by GSVA score. Similarly, why are some clusters of TAMs from the control group associated with pathways enriched in chemotherapy group?<br /> • Further explanation is necessary regarding the distinctions between malignant and non-malignant cells, as well as regarding the upregulation of metabolism-related pathways in fibroblasts from the NCT group. Additionally, clarification is needed regarding why certain TAMs from the control group are associated with pathways enriched in the chemotherapy group.<br /> • In the section titled 'Chemo-driven Pro-mac and Anti-mac Metabolic Reprogramming Exerted Diametrically Opposite Effects on Tumor Cells': The markers selected to characterize the anti- and pro-macrophages are commonly employed for describing M1 or M2 polarization. It is uncertain whether this new classification into anti- and pro-macrophages is necessary. Additionally, it should be noted that pro-macrophages are anti-inflammatory, while anti-macrophages are pro-inflammatory, which could lead to confusion. M2 macrophages are already recognized for their role in stimulating tumor relapse after chemotherapy.<br /> • The authors suggest that there is "reprogramming of CD8+ cytotoxic cells" following chemotherapy (Line 409). It remains unclear whether they imply the reprogramming of other CD8+ T cells into cytotoxic cells. While it is indicated that cytotoxic cells from the control group differ from those in the NCT group and that NCT cytotoxic T cells exhibit higher cytotoxicity, the authors did not assess the expression of NK and NK-like T cell markers (aside from NKG7), which may possess greater cytotoxic potential than CD8+ cytotoxic cells. This could also elucidate why cytotoxic cells from the NCT and control groups are positioned on separate branches in trajectory analysis. Overall, with 22.5k T cells in the dataset, only 3 subtypes were identified, suggesting a need for improved cell annotations by the authors.

Reviewer #2 (Public Review):

Summary:

The article uses a cell-based model to investigate how mutations and cells spread throughout a tumour. The paper uses published data and the proposed model to understand how growth and death mechanisms lead to the observed data. This work provides an insight into the early stages of tumour development. From the work provided here, the results are solid, showing a thorough analysis. However, the work has not fully specified the model, which can lead to some questions around the model's suitability. The article is well-written and presents a very suitable and rigorous analysis to describe the data. The authors did a particularly nice job of the discussion and decision of their "metrics of interest", though this is not the main aim of this work.

Strengths:

Due to the particularly nice and tractable cell-based model, the authors are able to perform a thorough analysis to compare the published data to that simulated with their model. They then used their computational model to investigate different growth mechanisms of volume growth and surface growth. With this approach, the authors are able to compare the metric of interest (here, the direction angle of a new mutant clone, the dispersion of mutants throughout the tumour) to quantify how the different growth models compare to the observed data. The authors have also used inference methods to identify model parameters based on the data observed. The authors performed a rigorous analysis and have chosen the metrics in an appropriate manner to compare the different growth mechanisms.

Weaknesses:

The work contained within this article considers a single cell-based model. While ideally, this is sufficient, results from simulated multi-cellular systems can often be sensitive to the model choice. Performing this work with various other standard models would strengthen the results significantly. This is, however, not an easy task.

Context:

Improved mechanistic understanding into the early developmental stages of tumours will further assist in disease treatment and quantification. Understanding how readily and quickly a tumour is evolving is key to understanding how it will develop and progress. This work provides a solid example as to how this can be achieved with data alongside simulated models.

Reviewer #2 (Public Review):

In this study, the authors assess whether selective pressure from drug chemotherapy influences the emergence of drug resistance through the acquisition of genetic mutations or phenotypic tolerance. I commend the authors on their approach of utilizing the mutation accumulation (MA) assay as a means to answer this and whole genome sequencing of clones from the assay convincingly demonstrates low mutation rates in Mycobacteria when exposed to sub-inhibitory concentrations of antibiotics. Also, quantitative PCR highlighted the upregulation of DNA repair genes in Mycobacteria following drug treatment, implying the preservation of genomic integrity via specific repair pathways.

Even though the findings stem from M. smegmatis exposure to antibiotics under in vitro conditions, this is still relevant in the context of the development of drug resistance so I can see where the authors' train of thought was heading in exploring this. However, I think important experiments to perform to more fully support the conclusion that resistance is largely associated with phenotypic rather than genetic factors would have been to either sequence clones from the ciprofloxacin tolerance assay (to show absence/ minimal genetic mutations) or to have tested the MIC of clones from the MA assay (to show an increase in MIC). There seems to be a disconnect between making these conclusions from experiments conducted under different conditions, or perhaps the authors can clarify why this was done. With regards to the sub-inhibitory drug concentration applied, there is significant variation in the viability as calculated by CFUs following the different treatments and there is evidence that cell death greatly affects the calculation of mutation rate (PMCID: PMC5966242). For instance, the COMBO treatment led to 6% viability whilst the INH treatment led to 80% cell viability. Are there any adjustments made to take this into account? It would also be useful to the reader to include a supplementary table of the SNPs detected from the lineages of each treatment - to determine if at any point rifampicin treatment led to mutations in rpoB, isoniazid to katG mutations, etc. Overall, while this study is tantalizingly suggestive of phenotypic tolerance playing a leading role in drug resistance (and perhaps genetic mutations a sub-ordinate role) a more substantial link is needed to clarify this.

Reviewer #2 (Public Review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology, and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

Reviewer #2 (Public Review):

Summary:

The paper entitled "Goal-directed motor actions drive acetylcholine dynamics in sensory cortex" aims to characterize the dynamics of cholinergic signaling in sensory cortex during perceptual behavior. The authors showed that acetylcholine release in S1 was linked to goal-directed motor actions rather than sensory input or reward delivery, a pattern also observed in the auditory cortex (A1). This release was specifically associated with whisking and licking and was potentiated by training. The results contribute to a better understanding of neuromodulator actions. That said, several aspects of the manuscript could benefit from improved writing, data presentation, and statistical analysis.

Strengths:

The evidence provided is clear to link ACh response to different task-related events. Implementing two different tasks to show generality is appreciated. Important control analysis is included.

Weaknesses:

The quantification of ACh signal differences across different trial types or between expert and early-training mice is lacking. Although statistical significance is occasionally mentioned, the indication of significance in figures seems rare. For example, in Figures 5A and E, it is difficult to tell when p is < 0.05. Based on the sentence "small, but significant increase on Hits over False Alarm trials (Figure 5A, S Figure 4A)" there is indeed a time point where the difference is significant, and more details should be added (when and the p-value).

For Figure 5D, it seems like there is no significant difference between Hit and False alarm trials, however, for the trials with 1 or 2 lick there appears to be a difference. Is it due to a lack of power? Moreover, in Figure 5 H the first licks also seem to differ.

Linear regression: the coefficient of determination (R²) is absent, in Figures 4E, F, and 6B, H, making it hard to evaluate the goodness of the fitting.

Similar comments apply to Figure 7: the lack of quantitative comparisons between the coefficients of first lick and other regressors, and between early and expert training, as well as the change in goodness of fit by removing a regressor.

The writing of the introduction and discussion could be improved to enhance readability, and the manuscript could improve its discussion on orofacial movement and acetylcholine release by citing relevant studies demonstrating the association between neuronal activity and orofacial/body movements.

Reviewer #2 (Public Review):

Summary:

While many studies have explored the impacts of pathogens on hosts, the effect of hosts on pathogens has received less attention. In this manuscript, Wang et al. utilize Drosophila melanogaster and an opportunistic pathogen, Serratia marcescens, to explore how the host impacts pathogenicity. Beginning with an observation that larval presence and density impacted microbial growth in fly vials (which they assess qualitatively as the amount of 'slick' and quantitatively as microbial load/CFUs), the authors focus on the impact of axenic/germ-free larvae on an opportunistic pathogen S. marcescens. Similar to their observations with general microbial load, they find that larvae reduce the presence of a pinkish slick of Sm, indicative of its secondary metabolite prodigiosin. The presence of larvae alters prodigiosin production, pathogen load, pathogen cellular morphology, and virulence, and this effect is through transcriptional and metabolic changes in the pathogen. Overall, they observe a loss of virulence factors/pathways and an increase in pathways contributing to growth. Given the important role the host plays in this lifestyle shift, the authors then examined host features that might influence these effects, focusing on the role of antimicrobial peptides (Amps). The authors combine the use of synthetic Amps and an Amp-deficient fly line and conclude much of the larval inhibitory effect is due to their production of AMPs.

