Reviewer #1 (Public review):

Summary:

This study uses single nucleus multi-omics to profile the transcriptome and chromatin accessibility of mouse XX and XY primordial germ cells (PGCs) at three time points spanning PGC sexual differentiation and entry of XX PGCs into meiosis (embryonic days 11.5-13.5). They find that PGCs can be clustered into sub-populations at each time point, with higher heterogeneity among XX PGCs and more switch-like developmental transitions evident in XY PGCs. In addition, they identify several transcription factors that appear to regulate sex-specific pathways as well as cell-cell communication pathways that may be involved in regulating XX vs XY PGC fate transitions. The findings are important and overall rigorous. The study could be further improved by better connection to the biological system, including putting the transcriptional heterogeneity of XX PGCs in the context of findings that meiotic entry is spatially asynchronous in the fetal ovary and further addressing the role of retinoic acid signaling. Overall, this study represents and advance in germ cell regulatory biology and will be a highly used resource in the field of germ cell development.

Strengths:

(1) The multi-omics data is mostly rigorously collected and carefully interpreted.

(2) The dataset is extremely valuable and helps to answer many long-standing questions in the field.

(3) In general, the conclusions are well anchored in the biology of the germ line in mammals.

Comments on revised version:

Most of my concerns have been addressed in the revised manuscript. I have one remaining concern but I believe this is important in order for the paper to be fully appreciated:

In Figures 2a, 2e, 3a, and 3e, the visualization scheme is very difficult to follow, and has not been updated or improved in the revised manuscript. It's very hard to see the colors corresponding to average expression for many genes because the circles are so small. The yellow color is hard to see and makes it hard to estimate the size of the circle. This issue is particularly egregious in Figure 2a for the data relating to ZKSCAN5, which is specifically highlighted in the text in lines 421-426. This data must be shown in a more convincing way in order to make the claims. An update to the visualization, including color scheme, is very strongly recommended; it is not difficult and would substantially improve the ability of these panels to communicate their message.

Reviewer #1 (Public review):

Summary:

Authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

Strengths and weaknesses:

(1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); this is acknowledged by the authors that no full mechanistic explanation can be given at this moment.

(2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? A RNA seq analysis is required to show the effect on cellular genes.

A RNA seq analysis was done in the revised manuscript comparing the effect of HSV-1 and deleted vector in J-LAT cells (Fig S5). More than 2000 genes are upregulated after transduction with the modified vector in comparison with the WT vector. Hence, the specificity of upregulation of SIV genes is questioned. Authors do NOT comment on these findings. In my view it questions the utility of this approach.

(3) The primate groups are too small and the results to variable to make averages. In Fig 5, the group with ART and saline has two slow rebounders. It is not correct to average those with the single quick rebounder. Here the interpretation is NOT supported by the data.

Although authors provided some promising SIV DNA data, no additional animals were added. Groups of 3 animals are too small to make any conclusion, especially since the huge variability in response. The average numbers out of 3 are still presented in the paper, which is not proper science.

No data are given of the effect of the deletion in primates. Now the deleted construct is compared with an empty vector containing no SIV genes. Authors provide new data in Fig S2 on the comparison of WT and modified vector in cells from PLWH, but data are not that convincing. A significant difference in reactivation is seen for LTR in only 2/4 donors and in Gag in 3/4 donors. (Additional question what is meaning of LTR mRNA, do authors relate to genomic RNA??)

Discussion

HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

The RNA seq data add on to this worry and should at least be discussed.

Comments on revisions:

The authors accept the limitations of the primate study (too small for strong conclusions). The new way of presenting the data clearly shows these limitations.

Reviewer #1 (Public review):

Summary:

By using the biophysical chromosome stretching, the authors measured the stiffness of chromosomes of mouse oocytes in meiosis I (MI) and meiosis II (MII). This study was the follow-up of previous studies in spermatocytes (and oocytes) by the authors (Biggs et al. Commun. Biol. 2020: Hornick et al. J. Assist. Rep. and Genet. 2015). They showed that MI chromosomes are much stiffer (~10 fold) than mitotic chromosomes of mouse embryonic fibroblast (MEF) cells. MII chromosomes are also stiffer than the mitotic chromosomes. The authors also found that oocyte aging increases the stiffness of the chromosomes. Surprisingly, the stiffness of meiotic chromosomes is independent of meiotic chromosome components, Rec8, Stag3, and Rad21L. and aging increases the stiffness.

Strengths

This provides a new insight into the biophysical property of meiotic chromosomes, that is chromosome stiffness. The stiffness of chromosomes in meiosis prophase I is ~10-fold higher than that of mitotic chromosomes, which is independent of meiotic cohesin. The increased stiffness during oocyte aging is a novel finding.

Weaknesses:

A major weakness of this paper is that it does not provide any molecular mechanism underlying the difference between MI and MII chromosomes (and/or prophase I and mitotic chromosomes).

Comments on revisions:

The main text lacks the first page with the authors' names and their affiliations (and corresponding authors etc).

Reviewer #2 (Public review):

Summary:

The authors used a yeast model for analyzing Parkinson's disease-associated synphilin-1 inclusion bodies (SY1 IBs). In this model system, large SY1 IBs are efficiently formed from smaller potentially more toxic SY1 aggregates. Using a genome-wide approach (synthetic genetic array, SGA, combined with a high content imaging approach), the authors identified the sphingolipid metabolic pathway as pivotal for SY1 IBs formation. Disturbances of this pathway increased SY1-triggered growth deficits, loss of mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and decreased cellular ATP levels pointing to an increased energy crisis within affected cells. Notably, SY1 IBs were found to be surrounded by mitochondrial membranes using state-of-the-art super-resolution microscopy. Finally, the effects observed in the yeast for SY1 IBs turned out to be evolutionary conserved in mammalian cells. Thus, sphingolipid metabolism might play an important role in the detoxification of misfolded proteins by large IBs formation at the mitochondrial outer membrane.

Strengths:

• The SY1 IB yeast model is very suitable for the analysis of genes involved in IB formation.<br /> • The genome-wide approach combining a synthetic genetic array (SGA) with a high content imaging approach is a compelling approach and enabled the reliable identification of novel genes. The authors tightly checked the output of the screen.<br /> • The authors clearly showed, including a couple of control experiments, that the sphingolipid metabolic pathway is crucial for SY1 IB formation and cytotoxicity.<br /> • The localization of SY1 IBs at mitochondrial membranes has been clearly demonstrated with state-of-the-art super-resolution microscopy and biochemical methods.<br /> • Pharmacological manipulation of the sphingolipid pathway influenced mitochondrial function and cell survival.<br /> • The authors have carefully redone critical experiments to avoid any misleading interpretation of data.

Weaknesses:

• It remains unclear how sphingolipids are involved in SY1 IB formation.

Comments on revisions: No further comments

Reviewer #1 (Public review):

Summary:

This paper introduces a new approach for modeling human behavioral responses using image-computable models. They create a model (VAM) that is a combination of a standard CNN coupled with a standard evidence accumulation model (EAM). The combined model is then trained directly on image-level data using human behavioral responses. This approach is original and can have wide applicability. However, many of the specific findings reported are less compelling.

Strengths:

(1) The manuscript presents an original approach of fitting an image-computable model to human behavioral data. This type of approach is sorely needed in the field.<br /> (2) The analyses are very technically sophisticated.<br /> (3) The behavioral data are large both in terms of sample size (N=75) and in terms of trials per subject.

Weaknesses:

(1) The main advance here thus appears to be methodological rather than conceptual. It's really cool that VAMs are image computable and are also fit to human data. But what we learn about the mind or brain is perhaps more modest.<br /> (2) In the approach here, a given stimulus is always processed in the same way through the core CNN to produce activations v_k. These v_k's are then corrupted by Gaussian noise to produce drift rates d_k, which can differ from trial to trial even for the same stimulus. In other words, the assumption built into VAM appears to be that the drift rate variability stems entirely from post-sensory (decisional) noise. In contrast, the typical interpretation of EAMs is that the variability in drift rates is sensory. In response to this concern, the authors responded that one can imagine an additional (unmodeled) sensory process that adds variability to the drift rates. However, this process remains unmodeled. The authors motivate their paper by saying "EAMs do not explain how the visual system extracts these representations in the first place" (second sentence of the Abstract). VAM is definitely a step in this direction but there's still a gap between the current VAM implementation and sensory systems.

Reviewer #1 (Public review):

Summary:

The authors aimed to modify the characteristics of the extracellular matrix (ECM) produced by immortalized mesenchymal stem cells (MSCs) by employing the CRISPR/Cas9 system to knock out specific genes. Initially, they established VEGF-KO cell lines, demonstrating that these cells retained chondrogenic and angiogenic properties. Additionally, lyophilized carriage tissues produced by these cells exhibited retained osteogenic properties.

Subsequently, the authors established RUNX2-KO cell lines, which exhibited reduced COLX expression during chondrogenic differentiation and notably diminished osteogenic properties in vitro. Transplantation of lyophilized carriage tissues produced by RUNX2-KO cell lines into osteochondral defects in rat knee joints resulted in the regeneration of articular cartilage tissues as well as bone tissues, a phenomenon not observed with tissues derived from parental cells. This suggests that gene-edited MSCs represent a valuable cell source for producing ECM with enhanced quality.

Strengths:

The enhanced cartilage regeneration observed with ECM derived from RUNX2-KO cells supports the authors' strategy of creating gene-edited MSCs capable of producing ECM with superior quality. Immortalized cell lines offer a limitless source of off-the-shelf material for tissue regeneration.

Weaknesses:

Most of the data align with anticipated outcomes, offering limited novelty to advance scientific understanding. Methodologically, the chondrogenic differentiation properties of immortalized MSCs appeared deficient, evidenced by Safranin-O staining of 3D tissues and histological findings lacking robust evidence for endochondral differentiation. This presents a critical limitation, particularly as authors propose the implantation of cartilage tissues for in vivo experiments. Instead, the bulk of data stemmed from type I collagen scaffold with factors produced by MSCs stimulated by TGFβ.

In the revised version, the authors presented Safranin-O staining results of pellets prior to lyophilization. The inset of figures showing entire pellets revealed that Safranin-O-positive areas were limited, suggesting that cells in the negative regions had not differentiated into chondrocytes. In Figure 3F, DAPI staining showed devitalized cells in the outer layer but was negative in the central part, indicating the absence of cells in these areas and incomplete differentiation induction.

The rationale for establishing VEGF-KO cell lines remains unclear, and the authors' explanation in the revised manuscript is still equivocal. While they mention that VEGF is a late marker for endochondral ossification, the data in Figures 1D and 1E clearly show that VEGF-KO affects the early phase of endochondral ossification.

Insufficient depth was given to elucidate the disparity in osteogenic properties between those observed in ectopic bone formation and those observed in transplantation into osteochondral defects.

In the ectopic bone formation study, most of the collagenous matrix observed at 2 weeks was resorbed by 6 weeks, with only a small amount contributing to bone formation in MSOD-B cells (Figs. 2I and 4C). This finding does not align with the micro-CT data presented in Figures 2H and 4B. For the micro-CT experiments, it would be more appropriate to use a standard window for bone and present the data accordingly.

While the regeneration of articular cartilage in RUNX2-KO ECM presents intriguing results, the study lacked an exploration into underlying mechanisms, such as histological analyses at earlier time points.

Reviewer #1 (Public review):

Summary:

In their previous publication (Dong et al. Cell Reports 2024), the authors showed that citalopram treatment resulted in reduced tumor size by binding to the E380 site of GLUT1 and inhibiting the glycolytic metabolism of HCC cells, instead of the classical citalopram receptor. Given that C5aR1 was also identified as the potential receptor of citalopram in the previous report, the authors focused on exploring the potential of the immune-dependent anti-tumor effect of citalopram via C5aR1. C5aR1 was found to be expressed on tumor-associated macrophages (TAMs) and citalopram administration showed potential to improve the stability of C5aR1 in vitro. Through macrophage depletion and adoptive transfer approaches in HCC mouse models, the data demonstrated the potential importance of C5aR1-expressing macrophage in the anti-tumor effect of citalopram in vivo. Mechanistically, their in vitro data suggested that citalopram may regulate the phagocytosis potential and polarization of macrophages through C5aR1. Next, they tried to investigate the direct link between citalopram and CD8+T cells by including an additional MASH-associated HCC mouse model. Their data suggest that citalopram may upregulate the glycolytic metabolism of CD8+T cells, probability via GLUT3 but not GLUT1-mediated glucose uptake. Lastly, as the systemic 5-HT level is down-regulated by citalopram, the authors analyzed the association between a low 5-HT and a superior CD8+T cell function against a tumor. Although the data is informative, the rationale for working on additional mechanisms and logical links among different parts is not clear. In addition, some of the conclusion is also not fully supported by the current data.

Strengths:

The idea of repurposing clinical-in-used drugs showed great potential for immediate clinical translation. The data here suggested that the anti-depression drug, citalopram displayed an immune regulatory role on TAM via a new target C5aR1 in HCC.

Weaknesses:

(1) The authors concluded that citalopram had a 'potential immune-dependent effect' based on the tumor weight difference between Rag-/- and C57 mice in Figure 1. However, tumor weight differences may also be attributed to a non-immune regulatory pathway. In addition, how do the authors calculate relative tumor weight? What is the rationale for using relative one but not absolute tumor weight to reflect the anti-tumor effect?

(2) The authors used shSlc6a4 tumor cell lines to demonstrate that citalopram's effects are independent of the conventional SERT receptor (Figure 1C-F). However, this does not entirely exclude the possibility that SERT may still play a role in this context, as it can be expressed in other cells within the tumor microenvironment. What is the expression profiling of Slc6a4 in the HCC tumor microenvironment? In addition, in Figure 1F, the tumor growth of shSlc6a4 in C57 mice displayed a decreased trend, suggesting a possible role of Slc6a4.

(3) Why did the authors choose to study phagocytosis in Figures 3G-H? As an important player, TAM regulates tumor growth via various mechanisms.

(4) The information on unchanged deposition of C5a has been mentioned in this manuscript (Figures 3D and 3F), the authors should explain further in the manuscript, for example, C5a could bind to receptors other than C5aR1 and/or C5a bind to C5aR1 by different docking anchors compared with citalopram.

(5) Figure 3I-M - the flow cytometry data suggested that citalopram treatment altered the proportions of total TAM, M1 and M2 subsets, CD4+ and CD8+T cells, DCs, and B cells. Why does the author conclude that the enhanced phagocytosis of TAM was one of the major mechanisms of citalopram? As the overall TAM number was regulated, the contribution of phagocytosis to tumor growth may be limited.

(6) Figure 4 - what is the rationale for using the MASH-associated HCC mouse model to study metabolic regulation in CD8+T cells? The tumor microenvironment and tumor growth would be quite different. In addition, how does this part link up with the mechanisms related to C5aR1 and TAM? The authors also brought GLUT1 back in the last part and focused on CD8+T cell metabolism, which was totally separated from previous data.

(7) Figure 5, the authors illustrated their mechanism that citalopram regulates CD8+T cell anti-tumor immunity through proinflammatory TAM with no experimental evidence. Using only CD206 and MHCII to represent TAM subsets obviously is not sufficient.

Reviewer #1 (Public review):

Summary:

In a previous work, Prut and colleagues had shown that during reaching, high-frequency stimulation of the cerebellar outputs resulted in reduced reach velocity. Moreover, they showed that the stimulation produced reaches that deviated from a straight line, with the shoulder and elbow movements becoming less coordinated. In this report, they extend their previous work by the addition of modeling results that investigate the relationship between the kinematic changes and torques produced at the joints. The results show that the slowing is not due to reductions in interaction torques alone, as the reductions in velocity occur even for movements that are single joints. More interestingly, the experiment revealed evidence for the decomposition of the reaching movement, as well as an increase in the variance of the trajectory.

Strengths:

This is a rare experiment in a non-human primate that assessed the importance of cerebellar input to the motor cortex during reaching.

Weaknesses:

My major concerns are described below.

If I understand the task design correctly, the monkeys did not need to stop their hand at the target. I think this design may be suboptimal for investigating the role of the cerebellum in control of reaching because a number of earlier works have found that the cerebellum's contributions are particularly significant as the movement ends, i.e., stopping at the target. For example, in mice, interposed nucleus neurons tend to be most active near the end of the reach that requires extension, and their activation produces flexion forces during the reach (Becker and Person 2019). Indeed, the inactivation of interposed neurons that project to the thalamus results in overshooting of reaching movements (Low et al. 2018). Recent work has also found that many Purkinje cells show a burst-pause pattern as the reach nears its endpoint, and stimulation of the mossy fibers tends to disrupt endpoint control (Calame et al. 2023). Thus, the fact that the current paper has no data regarding endpoint control of the reach is puzzling to me.

Because stimulation continued after the cursor had crossed the target, it is interesting to ask whether this disruption had any effects on the movements that were task-irrelevant. The reason for asking this is because we have found that whereas during task-relevant eye or tongue movements the Purkinje cells are strongly modulated, the modulations are much more muted when similar movements are performed but are task-irrelevant (Pi et al., PNAS 2024; Hage et al. Biorxiv 2024). Thus, it is interesting to ask whether the effects of stimulation were global and affected all movements, or were the effects primarily concerned with the task-relevant movements.

If the schematic in Figure 1 is accurate, it is difficult for me to see how any of the reaching movements can be termed single joint. In the paper, T1 is labeled as a single joint, and T2-T4 are labeled as dual-joint. The authors should provide data to justify this.

Because at least part of this work was previously analyzed and published, information should be provided regarding which data are new.

Reviewer #1 (Public review):

Summary:

Measurement of BOLD MR imaging has regularly found regions of the brain that show reliable suppression of BOLD responses during specific experimental testing conditions. These observations are to some degree unexplained, in comparison with more usual association between activation of the BOLD response and excitatory activation of the neurons (most tightly linked to synaptic activity) in the same brain location. This paper finds two patients whose brains were tested with both non-invasive functional MRI and with invasive insertion of electrodes, which allowed the direct recording of neuronal activity. The electrode insertions were made within the fusiform gyrus, which is known to process information about faces, in a clinical search for the sites of intractable epilepsy in each patient. The simple observation is that the electrode location in one patient showed activation of the BOLD response and activation of neuronal firing in response to face stimuli. This is the classical association. The other patient showed an informative and different pattern of responses. In this person, the electrode location showed a suppression of the BOLD response to face stimuli and, most interestingly, an associated suppression of neuronal activity at the electrode site.

Strengths:

Whilst these results are not by themselves definitive, they add an important piece of evidence to a long-standing discussion about the origins of the BOLD response. The observation of decreased neuronal activation associated with negative BOLD is interesting because, at various times, exactly the opposite association has been predicted. It has been previously argued that if synaptic mechanisms of neuronal inhibition are responsible for the suppression of neuronal firing, then it would be reasonable

Weaknesses:

The chief weakness of the paper is that the results may be unique in a slightly awkward way. The observation of positive BOLD and neuronal activation is made at one brain site in one patient, while the complementary observation of negative BOLD and neuronal suppression actually derives from the other patient. Showing both effects in both patients would make a much stronger paper.

Reviewer #1 (Public review):

Summary:

This study demonstrates that strip cropping enhances the taxonomic diversity of ground beetles across organically-managed crop systems in the Netherlands. In particular, strip cropping supported 15% more ground beetle species and 30% more individuals compared to monocultures.

Strengths:

A well-written study with well-analyzed data of a complex design. The data could have been analyzed differently e.g. by not pooling samples, but there are pros and cons for each type of analysis and I am convinced this will not affect the main findings. A strong point is that data were collected for 4 years. This is especially strong as most data on biodiversity in cropping systems are only collected for one or two seasons. Another strong point is that several crops were included.

Weaknesses:

This study focused on the biodiversity of ground beetles and did not examine crop productivity. Therefore, I disagree with the claim that this study demonstrates biodiversity enhancement without compromising yield. The authors should present results on yield or, at the very least, provide a stronger justification for this statement.

Reviewer #1 (Public review):

Summary:

Goal: Find downstream targets of cmk-1 phosphorylation, identify one that also seems to act in thermosensory habituation, test for genetic interactions between cmk-1 and this gene, and assess where these genes are acting in the thermosensory circuit during thermosensory habituation.

Methods: Two in vitro analyses of cmk-1 phosphorylation of C. elegans proteins. Thermosensory habituation of cmk-1 and tax-6 mutants and double mutants was assessed by measuring the rate of heat-evoked reversals (reversal probability) of C. elegans before and after 20s ISI repeated heat pulses over 60 minutes.

Conclusions: cmk-1 and tax-6 act in separate habituation processes, primarily in AFD, that interact complexly, but both serve to habituate the thermosensory reversal response. They found that cmk-1 primarily acts in AFD and tax-6 primarily acts in RIM (and FLP for naïve responses). They also identified hundreds of potential cmk-1 phosphorylation substrates in vitro.

Strengths:

The effect size in the genetic data is quite strong and a large number of genetic interaction experiments between cmk-1 and tax-1 demonstrate a complex interaction.

Weaknesses:

The major concern about this manuscript is the assumption that the process they are observing is habituation. The two previously cited papers using this (or a very similar) protocol, Lia and Glauser 2020 and Jordan and Glauser 2023, both use the word 'adaptation' to describe the observed behavioral decrement. Jordan and Glauser 2023 use the words 'habituation' or 'habituation-like' 10 times, however, they use 'adaptation' over 100 times. It is critical to distinguish habituation from sensory adaptation (or fatigue) in this thermal reversal protocol. These processes are often confused/conflated, however, they are very different; sensory adaptation is a process that decreases how much the nervous system is activated by a repeated stimulus, therefore it can even occur outside of the nervous system. Habituation is a learning process where the nervous system responds less to a repeated stimulus, despite (at least part of the nervous system) the nervous system still being similarly activated by the stimulus. Habituation is considered an attentional process, while adaptation is due to the fatigue of sensory transduction machinery. Control experiments such as tests for dishabituation (where the application of a different stimulus causes recovery of the decremented response) or rate of spontaneous recovery (more rapid recovery after short inter-stimulus intervals) are required to determine if habituation or sensory adaptation are occurring. These experiments will allow the results to be interpreted with clarity, without them, it isn't actually clear what biological process is actually being studied.

While the discrepancy between the in vitro phosphorylation experiments and the in silico predictions was discussed, the substantial discrepancy (over 85% of the substrates in the smaller in vitro dataset were not identified in the larger dataset) between the two different in vitro datasets was not discussed. This is surprising, as these approaches were quite similar, and it may indicate a measure of unreliability in the in vitro datasets (or high false negative rates). Additionally, the rationale for, and distinction between, the two separate in vitro experiments is not made clear.

Line 207: After reporting that both tax-6 and cnb-1 mutants have high spontaneous reversals, it is not made clear why cnb-1 is not further explored in the paper. Additionally, this spontaneous reversal data should be in a supplementary figure.

Figure 3 -S1: This model doesn't explain why the cmk-1(gf) group and the cmk-1(gf) +cyclo A group cause enhanced response decrement (presumably by reducing the inhibition by tax-6) but the +cyclo A group (inhibited tax-6) showed weaker response decrement, as here there is even further weakened inhibition of tax-6 on this process. Also, the cmk-1(lf) +cyclo A group is labeled as constitutive habituation, however, this doesn't appear to be the case in Figure 3 (seems like a similar initial level and response decrement phenotype to wildtype).

More discussion of the significance of the sites of cmk-1 and tax-6 function in the neural circuit should take place. Additionally, incorporating the suspected loci of cmk-1 and tax-6 in the neural circuit into the model would be interesting (using proper hypothetical language). For example, as it seems like AFD is not required for the naïve reversal response but just its reduction, cmk-1 activity in AFD might be generating inhibition of the reversal response by AFD. It certainly would be understandable if this isn't workable, given extrasynaptic signaling and other unknowns, but it potentially could also be helpful in generating a working model for these complex interactions. For example, cmk-1 induces AIZ inhibition of AVA (AIZ is electrically coupled to AFD), and tax-6 reduces RIM activation of AVA (these neurons are also electrically coupled according to the diagram). RIM is also a neuropeptide-rich neuron, so this could allow it to interact with the cmk-1-related process(es) in AFD. Some discussion of possibilities like this could be informative.

Provide an explanation for why some of the experiments in Figure 4 have such a high N, compared to other experiments.

Because the loss of function and gain of function mutations in cmk-1 have a similar effect, it is likely that this thermosensory plasticity phenotype is sensitive to levels of cmk-1 activity. Therefore, it is not surprising that the cmk-1 promoter failed to rescue very well as these plasmid-driven rescues often result in overexpression. Given this and that the cmk-1p rescue itself was so modest, these rescue experiments are not entirely convincing (and very hard to interpret; for example, is the AFD rescue or the ASER rescue more complete? The ASER one is actually closer to the cmk-1p rescue). Given the sensitivity to cmk-1 activity levels, a degradation strategy would be more likely to deliver clear results (or perhaps even the overactivation approach used for tax-6).

Reviewer #1 (Public review):

Summary:

This study presents convincing findings that oligodendrocytes play a regulatory role in spontaneous neural activity synchronisation during early postnatal development, with implications for adult brain function. Utilising targeted genetic approaches, the authors demonstrate how oligodendrocyte depletion impacts Purkinje cell activity and behaviours dependent on cerebellar function. Delayed myelination during critical developmental windows is linked to persistent alterations in neural circuit function, underscoring the lasting impact of oligodendrocyte activity.

Strengths:

(1) The research leverages the anatomically distinct olivocerebellar circuit, a well-characterized system with known developmental timelines and inputs, strengthening the link between oligodendrocyte function and neural synchronization.

(2) Functional assessments, supported by behavioral tests, validate the findings of in vivo calcium imaging, enhancing the study's credibility.

(3) Extending the study to assess the long-term effects of early-life myelination disruptions adds depth to the implications for both circuit function and behavior.

Weaknesses:

(1) The study would benefit from a closer analysis of myelination during the periods when synchrony is recorded. Direct correlations between myelination and synchronized activity would substantiate the mechanistic link and clarify if observed behavioral deficits stem from altered myelination timing.

(2) Although the study focuses on Purkinje cells in the cerebellum, neural synchrony typically involves cross-regional interactions. Expanding the discussion on how localized Purkinje synchrony affects broader behaviors - such as anxiety, motor function, and sociality - would enhance the findings' functional significance.

(3) The authors discuss the possibility of oligodendrocyte-mediated synapse elimination as a possible mechanism behind their findings, drawing from relevant recent literature on oligodendrocyte precursor cells. However, there are no data presented supporting this assumption. The authors should explain why they think the mechanism behind their observation extends beyond the contribution of myelination or remove this point from the discussion entirely.

(4) It would be valuable to investigate the secondary effects of oligodendrocyte depletion on other glial cells, particularly astrocytes or microglia, which could influence long-term behavioral outcomes. Identifying whether the lasting effects stem from developmental oligodendrocyte function alone or also involve myelination could deepen the study's insights.

(5) The authors should explore the use of different methods to disturb myelin production for a longer time, in order to further determine if the observed effects are transient or if they could have longer-lasting effects.

(6) Throughout the paper, there are concerns about statistical analyses, particularly on the use of the Mann-Whitney test or using fields of view as biological replicates.

Reviewer #1 (Public review):

Summary:

The paper by Lee and Ouellette explores the role of cyclic-d-AMP in chlamydial developmental progression. The manuscript uses a collection of different recombinant plasmids to up- and down-regulate cdAMP production, and then uses classical molecular and microbiological approaches to examine the effects of expression induction in each of the transformed strains.

Strengths:

This laboratory is a leader in the use of molecular genetic manipulation in Chlamydia trachomatis and their efforts to make such efforts mainstream is commendable. Overall, the model described and defended by these investigators is thorough and significant.