Strengths:

This is a very interesting question and the use of Drosophila-Serratia marcescens is a great model to explore these interactions and effects.

The authors have an interesting and compelling phenotype and are asking a unique question on the impact of the host on the pathogen. The use of microbial transcriptomics and metabolomics is a strength, especially in order to assess these impacts on the pathogen level and at single-cell level to capture heterogeneity.

Weaknesses:

Overall, the writing style in the manuscript makes it difficult to fully understand and appreciate the data and its interpretation.

The data on the role of AMPs would benefit from strengthening. Some of the arguments in the text of that section are also counterintuitive. The authors show that AMP larvae have a reduced impact on Sm as compared to wt larvae, but it seems less mild of an effect than that observed with wt excreta (assuming the same as secreta in Figures 7, should be corrected or harmonized). Higher doses of AMPs give a phenotype similar to wt larvae, but a lower dose (40 ng/ul) gives phenotypes more similar to controls. The authors argue that this data suggests AMPs are the factor responsible for much of the inhibition, but their data seems more to support that it's synergistic- you seem to still need larvae (or some not yet defined feature larvae make, although secreta/excreta was not sufficient) + AMPs to see similar effects as wt. Based on positioning and color scheme guessing that AMP 40ng/ul was used in Figures 7D-H, but could not find this detail in the text, methods, or figure legend and it should be indicated. This section does not seem to be well supported by the provided data, and this inconsistency greatly dampened this reviewer's enthusiasm for the paper.

Reviewer #2 (Public Review):

Summary:

The manuscript illustrates how spatial targeting (perisomatic vs distal, apical, and basal dendritic) and timing of inhibition are crucial to distinct effects on neuronal integration and show that beta and gamma oscillations differentially engage dendritic spiking mechanisms.

Strengths:

The strength of this study lies in the integrative biophysical modelling of a layer 5 pyramidal neuron by bringing together in vitro and in vivo observations.

Weaknesses:

The weaknesses are probably in some of the parameterizations of inhibitory synaptic dynamics. A unitary peak conductance of 1nS is very high for inhibitory synapses. This high value could invariably skew some of the network-level predictions. The authors could obtain specific parameters from the Neocortical Collaboration Portal (https://bbp.epfl.ch/nmc-portal/microcircuit.html), which is an incredible resource for cortical neurons and synapses.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors examine the question of whether discrete action sequences and coarticulated continuous sequential actions can be produced from the same controller, without having to derive separate control policies for each sequential movement. Using modeling and behavioral experiments, the authors demonstrate that this is indeed possible if the constraints of the policy are appropriately specified. These results are of interest to those interested in motor sequences, but it is unclear whether these findings can be interpreted to apply to the control of sequences more broadly (see weaknesses below).

Strengths:

The authors provide an interesting and novel extension of the stochastic optimal control model to demonstrate how different temporal constraints can lead to either individual or coarticulated movements. The authors use this model to make predictions about patterns of behavior (e.g., in response to perturbations), which they then demonstrate in human participants both by measuring movement kinematics as well as EMG. Together this work supports the authors' primary claims regarding how changes in task instructions (i.e., task constraints) can result in coarticulated or separated movement sequences and the extent to which the subsequent movement goal affects the planning and control of the previous movement.

Weaknesses:

I reviewed a prior version of this manuscript, and appreciate the authors addressing many of my previous comments. However, there are some concerns, particularly with regard to how the authors interpret their findings.

(1) It would be helpful for the authors to discuss whether they think there is a fundamental distinction between a coarticulated sequence and a single movement passing through a via point (or equivalently, avoiding an obstacle). The notion of a coarticulated sequence brings with it the notion of sequential (sub)movements and temporal structure, whereas the latter can be treated as more of a constraint on the production of a single continuous movement. If I am interpreting the authors' findings correctly it seems they are suggesting that these are not truly different kinds of movements at the level of a control policy, but it would be helpful for the authors to clarify this claim.

(2) The authors' model clearly shows that each subsequent target only influences the movement of one target back, but not earlier ones (page 7 lines 199-204). This stands in contrast to the paper they cite from Kashefi 2023, in which those authors clearly show that people account for at least 2 targets in the future when planning/executing the current movement. It would be useful to know whether this distinction arises because of a difference in experimental methodology, or because the model is not capturing something about human behavior.

(3) In my prior review I raised a concern that the authors seem to be claiming that because they can use a single control policy for both coarticulated and separated movement sequences, there need not be any higher-level or explicit specification of whether the movements are sequential. While much of that language has been removed, it still appears in a few places (e.g., p. 13, lines 403-404). As previously noted, the authors' control policy can generate both types of movements as long as the proper constraints are provided to the model. However, these constraints must be specified somewhere (potentially explicitly, as the authors do by providing them as task instructions). Moreover, in typical sequence tasks, although some movements become coarticulated, people also tend to form chunks with distinct chunk boundaries, which presumably means that there is at least some specification of the sequential ordering of these chunks that must exist (otherwise the authors' model might suggest that people can coarticulate forever without needing to exhibit any chunk boundaries). Hence the authors should limit themselves to the narrow claim that a single control policy can lead to separated or coarticulated movements given an appropriate set of constraints, but acknowledge that their work cannot speak to where or how those constraints are specified in humans (i.e., that there could still be an explicit sequence representation guiding coarticulation).

Reviewer #2 (Public Review):

Summary:

This manuscript aims to tackle the antimicrobial resistance through the development of vaccines. Specifically, the authors test the potential of the RSP protein as a vaccine candidate. The RSP protein contains bacterial Ig-like domains that are typically carried in IncHl1 plasmids like R27. The extracellular location of the RSP protein and its role in the conjugation process makes it a good candidate for a vaccine. The authors then use Salmonella carrying an IncHl plasmid to test the efficacy of the RSP protein as a vaccine antigen in providing protection against infection of antibiotic-resistant bacteria carrying the IncHl plasmid. The authors found no differences in total IgG or IgA levels, nor in pro-inflammatory cytokines between immunized and non-immunized mice. They however found differences in specific IgG and IgA, attenuated disease symptoms, and restricted systemic infection.

The manuscript also evaluates the potential use of nanobodies specifically targeting the RSP protein by expressing it in E. coli and evaluating their interference in the conjugation of IncHl plasmids. The authors found that E. coli strains expressing RSP-specific nanobodies bind to Salmonella cells carrying the R27 plasmid thereby reducing the conjugation efficacy of Salmonella.

Strengths:

- The main strength of this manuscript is that it targets the mechanism of transmission of resistance genes carried by any bacterial species, thus making it broad.

- The experimental setup is sound and with proper replication.

Weaknesses:

- The two main experiments, evaluating the potential of the RSP protein and the effects of nanobodies on conjugation, seem as parts of two different and unrelated strategies.

- The survival rates shown in Figure 1A and Figure 3A for Salmonella pHCM1 and non-immunized mice challenged with Salmonella, respectively, are substantially different. In the same figures, the challenge of immunized mice and Salmonella pHCM1 and mice challenged with Salmonella pHCM1 with and without ampicillin are virtually the same. While this is not the only measure of the effect of immunization, the inconsistencies in the resulting survival curves should be addressed by the authors more thoroughly as they can confound the effects found in other parameters, including total and specific IgG and IgA, and pro-inflammatory cytokines.

- Overall the results are inconsistent and provide only partial evidence of the effectiveness of the RSP protein as a vaccine target.

- The conjugative experiments use very long conjugation times, making it harder to asses if the resulting transconjugants are the direct result of conjugation or just the growth of transconjugants obtained at earlier points in time. While this could be assessed from the obtained results, it is not a direct or precise measure.

- While the potential outcomes of these experiments could be applied to any bacterial species carrying this type of plasmids, it is unclear why the authors use Salmonella strains to evaluate it. The introduction does a great job of explaining the importance of these plasmids but falls short in introducing their relevance in Salmonella.

Reviewer #2 (Public Review):

Summary:

The study is devoted to the deep investigation of the spermatogonial stem cell (SSC) niche in trans women after gender-affirming hormone therapy (GAHT). Both cellular structure and functionality of the niche were studied. The authors evidently demonstrated that all cellular components of SSC niche were affected by hormone therapy. Interestingly, the signs of "rejuvenation" within the niche were also observed indicating the possible reverse to the immature condition.

Strengths:

The obtained findings are important for the better understanding of hormonal regulation of testis and SSC niche and provide some clues for using the biomaterials from these specific and even unique donors for biomedical research.

Weaknesses:

This study has some limitations. Many studies can't be done using the testes cells of trans women, since their cells are significantly different from adult man cells and less from prepubertal and pubertal cells. The authors themselves identify some of the limitations: this material is suitable only for studying prepubertal processes in the testis. However, the authors also report large variability in data due to different hormonal therapy regimens and, apparently, age. Accordingly, not all material obtained from trans women can also be used for studies of prepubertal processes.

Reviewer #2 (Public Review):

Summary:

Bowman and colleagues have compiled a large comparative genomic dataset to examine the molecular evolution of genes in mammals, with the primary goal of identifying how changes in the gorilla mating system have shaped the evolution of spermatogenesis. They report several patterns pointing to signal of relaxed purifying selection on genes involved in male fertility, a pattern that they interpret as a response to changes in the mating system of gorillas. Many previous studies have used comparisons among species of primates and other mammals to understand how changes in mating systems have shaped the evolution or reproductive traits and genes. These collective works have provided some of the best evidence that changes in the form and intensity of sexual selection has had a strong effect on the evolution of male reproduction. The current study builds on this rich history by exploring molecular evolution of over 13,310 genes across 261 mammals. This very large phylogenetic dataset allows affords considerable power to characterize patterns of molecular evolution along the gorilla lineage. This allows for some added power relative to a previous study that interrogated the same lineage-specific patterns (Scally et al. 2021). They report a subset of genes showing evidence for either positive directional selection (less than 1% of genes) or relaxed purifying selection (4% of genes) in gorillas. Relaxed purifying selection is more common than positive selection, and genes showing signatures of relaxed constraint are enriched for spermatogenesis functions using various tests based on functional annotation or gene expression and infertility associations in humans and mice. The authors also report new functional data - the only original data in this study - using a high throughput genetic screen showing that some of these genes are also expressed in spermatogenesis in flies, and when perturbed they affect male fertility.

These results are interpreted as strong evidence that changes in mating system, specifically that loss of sperm competition, has shaped the evolution of male reproduction in gorillas. The authors argue that these discoveries illustrate, for the first time, the genome-wide effect of striking changes in mating behavior in gorillas on the genetic underpinnings of male reproduction and provide new candidates relevant to male fertility in humans. Support for these central conclusions is eroded by a lack of appropriate comparative contrasts needed clarify the uniqueness of these patterns to gorillas and, critically, establish a direct phylogenetic association with mating system or correlated reproductive traits.

Strengths:

The presentation is engaging, clear, and easy to follow throughout. I enjoyed reading the overall narrative and I think that the authors did a good job of presenting the details of male reproductive biology in an informative and accessible manner. Given the general interest in gorilla evolution, and the clear relevance to humans, studies of this scope on male reproductive biology are likely to be of broad interest to both evolutionary and reproductive biologists.

The reported signatures of molecular evolution in gorillas appear robust, well-executed, and supported by several lines of evidence that establish some links with male reproduction. The authors have presented a series of molecular evolution analyses that demonstrate both rigor and attention to analytical details and quality control. Although all the primary sequence data has been previously published by others, the compilation of a high-quality curated comparative dataset of this scale is impressive and inspires confidence in the underlying molecular results. Likewise, the incorporation of diverse other data from mice and humans helps shape the overall narrative. To my knowledge, this represents the most focused and detailed analysis of protein-coding evolution specific to gorillas to date (although parallel results from the landmark gorilla genome study - Scally et al. 2012 - are downplayed somewhat).

Likewise, the inclusion of new functional data from Drosophila establishes a subset of genes showing recent changes in molecular evolution in gorillas that appear to be both deeply conserved in animals and related to male fertility.

Weaknesses:

This study lacks the necessary comparative framework needed to ascribe any of the reported patterns to changes in the reproductive system of gorillas, or to really understand the uniqueness of these patterns relative to other species. Although wording is careful at times, the authors repeatedly ascribe the patterns they are finding directly to the specific changes in mating system biology that has occurred in gorillas. The general framing and significance rests on the central finding that "these data provide compelling evidence that reduced sperm competition in gorillas is associated with relaxed purifying selection on genes related to male reproductive function (Abstract)". No such association between variation in mating system or at any correlated reproductive traits and molecular evolution is ever directly tested let alone established as a clear statistical correlation. The massive comparative dataset is used to localize patterns of molecular evolution to the gorilla lineage and then these patterns are interpreted in the context of changes in mating system, as an assumption of the study not a direct result. Although basic information of the reproductive system (or correlates thereof) likely exists for many of the 261 species included here, this information is never used to test for a relationship between changes in positive or purifying selection and reproduction.

The lack of any such comparisons is especially curious given that there are many previous studies that have sought and established such connections for traits and/or genes in mammals (dozens now?), and especially great apes, before. This comparative approach is the gold standard to making claims linking mating system to molecular evolution and yet this is not pursued here. The authors are correct in that they provide a rigorous genome-wide analysis (but not at all for the first time, see Scally et al. 2012), but they skip this critical central step to rigorous inference in comparative genomics. This is essentially a broad comparative study, but the central conclusion (a direct link between mating system and molecular evolution) is speculative and not actually tested.

Note that despite the framing here, there are of course several aspects of lineage specific biology that undoubtedly shape molecular evolution of male reproduction and fertility but could be unrelated to sperm competition per se. For example, shift in operational sex ratios can have profound effects on effective population sizes and the efficacy of selection, which of course would be expected to change the intensity and direction of molecular evolution. Likewise, shifts in population size, structure, and diet all can affect molecular evolution and reproduction.

In the absence of a broad phylogenetically independent contrast (which would be really interesting here), the authors need to at least establish that there is indeed something noteworthy about the specific findings they report relative to other systems that have a different mating system. Such comparisons would be readily available within the great apes, especially compared to chimpanzees and bonobos (Pan). Most of the patterns are presented in such a way to suggest a clear connection between the result and the unique features of gorilla reproduction, but are these clearly outliers? Relaxed purifying selection is much more common than positive selection, is this result qualitatively or quantitatively unique to gorillas as implied (I would honestly be surprised if it was as this is a common outcome of these dn/ds-based tests)? Similar questions and the need for more context apply to the various enrichment tests. That genes involved in male reproduction evolve rapidly and that this reflects both relaxed constraint and positive selection is an exceptionally well-established pattern, as is enrichment for reproductive functions/expression of such genes in unbiased genome-wide screens (as cited by the authors, including in gorillas by Scally et al. 2012 who performed a very similar analysis albeit with some model advances used in the current study). Do chimpanzees or humans lack these specific signatures of relaxed constraint at reproductive genes or is it a much stronger enrichment in gorillas? Establishing these baseline comparisons would help a lot with interpretation of the core findings. A little bit of this is explored with the human comparisons but not in a parallel genome-wide manner that places the signatures in gorillas in context.

I had similar questions related to the high-throughput Drosophila screen. This is a creative and novel component of the study. However, I am unclear on how to interpret the results or the conclusions drawn from them. It is very interesting that a subset of genes showing relaxed constraint are conserved to Drosophila and that perturbation of some of these cause fertility issues. However, the conclusion that these genes reflect novel candidates not implicated in sperm biology is a bit overstated. Here implicated means genes with an annotated sterility phenotype in humans, mice, flies, or gorillas - specific annotations which are pretty limited at least in the mammalian systems. The entire design was conditioned on analyzing genes that were reliably expressed during Drosophila spermatogenesis, and then focusing on those. But the comparative set for the enrichment test was a random set of genes. Shouldn't the background be a random set of testis-expressed genes? I would say that genes that are reliably expressed during spermatogenesis in both mammals and flies are implicated in sperm biology and genetic manipulation of such genes would be expected to produce fertility phenotypes at some appreciable rate. So the result here adds some interesting data but it does not seem unexpected or significant as framed.