Weaknesses:

The biggest weakness in the document is their reliance on quantitative data that is statistically not significant, in the interpretation of results. These challenges can be addressed in a revision by the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors Eapen et al. investigated the peptide inhibitors of Cdc20. They applied a rational design approach, substituting residues found in the D-box consensus sequences to better align the peptides with the Cdc20-degron interface. In the process, the authors designed and tested a series of more potent binders, including ones that contain unnatural amino acids, and verified binding modes by elucidating the Cdc-20-peptide structures. The authors further showed that these peptides can engage with Cdc20 in the cellular context, and can inhibit APC/CCdc20 ubiquitination activity. Finally, the authors demonstrated that these peptides could be used as portable degron motifs that drive the degradation of a fused fluorescent protein.

Strengths:

This manuscript is clear and straightforward to follow. The investigation of different peptide variations was comprehensive and well-executed. This work provided the groundwork for the development of peptide drug modalities to inhibit degradation or apply peptides as portable motifs to achieve targeted degradation. Both of which are impactful.

Weaknesses:

A few minor comments:

(1) In my opinion, more attention to the solubility issue needs to be discussed and/or tested. On page 10, what is the solubility of D2 before a modification was made? The authors mentioned that position 2 is likely solvent exposed, it is not immediately clear to me why the mutation made was from one hydrophobic residue to another. What was the level of improvement in solubility? Are there any affinity data associated with the peptide that differ with D2 only at position 2?

(2) I'm not entirely convinced that the D19 density not observed in the crystal structure was due to crystal packing. This peptide is peculiar as it also did not induce any thermal stabilization of Cdc20 in the cellular thermal shift assay. Perhaps the binding of this peptide could be investigated in more detail (i.e., NMR?) Or at least more explanation could be provided.

Reviewer #1 (Public review):

This study investigates alterations in the autophagic-lysosomal pathway in the Q175 HD knock-in model crossed with the TRGL autophagy reporter mouse. The findings provide valuable insights into autophagy dynamics in HD and the potential therapeutic benefits of modulating this pathway. The study suggests that autophagy stimulation may offer therapeutic benefits in the early stages of HD progression, with mTOR inhibition showing promise in ameliorating lysosomal pathology and reducing mutant huntingtin accumulation.

However, the data raises concerns regarding the strength of the evidence. The observed changes in autophagic markers, such as autolysosome and lysosome numbers, are relatively modest, and the Western blot results do not fully match the quantitative results. These discrepancies highlight the need for further validation and more pronounced effects to strengthen the conclusions. While the study suggests the potential of autophagy regulation as a long-term therapeutic strategy, additional experiments and more reliable data are necessary to confirm the broader applicability of the TRGL/Q175 mouse model.

Furthermore, the 2004 publication by Ravikumar et al. demonstrated that inhibition of mTOR by rapamycin or the rapamycin ester CCI-779 induces autophagy and reduces the toxicity of polyglutamine expansions in fly and mouse models of Huntington's disease. mTOR is a key regulator of autophagy, and its inhibition has been explored as a therapeutic strategy for various neurodegenerative diseases, including HD. Studies suggest that inhibiting mTOR enhances autophagy, leading to the clearance of mHTT aggregates. Given that dysfunction of the autophagic-lysosomal pathway and lysosomal function in HD is already well-established, and that mTOR inhibition as a therapeutic approach for HD is also known, this study does not present entirely novel findings.

Major Concerns:

(1) In Figure 3A1 and A2, delayed and/or deficient acidification of AL causes deficits in the reformation of LY to replenish the LY pool. However, in Figure S2D, there is no difference in AL formation or substrate degradation, as shown by the Western blotting results for CTSD and CTSB. How can these discrepancies be explained?

(2) The results demonstrate that in the brain sections of 17-month-old TRGL/Q175 mice, there was an increase in the number of acidic autolysosomes (AL), including poorly acidified autolysosomes (pa-AL), alongside a decrease in lysosome (LY) numbers. These AL/pa-AL changes were not significant in 2-month-old or 7-month-old TRGL/Q175 mice, where only a reduction in lysosome numbers was observed. This indicates that these changes, representing damage to the autophagy-lysosome pathway (ALP), manifest only at later stages of the disease. Considering that the ALP is affected predominantly in the advanced stages of the disease (e.g., at 17 months), why were 6-month-old TRGL/Q175 mice selected for oral mTORi INK treatment, and why was the treatment duration restricted to just 3 weeks?

(3) Is the extent of motor dysfunction in TRGL/Q175 mice comparable to that in Q175 mice? Does the administration of mTORi INK improve these symptoms?

(4) Why is eGFP expression not visible in Fig. 6A in TRGL-Veh mice? Additionally, why do normal (non-poly-Q) mice have fewer lysosomes (LY) than TRGL/Q175-INK mice? IHC results also show that CTSD levels are lower in TRGL mice compared to TRGL/Q175-INK mice. Does this suggest lysosome dysfunction in TRGL-Veh mice?

(5) In Figure 5A, the phosphorylation of ATG14 (S29) shows minimal differences in Western blotting, which appears inconsistent with the quantitative results. A similar issue is observed in the quantification of Endo-LC3.

(6) In Figure S2A and Figure S2B, 17-month-old TRGL/Q175 mice show a decrease in p-p70S6K and the p-ULK1/ULK1 ratio, but no changes are observed in autophagy-related markers. Do these results indicate only a slight change in autophagy at this stage in TRGL/Q175 mice? Since the mTOR pathway regulates multiple cellular mechanisms, could mTOR also influence other processes? Is it possible that additional mechanisms are involved?

Reviewer #2 (Public review):

Summary:

In their manuscript, Quian and colleagues identified a novel mechanisms by which Pseudomonas control inflammatory responses upon inflammasome activation. They identified a caspase-11 substrates (VgrG2b) which, upon cleavage, binds and inhibit the NLRP3 to reduce the production of pro-inflammatory cytokines. This is a unique mechanism that allow for the tailoring of the innate immune response upon bacterial recognition.

Strengths:

The authors are presenting here a novel conceptual framework in host-pathogen interactions. Their work is supported by a range of approaches (biochemical, cellular immunology, microbiology, animal models) and their conclusions are supported by multiple independent evidences. The work is likely to have an important impact in the innate immunity field and host-pathogen interactions field and may guide the development of novel inhibitors.

Weaknesses:

Although quite exhaustive, a few of the authors conclusions are not fully supported (e.g, caspase-11 directly cleaving VgrG2b, the unique affinity of VgrG2b-C for NLRP3) and would require complementary approaches to validate their findings fully. This is minimal.

Comments on revisions:

I command the authors's effort to address my comments. They have addressed all my concerns.

Reviewer #1 (Public review):

Summary:

The authors aim to explore the effects of the electrogenic sodium-potassium pump (Na+/K+-ATPase) on the computational properties of highly active spiking neurons, using the weakly-electric fish electrocyte as a model system. Their work highlights how the pump's electrogenicity, while essential for maintaining ionic gradients, introduces challenges in neuronal firing stability and signal processing, especially in cells that fire at high rates. The study identifies compensatory mechanisms that cells might use to counteract these effects, and speculates on the role of voltage dependence in the pump's behavior, suggesting that Na+/K+-ATPase could be a factor in neuronal dysfunctions and diseases

Strengths:

(1) The study explores a less-examined aspect of neural dynamics-the effects of Na+/K+-ATPase electrogenicity. It offers a new perspective by highlighting the pump's role not only in ion homeostasis but also in its potential influence on neural computation.<br /> (2) The mathematical modeling used is a significant strength, providing a clear and controlled framework to explore the effects of the Na+/K+-ATPase on spiking cells. This approach allows for the systematic testing of different conditions and behaviors that might be difficult to observe directly in biological experiments.<br /> (3) The study proposes several interesting compensatory mechanisms, such as sodium leak channels and extracellular potassium buffering, which provide useful theoretical frameworks for understanding how neurons maintain firing rate control despite the pump's effects.

Weaknesses:

(1) While the modeling approach provides valuable insights, the lack of experimental data to validate the model's predictions weakens the overall conclusions.<br /> (2) The proposed compensatory mechanisms are discussed primarily in theoretical terms without providing quantitative estimates of their impact on the neuron's metabolic cost or other physiological parameters.

Reviewer #1 (Public review):

Summary:

The manuscript presents a short report investigating mismatch responses in the auditory cortex, following previous studies focused on visual cortex. By correlating mouse locomotion speed with acoustic feedback levels, the authors demonstrate excitatory responses in a subset of neurons to halts in expected acoustic feedback. They show a lack of responses to mismatch in he visual modality. A subset of neurons show enhanced mismatch responses when both auditory and visual modalities are coupled to the animal's locomotion.<br /> While the study is well-designed and addresses a timely question, several concerns exist regarding the quantification of animal behavior, potential alternative explanations for recorded signals, correlation between excitatory responses and animal velocity, discrepancies in reported values, and clarity regarding the identity of certain neurons.

Strengths:

(1) Well-designed study addressing a timely question in the field.<br /> (2) Successful transition from previous work focused on visual cortex to auditory cortex, demonstrating generic principles in mismatch responses.<br /> (3) Correlation between mouse locomotion speed and acoustic feedback levels provides evidence for prediction signal in the auditory cortex.<br /> (4) Coupling of visual and auditory feedback show putative multimodal integration in auditory cortex.

Weaknesses:

(1) Unclear correlation between excitatory responses and animal velocity during halts, particularly in closed-loop versus playback conditions.<br /> (2) Ambiguity regarding the identity of the [AM+VM] MM neurons.

Reviewer #1 (Public review):

Summary:

The study examines human biases in a regime-change task, in which participants have to report the probability of a regime change in the face of noisy data. The behavioral results indicate that humans display systematic biases, in particular, overreaction in stable but noisy environments and underreaction in volatile settings with more certain signals. fMRI results suggest that a frontoparietal brain network is selectively involved in representing subjective sensitivity to noise, while the vmPFC selectively represents sensitivity to the rate of change.

Strengths:

(1) The study relies on a task that measures regime-change detection primarily based on descriptive information about the noisiness and rate of change. This distinguishes the study from prior work using reversal-learning or change-point tasks in which participants are required to learn these parameters from experiences. The authors discuss these differences comprehensively.

(2) The study uses a simple Bayes-optimal model combined with model fitting, which seems to describe the data well.

(3) The authors apply model-based fMRI analyses that provide a close link to behavioral results, offering an elegant way to examine individual biases.

Weaknesses:

My major concern is about the correlational analysis in the section "Under- and overreactions are associated with selectivity and sensitivity of neural responses to system parameters", shown in Figures 5c and d (and similarly in Figure 6). The authors argue that a frontoparietal network selectively represents sensitivity to signal diagnosticity, while the vmPFC selectively represents transition probabilities. This claim is based on separate correlational analyses for red and blue across different brain areas. The authors interpret the finding of a significant correlation in one case (blue) and an insignificant correlation (red) as evidence of a difference in correlations (between blue and red) but don't test this directly. This has been referred to as the "interaction fallacy" (Niewenhuis et al., 2011; Makin & Orban de Xivry 2019). Not directly testing the difference in correlations (but only the differences to zero for each case) can lead to wrong conclusions. For example, in Figure 5c, the correlation for red is r = 0.32 (not significantly different from zero) and r = 0.48 (different from zero). However, the difference between the two is 0.1, and it is likely that this difference itself is not significant. From a statistical perspective, this corresponds to an interaction effect that has to be tested directly. It is my understanding that analyses in Figure 6 follow the same approach.

Relevant literature on this point is:

Nieuwenhuis, S, Forstmann, B & Wagenmakers, EJ (2011). Erroneous analyses of interactions in neuroscience: a problem of significance. Nat Neurosci 14, 1105-1107. https://doi.org/10.1038/nn.2886

Makin TR, Orban de Xivry, JJ (2019). Science Forum: Ten common statistical mistakes to watch out for when writing or reviewing a manuscript. eLife 8:e48175. https://doi.org/10.7554/eLife.48175

There is also a blog post on simulation-based comparisons, which the authors could check out: https://garstats.wordpress.com/2017/03/01/comp2dcorr/

I recommend that the authors carefully consider what approach works best for their purposes. It is sometimes recommended to directly compare correlations based on Monte-Carlo simulations (cf Makin & Orban). It might also be appropriate to run a regression with the dependent variable brain activity (Y) and predictors brain area (X) and the model-based term of interest (Z). In this case, they could include an interaction term in the model:

Y = \beta_0 + \beta_1 \cdot X + \beta_2 \cdot Z + \beta_3 \cdot X \cdot Z

The interaction term reflects if the relationship between the model term Z and brain activity Y is conditional on the brain area of interest X.

Another potential concern is that some important details about the parameter estimation for the system-neglect model are missing. In the respective section in the methods, the authors mention a nonlinear regression using Matlab's "fitnlm" function, but it remains unclear how the model was parameterized exactly. In particular, what are the properties of this nonlinear function, and what are the assumptions about the subject's motor noise? I could imagine that by using the inbuild function, the assumption was that residuals are Gaussian and homoscedastic, but it is possible that the assumption of homoscedasticity is violated, and residuals are systematically larger around p=0.5 compared to p=0 and p=1.

Relatedly, in the parameter recovery analyses, the authors assume different levels of motor noise. Are these values representative of empirical values?

The main study is based on N=30 subjects, as are the two control studies. Since this work is about individual differences (in particular w.r.t. to neural representations of noise and transition probabilities in the frontoparietal network and the vmPFC), I'm wondering how robust the results are. Is it likely that the results would replicate with a larger number of subjects? Can the two control studies be leveraged to address this concern to some extent?

It seems that the authors have not counterbalanced the colors and that subjects always reported the probability of the blue regime. If so, I'm wondering why this was not counterbalanced.

Reviewer #2 (Public review):

Summary:

The authors begin with the stated goal of gaining insight into the known repression of autophagy by Ezrin, a major membrane-actin linker that assembles signaling complexes on membranes. RNA and protein expression analysis is consistent with upregulation of lysosomal proteins in Ezrin-deficient MEFs, which the authors confirm by immunostaining and western blotting for lysosomal markers. Expression analysis also implicates EGF signaling as being altered downstream of Ezrin loss, and the authors demonstrate that Ezrin promotes relocalization of EGFR from the plasma membrane to endosomes. Ezrin loss reduces downstream MAPK and Akt signaling, and represses mTORC1 signaling by promoting lysosomal localization of the TSC complex. An Ezrin mutant Medaka fish line is then generated to test its role in retinal cells, which are known to be sensitive to changes in autophagy regulation. Phenotypes in this model appear generally consistent with observations made in cultured cells, though milder overall.

Strengths:

Data on the impact of Ezrin-loss on relocalization of EGFR from the plasma membrane are extensive, and thoroughly demonstrate that Ezrin is required for EGFR internalization in response to EGF.

A new Ezrin-deficient in vivo model (Medaka fish) is generated.

Strong data demonstrating that Ezrin loss suppresses Akt signaling and mTORC1 signaling by promoting TSC complex localization to the lysosome.

Weaknesses:

The authors have addressed all concerns

Reviewer #1 (Public review):

Summary:

This study examines the cortical modular functional organization of visual texture in comparison with that of color and disparity. While color, disparity, and orientation have been shown to exhibit clear functional organizations within the thin, thick, and thick/pale stripes of V2, whether the feature of texture is also organized within V2 is unknown. Using ultrahigh field 7T fMRI in humans viewing color-, disparity-, and texture-specific visual stimuli, the authors find that, unlike color and disparity, texture does not exhibit stripe-specific organization in V2. Moreover, using laminar imaging methods and calculations of informational connectivity, they find V2 color and disparity stripes exhibit the expected feedforward and feedback relationships with V1 & V4, and with V1 & V3ab, respectively. In contrast, texture activation, found predominantly in the deep layers of V2, is driven preferentially by feedback from V4. Based on these findings, the authors suggest that texture is a visual feature computed in higher-order areas and not generated by local intra-V2 computation.

Strengths:

This study poses an interesting and fundamental question regarding the relationship between functional modularity and hierarchical origin of computed properties. This question is thus highly significant and deserves study. The methodology is appropriate for the question and the areal and laminar resolution achieved across 10 subjects is commendable. The combination of high-resolution functional imaging and informational connectivity analysis introduces a useful way for examining feedforward and feedback relationships in mesoscale imaging data.

Comments on latest version:

The authors have responded adequately to my comments. The lack of texture organization in V2 is now strengthened by the apparently more clustered texture response in V4 (Fig. S9). The paired results in V2 and V4 make the study stronger. The authors may suggest that texture response, while present at the neural level, may not emerge as a primary organizational cue in V2, based on this texture stimulus paradigm. The negative results should still be presented cautiously. The connectivity inferences are interesting but should also be stated cautiously, as there are multiple assumptions. Overall, this study makes a contribution to emerging views about texture processing in the early visual pathways.

Reviewer #1 (Public review):

Summary:

This study investigated the role of PLECTIN, a cytoskeletal crosslinker protein, in liver cancer formation and progression. Using the liver-specific Plectin knockout mouse model, the authors convincingly showed that PLECTIN is critical for hepatocarcinogenesis, as functional inhibition of PLECTIN suppressed tumor formation in several models. They also provided evidence to show that inhibition of PLECTIN inhibited HCC cell invasion and reduced metastatic outgrowth in the lung. Mechanistically, they suggested that PLECTIN inhibition attenuated FAK, MAPK/ERK, and PI3K/AKT signaling.

Strengths:

The authors generated a liver-specific Plectin knockout mouse model. By using DEN and sgP53/MYC models, the authors convincingly demonstrated an oncogenic role of PLECTIN in HCC development. plecstatin-1 (PST), as a plectin inhibitor, showed promising efficacy in inhibiting HCC growth, which provides a basis for potentially treating HCC using PST.

The MIR images for tracking tumor growth in animal models were compelling. The high-quality confocal images and related qualifications convincingly showed the impact of plectin functional inhibition on contractility and adhesions in HCC cells.

Comments on latest version:

My concerns have been largely addressed. The authors did a good job in addressing the questions and clarifying the inconsistent results. I have two comments:

(1) The current data still cannot support the conclusion that plectin inactivation attenuates HCC oncogenic potential through FAK, Erk1/2, and PI3K/Akt axis, unless they can reactivate these signaling to restore the HCC congenic potential in plectin inactivated cells. It might be more appropriate to claim that plectin inactivation suppresses FAK, Erk1/2, and PI3K/Akt oncogenic signaling.

(2) I think it would be beneficial to include the H&E and HNF4α staining from lung tissue of mice inoculated with WT Huh7 cells indicated in the rebuttal letter.

Reviewer #2 (Public review):

Summary:

Tanaka et al. investigated the role of CCR4 in early atherosclerosis, focusing on the immune modulation elicited by this chemokine receptor under hypercholesterolemia. The study found that Ccr4 deficiency led to qualitative changes in atherosclerotic plaques, characterized by an increased inflammatory phenotype. The authors further analyzed the CD4 T cell immune response in para-aortic lymph nodes and atherosclerotic aorta, showing an increase mainly in Th1 cells and the Th1/Treg ratio in Ccr4-/-Apoe-/- mice compared to Apoe-/- mice. They then focused on Tregs, demonstrating that Ccr4 deficiency impaired their immunosuppressive function in in vitro assays. Authors also states that Ccr4-deficient Tregs had, as expected, impaired migration to the atherosclerotic aorta. Adoptive cell transfer of Ccr4-/- Tregs to Apoe-/- mice mimicked early atherosclerosis development in Ccr4-/-Apoe-/- mice. Therefore, this work shows that CCR4 plays an important role in early atherosclerosis but not in advanced stages.

Strengths:

Several in vivo and in vitro approaches were used to address the role of CCR4 in early atherosclerosis. Particularly, through the adoptive cell transfer of CCR4+ or CCR4- Tregs, the authors aimed to directly demonstrate the role of CCR4 in Tregs' protection against early atherosclerosis.

Weaknesses:

Flow cytometry experiments are not well controlled. Dead cells and doublets were not excluded from analysis.

Clinical relevance is unclear.

Reviewer #1 (Public review):

Summary:

In this manuscript, Liu et al have tried to dissect the neural and molecular mechanisms that C. elegans use to avoid digestion of harmful bacterial food. Liu et al show that C. elegans use the ON-OFF state of AWC olfactory neurons to regulate the digestion of harmful gram-positive bacteria S. saprophyticus (SS). The authors show that when C. elegans are fed on SS food, AWC neurons switch to OFF fate which prevents digestion of S. saprophyticus and this helps C. elegans avoid these harmful bacteria. Using genetic and transcriptional analysis as well as making use of previously published findings, Liu et al implicate the p38 MAPK pathway (in particular, NSY-1, the C. elegans homolog of MAPKKK ASK1) and insulin signaling in this process.

Strengths:

The authors have used multiple approaches to test the hypothesis that they present in this manuscript.

Weaknesses:

Overall, I am not convinced that the authors have provided sufficient evidence to support the various components of their hypothesis. While they present data that loosely align with their hypothesis, they fail to consider alternative explanations and do not use rigorous approaches to strengthen their overall hypothesis. The selective picking of genes from the RNA sequencing data and forcing the data to fit the proposed hypothesis based on previously published findings, without exploring other approaches, indicates a lack of thoroughness and rigor. These critical shortcomings significantly diminish enthusiasm for the manuscript in its totality. In my opinion, this is the biggest weakness in this manuscript.

Reviewer #1 (Public review):

Summary:

In their paper, Tutunji et al aim to investigate the dynamic effects of stress on activity of different brain networks (salience network, executive network, and default mode network). Crucially they differentiate between rapid (<1 h) and late (>1) effects of stress. Lastly, they connect acute changes in brain activity with inter-individual differences in stress reactivity in real-life assessed using EMA.

They first show the expected dynamics in stress-induced brain activity with a transient increase in salience network activity and a decrease in default mode network activity although in contrast to expectations, this did not disappear in the late phase. Notably, the increase in salience network activity was associated with a 'resilience index' derived from EMA that captures whether an individual responds with more or less reduction in positive effect than expected based on the number of above average stress events.

Linking acute stress to long-term affective stress reactivity is a crucial step to better understand how adaptive or maladaptive stress responses play out in the long term and how they might be related to mental health problems.

Strengths:

The link of the acute stress response to stress reactivity in daily life is highly relevant and a major strength of the paper. Moreover, the design of the EMA component assessing a week with low stress and one with high stress (exam week) in all participants and thus including a naturalistic manipulation enables a quantification of stress reactivity that captures 'real life'.

The authors do not only quantify the magnitude of the acute stress response but take into account an early as well as late response to disentangle the dynamic nature of the stress response. In that way, it is possible to establish which parts of the stress response are relevant for the affective response.

In addition to reporting changes in network activation, the authors also report behavioral outcomes of the tasks which is crucial to evaluate the meaning and relevance of the neural outcomes.

Weaknesses:

Although the authors assess multiple physiological outcomes to the stress task, only the cortisol response is analyzed with regard to its association with the stress-induced changes in network activity. Considering that it is mainly the salience network that shows an increase and this in the early phase that is characterized by the noradrenaline and not so much the cortisol response, an association with a marker of the NA response would be interesting.

To evaluate the association of the acute stress response with stress reactivity in real life more conclusively it would be interesting to see whether and how the affective response to the acute stress is related to stress reactivity in real life.

In the introduction, the authors hypothesize that all networks show distinct activation patterns during the stress response and expect all of them to be associated with the stress reactivity during EMA. However, no correction for multiple comparisons across the many tests (each network at two phases) is reported.

All stress-induced changes in activity are assessed by using other tasks since it is not trivial to measure changes in activation of specific regions without comparing different conditions of a task. Nonetheless, with the chosen approach it is not completely clear whether stress only modulates brain responses to other tasks or changes activation within those networks independently of any other tasks. Moreover, one of the tasks did not elicit the expected activation contrast and it is unclear whether this affects stress-effects.

Some of the less central results that are discussed in the paper such as the association of the real-life stress reactivity measure with neuroticism, the sex-effect of the cortisol response or the mediation and moderation models of the stress-induced changes in network activity and performance in the tasks seem slightly overinterpreted considering that they are either not quite significant or not hypothesized and thus it is not clear why for example once a mediation and in another outcome a moderation model was chosen.

Reviewer #1 (Public review):

Summary:

This is an important and interesting study that uses the split-GFP approach. Localization of receptors and correlating them to function is important in understanding the circuit basis of behavior.

Strengths:

The split-GFP approach allows visualization of subcellular enrichment of dopamine receptors in the plasma membrane of GAL4-expressing neurons allowing for high level of specificity.

The authors resolve the presynaptic localization of DopR1 and Dop2R, in "giant" Drosophila neurons differentiated from cytokinesis-arrested neuroblasts in culture as its not clear in the lobes and calyx.

Starvation induced opposite responses of dopamine receptor expression in the PPL1 and PAM DANs provides key insights into models of appetitive learning.<br /> Starvation induced increase in D2R allows for increased negative feedback that the authors test in D2R knockout flies where appetitive memory is diminished.<br /> This dual autoreceptor system is an attractive model for how amplitude and kinetics of dopamine release can be fine tunes and controlled depending on the cellular function and this paper presents a good methodology to do it and a good system where dynamics of dopamine release can be tested at the level of behavior.

Weaknesses:

Key weaknesses have been resolved: 

1) Receptor expression is consistent between time of the day and the authors picked two time points. The authors mention that the states of animals could affect LI (e.g. feeding state and anesthesia for sorting, see methods) were kept constant. These data and discussion are helpful. <br /> 2) Giant fiber system is argued to be a great model and authors have added additional references. However I am not very deeply familiar with these references or the giant fiber system so I am not completely clear but the argument seems reasonable. <br /> 3) The revised manuscript, shows data in the γ KCs (Figure 4C, Figure 5 - figure supplement 1) in addition to α/β KCs, so it appears there is consistency between lobes. <br /> 4) The new data for Dop1R1 and Dop2R in MBON-γ1pedc>αβ helps with thinking about dopamine receptor co-localization and it would be a herculean talk to do this for all the regions but still keeps room open for different scenarios. 

The papers discussion has been expanded to account for different possibilities which will help the readers get a more complete picture. I appreciate the review efforts and detailed response to reviewer comments.

Reviewer #1 (Public review):

Shen et al. conducted three experiments to study the cortical tracking of the natural rhythms involved in biological motion (BM), and whether these involve audiovisual integration (AVI). They presented participants with visual (dot) motion and/or the sound of a walking person. They found that EEG activity tracks the step rhythm, as well as the gait (2-step cycle) rhythm. The gait rhythm specifically is tracked superadditively (power for A+V condition is higher than the sum of the A-only and V-only condition, Experiments 1a/b), which is independent of the specific step frequency (Experiment 1b). Furthermore, audiovisual integration during tracking of gait was specific to BM, as it was absent (that is, the audiovisual congruency effect) when the walking dot motion was vertically inverted (Experiment 2). Finally, the study shows that an individual's autistic traits are negatively correlated with the BM-AVI congruency effect.

Reviewer #1 (Public review):

Summary:

This paper investigates the mechanism of axon growth directed by the conserved guidance cue UNC-6/Netrin. Experiments were designed to distinguish between alternative models in which UNC-6/Netrin functions as either a short range (haptotactic) cue or a diffusible (chemotactic) signal that steers axons to their final destinations. In each case, axonal growth cones execute ventrally directed outgrowth toward a proximal source of UNC-6/Netrin. This work concludes that UNC-6/Netrin functions as both a haptotactic and chemotactic cue to polarize the UNC-40/DCC receptor on the growth cone membrane facing the direction of growth. Ventrally directed axons initially contact a minor longitudinal nerve tract (vSLNC) at which UNC-6/Netrin appears to be concentrated before proceeding in the direction of the ventral nerve cord (VNC) from which UNC-6/Netrin is secreted. Time lapse imaging revealed that growth cones appear to pause at the vSLNC before actively extending ventrally directed filopodia that eventually contact the VNC. Growth cone contacts with the vSLNC were unstable in unc-6 mutants but were restored by expression of a membrane tethered UNC-6 in vSLNC neurons. In addition, expression of membrane tethered UNC-6/Netrin in the VNC was not sufficient to rescue initial ventral outgrowth in an unc-6 mutant. Finally, dual expression of membrane tethered UNC-6/Netrin in both vSLNC and VNC partially rescued the unc-6 mutant axon guidance defect, thus suggesting that diffusible UNC-6 is also required. This work is important because it potentially resolves the controversial question of how UNC-6/Netrin directs axon guidance by proposing a model in which both of the competing mechanisms, e.g., haptotaxis vs chemotaxis, are successively employed. The impact of this work is bolstered by its use of powerful imaging and genetic methods to test models of UNC-6/Netrin function in vivo thereby obviating potential artifacts arising from in vitro analysis.

Strengths:

A strength of this approach is the adoption of the model organism C. elegans to exploit its ready accessibility to live cell imaging and powerful methods for genetic analysis.

Weaknesses:

In the revised version of this manuscript, the authors have redressed the weaknesses highlighted in my review of the original paper.

Reviewer #1 (Public review):

Summary:

This interesting and well-written article by Tuckowski et al. summarizes work connecting the flavin-containing monooxygenase FMO-4 with increased lifespan through a mechanism involving calcium signaling in the nematode Caenorhabditis elegans.

The authors have previously studied another fmo in worms, FMO-2, prompting them to look at additional members of this family of proteins. They show that fmo-4 is up in dietary restricted worms and necessary for the increased lifespan of these animals as well as of rsks-1 (s6 kinase) knockdown animals. They then show that overexpression of fmo-4 is sufficient to significantly increase lifespan, as well as healthspan and paraquat resistance. Further, they demonstrate that overexpression of fmo-4 solely in the hypodermis of the animal recapitulates the entire effect of fmo-4 OE.

In terms of interactions between fmo-2 and fmo-4 they show that fmo-4 is necessary for the previously reported effects of fmo-2 on lifespan, while the effects of fmo-4 do not depend on fmo-2.

Next the authors use RNASeq to compare fmo-4 OE animals to wild type. Their analyses suggested the possibility that FMO-4 was modulating calcium signaling, and through additional experiments specifically identified the calcium signaling genes crt-1, itr-1, and mcu-1 as important fmo-4 interactors in this context. As previously published work has shown that loss of the worm transcription factor atf-6 can extend lifespan through crt-1, itr-1 and mcu-1, the authors asked about interactions between fmo-4 and atf-6. They showed that fmo-4 is necessary for both lifespan extension and increased paraquat resistance upon RNAi knockdown of atf-6.

Overall this clearly written manuscript summarizes interesting and novel findings of great interest in the biology of aging, and suggests promising avenues for future work in this area.

Strengths:

This paper contains a large number of careful, well executed and analysed experiments in support of its existing conclusions, and which also point toward significant future directions for this work. In addition it is clear and very well written.

Weaknesses:

Within the scope of the current work there are no major weaknesses. That said, the authors themselves note pressing questions beyond the scope of this study that remain unanswered. For instance, the mechanistic nature of the interactions between FMO-4 and the other players in this story, for example in terms of direct protein-protein interactions, is not at all understood yet. Further, powerful tools such as GCaMP expressing animals will enable a much more detailed understanding of what exactly is happening to calcium levels, and where and when it is happening, in these animals.

Reviewer #1 (Public review):

Hüppe and colleagues had already developed an apparatus and an analytical approach to capture swimming activity rhythms in krill. In a previous manuscript they explained the system, and here they employ it to show a circadian clock, supplemented by exogenous light, produces an activity pattern consistent with "twilight" diel vertical migration (DVM; a peak at sunset, a midnight sink, and a peak in the latter half of the night).

They used light:dark (LD) followed by dark:dark (DD) photoperiods at two times of the year to confirm the circadian clock, coupled with DD experiments at four times of year to show rhythmicity occurs throughout the year along with DVM in the wild population. The individual activity data show variability in the rhythmic response, which is expected. However, their results showed rhythmicity was sustained in DD throughout the year, although the amplitude decayed quickly. The interpretation of a weak clock is reasonable, and they provide a convincing justification for the adaptive nature of such a clock in a species that has a wide distributional range and experiences various photic environments. These data also show that exogenous light increases the activity response and can explain the morning activity bouts, with the circadian clock explaining the evening and late-night bouts. This acknowledgement that vertical migration can be driven by multiple proximate mechanisms is important.

The work is rigorously done, and the interpretations are sound. I see no major weaknesses in the manuscript. Because a considerable amount of processing is required to extract and interpret the rhythmic signals (see Methods and previous AMAZE paper), it is informative to have the individual activity plots of krill as a gut check on the group data.

The manuscript will be useful to the field as it provides an elegant example of looking for biological rhythms in a marine planktonic organism and disentangling the exogenous response from the endogenous one. Furthermore, as high latitude environments change, understanding how important organisms like krill have the potential to respond will become increasingly important. This work provides a solid behavioral dataset to complement the earlier molecular data suggestive of a circadian clock in this species.

Reviewer #1 (Public review):

Summary:

The authors' research group had previously demonstrated the release of large multivesicular body-like structures by human colorectal cancer cells. This manuscript expands on their findings, revealing that this phenomenon is not exclusive to colorectal cancer cells but is also observed in various other cell types, including different cultured cell lines, as well as cells in the mouse kidney and liver. Furthermore, the authors argue that these large multivesicular body-like structures originate from intracellular amphisomes, which they term "amphiectosomes." These amphiectosomes release their intraluminal vesicles (ILVs) through a "torn-bag mechanism." Finally, the authors demonstrate that the ILVs of amphiectosomes are either LC3B positive or CD63 positive. This distinction implicates that the ILVs either originate from amphisomes or multivesicular bodies, respectively.

Strengths:

The manuscript reports a potential origin of extracellular vesicle (EV) biogenesis. The reported observations are intriguing.

Weaknesses:

In their revised version, the authors have addressed the majority of my criticisms. I have no further concerns regarding this manuscript.

Reviewer #2 (Public review):

Summary:

Here Vogt et al., provide new insights into the need for sleep and the molecular and physiological response to sleep loss. The authors expand on their previously published work (Bjorness et al., 2020) and draw from recent advances in the field to propose a neuron-centric molecular model for the accumulation and resolution of sleep need and basis of restorative sleep function. While speculative, the proposed model successfully links important observations in the field and provides a framework to stimulate further research and advances on the molecular basis of sleep function. In my review, I highlight the important advances of this current work, the clear merits of the proposed model, and indicate areas of the model that can serve to stimulate further investigation.

Strengths: Reviewer comment on new data in Vogt et al., 2024<br /> Using classic slice electrophysiology, the authors conclude that wakefulness (sleep deprivation (SD)) drives a potentiation of excitatory glutamate synapses, mediated in large part by "un-silencing" of NMDAR-active synapses to AMPAR-active synapses. Using a modern single nuclear RNAseq approach the authors conclude that SD drives changes in gene expression primarily occurring in glutamatergic neurons. The two experiments combined highlight the accumulation and resolution of sleep need centered on the strength of excitatory synapses onto excitatory neurons. This view is entirely consistent with a large body of extant and emerging literature and provides important direction for future research.

Consistent with prior work, wakefulness/SD drives an LTP-type potentiation of excitatory synaptic strength on principle cortical neurons. It has been proposed that LTP associated with wake, leads to the accumulation of sleep need by increasing neuronal excitability, and by the "saturation" of LTP capacity. This saturation subsequently impairs the capacity for further ongoing learning. This new data provides a satisfying mechanism of this saturation phenomenon by introducing the concept of silent synapses. The new data show that in mice well rested, a substantial number of synapses are "silent", containing an NMDAR component but not AMPARs. Silent synapses provide a type of reservoir for learning in that activity can drive the un-silencing, increasing the number of functional synapses. SD depletes this reservoir of silent synapses to essentially zero, explaining how SD can exhaust learning capacity. Recovery sleep led to restoration of silent synapses, explaining how recovery sleep can renew learning capacity. In their prior work (Bjorness et al., 2020) this group showed that SD drives an increase in mEPSC frequency onto these same cortical neurons, but without a clear change in pre-synaptic release probability, implying a change in the number of functional synapses. This prediction is now born out in this new dataset.

The new snRNAseq dataset indicates the sleep need is primarily seen (at the transcriptional level) in excitatory neurons, consistent with a number of other studies. First, this conclusion is corroborated by two independent, contemporary snRNAseq analysis recently published in iScience 2024 doi: 10.1016/j.isci.2024.110752 and Neuroscience Research 2024 https://doi.org/10.1016/j.neures.2024.03.004. A recently published analysis on the effects of SD in drosophila imaged synapses in every brain region in a cell-type dependent manner (Weiss et al., PNAS 2024), concluding that SD drives brain wide increases in synaptic strength almost exclusively in excitatory neurons. Further, Kim et al., Nature 2022, heavily cited in this work, show that the newly described SIK3-HDAC4/5 pathway promotes sleep depth via excitatory neurons and not inhibitory neurons.

The new experiments provided in Fig1-3 are expertly conducted and presented. This reviewer has no comments of concern regarding the execution and conclusions of these experiments.

Reviewer comment on model in Vogt et al., 2024

To the view of this reviewer the new model proposed by Vogt et al., is an important contribution. The model is not definitively supported by new data, and in this regard should be viewed as a perspective, providing mechanistic links between recent molecular advances, while still leaving areas that need to be addressed in future work. New snRNAseq analysis indicates SD drives expression of synaptic shaping components (SSCs) consistent with the excitatory synapse as a major target for the restorative basis of sleep function. SD induced gene expression is also enriched for autism spectrum disorder (ASD) risk genes. As pointed out by the authors, sleep problems are commonly reported in ASD, but the emphasis has been on sleep amount. This new analysis highlights the need to understand the impact on sleep's functional output (synapses) to fully understand the role of sleep problems in ASD.

Importantly, SD induced gene expression in excitatory neurons overlap with genes regulated by the transcription factor MEF2C and HDAC4/5 (Fig. 4). In their prior work, the authors show loss of MEF2C in excitatory neurons abolished the SD transcriptional response and the functional recovery of synapses from SD by recovery sleep. Recent advances identified HDAC4/5 as major regulators of sleep depth and duration (in excitatory neurons) downstream of the recently identified sleep promoting kinase SIK3. In Zhou et al., and Kim et al., Nature 2022, both groups propose a model whereby "sleep-need" signals from the synapse activate SIK3, which phosphorylates HDAC4/5, driving cytoplasmic targeting, allowing for the de-repression and transcriptional activation of "sleep genes". Prior work shows that HDAC4/5 are repressors of MEF2C. Therefore, the "sleep genes" derepressed by HDAC4/5 may be the same genes activated in response to SD by MEF2C. The new model thereby extends the signaling of sleep need at synapses (through SIK3-HDAC4/5) to the functional output of synaptic recovery by expression of synaptic/sleep genes by MEF2C. The model thereby links aspects of expression of sleep need with the resolution of sleep need by mediating sleep function: synapse renormalization.

Weaknesses:

Areas for further investigation.<br /> In the discussion section Vogt et al., explore the links between excitatory synapse strength, arguably the major target of "sleep function", and NREM slow-wave activity (SWA), the most established marker of sleep need. SIK3-HDAC4/5 have major effects on the "depth" of sleep by regulation NREM-SWA. The effects of MEF2C loss of function on NREM SWA activity are less obvious, but clearly impact the recovery of glutamatergic synapses from SD. The authors point out how adenosine signaling is well established as a mediator of SWA, but the links with adenosine and glutamatergic strength are far from clear. The mechanistic links between SIK3/HDAC4/5, adenosine signaling, and MEF2C, are far from understood. Therefore, the molecular/mechanistic links between a synaptic basis of sleep need and resolution with NREM-SWA activity requires further investigation.

Additional work is also needed to understand the mechanistic links between SIK3-HDAC4/5 signaling and MEF2C activity. The authors point out that constitutively nuclear (cn) HDAC4/5 (acting as a repressor) will mimic MEF2C loss of function. This is reasonable, however, there are notable differences in the reported phenotypes of each. Notably, cnHDAC4/5 suppresses NREM amount and NREM SWA but had no effect on the NREM-SWA increase following SD (Zhou et al., Nature 2022). Loss of MEF2C in CaMKII neurons had no effect on NREM amount and suppressed the increase in NREM-SWA following SD (Bjorness et al., 2020). These instances indicate that cnHDAC4/5 and loss of MEF2C do not exactly match suggesting additional factors are relevant in these phenotypes. Likely HDAC4/5 have functionally important interactions with other transcription factors, and likewise for MEF2C, suggesting areas for future analysis.

One emerging theme may be that the SIK3-HDAC4/5 axis are major regulators of the sleep state, perhaps stabilizing the NREM state once the transition from wakefulness occurs. MEF2C is less involved in regulating sleep per se, and more involved in executing sleep function, by promoting the restorative synaptic modifications to resolve sleep need.

Finally, advances in the roles of the respective SIK3-HDAC4/5 and MEF2C pathways point towards transcription of "sleep genes", as clearly indicated in the model of Fig.4. Clearly more work is needed to understand how the expression of such genes ultimately lead to resolution of sleep need by functional changes at synapses. What are these sleep genes and how do they mechanistically resolve sleep need? Thus, the current work provides a mechanistic framework to stimulate further advances in understanding the molecular basis for sleep need and the restorative basis of sleep function.

Comments on revisions:

No further comments or concerns. I believe that the manuscript has been suitably revised, and the concerns raised by reviewers have been addressed. I am completely satisfied by the revisions and responses provided by the authors.

Reviewer #1 (Public review):

The study by Aguirre-Botero et al. shows the dynamics of 3D11 anti-CSP monoclonal antibody (mAb) mediated elimination of rodent malaria Plasmodium berghei (Pb) parasites in the liver. The authors show that the anti-CSP mAb could protect against intravenous (i.v.) Pb sporozoite challenge along with the cutaneous challenge, but requires higher concentration of antibody. Importantly, the study shows that the anti-CSP mAb not only affects sporozoite motility, sinusoidal extravasation, and cell invasion but also partially impairs the intracellular development inside the liver parenchyma, indicating a late effect of this antibody during liver stage development. While the study is interesting and conducted well, the only novel yet very important observation made in this manuscript is the effect of the anti-CSP mAb on liver stage development.

Comments on latest version:

No further comments.

Reviewer #1 (Public review):

Summary:

This manuscript by Kaya et al. studies the effect of food consumption on hippocampal sharp wave ripples (SWRs) in mice. The authors use multiple foods and forms of food delivery to show that the frequency and power of SWRs increase following food intake, and that this effect depends on the caloric content of food. The authors also studied the effects of the administration of various food-intake-related hormones on SWRs during sleep, demonstrating that ghrelin negatively affects SWR rate and power, but not GLP-1, insulin, or leptin. Finally, the authors use fiber photometry to show that GABAergic neurons in the lateral hypothalamus, increase activity during a SWR event.

Strengths:

The experiments in this study seem to be well performed, and the data are well presented, visually. The data support the main conclusions of the manuscript that food intake enhances hippocampal SWRs. Taken together, this study is likely to be impactful to the study of the impact of feeding on sleep behavior, as well as the phenomena of hippocampal SWRs in metabolism.

Weaknesses:

Details of experiments are missing in the text and figure legends. Additionally, the writing of the manuscript could be improved.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors identified that<br /> (1) CDK4/6i treatment attenuates the growth of drug-resistant cells by prolongation of the G1 phase;<br /> (2) CDK4/6i treatment results in an ineffective Rb inactivation pathway and suppresses the growth of drug-resistant tumors;<br /> (3) Addition of endocrine therapy augments the efficacy of CDK4/6i maintenance;<br /> (4) Addition of CDK2i with CDK4/6 treatment as second-line treatment can suppress the growth of resistant cell;<br /> (5) The role of cyclin E as a key driver of resistance to CDK4/6 and CDK2 inhibition.

Strengths:

To prove their complicated proposal, the authors employed orchestration of several kinds of live cell markers, timed in situ hybridization, IF and Immunoblotting. The authors strongly recognize the resistance of CDK4/6 + ET therapy and demonstrated how to overcome it.

Weaknesses:

The authors need to underscore their proposed results from what is to be achieved by them and by other researchers.

Reviewer #1 (Public review):

Summary:

The authors study the effect of the addition of synthetic amphiphile on the gating mechanisms of the mechano-sensitive channel MscL. They observe that the amphiphile reduces the membrane stretching and bending modulii, and increases the channel activation pressure. They then conclude that gating is sensitive to these two membrane parameters. This is explained by the effect of the amphiphile on the so-called membrane interfacial tension.

Strengths:

The major strength is that the authors found a way to tune the membrane's mechanical properties in a controlled manner, and find a progressive change of the suction pressure at which MscL gates. If analysed thoroughly, these results could give valuable information.

Weaknesses:

The weakness is the analysis and the discussion. I would like to have answers to some basic questions.

(1) The explanation of the phenomenon involves a difference between interfacial tension and tension, without the difference between these being precisely defined. In the caption of Figure 4, one can read "Under tension, the PEO groups adsorb to the bilayer, suggesting adsorption is a thermodynamically favorable process that lowers the interfacial tension." What does this mean? Under what tension is the interfacial tension lowered? The fact that the system's free energy could be lowered by putting it under mechanical tension would result in a thermodynamic unstable situation. Is this what the authors mean?

(2) From what I understand, a channel would feel the tension exerted by the membrane at its periphery, which is what I would call membrane tension. The fact that polymers may reorganise under membrane stretch to lower the system's free energy would certainly affect the membrane stretching modulus (as measured Figure 2E), but what the channel cares about is the tension (I would say). If the membrane is softer, a larger pipette pressure is required to reach the same level of tension, so it is not surprising that a given channel requires a larger activation pressure in softer membranes. To me, this doesn't mean that the channel feels the membrane stiffness, but rather that a given pressure leads to different tensions (which is what the channel feels) for different stiffnesses.

(3) In order to support the authors' claim, the micropipette suction pressure should be appropriately translated into a membrane tension. One would then see whether the gating tension is affected by the presence of amphiphiles. In the micropipette setup used here, one can derive a relationship between pressure and tension, that involves the shape of the membrane. This relationship is simple (tension=pressure difference times pipette radius divided by 2) only in the limit where the membrane tongue inside the pipette ends with a hemisphere of constant radius independent of the pressure, and the pipette radius is much smaller than the GUV radius. None of these conditions seem to hold in Figure 2C. On the other hand, the authors do report absolute values of tension in the y-axis of Figure 2D. It seems quite straightforward to plot the activation tension (rather than pressure) as a function of the amphiphile volume fraction in Figure 2B. This is what needs to be shown.

(4) The discussion needs to be improved. I could not find a convincing explanation of the role of interfacial tension in the discussion. The equation (p.14) distinguishes three contributions, which I understand to be (i) an elastic membrane deformation such as hydrophobic mismatch or other short-range effects, (ii) the protein conformation energy, and (iii) the work done by membrane tension. Apparently, the latter is where the effect is (which I agree with), but how this consideration leads to a gating energy difference (between lipid only and modified membrane) proportional to the interfacial tension is completely obscure (if not wrong).

(5) I am rather surprised at the very small values of stretching and bending modulii found under high-volume fraction. These quantities are obtained by fitting the stress-strain relationship (Figure 2D). Such a plot should be shown for all amphiphile volume fraction, so one can assess the quality of the fits.

Reviewer #1 (Public review):

Summary

In this manuscript, the authors generate an AAV-deliverable tool that generates action potentials in response to red light, but not blue light, when expressed in neurons. To do this, they screen some red light-excitatory/blue light-inhibitory opsin pairs to find ones that are spectrally and temporally matched. They first show that this works with Chrimson and GtACR2, however, they expand their search after finding that the tau-off (inactivation after light cessation) kinetics of these two opsins are not well-matched. They directly examine a small set of options based on a literature search and settle on a variant of red light-excitatory Chrimson and blue light-inhibitory ZipACR. To even more closely match the kinetics of this pair, the authors create a structure homology model of the ZipACR retinal binding pocket and use this to guide generation of a small mutant panel, leading to a more optimized ZipACR mutant. They then show that a bicistronically expressed fusion arrangement of these opsins, plus some functional peptides, can drive action potentials up to 20hz with red light and does not do so with blue light, in hippocampal cells transduced by AAV. They also show function in vivo, in a mouse, using a physiological readout. They conclude that their new tool may be useful for complex experimental designs requiring multiple optical channels for write-in/read-out.

The major advantage claimed by the authors over existing tools is the temporal time-locking of their inhibitory opsin - this is driven by the contrast between tau-off kinetics of their ZipACR variant compared to gtACR2, which is used by the leading competitor tool (BiPOLES).

Big thoughts<br /> While the authors were carefully thoughtful about the potential influence of temporal kinetics on the efficiency of a tool such as this one, there were no experiments conducted that make use of the unique properties of this molecular strategy (although the authors state that these experiments are now underway in their lab). They share some examples of how the tool could be useful in the discussion. Where do I think this could be useful?

First, experimental designs that require multiple optical channels of control. This appears to be aligned with the author's thoughts, as they state, correctly, that opsins utilizing retinal as a light-sensing chromophore are universally activated by blue light (the so-called 'blue shoulder'). Therefore, their tool may be useful for stimulating multiple populations using a blue excitatory opsin in neuron A and their tool for red excitation of neuron B - or, in the author's own words, "A potential solution to the problem of cross-talk...". In this manuscript, the authors provide state that there this is possible in theory and that there are no obvious reasons that it would not work, but do not present data that showcases their new tool for this purpose (e.g. Vierock, Johannes, et al. "BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons." Nature communications 12.1 (2021): 4527. Figure 4f-I; 6). The same set-up could be imagined for green GECI (or equivalent) imaging of cells in the same volume that their tool is being used in - for instance, interleaving red stimulation light and blue imaging light, (perhaps) without the typical concern of imaging light bleed-through activating the opsin itself. I agree that it will likely work for multi-channel control, but only time will tell, at this point.

Second, for high-frequency temporal control over both excitation and inhibition in the same neuron. Red light turns the cell on, and blue light turns the cell off (see, for instance, Zhang, Feng, et al. "Multimodal fast optical interrogation of neural circuitry." Nature 446.7136 (2007): 633-639. Figure 2; Vierock as above, Figure 4a,b). Again, here the authors are long on theory ("The new system...can drive time-locked high-frequency action potentials in response to red pulses") and short on explicit data. While they do show that red light = excitation and blue light = inhibition, they neither show 1) all-optical on/off modulation of the same cell; nor 2) high-frequency inhibition or excitation (max stim rate of 20hz, which is the same as the BiPOLES paper used for their LC stimulation paradigm; Vierock, as above, Figure 7a-d). They did provide a response to this critique that data showing excitation and inhibition spread across multiple panels were largely collected from the same cells.

Despite these major shortcomings, the further development and characterization of tandem opsins, such as this one, is of interest to the community. There is on-going work by the BiPOLES team to create new iterations (e.g. Wahid, J., et al. "P-15 BiPOLES2 is a bidirectional optogenetic tool with a narrow activation spectrum and low red-light excitability." Clinical Neurophysiology 148 (2023): e16.). The authors have collected a substantial amount of additional data along the course of review and, even aside from the final tool, the overall data and approaches shown are useful.

Reviewer #1 (Public review):

Summary:

Audio et al. measured cerebral blood volume (CBV) across cortical areas and layers using high-resolution MRI with contrast agents in non-human primates. While the non-invasive CBV MRI methodology is often used to enhance fMRI sensitivity in NHPs, its application for baseline CBV measurement is rare due to the complexities of susceptibility contrast mechanisms. The authors determined the number of large vessels and the areal and laminar variations of CBV in NHP, and compared those with various other metrics.

Strengths:

Noninvasive mapping of relative cerebral blood volume is novel for non-human primates. A key finding was the observation of variations in CBV across regions; primary sensory cortices had high CBV, whereas other higher areas had low CBV. The measured CBV values correlated with previously reported neuronal and receptor densities.

Weaknesses:

A weakness of this manuscript is that the quantification of CBV with postprocessing approaches to remove susceptibility effects from pial and penetrating vessels is not fully validated, especially on a laminar scale. Further specific comments follow.

(1) Baseline CBV indices were determined using contrast agent-enhanced MRI (deltaR2*). Although this approach is suitable for areal comparisons, its application at a laminar scale poses challenges due to significant contributions from large vessels including pial vessels. The primary concern is whether large-vessel contributions can be removed from the measured deltaR2* through processing techniques.

(2) High-resolution MRI with a critical sampling frequency estimated from previous studies (Weber 2008, Zheng 1991) was performed to separate penetrating vessels. However, this approach is still insufficient to accurately identify the number of vessels due to the blooming effects of susceptibility and insufficient spatial resolution. The reported number of penetrating vessels is only applicable to the experimental and processing conditions used in this study, which cannot be generalized.

(3) Baseline R2* is sensitive to baseline R2, vascular volume, iron content, and susceptibility gradients. Additionally, it is sensitive to imaging parameters; higher spatial resolution tends to result in lower R2* values (closer to the R2 value). Thus, it is difficult to correlate baseline R2* with physiological parameters.

(4) CBV-weighted deltaR2* is correlated with various other metrics (cytoarchitectural parcellation, myelin/receptor density, cortical thickness, CO, cell-type specificity, etc.). While testing the correlation between deltaR2* and these other metrics may be acceptable as an exploratory analysis, it is challenging for readers to discern a causal relationship between them. A critical question is whether CBV-weighted deltaR2* can provide insights into other metrics in diseased or abnormal brain states.

Reviewer #1 (Public review):

Summary:

Boldt et al test several possible relationships between trandiagnostically-defined compulsivity and cognitive offloading in a large online sample. To do so, they develop a new and useful cognitive task to jointly estimate biases in confidence and reminder-setting. In doing so, they find that over-confidence is related to less utilization of reminder-setting, which partially mediates the negative relationship between compulsivity and lower reminder-setting. The paper thus establishes that, contrary to the over-use of checking behaviors in patients with OCD, greater levels of transdiagnostically-defined compulsivity predicts less deployment of cognitive offloading. The authors offer speculative reasons as to why (perhaps it's perfectionism in less clinically-severe presentations that lowers the cost of expending memory resources), and sets an agenda to understand the divergence in cognitive between clinical and nonclinical samples. Because only a partial mediation had robust evidence, multiple effects may be at play, whereby compulsivity impacts cognitive offloading via overconfidence and also by other causal pathways.

Strengths:

The study develops an easy-to-implement task to jointly measure confidence and replicates several major findings on confidence and cognitive offloading. The study uses a useful measure of cognitive offloading - the tendency to set reminders to augment accuracy in the presence of experimentally manipulated costs. Moreover, the utilizes multiple measures of presumed biases -- overall tendency to set reminders, the empirically estimated indifference point at which people engage reminders, and a bias measure that compares optimal indifference points to engage reminders relative to the empirically observed indifference points. That the study observes convergenence along all these measures strengthens the inferences made relating compulsivity to the under-use of reminder-setting. Lastly, the study does find evidence for one of several a priori hypotheses and sets a compelling agenda to try to explain why such a finding diverges from an ostensible opposing finding in clinical OCD samples and the over-use of cognitive offloading.

Weaknesses:

Although I think this design and study are very helpful for the field, I felt that a feature of the design might reduce the tasks's sensitivity to measuring dispositional tendencies to engage cognitive offloading. In particular, the design introduces prediction errors, that could induce learning and interfere with natural tendencies to deploy reminder-setting behavior. These PEs comprise whether a given selected strategy will be or not be allowed to be engaged. We know individuals with compulsivity can learn even when instructed not to learn (e.g., Sharp, Dolan and Eldar, 2021, Psychological Medicine), and that more generally, they have trouble with structure knowledge (eg Seow et al; Fradkin et al), and thus might be sensitive to these PEs. Thus, a dispositional tendency to set reminders might be differentially impacted for those with compulsivity after an NPE, where they want to set a reminder, but aren't allowed to. After such an NPE, they may avoid moreso the tendency to set reminders. Those with compulsivity likely have superstitious beliefs about how checking behaviors lead to a resolution of catastrophes, that might in part originate from inferring structure in the presence of noise or from purely irrelevant sources of information for a given decision problem.<br /> It would be good to know if such learning effects exist, if they're modulated by PE (you can imagine PEs are higher if you are more incentivized - e.g., 9 points as opposed to only 3 points - to use reminders, and you are told you cannot use them), and if this learning effect confounds the relationship between compulsivity and reminder-setting.