Reviewer #2 (Public Review):

Summary:

The paper 'Sequence characteristic and an accurate model of abundant hyperactive loci in human genome' by Hydaiberdiev and Ovcharenko offers comprehensive analyses and insights about the 'high-occupancy target' (HOT) loci in the human genome. These are considered genomic regions that overlap with transcription factor binding sites. The authors provided very comprehensive analyses of the TF composition characteristics of these HOT loci. They showed that these HOT loci tend to overlap with annotated promoters and enhancers, GC-rich regions, open chromatin signals, and highly conserved regions, and that these loci are also enriched with potentially causal variants with different traits.

Strengths:

Overall, the HOT loci' definition is clear and the data of HOT regions across the genome can be a useful dataset for studies that use HepG2 or K562 as a model. I appreciate the authors' efforts in presenting many analyses and plots backing up each statement.

Weaknesses:

It is noteworthy that the HOT concept and their signature characteristics as being highly functional regions of the genome are not presented for the first time here. Additionally, I find the main manuscript, though very comprehensive, long-winded and can be put in a shorter, more digestible format without sacrificing scientific content.

The introduction's mention of the blacklisted region can be rather misleading because when I read it, I was anticipating that we are uncovering new regulatory regions within the blacklisted region. However, the paper does not seem to address the question of whether the HOT regions overlap, if any, with the ENCODE blacklisted regions afterward. This plays into the central assessment that this manuscript is long-winded.

The introduction also mentioned that HOT regions correspond to 'genomic regions that seemingly get bound by a large number of TFs with no apparent DNA sequence specificity' (this point of 'no sequence specificity' is reiterated in the discussion lines 485-486). However, later on in the paper, the authors also presented models such as convolutional neural networks that take in one-hot-encoded DNA sequence to predict HOT performed really well. It means that the sequence contexts with potential motifs can still play a role in forming the HOT loci. At the same time, lines 59-60 also cited studies that "detected putative drive motifs at the core segments of the HOT loci". The authors should edit the manuscript to clarify (or eradicate) contradictory statements.

Reviewer #2 (Public Review):

Summary:

In this study, Styer et al. impose artificial selection on root-associated microbiomes to increase drought tolerance in rice plants using different soils as starting microbiomes. Using NDVI and biomass as a proxy for plant health, they find that iterative passaging of the microbiomes of the best-performing plants increased plant resilience to drought stress in a soil-dependent manner. The study makes use of numerous controls. The authors survey the microbiota of the plants across generations, using an array of interesting analyses to characterize their observations. Firstly, the authors find that the acquired microbiomes are divergent towards the beginning of the selection experiment, but nearly converge later suggesting that the selected communities become more similar over time. One reason is that the diversity of the microbiomes severely decreases after only one or two generations of selection AND that microbes from each inoculation source appear to easily disperse across the experiment, leading to microbiome homogeneity. The authors then present an analysis to correlate ASVs with the NDVI and Biomass over the course of the experiment (using the rice soil selection lines) to develop hypotheses about which ASVs may impact plant traits.

Strengths:

The authors set out to refine the understanding of microbiome artificial selection, a topic of recent interest to the plant microbiome field. The authors use an established approach (Mueller et al), expanding upon it by including multiple starting soil inocula to ask whether the strength of selection varies by input microbiome. This is an important and novel question. Using drought resilience as measured by NDVI and plant biomass to select upon was a wise choice for this type of study, given their relative ease and quickness to assess. The inclusion of several types of controls, multiple selection lines, and several starting soil inocula showed a thoughtful experimental design. The analyses were diverse, non-standard, and attempted to address microbiome dynamics on multiple fronts. I am not necessarily convinced by some of the conclusions (see below), however, I think this study examines an important and exciting topic in the area of plant microbiomes. I predict the findings of the experiments will inform a wide audience of researchers attempting similar studies and be helpful in their designs.

Weaknesses:

Although the controls were well designed, the dispersal of the microbiomes erased the utility of the sterile inoculated (SI) controls, at least from my reading of the manuscript. Perhaps the original intent of the SI plants was to contrast the selected microbiomes vs axenic plants to show that plant resilience to drought increased generation after generation. If the controls had worked properly under my presumed scenario, this would allow the authors to account for batch variation across the generations (due to slight differences in MS media prep, water quality, etc.). Instead, the SI lines acquired microbes from the experiment and never appeared to significantly deviate from the SL plants. The dispersal of the microbes amongst soils and selection lines also minimizes any conclusions that can be made about the different starting inocula and how prone to selection they may be.

Reviewer #2 (Public Review):

This is a short and unpretentious paper. It is an interesting area and therefore, although much of this area of research was pioneered in flies, extending basic findings to butterflies would be worthwhile. Indeed, there is an intriguing observation but it is technically flawed and these flaws are serious.

The authors show that mirror is expressed at the back of the wing in butterflies (as in flies). They present some evidence that is required for the proper development of the back of the wing in butterflies (a region dubbed the vannus by the ancient guru Snodgrass). But there are problems with that evidence. First, concerning the method, using CRISP they treat embryos and the expectation is that the mirror gene will be damaged in groups of cell lineages, giving a mosaic animal in which some lines of cells are normal for mirror and others are not. We do not know where the clones or patches of cells that are defective for mirror are because they are not marked. Also, we do not know what part of the wing is wild type and what part is mutant for mirror. When the mirror mutant cells colonise the back of the wing and that butterfly survives (many butterflies fail to develop), the back of the wing is altered in some selected butterflies. This raises a second problem: we do not know whether the rear of the wing is missing or transformed. From the images, the appearance of the back of the wing is clearly different from the wild type, but is that due to transformation or not? And then I believe we need to know specifically what the difference is between the rear of the wing and the main part. What we see is a silvery look at the back that is not present in the main part, is it the structure of the scales? We are not told. There are other problems. Mirror is only part of a group of genes in flies and in flies both iroquois and mirror are needed to make the back of the wing, the alula (Kehl et al). What is known about iro expression in butterflies?

In flies, mirror regulates a late and local expression of dpp that seems to be responsible for making the alula. What happens in butterflies? Would a study of the expression of Dpp in wildtype and mirror compromised wings be useful?

Thus, I find the paper to be disappointing for a general journal as it does little more than claim what was discovered in Drosophila is at least partly true in butterflies. Also, it fails to explain what the authors mean by "wing domains" and "domain specification". They are not alone, butterfly workers, in general, appear vague about these concepts, their vagueness allowing too much loose thinking.

Since these matters are at the heart of the purpose and meaning of the work reported here, we readers need a paper containing more critical thought and information. I would like to have a better and more logical introduction and discussion.

The authors do define what they mean by the vannus of the wing. In flies the definition of compartments is clear and abundantly demonstrated, with gene expression and requirement being limited precisely to sets of cells that display lineage boundaries. It is true that domains of gene expression in flies, for example of the iroquois complex, which includes mirror, can only be related to patterns with difficulty. Some recap of what is known plus the opinion of the authors on how they interpret papers on possible lineage domains in butterflies might also be useful as the reader, is no wiser about what the authors might mean at the end of it!

The references are sometimes inappropriate. The discovery of the AP compartments should not be referred to Guillen et al 1995, but to Morata and Lawrence 1975. Proofreading is required.

Reviewer #2 (Public Review):

This paper reports investigations of chromosome stiffness in oocytes and spermatocytes. The paper shows that prophase I spermatocytes and MI/MII oocytes yield high Young Modulus values in the assay the authors applied. Deficiency in each one of three meiosis-specific cohesins they claim did not affect this result and increased stiffness was seen in aged oocytes but not in oocytes treated with the DNA-damaging agent etoposide.

The paper reports some interesting observations which are in line with a report by the same authors of 2020 where increased stiffness of spermatocyte chromosomes was already shown. In that sense, the current manuscript is an extension of that previous paper, and thus novelty is somewhat limited. The paper is also largely descriptive as it does neither propose a mechanism nor report factors that determine the chromosomal stiffness.

There are several points that need to be considered.

(1) Limitations of the study and the conclusions are not discussed in the "Discussion" section and that is a significant gap. Even more so as the authors rely on just one experimental system for all their data - there is no independent verification - and that in vitro system may be prone to artefacts.