A more subtle point, I think this study can be more said to be an exploration than a deductive of test of a particular model -> hypothesis -> experiment. Typically, when we test a hypothesis, we contrast it with competing models. Here, the tests were two-sided because multiple models, with mutually exclusive predictions (over-use or under-use of reminders) were tested. Moreover, it's unclear exactly how to make sense of what is called the direct mechanism, which is supported by the partial (as opposed to complete) mediation.

Reviewer #1 (Public review):

Summary:

The authors measured glutamate transients in the DMS of rats as they performed an action selection task. They identified diverse patterns of behavior and glutamate dynamics depending on the pre-existing behavioral phenotype of the rat (sign tracker or goal tracker). Using pathway-specific DREADDs, they showed that these behavioral phenotypes and their corresponding glutamate transients were differentially dependent on input from the prelimbic cortex to the DMS.

Strengths:

Overall there are some very interesting results that make an important contribution to the field. Notably, the results seem to point to differential recruitment of the PL-DMS pathway in goal-tracking vs sign-tracking behaviors.

Weaknesses:

(1) The controls for off-target effects of CNO are not given sufficient importance both in terms of power and in reporting of their results. There is precedent to accept that CNO at the dosage given is unlikely to disrupt the behaviour, this doesn't justify the assumption that glutamate transmission won't be affected, and this possibility hasn't been sufficiently ruled out.<br /> (2) The specificity of the viral approach needs to be clarified. Figure 8 indicates a large proportion of the PL neuron population that expresses mCherry in the absence of AAV-Cre. This infers that there are a large number of neurons inhibited by CNO administration that were outside the projection pathway, drawing into question the specificity of the effects.

Reviewer #1 (Public review):

Summary:

This paper investigates the effects of the explicit recognition of statistical structure and sleep consolidation on the transfer of learned structure to novel stimuli. The results show a striking dissociation in transfer ability between explicit and implicit learning of structure, finding that only explicit learners transfer structure immediately. Implicit learners, on the other hand, show an intriguing immediate structural interference effect (better learning of novel structure) followed by successful transfer only after a period of sleep.

Strengths:

This paper is very well written and motivated, and the data are presented clearly with a logical flow. There are several replications and control experiments and analyses that make the pattern of results very compelling. The results are novel and intriguing, providing important constraints on theories of consolidation. The discussion of relevant literature is thorough. In sum, this work makes an exciting and important contribution to the literature.

Weaknesses:

There have been several recent papers which have identified issues with alternative forced choice (AFC) tests as a method of assessing statistical learning (e.g. Isbilen et al. 2020, Cognitive Science). A key argument is that while statistical learning is typically implicit, AFC involves explicit deliberation and therefore does not match the learning process well. The use of AFC in this study thus leaves open the question of whether the AFC measure benefits the explicit learners in particular, given the congruence between knowledge and testing format, and whether, more generally, the results would have been different had the method of assessing generalization been implicit. Prior work has shown that explicit and implicit measures of statistical learning do not always produce the same results (eg. Kiai & Melloni, 2021, bioRxiv; Liu et al. 2023, Cognition).

The authors argued in their response to this point that this issue could have quantitative but not qualitative impacts on the results, but we see no reason that the impact could not be qualitative. In other words, it should be acknowledged that an implicit test could potentially result in the implicit group exhibiting immediate structure transfer.

Given that the explicit/implicit classification was based on an exit survey, it is unclear when participants who are labeled "explicit" gained that explicit knowledge. This might have occurred during or after either of the sessions, which could impact the interpretation of the effects and deserves discussion.

Reviewer #1 (Public review):

Summary:

The authors investigate the role of the melanocortin system in puberty onset. They conclude that POMC neurons within the arcuate nucleus of the hypothalamus provide important but differing input to kisspeptin neurons in the arcuate or rostral hypothalamus.

Strengths:

Innovative and novel<br /> Technically sound<br /> Well-designed<br /> Thorough

Weaknesses:

There were no major weaknesses identified.

Reviewer #2 (Public review):

This work describes a highly complex automated algorithm for analyzing vascular imaging data from two-photon microscopy. This tool has the potential to be extremely useful to the field and to fill gaps in knowledge of hemodynamic activity across a regional network. The biological application provided, however, has several problems that make many of the scientific claims in the paper questionable.

The authors have commented on my main concerns. They have provided some limited evidence in the literature of prolonged vascular signals - though still nothing close to the several hundred-second long vascular responses oscillating between dilation and constriction shown here. And they have added a nice experiment showing they can resolve small beads (though still quite bigger than their average capillary diameter) with their system. They have also added comparisons with other software which shows some modest but clear improvement in some aspects. All these make the paper stronger.

However, I still think the main overall problem from the biological interpretation side of the paper is still not fixed. Perhaps I am too skeptical but I have a hard time accepting the conclusions about dilators and constrictors (depth dependence, distance from nearest neuron, etc.) because the data are just too temporally sparse and too unconventional in their duration and fluctuation. Also, the differences are often very small compared to the variability.

Regarding the spatial resolution, I was more concerned that if the pixel size is about 1 micron, then detecting around 1 micron dilations (or even less) is really below the resolution of the system. While the bead imaging is good for showing they can extract these diameters very close to the real value, this is still not like a living brain with imaging and motion artifacts. Given the temporal resolution issues already mentioned, this makes me highly skeptical of the biological claims. I think the discussion should at least strongly emphasize that a major caveat in their analysis is that the diameters are only sampled every 42 seconds, and , given the fluctuation in vessel diameter above and below baseline, this makes classification of the vessel as constrictor/dilator and by how much highly dependent on what time point the vessel diameter was sampled.

Although the computational side of the paper is not my strong point, it seems there is potential for the pipeline to be useful in other applications. But given the limitations of the system they are using, I feel that it is a methods paper in its current form more than anything that should be making the biological claims included.

Reviewer #2 (Public review):

In this study, the authors measured extracellular electrical features of colliding APs travelling in different directions down an isolated earthworm axon. They then used these features to build a model of the potential ephaptic effects of AP annihilation, i.e. the electrical signals produced by colliding/annihilating APs that may influence neighbouring tissue. The model was then applied to some different hypothetical scenarios involving synaptic connections. In a revised version of the manuscript, it was also applied, with success, to published experimental data on the cerebellar basket cell-to-Purkinje cell pinceau connection. The conclusion is that an annihilating AP at a presynaptic terminal can emphatically influence the voltage of a postsynaptic cell (the 'electrical coupling between neurons' of the title), and that the nature of this influence depends on the physical configuration of the synapse.

As an experimental neuroscientist who has never used computational approaches, I am unable to comment on the rigour of the analytical approaches that form the bulk of this paper. The experimental approaches appear very well carried out, and the data showing equal conduction velocity of anti- and orthodromically propagating APs in every preparation are convincing.

The conclusions drawn from the synaptic modelling are considerably strengthened by the data in Figure 5. Here, the authors' model - including AP annihilation at a synaptic terminal - is used to predict the amplitude and direction of experimentally observed effects at the cerebellar basket cell-to-Purkinje cell synapse (Blot & Barbour 2014). One particular form of the model (RTM with tau=0.5ms and realistic non-excitability of the terminal) matches the experimental data extremely well. The authors also include a convincing demonstration (Panel A) that a propagating but not annihilating AP has almost no effect on a neighbouring neuron's activity. Given that the authors' model of ephaptic effects can quantitatively explain key features of experimental data pertaining to synaptic function, the implications for the relevance of ephaptic coupling at different synaptic contacts may be widespread and important.

Reviewer #1 (Public review):

Summary:

The authors of this study investigated the development of interoceptive sensitivity in the context of cardiac and respiratory interoception in 3-, 9-, and 18-month-old infants using a combination of both cross-sectional and longitudinal designs. They utilised the cardiac interoception paradigm developed by Maister et al (2017) and also developed a new paradigm to investigate respiratory interoception in infants. The main findings of this research are that 9-month-old infants displayed a preference for stimuli presented synchronously with their own heartbeat and respiration. The authors found less reliable effects in the 18-month-old group, and this was especially true for the respiratory interoceptive data. The authors replicated a visual preference for synchrony over asynchrony for the cardiac domain in 3-month-old infants, while they found inconclusive evidence regarding the respiratory domain. Considering the developmental nature of the study, the authors also investigated the presence of developmental trajectories and associations between the two interoceptive domains. They found evidence for a relationship between cardiac and respiratory interoceptive sensitivity at 18 months only and preliminary evidence for an increase in respiratory interoception between 9 and 18 months.

Strengths:

The conclusions of this paper are mostly well supported by data, and the data analysis procedures are rigorous and well-justified. The main strengths of the paper are:

- A first attempt to explore the association between two different interoceptive domains. How different organ-specific axes of interoception relate to each other is still open and exploring this from a developmental lens can help shed light into possible relationships. The authors have to be commended for developing a novel interoceptive tasks aimed at assessing respiratory interoceptive sensitivity in infants and toddlers, and for trying to assess the relationship between cardiac and respiratory interoception across developmental time.<br /> - A thorough justification of the developmental ages selected for the study. The authors provide a rationale behind their choice to examine interoceptive sensitivity at 3, 9, and 18-months of age. These are well justified based on the literature pertaining to self- and social development. Sometimes, I wondered whether explaining the link between these self and social processes and interoception would have been beneficial as a reader not familiar with the topics may miss the point.<br /> - An explanation of direction of looking behaviour using latent curve analysis. I found this additional analysis extremely helpful in providing a better understanding of the data based on previous research and analytical choices. As the authors explain in the manuscript, it is often difficult to interpret the direction of infant looking behaviour as novelty and familiarity preferences can also be driven by hidden confounders (e.g. task difficulty). The authors provide compelling evidence that analytical choices can explain some of these effects. Beyond the field of interoception, these findings will be relevant to development psychologists and will inform future studies using looking time as a measure of infants' ability to discriminate among stimuli.<br /> - The use of simulation analysis to account for small sample size. The authors acknowledge that some of the effects reported in their study could be explained by a small sample size (i.e. the 3-month-olds and 18-month-olds data). Using a simulation approach, the authors try to overcome some of these limitations and provide convincing evidence of interoceptive abilities in infancy and toddlerhood (but see also my next point).

Comments on revision:

The authors have clearly addressed the comments on the previous version of this manuscript. I have no further comments.

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging.

Joint Public Review:

Engineered artificial gene regulatory networks ("circuits") have a wide range of applications, but their design is often hindered by unforeseen interactions between the host and circuit processes. This manuscript employs computational modeling to investigate how growth feedback influences the performance of synthetic gene circuits capable of adaptation. By analyzing 425 hypothetical circuits previously identified as achieving nearly perfect adaptation (Ma et al., 2009; Shi et al., 2017), the authors introduce growth feedback into their models using additional terms in ordinary differential equations. Their simulations reveal that growth feedback can disrupt adaptation dynamics in diverse ways but also identify core motifs that ensure robust performance under such conditions. Additionally, they establish a scaling law linking circuit robustness to the strength of growth feedback. The findings have important implications for synthetic biology, where host-circuit interactions frequently compromise desired behaviors, and for systems biology, by advancing the understanding of network motif dynamics. The authors' classification schemes will be highly valuable to the community, offering a framework for addressing growth-related challenges in circuit design.

Strengths<br /> - A detailed investigation into the reasons for adaptation failure upon the introduction of cell growth was conducted, distinguishing this work from other studies of functional screening in gene regulatory network topologies. The comprehensiveness of the analysis is particularly noteworthy.<br /> - Approaches for assessing robustness, such as the survival ratio Q, were employed, providing tools that may be applicable to a broad range of network topologies beyond adaptation. The scaling law derived from these approaches is both novel and insightful.<br /> - A thorough numerical analysis of three gene regulatory networks exhibiting adaptation was performed. For each of the 425 topologies analyzed, approximately 2e5 circuits were sampled using Latin hypercube sampling, ensuring robust coverage of the parameter space. Among these, 1.5e5 circuits were identified as showing adaptation and subsequently subjected to further analysis, yielding approximately 350 parametric designs per topology for deeper investigation.<br /> - The systematic approach and depth of the analysis position this study as a significant contribution to the understanding of gene regulatory networks and their response to growth feedback. The combination of detailed investigation, novel robustness metrics, and rigorous computational techniques enhances the impact of this work within the field.

Weaknesses<br /> - The study focuses exclusively on a preselected set of 425 topologies previously shown to achieve adaptation, limiting the exploration of whether growth feedback could enable adaptation in circuits not inherently adaptive. While the authors have discussed and justified this choice, the focus restricts the generality of the conclusions, as the potential for growth feedback to induce adaptation in non-adaptive circuits remains unaddressed. The analysis includes scenarios where higher growth feedback restores adaptation in circuits that lose it at intermediate levels, but further elaboration on the implications for circuit design would strengthen the impact. The numerical framework and parameter choices align well with established methods, and an overview of the selected topologies has been provided. However, offering detailed information in supplementary materials or a public repository would further enhance the paper's accessibility and reproducibility.

- The model fails to capture the influence of protein levels on growth. To ensure accurate modeling of protein-level effects on growth, the b(t) term should be scaled appropriately, similar to Tan et al. Nature Chemical Biology 5:842-848 (2009).

- The authors propose bistability or multistability as the primary mechanisms behind different types of adaptation failure, explaining why the failures do not occur precisely at bifurcation points. They argue that their ODE simulations provide evidence for oscillation-related bifurcations, and an included appendix explores this phenomenon further, detailing how it can be observed in their results. While the authors choose not to apply semi-analytic methods, such as numerical continuation and eigenvalue analysis, to validate the existence of bifurcations, their approach offers valuable insights into the underlying dynamics of adaptation failures.

- The analysis in this work is carried out exclusively in a deterministic regime, as the focus is on scenarios where the effects of noise are assumed to be minimal. This approach is justified, and the authors acknowledge the complexity of extending their analysis to include stochasticity, which they suggest as an avenue for future research. The discussion has been expanded to address the potential impact of noise, its handling, and the assumptions underlying its exclusion. It is important to note, however, that noise can significantly alter system behavior-for instance, stabilizing trajectories and removing oscillations, as shown in prior studies (e.g., 10.1016/j.cels.2016.01.004). Additionally, variability in experimental implementations may influence the dynamics beyond what is predicted in deterministic models. These factors should be considered when interpreting the results.

Reviewer #2 (Public review):

Summary:

This manuscript provides a comprehensive overview of potential resistance mutations within MET Receptor Tyrosine Kinase and defines how specific mutations affect different inhibitors and modes of target engagement. The goal is to identify inhibitor combinations with the lowest overlap in their sensitivity to resistant mutations and determine if certain resistance mutations/mechanisms are more prevalent for specific modes of ATP-binding site engagement. To achieve this, the authors measured the ability of ~6000 single mutants of MET's kinase domain (in the context of a cytosolic TPR fusion) to drive IL-3-independent proliferation (used as a proxy for activity) of Ba/F3 cells (deep mutational profiling) in the presence of 11 different inhibitors. The authors then used co-crystal and docked structures of inhibitor-bound MET complexes to define the mechanistic basis of resistance and applied a protein language model to develop a predictive model of inhibitor sensitivity/resistance.

Strengths:

The major strengths of this manuscript are the comprehensive nature of the study and the rigorous methods used to measure the sensitivity of ~6000 MET mutants in a pooled format. The dataset generated will be a valuable resource for researchers interested in understanding kinase inhibitor sensitivity and, more broadly, small molecule ligand/protein interactions. The structural analyses are systematic and comprehensive, providing interesting insights into resistance mechanisms. Furthermore, the use of machine learning to define inhibitor-specific fitness landscapes is a valuable addition to the narrative. Although the ESM1b protein language model is only moderately successful in identifying the underlying mechanistic basis of resistance, the authors' attempt to integrate systematic sequence/function datasets with machine learning serves as a foundation for future efforts.

Weaknesses:

The main limitation of this study is that the authors' efforts to define general mechanisms between inhibitor classes were only moderately successful due to the challenge of uncoupling inhibitor-specific interaction effects from more general mechanisms related to the mode of ATP-binding site engagement. However, this is a minor limitation that only minimally detracts from the impressive overall scope of the study.

Reviewer #1 (Public review):

Summary:

In this study from Zhu and colleagues, a clear role for MED26 in mouse and human erythropoiesis is demonstrated that is also mapped to amino acids 88-480 of the human protein. The authors also show the unique expression of MED26 in later-stage erythropoiesis and propose transcriptional pausing and condensate formation mechanisms for MED26's role in promoting erythropoiesis. Despite the author's introductory claim that many questions regarding Pol II pausing in mammalian development remain unanswered, the importance of transcriptional pausing in erythropoiesis has actually already been demonstrated (Martell-Smart, et al. 2023, PMID: 37586368, which the authors notably did not cite in this manuscript). Here, the novelty and strength of this study is MED26 and its unique expression kinetics during erythroid development.

Strengths:

The widespread characterization of kinetics of mediator complex component expression throughout the erythropoietic timeline is excellent and shows the interesting divergence of MED26 expression pattern from many other mediator complex components. The genetic evidence in conditional knockout mice for erythropoiesis requiring MED26 is outstanding. These are completely new models from the investigators and are an impressive amount of work to have both EpoR-driven deletion and inducible deletion. The effect on red cell number is strong in both. The genetic over-expression experiments are also quite impressive, especially the investigators' structure-function mapping in primary cells. Overall the data is quite convincing regarding the genetic requirement for MED26. The authors should be commended for demonstrating this in multiple rigorous ways.

Weaknesses:

(1) The authors state that MED26 was nominated for study based on RNA-seq analysis of a prior published dataset. They do not however display any of that RNA-seq analysis with regards to Mediator complex subunits. While they do a good job showing protein-level analysis during erythropoiesis for several subunits, the RNA-seq analysis would allow them to show the developmental expression dynamics of all subunit members.

(2) The authors use an EpoR Cre for red cell-specific MED26 deletion. However, other studies have now shown that the EpoR Cre can also lead to recombination in the macrophage lineage, which clouds some of the in vivo conclusions for erythroid specificity. That being said, the in vitro erythropoiesis experiments here are convincing that there is a major erythroid-intrinsic effect.

(3) The donor chimerism assessment of mice transplanted with MED26 knockout cells is a bit troubling. First, there are no staining controls shown and the full gating strategy is not shown. Furthermore, the authors use the CD45.1/CD45.2 system to differentiate between donor and recipient cells in erythroblasts. However, CD45 is not expressed from the CD235a+ stage of erythropoiesis onwards, so it is unclear how the authors are detecting essentially zero CD45-negative cells in the erythroblast compartment. This is quite odd and raises questions about the results. That being said, the red cell indices in the mice are the much more convincing data.

(4) The authors make heavy use of defining "erythroid gene" sets and "non-erythroid gene" sets, but it is unclear what those lists of genes actually are. This makes it hard to assess any claims made about erythroid and non-erythroid genes.

(5) Overall the data regarding condensate formation is difficult to interpret and is the weakest part of this paper. It is also unclear how studies of in vitro condensate formation or studies in 293T or K562 cells can truly relate to highly specialized erythroid biology. This does not detract from the major findings regarding genetic requirements of MED26 in erythropoiesis.

(6) For many figures, there are some panels where conclusions are drawn, but no statistical quantification of whether a difference is significant or not.

Reviewer #2 (Public review):

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

The revised manuscript has addressed all of the previous comments. The re-analysis of flow cytometry and WB data by authors makes the results and conclusion more complete and convincing.

Reviewer #1 (Public review):

Summary:

TMEM16, OSCA/TMEM63, and TMC belong to a large superfamily of ion channels where TMEM16 members are calcium activated lipid scramblases and chloride channels, whereas OSCA/TMEM63 and TMCs are mechanically activated ion channels. In the TMEM16 family, TMEM16F is a well characterized calcium activated lipid scramblase that play an important role in processes like blood coagulation, cell death signaling, and phagocytosis. In a previous study the group has demonstrated that lysine mutation in TM4 of TMEM16A can enable the calcium activated chloride channel to permeate phospholipids too. Based on this they hypothesize that the energy barrier for lipid scramblase in these ion channels is low, and that modification in the hydrophobic gate region by introducing a charged side chain between TM4/6 interface in TMEM16 and OSCA/TMEM63 family can allow lipid scramblase. In this manuscript, using scramblase activity via Annexin V binding to phosphatidylserine, and electrophysiology, the authors demonstrate that lysine mutation in TM4 of TMEM16F and TMEM16A can cause constitutive lipid scramblase activity. The authors then go on to show that analogous mutations in OSCA1.2 and TMEM63A can lead to scramblase activity. The revised version does a thorough characterization of residues that form the hydrophobic gate region in TM4/6 of this superfamily of channels. Their results indicated that disrupting the TM4/6 interaction can reduce energy barrier for this channels to scramblase lipids.

Strengths:

Overall, the authors introduce an interesting concept that this large superfamily can permeate ions and lipids.

Weaknesses:

none noted in the revised version.

Reviewer #1 (Public review):

Summary:

The authors addressed the influence of DKK2 on colorectal cancer (CRC) metastasis to the liver using an orthotopic model transferring AKP-mutant organoids into the spleens of wild-type animals. They found that DKK2 expression in tumor cells led to enhanced liver metastasis and poor survival in mice. Mechanistically, they associate Dkk2-deficiency in donor AKP tumor organoids with reduced Paneth-like cell properties, particularly Lz1 and Lyz2, and defects in glycolysis. Quantitative gene expression analysis showed no significant changes in Hnf4a1 expression upon Dkk2 deletion. Ingenuity Pathway Analysis of RNA-Seq data and ATAC-seq data point to a Hnf4a1 motif as a potential target. They also show that HNF4a binds to the promoter region of Sox9, which leads to LYZ expression and upregulation of Paneth-like properties. By analyzing available scRNA data from human CRC data, the authors found higher expression of LYZ in metastatic and primary tumor samples compared to normal colonic tissue; reinforcing their proposed link, HNF4a was highly expressed in LYZ+ cancer cells compared to LYZ- cancer cells.

Strengths:

Overall, this study contributes a novel mechanistic pathway that may be related to metastatic progression in CRC.

Weaknesses:

The main concerns are related to incremental gains, missing in vivo support for several of their conclusions in murine models, and missing human data analyses.

Main comments

Novelty:<br /> The authors previously described the role of DKK2 in primary CRC, correlating increased DKK2 levels to higher Src phosphorylation and HNF4a1 degradation, which in turn enhances LGR5 expression and "stemness" of cancer cells, resulting in tumor progression (PMID: 33997693). A role for DKK2 in metastasis has also been previously described (sarcoma, PMID: 23204234)

Mouse data:<br /> (a) The authors analyzed liver mets, but the main differences between AKT and AKP/Dkk2 KO organoids could arise during the initial tumor cell egress from the intestinal tissue (which cannot be addressed in their splenic injection model), or during pre-liver stages, such as endothelial attachment. While the analysis of liver mets is interesting, given that Paneth cells play a role in the intestinal stem cell niche, it is questionable whether a study that does not involve the intestine can appropriately address this pathway in CRC metastasis.<br /> (b) The overall number of Paneth cells found in the scRNA-seq analysis of liver mets was low (17 cells, Fig.3), and assuming that these cells are driving the differences seems somewhat far-fetched.<br /> (c) Fig. 6 suggests a signaling cascade in which the absence of DKK2 leads to enhanced HNF4A expression, which in turn results in reduced Sox9 expression and hence reduced expression of Paneth cell properties. It is therefore crucial that the authors perform in vivo (splenic organoid injection) loss-of-function experiments, knockdown of Sox9 expression in AKP organoids, and Sox9 overexpression experiments in AKP/Dkk2 KO organoids to demonstrate Sox9 as the central downstream transcription factor regulating liver CRC metastasis.<br /> (d) Given the previous description of the role of DKK2 in primary CRC, it is important to define the step of liver metastasis affected by Dkk2 deficiency in the metastasis model. Does it affect extravasation, liver survival, etc.?

Human data:<br /> Can the authors address whether the expression of Dkk2 changes in human CRC and whether mutations in Dkk2 as correlated with metastatic disease or CRC stage?

Bioinformatic analysis<br /> GEO repositories remain not open (at the time of the re-review) and SRA links for raw data are still unavailable. Without access to raw data, it is not possible to verify the analyses or fully assess the results. A part of the article was made by re-analyzing public data so the authors should make even the raw available and not just the count tables

Reviewer #1 (Public review):

Summary:

The author developed a new device to overcome current limitations in the imaging process of 3D spheroidal structures. In particular, they created a system to follow in real-time tumour spheroid formation, fusion and cell migration without disrupting their integrity. The system has also been exploited to test the effects of a therapeutic agent (chemotherapy) and immune cells.

Comments on revised version:

The authors well addressed all my concerns. It is a wonderful design to view the 3D cell spheroids.

Reviewer #2 (Public review):

This study presents valuable insight on how neurons within the central amygdala may broadly encode the valence of emotional stimuli. The evidence supporting most of the authors' conclusion is solid, although some of the claims should be treated with caution due to potential alternative interpretation of the data.

In this revised manuscript the authors have addressed the reviewers' critiques in a way that acknowledges the feedback but does not fully embrace or rigorously address the reviewers' core concerns. Here are the main observations that support this impression:

(1) The authors repeatedly acknowledge the ambiguity in defining "valence" and "salience" in the literature, but their responses don't clarify how they address these terms more rigorously. They seem to justify their operational definitions by citing previous studies but do not address how their definitions impact the clarity and robustness of their findings.

(2) The reviewers highlighted that using stimuli from different sensory modalities without scaling them or including neutral cues limits the ability to distinguish between valence and salience. The authors acknowledge this but argue that using same-modality stimuli would not produce distinct responses. This response doesn't address the reviewers' point about how these design limitations could weaken the conclusions. They seem to rely on citations of similar experimental designs instead of addressing the core critique or proposing additional experiments.

(3) In response to the low number of cue-responsive units and the call for more rigorous behavioral measures (like licking or orienting), the authors provide some data but emphasize statistical rigor over behavioral insights, which was questioned during the initial review. They don't propose any methodological adjustments or consider alternative explanations.

(4) The reviewers suggested clustering or other population-level analyses to understand functional diversity within the central amygdala. The authors argue that their statistical approach was sufficient and don't believe additional clustering analyses would add value. This response seems dismissive, as they don't consider whether population-level insights might reveal patterns that single-cell responses overlook.

Overall, while the authors have responded to each concern, their rebuttals often reference other studies to justify their choices rather than addressing the specific limitations highlighted by the reviewers.

Reviewer #1 (Public review):

The authors present an important work where they model some of the complex interactions between immune cells, fibroblasts and cancer cells. The model takes into account the increased ECM production of cancer-associated fibroblasts. These fibres trap the cancer but also protect it from immune system cells. In this way, these fibroblasts' actions both promote and hinder cancer growth. By exploring different scenarios, the authors can model different cancer fates depending on the parameters regulating cancer cells, immune system cells and fibroblasts. In this way, the model explores non-trivial scenarios. An important weakness of this study is that, though it is inspired by NSCLC tumors, it is still far from modelling tumor lesions with morphologies similar to NSCLC tumors and does not explore the formation of ramified tumors. In this way, is a general model and it is challenging how it can be adapted to simulate more realistic tumor morphologies.

Comments on revisions:

The authors have improved the manuscript and addressed my concerns.

Reviewer #1 (Public review):

Summary:

The authors created a transgenic mouse line to read out integrated stress responses with single-cell resolution.

Strengths:

ISR plays an important role in the development, maintenance, and degeneration of the nervous system. This mouse line represents a potentially important tool to understand ISR in situ.

Weaknesses:

The current manuscript is clearly written. However, more validation experiments should be performed to understand the exact meaning of the fluorescence intensity of GFP and RFP channels. This is important because these results will define how this tool will be used in the future and in the field.

Reviewer #1 (Public review):

Summary:

In organisms with an open mitosis, nuclear envelope breakdown at mitotic entry and re-assembly of the nuclear envelope at the end of mitosis are important, highly regulated processes. One key regulator of nuclear envelope re-assembly is the BAF (Barrier-to-Autointegration) protein, which contributes to cross-linking of chromosomes to the nuclear envelope. Crucially, BAF has to be in a dephosphorylated form to carry out this function, and PP2A has been shown to be the phosphatase which dephosphorylates BAF. The Ankle2/LEM4 protein has previously been identified as an important regulator of PP2A in the dephosphorylation of BAF but its precise function is not fully understood, and Li and colleagues set out to investigate the function of Ankle2/LEM4 in both Drosophila flies and Drosophila cell lines.

Strengths:

The authors use a combination of biochemical and imaging techniques to understand the biology of Ankle2/LEM4. On the whole the experiments are well conducted and the results look convincing. A particular strength of this manuscript is that the authors are able to study both cellular phenotypes and organismal effects of their mutants by studying both Drosophila D-mel cells and whole flies.<br /> The work presented in this manuscript significantly enhances our understanding of how Ankle2/LEM4 supports BAF dephosphorylation at the end of mitosis. Particularly interesting is finding that Ankle2/LEM4 appears to be a bona fide PP2A regulatory protein in Drosophila, as well as the localisation of Ankle2/LEM4 and how this is influenced by the interaction between Ankle2 and the ER protein Vap33. It would be interesting to see, though, whether these insights are conserved in mammalian cells, e.g. does mammalian Vap33 also interact with LEM4? Is LEM4 also a part of the PP2A holoenzyme complex in mammalian cells?

Weaknesses:

This work is certainly impactful but more discussion and comparison of the Drosophila versus mammalian cell system would be helpful. Also, to attract the largest possible readership, the Ankle2 protein should be referred to as Ankle2/LEM4 throughout the paper to make it clear that this is the same molecule.

A schematic model at the end of the final figure would be very useful to summarise the findings.

Comments on revisions:

The authors have carefully revised the manuscripts and have satisfactorily addressed the issues that were raised by the reviewers.

Reviewer #1 (Public review):

The hypothesis is based on the idea that inversions capture genetic variants that have antagonistic effects on male sexual success (via some display traits) and survival of females (or both sexes) until reproduction. Furthermore, a sufficiently skewed distribution of male sexual success will tend to generate synergistic epistasis for male fitness even if the individual loci contribute to sexually selected traits in an additive way. This should favor inversions that keep these male-beneficial alleles at different loci together at a cis-LD. A series of simulations are presented and show that the scenario works at least under some conditions. While a polymorphism at a single locus with large antagonistic effects can be maintained for a certain range of parameters, a second such variant with somewhat smaller effects tends to be lost unless closely linked. It becomes much more likely for genomically distant variants that add to the antagonism to spread if they get trapped in an inversion; the model predicts this should drive accumulation of sexually antagonistic variants on the inversion versus standard haplotype, leading to the evolution of haplotypes with very strong cumulative antagonistic pleiotropic effects. This idea has some analogies with one of predominant hypotheses for the evolution of sex chromosomes, and the authors discuss these similarities. The model is quite specific, but the basic idea is intuitive and thus should be robust to the details of model assumption. It makes perfect sense in the context of the geographic pattern of inversion frequencies. One prediction of the models (notably that leads to the evolution of nearly homozygously lethal haplotypes) does not seem to reflect the reality of chromosomal inversions in Drosophila, as the authors carefully discuss, but it is the case of some other "supergenes", notably in ants. So the theoretical part is a strong novel contribution,

To provide empirical support for this idea, the authors study the dynamics of inversions in population cages over one generation, tracking their frequencies through amplicon sequencing at three time points: (young adults), embryos and very old adult offspring of either sex (>2 months from adult emergence). Out of four inversions included in the experiment, two show patterns consistent with antagonistic effects on male sexual success (competitive paternity) and the survival of offspring, especially females, until an old age, which the authors interpret as consistent with their theory.

As I have argued in my comments on previous versions, the experiment only addresses one of the elements of the theoretical hypothesis, namely antagonistic effects of inversions on male reproductive success and other fitness components, in particular of females. Furthermore, the design of this experiment is not ideal from the viewpoint of the biological hypothesis it is aiming to test. This is in part because, rather than testing for the effects of inversion on male reproductive success versus the key fitness components of survival to maturity and female reproductive output, it looks at the effects on male reproductive success versus survival to a rather old age of 2 months. The relevance of survival until old age to fitness under natural conditions is unclear, as the authors now acknowledge. Furthermore, up to 15% of males that may have contributed to the next generation did not survive until genotyping, and thus the difference between these males' inversion frequency and that in their offspring may be confounded by this potential survival-based sampling bias. The experiment does not test for two other key elements of the proposed theory: the assumption of frequency-dependence of selection on male sexual success, and the prediction of synergistic epistasis for male fitness among genetic variants in the inversion. To be fair, particularly testing for synergistic epistasis would be exceedingly difficult, and the authors have now included a discussion of the above caveats and limitations, making their conclusions more tentative. This is good but of course does not make these limitations of the experiment go away. These limitations mean that the paper is stronger as a theoretical than as an empirical contribution.

Reviewer #3 (Public review):

The manuscript by Goyal et al report substrate-bound and substrate-free structures of a tripartite ATP independent periplasmic (TRAP) transporter from a previously uncharacterized homolog, F. nucleatum. This is one of most mechanistically fascinating transporter families, by means of its QM domain (the domain reported in his manuscript) operating as a monomeric 'elevator', and its P domain functioning as a substrate-binding 'operator' that is required to deliver the substrate to the QM domain; together, this is termed an 'elevator with an operator' mechanism. Remarkably, previous structures had not demonstrated the substrate Neu5Ac bound. In addition, they confirm the previously reported Na+ binding sites, and report a new metal binding site in the transporter, which seems to be mechanistically relevant. Finally, they mutate the substrate binding site and use proteoliposomal uptake assays to show the mechanistic relevance of the proposed substrate binding residues.

Strengths:

The structures are of good quality, the presentation of the structural data has improved, the functional data is robust, the text is well-written, and the authors are appropriately careful with their interpretations. Determination of a substrate bound structure is an important achievement and fills an important gap in the 'elevator with an operator' mechanism.

Weaknesses:

Although the possibility of the third metal site is compelling, I do not feel it is appropriate to model in a publicly deposited PDB structure without directly confirming experimentally. The authors do not extensively test the binding sites due to technical limitations of producing relevant mutants; however, their model is consistent with genetic assays of previously characterized orthologs, which will be of benefit to the field.

Reviewer #1 (Public review):

Summary:

In this paper, the authors present an interesting strategy to interfere with the HBV life cycle: the preparation of geranyl and peptides' dimers that could impede the correct assembly of hepatitis B core protein HBc into viable capsids. These dimers are of different nature, depending on the HBc site the authors plan to target. A preliminary study with geranyl dimers (targeting a hydrophobic site of HBc) was first investigated. The second series deals with peptide-PEG linker-peptide dimers, targeting the tips of HBc dimer spikes.

Strengths:

This work is very well conducted, combining ITC experiments (for determination of dimers' KD), cellular effects (thanks to the grafting of previously developed dimers with polyarginine-based cell penetrating peptide) HBV infected HEK293 cells and Cryo-EM studies.<br /> The findings of these research teams unambiguously demonstrated the interest of such dimeric structures in impeding the correct HBV life cycle and thus, could bring solutions in the control of its development. Ultimately, a new class of HBV Capside Assembly Modulators could arise from this study.<br /> There is no doubt that this work could bring very interesting information for people working on VHB.

Comments on revisions:

Minor corrections have been made in this revised version of this work, according to the remarks of the reviewers.

Reviewer #1 (Public Review):

Summary:

In this manuscript, "PAbFold: Linear Antibody Epitope Prediction using AlphaFold2", the authors generate a python wrapper for the screening of antibody-peptide interactions using AlphaFold, and test the performance of AlphaFold on 3 antibody-peptide complexes. In line with previous observations regarding the ability of AlphaFold to predict antibody structures and antigen binding, the results are mixed. While the authors are able to use AlphaFold to identify and experimentally validate a previously characterized broad binding epitope with impressive precision, they are unable to consistently identify the proper binding registers for their control [Myc-tag, HA-tag] peptides. Further, it appears that the reproducibility and generality of these results are low, with new versions of AlphaFold negatively impacting the predictive power. However, if this reproducibility issue is solved, and the test set is greatly increased, this manuscript could contribute strongly towards our ability to predict antibody-antigen interactions.

Strengths:

Due to the high significance, but difficulty, of the prediction of antibody-antigen interactions, any attempts to break down these predictions into more tractable problems should be applauded. The authors' approach of focusing on linear epitopes (peptides) is clever, reducing some of the complexities inherent to antibody binding. Further, the ability of AlphaFold to narrow down a previously broadly identified experimental epitope is impressive. The subsequent experimental validation of this more precisely identified epitope makes for a nice data point in the assessment of AlphaFold's ability to predict antibody-antigen interactions.

Weaknesses:

Without a larger set of test antibody-peptide interactions, it is unclear whether or not AlphaFold can precisely identify the binding register of a given antibody to a given peptide antigen. Even within the small test set of 3 antibody-peptide complexes, performance is variable and depends upon the scFv scaffold used for unclear reasons. Lastly, the apparent poor reproducibility is concerning, and it is not clear why the results should rely so strongly on which multi-sequence alignment (MSA) version is used, when neither the antibody CDR loops nor the peptide are likely to strongly rely on these MSAs for contact prediction.

Major Point-by-Point Comments:

(1) The central concern for this manuscript is the apparent lack of reproducibility. The way the authors discuss the issue (lines 523-554) it sounds as though they are unable to reproduce their initial results (which are reported in the main text), even when previous versions of AlphaFold2 are used. If this is the case, it does not seem that AlphaFold can be a reliable tool for predicting antibody-peptide interactions.

(2) Aside from the fundamental issue of reproducibility, the number of validating tests is insufficient to assess the ability of AlphaFold to predict antibody-peptide interactions. Given the authors' use of AlphaFold to identify antibody binding to a linear epitope within a whole protein (in the mBG17:SARS-Cov-2 nucleocapsid protein interaction), they should expand their test set well beyond Myc- and HA-tags using antibody-antigen interactions from existing large structural databases.

(3) As discussed in lines 358-361, the authors are unsure if their primary control tests (antibody binding to Myc-tag and HA-tag) are included in the training data. Lines 324-330 suggest that even if the peptides are not included in the AlphaFold training data because they contain fewer than 10 amino acids, the antibody structures may very well be included, with an obvious "void" that would be best filled by a peptide. The authors must confirm that their tests are not included in the AlphaFold training data, or re-run the analysis with these templates removed.

(4) The ability of AlphaFold to refine the linear epitope of antibody mBG17 is quite impressive and robust to the reproducibility issues the authors have run into. However, Figure 4 seems to suggest that the target epitope adopts an alpha-helical structure. This may be why the score is so high and the prediction is so robust. It would be very useful to see along with the pLDDT by residue plots a structure prediction by residue plot. This would help to see if the high confidence pLDDT is coming more from confidence in the docking of the peptide or confidence in the structure of the peptide.

(5) Related to the above comment, pLDDT is insufficient as a metric for assessing antibody-antigen interactions. There is a chance (as is nicely shown in Figure S3C) that AlphaFold can be confident and wrong. Here we see two orange-yellow dots (fairly high confidence) that place the peptide COM far from the true binding region. While running the recommended larger validation above, the authors should also include a peptide RMSD or COM distance metric, to show that the peptide identity is confident, and the peptide placement is roughly correct. These predictions are not nearly as valuable if AlphaFold is getting the right answer for the wrong reasons (i.e. high pLDDT but peptide binding to a non-CDR loop region). Eventual users of the software will likely want to make point mutations or perturb the binding regions identified by the structural predictions (as the authors do in Figure 4).

Comments on revisions:

I have read the author's responses and the revised manuscript. The authors did not sufficiently address my comments, nor the fundamental issue with the manuscript.

By the authors' own admission, many of the results presented in the current version of the manuscript cannot be reproduced without relying on locally saved MSAs. In other words, there is almost no evidence presented that this pipeline will predict antibody-antigen interactions using currently publicly available software. This manuscript is reduced to essentially a case study (N=1) in how one might go about making such predictions coupled with pretty good experimental evidence backing up this singular prediction.

Reviewer #1 (Public review):

Summary:

Rigor in the design and application of scientific experiments is an ongoing concern in preclinical (animal) research. Because findings from these studies are often used in the design of clinical (human) studies, it is critical that the results of the preclinical studies are valid and replicable. However, several recent peer-reviewed published papers have shown that some of the research results in cardiovascular research literature may not be valid because their use of key design elements is unacceptably low. The current study is designed to expand on and replicate previous preclinical studies in nine leading scientific research journals. Cardiovascular research articles that were used for examination were obtained from a PubMed Search. These articles were carefully examined for four elements that are important in the design of animal experiments: use of both biological sexes, randomization of subjects for experimental groups, blinding of the experimenters, and estimating the proper size of samples for the experimental groups. The findings of the current study indicate that the use of these four design elements in the reported research in preclinical research is unacceptably low. Therefore, the results replicate previous studies and demonstrate once again that there is an ongoing problem in the experimental design of preclinical cardiovascular research.

Strengths:

This study selected four important design elements for study. The descriptions in the text and figures of this paper clearly demonstrate that the rate of use of all four design elements in the examined research articles was unacceptably low. The current study is important because it replicates previous studies and continues to call attention once again to serious problems in the design of preclinical studies, and the problem does not seem to lessen over time.

Weaknesses:

Weaknesses from the first review were adequately addressed.

Reviewer #1 (Public review):

Summary:

Authors of this article have previously shown the involvement of the transcription factor Zinc finger homeobox-3 (ZFHX3) in the function of the circadian clock and the development/differentiation of the central circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. Here, they show that ZFHX3 plays a critical role in the transcriptional regulation of numerous genes in the SCN. Using inducible knockout mice, they further demonstrate that the deletion Of Zfhx3 induces a phase advance of the circadian clock, both at the molecular and behavioral levels.

Strengths:

- Inducible deletion of Zfhx3 in adults<br /> - Behavioral analysis<br /> - Properly designed and analyzed ChIP-Seq and RNA-Seq supporting the conclusion of the behavioral analysis

Weaknesses:

- Further characterization of the disruption of the activity of the SCN is required.<br /> - The description of the controls needs some clarification.

Reviewer #1 (Public review):

Summary:

Migration of the primordial germ cells (PGCs) in mice is asynchronous, such that leading and lagging populations of migrating PGCs emerge. Prior studies found that interactions between the cells the PGCs encounter along their migration routes regulates their proliferation. In this study, the authors used single cell RNAseq to investigate PGC heterogeneity and to characterize their niches during their migration along the AP axis. Unlike prior scRNAseq studies of mammalian PGCs, the authors conducted a time course covering 3 distinct stages of PGC migration (pre, mid, and post migration) and isolated PGCs from defined somite positions along the AP axis. In doing so, this allowed the authors to uncover differences in gene expression between leading and lagging PGCs and their niches and to investigate how their transcript profiles change over time. Among the pathways with the biggest differences were regulators of actin polymerization and epigenetic programming factors and Nodal response genes. In addition, the authors report changes in somatic niches, specifically greater non-canonical WNT in posterior PGCs compared to anterior PGCs. This relationship between the hindgut epithelium and migrating PGCs was also detected in reanalysis of a previously published dataset of human PGCs. Using whole mount immunofluorescence, the authors confirmed elevated Nodal signaling based on detection of the LEFTY antagonists and targets of Nodal during late stage PGC migration. Taken together, the authors have assembled a temporal and spatial atlas of mouse PGCs and their niches. This resource and the data herein provide support for the model that interactions of migrating mouse PGCs with their niches influences their proliferation, cytoskeletal regulation, epigenetic state and pluripotent state.

Overall, the findings provide new insights into heterogeneity among leading and lagging PGC populations and their niches along the AP axis, as well as comparisons between mouse and human migrating PGCs. The data are clearly presented, and the text is clear and well-written. This atlas resource will be valuable to reproductive and developmental biologists as a tool for generating hypotheses and for comparisons of PGCs across species.

Strengths:

(1) High quality atlas of individual PGCs prior to, during and post migration and their niches at defined positions along the AP axis.<br /> (2) Comparisons to available datasets, including human embryos, provide insight into potentially conserved relationships among PGCs and the identified pathways and gene expression changes.<br /> (3) Detailed picture of PGC heterogeneity.<br /> (4) Valuable resource for the field.<br /> (5) Some validation of Nodal results and further support for models in the literature based on less comprehensive expression analysis.

Weaknesses:

(1) No indication of which sex(es) were used for the mouse data and whether or not sex-related differences exist or can excluded at the stages examined. This should be clarified.

Reviewer #1 (Public review):

This manuscript introduces a useful curation pipeline of antibody-antigen structures downloaded from the PDB database. The antibody-antigen structures are presented in a new database called AACDB, alongside annotations that were either corrected from those present in the PDB database or added de-novo with a solid methodology. Sequences, structures, and annotations can be very easily downloaded from the AACDB website, speeding up the development of structure-based algorithms and analysis pipelines to characterize antibody-antigen interactions. However, AACDB is missing some key annotations that would greatly enhance its usefulness.

Here are detailed comments regarding the three strengths above:

(1) I think potentially the most significant contribution of this database is the manual data curation to fix errors present in the PDB entries, by cross-referencing with the literature. However, as a reviewer, validating the extent and the impact of these corrections is hard, since the authors only provided a few anecdotal examples in their manuscript.

I have personally verified some of the examples presented by the authors and found that SAbDab appears to fix the mistakes related to the misidentification of antibody chains, but not other annotations.

(a) "the species of the antibody in 7WRL was incorrectly labeled as "SARS coronavirus B012" in both PDB and SabDab" → I have verified the mistake and fix, and that SAbDab does not fix is, just uses the pdb annotation.<br /> (b) "1NSN, the resolution should be 2.9 , but it was incorrectly labeled as 2.8" → I have verified the mistake and fix, and that saabdab does not fix it, just uses the PDB annotation.<br /> (c) "mislabeling of antibody chains as other proteins (e.g. in 3KS0, the light chain of B2B4 antibody was misnamed as heme domain of flavocytochrome b2)" → SAbDab fixes this as well in this case.<br /> (d) "misidentification of heavy chains as light chains (e.g. both two chains of antibody were labeled as light chain in 5EBW)" → SAbDab fixes this as well in this case.

I personally believe the authors should make public the corrections made, and describe the procedures - if systematic - to identify and correct the mistakes. For example, what was the exact procedure (e.g. where were sequences found, how were the sequences aligned, etc.) to find mutations? Was the procedure run on every entry?

(2) I believe the splitting of the pdb files is a valuable contribution as it standardizes the distribution of antibody-antigen complexes. Indeed, there is great heterogeneity in how many copies of the same structure are present in the structure uploaded to the PDB, generating potential artifacts for machine learning applications to pick up on. That being said, I have two thoughts both for the authors and the broader community. First, in the case of multiple antibodies binding to different epitopes on the same antigen, one should not ignore the potentially stabilizing effect that the binding of one antibody has on the complex, thereby enabling the binding of the second antibody. In general, I urge the community to think about what is the most appropriate spatial context to consider when modeling the stability of interactions from crystal structure data. Second, and in a similar vein, some antigens occur naturally as homomultimers - e.g. influenza hemagglutinin is a homotrimer. Therefore, to analyze the stability of a full-antigen-antibody structure, I believe it would be necessary to consider the full homo-trimer, whereas, in the current curation of AACDB with the proposed data splitting, only the monomers are present.

(3) I think the annotation of interface residues is a useful addition to structural datasets, but their current presentation is lacking on several fronts.

I think the manuscript is lacking in justification about the numbers used as cutoffs (1A^2 for change in SASA and 5A for maximum distance for contact) The authors just cite other papers applying these two types of cutoffs, but the underlying physico-chemical reasons are not explicit even in these papers. I think that, if the authors want AACDB to be used globally for benchmarks, they should provide direct sources of explanations of the cutoffs used, or provide multiple cutoffs. Indeed, different cutoffs are often used (e.g. ATOM3D uses 6A instead of 5A to determine contact between a protein and a small molecule https://datasets-benchmarks-proceedings.neurips.cc/paper/2021/hash/c45147dee729311ef5b5c3003946c48f-Abstract-round1.html)

I think the authors should provide a figure with statistics pertaining to the interface atoms. I think showing any distribution differences between interface atoms determined according to either strategy (number of atoms, correlation between change in SASA and distance...) would be fundamental to understanding the two strategies. I think other statistics would constitute an enhancement as well (e.g. proportion of heavy vs. light chain residues).

Some obvious limitations of AACDB in its current form include:

AACDB only contains entries with protein-based antigens of at most 50 amino acids in length. This excludes non-protein-based antigens, such as carbohydrate- and nucleotide-based, as well as short peptide antigens.

AACDB does not include annotations of binding affinity, which are present in SAbDab and have been proven useful both for characterizing drivers of antibody-antigen interactions (cite https://www.sciencedirect.com/science/article/pii/S0969212624004362?via%3Dihub) and for benchmarking antigen-specific antibody-design algorithms (cite https://www.biorxiv.org/content/10.1101/2023.12.10.570461v1)).

In conclusion, I believe AACDB has the potential to be a more standardized and error-light database for antibody-antigen complex structures. It is, however, hard to evaluate the extent to which errors have been corrected since the authors do not provide a list of the errors or a step-by-step procedure for fixing the errors. Unfortunately, AACDB is currently missing binding affinity annotations, which hinders its usefulness.

Reviewer #1 (Public review):

Summary:

Mackie and colleagues compare chemosensory preferences between C. elegans and P. pacificus, and the cellular and molecular mechanisms underlying them. The nematodes have overlapping and distinct preferences for different salts. Although P. pacificus lacks the lsy-6 miRNA important for establishing asymmetry of the left/right ASE salt-sensing neurons in C. elegans, the authors find that P. pacificus ASE homologs achieve molecular (receptor expression) and functional (calcium response) asymmetry by alternative means. This work contributes an important comparison of how these two nematodes sense salts and highlights that evolution can find different ways to establish asymmetry in small nervous systems to optimize the processing of chemosensory cues in the environment.

Strengths:

The authors use clear and established methods to record the response of neurons to chemosensory cues. They were able to show clearly that ASEL/R are functionally asymmetric in P. pacificus, and combined with genetic perturbation establish a role for che-1-dependent gcy-22.3 in in the asymmetric response to NH4Cl.

Weaknesses:

The mechanism of lsy-6-independent establishment of ASEL/R asymmetry in P. pacificus remains uncharacterized.

Reviewer #1 (Public review):

Summary:

In this paper Kawasaki et al describe a regulatory role for the PIWI/piRNA pathway in rRNA regulation in Zebrafish. This regulatory role was uncovered through a screen for gonadogenesis defective mutants, which identified a mutation in the meioc gene, a coiled-coil germ granule protein. Loss of this gene leads to redistribution of Piwil1 from germ granules to the nucleolus, resulting in silencing of rRNA transcription.

Strengths:

Most of the experimental data provided in this paper is compelling. It is clear that in the absence of meioc, PiwiL1 translocates in to the nucleolus and results in down regulation of rRNA transcription. the genetic compensation of meioc mutant phenotypes (both organismal and molecular) through reduction in PiwiL1 levels are evidence for a direct role for PiwiL1 in mediating the phenotypes of meioc mutant.

Weaknesses:

Questions remain on the mechanistic details by which PiwiL1 mediated rRNA down regulation, and whether this is a function of Piwi in an unperturbed/wildtype setting. There is certainly some evidence provided in support of the natural function for piwi in regulating rRNA transcription (figure 5A+5B). However, the de-enrichment of H3K9me3 in the heterozygous (Figure 6F) is very modest and in my opinion not convincingly different relative to the control provided. It is certainly possible that PiwiL1 is regulating levels through cleavage of nascent transcripts. Another aspect I found confounding here is the reduction in rRNA small RNAs in the meioc mutant; I would have assumed that the interaction of PiwiL1 with the rRNA is mediated through small RNAs but the reduction in numbers do not support this model. But perhaps it is simply a redistribution of small RNAs that is occurring. Finally, the ability to reduce PiwiL1 in the nucleolus through polI inhibition with actD and BMH-21 is surprising. What drives the accumulation of PiwiL1 in the nucleolus then if in the meioc mutant there is less transcription anyway?

Despite the weaknesses outlined, overall I find this paper to be solid and valuable, providing evidence for a consistent link between PIWI systems and ribosomal biogenesis. Their results are likely to be of interest to people in the community, and provide tools for further elucidating the reasons for this link.

Reviewer #1 (Public review):

Summary:

By way of background, the Jiang lab has previously shown that loss of the type II BMP receptor Punt (Put) from intestinal progenitors (ISCs and EBs) caused them to differentiate into EBs, with a concomitant loss of ISCs (Tian and Jiang, eLife 2014). The mechanism by which this occurs was activation of Notch in Put-deficient progenitors. How Notch was upregulated in Put-deficient ISCs was not established in this prior work. In the current study, the authors test whether a very low level of Dl was responsible. But co-depletion of Dl and Put led to a similar phenotype as depletion of Put alone. This result suggested that Dl was not the mechanism. They next investigate genetic interactions between BMP signaling and Numb, an inhibitor of Notch signaling. Prior work from Bardin, Schweisguth and other labs has shown that Numb is not required for ISC self-renewal. However the authors wanted to know whether loss of both the BMP signal transducer Mad and Numb would cause ISC loss. This result was observed for RNAi depletion from progenitors and for mad, numb double mutant clones. Of note, ISC loss was observed in 40% of mad, numb double mutant clones, whereas 60% of these clones had an ISC. They then employed a two-color tracing system called RGT to look at the outcome of ISC divisions (asymmetric (ISC/EB) or symmetric (ISC/ISC or EB/EB)). Control clones had 69%, 15% and 16%, respectively, whereas mad, numb double mutant clones had much lower ISC/ISC (11%) and much higher EB/EB (37%). They conclude that loss of Numb in moderate BMP loss of function mutants increased symmetric differentiation which lead caused ISC loss. They also reported that numb15 and numb4 clones had a moderate but significant increase in ISC-lacking clones compared to control clones, supporting the model that Numb plays a role in ISC maintenance. Finally, they investigated the relevance of these observation during regeneration. After bleomycin treatment, there was a significant increase in ISC-lacking clones and a significant decrease in clone size in numb4 and numb15 clones compared to control clones. Because bleomycin treatment has been shown to cause variation in BMP ligand production, the authors interpret the numb clone under bleomycin results as demonstrating an essential role of Numb in ISC maintenance during regeneration.

Strengths:

(i) Most data is quantified with statistical analysis<br /> (ii) Experiments have appropriate controls and large numbers of samples<br /> (iii) Results demonstrate an important role of Numb in maintaining ISC number during regeneration and a genetic interaction between Mad and Numb during homeostasis.