(2) It is somewhat unfortunate that they jump between oocytes and spermatocytes to address the cohesin question. Prophase I (pachytene) spermatocytes chromosomes are not directly comparable to MI or MII oocyte chromosomes. In fact, the authors report Young Modulus values of 3700 for MI oocytes and only 2700 for spermatocyte prophase chromosomes, illustrating this difference. Why not use oocyte-specific cohesin deficiencies?

(3) It remains unclear whether the treatment of oocytes with the detergent TritonX-100 affects the spindle and thus the chromosomes isolated directly from the Triton-lysed oocytes. In fact, it is rather likely that the detergent affects chromatin-associated proteins and thus structural features of the chromosomes.

(4) Why did the authors use mouse strains of different genetic backgrounds, CD-1, and C57BL/6? That makes comparison difficult. Breeding of heterozygous cohesin mutants will yield the ideal controls, i.e. littermates.

(5) How did the authors capture chromosome axes from STAG3-deficienct spermatocytes which feature very few if any axes? How representative are those chromosomes that could be captured?

Reviewer #2 (Public Review):

Summary:<br /> The paper explores a mathematical model of subsecond time perception, engaging with established theories such as the linear psychophysical law, Weber's law, and dopaminergic modulation of subjective durations. While it ambitiously attempts to confirm specific mechanisms of time perception and presents a comprehensive description of these mechanisms, the work is presented as data-driven but its empirical backing and model generalization capabilities are questionable. The title's implication of a robust empirical foundation is misleading, as the main figures do not reflect empirical data directly but rather model outputs aligned with general trends in psychophysical studies. This disjunction raises concerns about the model's applicability and the strength of the claims made regarding time perception mechanisms.

Strengths:<br /> (1) The paper describes specific mechanisms of time perception, providing a theoretical examination of linear psychophysical law, Weber's law, and dopaminergic modulation. This aspect is valuable for readers seeking a theoretical understanding of temporal perception.

(2) The authors describe a range of psychophysical studies and theories, attempting to position their model within the broader scientific discourse on time perception.

Weaknesses:<br /> (1) Lack of Empirical Data: The absence of two things: 1) quantification of error between model and empirical data with interpretation of what this degree of error means, and 2) clear comparisons between model and empirical data in all figures and tables, to substantiate the model's predictions stands out. The reliance on general trends rather than specific empirical studies undermines the strength and reliability of the model's claims. The paper would benefit from quantitative and qualitative simulations of results from specific, large-sample studies to anchor the model's predictions in concrete empirical evidence.

(2) Methodological Ambiguities: The training and testing procedures lack robust checks for generalization, leading to potential overfitting issues. Clarifications are needed on whether and how the model reaches a steady state before stimulation and the implications of the chosen model time constants in the absence of stimulation. The overlap between training (50ms) and testing (25ms) steps and the implications for model generalization need validation with "traditional" parameter fitting protocols, such as formal model cross-validation across well-defined datasets and splits, as well as evaluations to understand and assess potential overfitting.

(3) Inadequate Visualization of Empirical Data: References to empirical data are vague and not directly visualized alongside model outputs. Future iterations should include empirical data, not general trends from psychophysics, in figures for a clear comparison.

(4) Limitations in Model Scope and Dynamics: The exploration of limitations is narrowly focused on interval length and noise. Expanding the model limitations to consider isochronous pulse processing and the emergence of limit-cycle behaviors after prolonged stimulation would provide a more comprehensive understanding of the model's capabilities and limitations. Additionally, the justification for using \(N_{Poisson}\) as a proxy for more connections is unclear and warrants a more direct approach. Adding more units to a truly data-driven model should be trivial.

(5) Omissions and Redundancies: Certain omissions, such as the lack of a condition in Figure 7A or missing references to relevant models and reviews, detract from the paper's thoroughness. Moreover, some statements and terms like "internal clock" are used without a clear mechanistic definition within the model.

Guidance for Readers<br /> Readers should approach this paper as a theoretical exploration into the mechanisms of subsecond-time perception. The model offers a detailed theoretical framework that engages with established laws and theories in time perception. However, it's crucial to note the model's reliance on general trends and its lack of direct empirical backing. The findings should be interpreted as a hypothesis-generating exercise rather than conclusive evidence.

Reviewer #3 (Public Review):

Summary:

The authors are trying to find a vaccine solution for invasive candidiasis.

Strengths:

The testing of the antifungal activity of EDTA on Candida is not new as many other papers have examined this effect. The novelty here is on the use of this such EDTA treated strain as a vaccine to protect against a secondary challenge with wild-type Candida.

Weaknesses:

However, data presented in Fig. 5 and in Fig. 6 are not convincing and need further experimental controls and analysis as the authors do not show a time-dependent effect on the CFU of their vaccine formulation. Specific points are below.

Methodology used is also an issue. As it stands, the impact is minor, if any.

Comments on revised version:

The data provided in the revised paper are simply not satisfactory and do not give confidence that a rigorous design and methodologies were used to obtain the results illustrated in this paper.

Reviewer #2 (Public Review):

Microbial degradation of synthetic organic compounds is the basis of bioremediation. Biodegradation of 1NA has not been previously reported. The report describes a complete study of 1NA biodegradation by a new isolate Pseudomonas sp. strain JS3066. The study includes the enrichment and isolation of the 1NA-degrading bacterium Pseudomonas sp. strain JS3066, the identification of the genes and enzymes involved in 1NA degradation, and the detailed characterization of γ-glutamylorganoamide synthetase by using biochemical and structural analysis. In the discussion, the potential evolution of 1NA degradation pathway, the similarity and difference between γ-glutamylorganoamide synthetase and glutamine synthetase, and the significance were explained. The conclusions were well supported by the results presented.

Reviewer #2 (Public Review):

The paper by Gilbert et al. is well-written in a detailed format and the authors are candid in their data interpretation by acknowledging that the described ninein bone defects are mild, transient, and do not lead to major long-lasting defects in adulthood.

The main strength of the study is presenting a novel link between a centrosomal protein and osteoclasts in the mouse. However, the majority of the work is dedicated to describing the premature ossification phenotype and less attention is paid to how a centrosomal protein affects osteoclast proliferation, survival, and/or differentiation into mature osteoclasts.

Based on the decrease in the number of osteoclasts (Fig 5E, G, and also per coverslip after 2 days in culture), the authors suggest that the loss of ninein impacts osteoclast proliferation. First, proliferation can be directly quantified using Ki67 staining or EdU incorporation. Second, other interpretations are also plausible and can also be experimentally tested. These include less adhesion and attachment of the mutants to the coverslips, but perhaps more relevant in vivo is cell death of the ninein mutant osteoclasts. It has been established that the loss of centrosome function activates p53-dependent cell death and osteoclasts might be a vulnerable cell population. Quantifying p53 immunoreactivity and/or cell death in osteoclasts might help clarify the phenotype of osteoclast reduction.

Given many updates are merged at the same time pointers are = many. But given we merge right away having learned an update - we always merge two updates - hence two pointers.

Reviewer #2 (Public Review):

The research uses a large collection of Arabidopsis thaliana accessions from various geographic scales to investigate the natural genetic variation underlying the response of ionome (elemental) composition to elevated CO2 (eCO2), a concern for future food security. While most accessions show a decrease in elemental accumulation, the authors demonstrate a wide variety of responses to eCO2 across the diversity of Arabidopsis, including lines that increase elemental content in eCO2. The demonstration of genetic diversity in eCO2 response is a significant contribution to our understanding of this important phenomenon.

Comments on revised version:

The authors made significant improvements in the manuscript from the original preprint, and the conclusions are now well supported by the evidence presented.

Reviewer #3 (Public Review):

Summary:

Zai et al. test whether birds can modify their vocal behavior in a manner consistent with planning. They point out that while some animals are known to be capable of volitional control of vocalizations, it has been unclear if animals are capable of planning vocalizations-that is, modifying vocalizations towards a desired target without the need to learn this modification by practising and comparing sensory feedback of practised behavior to the behavioral target. They study zebra finches that have been trained to shift the pitch of song syllables away from their baseline values. It is known that once this training ends, zebra finches have a drive to modify pitch so that it is restored back to its baseline value. They take advantage of this drive to ask whether birds can implement this targeted pitch modification in a manner that looks like planning, by comparing the time course and magnitude of pitch modification in separate groups of birds who have undergone different manipulations of sensory and motor capabilities. A key finding is that birds who are deafened immediately before the onset of this pitch restoration paradigm, but after they have been shifted away from baseline, are able to shift pitch partially back towards their baseline target. In other words, this targeted pitch shift occurs even when birds don't have access to auditory feedback, which argues that this shift is not due to reinforcement-learning-guided practice, but is instead planned based on the difference between an internal representation of the target (baseline pitch) and current behavior (pitch the bird was singing immediately before deafening).