Weaknesses:

(i) No quantification for Fig. 1<br /> (ii) The premise is a bit unclear. Under homeostasis, strong loss of BMP (Put) leads to loss of ISCs, presumably regardless of Numb level (which was not tested). But moderate loss of BMP (Mad) does not show ISC loss unless Numb is also reduced. I am confused as to why numb does not play a role in Put mutants. Did the authors test whether concomitant loss of Put and Numb leads to even more ISC loss than Put-mutation alone.<br /> (iii) I think that the use of the word "essential" is a bit strong here. Numb plays an important role but in either during homeostasis or regeneration, most numb clones or mad, numb double mutant clones still have ISCs. Therefore, I think that the authors should temper their language about the role of Numb in ISC maintenance.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors set out to determine how a DNA demethylation enzyme TET2 regulates beta cell senescence in the context of Type 2 Diabetes and aging. They analyze public RNA-seq data and found upregulation of TET2 coincident with downregulation of MOF and PTEN, genes involved in chromatin regulation and cell cycle. TET2 is upregulated during aging, high-fat diet feeding, high glucose on rat beta cell line INS1E, and in leptin receptor deficient (db/db) mice islets. This was not found for TET1 and TET3. TET2 global KO mice show improved glucose tolerance during aging, but not TET1 or TET3. The authors show improved beta cell identity genes in TET2 KO islets. They they performed DNA methyalation/hydroxymethylation analyses of TET2 KO transformed rat beta cell line INS1E followed by ChIP-seq of Histone H4K16 acetylation to find this mark relies on TET2 expression. Finally they demonstrate in the cell lines that overexpressing TET2 leads to loss of MOF and increased PTEN and p16, linking TET2 to a regulatory mechanism with these factors that may influence senescence.

Strengths:

The study uses a number of orthogonal approaches and evidence from cell lines and the genetic TET2 KO as well as primary islets. The concept is interesting and potentially useful to the field. Efforts were made to examine TET1 and TET3 paralogues to rule out their compensation.

Weaknesses:

The study has several major weaknesses that mean the data presented did not fully support the main conclusions. These include the following:

(1) From the beginning of the manuscript the authors first sentence does not seem to indicate which datasets were analysed, the rationale behind why public datasets were used and what the main conclusions are being drawn from the plots shown throughout Fig. 1. This section of the manuscript was very hard to follow, and lacked rationale and explanation as to what these data show.

(2) All of the metabolic phenotypic data come from global TET2 KO mice, where TET2 is lost from all cells. The authors need to use a beta cell-specific KO of TET2 to ensure that metabolic changes are not due to cross-talk with other tissues (e.g. liver, adipose, even effects on central control of metabolism). No insulin tolerance tests were done to ascertain phenotypes in other metabolic tissues. This was a major weakness of the study. The authors should also provide clear validation of their global TET2 KO mice demonstrating a total lack of protein in islets and metabolic tissues.

(3) TET2 localization and expression pattern in islets was not clearly demonstrated and the data shown are not convincing from Fig 3 and Fig 4. In Fig 3e the staining for TET2 in green looks ubiquitous in acinar tissue (not nuclear) and not in the islet. In Fig 4d there is an increase in nuclear stain shown during aging, but no INS stain is used to show specificity to beta cells. Thus there is not sufficient data to support the expression pattern and localization of TET2 and specificity of the antibody.

(4) In Fig. 5: The effect sizes for the beta cell identity gene expression differences by qRT-PCR between WT and TET2 KO islets shown in Fig 5 are extremely modest so as to be questionable whether they are biologically meaningful. The same is true of the senescence markers quantified from isolated islets by qRT-PCR in Fig 5f. The immunostains for Pdx1 are hard to see and signal should be quantified. The SA-Bgal staining is quantified but no representative image is shown. The p16 immunostaining is not clear and should be quantified. Given that a lack of truly specific p16 antibodies in mouse immunostainings have been a major issue for the field, the authors would be advised to demonstrate specificity of the antibody if possible on mouse KO tissue, or to at least validate the predicted increase in p16 staining comparing young versus old pancreas as has been shown in other studies.

(5) Throughout the manuscript the figures colors are difficult to see and text difficult to read. Text in the p-values above the bars on most Figures is not legible (particularly Figs 4, 5, and 9). The legends simply do not contain sufficient information to interpret the data panels. This is true from Figures 1 through 9. P-value and specific statistical tests are missing from legends as well. For instance, in Fig 6c, what is being shown in LV-Ctrl vs LV-TET2 and why are these sample labels the same for two sets of images with two different outcomes of the staining? How many cells were quantified here?

(6) There is an over-reliance on cell lines throughout the manuscript. INS1E and BTC6 are not truly representative of mature adult mouse or rat beta cells, and hence the connections between H4K16ac/MOF/PTEN and TET2 must be assessed in primary mouse or rat islets to confirm these phenotypes.

(7) In the in vitro studies of senescence markers, it is not convincingly shown that the cells are actually senescent. Even though there changes found in expression of p16 and SA-Bgal in the cultures, the authors did not evaluate key senescence phenotypes such as the actual cell cycle arrest, SASP proteins or apoptosis resistance. Are the cells actually senescent or are these markers simply increasing? Hence much of the changes driven by TET2 overexpression in the in vitro cell lines could likely changes in p16 protein but not actually a senescence phenotype. BTC6, INS1E, and MIN6 are cell lines that are transformed, and while they can undergo some senescence-like changes in response to specific stressors like lipotoxicity, DNA damage, or oxidative stress, the authors did not evaluate these, only senescence genes/proteins in otherwise unstressed cells. Thus the claim that TET2 modifies senescence of beta cells remains unsubstantiated from the in vitro studies. It was not clear how any of these studies related to beta cell senescence in T2DM where there is metabolic and/or gluco-lipotoxic stress. Although it is claimed from Fig 9 that TET2 regulates PTEN/MOF axis to regulate beta cell function, no functional data (e.g. GSIS) are shown.

(8) There were issues and difficulties with the writing in the introduction and discussion in that they did not clearly or adequately describe, discuss or interpret the main conclusions and their significance. The work is not positioned within the current state of the field and it is very difficult to follow the rationales for the study and the advances in knowledge provided.

Reviewer #1 (Public review):

Summary

This very interesting article describes extensive work by the authors connecting topoisomerase 2 to aging across multiple model systems. The authors began by analyzing published transcriptomes for genes previously reported to be connected to increased lifespan in S. cerevisiae, focusing on genes whose downregulation is highly correlated with increased lifespan. One of these candidates was topoisomerase 2, which had previously been shown to be connected to lifespan in yeast.

The authors here show that reduction in topoisomerase 2 levels can significantly extend lifespan in yeast (by damp), C. elegans (by RNAi), and mice (by CRISPR CasRx).

Next, the authors demonstrate in both C. elegans and mice that in addition to increased survival times, animals with decreased top2 levels also show increased healthspan, as measured by using rates of body bends and of pharyngeal pumping in C. elegans, and using the Frailty Index (FI) for mice. Further, they report that lowered top2 levels result in less aged tissue phenotypes in multiple tissues in mice as assayed by histology, and positively affect multiple hallmarks of aging in both mouse tissues and human IMR-90 cells.

The authors go on to perform thorough transcriptomic analysis of reduced top2 animals in both C. elegans and mice. Many interesting GO terms are highly overrepresented among both up- and down-regulated transcripts from these experiments, and the authors conclude that in the case of mice there is significant tissue specific biology based on differing results in the tissues they examined.

Given the previously known biological roles of top2, the authors looked at changes in the epigenetic landscape of reduced top2 organisms as evidenced by changes in H3K4me3, H3K9me3 and H3K27me3. Overall, the authors conclude from these data that reduction of top2 "differentially down-regulates genes with active promoters/high abundance".

Overall this well-written manuscript summarizes a great deal of new data that will be of great interest to aging researchers broadly.

The figures and tables are all very clear and well-designed, and all add greatly to the manuscript overall including the use of color which is in all cases justified.

Reviewer #1 (Public review):

Koren et al. derive and analyse a spiking network model optimised to represent external signals using the minimum number of spikes. Unlike most prior work using a similar setup, the network includes separate populations of excitatory and inhibitory neurons. The authors show that the optimised connectivity has a like-to-like structure, which leads to the experimentally observed phenomenon of feature competition. The authors also examine how various (hyper)parameters-such as adaptation timescale, the excitatory-to-inhibitory cell ratio, regularization strength, and background current-affect the model. These findings add biological realism to a specific implementation of efficient coding. They show that efficient coding explains, or at least is consistent with, multiple experimentally observed properties of excitatory and inhibitory neurons.

As discussed in the first round of reviews, the model's ability to replicate biological observations such as the 4:1 ratio of excitatory vs. inhibitory neurons hinges on somewhat arbitrary hyperparameter choices. Although this may limit the model's explanatory power, the authors have made significant efforts to explore how these parameters influence their model. It is an empirical question whether the uncovered relationships between, e.g., metabolic cost and the fraction of excitatory neurons are biologically relevant.

The revised manuscript is also more transparent about the model's limitations, such as the lack of excitatory-excitatory connectivity. Further improvements could come from explicitly acknowledging additional discrepancies with biological data, such as the widely reported weak stimulus tuning of inhibitory neurons in the primary sensory cortex of untrained animals.

Reviewer #1 (Public review):

Summary:

The Authors investigated the anatomical features of the excitatory synaptic boutons in layer 1 of the human temporal neocortex. They examined the size of the synapse, the macular or the perforated appearance and the size of the synaptic active zone, the number and volume of the mitochondria, the number of the synaptic and the dense core vesicles, also differentiating between the readily releasable, the recycling and the resting pool of synaptic vesicles. The coverage of the synapse by astrocytic processes was also assessed, and all the above parameters were compared to other layers of the human temporal neocortex. The Authors conclude that the subcellular morphology of the layer 1 synapses is suitable for the functions of the neocortical layer, i.e. the synaptic integration within the cortical column. The low glial coverage of the synapses might allow the glutamate spillover from the synapses enhancing synpatic crosstalk within this cortical layer.

Strengths:

The strengths of this paper are the abundant and very precious data about the fine structure of the human neocortical layer 1. Quantitative electron microscopy data (especially that derived from the human brain) are very valuable, since this is a highly time- and energy consuming work. The techniques used to obtain the data, as well as the analyses and the statistics performed by the Authors are all solid, strengthen this manuscript, and mainly support the conclusions drawn in the discussion.

Comments on latest version:

The corrected version of the article titled „Ultrastructural sublaminar specific diversity of excitatory synaptic boutons in layer 1 of the adult human temporal lobe neocortex" has been improved thanks to the comments and suggestions of the reviewers. The Authors implemented several of my comments and suggestions. However, many of them were not completed. It is understandable that the Authors did not start a whole new series of experiment investigating inhibitory synapses (as it was a misunderstanding affecting 2 reviewers from the three). But the English text is still very hard to understand and has many mistakes, although I suggested to extensively review the use of English. Furthermore, my suggestion about avoiding many abbreviations in the abstract, analyse and discuss more the perforated synapses, the figure presentation (Figure 3) and including data about the astrocytic coverage in the Results section were not implemented. My questions about the number of docked vesicles and p10 vesicles, as well as about the different categories of the vesicle pools have not been answered neither. Many other minor comments and suggestions were answered, corrected and implemented, but I think it could have been improved more if the Authors take into account all of the reviewers' suggestions, not only some of them. I still have several main and minor concerns, with a few new ones as well I did not realized earlier, but still think it is important.

Main concerns:

(1) Epileptic patients:<br /> As all patients were epileptic, it is not correct to state in the abstract that non-epileptic tissue was investigated. Even if the seizure onset zone was not in the region investigated, seizures usually invade the temporal lobe in TLE. If you can prove that no spiking activity occured in the sample you investigated and the seizures did not invade that region, then you can write that it is presumably non-epileptic. I would suggest to write „L1 of the human temporal lobe neocortical biopsy tissue". See also Methods lines 608-612. Write only „non-epileptic" or „non-affected" if you verified it with EcoG. If this was the case, please write a few sentences about it in the Methods.

(2) About the inhibitory/excitatory synapses.<br /> Since our focus was on excitatory synaptic boutons as already stated in the title we have not analyzed inhibitory SBs.<br /> Now, I do understand that only excitatory synapses were investigated. Although it was written in the title, I did not realized, since all over the manuscript the Authors were writing synapses, and were distinguishing between inhibitory and excitatory syanpses in the text and showing numerous excitatory and inhibitory synapses on Figure 2 and discussing inhibitory interneurons in the Discussion as well. Maybe this was the reason why two reviewers out of the three (including myself) thought you investigated both types of synapses but did not differentiated between them. So, please, emphasize in the Abstract (line 40), Introduction (for ex. line 92-97) and the Discussion (line 369) that only excitatory synaptic boutons were investigated.<br /> As this paper investigated only excitatory synaptic boutons, I think it is irrelevant to write such a long section in the Discussion about inhibitory interneurons and their functions in the L1 of the human temporal lobe neocortex. Same applies to the schematic drawing of the possible wiring of L1 (Figure 7). As no inhibitory interneurons were examined, neither the connection of the different excitatory cells, only the morphology of single synaptic boutons without any reference on their origin, I think this figure does not illustrate the work done in this paper. This could be a figure of a review paper about the human L1, but is is inappropriate in this study.

(3) Perforated synapses<br /> "the findings of the Geinismann group suggesting that perforated synapses are more efficient than non-perforated ones is nowadays very controversially discussed"<br /> I did not ask the Authors to say that perforated synapses are more efficient. However, based on the literature (for ex. Harris et al, 1992; Carlin and Siekievitz, 1982; Nieto-Sampedro et al., 1982) the presence of perforated synapses is indeed a good sign of synapse division/formation - which in turn might be coupled to synaptic plasticity (Geinisman et al, 1993), increased synaptic activity (Vrensen and Cardozo, 1981), LTP (Geinisman et al, 1991, Harris et al, 2003), pathological axonal sprouting (Frotscher et al, 2006), etc. I think it is worth mentioning this at least in the Discussion.

(4) Question about the vesicle pools<br /> Results, Line 271: Still not understandable, why the RRP was defined as {less than or equal to}10 nm and {less than or equal to}20nm. Why did you use two categories? One would be sufficient (for example {less than or equal to}20nm). Or the vesicles between 10 and 20nm were considered to be part of RRP? In this case there is a typo, it should be {greater than or equal to}10 nm and {less than or equal to}20nm.<br /> The answer of the Authors was to my question raised: We decided that also those very close within 10 and 20 nm away from the PreAZ, which is less than a SV diameter may also contribute to the RRP since it was shown that SVs are quite mobile.<br /> This does not clarify why did you use two categories. Furthermore, I did not receive answer (such as Referee #2) for my question on how could you have 3x as many docked vesicles than vesicles {less than or equal to}10nm. The category {less than or equal to}10nm should also contain the docked vesicles. Or if this is not the case, please, clarify better what were your categories.

(5) Astrocytic coverage<br /> On Fig. 6 data are presented on the astrocytic coverage derived from L1 and L4. In my previous review I asked to include this in the text of the Results as well, but I still do not see it. It is also lacking from the Results how many samples from which layer were investigated in this analysis. Only percentages are given, and only for L1 (but how many patients, L1a and/or L1b and/or L4 is not provided). In contrast, Figure 6 and Supplementary Table 2 (patient table) contains the information that this analysis has been made in L4 as well. Please, include this information in the text as well (around lines 348-360).<br /> About how to determine glial elements. I cannot agree with the Authors that glial elements can be determined with high certainty based only on the anatomical features of the profiles seen in the EM. „With 25 years of experience in (serial) EM work" I would say, that glial elements can be very similar to spine necks and axonal profiles.<br /> All in all, if similar methods were used to determine the glial coverage in the different layers of the human neocortex, than it can be compared (I guess this is the case). However, I would say in the text that proper determination would need immunostaining and a new analysis. This only gives an estimatation with the possibility of a certain degree of error.

(6) Large interindividual differences in the synapse density should be discussed in the Discussion.

Reviewer #1 (Public review):

Summary:

Trutti and colleagues used 7T fMRI to identify brain regions involved in subprocesses of updating the content of working memory. Contrary to past theoretical and empirical claims that the striatum serves a gating function when new information is to be entered into working memory, the relevant contrast during a reference-back task did not reveal significant subcortical activation. Instead, the experiment provided support for a role of subcortical (and cortical) regions in other subprocesses.

Strengths

The use of high-field imaging optimized for subcortical regions in conjunction with the theory-driven experimental design mapped well to the focus on a hypothetical striatal gating mechanism.

Consideration of multiple subprocesses and the transparent way of identifying these, summarized in a table, will make it easy for future studies to replicate and extend the present experiment.

Weaknesses:

The reference-back paradigm seems to only require holding a single letter in working memory (X or O; Fig 1). It remains unclear how such low demand on working memory influences associated fMRI updating responses. It is also not clear whether reference-switch trials with 'same' response truly taxes working-memory updating (and gate opening), as the working-memory content/representation does not need to be updated in this case. These potential design issues, together with the rather low number of experimental trials, raise concerns about the demonstrated absence of evidence for striatal gate opening. Adding an experiment with higher working-memory demand and additional trials could strengthen the evidence for the authors present claim

The authors provide a motivation for their multi-step approach to fMRI analyses. Still, the three subsections of fMRI results (3.2.1; 3.2.2; 3.3.3) for 4 subprocesses each (gate opening, gate closing, substitution, updating mode) made the Results section complex and it was not always easy to understand why some but not other approaches revealed significant effects (as the midbrain in gate opening).<br /> It could be helpful to readers to further revise the Results section and/or more clearly convey the analytic strategy.

The many references to the role of dopamine are interesting, but the discussion of dopaminergic pathways and signals remains speculative and must be confirmed in future studies (e.g., with PET imaging).

Several relevant studies were not cited (e.g., Dahlin et al., 2008, Science; Bäckman et al., 2011, Science).

Reviewer #1 (Public review):

Summary:

This study presents compelling evidence for a novel treatment approach in a challenging patient population with MSS/pMMR mCRC, where traditional immunotherapy has often fallen short. The combination of SBRT and tislelizumab not only yielded a high disease control rate but also indicated significant improvements in the tumor's immune landscape. The safety profile appears favorable, which is crucial for patients who have already undergone multiple lines of therapy.

Strengths:

The results underscore the potential of leveraging radiation therapy to enhance the effectiveness of immunotherapy, especially in tumor environments previously deemed hostile to immune interventions. Future research should focus on larger cohorts to validate these findings and explore the underlying mechanisms of immune modulation post-treatment.

Weaknesses:

I believe the author's work is commendable and should be considered with some minor modifications:

(1) While the author categorized patients based on the type of RAS mutation and the location of colorectal cancer metastasis, the article does not adequately address how these classifications influence treatment outcomes. Such as whether KRAS or NRAS mutations, as well as the type of metastatic lesions, affect the sensitivity to gamma-ray treatment and lead to varying responses.

(2) In Figure 2, clarification is needed on how the author differentiated between on-target and off-target lesions. I observed that some images depicted both lesion types at the same level, which could lead to confusion.

(3) The author performed only a basic difference analysis. A more comprehensive analysis, including calculations of markers related to treatment efficacy, could offer additional insights for clinical practice.

(4) The transcriptome sequencing analysis provides insights into how stereotactic radiotherapy sensitizes immunotherapy; however, it currently relies on a simple pre- and post-treatment group comparison. It would be beneficial to include additional subgroups to explore more nuanced findings.

(5) The author briefly discusses the effects of changes in tumor fibrosis and angiogenesis on treatment outcomes. Further experiments may be necessary to validate these findings and investigate the underlying mechanisms of immune regulation following treatment.

Reviewer #1 (Public review):

This study uses a variety of approaches to explore the role of cerebellum, and in particular Purkinje cells (PCs), in the development of postural control in larval zebrafish. A chemogenetic approach is used to either ablate PCs or disrupt their normal activity and a powerful, high-throughput behavioural tracking system then enables quantitative assessment of swim kinematics. Using this strategy, convincing evidence is presented that PCs are required for normal postural control in the pitch axis. Calcium imaging further shows that PCs encode tilt direction. Evidence is also presented that suggests the role of the cerebellum changes over the course of early development, although this claim is less robust. Finally, the authors build on their prior work showing that both axial muscles and pectoral fins contribute to "climbs" and show convincing evidence that PCs are required for speed-dependent engagement of the fins during this behavior. Overall, establishing a role for cerebellum in postural control is not very surprising. However, a clear motivation of this study was to establish a robust experimental platform to investigate the changing role of cerebellar circuits in the development of postural control in the highly experimentally accessible zebrafish larvae and in this regard the authors have certainly succeeded.

This revised version of the manuscript incorporates several improvements. In particular, additional analysis and methodological detail is provided regarding the chemogenetic manipulation, there is expanded analysis of the speed-dependency of pectoral fin engagement, and aspects of the decoding analysis are clearer. However, it is still not certain that the emergence of a dive phenotype over development (from 7 to 14 day post fertilisation) really represents changing role for the cerebellum as opposed to changing sensitivity of Purkinje cells to the chemogenetic treatment.

Reviewer #1 (Public review):

Summary:

In this paper, Bose et al. investigated the role of Foxg1 transcription factor in the progenitors at late stages of cerebral cortex development.

They discover that Foxg1 is a repressor of gliogenesis and has a dual function, first as a repressor of Fgfr3 receptor in progenitors, and second as a suppressor of the Fgf ligands in young neurons.

They found that the inactivation of Foxg1 in cortical progenitors causes premature astrogliogenesis at the expense of neurogenesis. They identify Fgfr3 as a novel FOXG1 target. They show that suppression of Fgfr3 by FOXG1 in progenitors is required to maintain neurogenesis. On the other hand, they also show that FOXG1 negatively regulates the expression of Fgf gliogenic secreted factors in young neurons suppressing gliogenesis cells extrinsically.

Strengths:

The authors used time-consuming in vivo experiments utilizing several mouse strains including Foxg1-MADM in combination with RNA-Seq and ChIP to convincingly show that Foxg1 acts upstream of FGF signalling in the control of gliogenesis onset. The conclusions of this paper are mostly well supported by data.

Reviewer #3 (Public review):

Summary:

The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.

Strengths:

The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.

Weaknesses:

As in previous revisions, there remains concerning ambiguity in the methodology used for microbiota sequence analysis and it would be difficult to replicate the analysis in any meaningful way. In this revision, concerns about the rigor and reproducibility of this component of the manuscript have been increased. Readers should be cautious with interpretation of this data.

(1) In previous versions of the manuscript it would appear the correct bioproject accession was listed but, the actual link went to an unrelated project. The updated accession link appears to contain raw data; however, the authors state they used an Illumina HiSeq 2500. This would be an unusual choice for V3-V4 as it would not have read lengths long enough to overlap. Inspection of the first sample (SRR19164796) demonstrates that this is absolutely not the raw data, as there is a ~400 nt forward read, and a 0 length reverse read. All quality scores are set to 30. There is no logical way to go from HiSeq 2500 raw data and read lengths to what was uploaded to the SRA and it was certainly not described in the manuscript.

(2) No multiple testing correction was applied to the microbiome data.

Reviewer #1 (Public review):

Summary:

The authors investigate the effects of aging on auditory system performance in understanding temporal fine structure (TFS), using both behavioral assessments and physiological recordings from the auditory periphery, specifically at the level of the auditory nerve. This dual approach aims to enhance understanding of the mechanisms underlying observed behavioral outcomes. The results indicate that aged animals exhibit deficits in behavioral tasks for distinguishing between harmonic and inharmonic sounds, which is a standard test for TFS coding. However, neural responses at the auditory nerve level do not show significant differences when compared to those in young, normal-hearing animals. The authors suggest that these behavioral deficits in aged animals are likely attributable to dysfunctions in the central auditory system, potentially as a consequence of aging. To further investigate this hypothesis, the study includes an animal group with selective synaptic loss between inner hair cells and auditory nerve fibers, a condition known as cochlear synaptopathy (CS). CS is a pathology associated with aging and is thought to be an early indicator of hearing impairment. Interestingly, animals with selective CS showed physiological and behavioral TFS coding similar to that of the young normal-hearing group, contrasting with the aged group's deficits. Despite histological evidence of significant synaptic loss in the CS group, the study concludes that CS does not appear to affect TFS coding, either behaviorally or physiologically.

Strengths:

This study addresses a critical health concern, enhancing our understanding of mechanisms underlying age-related difficulties in speech intelligibility, even when audiometric thresholds are within normal limits. A major strength of this work is the comprehensive approach, integrating behavioral assessments, auditory nerve (AN) physiology, and histology within the same animal subjects. This approach enhances understanding of the mechanisms underlying the behavioral outcomes and provides confidence in the actual occurrence of synapse loss and its effects. The study carefully manages controlled conditions by including five distinct groups: young normal-hearing animals, aged animals, animals with CS induced through low and high doses, and a sham surgery group. This careful setup strengthens the study's reliability and allows for meaningful comparisons across conditions. Overall, the manuscript is well-structured, with clear and accessible writing that facilitates comprehension of complex concepts.

Weaknesses:

The stimulus and task employed in this study are very helpful for behavioral research, and using the same stimulus setup for physiology is advantageous for mechanistic comparisons. However, I have some concerns about the limitations in auditory nerve (AN) physiology. Due to practical constraints, it is not feasible to record from a large enough population of fibers that covers a full range of best frequencies (BFs) and spontaneous rates (SRs) within each animal. This raises questions about how representative the physiological data are for understanding the mechanism in behavioral data. I am curious about the authors' interpretation of how this stimulus setup might influence results compared to methods used by Kale and Heinz (2010), who adjusted harmonic frequencies based on the characteristic frequency (CF) of recorded units. While, the harmonic frequencies in this study are fixed across all CFs, meaning that many AN fibers may not be tuned closely to the stimulus frequencies. If units are not responsive to the stimulus further clarification on detecting mistuning and phase locking to TFS effects within this setup would be valuable. Given the limited number of units per condition-sometimes as few as three for certain conditions - I wonder if CF-dependent variability might impact the results of the AN data in this study and discussing this factor can help with better understanding the results. While the use of the same stimuli for both behavioral and physiological recordings is understandable, a discussion on how this choice affects interpretation would be beneficial. In addition a 60 dB stimulus could saturate high spontaneous rate (HSR) AN fibers, influencing neural coding and phase-locking to TFS. Potentially separating SR groups, could help address these issues and improve interpretive clarity.

A deeper discussion on the role of fiber spontaneous rate could also enhance the study. How might considering SR groups affect AN results related to TFS coding? While some statistical measures are included in the supplement, a more detailed discussion in the main text could help in interpretation.

Although Figure S2 indicates no change in median SR, the high-dose treatment group lacks LSR fibers, suggesting a different distribution based on SR for different animal groups, as seen in similar studies on other species. A histogram of these results would be informative, as LSR fiber loss with CS-whether induced by ouabain in gerbils or noise in other animals-is well documented (e.g., Furman et al., 2013).

Although ouabain effects on gerbils have been explored in previous studies, since these data already seems to be recorded for the animal in this study, a brief description of changes in auditory brainstem response (ABR) thresholds, wave 1 amplitudes, and tuning curves for animals with cochlear synaptopathy (CS) in this study would be beneficial. This would confirm that ouabain selectively affects synapses without impacting outer hair cells (OHCs). For aged animals, since ABR measurements were taken, comparing hearing differences between normal and aged groups could provide insights into the pathologies besides CS in aged animals. Additionally, examining subject variability in treatment effects on hearing and how this correlates with behavior and physiology would yield valuable insights. If limited space maybe a brief clarification or inclusion in supplementary could be good enough.

Another suggestion is to discuss the potential role of MOC efferent system and effect of anesthesia in reducing efferent effects in AN recordings. This is particularly relevant for aged animals, as CS might affect LSR fibers, potentially disrupting the medial olivocochlear (MOC) efferent pathway. Anesthesia could lessen MOC activity in both young and aged animals, potentially masking efferent effects that might be present in behavioral tasks. Young gerbils with functional efferent systems might perform better behaviorally, while aged gerbils with impaired MOC function due to CS might lack this advantage. A brief discussion on this aspect could potentially enhance mechanistic insights.