The authors present additional behavioral studies arguing that this pitch shift requires auditory experience of song in its state after it has been shifted away from baseline (birds deafened early on, before the initial pitch shift away from baseline, do not exhibit any shift back towards baseline), and that a full shift back to baseline requires auditory feedback. The authors synthesize these results to argue that different mechanisms operate for small shifts (planning, which does not need auditory feedback) and large shifts (through a mechanism that requires auditory feedback).

The authors also make a distinction between two kinds of planning: covert-not requiring any motor practice and overt-requiring motor practice but without access to auditory experience from which target mismatch could be computed. They argue that birds plan overtly, based on these deafening experiments as well as an analogous experiment involving temporary muting, which suggests that indeed motor practice is required for pitch shifts.

Strengths:

The primary finding (that partially restorative pitch shift occurs even after deafening) rests on strong behavioral evidence. It is less clear to what extent this shift requires practice, since their analysis of pitch after deafening takes the average over within the first two hours of singing. If this shift is already evident in the first few renditions then this would be evidence for covert planning. Technical hurdles, such as limited sample sizes and unstable song after surgical deafening, make this difficult to test. (Similarly, the authors could test whether the first few renditions after recovery from muting already exhibit a shift back towards baseline.)

This work will be a valuable addition to others studying birdsong learning and its neural mechanisms. It documents features of birdsong plasticity that are unexpected in standard models of birdsong learning based on reinforcement and are consistent with an additional, perhaps more cognitive, mechanism involving planning. As the authors point out, perhaps this framework offers a reinterpretation of the neural mechanisms underlying a prior finding of covert pitch learning in songbirds (Charlesworth et al., 2012).

A strength of this work is the variety and detail in its behavioral studies, combined with sensory and motor manipulations, which on their own form a rich set of observations that are useful behavioral constraints on future studies.

Weaknesses:

The argument that pitch modification in deafened birds requires some experience hearing their song in its shifted state prior to deafening (Fig. 4) is solid but has an important caveat. Their argument rests on comparing two experimental conditions: one with and one without auditory experience of shifted pitch. However, these conditions also differ in the pitch training paradigm: the "with experience" condition was performed using white noise training, while the "without experience" condition used "lights off" training (Fig. 4A). It is possible that the differences in ability for these two groups to restore pitch to baseline reflects the training paradigm, not whether subjects had auditory experience of the pitch shift. Ideally, a control study would use one of the training paradigms for both conditions, which would be "lights off" or electrical stimulation (McGregor et al. 2022), since WN training cannot be performed in deafened birds. In the Discussion, in response to this point, the authors point out that birds are known to recover their pitch shift if those shifts are driven using electrical stimulation as reinforcement (McGregor et al. 2022); however, it is arguably still relevant to know whether a similar recovery occurs for the "lights off" paradigm used here.

Reviewer #2 (Public Review):

Summary:

Several publications during the past years provided evidence that NMD protects tumor cells from being recognized by the immune system by suppressing the display of neoantigens, and hence NMD inhibition is emerging as a promising anti-cancer approach. However, the lack of an efficacious and specific small-molecule NMD inhibitor with suitable pharmacological properties is currently a major bottleneck in the development of therapies that rely on NMD inhibition. In this manuscript, the authors describe their screen for identifying NMD inhibitors, which is based on isogenic cell lines that either express wild-type or NMD-sensitive transcript isoforms of p53 and STAG2. Using this setup, they screened a library of 2658 FDA-approved or late-phase clinical trial drugs and had 8 hits. Among them they further characterized LY3023414, showing that it inhibits NMD in cultured cells and in a mouse xenograft model, where it, however, was very toxic. Because LY3023414 was originally developed as a PI3K inhibitor, the authors claim that it inhibits NMD by inhibiting SMG1. While this is most likely true, the authors do not provide experimental evidence for this claim. Instead, they use this statement to switch their attention to another previously developed SMG1 inhibitor (SMG1i-11), of which they design and test several derivatives. Of these derivatives, KVS0001 showed the best pharmacological behavior. It upregulated NMD-sensitive transcripts in cultured cells and the xenograft mouse model and two predicted neoantigens could indeed be detected by mass spectrometry when the respective cells were treated with KVS0001. A bispecific antibody targeting T cells to a specific antigen-HLA complex led to increased IFN-gamma release and killing of cancer cells expressing this antigen-HLA complex when they were treated with KVS0001. Finally, the authors show that renal (RENCA) or lung cancer cells (LLC) were significantly inhibited in tumor growth in immunocompetent mice treated with KVS0001. Overall, this establishes KVS0001 as a novel and promising ant-cancer drug that by inhibiting SMG1 (and therewith NMD) increases the neoantigen production in the cancer cells and reveals them to the body's immune system as "foreign".

Strengths:

The novelty and significance of this work consists in the development of a novel and - judging from the presented data - very promising NMD inhibiting drug that is suitable for applications in animals. This is an important advance for the field, as previous NMD inhibitors were not specific, lacked efficacy, or were very toxic and hence not suitable for animal application. It will be still a long way with many challenges ahead towards an efficacious NMD inhibitor that is safe for use in humans, but KVS0001 appears to be a molecule that bears promise for follow-up studies. In addition, while the idea of inhibiting NMD to trigger neoantigen production in cancer cells and so reveal them to the immune system has been around for quite some time, this work provides ample and compelling support for the feasibility of this approach, at least for tumors with a high mutational burden.

Main weaknesses:

There is a disconnect between the screen and the KVS0001 compound, that they describe and test in the second part of the manuscript since KVS0001 is a derivative of the SMG1 inhibitors developed by Gopalsamy et al. in 2012 and not of the lead compound identified in the screen (LY3023414). Because of high toxicity in the mouse xenograft experiments, the authors did not follow up LY3023414 but instead switched to the published SMG1i-11 drug of Gopalsamy and colleagues, a molecule that is widely used among NMD researchers for NMD inhibition in cultured cells. Therefore, in my view, the description of the screen is obsolete, and the paper could just start with the optimization of the pharmacological properties of SMG1i-11 and the characterization of KVS0001. Even though the screen is based on an elegant setup and was executed successfully, it was ultimately a failure as it didn't reveal a useful lead compound that could be further optimized.

Additional points:

- Compared to SMG1i-11, KVS0001 seems less potent in inhibiting SMG1 (higher IC50). It would therefore be important to also compare the specificity of both drugs for SMG1 over other kinases at the applied concentrations (1 uM for SMG1i-11, 5 uM for KVS0001). The Kinativ Assay (Fig. S13) was performed with 100 nM KVS0001, which is 50-fold less than the concentration used for functional assays and hence not really meaningful. In addition, more information on the pharmacokinetic properties and toxicology of KVS0001 would allow a better judgment of the potential of this molecule as a future therapeutic agent.

- On many figures, the concentrations of the used drugs are missing. Please ensure that for every experiment that includes drugs, the drug concentration is indicated.

- Do the authors have an explanation for why LY3023414 has a much stronger effect on the p53 than on the STAG2 nonsense allele (Figure 1B, S8), whereas emetine upregulates the STAG2 nonsense alleles more than the p53 nonsense allele (Figure S5). I find this curious, but the authors do not comment on it.

- While it is a strength of the study that the NMD inhibitors were validated on many different truncation mutations in different cell lines, it would help readers if a table or graphic illustration was included that gives an overview of all mutant alleles tested in this study (which gene, type of mutation, in which cell type). In the current version, this information is scattered throughout the manuscript.

- Lines 194 and 302: That SMG1i-11 was highly insoluble in the hands of the authors is surprising. It is unclear why they used variant 11j, since variant 11e of this inhibitor is widely used among NMD researchers and readily dissolves in DMSO.