Lastly, although synapse counts did not differ between the low-dose treatment and NH I sham groups, separating these groups rather than combining them with the sham might reveal differences in behavior or AN results, particularly regarding the significance of differences between aged/treatment groups and the young normal-hearing group.

Reviewer #1 (Public review):

Turi, Teng and the team used state-of-the-art techniques to provide convincing evidence on the infraslow oscillation of DG cells during NREM sleep, and how serotonergic innervation modulates hippocampal activity pattern during sleep and memory. First, they showed that the glutamatergic DG cells become activated following an infraslow rhythm during NREM sleep. In addition, the infraslow oscillation in the DG is correlated with rhythmic serotonin release during sleep. Finally, they found that specific knockdown of 5-HT receptors in the DG impairs the infraslow rhythm and memory, suggesting that serotonergic signaling is crucial for regulating DG activity during sleep. Given that the functional role of infraslow rhythm still remains to be studied, their findings deepen our understanding on the role of DG cells and serotonergic signaling in regulating infraslow rhythm, sleep microarchitecture and memory.

Reviewer #2 (Public review):

Summary:

This computational modeling study addresses the observation that variable observations are interpreted differently depending on how much uncertainty an agent expects from its environment. That is, the same mismatch between a stimulus and an expected stimulus would be less significant, and specifically would represent a smaller prediction error, in an environment with a high degree of variability than in one where observations have historically been similar to each other. The authors show that if two different classes of inhibitory interneurons, the PV and SST cells, (1) encode different aspects of a stimulus distribution and (2) act in different (divisive vs. subtractive) ways, and if (3) synaptic weights evolve in a way that causes the impact of certain inputs to balance the firing rates of the targets of those inputs, then pyramidal neurons in layer 2/3 of canonical cortical circuits can indeed encode uncertainty-modulated prediction errors. To achieve this result, SST neurons learn to represent the mean of a stimulus distribution and PV neurons its variance.

The impact of uncertainty on prediction errors in an understudied topic, and this study provides an intriguing and elegant new framework for how this impact could be achieved and what effects it could produce. The ideas here differ from past proposals about how neuronal firing represents uncertainty. The developed theory is accompanied by several predictions for future experimental testing, including the existence of different forms of coding by different subclasses of PV interneurons, which target different sets of SST interneurons (as well as pyramidal cells). The authors are able to point to some experimental observations that are at least consistent with their computational results. The simulations shown demonstrate that if we accept its assumptions, then the authors' theory works very well: SSTs learn to represent the mean of a stimulus distribution, PVs learn to estimate its variance, firing rates of other model neurons scale as they should, and the level of uncertainty automatically tunes the learning rate, so that variable observations are less impactful in a high uncertainty setting.

Strengths:

The ideas in this work are novel and elegant, and they are instantiated in a progression of simulations that demonstrate the behavior of the circuit. The framework used by the authors is biologically plausible and matches some known biological data. The results attained, as well as the assumptions that go into the theory, provide several predictions for future experimental testing. The authors have taken into account earlier review comments to revise their paper in ways that enhance its clarity.

Weaknesses:

One weakness could be that the proposed theory does rely on a fairly large number of assumptions. However, there is at least some biological support for these. Importantly, the authors do lay out and discuss their key assumptions in the Discussion section, so readers can assess their validity and implications for themselves.

Comments on revisions:

I have no further suggestions for the authors.

Reviewer #1 (Public review):

The focus of this manuscript was to investigate the role of Cldn9 in the development of the mammalian cochlea. The main rationale of the study is the fact that cochlear hair cells do not regenerate, so when damaged they are lost forever, causing irreparable hearing loss. The authors have attempted to address this problem by inducing the ectopic production of additional hair cells and test whether they acquire the morphological and functional characteristics of native hair cells. They show that downregulation of Cldn9 using a well-established genetic manipulation of transgenic mice led to the production of extra numerary inner hair cells, which were able to survive for several months. By performing a large battery of experiments, the authors were able to determine that the native and ectopic inner hair cells have comparable morphological and physiological characteristics. There are several conclusions highlighted by the authors in different parts of the manuscript, including the key role of Cldn9 in coordinating embryonic and postnatal development, the differentiation of supporting cells into inner hair cells, and the possible use of Cldn9 to induce inner hair cell differentiation following deafness induced by hair cell loss.

Comments on revised version:

The authors have addressed the following points raised during the first submission: statistical analysis and wave 1 analysis. However, very little was done to address the other key aspects of my report, which are essential for the interpretation of the results. As mentioned in my previous report, some aspects of the work are not justified by the current data and will require either a tone-down of the claims or further experiments.

For example, one puzzling finding that is not addressed in the manuscript is the lack of functional benefit from these additional inner hair cells. In fact, it appears to be detrimental based on the increased ABR thresholds and EP. So, it is not clear to this reviewer the advantage of this approach.

It is not clear what direct evidence there is, apart from some immunostaining, indicating that the ectopic inner hair cells derive from the supporting cells. This part would benefit from a more careful consideration and maybe an attempt at a more direct experimental approach. Alternatively, the text should be modified accordingly.

One point that should be made clear throughout the manuscript is that the ectopic inner hair cells are generated in a cochlea that is undergoing normal maturation. Thus, there is no guarantee that modulating the expression levels of Cldn9 in a deaf mouse lacking hair cells would produce the same result as that shown in this study. This point should be at least discussed.

Joint Public Review:

Summary:

The authors aimed to identify the neural sources of behavioral variation in fruit flies deciding between odor and air, or between two odors.

Strengths:

- The question is of fundamental importance.<br /> - The behavioral studies are automated, and high-throughput.<br /> - The data analyses are sophisticated and appropriate.<br /> - The paper is clear and well-written aside from some initially strong wording.<br /> - The figures beautifully illustrate their results.<br /> - The modeling efforts mechanistically ground observed data correlations.

Weaknesses:

-The correlations between behavioral variations and neural activity/synapse morphology are statistically significant but relatively weak.

Reviewer #1 (Public review):

Summary:

This manuscript details the results of a small pilot study of neoadjuvant radiotherapy followed by combination treatment with hormone therapy and dalpiciclib for early-stage HR+/HER2-negative breast cancer.

Strengths:

The strengths of the manuscript include the scientific rationale behind the approach and the inclusion of some simple translational studies.

Weaknesses:

The main weakness of the manuscript is that overly strong conclusions are made by the authors based on a very small study of twelve patients. A study this small is not powered to fully characterize the efficacy or safety of a treatment approach, and can, at best, demonstrate feasibility. These data need validation in a larger cohort before they can have any implications for clinical practice, and the treatment approach outlined should not yet be considered a true alternative to standard evidence-based approaches.

I would urge the authors and readers to exercise caution when comparing results of this 12-patient pilot study to historical studies, many of which were much larger, and had different treatment protocols and baseline patient characteristics. Cross-trial comparisons like this are prone to mislead, even when comparing well powered studies. With such a small sample size, the risk of statistical error is very high, and comparisons like this have little meaning.

Reviewer #1 (Public review):

Summary:

The authors set out to evaluate the regulation of interferon (IFN) gene expression in fish, using mainly zebrafish as a model system. Similar to more widely characterized mammalian systems, fish IFN is induced during viral infection through the action of the transcription factor IRF3 which is activated by phosphorylation by the kinase TBK1. It has been previously shown in many systems that TBK1 is subjected to both positive and negative regulation to control IFN production. In this work, the authors find that the cell cycle kinase CDK2 functions as a TBK1 inhibitor by decreasing its abundance through recruitment of the ubiquitinylation ligase, Dtx4, which has been similarly implicated in the regulation of mammalian TBK1. Experimental data are presented showing that CDK2 interacts with both TBK1 and Dtx4, leading to TBK1 K48 ubiqutinylation on K567 and its subsequent degradation by the proteasome.

Strengths:

The strengths of this manuscript are its novel demonstration of the involvement of CDK2 in a process in fish that is controlled by different factors in other vertebrates and its clear and supportive experimental data.

Weaknesses:

The weaknesses of the study include the following. 1) It remains unclear how CDK is regulated during viral infection and how it specifically recruits E3 ligase to TBK1. 2) The implications and mechanisms for a relationship between the cell cycle and IFN production will be a fascinating topic for future studies.

Reviewer #1 (Public review):

Summary:

Lodhiya et al. demonstrate that antibiotics with distinct mechanisms of action, norfloxacin and streptomycin, cause similar metabolic dysfunction in the model organism Mycobacterium smegmatis. This includes enhanced flux through the TCA cycle and respiration as well as a build-up of reactive oxygen species (ROS) and ATP. Genetic and/or pharmacologic depression of ROS or ATP levels protect M. smegmatis from norfloxacin and streptomycin killing. Because ATP depression is protective, but in some cases does not depress ROS, the authors surmise that excessive ATP is the primary mechanism by which norfloxacin and streptomycin kill M. smegmatis. In general, the experiments are carefully executed; alternative hypotheses are discussed and considered; the data are contextualized within the existing literature.

Strengths:

The authors tackle a problem that is both biologically interesting and medically impactful, namely, the mechanism of antibiotic-induced cell death.

Experiments are carefully executed, for example, numerous dose- and time-dependency studies; multiple, orthogonal readouts for ROS; and several methods for pharmacological and genetic depletion of ATP.

There has been a lot of excitement and controversy in the field, and the authors do a nice job of situating their work in this larger context.

Inherent limitations to some of their approaches are acknowledged and discussed e.g., normalizing ATP levels to viable counts of bacteria.

Weaknesses:

All of the experiments performed here were in the model organism M. smegmatis. As the authors point out, the extent to which these findings apply to other organisms (most notably, slow-growing pathogens like M. tuberculosis) is to be determined.

At first glance, some of the results in the manuscript seem to conflict with what has been previously reported in the (referenced) literature. In their response to reviewers, the authors addressed these concerns. Ideally they would have addressed them in the main manuscript too.

Figs. 9 and 10A-B and associated text make the manuscript significantly longer and more descriptive. They are more appropriate to the beginning of a new story rather than the end of the current one.

Reviewer #1 (Public review):

Summary:

This comprehensive study employed molecular, optical, electrophysiological and tonometric strategies to establish the role of TGFβ2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels, and intraocular pressure regulation in a mouse model of TGFβ2 -induced ocular hypertension. TGFβ2 upregulated expression of TRPV4 and PIEZO1 transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 activation induced TM contractility whereas pharmacological inhibition suppressed TGFβ2-induced hypercontractility and abrogated ocular hypertension in eyes overexpressing TGFβ2. Trpv4-/- mice resisted TGFβ2-driven increases in IOP. These data establish a fundamental role of TGFβ as a modulator of mechanosensing and identifies TRPV4 channel as a common mechanism for TM contractility and pathological ocular hypertension.

Strengths:

The manuscript is very well written and details the important function of TRPV4 in TM cell function. These data provide novel therapeutic targets and potential for disease-altering therapeutics.

Weaknesses:

The experimental rigor and design of the noctural IOP experiments was weak with low n values and differing methods of IOP measurement (conscious versus anesthetized). The same method of IOP measurement needs to be used for all measurements to make any conclusions on the circadian patterns of IOP in each condition.

Reviewer #1 (Public review):

Summary:

The authors use a sophisticated task design and Bayesian computational modeling to test their hypothesis that information generalization (operationalized as a combination of self-insertion and social contagion) in social situations is disrupted in Borderline Personality Disorder. Their main finding relates to the observation that two different models best fit the two tested groups: While the model assuming both self-insertion and social contagion to be present when estimating others' social value preferences fit the control group best, a model assuming neither of these processes provided the best fit to BPD participants.

Strengths:

The strengths of the presented work lie in the sophisticated task design and the thorough investigation of their theory by use of mechanistic computational models to elucidate social decision-making and learning processes in BPD.

Weaknesses:

The manuscript's primary weakness relates to the number of comparisons conducted and a lack of clarity in how those comparisons relate to the authors' hypotheses. The authors specify a primary prediction about disruption to information generalization in social decision making & learning processes, and it is clear from the text how their 4 main models are supposed to test this hypothesis. With regards to any further analyses however (such as the correlations between multiple clinical scales and eight different model parameters, but also individual parameter comparisons between groups), this is less clear. I recommend the authors clearly link each test to a hypothesis by specifying, for each analysis, what their specific expectations for conducted comparisons are, so a reader can assess whether the results are/aren't in line with predictions. The number of conducted tests relating to a specific hypothesis also determines whether multiple comparison corrections are warranted or not. If comparisons are exploratory in nature, this should be explicitly stated.

Furthermore, the authors present some measures for external validation of the models, including comparison between reaction times and belief shifts, and correlations between model predicted accuracy and behavioural accuracy/total scores. However it would be great to see some more formal external validation of how the model parameters relate to participant behaviour, e.g., the correlation between the number of pro-social choices and ß-values, or the correlation between the change in absolute number of pro-social choices and the change in ß. From comparing the behavioural and computational results it looks like they would correlate highly, but it would be nice to see this formally confirmed.

The statement in the abstract that 'Overall, the findings provide a clear explanation of how self-other generalisation constrains and assists learning, how childhood adversity disrupts this through separation of internalised beliefs' makes an unjustified claim of causality between childhood adversity and separation of self - and other beliefs, although the authors only present correlations. I recommend this should be rephrased to reflect the correlational nature of the results.

Currently, from the discussion the findings seem relevant in explaining certain aberrant social learning and -decision making processes in BPD. However, I would like to see a more thorough discussion about the practical relevance of their findings in light of their observation of comparable prediction accuracy between the two groups.

Relatedly, the authors mention that a primary focus of mentalization based therapy for BPD is 'restoring a stable sense of self' and 'differentiating the self from the other'. These goals are very reminiscent of the findings of the current study that individuals with BPD show lower uncertainty over their own and relative reward preferences, and that they are less susceptible to social contagion. Could the observed group differences therefore be a result of therapy rather than adverse early life experiences?

Regarding partner similarity: It was unclear to me why the authors chose partners that were 50% similar when it would be at least equally interesting to investigate self-insertion and social contagion with those that are more than 50% different to ourselves? Do the authors have any assumptions or even data that shows the results still hold for situations with lower than 50% similarity?

Reviewer #1 (Public review):

Summary:

This manuscript reports a very interesting, novel and important research angle to add to the now enormous interest in how pesticides can be toxic to beneficial insects like the honey bee. Many studies have reported on how pesticides in standard use formulations show both lethality as well as sublethal negative effects on behavior and reproduction. The authors propose to use machine learning algorithms to identify new volatile compounds that can be tested for repellency. They use as input chemical structures that are derived from chemicals that have known repellent effects as identified in their initial behavioral assays.

Strengths:

The conclusion is that such chemicals specific to repelling bees and not pest insects (using the fruit fly as a model for the latter) can be identified using the ML approach. Have a list of such chemicals that can be rotated among in any field application would be a benefit because of the honey bees' ability to learn its way around any kind of stimulus designed to keep it from nectar and pollen, even when they may be tainted by pesticide.

Weaknesses:

The use of machine learning seems well-executed and legitimate. But this is beyond my expertise. So other reviewers can maybe comment more on that.

The behavioral data report on the use of a two-choice assay for bees in small Petrie plates. Bess can feed from two small wells place of filter paper impregnated with control or the control containing a chemical. The primary behavior, for ex in Fig 2C, is the first choice by one of the five bees in the plate of which well to feed from. For some chemical compound, there seems to be a 50:50 choice, indicating no repellent effects. In other cases the first bee making the choice chose the control, indicating possible repellent effects of the test chemical. Choices in this assay were validated in a free flying assay.

Concerns with the choice assay:<br /> - 50-70 microliters amounts to what one hungry bee will drink. Did the first bee drink most of it, such that measures of bait consumed reflect a single bee or multiple bees?<br /> - How many bees were repelled to the control side? Was it just the one bee? Were other measures considered? E.g. time to first approach; the number of bees feeding at different time points; the total number of bees observed feeding per unit time.

Reviewer #1 (Public review):

Summary:

This work is meant to help create a foundation for future studies of the Central Complex, which is a critical integrative center in the fly brain. The authors present a systematic description of cellular elements, cell type classifications, behavioral evaluations and genetic resources available to the Drosophila neuroscience community.

Strengths:

The work contributes new, useful and systematic technical information in compelling fashion to support future studies of the fly brain. It also continues to set a high and transparent standard by which large-scale resources can be defined and shared.

Weaknesses:

manuscript p. 1<br /> "The central complex (CX) of the adult Drosophila melanogaster brain consists of approximately 2,800 cells that have been divided into 257 cell types based on morphology and connectivity (Scheer et al., 2020; Hulse et al. 2021; Wolff et al., 2015)."<br /> The 257 accumulated cell types have informational names (e.g., PBG2‐9.s‐FBl2.b‐NO3A.b) in addition to their associations with specific Gal4 lines and specific EM Body IDs. All this is very useful. I have one suggestion to help a reader trying to get a "bird's eye view" of such a large amount of detailed and multi-layered information. Give each of the 257 CX cell types an arbitrary number: 1 to 257. In fact, Supplemental File 2 lists ~277 cell types each with a number in sequence, so perhaps in principle, it is there. This could expedite the search function when a reader is trying to cross-reference CX cell type information from the text, to the Figures and/or to the Supplemental Figures. Also, the use of (arbitrary) cell type numbers could expedite the explanation of which cell types are included in any compilation of information (e.g., which ones were tested for specific NT expression).

manuscript p 2<br /> "Figure 2 and Figure 2-figure supplements 1-4 show the expression of 52 new split-GAL4 lines with strong GAL4 expression that is largely limited to the cell type of interest. .... We also generated lines of lesser quality for other cell types that in total bring overall coverage to more than three quarters of CX cell types."<br /> This section describes the generation and identification of specific split Gal4 lines, and the presentation is generally excellent. It represents an outstanding compendium of information. My reading of the text suggests ~200 cell types have Gal4 lines that are of immediate use (having high specificity or v close-to-high). Use of an arbitrary number system (mentioned above) could augment that description for the reasons stated. For example, which of the 257 cell types are represented by split Gal4 lines that constitute the ~1/3 representing "high-quality lines "? A second comment relates to this study 's functional analysis of the contributions of CX cell types to sleep physiology. The recent literature contains renewed interest in the specific expression patterns of Gal4 lines that can promote sleep-like behaviors. In particular Gal4 line expression outside the brain (in the VNC and outside the CNS) have been raised as important elements that need be included for interpretation interpretation of sleep regulation. This present study offers useful information about a large number of expression patterns, as well as a basis with which to seek additional information., including mention of VNC expression in many cases However, perhaps I missed it, but I could not find a short description of the over-all strategy used to describe the expression patterns and feel that could be helpful. Were all Gal4 lines studied for expression in the VNC? and in the peripheral NS? It is probably published elsewhere, but even a short reprise would still be useful.

manuscript p 9<br /> Neurotransmitter expression in CX cell types<br /> "To determine what neurotransmitters are used by the CX cell types, we carried out fluorescent in situ hybridization using EASI-FISH (Eddison and Irkhe, 2022; Close et al., 2024) on brains that also expressed GFP driven from a cell-type-specific split GAL4 line. In this way, we could determine what neurotransmitters were expressed in over 100 different CX cell types based on ...."<br /> Reading this description, I was uncertain whether the >100 cell types mentioned were tested with all the NT markers by EASI-FISH? Also, assigning arbitrary numbers to the cell types (same suggestion as above) could help the reader more readily ascertain which were the ~100 cell types classified in this context.

manuscript p 10<br /> "Our full results are summarized below, together with our analysis of neuropeptide expression in the same cell types."<br /> I recommend specifying which Figures and Tables contain the "full results" indicated.

NP expression in CX cell types<br /> Similar to the comments regarding studies of NT expression: were all ~100 cell types tested with each of the 17 selected NPs? Arbitrary numerical identifies could be useful for the reader to determine which cell types/ lines were tested and which were not yet tested

manuscript p. 11<br /> "The neuropeptide expression patterns we observed fell into two broad categories."<br /> This section presents information that is extensive and extremely useful. It supports consideration of peptidergic cell signaling at a circuits level and in a systematic fashion that will promote future progress in this field. I have two comments. First, regarding the categorization of two NP expression patterns, discernible by differences in cell number: this idea mirrors one present in prior literature. Recently the classification of the transcription factor DIMM summarizes this same two-way categorization (e.g., doi: 10.1371/journal.pone.0001896). That included the fact that a single NP can be utilized by cell of either category.

Second, regarding this comment:<br /> "In contrast, neuropeptides like those shown in Figure 6 appear to be expressed in dozens to hundreds of cells and appear poised to function by local volume transmission in multiple distinct circuits."<br /> Signaling by NPs in this second category (many small cells) suggests more local diffusion, a smaller geographic expanse compared to "volume" signaling by the sparser larger peptidergic cells. Given this, I suggest re-consideration in using the term "volume" in this instance, perhaps in favor of "local" or "paracrine". This is only a suggestion and in fact rests almost entirely on speculation/ interpretation, as the field lacks a strong empirical basis to say how far NPs diffuse and act. A recent study in the fly brain of peptide co-transmitters (doi: 10.1016/j.cub.2020.04.025) provides an instructive example in which differences between the spatial extents of long-range (peptide 1) versus short-range (peptide 2) NP signaling may be inferred in vivo.

Spab was mentioned (Figure 6 legend) but discarded as a candidate NP to include based on a personal communication, as was Nplp1. The manuscript did not include reasons to do so, nor include a reference to spab peptide. I suggest including explicit reasons to discard candidate NPs.

In Fig 9-supplement 1, neurotransmitter biosynthetic enzymes were measured by RNA-seq for given CX cell types to augment the cell type classification. The same methods could be used to support cell type classification regarding putative peptidergic character (in Figure 9 supplement 2) by measuring expression levels of critical, canonical neuropeptide biosynthetic enzymes. These include the proprotein convertase dPC2 (amon); the carboxypeptidase dCPD/E (silver); and the amidating enzymes dPHM; dPal1; dPal2. PHM is most related to DBM (dopamine beta monooxygenase), the rate limiting enzyme for DA production, and greater than 90% of Drosophila neuropeptides are amidated. If the authors are correct in surmising widespread use of NPs by CX cell types (and I expect they are), there could be diagnostic value to report expression levels of this enzyme set across many/most CX cell types.

Comment #6<br /> Screen of effects on Sleep behavior<br /> This work is large in scope and as suggested likely presents excellent starting points for many follow-up studies. I again suggest assigning stable number identities to the elements described. In this case, not cell types, but split Gal4 lines. This would expedite the cross-referencing of results across the four Supplemental Files 3-6. For example, line SS00273 is entry line #27 in S Files 3 and 4, but line entry #18 in S Files 5 and 6.

manuscript p 26<br /> Clock to CX<br /> "Not surprisingly, the connectome reveals that many of the intrinsic CX cell types with sleep phenotypes are connected by wired pathways (Figure 12 and Figure 12-figure supplement 1)."<br /> Do intrinsic CX cells with sleep phenotypes also connect by wired pathways to CX cells that do not have sleep phenotypes?

"The connectome also suggested pathways from the circadian clock to the CX. Links between clock output DN1 neurons to the ExR1 have been described in Lamaze et al. (2018) and Guo et al. (2018), and Liang et al. (2019) described a connection from the clock to ExR2 (PPM3) dopaminergic neurons."<br /> The introduction to this section indicates a focus on connectome-defined synaptic contacts. Whereas the first two studies cited featured both physiological and anatomic evidence to support connectivity from clock cells to CX, the third did not describe any anatomical connections, and that connection may in fact be due to diffuse not synaptic signaling

I could not easily discern the difference between Figs 12 and 12-S1? These appear to be highly-related circuit models, wherein the second features more elements. Perhaps spell out the basis for the differences between the two models to avoid ambiguity.

"...the cellular targets of Dh31 released from ER5 are unknown, however previous work (Goda et al., 2017; Mertens et al., 2005) has shown that Dh31 can activate the PDF receptor raising the possibility of autocrine signaling."<br /> Regarding pharmacological evidence for Dh31 activation of Pdfr: strong in vivo evidence was developed in doi: 10.1016/j.neuron.2008.02.018: a strong pdfr mutation greatly reduces response to synthetic dh31 in neurons that normally express Pdfr

manuscript p 30<br /> "Unexpectedly, we found that all neuropeptide-expressing cell types also expressed a small neurotransmitter."<br /> Did this conclusion apply only to CX cell types? - or was it also true for large peptidergic neurons? Prior evidence suggests the latter may not express small transmitters (doi: 10.1016/j.cub.2009.11.065). The question pertains to the broader biology of peptidergic neurons, and is therefore outside the strict scope of the main focus area - the CX. However, the text did initially consider peptidergic neurons outside the CX, so the information may be pertinent to many readers.

Reviewer #1 (Public review):

Summary:

Recent work has demonstrated that the hummingbird hawkmoth, Macroglossum stellatarum, like many other flying insects, use ventrolateral optic flow cues for flight control. However, unlike other flying insects, the same stimulus presented in the dorsal visual field elicits a directional response. Bigge et al., use behavioral flight experiments to set these two pathways in conflict in order to understand whether these two pathways (ventrolateral and dorsal) work together to direct flight and if so, how. The authors characterize the visual environment (the amount of contrast and translational optic flow) of the hawkmoth and find that different regions of the visual field are matched to relevant visual cues in their natural environment and that the integration of the two pathways reflects a priortiziation for generating behavior that supports hawkmoth safety rather than than the prevalence for a particular visual cue that is more prevalent in the environment.

Strengths:

This study creatively utilizes previous findings that the hawkmoth partitions their visual field as a way to examine parallel processing. The behavioral assay is well-established and the authors take the extra steps to characterize the visual ecology of the hawkmoth habitat to draw exciting conclusions about the hierarchy of each pathway as it contributes to flight control.

Weaknesses:

The work would be further clarified and strengthened by additional explanation included in the main text, figure legends, and methods that would permit the reader to draw their own conclusions more feasibly. It would be helpful to have all figure panels referenced in the text and referenced in order, as they are currently not. In addition, it seems that sometimes the incorrect figure panel is referenced in the text, Figure S2 is mislabeled with D-E instead of A-C and Table S1 is not referenced in the main text at all. Table S1 is extremely important for understanding the figures in the main text and eliminating acronyms here would support reader comprehension, especially as there is no legend provided for Table S1. For example, a reader that does not specialize in vision may not know that OF stands for optic flow. Further detail in figure legends would also support the reader in drawing their own conclusions. For example, dashed red lines in Figures 3 and 4 A and B are not described and the letters representing statistical significance could be further explained either in the figure legend or materials to help the reader draw their own conclusions.

Reviewer #1 (Public review):

Summary:

The authors investigate the neuroprotective effect of reserpine in a retinitis pigmentosa (P23H-1) model, characterized by a mutation in the rhodopsin gene. Their results reveal that female rats show better preservation of both rod and cone photoreceptors following reserpine treatment compared to males.

Strengths:

This study effectively highlights the neuroprotective potential of reserpine and underscores the value of drug repositioning as a strategy for accelerating the development of effective treatments. The findings are significant for their clinical implications, particularly in demonstrating sex-specific differences in therapeutic response.

Weaknesses:

The main limitation is the lack of precise identification of the specific pathway through which reserpine prevents photoreceptor death.

Reviewer #1 (Public review):

Summary:

The study addresses the growing threat of multi-drug-resistant (MDR) pathogens, focusing on the efficacy of colistin (COL), a last-resort antibiotic, and its enhanced activity when combined with artesunate (AS) and ethylenediaminetetraacetic acid (EDTA) against colistin-resistant Salmonella strains. The researchers aim to explore whether these combinations can restore the effectiveness of colistin and understand the underlying mechanisms. The study used a combination of microbiological and molecular techniques to evaluate the antibacterial activity and mechanisms of action of COL, AS, and EDTA. Key methods included: (i) Antimicrobial Susceptibility Testing: Determining minimum inhibitory concentrations (MICs) of COL, AS, and EDTA, both alone and in combination, against various Salmonella strains; (ii) Time-Kill Assays: Measuring bacterial growth inhibition over time with different drug combinations; (iii) Fluorescent Probe-Permeability Assays: Assessing cell membrane integrity using fluorescent dyes; (iv) Proton Motive Force Assay: Evaluating the impact on the electrochemical proton gradient (PMF); (v) Reactive Oxygen Species (ROS) Measurement: Quantifying intracellular ROS levels; (vi) Scanning Electron Microscopy (SEM): Observing morphological changes in bacterial cells; and (vii) Omics Analysis: Transcriptome and metabolome profiling to identify differentially expressed genes (DEGs) and significant differential metabolites (SDMs). The combination of COL, AS, and EDTA (AEC) showed significant antibacterial activity against colistin-resistant Salmonella strains, reducing the MICs and enhancing bacterial killing compared to individual treatments. The AEC treatment caused extensive damage to both the outer and inner bacterial membranes, as evidenced by increased fluorescence of membrane-impermeant dyes and SEM images showing deformed cell membranes. AEC treatment selectively collapsed the Δψ component of PMF, indicating disruption of vital cellular processes. The combination therapy increased intracellular ROS levels, contributing to bacterial killing. Transcriptome data revealed changes in genes related to two-component systems, flagellar assembly, and ABC transporters. Metabolome analysis highlighted disruptions in pathways such as arachidonic acid metabolism. The findings suggest that AS and EDTA can potentiate the antibacterial effects of colistin by disrupting bacterial membranes, collapsing PMF, and increasing ROS levels. This combination therapy could serve as a promising approach to combat colistin-resistant Salmonella infections.

Strengths:

- The study employs a wide range of techniques to thoroughly investigate the antibacterial mechanisms and efficacy of the drug combinations.<br /> - The results are consistent across multiple assays and supported by both in vitro and in vivo data.<br /> - Combining AS and EDTA with COL represents a novel strategy to tackle antibiotic resistance.

Weaknesses:

- The methodology used for interpreting and reporting time-kill assay results.

Comments on revised version:

Overall, the authors have adequately addressed the suggestions provided.

Reviewer #1 (Public review):

Summary:

SARS-CoV-2 infection induces syncytia formation, which promotes viral transmission. In this paper, the authors aimed to understand how host-derived inflammatory cytokines IL-1α/β combat SARS-CoV-2 infection.

Strengths:

First, they used a cell-cell fusion assay developed previously to identify IL-1α/β as the cytokines that inhibit syncytia formation. They co-cultured cells expressing the spike protein and cells expressing ACE2 and found that IL-1β treatment decreased syncytia formation and S2 cleavage.

Second, they investigated the IL-1 signaling pathway in detail, using knockouts or pharmacological perturbation to understand the signaling proteins responsible for blocking cell fusion. They found that IL-1 prevents cell-cell fusion through MyD88/IRAK/TRAF6 but not TAK1/IKK/NF-κB, as only knocking out MyD88/IRAK/TRAF6 eliminates the inhibitory effect on cell-cell fusion in response to IL-1β. This revealed that the inhibition of cell fusion did not require a transcriptional response and was mediated by IL-1R proximal signaling effectors.

Third, the authors identified RhoA/ROCK activation by IL-1 as the basis for this inhibition of cell fusion. By visualizing a RhoA biosensor and actin, they found a redistribution of RhoA to the cell periphery and cell-cell junctions after IL-1 stimulation. This triggered the formation of actin bundles at cell-cell junctions, preventing fusion and syncytia formation. The authors confirmed this molecular mechanism by using constitutively active RhoA and an inhibitor of ROCK.<br /> Diverse Cell types and in vivo models were used, and consistent results were shown across diverse models. These results were convincing and well-presented.

In summary, the authors have provided compelling evidence regarding how IL-1 signaling induces a prophylactic response to viral infection. While the mechanistic details of how IL-1R and MyD88 induce RhoA/Rock pathway to mediate actin remodeling remain unclear, this manuscript serves as the basis for future studies.

Reviewer #1 (Public review):

Summary:

In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and pro-invasive phenotype.

In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10CA) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasmamembrane localization correlates with high-grade DCIS cells in patient tissue samples. Specifically in invasive MCF10DCIS.com cells they showed that overcrowding or over-confluence leads to a decrease in cell volume and intracellular calcium levels. This condition also triggers the trafficking of TRPV4 channels from intracellular stores (nucleus and potentially endosomes), to the plasma membrane (PM). When these over-confluent cells are incubated with a TRPV4 activator, there is an acute and substantial influx of calcium, attesting the fact that there are high number of TRPV4 channels present on the PM. Long-term incubation of these over-confluent cells with the TRPV4 activator results in the internalization of the PM-localized TRPV4 channels.

In contrast, cells plated at lower confluence primarily have TRPV4 channels localized in the nucleus and cytosol. Long-term incubation of these cells at lower confluence with a TRPV4 inhibitor leads to the relocation of TRPV4 channels to the plasma membrane from intracellular stores and a subsequent reduction in cell volume. Similarly, incubation of these cells at low confluence with PEG 3000 (a hyperosmotic agent) promotes the trafficking of TRPV4 channels from intracellular stores to the plasma membrane.

Strengths:

The study is elegantly designed and the findings are novel. Their findings on this mechano-transduction pathway involving TRPV4 channels, calcium homeostasis, cell volume plasticity, motility and invasiveness will have a great impact in the cancer field and potentially applicable to other fields as well. Experiments are well-planned and executed, and the data is convincing. Authors investigated TRVP4 dynamics using multiple different strategies- overcrowding, hyperosmotic stress, pharmacological and genetic means, and showed a good correlation between different phenomena.

All of my previous concerns have been addressed. The quality of the manuscript has improved significantly.

Reviewer #1 (Public review):

Summary:

The authors report a study aimed at understanding the brain's representations of viewed actions, with a particular aim to distinguish regions that encode observed body movements, from those that encode the effects of actions on objects. They adopt a cross-decoding multivariate fMRI approach, scanning adult observers who viewed full-cue actions, pantomimes of those actions, minimal skeletal depictions of those actions, and abstract animations that captured analogous effects to those actions. Decoding across different pairs of these action conditions allowed the authors to pull out the contributions of different action features in a given region's representation. The main hypothesis, which was largely confirmed, was that the superior parietal lobe (SPL) more strongly encodes movements of the body, whereas the anterior inferior parietal lobe (aIPL) codes for action effects of outcomes. Specifically, region of interest analyses showed dissociations in the successful cross-decoding of action category across full-cue and skeletal or abstract depictions. Their analyses also highlight the importance of the lateral occipito-temporal cortex (LOTC) in coding action effects. They also find some preliminary evidence about the organisation of action kinds in the regions examined, and take some steps to distinguishing the differences and similarities of action-evoked patterns in primary visual cortex and the other examined regions.

Strengths:

The paper is well-written, and it addresses a topic of emerging interest where social vision and intuitive physics intersect. The use of cross-decoding to examine actions and their effects across four different stimulus formats is a strength of the study. Likewise the a priori identification of regions of interest (supplemented by additional full-brain analyses) is a strength. Finally, the authors successfully deployed a representational-similarity approach that provides more detailed evidence about the different kinds of action features that seem to be captured in each of the regions that were examined.

Weaknesses:

Globally, the findings provide support for the predicted anatomical distinctions, and for the distinction between body-focused representations of actions and more abstract "action effect structures". Viewed more narrowly, the picture is rather complex, and the patterns of (dis)similarity in the activity evoked by different action kinds do not always divide neatly. Probably, examining many more kinds of actions with the multi-format decoding approach developed here will be needed to more effectively disentangle the various contributions of movement, posture, low-level visual properties, and action outcomes/effects.

Reviewer #1 (Public review):

Summary:

Shen et al. conducted three experiments to study the cortical tracking of the natural rhythms involved in biological motion (BM), and whether these involve audiovisual integration (AVI). They presented participants with visual (dot) motion and/or the sound of a walking person. They found that EEG activity tracks the step rhythm, as well as the gait (2-step cycle) rhythm. The gait rhythm specifically is tracked superadditively (power for A+V condition is higher than the sum of the A-only and V-only condition, Experiments 1a/b), which is independent of the specific step frequency (Experiment 1b). Furthermore, audiovisual integration during tracking of gait was specific to BM, as it was absent (that is, the audiovisual congruency effect) when the walking dot motion was vertically inverted (Experiment 2). Finally, the study shows that an individual's autistic traits are negatively correlated with the BM-AVI congruency effect.

Strengths:

The three experiments are well designed and the various conditions are well controlled. The rationale of the study is clear, and the manuscript is pleasant to read. The analysis choices are easy to follow, and mostly appropriate.

Weaknesses:

On revision, the authors are careful not to overinterpret an analysis where the statistical test is not independent from the data (channel) selection criterion.

Reviewer #1 (Public review):

Summary:

"Neural noise", here operationalized as an imbalance between excitatory and inhibitory neural activity, has been posited as a core cause of developmental dyslexia, a prevalent learning disability that impacts reading accuracy and fluency. This is study is the first to systematically evaluate the neural noise hypothesis of dyslexia. Neural noise was measured using neurophysiological (electroencephalography [EEG]) and neurochemical (magnetic resonance spectroscopy [MRS]) in adolescents and young adults with and without dyslexia. The authors did not find evidence of elevated neural noise in the dyslexia group from EEG or MRS measures, and Bayes factors generally informed against including the grouping factor in the models. Although the comparisons between groups with and without dyslexia did not support the neural noise hypothesis, a mediation model that quantified phonological processing and reading abilities continuously revealed that EEG beta power in the left superior temporal sulcus was positively associated with reading ability via phonological awareness. This finding lends support for analysis of associations between neural excitatory/inhibitory factors and reading ability along a continuum, rather than as with a case/control approach, and indicates the relevance of phonological awareness as an intermediate trait that may provide a more proximal link between neurobiology and reading ability. Further research is needed across developmental stages and over a broader set of brain regions to more comprehensively assess the neural noise hypothesis of dyslexia, and alternative neurobiological mechanisms of this disorder should be explored.

Strengths:

The inclusion of multiple methods of assessing neural noise (neurophysiological and neurochemical) is a major advantage of this paper. MRS at 7T confers an advantage of more accurately distinguishing and quantifying glutamate, which is a primary target of this study. In addition, the subject-specific functional localization of the MRS acquisition is an innovative approach. MRS acquisition and processing details are noted in the supplementary materials using according to the experts' consensus recommended checklist (https://doi.org/10.1002/nbm.4484). Commenting on rigor the EEG methods is beyond my expertise as a reviewer.<br /> Participants recruited for this study included those with a clinical diagnosis of dyslexia, which strengthens confidence in the accuracy of the diagnosis. The assessment of reading and language abilities during the study further confirms the persistently poorer performance of the dyslexia group compared to the control group.<br /> The correlational analysis and mediation analysis provide complementary information to the main case-control analyses, and the examination of associations between EEG and MRS measures of neural noise is novel and interesting.<br /> The authors follow good practice for open science, including data and code sharing. They also apply statistical rigor, using Bayes Factors to support conclusions of null evidence rather than relying only on non-significant findings. In the discussion, they acknowledge the limitations and generalizability of the evidence and provide directions for future research on this topic.

Appraisal:

The authors present a thorough evaluation of the neural noise hypothesis of developmental dyslexia in a sample of adolescents and young adults using multiple methods of measuring excitatory/inhibitory imbalances as an indicator of neural noise. The authors concluded that there was not support for the neural noise hypothesis of dyslexia in their study based on null significance and Bayes factors. This conclusion is justified, and further research is called for to more broadly evaluate the neural noise hypothesis in developmental dyslexia.

Impact:

This study provides an exemplar foundation for the evaluation of the neural noise hypothesis of dyslexia. Other researcher may adopt the model applied in this paper to examine neural noise in various populations with/without dyslexia, or across a continuum of reading abilities, to more thoroughly examine evidence (or lack thereof) for this hypothesis. Notably, the lack of evidence here does not rule out the possibility for a role of neural noise in dyslexia, and the authors point out that presentation with co-occurring conditions, such as ADHD, may contribute to neural noise in dyslexia. Dyslexia remains a multi-faceted and heterogenous neurodevelopmental condition, and many genetic, neurobiological and environmental factors play a role. This study demonstrates one step toward evaluating neurobiological mechanisms that may contribute to reading difficulties.

Reviewer #1 (Public review):

Summary:

This study by Wang et al. identifies a new type of deacetylase, CobQ, in Aeromonas hydrophila. Notably, the identification of this deacetylase reveals a lack of homology with eukaryotic counterparts, thus underscoring its unique evolutionary trajectory within the bacterial domain.

Strengths:

The manuscript convincingly illustrates CobQ's deacetylase activity through robust in vitro experiments, establishing its distinctiveness from known prokaryotic deacetylases. Additionally, the authors elucidate CobQ's potential cooperation with other deacetylases in vivo to regulate bacterial cellular processes. Furthermore, the study highlights CobQ's significance in the regulation of acetylation within prokaryotic cells.

Weaknesses:

The problem I raised has been well resolved. I have no further questions.

Reviewer #1 (Public review):

Summary:

The authiors show that SVZ derived astrocytes respond to a middle carotid artery occlusion (MCAO) hypoxia lesion by secreting and modulating hyaluronan at the edge of the lesion (penumbra) and that hyaluronin is a chemoattractant to SVZ astrocytes. They use lineage tracing of SVZ cells to determine their origin. They also find that SVZ derived astrocytes express Thbs-4 but astrocytes at the MCAO-induced scar do not. Also, they demonstrate that decreased HA in the SVZ is correlated with gliogenesis. While much of the paper is descriptive/correlative they do overexpress Hyaluronan synthase 2 via viral vectors and show this is sufficient to recruit astrocytes to the injury. Interestingly, astrocytes preferred to migrate to the MCAO than to the region of overexpressed HAS2.

Strengths:

The field has largely ignored the gliogenic response of the SVZ, especially with regards to astrocytic function. These cells and especially newborn cells may provide support for regeneration. Emigrated cells from the SVZ have been shown to be neuroprotective via creating pro-survival environments, but their expression and deposition of beneficial extracellular matrix molecules is poorly understood. Therefore, this study is timely and important. The paper is very well written and the flow of results logical.

Comments on revised version:

The authors have addressed my points and the paper is much improved. Here are the salient remaining issues that I suggest be addressed.

The authors have still not shown, using loss of function studies, that Hyaluronan is necessary for SVZ astrogenesis and or migration to MCAO lesions.

(1) The co-expression of EGFr with Thbs4 and the literature examination is useful.

(2) Too bad they cannot explain the lack of effect of the MCAO on type C cells. The comparison with kainate-induced epilepsy in the hippocampus may or may not be relevant.

(3) Thanks for including the orthogonal confocal views in Fig S6D.

(4) The statement that "BrdU+/Thbs4+ cells mostly in the dorsal area" and therefore they mostly focused on that region is strange. Figure 8 clearly shows Thbs4 staining all along the striatal SVZ. Do they mean the dorsal segment of the striatal SVZ or the subcallosal SVZ? Fig. 4b and Fig 4f clearly show the "subcallosal" area as the one analysed but other figures show the dorsal striatal region (Fig. 2a). This is important because of the well-known embryological and neurogenic differences between the regions.

(5) It is good to know that the harsh MCAO's had already been excluded.

(6) Sorry for the lack of clarity - in addition to Thbs4, I was referring to mouse versus rat Hyaluronan degradation genes (Hyal1, Hyal2 and Hyal3) and hyaluronan synthase genes (HAS1 and HAS2) in order to address the overall species differences in hyaluronan biology thus justifying the "shift" from mouse to rat. You examine these in the (weirdly positioned) Fig. 8h,i. Please add a few sentences on mouse vs rat Thbs4 and Hyaluronan relevant genes.

(7) Thank you for the better justification of using the naked mole rat HA synthase.

Reviewer #1 (Public review):

When you search for something, you need to maintain some representation (a "template") of that target in your mind/brain. Otherwise, how would you know what you were looking for? If your phone is in a shocking pink case, you can guide your attention to pink things based on a target template that includes the attribute 'pink'. That guidance should get you to the phone pretty effectively if it is in view. Most real-world searches are more complicated. If you are looking for the toaster, you will make use of your knowledge of where toasters can be. Thus, if you are asked to find a toaster, you might first activate a template of a kitchen or a kitchen counter. You might worry about pulling up the toaster template only after you are reasonably sure you have restricted your attention to a sensible part of the scene.

Zhou and Geng are looking for evidence of this early stage of guidance by information about the surrounding scene in a search task. They train Os to associate four faces with four places. Then, with Os in the scanner, they show one face - the target for a subsequent search. After an 8 sec delay, they show a search display where the face is placed on the associated scene 75% of the time. Thus, attending to the associated scene is a good idea. The questions of interest are "When can the experimenters decode which face Os saw from fMRI recording?" "When can the experimenters decode the associated scene?" and "Where in the brain can the experimenters see evidence of this decoding? The answer is that the face but not the scene can be read out during the face's initial presentation. The key finding is that the scene can be read out (imperfectly but above chance) during the subsequent delay when Os are looking at just a fixation point. Apparently, seeing the face conjures up the scene in the mind's eye.

This is a solid and believable result. The only issue, for me, is whether it is telling us anything specifically about search. Suppose you trained Os on the face-scene pairing but never did anything connected to the search. If you presented the face, would you not see evidence of recall of the associated scene? Maybe you would see the activation of the scene in different areas and you could identify some areas as search specific. I don't think anything like that was discussed here.

You might also expect this result to be asymmetric. The idea is that the big scene gives the search information about the little face. The face should activate the larger useful scene more than the scene should activate the more incidental face, if the task was reversed. That might be true if the finding is related to a search where the scene context is presumed to be the useful attention guiding stimulus. You might not expect an asymmetry if Os were just learning an association.

It is clear in this study that the face and the scene have been associated and that this can be seen in the fMRI data. It is also clear that a valid scene background speeds the behavioral response in the search task. The linkage between these two results is not entirely clear but perhaps future research will shed more light.

It is also possible that I missed the clear evidence of the search-specific nature of the activation by the scene during the delay period. If so, I apologize and suggest that the point be underlined for readers like me.

Reviewer #1 (Public review):

Summary:

Ren et al developed a novel computational method to investigate cell evolutionary trajectory for scRNA-seq samples. This method, MGPfact, estimates pseudotime and potential branches in the evolutionary path through explicitly modeling the bifurcations in a Gaussian process. They benchmarked this method using synthetic as well as real world samples and showed superior performance for some of the tasks in cell trajectory analysis. They further demonstrated the utilities of MGPfact using single cell RNA-seq samples derived from microglia or T cells and showed that it can accurately identify the differentiation timepoint and uncover biologically relevant gene signatures.

Strengths:

Overall I think this is a useful new tool that could deliver novel insights for the large body of scRNA-seq data generated in the public domain. The manuscript is written is a logical way and most parts of the method are well described.

Comments on revisions:

In this revision, the authors have sufficiently addressed all of my concerns. I don't have any follow-up comments.

Reviewer #1 (Public review):

Summary:

Kang et al. provide the first experimental insights from holographic stimulation of auditory cortex. Using stimulation of functionally-defined ensembles, they test whether overactivation of a specific subpopulation biases simultaneous and subsequent sensory-evoked network activations.

Strengths:

The investigators use a novel technique to investigate the sensory response properties in functionally defined cell assemblies in auditory cortex. These data provide the first evidence of how acutely perturbing specific frequency-tuned neurons impacts the tuning across a broader population.

Weaknesses:

I have several main concerns about the interpretation of these data:<br /> (1) The premise of the paper suggests that sensory responses are noisy at the level of neurons, but that population activity is reliable and that different neurons may participate in sensory coding on different trials. However, no analysis related to single trial variance or overall stability of population coding is provided. Specifically, showing that population activity is stable across trials in terms of total activity level or in some latent low dimensional representation would be required to support the concept of "homeostatic balancing".<br /> (2) Rebalancing would predict either that the responses of stimulated neurons would remain A) elevated after stimulation due to a hebbian mechanism or B) suppressed due to high activity levels on previous trials, a homeostatic mechanism. The authors report suppression in targeted neurons after stimulation blocks, but this appears similar to all other non-stimulated neurons. How do the authors interpret the post-stimulation effect in stimulated neurons?<br /> (3) The authors suggest that ACtx is different from visual cortex in that neurons with different tuning properties are intermingled. While that is true at the level of individual neurons, there is global order, as demonstrated by the authors own widefield imaging data and others at the single cell level (e.g. Tischbirek et al. 2019). Generally, distance is dismissed as a variable in the paper, but this is not convincing. Work across multiple sensory systems, including the authors own work, has demonstrated that cortical neuron connectivity is not random but varies as a function of distance (e.g. Watkins et al. 2014). Better justification is needed for the spatial pattern of neurons that were chosen for stimulation. Further, analyses that account for center of mass of stimulation, rather than just the distance from any stimulated neuron would be important to any negative result related to distance.<br /> (4) Data curation and presentation: Broadly, the way the data were curated and plotted makes it difficult to determine how well-supported the authors claims are. In terms of curation, the removal of outliers 3 standard deviations above the mean in the analysis of stimulation effects is questionable. Given the single-cell stimulation data presented in Figure 1, the reader is led to believe that holographic stimulation is quite specific. However, the justification for removing these outliers is that there may be direct stimulation 20-30 um from the target. Without plotting and considering the outliers as well, it is difficult to understand if these outsized responses are due to strong synaptic connections with neighboring neurons or rather just direct off-target stimulation. Relatedly, data presentation is limited to the mean + SEM for almost all main effects and pre-post stimulation effects are only compared indirectly. Whether stimulation effects are driven by just a few neurons that are particularly suppressed or distinct populations which are suppressed or enhanced remains unclear.

Reviewer #1 (Public review):

Summary:<br /> This work done by Huang et.al. revealed the complex regulatory functions and transcription network of 172 unknown transcription factors of Pseudomonas aeruginosa PAO1. The authors utilized ChIP-seq to profile TFs binding site information across the genome, demonstrating diverse regulatory relationships among them via hierarchical networks with three levels. They further constructed thirteen ternary regulatory motifs in small subs and co-association atlas with 7 core associated clusters. The study also uncovered 24 virulence-related master regulators. The pan-genome analysis uncovered both the conservation and evolution of TFs with P. aeruginosa complex and related species. Furthermore, they established a web-based database combining both existing and novel data from HT-SELEX and ChIP-seq to provide TF binding site information. This study offered valuable insights into studying transcription regulatory networks in P. aeruginosa and other microbes.

Strengths:<br /> The results are presented with clarity, supported by well-organized figures and tables that not only illustrate the study's findings but also enhance the understanding of complex data patterns.

Weaknesses:<br /> The results of this manuscript are mainly presented in systematic figures and tables. Some of the results need to be discussed as an illustration how readers can utilize these datasets.

Reviewer #1 (Public review):

In this work, Urbanska and colleagues use a machine-learning based crossing of mechanical characterisations of various cells in different states and their transcriptional profiles. Using this approach, they identify a core set of five genes that systematically vary together with the mechanical state of the cells, although not always in the same direction depending on the conditions. They show that the combined transcriptional changes in this gene set is strongly predictive of a change in the cell mechanical properties, in systems that were not used to identify the genes (a validation set). Finally, they experimentally after the expression level of one of these genes, CAV1, that codes for the caveolin 1 protein, and show that, in a variety of cellular systems and contexts, perturbations in the expression level of CAV1 also induce changes in cell mechanics, cells with lower CAV1 expression being generally softer.

Overall the approach seems accessible, sound and is well described. My personal expertize is not suited to judge its validity, novelty or relevance, so I do not make comments on that. The results it provides seem to have been thoroughly tested by the authors (using different types of mechanical characterisations of the cells) and to be robust in their predictive value. The authors also show convincingly that one of the genes they identified, CAV1, is not only correlated with the mechanical properties of cells, but also that changing its expression level affects cell mechanics. At this stage, the study appears mostly focused on the description and validation of the methodological approach, and it is hard to really understand what the results obtain really mean, the importance of the biological finding - what is this set of 5 genes doing in the context of cell mechanics? Is it really central, or is it just one of the set of knobs on which the cell plays - and it is identified by this method because it is systematically modulated but maybe, for any given context, it is not the dominant player - all these fundamental questions remain unanswered at this stage. On one hand, it means that the study might have identified an important novel module of genes in cell mechanics, but on the other hand, it also reveals that it is not yet easy to interpret the results provided by this type of novel approach.

Comments on revisions:

In their point-by-point answer, the authors did a great effort to provide pedagogical answers that clarified most of the points I had raised. They also did more analysis, some of which are included as supplementary data, and added a few sentences to the main text and discussion. As far as I am concerned, I see no particular issue with the revised article. I think it will be interesting both as a new type of approach in mechanobiology, and also as a motivation for more experimentally oriented labs to test the hypothesis proposed in the article and the 'module' they found.

Reviewer #1 (Public review):

Strengths:

This is an interesting topic and a novel theme. The visualisations and presentation are to a very high standard. The Introduction is very well-written and introduces the main concepts well, with a clear logical structure and good use of the literature. The methods are detailed and well described and written in such a fashion that they are transparent and repeatable.

Weaknesses:

I only have one major issue, which is possibly a product of the structure requirements of the paper/journal. This relates to the Results and Discussion, line 91 onwards. I understand the structure of the paper necessitates delving immediately into the results, but it is quite hard to follow due to a lack of background information. In comparison to the Methods, which are incredibly detailed, the Results in the main section reads as quite superficial. They provide broad overviews of broad findings but I found it very hard to actually get a picture of the main results in its current form. For example, how the different species factor in, etc.

Reviewer #1 (Public review):

Summary:

The mechanism by which WNT signals are received and transduced into the cell has been the topic of extensive research. Cell surface levels of the WNT receptors of the FZD family are subject to tight control and it's well established that the transmembrane ubiquitin ligases ZNRF3 and RNF43 target FZDs for degradation and that proteins of the R-spondin family block this effect. This manuscript explores the role that WNT proteins play in receptor internalization, recycling and degradation, and the authors provide evidence that WNTs promote interactions of FZD with the ubiquitin ligases. Using cells mutant in all 3 DVL genes, the authors demonstrate that this effect of WNT on FZD is DVL-independent.

Strengths:

Overall, the data are of good quality and support the authors' hypothesis. Strengths of this study are the use of CRISPR-mutated cell lines to establish genetic requirements for the various components. The finding that FZD internalization and degradation is WNT dependent and does not involve DVL is novel.

Weaknesses:

Weaknesses of the work include a heavy reliance on overexpression and monitoring the effects in a single cell line, HEK293. In addition, the claim of specificity - only FZD5 and FZD8 participate in this process - is not strongly supported.

Reviewer #1 (Public review):

Summary:

This manuscript aimed to study the role of Rudhira (also known as Breast Carcinoma Amplified Sequence 3), an endothelium-restricted microtubules-associated protein, in regulating of TGFβ signaling. The authors demonstrate that Rudhira is a critical signaling modulator for TGFβ signaling by releasing Smad2/3 from cytoskeletal microtubules and how that Rudhira is a Smad2/3 target gene. Taken together, the authors provide a model of how Rudhira contributes to TGFβ signaling activity to stabilize the microtubules, which is essential for vascular development.

Strengths:

The study used different methods and techniques to achieve aims and support conclusions, such as Gene Ontology analysis, functional analysis in culture, immunostaining analysis, and proximity ligation assay. This study provides unappreciated additional layer of TGFβ signaling activity regulation after ligand-receptor interaction.

Weaknesses:

(1) It is unclear how current findings provide a better understanding of Rudhira KO mice, which the authors published some years ago.

(2) Why do they use HEK cells instead of SVEC cells in Fig 2 and 4 experiments?

(3) A model shown in Fig 5E needs improvement to grasp their findings easily.

Comments on revised version:

The authors have adequately responded to the reviewers' concerns.