- Line 296: The authors claim that they were able to show that LY3023414 inhibited the SMG1 kinase, which is not true. To show this, they would have for example to show that LY3023414 prevents SMG1-mediated UPF1 phosphorylation, as they did for KVS0001 and SMG1i-11 in Fig. 3F. Unless the authors provide this data, the statement should be deleted or modified.

Reviewer #3 (Public Review):

This study seeks to investigate one aspect of disparity in academia: how gender balance in a discipline is valued in terms of evaluated research quality score and funding success. This is important in understanding disparities within academia.<br /> This study uses publicly available data to investigate covariation between gender balance in an academic discipline and:<br /> i) Individual research quality scores of New Zealand academics as evaluated by one of 14 broader subject panels.<br /> ii) Funding success in Australia, Canada, Europe, UK.

The study would benefit from further discussion of it limitations, and from the clarification of some technical points (as described in the recommendations for the authors).

Reviewer #2 (Public Review):

Summary

Recent evidence indicates that cells of the navigation system representing different directions and whole spatial routes fire in a rhythmic alternation during 5-10 Hz (theta) network oscillation (Brandon et al., 2013, Kay et al., 2020). This phenomenon of theta cycle skipping was also reported in broader circuitry connecting the navigation system with the cognitive control regions (Jankowski et al., 2014, Tang et al., 2021). Yet nothing was known about the translation of these temporally separate representations to midbrain regions involved in reward processing as well as the hypothalamic regions, which integrate metabolic, visceral, and sensory signals with the descending signals from the forebrain to ensure adaptive control of innate behaviors (Carus-Cadavieco et al., 2017). The present work aimed to investigate theta cycle skipping and alternating representations of trajectories in the lateral septum, neurons of which receive inputs from large number of CA1 and nearly all CA3 pyramidal cells (Risold and Swanson, 1995). While spatial firing has been reported in the lateral septum before (Leutgeb and Mizumori, 2002, Wirtshafter and Wilson, 2019), its dynamic aspects have remained elusive. The present study replicates the previous findings of theta-rhythmic neuronal activity in the lateral septum and reports a temporal alternation of spatial representations in this region, thus filling an important knowledge gap and significantly extending the understanding of the processing of spatial information in the brain. The lateral septum thus propagates the representations of alternative spatial behaviors to its efferent regions. The results can instruct further research of neural mechanisms supporting learning during goal-oriented navigation and decision-making in the behaviourally crucial circuits entailing the lateral septum.

Strengths

To this end, cutting-edge approaches for high-density monitoring of neuronal activity in freely behaving rodents and neural decoding were applied. Strengths of this work include comparisons of different anatomically and probably functionally distinct compartments of the lateral septum, innervated by different hippocampal domains and projecting to different parts of the hypothalamus; large neuronal datasets including many sessions with simultaneously recorded neurons; consequently, the rhythmic aspects of the spatial code could be directly revealed from the analysis of multiple spike trains, which were also used for decoding of spatial trajectories; and comparisons of the spatial coding between the two differently reinforced tasks.

Weaknesses

Without using perturbation techniques, the present approach could not identify the aspects of the spatial code actually influencing the generation of behaviors by downstream regions.

Reviewer #2 (Public Review):

Summary:

This manuscript builds on previous work suggesting that the CCK peptide is the releasing hormone for FSH in fishes, which is different than that observed in mammals where both LH and FSH release are under the control of GnRH. Based on data using calcium imaging as a readout for stimulation of the gonadotrophs, the researchers present data supporting the hypothesis that CCK stimulates FSH-containing cells in the pituitary. In contrast, LH-containing cells show a weak and variable response to CCK but are highly responsive to GnRH. Data are presented that support the role of CCK in the release of FSH. Researchers also state that functional overlap exists in the potency of GnRH to activate FSH cells, thus the two signalling pathways are not separate.

The results are of interest to the field because for many years the assumption has been that fishes use the same signalling mechanism. These data present an intriguing variation where a hormone involved in satiation acts in the control of reproduction.

Strengths:

The strengths of the manuscript are that researchers have shed light on different pathways controlling reproduction in fishes.

Weaknesses:

Weaknesses are that it is not clear if multiple ligand/receptors are involved (more than one CCK and more than one receptor?). The imaging of the CCK terminals and CCK receptors needs to be reinforced.

Reviewer consultation summary:

- The data presented establish sufficiency, but not necessity of CCK in FSH regulation. The paper did not show that CCK endogenously regulates FSH in fish. This has not been established yet.

- The paper presents the pharmacological effects of CCK on ex vivo preparations but does not establish the in vivo physiological function of the peptide. The current evidence for a novel physiological regulatory mechanism is incomplete and would require further physiological experiments. These could include the use of a CCK receptor antagonist in adult fish to see the effects on FSH and LH release, the generation of a CCK knockout, or cell-specific genetic manipulations.

- Zebrafish have two CCK ligands: ccka, cckb and also multiple receptors: cckar, cckbra and cckbrb. There is ambiguity about which CCK receptor and ligand are expressed and which gene was knocked out.

- Blocking CCK action in fish (with receptor KO) affects FSH and LH. Therefore, the work did not demonstrate a selective role for CCK in FSH regulation in vivo and any claims to have discovered FSHRH need to be more conservative.

- The labelling of the terminals with anti-CCK looks a lot like the background and the authors did not show a specificity control (e.g. anti-CCK antibody pre-absorbed with the peptide or anti-CCK in morphant/KO animals).

Reviewer #2 (Public Review):

Summary:

The physiology and behaviour of animals are regulated by a huge variety of neuropeptide signalling systems. In this paper, the authors focus on the neuropeptide ion transport peptide (ITP), which was first identified and named on account of its effects on the locust hindgut (Audsley et al. 1992). Using Drosophila as an experimental model, the authors have mapped the expression of three different isoforms of ITP (Figures 1, S1, and S2), all of which are encoded by the same gene.

The authors then investigated candidate receptors for isoforms of ITP. Firstly, Drosophila orthologs of G-protein coupled receptors (GPCRs) that have been reported to act as receptors for ITPa or ITPL in the insect Bombyx mori were investigated. Importantly, the authors report that ITPa does not act as a ligand for the GPCRs TkR99D and PK2-R1 (Figure S3). Therefore, the authors investigated other putative receptors for ITPs. Informed by a previously reported finding that ITP-type peptides cause an increase in cGMP levels in cells/tissues (Dircksen, 2009, Nagai et al., 2014), the authors investigated guanylyl cyclases as candidate receptors for ITPs. In particular, the authors suggest that Gyc76C may act as an ITP receptor in Drosophila.

Evidence that Gyc76C may be involved in mediating effects of ITP in Bombyx was first reported by Nagai et al. (2014) and here the authors present further evidence, based on a proposed concordance in the phylogenetic distribution ITP-type neuropeptides and Gyc76C (Figure 2). Having performed detailed mapping of the expression of Gyc76C in Drosophila (Figures 3, S4, S5, S6), the authors then investigated if Gyc76C knockdown affects the bioactivity of ITPa in Drosophila. The inhibitory effect of ITPa on leucokinin- and diuretic hormone-31-stimulated fluid secretion from Malpighian tubules was found to be abolished when expression of Gyc76C was knocked down in stellate cells and principal cells, respectively (Figure 4). However, as discussed below, this does not provide proof that Gyc76C directly mediates the effect of ITPa by acting as its receptor. The effect of Gyc76C knockdown on the action of ITPa could be an indirect consequence of an alteration in cGMP signalling.

Having investigated the proposed mechanism of ITPa in Drosophila, the authors then investigated its physiological roles at a systemic level. In Figure 5 the authors present evidence that ITPa is released during desiccation and accordingly, overexpression of ITPa increases survival when animals are subjected to desiccation. Furthermore, knockdown of Gyc76C in stellate or principal cells of Malphigian tubules decreases survival when animals are subject to desiccation. However, whilst this is correlative, it does not prove that Gyc76C mediates the effects of ITPa. The authors investigated the effects of knockdown of Gyc76C in stellate or principal cells of Malphigian tubules on i). survival when animals are subject to salt stress and ii). time taken to recover from of chill coma. It is not clear, however, why animals over-expressing ITPa were also not tested for its effect on i). survival when animals are subject to salt stress and ii). time taken to recover from of chill coma. In Figures 6 and S8, the authors show the effects of Gyc76C knockdown in the female fat body on metabolism, feeding-associated behaviours and locomotor activity, which are interesting. Furthermore, the relevance of the phenotypes observed to potential in vivo actions of ITPa is explored in Figure 7. The authors conclude that "increased ITPa signaling results in phenotypes that largely mirror those seen following Gyc76C knockdown in the fat body, providing further support that ITPa mediates its effects via Gyc76C." Use of the term "largely mirror" seems inappropriate here because there are opposing effects- e.g. decreased starvation resistance in Figure 6A versus increased starvation resistance in Figure 7A. Furthermore, as discussed above, the results of these experiments do not prove that the effects of ITPa are mediated by Gyc76C because the effects reported here could be correlative, rather than causative.

Lastly, in Figures 8, S9, and S10 the authors analyse publicly available connectomic data and single-cell transcriptomic data to identify putative inputs and outputs of ITPa-expressing neurons. These data are a valuable addition to our knowledge ITPa expressing neurons; but they do not address the core hypothesis of this paper - namely that Gyc76C acts as an ITPa receptor.

Strengths:

(1) The main strengths of this paper are i) the detailed analysis of the expression and actions of ITP and the phenotypic consequences of over-expression of ITPa in Drosophila. ii). the detailed analysis of the expression of Gyc76C and the phenotypic consequences of knockdown of Gyc76C expression in Drosophila.

(2) Furthermore, the paper is generally well-written and the figures are of good quality.

Weaknesses:

(1) The main weakness of this paper is that the data obtained do not prove that Gyc76C acts as a receptor for ITPa. Therefore, the following statement in the abstract is premature: "Using a phylogenetic-driven approach and the ex vivo secretion assay, we identified and functionally characterized Gyc76C, a membrane guanylate cyclase, as an elusive Drosophila ITPa receptor." Further experimental studies are needed to determine if Gyc76C acts as a receptor for ITPa. In the section of the paper headed "Limitations of the study", the authors recognise this weakness. They state "While our phylogenetic analysis, anatomical mapping, and ex vivo and in vivo functional studies all indicate that Gyc76C functions as an ITPa receptor in Drosophila, we were unable to verify that ITPa directly binds to Gyc76C. This was largely due to the lack of a robust and sensitive reporter system to monitor mGC activation." It is not clear what the authors mean by "the lack of a robust and sensitive reporter system to monitor mGC activation". The discovery of mGCs as receptors for ANP in mammals was dependent on the use of assays that measure GC activity in cells (e.g. by measuring cGMP levels in cells). Furthermore, more recently cGMP reporters have been developed. The use of such assays is needed here to investigate directly whether Gyc76C acts as a receptor for ITPa. In summary, insufficient evidence has been obtained to conclude that Gyc76C acts as a receptor for ITPa. Therefore, I think there are two ways forward, either:<br /> (a) The authors obtain additional biochemical evidence that ITPa is a ligand for Gyc76C.<br /> or<br /> (b) The authors substantially revise the conclusions of the paper (in the title, abstract, and throughout the paper) to state that Gyc76C MAY act as a receptor for ITPa, but that additional experiments are needed to prove this.

(2) The authors state in the abstract that a phylogenetic-driven approach led to their identification of Gyc76C as a candidate receptor for ITPa. However, there are weaknesses in this claim. Firstly, because the hypothesis that Gyc76C may be involved in mediating effects of ITPa was first proposed ten years ago by Nagai et al. 2014, so this surely was the primary basis for investigating this protein. Nevertheless, investigating if there is correspondence in the phylogenetic distribution of ITP-type and Gyc76C-type genes/proteins is a valuable approach to addressing this issue. Unfortunately, the evidence presented is rather limited in scope. Essentially, the authors report that they only found ITP-type and Gyc76C-type genes/proteins in protostomes, but not in deuterostomes. What is needed is a more fine-grained analysis at the species level within the protostomes. Thus, are there protostome species in which both ITP-type and Gyc76C-type genes/proteins have been lost? Furthermore, are there any protostome species in which an ITP-type gene is present but an Gyc76C-type gene is absent, or vice versa? If there are protostome species in which an ITP-type gene is present but a Gyc76C-type gene is absent or vice versa, this would argue against Gyc76C being a receptor for ITPa. In this regard, it is noteworthy that in Figure 2A there are two ITP-type precursors in C. elegans, but there are no Gyc76C-type proteins shown in the tree in Figure 2B. Thus, what is needed is a more detailed analysis of protostomes to investigate if there really is correspondence in the phylogenetic distribution of Gyc76C-type and ITP-type genes at the species level.

(3) The manuscript would benefit from a more comprehensive overview and discussion of published literature on Gyc76C in Drosophila, both as a basis for this study and for interpretation of the findings of this study.

Reviewer #2 (Public Review):

Summary:

Latham A.P. et al. apply simulations and FLIM to analyse several di-block elastin-like polypetides and connect their sequence to the micro-structure of coacervates resulting from their phase-separation.

Strengths:

Understanding the molecular grammar of phase separating proteins and the connection with mesoscale properties of the coacervates is highly relevant. This work provides insights into micro-structures of coacervates resulting from di-block polypetides.

Weaknesses:

The results apply to a very specific architecture (di-block polypetides) with specific sequences.

Reviewer #2 (Public Review):

Summary:

Thakare et al. present the DIETS assay for quantifying food consumption in adult Drosophila. DIETS measures food intake by weighing fly food before and after feeding. Technically, this is a well-designed, executed, and analyzed study. The interpretations are generally conservative and justified by the results. Although the results aren't always consistent with other published studies, which might reflect some of the unique conditions of the DIETS assay, the technique can clearly distinguish between some expected differences in food intake. Although lifespan is shortened in the DIETS chamber, the method seems robust for various time scales up to a week. DIETS adds another useful and versatile tool for fly researchers interested in studying feeding behavior.

Strengths:

The authors test various conditions, including food presentation, surface area, and humidity (by changing the food cup distance to an agar base) to demonstrate an optimized technique for quantifying consumption. Under these conditions, evaporation is generally limited to <10%.

The authors use DIETS to validate diverse feeding paradigms, including the published effects of temperature, food dilution, and intermittent fasting on food intake.

Weaknesses:

The studies to optimize and test the DIETS assay are technically rigorous and well-designed. However, the results reveal some weaknesses or potential caveats of the assay. As highlighted below, how much nutrition flies are actually obtaining may be misestimated due to vapor diffusion, and crowding/competition for food. This appears largely acceptable though, since the 'group' measurement can still clearly distinguish between expected feeding differences under different conditions, but it likely reduces accuracy, which may be important in some studies, and probably nullifies the effectiveness of using DIETS to restrict caloric intake.

It is my understanding that flies suck out nutrients from the medium, leaving behind the agar/cornmeal matrix. This seems consistent with the images in Figure S2B, where the spheroidal medium in the food cup maintains its shape as it shrinks, but there don't seem to be any pits or holes from fly consumption. Given that flies in DIETS consume a significant portion of the available food, it seems that the food concentration at the medium surface may be changing throughout the experiment. This may also make it challenging to use other common fly food ingredients, such as cornmeal, much of which is indigestible.

Similarly, vapor diffusion is expected between the agar bed and food cup (which the authors observed; in line 385), which may further affect assay accuracy, especially in comparisons between foods with different osmolarity.

In DIETS, increased feeding is observed with increased flies per chamber, but this is not observed in other techniques, such as EX-Q (Wu et al. 2020). It is unclear whether sensitivity to adult density is a DIETS-specific feature, or if adult density instead directly affects food intake estimates using DIETS (e.g., by affecting chamber humidity).

In another example, there is a ~300% difference in absolute feeding when the DIETS food cup is presented in different formats (Figure 3C). Again, it is unclear whether food presentation has an inherently greater effect in DIETS, or if the measurements themselves are highly sensitive to the environment.

Although the control of total food mass given to the animals is a novel feature of the assay, the likely differences in nutrient intake between individuals (and shortened lifespan) in a DIETS chamber makes this a challenging method to use to study caloric restriction. The shortened lifespan likely stems from the high adult density per vial, which has been explored in previous publications (e.g., Pearl in the 1920s; Mueller in the 1990s).